The HPA Axis Clock: Circadian Regulation of Glucocorticoids and ACTH in Health and Disease

Abstract

This article synthesizes current knowledge on the intricate circadian regulation of the hypothalamic-pituitary-adrenal (HPA) axis, focusing on adrenocorticotropic hormone (ACTH) and glucocorticoids. It explores the molecular mechanisms driving their 24-hour rhythms, from core clock genes to systemic synchronizers. The review details methodological approaches for assessing HPA axis dynamics, including ACTH stimulation tests and mathematical modeling, and examines the profound consequences of circadian disruption in conditions like Cushing syndrome and metabolic disorders. Finally, it discusses emerging diagnostic strategies and therapeutic interventions aimed at correcting circadian HPA axis dysfunction, providing a comprehensive resource for researchers and drug development professionals in chronobiology and endocrinology.

Unraveling the Core Clock: Molecular and Systemic Regulation of HPA Axis Rhythmicity

The suprachiasmatic nucleus (SCN) of the hypothalamus functions as the principal circadian pacemaker in mammals, coordinating near-24-hour rhythms in physiology and behavior. This review details the molecular, cellular, and circuit mechanisms by which the SCN integrates environmental light signals to synchronize peripheral oscillators throughout the body, with a specialized focus on the hypothalamic-pituitary-adrenal (HPA) axis. We explore how the SCN orchestrates the circadian rhythm of glucocorticoid release—a critical hormonal output that peaks at the onset of the active phase to mobilize energy and prepare the organism for daily challenges. The synthesis of recent research presented herein highlights the hierarchical organization of the circadian system, the specific neural pathways connecting the SCN to glucocorticoid release, and the experimental methodologies enabling these discoveries. Furthermore, we discuss the translational implications of this knowledge for the development of chronotherapeutic strategies targeting conditions of circadian and HPA axis dysregulation.

Circadian rhythms are endogenous, self-sustained oscillations with a period of approximately 24 hours that persist in the absence of external time cues [1]. In mammals, the circadian system is organized in a hierarchical manner, with the SCN serving as the master pacemaker that synchronizes subordinate clocks in peripheral tissues and other brain regions [2]. This temporal coordination is essential for health, as desynchronization between internal clocks and the external environment is linked to various disorders, including metabolic syndrome, cardiovascular disease, and neuropsychiatric conditions [1] [3].

The SCN achieves its synchronizing function through a combination of neuronal and hormonal outputs. It receives direct photic input from the retina, which allows it to entrain to the external light-dark cycle. In turn, it coordinates critical physiological rhythms, including the sleep-wake cycle, body temperature, and hormone secretion [1] [4]. Among these rhythmic hormonal outputs, the daily secretion of glucocorticoids (cortisol in humans, corticosterone in rodents) is a key signal for communicating timing information from the SCN to peripheral tissues [5] [6].

Neuroanatomy and Molecular Timekeeping of the SCN

Functional Organization of the SCN

The SCN is a bilaterally paired nucleus located in the anterior hypothalamus, above the optic chiasm, and consists of approximately 20,000 neurons on each side of the third ventricle [2] [4]. It is functionally and anatomically divided into two primary subregions:

• Ventral Core (Retinorecipient Zone): This region receives direct photic input from the retina via the retinohypothalamic tract (RHT). The primary neurons in this core are characterized by the expression of vasoactive intestinal peptide (VIP). These neurons are crucial for the entrainment and synchronization of the entire SCN network [2].
• Dorsal Shell: This region contains neurons that primarily express arginine vasopressin (AVP). These cells show robust autonomous circadian activity and are major sources of rhythmic output from the SCN to other brain regions [2].

Beyond this neuronal framework, recent research has highlighted the essential role of SCN astrocytes. These glial cells exhibit circadian rhythms in calcium activity and clock gene expression that are antiphase to neuronal rhythms. Crucially, astrocyte clocks can sustain behavioral rhythms even when the clock in neurons is genetically deficient, establishing them as active participants in the SCN timekeeping machinery [2].

The Molecular Clockwork

At the core of the SCN's timekeeping ability is the transcriptional-translational feedback loop (TTFL), which generates approximately 24-hour molecular oscillations [4].

Figure 1: The Core Molecular Clockwork. The TTFL consists of interacting positive and negative feedback loops. The CLOCK-BMAL1 heterodimer activates transcription of Per, Cry, and Rev-Erbα genes. PER and CRY proteins form a complex that translocates to the nucleus to inhibit CLOCK-BMAL1 activity. REV-ERBα represses Bmal1 transcription, creating a stabilizing loop. This cycle takes approximately 24 hours.

The molecular clock is not perfectly 24 hours and requires daily resetting. The primary zeitgeber for this entrainment is light. Photons captured by intrinsically photosensitive retinal ganglion cells (ipRGCs) containing melanopsin trigger glutamate release from the RHT onto SCN VIP neurons. This activates a Ca2+/cAMP signaling cascade, leading to CREB-mediated transcription of core clock genes like Per1 and Per2, thereby adjusting the phase of the molecular clock [2] [4].

Neural Circuitry of Systemic Synchrony: The SCN's Control of Glucocorticoid Rhythms

The SCN regulates the circadian release of glucocorticoids through a sophisticated dual-route circuitry that converges on the adrenal gland. This system ensures that glucocorticoid levels peak at the beginning of the active phase (morning in humans, evening in nocturnal rodents) to mobilize energy resources [5] [7] [6].

The HPA Axis Route

The classic hypothalamic-pituitary-adrenal (HPA) axis is under direct circadian control from the SCN. The pivotal node in this pathway is the paraventricular nucleus of the hypothalamus (PVN), which contains corticotropin-releasing hormone (CRH) neurons [5] [7].

• Neural Projections: The SCN, primarily through its AVP-expressing neurons, sends inhibitory projections to the PVN. These connections rhythmically inhibit CRH neurons, contributing to the low trough of glucocorticoids during the rest phase [5] [7].
• PVNCRH Neurons as a Circadian Node: Recent research has demonstrated that PVNCRH neurons themselves possess intrinsic circadian properties. They exhibit daily rhythms in Per2 expression (peaking around midday) and calcium activity (peaking about three hours later). The loss of the core clock gene Bmal1 specifically in CRH neurons results in arrhythmic calcium activity and dramatically reduces the amplitude and precision of the daily corticosterone rhythm [7].

The Autonomic Nervous System Route

In parallel to the HPA axis, the SCN regulates adrenal sensitivity through pre-autonomic neurons in the PVN that project to the intermediolateral column of the spinal cord. This multisynaptic autonomic pathway ultimately innervates the adrenal gland, setting its sensitivity to adrenocorticotropic hormone (ACTH) [5] [6]. This explains why the adrenal can respond to ACTH with varying efficacy across the 24-hour day.

Figure 2: Neural Circuitry of Circadian Glucocorticoid Control. The SCN integrates light information and uses dual pathways to regulate glucocorticoid rhythm. It directly inhibits PVN CRH neurons via AVP+ projections (HPA axis route) and simultaneously regulates adrenal sensitivity via a pre-autonomic pathway. The resulting rhythmic glucocorticoid release then helps entrain peripheral clocks.

The rhythmic glucocorticoid output, in turn, functions as a potent entrainment signal for peripheral clocks in tissues like the liver, thus completing a feedback loop that ensures systemic circadian synchrony [5] [4].

Experimental Protocols for Investigating SCN Function

Understanding the SCN's role has been propelled by advanced methodological approaches. The following table summarizes key experimental models and their applications in circadian research.

Table 1: Key Experimental Models in Circadian Rhythm Research

Model/System	Key Application	Principal Findings	Reference
Prenatal GC Exposure (Mouse)	Modeling developmental programming of HPA axis & circadian system	Alters hippocampal neurogenesis; induces depression-like behavior; disrupts circadian activity patterns.	[3]
SCN-VIP Neuron Manipulation (Mouse)	Dissecting SCN output to PVNCRH neurons	Chemogenetic inhibition increases corticosterone; activation entrains PVN clock gene rhythms by inhibiting PVNCRH neurons.	[7]
Bmal1 Knockdown in CRH Neurons (Mouse)	Determining role of local clock in PVNCRH neurons	Abolishes PVNCRH calcium rhythm and reduces amplitude/precision of corticosterone peak.	[7]
DNA Methylation Inhibition (Rodent)	Probing epigenetic plasticity of SCN clock	DNMT inhibitors (RG108, SGI-1027) block period after-effects of light, showing role of DNA methylation in long-term clock plasticity.	[8]

Detailed Protocol: In Vivo Fiber Photometry for Recording PVNCRH Neuron Activity

This protocol, adapted from a key study [7], allows for longitudinal, cell-type-specific monitoring of circadian neuronal activity in vivo.

Objective: To record daily rhythms in clock gene expression and calcium activity from PVNCRH neurons in freely-behaving mice.

Materials:

• Animals: Heterozygous Crh-IRES-Cre knock-in mice.
• Viral Vectors:
  • For gene expression: Cre-dependent AAV encoding Per2.DIO.Venus fluorescent reporter.
  • For calcium activity: Cre-dependent AAV encoding GCaMP6s.
• Surgical Equipment: Stereotaxic apparatus, fiber optic cannula.
• Recording System: Fiber photometry system capable of dual-wavelength excitation (for calcium-dependent and isosbestic signals).

Methodology:

• Stereotaxic Surgery: Inject the appropriate Cre-dependent viral vector into the PVN of Crh-IRES-Cre mice. Implant a fiber optic cannula above the PVN.
• Post-operative Recovery: Allow ≥2 weeks for viral expression and recovery.
• Longitudinal Recording: House mice in a controlled light cycle (e.g., 12:12 Light/Dark). Record signals continuously over multiple days.
  • For Per2 rhythm: Measure Venus fluorescence every 15 minutes for 60 seconds.
  • For calcium rhythm: Record GCaMP6s signals for 10 minutes every hour, using both calcium-dependent and isosbestic excitation wavelengths to control for motion artifacts and photobleaching.
• Data Analysis: Calculate the rhythm period, amplitude, and phase (peak time) of the fluorescence signals. For calcium data, analyze event frequency and integrated calcium levels.

Key Insight: This approach revealed that PVNCRH neurons exhibit synchronous daily rhythms, with Per2 expression peaking around midday (ZT5.9) and calcium activity peaking in the mid-afternoon (ZT7.1) [7].

The Scientist's Toolkit: Essential Research Reagents

Table 2: Key Research Reagent Solutions for Circadian and SCN Research

Reagent / Tool	Category	Primary Function / Application	Example Use
Crh-IRES-Cre Mice	Genetic Model	Enables cell-type-specific targeting and manipulation of CRH-expressing neurons.	Studying PVNCRH neuron-specific functions [7].
DIO (Double-floxed Inverted Orientation) Vectors	Viral Vector	Ensures Cre-dependent expression of transgenes (e.g., reporters, actuators).	Expressing GCaMP6s or Per2.Venus specifically in CRH neurons [7].
GCaMP6s	Biosensor	Genetically encoded calcium indicator for monitoring neuronal activity in real-time.	Recording daily rhythms in PVNCRH neuron calcium dynamics [7].
Chemogenetic Tools (DREADDs)	Neuromodulation	Chemically activate or inhibit specific neuronal populations with high temporal precision.	Probing the causal role of SCNVIP neurons in glucocorticoid rhythm [7].
DNA Methyltransferase Inhibitors (e.g., RG108)	Pharmacological Tool	Inhibits DNA methylation to probe epigenetic mechanisms of circadian plasticity.	Blocking light-induced period after-effects in the SCN [8].
Nanomaterial Carriers (e.g., Liposomes, PNPs)	Drug Delivery	Enables targeted, sustained, or stimuli-responsive drug release for chronotherapy.	Potential for circadian-timed drug delivery to specific tissues [9].

Implications for Chronotherapy and Drug Development

The intricate relationship between the SCN, glucocorticoid rhythms, and peripheral physiology provides a compelling rationale for circadian medicine. This field comprises two main strategies:

• Direct Rhythm Modulation: Correcting the underlying circadian disruption using chronobiotics (e.g., melatonin agonists) or by targeting core clock components [9].
• Chronotherapy: Aligning the timing of conventional drug administration with circadian rhythms in drug pharmacokinetics, pharmacodynamics, and disease processes to maximize efficacy and minimize side effects [9] [10].

Emerging technologies are poised to revolutionize this field. Nanomaterial-enabled drug delivery systems (e.g., liposomes, polymeric nanoparticles) offer the potential for sustained and targeted release of chronobiotics or conventional drugs, overcoming the limitations of complex dosing schedules [9]. Furthermore, the development of programmable chronogenetic gene circuits represents a frontier in autonomous therapy. For example, engineered cells can be designed to produce a biologic drug like interleukin-1 receptor antagonist (IL-1Ra) with a specific circadian phase, protecting against inflammatory damage in conditions like arthritis in a self-regulated manner [10].

The SCN stands as the central node in a vast network of biological clocks, integrating light information to coordinate systemic physiology. Its regulation of the glucocorticoid rhythm via specific neural circuits to the PVN and adrenal gland is a paradigm of how the brain translates time of day into hormonal action. Disruptions to this precise system, whether through genetic, environmental, or developmental insults, can have profound consequences for physical and mental health. The continued elucidation of the SCN's mechanisms, powered by the advanced experimental tools detailed in this review, is paving the way for a new era of chronotherapeutics that respect the intrinsic temporal order of human biology.

The hypothalamic-pituitary-adrenal (HPA) axis represents a fundamental neuroendocrine system that orchestrates the body's stress response and maintains physiological homeostasis. This sophisticated axis operates through a precise cascade of hormonal signals, initiating in the hypothalamus and culminating in the adrenal production of glucocorticoids (GCs)—cortisol in humans and corticosterone in rodents. The HPA axis is distinguished by its intricate feedback mechanisms, which ensure regulated hormone production and release, preventing both excessive and insufficient activity. Beyond its role in stress adaptation, the HPA axis is intrinsically coupled with the circadian system, creating a dynamic interface between endocrine function and daily biological rhythms that optimizes physiological processes according to anticipatory demands throughout the 24-hour cycle [11] [12].

The circadian regulation of glucocorticoids represents a critical dimension of HPA axis function, with GC secretion exhibiting robust 24-hour oscillations that peak at the onset of the active phase. This temporal patterning is not merely reactive but anticipatory, preparing the organism for upcoming metabolic, immune, and cognitive challenges associated with the sleep-wake cycle. The suprachiasmatic nucleus (SCN), the master circadian pacemaker in the hypothalamus, coordinates this rhythmicity through complex neural and hormonal outputs that synchronize the HPA axis with external light-dark cycles [12] [4]. Understanding the mechanistic interplay between the core HPA axis feedback loop and its circadian regulation provides crucial insights for developing chronotherapeutic interventions for endocrine disorders, mood disorders, and metabolic conditions where HPA axis dysregulation is implicated [11] [3].

Core Components of the HPA Axis Feedback Loop

The HPA axis operates through a sequential signaling cascade involving specific anatomical structures and their hormonal products, each subject to precise regulatory controls.

Hierarchical Hormonal Cascade

The HPA axis activation begins with corticotropin-releasing hormone (CRH) synthesis and secretion from parvocellular neurons of the paraventricular nucleus (PVN) of the hypothalamus. CRH is released into the hypophyseal portal system and transported to the anterior pituitary, where it stimulates corticotropes to produce and release adrenocorticotropic hormone (ACTH) into systemic circulation [12] [13]. ACTH then acts on the adrenal cortex (specifically the zona fasciculata) to stimulate the synthesis and secretion of glucocorticoids (GCs), completing the efferent limb of the axis [14] [15].

Table 1: Core Components of the HPA Axis

Anatomical Structure	Key Secretory Products	Primary Functions
Hypothalamus (PVN)	Corticotropin-Releasing Hormone (CRH)	Initiates HPA axis activation; stimulated by stress and circadian signals
Anterior Pituitary	Adrenocorticotropic Hormone (ACTH)	Stimulates glucocorticoid production and secretion from adrenal cortex
Adrenal Cortex	Glucocorticoids (Cortisol in humans)	Regulates metabolism, immune function, and stress response; provides negative feedback

Feedback Regulation Mechanisms

The HPA axis employs a sophisticated negative feedback system to maintain homeostasis. Circulating glucocorticoids act at multiple levels to inhibit further HPA axis activity:

• Fast feedback (non-genomic): Occurs within minutes via membrane-associated glucocorticoid receptors [5]
• Delayed feedback (genomic): Occurs over hours via classical nuclear glucocorticoid receptor (GR) mechanisms, reducing CRH and ACTH synthesis [5] [13]

Glucocorticoids exert negative feedback primarily through glucocorticoid receptors (GR) in the hippocampus, hypothalamus, and pituitary, suppressing CRH and ACTH production and maintaining physiological set-points for axis activity [13]. This regulatory loop ensures that GC levels remain within an appropriate range for current physiological demands, preventing the detrimental effects of chronic elevation associated with conditions like Cushing's syndrome or depression [11] [13].

Circadian Regulation of the HPA Axis

The HPA axis exhibits robust circadian rhythmicity that is critically entrained by the central circadian pacemaker in the suprachiasmatic nucleus (SCN). This temporal regulation ensures that glucocorticoid secretion aligns with anticipated daily demands.

The Suprachiasmatic Nucleus (SCN) as Master Regulator

The SCN serves as the master circadian clock, receiving direct photic input from the retina via the retinohypothalamic tract and synchronizing peripheral oscillators throughout the body [4]. The SCN regulates HPA axis activity through neural projections to the PVN, primarily using arginine-vasopressin (AVP) as a neurotransmitter to influence CRH release and consequently modulate the rhythmic pattern of glucocorticoid secretion [12] [4]. This neural connection allows the light-dark cycle to directly shape the timing of HPA axis activity, with the SCN generating a circadian signal that promotes increased HPA axis activity before the active phase—morning in diurnal humans and evening in nocturnal rodents [12].

The Adrenal Gland as a Peripheral Clock

The adrenal gland contains an intrinsic circadian clock that gates its sensitivity to ACTH [15]. This local timing mechanism explains why similar ACTH concentrations can produce different cortisol outputs depending on the time of day, a phenomenon known as ACTH gating [15]. The adrenal clock regulates the expression of genes involved in steroid biosynthesis, creating a daily window of heightened responsiveness to ACTH that coincides with the pre-active phase peak in GC secretion [15]. This gating mechanism ensures appropriately timed glucocorticoid release even in the absence of strong ACTH rhythms [15].

Diagram: Circadian Regulation of HPA Axis. The SCN integrates light information and regulates HPA axis activity via neural and hormonal pathways. The adrenal gland's intrinsic clock gates ACTH sensitivity.

Ultradian Rhythms in Glucocorticoid Secretion

Superimposed on the 24-hour circadian rhythm are ultradian rhythms characterized by pulsatile GC secretion approximately every 60-90 minutes [12] [16]. These high-frequency oscillations are not merely episodic release but represent a fundamental mode of HPA axis operation with significant implications for gene regulation and cellular responses to glucocorticoids [12]. The pulsatile pattern is thought to prevent receptor desensitization and allow for more dynamic regulation of target gene expression compared to sustained hormone exposure [12].

Molecular Mechanisms of Glucocorticoid Signaling and Clock Interactions

The interplay between glucocorticoid signaling and the molecular circadian clock occurs at multiple levels, creating a bidirectional relationship that ensures temporal coordination of physiological processes.

Glucocorticoid Receptor Signaling

Glucocorticoids exert their effects primarily through the glucocorticoid receptor (GR), a ligand-activated transcription factor that resides in the cytoplasm complexed with chaperone proteins including HSP90 and FKBP5 in its unactivated state [5]. Upon glucocorticoid binding, GR undergoes conformational changes, dissociates from chaperone proteins, dimerizes, and translocates to the nucleus where it binds to glucocorticoid response elements (GREs) in target genes to regulate their transcription [5] [16]. GR signaling can also occur through non-genomic mechanisms via membrane-associated receptors and through protein-protein interactions with other transcription factors such as NF-κB and AP-1, providing rapid effects and contextual regulation of inflammatory pathways [16].

The mineralocorticoid receptor (MR) also binds glucocorticoids with high affinity and is primarily occupied at basal glucocorticoid levels, particularly in the hippocampus where it tonically influences HPA axis tone [5]. The relative balance between MR and GR activation helps determine the set-point for HPA axis feedback sensitivity, with MR mediating pro-active feedback and GR mediating reactive feedback [5].

Bidirectional Interaction with Clock Genes

The molecular circadian clock consists of interlocking transcriptional-translational feedback loops centered on core clock genes. The CLOCK-BMAL1 heterodimer activates transcription of Per and Cry genes, whose protein products subsequently repress CLOCK-BMAL1 activity, completing a approximately 24-hour cycle [4] [16].

Glucocorticoids directly influence this molecular clock through GREs present in promoter regions of clock genes including Per1, Per2, and Nfil3 [16]. This allows glucocorticoids to reset or synchronize peripheral clocks, positioning them as key zeitgebers (time-giving signals) for tissues throughout the body [12]. Conversely, clock components can regulate glucocorticoid signaling, as demonstrated by REV-ERBα influencing expression of GR chaperone proteins and thereby modulating cellular sensitivity to glucocorticoids [16].

Table 2: Molecular Interactions Between Glucocorticoid Signaling and Clock Components

Clock Element	Interaction with Glucocorticoid Signaling	Functional Outcome
CLOCK-BMAL1	Regulates expression of steroidogenic enzymes (StAR)	Modulates adrenal GC production capacity
PER2	Mutation alters corticosterone response to immune challenge	Disrupted circadian GC rhythm and stress response
REV-ERBα	Regulates expression of GR chaperone proteins	Influences cellular sensitivity to GCs
NFIL3	Contains GREs in promoter region	Directly regulated by GC signaling

Experimental Models and Research Methodologies

Advancements in understanding HPA axis function and circadian regulation have relied on sophisticated experimental models and methodological approaches that enable precise dissection of this complex system.

In Vivo and In Vitro Models

Animal models have been instrumental in elucidating HPA axis regulation. Transgenic mouse models with tissue-specific deletion of GR have revealed its critical role in feedback regulation [16]. Studies in Per2 mutant mice demonstrated altered corticosterone responses to immune challenge and disrupted circadian GC rhythms, highlighting the importance of clock genes in HPA axis function [16]. Prenatal glucocorticoid exposure models have provided insights into developmental programming of the HPA axis, showing long-term effects on stress responsiveness and behavior [3].

The recent development of patient-derived organoids (PDOs) represents a significant advancement for studying human HPA axis pathology. PDOs from ACTH-secreting pituitary neuroendocrine tumors (PitNETs) have enabled drug screening and mechanistic studies, leading to identification of ceritinib as a potential therapeutic that suppresses ACTH production through Akt1/Nur77 signaling [14]. This model system provides unprecedented access to human tissue while maintaining pathophysiological characteristics of the original tumors.

Assessment Techniques and Methodologies

• Diurnal salivary cortisol profiling: This non-invasive method tracks free cortisol levels across multiple timepoints, providing insight into circadian rhythm patterns and feedback regulation in clinical populations [11]

• Circadian activity monitoring: Automated tracking of spontaneous locomotor activity reveals circadian rhythm alterations that may predict depression onset and treatment response [3]

• Neural tracing and optogenetics: These approaches map specific neural circuits connecting the SCN to PVN and adrenal gland, elucidating pathways for circadian regulation [4]

• Transcriptome profiling: RNA sequencing of adrenal tissue across the circadian cycle identifies rhythmically expressed genes in steroid biogenesis pathways [15]

• Multi-omics analyses: Integrated genomics, transcriptomics, and proteomics in PDO models reveal signaling pathways such as PI3K-Akt in ACTH regulation [14]

Diagram: Experimental Workflow for HPA Axis Research. Comprehensive approach integrating multiple model systems, interventions, and analytical methods.

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Research Reagents for HPA Axis and Circadian Research

Reagent/Category	Specific Examples	Research Applications
Receptor Antagonists	RU486 (GR antagonist), spironolactone (MR antagonist)	Dissecting receptor-specific contributions to HPA axis feedback
ACTH Detection Assays	ELISA, RIA, immunometric assays	Quantifying ACTH levels in plasma, tissue, and cell culture
Circadian Reporters	PER2::LUCIFERASE, Rev-Erbα-GFP	Real-time monitoring of circadian clock function in cells and tissues
CRH/AVP Ligands	CRF receptor agonists/antagonists, AVP analogs	Probing hypothalamic regulation of pituitary function
Kinase Pathway Modulators	Ceritinib (ALK inhibitor), Akt inhibitors	Investigating signaling pathways regulating ACTH production and cell growth [14]
Biological Rhythms Monitoring	Salivary cortisol kits, telemetry systems, activity monitoring	Assessing circadian patterns of HPA axis activity in humans and animals

Clinical Implications and Therapeutic Applications

Dysregulation of the HPA axis and its circadian rhythm contributes to numerous pathological conditions, making it a valuable target for therapeutic intervention.

Pathological Disruptions of HPA Axis Rhythmicity

Cushing's disease represents a state of HPA axis hyperactivity driven primarily by ACTH-secreting pituitary tumors (PitNETs), resulting in loss of circadian cortisol rhythm and pathological hypercortisolism [14]. Conversely, major depressive disorder (MDD) is frequently associated with HPA axis hyperactivity characterized by elevated cortisol, impaired feedback inhibition, and circadian rhythm disturbances [3] [13]. The developmental origins of health and disease (DOHaD) hypothesis posits that prenatal exposure to excess glucocorticoids or stress programs HPA axis function, increasing susceptibility to mood disorders, cognitive deficits, and metabolic conditions in adulthood [3].

Circadian disruption itself can drive HPA axis pathology, as evidenced by increased depression rates in shift workers and the association between blunted circadian activity rhythms and mood disorders [3]. The gut-brain axis further modulates HPA axis function, with gut dysbiosis promoting systemic inflammation that can impair feedback regulation and contribute to depressive symptomatology [13].

Chronotherapeutic Approaches and Drug Development

Understanding the circadian regulation of the HPA axis enables chronotherapeutic approaches that optimize treatment timing for enhanced efficacy and reduced side effects. Circadian-based interventions for depression include wake therapy, light therapy, and scheduled social rhythms that help resynchronize disrupted biological rhythms [3].

Recent drug development targeting ACTH-secreting PitNETs has identified ceritinib as a promising therapeutic candidate that suppresses both tumor growth and ACTH production by inhibiting Akt1-mediated regulation of Nur77, a transcriptional activator of POMC (ACTH precursor) [14]. This discovery emerged from patient-derived organoid screening approaches that more accurately model human disease, highlighting the power of advanced experimental systems for identifying targeted therapies.

The global ACTH market, projected to reach USD 1524.8 million in 2025, reflects ongoing therapeutic applications of ACTH for conditions including rheumatological, neurological, and nephrological disorders [17]. Advances in understanding ACTH regulation and HPA axis function may expand these clinical applications and inspire novel therapeutic strategies.

The HPA axis feedback loop represents a sophisticated neuroendocrine system that integrates neural, endocrine, and circadian signals to maintain physiological homeostasis and coordinate adaptive responses to stress. The bidirectional interaction between glucocorticoid signaling and the molecular circadian clock creates a temporal framework that optimizes physiological function across the 24-hour cycle. Disruption of this precise regulatory system contributes to numerous pathological conditions, highlighting the clinical importance of understanding HPA axis function in health and disease.

Future research directions include elucidating the specific neural circuits that connect the SCN to HPA axis components, developing more sophisticated tissue-specific and temporally controlled genetic models to dissect receptor function, and exploring the therapeutic potential of chronotherapeutics for endocrine and mood disorders. The application of multi-omics approaches and advanced bioengineering models such as patient-derived organoids will continue to provide unprecedented insights into HPA axis regulation and facilitate development of targeted therapies for conditions characterized by HPA axis dysregulation. As our understanding of this complex system deepens, so too will our ability to manipulate it for therapeutic benefit while preserving its essential functions in health and adaptation.

The circadian rhythm, an endogenous ~24-hour biological clock, is a fundamental physiological regulator that allows organisms to anticipate and adapt to daily environmental changes. At its core, this rhythm is generated by a cell-autonomous transcription-translation feedback loop (TTFL) involving a set of clock genes [18]. This molecular oscillator is hierarchically organized, with a master pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus synchronizing subsidiary clocks in nearly all peripheral tissues [19] [5] [20].

The circadian system exerts profound control over complex physiological functions, including the sleep-wake cycle, energy metabolism, and hormone secretion [18] [21]. A key hormonal output is the circadian rhythm of glucocorticoids (cortisol in humans, corticosterone in rodents) and their principal regulator, adrenocorticotropic hormone (ACTH) [5] [22] [23]. The SCN coordinates the rhythmic release of glucocorticoids through direct and indirect control of the hypothalamic-pituitary-adrenal (HPA) axis [5]. This intricate coupling ensures that glucocorticoid levels peak at the onset of the active phase, mobilizing energy resources and priming the body for anticipated activity [5].

Understanding the TTFL is therefore not merely a molecular biological exercise but is essential for grasping the temporal architecture of physiology. Disruption of these rhythms is increasingly linked to metabolic diseases, aging, and mood disorders, highlighting the translational importance of this field for therapeutic development [19] [20]. This whitepaper details the mechanisms of the TTFL, its relationship to glucocorticoid rhythms, and the experimental tools used to investigate it.

Molecular Architecture of the Core Circadian Clock

The self-sustaining circadian rhythm within individual cells arises from an autoregulatory transcriptional feedback loop. This TTFL consists of a primary negative feedback loop and a stabilizing auxiliary loop [20].

The Primary Negative Feedback Loop

The core TTFL is driven by a set of clock genes and their protein products:

• Positive Elements: The transcription factors CLOCK and BMAL1 (encoded by ARNTL) form a heterodimer (the BMAL1-CLOCK complex). This complex binds to E-box enhancer elements in the promoter regions of target genes, activating their transcription [20] [24].
• Negative Elements: Among the genes activated by BMAL1-CLOCK are Period (Per1, Per2, Per3) and Cryptochrome (Cry1, Cry2). After transcription and translation, PER and CRY proteins form heterodimers in the cytoplasm. Following post-translational modifications, these complexes translocate back into the nucleus, where they directly inhibit the transcriptional activity of the CLOCK-BMAL1 complex, thereby repressing their own transcription [20] [21].

This cycle of activation and repression generates approximately 24-hour oscillations in the transcription of clock-controlled genes (CCGs) and in the abundance of core clock proteins [18].

The Stabilizing Auxiliary Loop

A second, interlocking loop provides stability and allows for additional input signals.

• The CLOCK-BMAL1 complex also activates the transcription of nuclear receptor genes Rev-Erbα/β (Nr1d1/2) and Rorα/β/γ [20] [21].
• Their protein products, REV-ERB and ROR, compete to bind ROR response elements (RREs) in the promoter of the Bmal1 gene.
• ROR acts as a transcriptional activator, promoting Bmal1 expression.
• REV-ERB acts as a transcriptional repressor, suppressing Bmal1 expression. This RRE-mediated regulation creates a second feedback loop that stabilizes the core oscillator and fine-tunes the expression rhythm of Bmal1 [20].

Table 1: Core Components of the Mammalian Circadian TTFL

Component	Gene Symbol(s)	Function in TTFL	Phase of Peak Expression
BMAL1	ARNTL	Forms positive limb heterodimer; activates Per, Cry, Rev-Erb, Ror transcription	Dark/Light transition [18]
CLOCK	CLOCK	Forms positive limb heterodimer with BMAL1	Dark/Light transition [18]
Period	PER1, PER2, PER3	Forms negative limb; inhibits CLOCK-BMAL1 activity	Light/Dark transition [18]
Cryptochrome	CRY1, CRY2	Forms negative limb with PER; inhibits CLOCK-BMAL1	Light/Dark transition [18]
REV-ERB	NR1D1, NR1D2	Represses Bmal1 transcription; stabilizes loop	Light/Dark transition [18]
ROR	RORA, RORB, RORC	Activates Bmal1 transcription; stabilizes loop	Dark/Light transition [18]

The following diagram illustrates the core TTFL and its auxiliary loop:

Systemic Integration: The Circadian Rhythm of Glucocorticoids and ACTH

The molecular TTFL is embedded within a complex physiological system. One of the most crucial outputs of the circadian system is the rhythmic secretion of glucocorticoids, which in turn serves as a key systemic synchronizer for peripheral clocks [5].

The Hypothalamic-Pituitary-Adrenal (HPA) Axis

The SCN, the central pacemaker, regulates glucocorticoid release through multi-faceted control of the HPA axis:

• SCN to PVN: The SCN projects to the paraventricular nucleus (PVN) of the hypothalamus. It provides both inhibitory and stimulatory signals, involving neurotransmitters like arginine vasopressin (AVP), to regulate the release of corticotropin-releasing hormone (CRH) from the PVN [5].
• Pituitary and Adrenal Glands: CRH stimulates the anterior pituitary to secrete ACTH, which then acts on the adrenal cortex to stimulate the production and release of glucocorticoids [22].
• Autonomic Pathway: The SCN also influences the adrenal gland via autonomic nervous system pathways, which rhythmically modulate the adrenal's sensitivity to ACTH [5].

This multi-layered control results in a characteristic daily cortisol profile in humans, with levels peaking in the early morning around the wake-up time and reaching a nadir around midnight [22].

Bidirectional Interaction and Molecular Cross-Talk

The relationship between the clock and the HPA axis is profoundly bidirectional. Glucocorticoids are not merely a passive output; they function as potent entraining signals for peripheral clocks. The intracellular glucocorticoid receptor (GR), upon binding cortisol, dimerizes and translocates to the nucleus. There, it binds to glucocorticoid response elements (GREs) in the genome, including within clock genes such as Per1 and Per2, thereby resetting and synchronizing peripheral circadian rhythms [5].

This creates a critical metabolic-epigenetic link. Furthermore, clock components and GR signaling interact directly; for example, the clock protein BMAL1 can regulate GR expression, and REV-ERB can influence HPA axis activity [5] [21].

Table 2: Key Features of Glucocorticoid and ACTH Circadian Rhythms

Feature	Description	Physiological Impact
ACTH Rhythm	Driven by SCN via CRH release from PVN; subject to negative feedback by cortisol [22].	Directly controls rhythmic glucocorticoid production from adrenal cortex.
Glucocorticoid Rhythm	Peak at onset of active phase (morning in humans, evening in rodents) [5] [22].	Promotes glucose mobilization (gluconeogenesis, lipolysis) and primes cognitive function for active period [5].
Adrenal Sensitivity	Rhythmic sensitivity to ACTH, gated by local adrenal clock and autonomic innervation from SCN [5].	Sharpens the rhythm of glucocorticoid release, ensuring precise timing.
Feedback on Clocks	Glucocorticoids act via GR to entrain peripheral clocks by regulating clock gene expression (e.g., Per1) [5].	Synchronizes metabolic tissues (liver, muscle, fat) to the central SCN clock and feeding time.

The following diagram summarizes the regulatory pathways connecting the central clock to glucocorticoid release:

Investigating the Loop: Key Experimental Methodologies

Research into the circadian TTFL and its outputs relies on a suite of sophisticated molecular, cellular, and physiological techniques.

Transcriptomic Analysis of Circadian Gene Expression

A foundational method involves measuring time-dependent gene expression across tissues.

• Protocol Overview: Experimental subjects (e.g., rats, mice) are housed in controlled light-dark cycles. Multiple tissues (e.g., liver, muscle, adipose, lung) are collected at regular intervals (e.g., every 4-6 hours) over at least 24 hours. RNA is extracted from the tissues and analyzed using microarrays or RNA sequencing (RNA-Seq) [18].
• Key Insights: This approach has demonstrated that core clock genes oscillate synchronously across diverse peripheral tissues, while the majority of rhythmic gene expression is tissue-specific, dictated by local clock-controlled genes (CCGs) [18].

Measuring Hormonal Outputs (ACTH and Cortisol)

Assessing the rhythmic output of the HPA axis is critical for clinical and basic research.

• Sampling Protocol: Blood samples are collected at specific times, crucially considering the circadian phase (e.g., 8:00 AM for peak cortisol and a late-evening nadir). For human studies, salivary cortisol offers a non-invasive alternative [22] [23].
• Analysis: ACTH and cortisol levels are typically measured via immunoassay or electrochemiluminescence (e.g., Roche Cobas e601) [23]. The ACTH stimulation test is a diagnostic tool where synthetic ACTH is administered and the cortisol response is measured, used to assess adrenal function [22]. Recent research, however, has revealed significant flaws in this test's diagnostic value during sepsis, as it may fail to correctly identify adrenal insufficiency and can even trigger a lethal inflammatory response in mouse models [25].

Single-Timepoint Gene Expression in Chronotype Studies

To explore individual variations in circadian timing (chronotype), studies analyze clock gene expression at a fixed time.

• Protocol: As performed in the ChroNu study, participants are grouped by chronotype using questionnaires (e.g., Munich Chronotype Questionnaire). Blood is drawn at a fixed time (e.g., 7:00 AM), and RNA is isolated from specific immune cells like CD14+ monocytes. The expression of key clock genes (e.g., PER1, PER2, NR1D1) is quantified by reverse transcription quantitative PCR (RT-qPCR) [26].
• Application: This method tests whether individuals with late chronotypes exhibit a phase-delay in their peripheral clock gene expression compared to early types, even at a single timepoint [26].

The Scientist's Toolkit: Essential Research Reagents and Models

Table 3: Key Reagents and Models for Circadian Rhythm Research

Category / Item	Specific Examples	Function and Application
In Vivo Models	Wild-type C57BL/6J mice, SR-BI (Scarb1) null mice [25], Bmal1 knockout mice [21].	Used to study circadian behavior, metabolism, and gene function. SR-BI null mice are a model for relative adrenal insufficiency (RAI) [25].
Disease Induction	Cecal Ligation and Puncture (CLP) [25].	Standardized model for inducing polymicrobial sepsis, used to study circadian-immune-stress interactions and adrenal stress response.
Hormonal Assays	ACTH (Chemiluminescence Immunoassay) [25], Cortisol/Corticosterone (ELISA, ECLIA) [23], Melatonin (ELISA) [26].	Quantify circadian hormonal rhythms in plasma, serum, or saliva.
Gene Expression	RNA Isolation Kits (e.g., NucleoSpin RNA) [26], RT-qPCR reagents, Gene Expression Microarrays, RNA-Seq.	Measure rhythmic transcription of clock genes and CCGs in tissues and sorted cells.
Cell Separation	CD14+ Microbeads (e.g., Miltenyi Biotec) [26], AutoMACS Pro Separator.	Isolate specific cell populations (e.g., monocytes) from whole blood for cell-type-specific circadian analysis.
Pharmacological Tools	REV-ERB Agonists (e.g., SR9009) [20] [21], Synthetic ACTH (Cosyntropin) [25] [22], Glucocorticoid Receptor Modulators.	Probe clock function and HPA axis physiology; potential chronotherapeutic agents.
Mathematical Modeling	Differential Equation-based Models (e.g., Kim-Forger model) [18] [21].	Simulate TTFL dynamics, predict effects of perturbations, and integrate multi-scale data.

The transcription-translation feedback loop of clock genes represents a fundamental biological mechanism for timing cellular and physiological processes. Its intricate connection with the HPA axis, governing the circadian rhythm of glucocorticoids and ACTH, exemplifies how molecular cycles regulate systemic physiology. Disruption of this interplay is implicated in aging, metabolic syndrome, and mood disorders [19] [20]. Future research, leveraging the experimental tools and models detailed herein, will continue to decipher the complexities of this system, paving the way for novel chronotherapeutic strategies that align treatments with the body's internal clock to maximize efficacy and minimize side effects.

The circadian rhythm of glucocorticoids (GCs), including cortisol in humans and corticosterone in rodents, represents one of the most robust endocrine oscillations in mammalian physiology. This rhythm is characterized by a daily surge preceding the active phase, preparing the organism for anticipated metabolic and cognitive demands [5] [7]. The precise temporal control of GC secretion is orchestrated by a sophisticated hierarchical system integrating neural and hormonal signals across multiple anatomical sites. This review synthesizes current understanding of the multifaceted control mechanisms governing GC rhythmicity, focusing on the integrated roles of suprachiasmatic nucleus (SCN) signaling, adrenocorticotropic hormone (ACTH) dynamics, and the intrinsic adrenal clock. The implications of this regulatory network extend to fundamental physiology, chronopharmacology, and the development of novel therapeutics for endocrine disorders.

Systemic Organization of the Circadian Glucocorticoid Axis

The mammalian circadian system is organized hierarchically, with the central pacemaker in the SCN synchronizing subordinate oscillators throughout the body [5] [4]. The SCN, located in the anterior hypothalamus, receives photic input directly from the retina via the retinohypothalamic tract, aligning its intrinsic ~24-hour rhythm with the external light-dark cycle [4]. This master clock then coordinates the timing of GC release through dual pathways: a neural circuit to the paraventricular nucleus (PVN) of the hypothalamus and an endocrine cascade through the hypothalamic-pituitary-adrenal (HPA) axis [5] [7].

The SCN communicates with the PVN through both direct and indirect neural projections, with vasoactive intestinal peptide (VIP)-producing SCN neurons playing a critical role in this circuit [7]. PVN corticotropin-releasing hormone (CRH) neurons subsequently stimulate pituitary ACTH secretion, which drives glucocorticoid production from the adrenal cortex [27]. Additionally, the SCN regulates adrenal sensitivity to ACTH via autonomic nervous system outputs, creating a pre-emptive mechanism that primes the adrenal gland for subsequent hormonal stimulation [5]. This multi-layered regulation ensures that GC secretion peaks at the onset of the active phase (morning in humans, evening in nocturnal rodents), facilitating optimal metabolic and cognitive performance.

Table 1: Core Components of the Circadian Glucocorticoid System

Component	Location	Primary Function in GC Rhythm	Key Regulatory Molecules
SCN (Master Clock)	Hypothalamus	Generates and synchronizes circadian timing to light-dark cycle	VIP, AVP, PER1/2, CRY1/2, BMAL1, CLOCK
PVN CRH Neurons	Hypothalamus	Integrate SCN signals and stress inputs to initiate HPA axis activation	CRH, AVP, BMAL1 (intrinsic clock)
Anterior Pituitary	Sella turcica	Transduces hypothalamic signals into endocrine output	ACTH, POMC, MC2R
Adrenal Cortex	Adrenal Gland	Final site of glucocorticoid synthesis and secretion, contains peripheral clock	StAR, Cortisol/Corticosterone, PER2, BMAL1, GR, MR

Molecular Mechanisms of Circadian Timekeeping

The molecular machinery governing circadian rhythms consists of interlocking transcriptional-translational feedback loops (TTFL) that generate ~24-hour oscillations in clock gene expression [5] [4]. The core positive regulators CLOCK and BMAL1 form heterodimers that activate transcription of period (Per1, Per2) and cryptochrome (Cry1, Cry2) genes by binding to E-box elements in their promoter regions. After translation and post-translational modification, PER and CRY proteins accumulate in the cytoplasm, heterodimerize, and translocate back to the nucleus to repress CLOCK:BMAL1-mediated transcription, thereby completing the negative feedback loop [4].

A stabilizing auxiliary loop involves REV-ERBα, which is also activated by CLOCK:BMAL1 and subsequently represses Bmal1 transcription. This network of oscillating gene expression regulates the transcription of clock-controlled genes, including those involved in steroidogenesis [5]. In the adrenal gland, this molecular clock directly gates sensitivity to ACTH by rhythmically regulating the expression of steroidogenic acute regulatory protein (StAR), a rate-limiting factor in glucocorticoid synthesis [28]. The core clock components are expressed in all major tissues, but their specific output functions are tissue-specific.

Diagram Title: Molecular Circadian Clock Mechanism

SCN-to-PVN Neural Circuitry: Precision Timing of GC Secretion

Recent research has elucidated the critical neural pathway through which the SCN communicates with PVN CRH neurons to precisely time the daily corticosterone surge. SCN VIP-producing neurons project directly to PVN CRH neurons, forming a dedicated circuit for circadian GC regulation [7]. In vivo monitoring of PVN CRH neurons has revealed robust circadian rhythms in both clock gene expression and calcium activity. Specifically, Per2 expression in PVN CRH neurons peaks around midday (ZT5.9), while calcium activity, reflecting neuronal firing, peaks approximately 2-3 hours later in the mid-afternoon (ZT7.1) [7].

The functional importance of this rhythmicity was demonstrated through cell-specific knockout of the core clock gene Bmal1 in CRH neurons, which resulted in arrhythmic PVN CRH calcium activity and dramatically reduced the amplitude and precision of the daily corticosterone rhythm [7]. Optogenetic manipulation studies further established that acute activation of SCN VIP neurons inhibits PVN CRH neurons and reduces corticosterone release, while their inactivation has the opposite effect [7]. This indicates that SCN VIP neurons provide primarily inhibitory input to PVN CRH neurons, with their rhythmic activity patterning the daily GC surge through phasic disinhibition.

Table 2: Experimental Evidence for SCN-PVN Circuit Function

Experimental Approach	Key Findings	Technical Methods
In vivo fiber photometry	PVN^CRH^ neurons show daily Per2 expression (peak: ZT5.9) and calcium activity (peak: ZT7.1) rhythms	Cre-dependent Per2.Venus or GCaMP6s expression in CRH-IRES-Cre mice [7]
CRH neuron-specific Bmal1 knockout	Arrhythmic PVN^CRH^ calcium activity and blunted corticosterone rhythm	Conditional deletion of Bmal1 in CRH neurons [7]
SCN^VIP^ neuron manipulation	Activation inhibits, while inactivation stimulates, PVN^CRH^ neurons and corticosterone release	Chemogenetics and optogenetics [7]
Circuit tracing	Direct monosynaptic connection from SCN^VIP^ to PVN^CRH^ neurons	Anterograde and retrograde tracing techniques [7]

ACTH as an Entraining Signal for the Adrenal Clock

While ACTH's steroidogenic function is well-established, emerging evidence indicates it also serves as a key entraining signal for the intrinsic adrenal clock. The adrenal gland possesses its own molecular circadian clock that regulates the temporal gating of glucocorticoid synthesis [28] [29]. This peripheral oscillator can be directly reset by ACTH in a phase-dependent manner.

In vitro studies using mPER2::LUCIFERASE knockin mouse adrenal explants demonstrated that ACTH administration produces phase-dependent phase shifts in the PER2 rhythm, with maximal phase delays occurring at CT18 [28]. This phase-shifting effect is concentration-dependent, requires activation of the melanocortin 2 receptor (MC2R), and is mimicked by forskolin, indicating involvement of the cAMP signaling pathway [28]. The physiological relevance of these in vitro findings was confirmed by in vivo experiments showing that acute restraint stress or ACTH injection at different circadian times induces phase-dependent shifts in the adrenal PER2 rhythm [29]. Specifically, stress or ACTH administration at ZT2 (early subjective day) causes phase advances, while the same stimuli at ZT16 (early subjective night) causes phase delays [29].

This phase-dependent resetting mechanism allows stressful experiences to dynamically adjust the phase of the adrenal clock, potentially optimizing glucocorticoid production to anticipated future demands. The adrenal clock, in turn, gates steroidogenic sensitivity to ACTH by rhythmically regulating expression of StAR protein, creating a bidirectional relationship between the adrenal clock and ACTH signaling [28].

Diagram Title: ACTH-Adrenal Clock Entrainment Pathway

Glucocorticoid-Mediated Feedback and Peripheral Entrainment

Glucocorticoids themselves function as critical entraining signals for peripheral circadian oscillators throughout the body. This function is mediated through genomic actions involving glucocorticoid receptors (GR) and mineralocorticoid receptors (MR) [30] [27]. GRs are widely expressed in peripheral tissues and have a lower affinity for GCs, becoming activated primarily during circadian peaks and stress responses, while MRs have a higher affinity and are occupied even at basal GC levels [27].

Adrenalectomy experiments have demonstrated the importance of GCs in maintaining phase coherence in peripheral clocks. Adrenalectomy (ADX) significantly phase-shifts Per1-luc expression rhythms in liver, kidney, and cornea, and causes severe phase desynchrony and rhythm dampening in corneal explants [30]. These effects were reversed by hydrocortisone replacement, confirming the specific role of GCs in peripheral clock entrainment [30]. Interestingly, adrenalectomy also accelerated reentrainment to shifted light cycles in most peripheral tissues, suggesting that GC rhythms normally provide a stabilizing influence that resists rapid phase adjustments [30].

The molecular mechanism underlying GC-mediated entrainment involves direct genomic actions through glucocorticoid response elements (GREs) in clock gene promoters. Ligand-bound GR translocates to the nucleus where it can dimerize and bind to GREs, modulating the transcription of core clock genes, including Per1 and Per2 [5]. This provides a direct pathway for GCs to adjust the phase of peripheral clocks, synchronizing metabolic and physiological processes across tissues to align with the central SCN pacemaker and anticipated behavioral cycles.

Experimental Models and Methodologies

In Vivo Circadian Phenotyping

State-of-the-art approaches for investigating circadian glucocorticoid rhythms combine longitudinal monitoring in freely behaving animals with precise circuit manipulation techniques. In vivo fiber photometry enables real-time monitoring of both clock gene expression and neuronal activity in specific cell populations over multiple days [7]. This methodology involves implanting a fiber optic cannula above the target region (e.g., PVN) in transgenic mice expressing fluorescent reporters (e.g., Per2-Venus for gene expression or GCaMP6s for calcium activity) under cell-type-specific promoters (e.g., CRH-IRES-Cre). Measurements are typically taken at high temporal resolution (e.g., every 15 minutes for gene expression, 10 minutes/hour for calcium activity) under various lighting conditions [7].

For assessing the functional output of specific neuronal populations, chemogenetic (DREADDs) and optogenetic approaches allow precise temporal control of neuronal activity. These techniques have been instrumental in establishing the causal relationship between SCN VIP neuron activity and PVN CRH neuronal dynamics [7]. Circulating glucocorticoid levels can be monitored through serial blood sampling or automated tail-blood collection systems, while tissue-specific clock function is assessed by ex vivo bioluminescence recording of tissues from clock reporter mice (e.g., mPER2::LUC) [28] [29].

In Vitro Adrenal Clock Assays

The isolated adrenal explant culture system has been pivotal for characterizing direct effects of entraining signals on the adrenal clock. This methodology involves harvesting adrenals from PER2::LUC reporter mice during the subjective day (ZT8-8.5), cleaning and hemisectioning the glands, and culturing them on organotypic inserts in photomultiplier tube chambers [28] [29]. Bioluminescence rhythms are recorded under constant conditions, and potential entraining agents (e.g., ACTH, corticosteroids) are applied at specific circadian times to assess phase-shifting effects [28]. Data analysis involves detrending raw bioluminescence records, fitting damped sine waves to determine period and phase, and comparing treatment groups to vehicle controls using circular statistics [28].

Table 3: Key Research Reagents and Experimental Tools

Reagent/Tool	Application	Function/Mechanism	Example Use
mPER2::LUC mice	Real-time monitoring of peripheral clock rhythms	Luciferase reporter fused to mPER2 protein	Monitoring adrenal clock phase shifts in vitro [28] [29]
Crh-IRES-Cre mice	Cell-type-specific targeting of PVN^CRH^ neurons	Enables Cre-dependent recombination in CRH-expressing cells	Specific monitoring/manipulation of PVN^CRH^ neurons [7]
Fiber photometry	In vivo monitoring of neural activity or gene expression	Records fluorescence from GCaMP or Venus reporters	Longitudinal Per2 or calcium rhythms in PVN^CRH^ neurons [7]
Chemogenetics (DREADDs)	Remote control of neuronal activity	Modified GPCRs activated by inert ligands (CNO)	Acute manipulation of SCN^VIP^ neuron activity [7]
ACTH (1-39)	Adrenal clock entrainment studies	Native ACTH peptide activating MC2 receptors	Phase-shifting adrenal PER2 rhythms in vitro [28]

Chronopharmacology and Therapeutic Implications

Understanding the circadian regulation of glucocorticoids has profound implications for clinical therapy and drug development. Chronopharmacology principles leverage knowledge of endogenous GC rhythms to optimize dosing schedules, maximizing therapeutic efficacy while minimizing adverse effects [31]. Synthetic glucocorticoids, widely used for inflammatory and autoimmune conditions, can disrupt the natural cortisol rhythm when administered without regard to circadian timing, leading to HPA axis suppression and metabolic complications [31].

Experimental and clinical evidence supports timed administration of glucocorticoid therapies. For instance, dosing in the early morning aligns better with the physiological cortisol peak and causes less HPA axis disruption than evening administration [31]. Novel drug development approaches include delayed-release formulations that target specific phases of the circadian cycle and compounds designed to dissociate anti-inflammatory effects from metabolic side effects [31].

Recent therapeutic advances for conditions involving glucocorticoid dysregulation target upstream components of the HPA axis. Atumelnant, an oral ACTH antagonist that blocks the melanocortin type 2 receptor (MC2R) in the adrenal cortex, has shown promise in clinical trials for congenital adrenal hyperplasia (CAH) and ACTH-dependent Cushing's syndrome [32]. Similarly, crinecerfont, a CRF1 receptor antagonist, reduces ACTH levels and has demonstrated efficacy in enabling glucocorticoid dose reduction in CAH patients while maintaining androgen control [33]. These approaches represent a paradigm shift from directly replacing glucocorticoids to modulating the regulatory pathways that control their production.

The multifaceted control of glucocorticoid rhythm involves sophisticated integration of SCN-derived neural signals, pituitary ACTH release, and the intrinsic adrenal clock. This hierarchical system ensures precise temporal coordination of GC secretion with anticipated metabolic and behavioral demands. The SCN-PVN circuit provides the primary timing signal, ACTH serves as both a steroidogenic stimulus and adrenal clock entrainer, and glucocorticoids themselves function as entraining signals for peripheral oscillators while providing feedback regulation to higher centers.

Future research directions include elucidating the precise molecular mechanisms by which stress experience induces lasting changes in this regulatory network, understanding how misalignment between central and peripheral clocks contributes to disease pathogenesis, and developing more sophisticated chronotherapeutic approaches that respect the inherent temporal architecture of the HPA axis. The continued development of targeted therapeutics like ACTH antagonists and CRF receptor blockers holds promise for treating endocrine disorders with greater precision and fewer side effects than conventional glucocorticoid replacement therapies.

The circadian rhythm of glucocorticoid secretion is a fundamental biological process critical for coordinating metabolism, immune function, and cardiovascular health with daily behavioral cycles. While the hypothalamic-pituitary-adrenal (HPA) axis has long been recognized as the primary regulator of glucocorticoid production, recent research has elucidated that a local circadian clock within the adrenal cortex itself plays a pivotal role in gating the gland's sensitivity to adrenocorticotropic hormone (ACTH). This in-depth technical review synthesizes current understanding of the molecular mechanisms whereby the adrenal intrinsic clock regulates transcriptional programming, steroidogenic capacity, and ACTH receptor signaling to generate temporally restricted glucocorticoid output. We present structured experimental data, detailed methodologies, and visualization of signaling pathways to provide researchers and drug development professionals with a comprehensive resource on this sophisticated regulatory system and its implications for circadian physiology and therapeutic innovation.

The traditional view of glucocorticoid regulation has centered on the hypothalamic-pituitary-adrenal (HPA) axis, wherein hypothalamic corticotropin-releasing hormone (CRH) stimulates pituitary ACTH secretion, which in turn drives adrenal cortisol production [34]. However, this linear model fails to fully explain the robust circadian rhythm of glucocorticoid secretion, particularly the observation that circulating ACTH levels exhibit much lower amplitude oscillations than cortisol, and that adrenocortical tissues maintain intrinsic rhythmicity even when isolated from upstream regulators [34] [35] [6].

The discovery of local circadian clocks in peripheral tissues has transformed our understanding of glucocorticoid regulation. The adult mammalian adrenal cortex possesses an intrinsic circadian clock that enables it to anticipate and respond to predictable daily demands by gating its sensitivity to ACTH [12] [36] [6]. This gating mechanism ensures optimal temporal coordination between glucocorticoid-mediated processes and environmental cycles, representing a crucial adaptation for metabolic homeostasis and stress resilience.

This whitepaper examines the molecular architecture and functional significance of this local regulatory system, framing it within the broader context of circadian glucocorticoid research. We synthesize evidence from genetic, molecular, and physiological studies to provide a comprehensive technical resource for investigators exploring circadian endocrinology and developing chronotherapeutic interventions.

Molecular Architecture of the Adrenal Circadian Clock

Core Clock Machinery in Adrenocortical Cells

The adrenal circadian clock operates through the same transcriptional-translational feedback loop (TTFL) that functions in the suprachiasmatic nucleus (SCN) and other peripheral tissues. This molecular oscillator consists of core clock genes and their protein products that interact to generate approximately 24-hour rhythms in gene expression [12] [4].

The primary feedback loop involves activation of Period (Per1, Per2, Per3) and Cryptochrome (Cry1, Cry2) genes by heterodimers of CLOCK (Circadian Locomotor Output Cycles Kaput) and BMAL1 (Brain and Muscle ARNT-Like 1), which bind to E-box elements in promoter regions. After translation and post-translational modification, PER and CRY proteins form complexes that translocate back to the nucleus and inhibit CLOCK:BMAL1-mediated transcription, thereby repressing their own expression [12] [4]. This negative feedback loop generates antiphasic oscillations of Bmal1 and Per/Cry mRNA levels.

An auxiliary feedback loop involves REV-ERBα, which is also activated by CLOCK:BMAL1 and subsequently represses Bmal1 transcription by binding to ROR elements (ROREs) in its promoter. This stabilization of the core oscillator results in a self-sustaining transcriptional rhythm with a period of approximately 24 hours [4].

The following diagram illustrates this core molecular clock machinery:

Synchronization of the Peripheral Adrenal Clock

While the adrenal intrinsic clock can generate autonomous rhythms, its phase must be synchronized with the central pacemaker in the SCN and external environmental cycles. This synchronization occurs through multiple neurohumoral pathways:

• Neuroendocrine Signaling via the HPA Axis: The SCN transmits circadian signals to the paraventricular nucleus (PVN) of the hypothalamus, leading to rhythmic release of CRH and arginine-vasopressin (AVP). These neuropeptides drive pulsatile ACTH secretion from the pituitary, which provides a hormonal signal to the adrenal gland [34] [12].

• Autonomic Innervation: Direct neural connections from the SCN to the adrenal gland via the sympathetic nervous system (splanchnic nerve) convey photic information independently of ACTH. This innervation modulates adrenal sensitivity to ACTH and can directly stimulate glucocorticoid production [34] [35] [6].

• Systemic Cues: Other circulating factors with circadian rhythms, including glucocorticoids themselves (via feedback regulation), body temperature, and metabolic signals, provide additional synchronizing inputs to the adrenal clock [12] [4].

The integration of these signals ensures coherent timing between the central and peripheral circadian systems, optimizing physiological coordination across tissues.

Mechanisms of ACTH Sensitivity Gating

Transcriptional Regulation of Steroidogenic Enzymes

The adrenal clock gates glucocorticoid output primarily by regulating the expression and activity of key steroidogenic enzymes in a time-dependent manner. This temporal control occurs at multiple levels in the steroidogenesis pathway, creating a coordinated system that maximizes cortisol production during the active phase and minimizes it during rest.

Table 1: Circadian Regulation of Key Steroidogenic Components

Component	Regulatory Mechanism	Phase of Peak Expression/Activity	Functional Impact
MC2R (ACTH Receptor)	Clock-controlled expression; modulation by MRAP	Early active phase	Increases ACTH sensitivity and signaling efficiency
StAR (Steroidogenic Acute Regulatory Protein)	Transcriptional regulation via clock-controlled transcription factors	Active phase	Enhances cholesterol transport into mitochondria
CYP11A1 (Cholesterol side-chain cleavage enzyme)	E-box mediated circadian transcription	Active phase	Increases conversion of cholesterol to pregnenolone
CYP17A1 (17α-hydroxylase/17,20-lyase)	BMAL1:CLOCK regulation	Active phase	Directs steroidogenesis toward cortisol production
HSD3B2 (3β-hydroxysteroid dehydrogenase)	Circadian transcriptional control	Active phase	Enhances pregnenolone to progesterone conversion

The adrenal clock controls the transcription of these steroidogenic enzymes through several mechanisms, including direct binding of clock components to E-box elements in their promoter regions, regulation of intermediary transcription factors, and epigenetic modifications that rhythmically alter chromatin accessibility [34] [6].

The following diagram illustrates the integrated signaling pathway through which the adrenal clock gates ACTH sensitivity and regulates steroidogenesis:

Regulation of MC2R Expression and Signaling

A critical mechanism of ACTH sensitivity gating involves the rhythmic regulation of the melanocortin 2 receptor (MC2R), the specific receptor for ACTH in zona fasciculata cells. The adrenal clock controls MC2R expression levels, resulting in higher receptor availability during the early active phase when ACTH stimulation typically occurs [34] [6].

Furthermore, MC2R function requires the melanocortin 2 receptor accessory protein (MRAP), which facilitates receptor trafficking to the cell surface and enhances ACTH binding affinity. Mutations in either MC2R or MRAP cause familial glucocorticoid deficiency type 1 and 2, respectively, underscoring their essential role in ACTH signaling [34]. The adrenal clock may rhythmically regulate MRAP expression or function, adding another layer of temporal control over ACTH responsiveness.

Upon ACTH binding, MC2R activates Gs-protein-mediated cAMP production, which in turn activates protein kinase A (PKA). This signaling cascade ultimately stimulates cholesterol uptake (via increased LDL receptor expression), cholesterol transport into mitochondria (via StAR protein), and the sequential enzymatic conversions that produce cortisol from cholesterol [34]. The adrenal clock gates multiple steps in this pathway, creating a coordinated system that maximizes steroidogenic efficiency during anticipated periods of demand.

Chromatin Remodeling and Epigenetic Regulation

Emerging evidence indicates that the adrenal clock regulates steroidogenic capacity through epigenetic mechanisms, including rhythmic chromatin modifications that alter accessibility of steroidogenic gene promoters. Clock-controlled proteins recruit histone acetyltransferases (HATs) and deacetylases (HDACs) that rhythmically modify histone tails, creating permissive or repressive chromatin states at key steroidogenic loci [6].

Additionally, clock components interact with other transcription factors that regulate steroidogenesis, including NUR77 (NR4A1), SF-1 (NR5A1), and DAX-1 (NR0B1), creating a complex regulatory network that integrates temporal information with tissue-specific steroidogenic programming. This epigenetic regulation creates a "molecular memory" that maintains circadian steroidogenic capacity even in the face of transient fluctuations in ACTH levels.

Experimental Approaches and Methodologies

In Vivo Models for Studying Adrenal Clock Function

Several well-established experimental approaches enable investigators to dissect the mechanisms of adrenal clock gating of ACTH sensitivity:

Circadian Gene Expression Profiling: This methodology involves collecting adrenal glands at multiple time points throughout the 24-hour cycle to analyze temporal patterns of clock gene and steroidogenic enzyme expression.

Table 2: Key Research Reagents for Adrenal Circadian Research

Reagent/Category	Specific Examples	Research Application	Technical Function
Animal Models	Bmal1-KO; Per2::Luc reporter mice; Adrenal-specific clock gene mutants	In vivo functional studies	Enables tissue-specific disruption of clock components
Cell Lines	H295R adrenocortical cells; Primary adrenocortical cultures	In vitro mechanistic studies	Provides model system for signaling and gene expression
Antibodies	Anti-BMAL1, anti-CLOCK, anti-STAR, anti-MC2R, anti-CYP11A1	Immunodetection and protein localization	Enables protein quantification and cellular localization
Molecular Reagents	Clock gene siRNA/shRNA; Luciferase reporter constructs; cAMP analogs	Molecular manipulation and signaling studies	Modulates specific pathways and measures activity
Hormone Assays	ACTH ELISA; Corticosterone/Cortisol RIA or ELISA	Hormonal measurement	Quantifies endocrine parameters in plasma and media

Protocol:

• Sacrifice animals in 4-6 hour intervals over at least 48 hours under controlled light-dark conditions
• Collect adrenal glands, quickly freeze in liquid nitrogen, and store at -80°C
• Extract total RNA and perform quantitative RT-PCR for core clock genes (Bmal1, Per1, Per2, Cry1, Rev-erbα) and steroidogenic genes (StAR, Cyp11a1, Cyp17a1, Mc2r)
• Analyze data for circadian rhythmicity using appropriate statistical methods (COSINOR analysis, JTK_CYCLE)

Ex Vivo Adrenal Explant Cultures: This approach assesses intrinsic adrenal rhythmicity independent of systemic influences.

Protocol:

• Culture adrenal glands from PER2::LUC reporter mice in DMEM/F12 medium supplemented with 10% FBS, penicillin/streptomycin, and 0.1 mM luciferin
• Maintain cultures at 37°C in 5% CO2 and measure bioluminescence rhythms in real-time using a photomultiplier tube or CCD camera system
• Treat with ACTH (10-100 nM) at different circadian times to assess temporal variations in response
• Analyze phase, period, and amplitude of bioluminescence rhythms using Chronostar or similar software

Assessing ACTH Sensitivity Across the Circadian Cycle

Timed ACTH Stimulation Tests: This in vivo approach directly measures adrenal sensitivity to ACTH at different circadian phases.

Protocol:

• Administer ACTH (1-5 μg/kg) to animals at 4-6 different time points across the 24-hour cycle
• Collect blood samples via jugular or tail vein catheter before and at 30, 60, and 120 minutes post-injection
• Measure plasma corticosterone/cortisol levels by RIA or ELISA
• Calculate response magnitude as area under the curve (AUC) or peak level for each time point
• Compare responses across circadian phases to identify periods of heightened and reduced sensitivity

Molecular Analysis of Signaling Components:

Protocol:

• Isolate adrenocortical cells or tissue fragments at different circadian times
• Measure MC2R membrane expression by surface biotinylation and Western blotting
• Assess ACTH-stimulated cAMP production using ELISA-based assays
• Evaluate StAR phosphorylation and translocation to mitochondria via subcellular fractionation and immunoblotting
• Correlate signaling component rhythms with corticosteroid output

Pathophysiological and Clinical Implications

Circadian Disruption and Metabolic Disease

Disturbances in the adrenal clock and its gating of ACTH sensitivity contribute to various pathological conditions. Circadian misalignment, such as occurs in shift work, jet lag, and social jet lag, disrupts the precise temporal coordination between the HPA axis and the adrenal clock, leading to altered glucocorticoid rhythms [37] [36] [38].

Chronic circadian disruption is associated with flattened glucocorticoid rhythms, elevated evening cortisol levels, and eventually reduced overall cortisol exposure, which may contribute to the development of metabolic syndrome, insulin resistance, and cardiovascular disease [37] [36]. The mechanisms underlying these associations include:

• Loss of appropriate timing of glucocorticoid receptor-mediated gene expression in metabolic tissues
• Misalignment between cortisol rhythms and feeding-fasting cycles
• Altered interplay between cortisol and other hormonal rhythms (e.g., insulin, melatonin)

Implications for Glucocorticoid Therapy

Understanding adrenal clock gating has profound implications for glucocorticoid replacement therapy and chronotherapeutic approaches. Conventional glucocorticoid replacement often fails to replicate the natural circadian rhythm, leading to suboptimal metabolic outcomes and increased morbidity [6].

Chronotherapeutic strategies that align glucocorticoid administration with the endogenous rhythm of adrenal sensitivity may improve efficacy and reduce adverse effects. Specifically, timed-release hydrocortisone preparations that deliver higher doses in the early morning and lower doses in the evening better replicate the physiological cortisol rhythm and have shown improved metabolic outcomes compared to conventional replacement regimens.

Furthermore, understanding adrenal clock gating informs optimal timing for diagnostic tests, such as the ACTH stimulation test, which may yield different results depending on the circadian phase of administration due to variations in adrenal sensitivity.

Future Research Directions and Therapeutic Opportunities

Several promising research directions emerge from our current understanding of adrenal clock gating mechanisms:

Targeting Clock-Controlled Steroidogenic Regulators: Identification of specific clock-output regulators that control steroidogenesis could yield novel therapeutic targets for conditions of glucocorticoid excess or deficiency without disrupting the core clock mechanism itself.

Development of Chronotherapeutic Agents: Compounds that specifically modulate the phase or amplitude of the adrenal clock could help realign disrupted glucocorticoid rhythms in shift workers, individuals with jet lag, or patients with circadian rhythm sleep-wake disorders.

Personalized Chronotherapy: Genetic profiling of clock gene polymorphisms may identify individuals with specific adrenal clock properties, enabling personalized timing of glucocorticoid therapies for optimal efficacy.

Non-invasive Assessment of Adrenal Clock Function: Development of biomarkers that reflect adrenal clock phase and function would facilitate clinical assessment and timing of interventions without requiring invasive procedures.

The intricate gating mechanism whereby the adrenal cortical clock regulates sensitivity to ACTH represents a sophisticated biological system that optimizes temporal coordination of glucocorticoid function with daily environmental cycles. Understanding this system at molecular, cellular, and physiological levels provides not only fundamental biological insights but also promising avenues for therapeutic innovation in a wide range of glucocorticoid-related disorders.

From Bench to Bedside: Assessing HPA Axis Dynamics and Clinical Testing Protocols

The adrenocorticotropic hormone (ACTH) stimulation test represents a cornerstone diagnostic tool in endocrinology for assessing adrenal gland function. This dynamic test evaluates the functional integrity of the hypothalamic-pituitary-adrenal (HPA) axis by measuring the adrenal cortisol response to exogenous ACTH administration [39]. The test's clinical utility extends across multiple endocrine disorders, primarily focusing on the diagnosis of adrenal insufficiency (AI) in its various forms—primary, secondary, and tertiary [39] [40].

The physiological principle underpinning this test lies in the HPA axis's fundamental operational logic. Under normal conditions, the hypothalamus secretes corticotropin-releasing hormone (CRH), which stimulates the pituitary gland to release ACTH [41]. ACTH then binds to melanocortin-2 receptors in the adrenal cortex, triggering the synthesis and secretion of cortisol [40] [12]. This cascade operates within a robust circadian framework, with cortisol levels typically peaking in the early morning and reaching their nadir around midnight [12] [4]. The circadian rhythmicity of glucocorticoid secretion is regulated by the suprachiasmatic nucleus (SCN), which synchronizes peripheral tissue clocks through neuronal, hormonal, and behavioral signals [4]. The adrenal gland itself possesses an intrinsic circadian clock that gates its sensitivity to ACTH, contributing to the robust diurnal rhythm of cortisol secretion [12] [42]. This intricate temporal organization means that diagnostic interpretation of ACTH stimulation tests must consider the timing of administration, as adrenal responsiveness demonstrates phase dependency [41].

Test Methodology and Experimental Protocols

Standard Test Procedure

The ACTH stimulation test follows a standardized protocol with minimal variation between institutions. The procedure begins with the establishment of a baseline cortisol level. A phlebotomist draws an initial blood sample from a vein, typically in the patient's arm [39] [43]. Following this baseline measurement, a healthcare professional administers synthetic ACTH (cosyntropin), which contains the biologically active first 24 amino acids of the native ACTH molecule [40]. This compound is typically injected either intramuscularly or intravenously; for intravenous administration, the cosyntropin is diluted in 2-5 mL of normal saline and injected over two minutes [40]. Subsequent blood samples are collected at specified intervals post-injection—usually at 30 and 60 minutes—to measure the cortisol response to ACTH stimulation [39] [44].

Preparation and Precautions

Patient preparation is crucial for obtaining accurate results. Protocols generally recommend limiting physical activities and consuming a high-carbohydrate diet 12-24 hours before testing [39] [45]. Patients are typically instructed to fast for six hours prior to the procedure, consuming only water if needed [39] [43]. Since cortisol exhibits diurnal variation, the test is optimally performed in the morning (between 8:00-9:00 AM) to minimize the risk of false-positive results and to align with established normative data [39] [40]. Perhaps most critically, clinicians must carefully manage medications that could interfere with test results; glucocorticoids such as hydrocortisone, prednisone, or dexamethasone should be temporarily discontinued under medical supervision, as recent use represents a common cause of false-positive tests [39] [43]. Other medications including estrogens and spironolactone may also interfere with cortisol assays and require consideration [40].

Dosing Variations

Two primary dosing regimens exist for the ACTH stimulation test, each with distinct diagnostic applications:

• High-Dose Test (HDST): Utilizes 250 μg of cosyntropin for patients weighing ≥37 pounds and is considered the gold standard for diagnosing primary adrenal insufficiency [40] [44]. Blood samples are typically drawn at 0, 30, and 60 minutes post-injection [44].

• Low-Dose Test (LDST): Employs 1 μg of cosyntropin and is often preferred for evaluating secondary or tertiary adrenal insufficiency, as it provides a more physiological stimulus and may be more sensitive for detecting partial ACTH deficiency [41] [44]. The low-dose test is typically completed in about one hour, while the high-dose version may extend to two hours [42].

Table 1: Key Components of ACTH Stimulation Test Protocols

Component	Standard High-Dose Test	Low-Dose Test
ACTH Dose	250 μg (for patients ≥37 lbs)	1 μg
Administration	IV or IM	IV
Blood Sampling	Baseline, 30, 60 minutes	Baseline, 30, 60 minutes
Primary Use	Diagnosis of primary AI	Evaluation of secondary/tertiary AI
Test Duration	~2 hours	~1 hour

Diagnostic Interpretation and Criteria

Normal and Abnormal Responses

Interpretation of the ACTH stimulation test centers on evaluating the magnitude of the cortisol response to cosyntropin administration. A normal adrenal response is characterized by a significant rise in cortisol levels following stimulation. Current diagnostic thresholds define a normal response as a peak cortisol level exceeding 18-20 μg/dL (497-552 nmol/L) after ACTH administration [39] [45] [44]. Some institutions use a slightly more conservative cutoff of >12.6 μg/dL [39]. The magnitude of increase is also diagnostically significant, with a rise of ≥7-10 μg/dL from baseline considered normal [40].

Abnormal responses manifest as blunted or absent cortisol increases following ACTH stimulation and indicate various forms of adrenal dysfunction. In primary adrenal insufficiency (such as Addison's disease), the adrenal glands cannot produce adequate cortisol regardless of ACTH stimulation, resulting in little to no increase in cortisol levels [39]. In prolonged secondary adrenal insufficiency (due to pituitary dysfunction), adrenal atrophy from chronic ACTH deficiency similarly leads to a diminished cortisol response [39]. Recent or partial pituitary dysfunction may yield more variable responses, sometimes falling within the normal range despite clinical suspicion of insufficiency [39] [40].

Table 2: Diagnostic Interpretation of ACTH Stimulation Test Results

Condition	Baseline Cortisol	Post-ACTH Cortisol	Baseline ACTH	Physiological Basis
Normal	Normal diurnal variation	>18-20 μg/dL	Normal	Intact HPA axis and adrenal responsiveness
Primary AI	Low	Subnormal response	High	Adrenal gland destruction/dysfunction
Secondary AI	Low	Subnormal response	Low	Pituitary dysfunction causing adrenal atrophy
Tertiary AI	Low	Variable; may be normal	Low	Hypothalamic dysfunction

Advanced Interpretation in Circadian Context

Emerging research emphasizes that interpretation of ACTH stimulation tests must account for the dynamic, adaptive nature of the HPA axis. Mathematical modeling reveals that cortisol responses are highly sensitive to both the magnitude and timing of stress exposure, with ACTH responsiveness demonstrating phase dependency [41]. The adrenal gland's sensitivity to ACTH is gated by its intrinsic circadian clock, meaning that identical tests administered at different times of day may yield different results [12] [41]. Furthermore, chronic stress induces functional adaptations including glandular remodeling and glucocorticoid receptor resistance, which can alter test outcomes without indicating permanent adrenal pathology [41]. These dynamics may explain why blunted responses sometimes persist during recovery phases despite clinical improvement.

Circadian Rhythm Integration and Research Applications

The HPA Axis as a Circadian System

The HPA axis operates as a quintessential circadian system, with glucocorticoid secretion following a robust 24-hour rhythm that peaks around wake-up time in both diurnal and nocturnal species [12] [4]. This rhythm results from the integration of three distinct mechanisms: circadian input from the SCN to the hypothalamus, adrenal sensitivity to ACTH modulated by autonomic nervous system signals, and the adrenal gland's intrinsic circadian clock [12] [4]. The SCN transmits timing information through arginine-vasopressin projections to the paraventricular nucleus, generating rhythmic CRH and ACTH release [4]. However, the striking amplitude of cortisol rhythm cannot be fully explained by ACTH fluctuations alone, highlighting the contribution of direct neural control and local adrenal clock gating of steroidogenic capacity [12] [42].

Glucocorticoids themselves function as potent circadian zeitgebers (synchronizing cues) for peripheral tissues by binding to glucocorticoid response elements in the promoter regions of various clock genes, including Per1 and Per2 [12] [4]. This creates a bidirectional relationship wherein the central clock regulates glucocorticoid secretion, and glucocorticoids in turn synchronize peripheral clocks throughout the body. This feedback mechanism ensures temporal coordination between physiological systems but also creates vulnerability when circadian organization is disrupted.

Research Implications and Diagnostic Innovations

Within circadian research, the ACTH stimulation test serves as a critical tool for probing HPA axis dynamics under various physiological and pathological conditions. Recent studies utilizing chronic ACTH infusion models have demonstrated that glucocorticoid excess disrupts diurnal patterns of blood pressure and sodium excretion, inducing non-dipping hypertension and salt sensitivity [46]. These findings illuminate the pathophysiology of Cushing syndrome and iatrogenic glucocorticoid excess, conditions associated with significant cardiovascular risk.

Advanced modeling frameworks now integrate circadian rhythmicity, stress-induced adaptation, and feedback dynamics to enhance test interpretation [41]. These models demonstrate that during chronic stress, glucocorticoid receptor resistance develops, leading to delayed feedback recovery and altered ACTH test responses that may not reflect true adrenal capacity [41]. Such insights are refining diagnostic approaches, suggesting that low-dose ACTH testing may more reliably detect partial adrenal adaptation than high-dose protocols, which can mask dysfunction through supraphysiological stimulation [41] [44].

Emerging diagnostic innovations include re-evaluating traditional cortisol cut-offs and incorporating additional parameters like cortisol increment (Δ cortisol). Recent research proposes baseline cortisol values ≤5.35 μg/dL as highly specific for ruling in adrenal insufficiency, while values ≥12.4 μg/dL effectively rule it out [44]. The cortisol increment, with an optimal cut-off of 6.35 μg/dL for diagnosing AI, provides complementary diagnostic value [44]. Some studies suggest that a single 30-minute cortisol measurement may offer comparable diagnostic accuracy to traditional multiple timepoint sampling, potentially streamlining clinical protocols [44].

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Research Reagents for ACTH Stimulation Studies

Reagent/Resource	Function and Research Application
Cosyntropin (ACTH 1-24)	Synthetic ACTH fragment; biologically active with reduced allergenicity; used for stimulation at high (250 μg) or low (1 μg) doses [40].
Cortisol Immunoassays	Various platforms (e.g., RIA, CLIA) for quantifying serum cortisol levels; must account for assay-specific differences in reference ranges [44].
ACTH Assays	Measuring baseline ACTH levels; crucial for differentiating primary (high ACTH) from secondary/tertiary (low ACTH) adrenal insufficiency [40].
Corticosterone ELISA	For rodent studies; measures corticosterone (the primary rodent glucocorticoid) in response to ACTH stimulation [46].
Osmotic Minipumps	Enables continuous subcutaneous infusion of ACTH in animal models to simulate chronic exposure and study HPA axis adaptation [46].
CRH/ACTH Precursor Assays	Research tools for mapping comprehensive HPA axis dynamics beyond standard diagnostic needs [41].

Visualizing HPA Axis Dynamics and Experimental Workflow

HPA Axis Circadian Signaling Pathway

ACTH Stimulation Test Experimental Workflow

The ACTH stimulation test remains an indispensable tool in both clinical endocrinology and circadian research. Its principles, rooted in the fundamental physiology of the HPA axis, provide critical insights into adrenal function across the spectrum of adrenal disorders. Contemporary understanding emphasizes that test interpretation must evolve beyond static diagnostic thresholds to incorporate the dynamic, circadian nature of glucocorticoid regulation. The integration of mathematical modeling, refined diagnostic criteria, and circadian perspectives promises to enhance both the clinical utility of this procedure and its research applications in understanding the complex temporal organization of endocrine systems. As our comprehension of HPA axis dynamics deepens, the ACTH stimulation test continues to offer a window into the intricate relationship between stress physiology, circadian biology, and metabolic health.

The adrenocorticotropic hormone (ACTH) stimulation test, a cornerstone in evaluating hypothalamic-pituitary-adrenal (HPA) axis integrity, exists primarily in two pharmacological forms: the standard-dose (250μg) and low-dose (1μg) Synacthen challenges. These diagnostic protocols function as dynamic proxies for the body's natural stress response system, which follows a precise circadian rhythm characterized by peak ACTH and cortisol secretion in the early morning (around 6-8 AM) and a nadir around midnight [47]. The physiological basis for the test rests upon the HPA axis's hierarchical organization, wherein corticotropin-releasing hormone (CRH) from the hypothalamus stimulates pituitary ACTH secretion, which in turn drives cortisol production from the adrenal cortex [47].

The standard-dose test employs a pharmacological dose of ACTH (250μg) that profoundly stimulates the adrenal glands, while the low-dose test utilizes a more physiological stress-mimicking dose (1μg) designed to be more sensitive in detecting partial or recent-onset ACTH deficiency [48]. Understanding the distinction between these protocols is crucial for researchers and clinicians investigating HPA axis dynamics, particularly in conditions like pituitary dysfunction, critical illness, and circadian rhythm disorders affecting glucocorticoid secretion. The accurate assessment of adrenal reserve is vital, as adrenal insufficiency can be life-threatening, especially during physiological stress, and both over- and under-diagnosis carry significant clinical consequences [48] [49].

Comparative Test Characteristics and Diagnostic Performance

The fundamental distinction between the two tests lies in the administered dose of synthetic ACTH (cosyntropin) and the resulting physiological response. The standard 250μg dose represents a supraphysiological stimulus, producing maximal adrenal cortisol output and effectively assessing adrenal gland reserve capacity. In contrast, the 1μg low-dose is designed to approximate the peak plasma ACTH concentration observed during major physiological stress (approximately 100-150 pg/mL), making it a more sensitive probe for detecting subtle impairments in the HPA axis, particularly at the pituitary level [48].

Table 1: Key Characteristics of Low-Dose vs. Standard-Dose ACTH Tests

Parameter	Low-Dose (1μg) ACTH Test	Standard-Dose (250μg) ACTH Test
ACTH Dose	1μg cosyntropin (IV bolus)	250μg cosyntropin (IV or IM)
Physiological Basis	Mimics peak physiological stress-level ACTH	Pharmacological, supraphysiological stimulus
Primary Application	Detection of secondary/tertiary AI, recent-onset deficiency	General assessment of adrenal glucocorticoid reserve
Diagnostic Threshold (Peak Cortisol)	~500 nmol/L (≈18 μg/dL) [48]	~500 nmol/L (≈18 μg/dL) [48]
Reported Sensitivity	Higher for central AI	Lower for central AI
Reported Specificity	Slightly lower	Slightly higher
Advantages	Greater physiological relevance, higher sensitivity for partial deficiencies	Well-established, robust, less technically sensitive to preparation errors
Limitations	Requires precise dilution, shorter plasma cortisol half-life	May miss mild/early central AI, false negatives in recent pituitary damage

The diagnostic cutoff for a normal response—a peak serum cortisol concentration exceeding 500 nmol/L (≈18 μg/dL)—is commonly applied to both tests, though it is crucial to note that this threshold can be assay-dependent [48]. The high-dose test's primary strength is its robustness and standardized procedure, making it a reliable tool for excluding primary adrenal insufficiency. However, its primary weakness is a lower sensitivity for detecting partial or recent secondary adrenal insufficiency (SAI). This is because a recently dysfunctional pituitary may not have caused significant adrenal atrophy, and the potent 250μg stimulus can still elicit a normal cortisol response, yielding a false negative [48]. The low-dose test, being a more physiological stressor, is more likely to be sub-normal in such scenarios, thus offering superior sensitivity for diagnosing SAI.

Experimental Protocols and Methodological Considerations

Standard-Dose (250μg) ACTH Test Protocol

The standard high-dose Synacthen test is widely used for its simplicity and excellent reliability for assessing adrenal reserve. The test procedure is as follows:

• Baseline Sample: At time zero (typically between 8-9 AM to account for circadian rhythm), a baseline blood sample is drawn for plasma cortisol and potentially ACTH measurement.
• ACTH Administration: 250μg of cosyntropin is administered via intravenous (IV) or intramuscular (IM) injection.
• Post-Stimulation Samples: Blood samples for cortisol measurement are collected at 30 and 60 minutes after injection [48].
• Interpretation: A normal adrenal response is typically defined by a peak cortisol level (at either 30 or 60 minutes) above 500 nmol/L (18 μg/dL). A response below this level is indicative of adrenal insufficiency [48].

A critical methodological consideration is the test's potential to produce false-negative results in patients with very recent pituitary injury (<4-6 weeks), as the adrenal glands may not have atrophied yet and remain responsive to a potent supraphysiological stimulus [48].

Low-Dose (1μg) ACTH Test Protocol

The low-dose test is designed to be more sensitive and requires meticulous attention to preparation detail. The procedural workflow is outlined in the diagram below.

The protocol requires precise preparation of the 1μg dose, typically by serial dilution of the standard 250μg vial [48]. The sampling points are more focused, often at 0 and 30 minutes post-injection, reflecting the rapid clearance of the low-dose ACTH. The same diagnostic cutoff of 500 nmol/L is often used, though its validation against the gold-standard insulin tolerance test (ITT) is more established for the low-dose test in many clinical studies. A significant confounder, especially in research settings involving pre-menopausal women, is the mode of estrogen replacement. Oral estrogen significantly increases cortisol-binding globulin (CBG), thereby elevating total plasma cortisol levels without necessarily changing free, bioactive cortisol. This can lead to misinterpretation of test results, whereas transdermal estrogen or the use of salivary cortisol (which reflects free cortisol) is less affected [50].

Signaling Pathways and HPA Axis Physiology

The ACTH stimulation test directly probes the final limb of the HPA axis. The underlying physiology and the point of intervention for the diagnostic test are illustrated in the following pathway diagram.

This diagram illustrates the core regulatory circuit: stress triggers hypothalamic CRH release, which stimulates pituitary ACTH secretion, leading to cortisol production from the adrenal cortex. Circulating cortisol then completes the loop by inhibiting CRH and ACTH release via negative feedback. The ACTH stimulation test (both low and standard dose) bypasses the upper portions of the axis to directly stimulate the adrenal cortex. An intact response indicates functional adrenal glands and a certain level of pre-existing trophic support from endogenous ACTH.

The Scientist's Toolkit: Essential Research Reagents

For researchers developing and validating ACTH stimulation tests and related HPA axis assays, a suite of high-quality reagents is essential. The following table details key materials and their specific functions in this field.

Table 2: Key Research Reagents for HPA Axis and ACTH Test Research

Reagent / Material	Primary Function in Research	Technical Notes
Synthetic ACTH (Cosyntropin)	The core reagent for performing stimulation tests; used in both 1μg and 250μg protocols.	Requires precise dilution for low-dose test; stability and sourcing are key.
ACTH Antibodies	Essential for developing immunoassays (ELISA, CLIA) to measure endogenous ACTH levels.	Specificity to ACTH (1-39) and Synacthen (1-24) is critical; minimal cross-reactivity with CLIP or β-LPH is required [51].
Cortisol Antibodies	Used to develop assays for quantifying total cortisol in serum/plasma, the primary test outcome.	Must be validated for the specific matrix (serum, saliva).
Cortisol-Binding Globulin (CBG)	Important for studying cortisol bioavailability and transport, especially in states of estrogen effect or inflammation.	Relevant for interpreting total vs. free cortisol levels [50] [49].
CRH & CRH Receptor Antagonists	Tools for probing the upper HPA axis; CRH for CRH stimulation tests, antagonists for blocking signaling.	Used to investigate central defects and develop novel therapeutics [52].
Cortisol ELISA/EIA/Kits	Ready-to-use kits for high-throughput measurement of cortisol in research samples.	Performance characteristics (sensitivity, dynamic range) must be suited to expected concentrations.
Salivary Cortisol Collection Kits	Non-invasive method to measure free, bioavailable cortisol, avoiding CBG confounders.	Highly valuable for circadian rhythm studies and in populations on oral estrogen [50].

The choice between the low-dose (1μg) and standard-dose (250μg) ACTH stimulation tests represents a critical methodological decision in HPA axis research. The low-dose test offers superior sensitivity for detecting central adrenal insufficiency and is more physiologically relevant, making it preferable for diagnosing secondary causes and studying partial deficiencies. The standard-dose test remains a robust, well-validated tool for assessing overall adrenal reserve, particularly in primary adrenal insufficiency.

Future research directions should focus on further standardizing the low-dose test protocol, establishing population- and assay-specific reference ranges, and exploring the role of free versus total cortisol measurement, particularly in special populations. Furthermore, the integration of novel functional molecular imaging techniques may provide complementary anatomical and functional data, enhancing our overall understanding of adrenal physiology and pathology [53]. A thorough grasp of these distinct test protocols, their underlying mechanisms, and associated research tools is indispensable for advancing both clinical diagnostics and fundamental research into the circadian dynamics of the HPA axis.

Chronopharmacology represents a transformative approach in pharmacology, founded on the principle that the effects of medications are significantly influenced by the body's endogenous circadian rhythms. The timing of drug administration can alter pharmacokinetics and pharmacodynamics by up to tenfold, directly impacting therapeutic efficacy and toxicity profiles [54] [55]. This technical review examines the core mechanisms of chronopharmacology, with a specific focus on the hypothalamic-pituitary-adrenal (HPA) axis and glucocorticoid signaling as primary regulators of circadian drug responses. We synthesize current experimental methodologies, quantitative findings, and practical research tools to provide a comprehensive framework for integrating chronopharmacological principles into drug development and therapeutic optimization. The intricate coordination between the central circadian clock and peripheral tissue clocks creates time-dependent windows of vulnerability and responsiveness to pharmaceutical agents, offering substantial opportunities for enhancing treatment precision while minimizing adverse effects across diverse therapeutic areas including oncology, cardiology, and psychiatry [56] [57] [58].

Chronopharmacology investigates how biological rhythms influence drug effects, encompassing two primary domains: chronopharmacokinetics (time-dependent variations in drug absorption, distribution, metabolism, and excretion) and chronopharmacodynamics (rhythmic changes in drug effects on target tissues and receptors) [58]. These temporal variations are governed by the circadian timing system, a hierarchical network of molecular clocks throughout the body. The master pacemaker resides in the suprachiasmatic nucleus (SCN) of the hypothalamus, which synchronizes peripheral clocks in various organs via neuronal, endocrine, and behavioral outputs [12] [4].

The clinical significance of chronopharmacology is profound, with research demonstrating that aligning medication schedules with circadian cycles can substantially improve outcomes. For example, the toxicity and efficacy of numerous anticancer drugs vary dramatically depending on administration time, while psychiatric medications show altered effectiveness based on dosing schedules relative to circadian physiology [57] [54]. The glucocorticoid system, through its robust circadian secretion pattern and widespread regulatory functions, serves as a critical mediator of these temporal variations in drug response, making it a focal point for chronopharmacological research and application [12] [46].

Molecular Mechanisms of Circadian Rhythms

Core Clock Gene Machinery

The molecular foundation of circadian rhythms consists of interlocked transcriptional-translational feedback loops (TTFL) that generate approximately 24-hour oscillations in gene expression. The core negative feedback loop involves heterodimers of CLOCK (or NPAS2) and BMAL1 proteins that activate transcription of Period (Per1, Per2, Per3) and Cryptochrome (Cry1, Cry2) genes by binding to E-box enhancer elements [4] [54]. After translation and post-translational modification, PER and CRY proteins form complexes that translocate back to the nucleus and inhibit CLOCK-BMAL1-mediated transcription, thereby repressing their own expression.

An auxiliary feedback loop involves the nuclear receptors REV-ERBα and RORα, which are also activated by CLOCK-BMAL1 and compete for binding to ROR elements (RREs) in the Bmal1 promoter. REV-ERBα represses while RORα activates Bmal1 transcription, creating stabilizing oscillations that enhance the robustness of the core clock [4]. This molecular oscillator is present in virtually all cells and regulates numerous clock-controlled genes (CCGs) through E-boxes and other regulatory elements in their promoters, ultimately governing circadian rhythms in cellular function, metabolism, and drug responsiveness.

Figure 1: Core Circadian Clock Mechanism. The molecular clock consists of interlocked transcriptional-translational feedback loops. CLOCK-BMAL1 heterodimers activate Per, Cry, and Rev-Erbα transcription. PER-CRY complexes accumulate and eventually inhibit CLOCK-BMAL1 activity. REV-ERBα and RORα compete to regulate Bmal1 expression, creating stabilizing oscillations [4] [54].

Glucocorticoid-Mediated Circadian Coordination

Glucocorticoids (GCs), primarily cortisol in humans and corticosterone in rodents, serve as critical systemic synchronizers of peripheral circadian clocks. These steroid hormones exhibit a robust circadian rhythm with peak secretion occurring just before the active phase (morning in humans, evening in nocturnal rodents) [12] [4]. This rhythm originates from a multi-layered regulatory system involving: (1) SCN control of the HPA axis via arginine-vasopressin (AVP) projections to the paraventricular nucleus (PVN); (2) autonomic innervation of the adrenal gland modulating sensitivity to adrenocorticotropic hormone (ACTH); and (3) an intrinsic adrenal clock that gates glucocorticoid production [12].

Glucocorticoids regulate circadian physiology through both genomic and non-genomic mechanisms. They bind to glucocorticoid receptors (GR) and mineralocorticoid receptors (MR), which translocate to the nucleus and interact with glucocorticoid response elements (GREs) in target genes. Notably, multiple clock genes including Per1 and Per2 contain GREs, allowing glucocorticoids to directly reset peripheral clocks [12]. This positions glucocorticoids as crucial zeitgebers (time-giving signals) that synchronize peripheral oscillators with the central SCN pacemaker and with each other, creating temporal coordination across tissues that profoundly influences drug metabolism, distribution, and action.

Chronopharmacokinetics: Temporal Variations in Drug Disposition

Absorption, Distribution, Metabolism, and Excretion Rhythms

Circadian rhythms significantly influence all phases of drug disposition, creating predictable temporal patterns in drug bioavailability and clearance. These variations are driven by circadian regulation of enzyme systems, transport proteins, and physiological processes throughout the body [58] [55].

Drug absorption exhibits circadian dependence due to rhythmic changes in gastric pH, gastrointestinal motility, blood flow, and transporter expression. For example, gastric pH is typically lowest just before midnight, potentially reducing the bioavailability of alkaline drugs administered during this period. Gastrointestinal motility and gastric emptying are generally higher during the daytime, leading to increased bioavailability of lipophilic drugs [58].

Drug distribution fluctuates with circadian changes in cardiac output, blood flow, and plasma protein binding. Cardiac output is typically higher during the day, potentially enhancing drug distribution. Additionally, expression of efflux transporters like P-glycoprotein (P-gp) shows circadian variation at critical barriers including the blood-brain barrier (BBB), creating time-dependent windows for drug penetration into protected compartments [58] [55].

Drug metabolism demonstrates particularly robust circadian regulation, primarily through rhythmic expression of cytochrome P450 (CYP) enzymes and phase II conjugation systems. The molecular clock directly regulates transcription of these enzymes in the liver and intestines, creating temporal metabolic windows. For instance, research indicates that approximately 50% of drug-metabolizing enzymes exhibit circadian fluctuations in expression or activity [55].

Drug excretion via renal and hepatic routes also follows circadian patterns. Glomerular filtration rate (GFR), renal blood flow, and tubular secretion/show circadian variation, leading to time-dependent differences in drug elimination. Similarly, biliary excretion fluctuates with circadian regulation of transport proteins like ABCG2/BCRP [58] [55].

Table 1: Circadian Regulation of Pharmacokinetic Processes

Process	Key Circadian Influences	Example Compounds Affected	Timing of Peak Activity/Favorable Administration
Absorption	Gastric pH, GI motility, blood flow, transporter expression (P-gp, BCRP)	Alkaline drugs, lipophilic drugs	Daytime (for lipophilic drugs); varies by compound [58]
Distribution	Cardiac output, blood flow, plasma protein binding, BBB permeability	CNS drugs, highly protein-bound drugs	Daytime (for increased distribution) [58]
Metabolism	CYP450 enzymes (CYP3A4, CYP2D6, etc.), phase II conjugation	>50 anticancer drugs, statins, psychotropics	Compound-specific; often nighttime for certain CYP3A4 substrates [55]
Excretion	Renal blood flow, GFR, tubular function, biliary transport	Renal-cleared drugs, methotrexate	Daytime (for renal excretion) [58]

Glucocorticoid Influence on Drug Metabolism and Transport

Glucocorticoids significantly contribute to the circadian regulation of drug metabolism and transport through multiple mechanisms. As major outputs of the circadian system, glucocorticoids rhythmically activate GR-mediated transcription of numerous metabolic enzymes and transporters. For example, several CYP enzymes contain GREs in their promoter regions, creating direct genomic regulation by glucocorticoids [12]. Additionally, glucocorticoids indirectly influence drug disposition by modulating the expression of clock genes that subsequently regulate metabolic pathways.

This glucocorticoid-mediated regulation creates a hierarchical system wherein the SCN controls glucocorticoid rhythm, which in turn synchronizes metabolic capacity across tissues. This has profound implications for chronopharmacology, as demonstrated in conditions of glucocorticoid excess (Cushing's syndrome) or deficiency (Addison's disease), where the normal circadian variation in drug metabolism is disrupted. Furthermore, exogenous glucocorticoid administration can reset metabolic rhythms, potentially optimizing drug efficacy or minimizing toxicity when timed appropriately [12] [46].

Chronopharmacodynamics: Temporal Variations in Drug Effects

Target Pathway Rhythmicity

Beyond influencing drug concentrations, circadian rhythms directly modulate drug targets, creating temporal windows of enhanced or diminished pharmacodynamic response. Nearly all physiological systems exhibit circadian regulation, including cardiovascular function, immune activity, cell cycle progression, and neuronal signaling—each representing potential targets for chronotherapeutic optimization [57] [58].

In oncology, numerous critical pathways involved in cell proliferation, DNA repair, and apoptosis demonstrate circadian regulation. For example, the expression and activity of thymidylate synthase, dihydrofolate reductase, and topoisomerases oscillate daily, creating time-dependent susceptibility to chemotherapeutic agents. Research shows that aligning drug administration with these rhythmic pathways can significantly improve the therapeutic index of anticancer treatments [57].

In cardiovascular medicine, blood pressure, heart rate, coagulation parameters, and vascular tone all exhibit circadian patterns. These rhythms contribute to the morning peak in cardiovascular events and create temporal variation in responses to antihypertensives, anticoagulants, and other cardiovascular drugs. For instance, angiotensin-converting enzyme (ACE) inhibitors administered in the evening demonstrate superior blood pressure control compared to morning dosing for some patients [58].

In psychiatry, neurotransmitter systems, receptor sensitivity, and second messenger pathways show circadian regulation. This is particularly relevant given the well-established circadian disruptions in mood disorders and the circadian influence on drug targets for psychotropic medications. Melatonin and cortisol rhythms serve as particularly important biomarkers for optimizing dosing schedules of psychiatric medications [54].

Glucocorticoid Modulation of Drug Targets

Glucocorticoids exert widespread influences on drug target pathways through their roles as primary circadian regulators. Approximately 20% of the transcriptome in various tissues may be under direct or indirect glucocorticoid control, creating broad temporal regulation of cellular responsiveness [12]. Key mechanisms include:

• Receptor expression rhythms: Numerous neurotransmitter and hormone receptors display circadian expression patterns influenced by glucocorticoid signaling.
• Signal transduction modulation: Glucocorticoids rhythmically regulate components of intracellular signaling cascades, including G proteins, kinases, and transcription factors.
• Cell cycle regulation: Glucocorticoids influence the timing of cell cycle progression, particularly relevant for cytotoxic cancer therapies.
• Immune function coordination: As potent immunomodulators, glucocorticoids create circadian variation in inflammatory responses and immune cell activity, affecting responses to immunotherapies and anti-inflammatory drugs.

This glucocorticoid-mediated regulation creates a scenario where target tissue sensitivity oscillates independently of drug concentrations, necessitating coordinated timing of administration to maximize therapeutic effects while minimizing adverse reactions.

Experimental Approaches in Chronopharmacology Research

Circadian Phenotyping Methodologies

Comprehensive chronopharmacology research requires precise characterization of circadian rhythms in model systems. Recent advances enable high-throughput deep phenotyping of circadian parameters, clock strength, and time-of-day drug responses [57].

Reporter Systems: Engineered cell lines expressing luciferase reporters under control of clock gene promoters (e.g., Bmal1-Luc, Per2-Luc) enable real-time monitoring of molecular clock dynamics. These systems allow longitudinal tracking of circadian parameters without disrupting cells, facilitating drug screening across circadian phases [57].

Multiparametric Rhythm Analysis: Advanced computational approaches provide robust assessment of circadian characteristics:

• Autocorrelation: Quantifies rhythm strength and period from the regularity of oscillations
• Continuous Wavelet Transform (CWT): Identifies time-dependent changes in period and amplitude
• Multiresolution Analysis (MRA): Decomposes signals into circadian, ultradian, and noise components to calculate "circadianicity" indices [57]

High-Throughput Drug Screening: Automated systems now enable evaluation of drug sensitivity across multiple time points, identifying optimal treatment windows. This approach typically involves establishing synchronized cellular models, treating at different circadian phases, and assessing viability, apoptosis, or other endpoints to construct time-response profiles [57].

Table 2: Experimental Protocols for Chronopharmacology Research

Method	Key Procedures	Output Parameters	Applications
Reporter Cell Line Assay	1. Synchronize cells (serum shock, dexamethasone, temperature) 2. Record bioluminescence rhythms 3. Analyze period, phase, amplitude	Circadian period, phase, amplitude, damping rate	Screening clock effects of compounds, testing tissue-specific rhythms [57]
Time-of-Day Drug Screening	1. Establish synchronized cell cultures 2. Administer compounds at different circadian times 3. Measure endpoint responses (viability, apoptosis, etc.) 4. Construct chronotoxicity/efficacy profiles	IC50/EC50 timing, therapeutic index timing, chronotherapeutic index	Identifying optimal dosing times for anticancer drugs, neuroprotective agents [57]
HPA Axis Manipulation (ACTH infusion)	1. Implant osmotic minipumps for continuous ACTH delivery 2. Monitor blood pressure via telemetry 3. Assess circadian parameters (rhythm amplitude, phase) 4. Evaluate salt sensitivity, pressure-natriuresis	Circadian rhythm disruption, non-dipping blood pressure, salt sensitivity	Modeling Cushing syndrome, glucocorticoid-dependent hypertension [46]
Mathematical Modeling of HPA Axis	1. Develop ODE-based models incorporating circadian inputs 2. Parameterize with experimental data 3. Simulate chronic stress and recovery phases 4. Predict ACTH test responses across phases	Hormonal dynamics, glandular adaptation, feedback resistance, test outcome predictions	Understanding HPA adaptation states, optimizing diagnostic testing [41]

In Vivo Chronopharmacology Models

Animal models remain essential for translational chronopharmacology research, particularly for understanding complex systemic interactions like HPA axis regulation:

Chronic ACTH Infusion Model: Subcutaneous osmotic minipumps delivering ACTH (2.5 μg/day) for up to 28 days effectively model Cushing syndrome in rodents. This approach induces glucocorticoid excess, leading to non-dipping blood pressure, salt-sensitive hypertension, and disrupted sodium excretion rhythms—key features of circadian disruption in hypercortisolemic states [46].

Telemetric Monitoring: Radiotelemetry devices enable continuous measurement of physiological parameters (blood pressure, heart rate, activity, temperature) without disturbance artifacts, providing precise characterization of circadian rhythms and their manipulation by drugs or genetic interventions.

Timed Tissue Collection: Systematic sacrifice at multiple circadian time points permits assessment of molecular rhythms (gene expression, protein levels, enzyme activity) across tissues, revealing organ-specific circadian regulation of drug targets and metabolic pathways.

Figure 2: Glucocorticoid-Mediated Chronopharmacology. The hypothalamic-pituitary-adrenal (HPA) axis regulates glucocorticoid secretion, which synchronizes peripheral clocks. These clocks subsequently regulate drug metabolism enzymes, transport proteins, and target pathways, collectively determining time-dependent drug responses [12] [46] [4].

Table 3: Research Reagent Solutions for Chronopharmacology

Reagent/Category	Specific Examples	Research Applications	Key Functions
Circadian Reporter Systems	Bmal1-Luc, Per2-Luc reporter cell lines	Real-time monitoring of molecular clock dynamics	Non-invasive tracking of circadian phase, period, and amplitude in live cells [57]
Hormones & Peptides	ACTH (1-24), corticosterone, cortisol, dexamethasone	HPA axis manipulation, peripheral clock resetting	Experimentally control glucocorticoid signaling; synchronize cellular clocks [46]
Chronobiotic Compounds	Melatonin, melatonin receptor agonists, REV-ERB agonists/antagonists, CRY stabilizers	Modifying clock function, testing chronopharmacological principles	Probe clock-controlled physiological processes; potential adjuvants for chronotherapy [59]
Computational Resources	ChronobioticsDB, mathematical modeling frameworks	Data integration, prediction of optimal dosing times	Database of chronobiotic compounds; simulation of HPA axis dynamics and drug responses [59] [41]

Chronopharmacology represents a paradigm shift in pharmacological science, recognizing that drug effects are not static but vary predictably with biological time. The glucocorticoid system, as a key output of the central circadian pacemaker, serves a critical role in synchronizing circadian drug metabolism, distribution, and action across tissues. The experimental approaches and mechanistic insights detailed in this review provide a foundation for systematically incorporating temporal considerations into drug development and therapeutic optimization.

Future progress in chronopharmacology will require advances in several key areas: (1) development of minimally invasive biomarkers for assessing individual circadian phase in clinical settings; (2) refinement of mathematical models that predict temporal variation in drug responses based on underlying circadian physiology; (3) expanded clinical trials that systematically evaluate timing-dependent efficacy and toxicity; and (4) regulatory frameworks that accommodate chronotherapeutic optimization in drug labeling and prescribing guidelines.

As these developments unfold, chronopharmacology will increasingly transform from a specialized research area into an integral component of precision medicine, enabling drug therapies that are synchronized not only to disease pathophysiology but also to the innate temporal architecture of human physiology.

The Hypothalamic-Pituitary-Adrenal (HPA) axis is a central neuroendocrine system that regulates the body's response to stress and maintains homeostasis through the coordinated release of hormones. Its activity exhibits complex circadian rhythms, crucial for health, and disruption of these rhythms is implicated in numerous pathologies [60] [61]. Mathematical modeling provides a powerful framework to understand this complex system, complementing experimental findings by simulating its dynamics, predicting behaviors under various conditions, and elucidating the consequences of circadian disruption [60] [61]. This review explores how mathematical modeling, from ordinary differential equations to discrete logical networks, enhances our understanding of HPA axis dynamics. We focus on its role in simulating circadian rhythms and pathological states, framed within broader research on glucocorticoid and ACTH chronobiology, and provide a practical toolkit for researchers.

Core Circadian Dynamics of the HPA Axis

The HPA axis is hierarchically controlled by the central pacemaker in the suprachiasmatic nucleus (SCN). The SCN synchronizes internal physiology to the external light-dark cycle, signaling the HPA axis to release hormones in a pulsatile and circadian manner [60] [62] [61].

Physiological Cortisol Rhythm

In healthy individuals, cortisol secretion follows a distinct and reproducible pattern [62]:

• Nadir: Lowest levels occur around midnight.
• Morning Rise: Levels begin to ascend between 02:00 and 03:00.
• Peak: Maximum concentration is reached at approximately 08:30.
• Diurnal Decline: Levels gradually decrease throughout the day to complete the cycle.

Quantitatively, the peak cortisol level attains approximately 399 nmol/L, while the nadir falls below 50 nmol/L [62]. This rhythm is regulated by a core transcriptional-translational feedback loop (TTFL) of clock genes within SCN neurons [4].

The HPA Axis as a Coupled Oscillator System

The HPA axis functions as a system of coupled oscillators with integrated feedback [60] [61]:

• Central Drive: The SCN provides the primary entraining signal via neuronal projections to CRH-secreting neurons in the hypothalamic paraventricular nucleus (PVN) and through circadian release of arginine-vasopressin (AVP) [3] [4].
• Hormonal Cascade: CRH release stimulates pituitary secretion of ACTH, which in turn prompts cortisol release from the adrenal cortex.
• Feedback Loops: Cortisol exerts negative feedback on the hypothalamus and pituitary to suppress further CRH and ACTH secretion. This feedback occurs through glucocorticoid (GR) and mineralocorticoid (MR) receptors [63] [61].
• Ultradian Rhythms: Superimposed on the circadian rhythm are ultradian oscillations with a period of 60–90 minutes, representing 12–18 secretory episodes throughout a 24-hour period [63].

caption: The core signaling pathway of the HPA axis, showing the influence of the central SCN clock and the hormonal cascade with its feedback loops.

Mathematical Modeling Approaches for the HPA Axis

Mathematical models of the HPA axis range from detailed continuous differential equations to abstract discrete logical networks, each offering unique insights.

Ordinary Differential Equation (ODE) Models

ODE models are the most prevalent approach, describing the kinetics of hormone concentrations.

Goodwin-Type Oscillator Models One of the earliest and most influential frameworks, the Goodwin oscillator, uses a delayed negative-feedback loop to generate oscillations [61]. In the HPA context:

• ( X_1 ) represents CRH concentration
• ( X_2 ) represents ACTH concentration
• ( X_3 ) represents Cortisol concentration
• Cortisol (( X3 )) inhibits the production of CRH (( X1 ))

This model has been expanded to include Michaelian degradation kinetics, avoiding unrealistically high Hill coefficients and improving biological fidelity [61]. Such models successfully capture essential properties like phase response curves and time-dependent stress responses [61].

Mechanistic Models Integrating Circadian and Feedback Dynamics Recent models incorporate greater physiological detail. A notable mechanistic framework integrates [41]:

• Circadian Input: A sinusoidal stress input ( u(t) ) with a 24-hour period.
• Hormonal Kinetics: Differential equations for CRH (( s1 )), ACTH (( s2 )), and cortisol (( s_3 )) dynamics.
• Glandular Plasticity: Long-term adaptation through changes in functional corticotroph (( C )) and adrenal (( A )) cell mass under chronic stress.
• Dynamic GR Resistance: A time-varying function to simulate feedback desensitization and its resolution.

Discrete and Logical Modeling

When precise kinetic parameters are unavailable, discrete formalisms offer a valuable alternative by focusing on network connectivity and qualitative dynamics.

Generalized Discrete Formalism This parsimonious framework uses multi-valued logic (e.g., cortisol and receptor expression levels can be 0, 1, or 2) and requires minimal parameterization [64]. The model's topology is analyzed to identify functional positive and negative feedback loops, which are necessary for multi-stability and oscillations, respectively [64]. A key finding is that this simple model can reproduce bi-stable cyclic attractors, corresponding to normal and pathological circadian cycles, as predicted by more complex ODE models [64]. The system's state can be shifted between these attractors by external stressors or therapeutic interventions, demonstrating regulatory plasticity [64].

Key Parameters and Variables in HPA Axis Models

The following table summarizes critical components and parameters used in various HPA axis models.

Table 1: Key Variables and Parameters in HPA Axis Mathematical Models

Component/Parameter	Description	Typical Formulation/Value
CRH (( s_1 ))	Hypothalamic Corticotropin-Releasing Hormone concentration	ODE: ( \frac{ds_1}{dt} = \text{Secretion} - \text{Degradation} ) [41]
ACTH (( s_2 ))	Pituitary Adrenocorticotropic Hormone concentration	ODE: ( \frac{ds2}{dt} = f(CRH, Cortisol Feedback) - k2 \cdot s_2 ) [41]
Cortisol (( s_3 ))	Adrenal Cortisol concentration	ODE: ( \frac{ds3}{dt} = g(ACTH, Adrenal Mass) - k3 \cdot s_3 ) [41]
Circadian Input (( u(t) ))	SCN-derived drive on the HPA axis	Sinusoidal function with 24-hour period [41]
GR Resistance	Time-varying sensitivity to glucocorticoid feedback	Function mimicking desensitization and resolution [41]
Logical Parameters (K)	Relative contextual weight of signals in discrete models	Tuned to ensure functional feedback loops (e.g., ( K_{43}=2 ) for ACTH effect on CORT) [64]

Simulating Pathological States and Therapeutic Interventions

Mathematical models are instrumental in elucidating the mechanisms of HPA axis dysfunction in disease and evaluating treatments.

Major Depressive Disorder (MDD) and Chronic Stress

MDD, particularly the melancholic subtype, is often associated with HPA axis dysregulation [63] [41].

• Hypercortisolism and GR Resistance: A core hypothesis is that MDD involves decreased sensitivity and density of GRs, leading to impaired negative feedback and sustained high cortisol levels [63] [41]. Mechanistic models incorporate dynamic GR resistance to show how this leads to hypercortisolism and a blunted response to stress, with recovery often being protracted [41].
• Allostatic Load: Chronic stress leads to repeated activation of the HPA axis, resulting in accumulated "wear and tear" known as allostatic load. This decreases the system's flexibility to respond to new stressors and can be simulated as a migration from a healthy to a pathological stable state in discrete models [60] [64].

Prenatal Programming of Disease

Adverse conditions in utero, such as exposure to synthetic glucocorticoids, can reprogram HPA axis function and increase lifelong risk for neuropsychiatric disorders [3].

• Mechanisms: Prenatal GC exposure alters hippocampal neurogenesis and can lead to epigenetic modifications in genes regulating proliferation, differentiation, and migration [3].
• Circadian Alterations: In mouse models, prenatal GC exposure alters circadian activity patterns and weakens the coupling between the central and peripheral clocks, preceding the onset of depression-like behavior [3].

Evaluating Diagnostic Tests: The ACTH Stimulation Test

The ACTH test is a standard diagnostic tool, but its interpretation is complex under dynamic HPA states. Modeling provides insights:

• Simulating the Test: Models can integrate an exogenous ACTH input term into the cortisol production equation, ( \text{Production}{s3} = A \cdot (s2 + \text{ACTH}{ex}) / (1 + \text{ACTH}_{ex}) ), to simulate both low-dose (1 µg) and high-dose (250 µg) tests [41].
• Model Findings: Simulations reveal that cortisol responses are phase-dependent. During recovery from chronic stress, ACTH responsiveness is often blunted due to persistent GR resistance and glandular remodeling. Low-dose ACTH may more reliably detect partial adrenal adaptation than high-dose tests [41].

caption: A workflow for using mechanistic modeling to simulate and interpret ACTH stimulation tests across different physiological states.

The Scientist's Toolkit: Research Reagent Solutions

This table details key reagents, computational tools, and protocols essential for experimental and computational research on the HPA axis.

Table 2: Essential Research Tools for HPA Axis Investigation

Tool/Reagent	Function/Description	Application Example
Synthetic GCs (e.g., Dexamethasone)	Potent synthetic glucocorticoid receptor agonist	Used in experiments to study negative feedback, and prenatally in animal models of programming [3].
ACTH (1 µg & 250 µg)	Synthetic adrenocorticotropic hormone	Diagnostic stimulation test to assess adrenal gland responsiveness and reserve [41].
GR Antagonists (e.g., Mifepristone)	Blocker of glucocorticoid receptors	Used in models to disrupt negative feedback; discrete models predict this can shift the system from a pathological to a healthy attractor [64].
VeVaPy (Python Library)	Computational framework for model Verification & Validation (V&V)	Contains modules (dataImport, DEsolver, optimize, visualize) to benchmark HPA models against experimental data [63].
Trier Social Stress Test (TSST)	Standardized protocol for psychosocial stress induction	Used to elicit a reliable cortisol response in human subjects; movement during TSST can predict cortisol reactivity via machine learning [65].
Modified-Release Hydrocortisone	Pharmaceutical formulation designed to mimic the circadian cortisol rhythm	Used in replacement therapy for adrenal insufficiency to improve biochemical control and quality of life compared to immediate-release formulations [62].

Mathematical modeling is an indispensable tool for deciphering the complex, multi-scale dynamics of the HPA axis. Through ODEs, discrete logical networks, and other frameworks, researchers can simulate the system's robust circadian rhythms, understand its vulnerability to pathological states like major depression, and evaluate the principles of chronotherapy. The future of this field lies in developing even more integrated models that incorporate genetic and epigenetic factors, the role of other brain regions like the amygdala and hippocampus, and the complex crosstalk with the immune and metabolic systems. As models become more refined and are rigorously validated against clinical data, they will play an increasingly critical role in personalizing chronotherapeutic strategies for patients with HPA axis disorders.

Circadian rhythms are endogenous, near-24-hour cycles that govern a vast array of physiological processes, from sleep-wake patterns to hormone secretion and metabolism [66]. The master circadian pacemaker, located in the suprachiasmatic nucleus (SCN) of the hypothalamus, synchronizes peripheral clocks found in virtually every tissue and cell in the body [67]. The reliable assessment of circadian phase is crucial for both research and the emerging field of circadian medicine, particularly in the context of glucocorticoid and ACTH research, as their secretion is under strong circadian control [67] [66]. This technical guide provides an in-depth examination of established and novel circadian biomarkers, their measurement methodologies, and their application in clinical and research settings.

Core Circadian Biomarkers

The most established biomarkers for assessing the human circadian phase are the hormones melatonin and cortisol. Their distinct diurnal rhythms provide robust, measurable outputs of the SCN's activity.

Melatonin and the Dim Light Melatonin Onset (DLMO)

Melatonin, secreted by the pineal gland, signals the onset of the biological night. Its production is suppressed by light and peaks during the dark phase [66]. The Dim Light Melatonin Onset (DLMO) is the most reliable marker of internal circadian timing [68] [66].

• Physiological Role: Promotes sleep and influences nearly every organ system, including antioxidant activity, bone formation, and immune function [66].
• Sampling Protocol: DLMO assessment typically requires a 4–6 hour sampling window, from 5 hours before to 1 hour after habitual bedtime. Sampling must occur under dim light conditions (<10–30 lux) to prevent suppression of melatonin secretion [66].
• Determination Methods:
  • Fixed Threshold: DLMO is defined as the time when interpolated melatonin concentrations cross an absolute threshold (e.g., 10 pg/mL in serum or 3–4 pg/mL in saliva).
  • Variable Threshold: The time when levels exceed two standard deviations above the mean of three or more baseline (pre-rise) values.
  • "Hockey-Stick" Algorithm: An automated, objective method that estimates the point of change from baseline to the rising phase of melatonin [66].

Cortisol and the Cortisol Awakening Response (CAR)

Cortisol, a glucocorticoid, exhibits a diurnal rhythm opposite to melatonin, peaking shortly after awakening and reaching its nadir around midnight. The Cortisol Awakening Response (CAR) is a sharp increase in cortisol levels within 30–45 minutes of waking and serves as an index of hypothalamic-pituitary-adrenal (HPA) axis activity [66].

• Physiological Role: A key stress hormone that regulates metabolism, immune function, and the response to stress. Its onset of quiescence is phase-locked to melatonin onset [66].
• Sampling Protocol: To capture CAR, samples are typically collected immediately upon awakening, and then at 30-minute intervals for the next 1–1.5 hours. Timing must be strictly adhered to and recorded by participants [66].
• Precision Note: While useful, cortisol is a less precise marker of SCN phase than melatonin. Melatonin allows phase determination with a standard deviation of 14–21 minutes, compared to about 40 minutes for cortisol [66].

Table 1: Comparison of Core Circadian Biomarkers

Feature	Melatonin (DLMO)	Cortisol (CAR)
Primary Role	Marker of biological night onset	Marker of HPA axis activity & morning awakening
Peak Time	Biological night (early morning)	30-45 minutes after awakening
Gold Standard	Plasma or saliva under dim light	Saliva upon and after awakening
Key Metric	Dim Light Melatonin Onset (DLMO)	Cortisol Awakening Response (CAR)
Phase Precision	High (SD: 14-21 min) [66]	Moderate (SD: ~40 min) [66]
Common Matrices	Blood (plasma/serum), saliva, urine	Blood (plasma/serum), saliva, urine
Major Confounders	Ambient light, beta-blockers, NSAIDs [66]	Stress, sleep quality, exact timing of awakening

Molecular Architecture of the Circadian Clock

The rhythmicity of hormonal outputs is generated by a conserved molecular feedback loop within cells.

The core mechanism involves transcription-translation feedback loops (TTFLs):

• Core Negative Feedback Loop: The BMAL1 and CLOCK proteins form a heterodimer that activates the transcription of Period (Per) and Cryptochrome (Cry) genes. PER and CRY proteins accumulate in the cytoplasm, form a complex, translocate to the nucleus, and inhibit BMAL1:CLOCK activity, closing the feedback loop [67].
• Auxiliary Stabilizing Loop: The BMAL1:CLOCK complex also activates transcription of nuclear receptors REV-ERB and ROR. These compete for binding sites on the Bmal1 promoter; ROR activates while REV-ERB represses Bmal1 transcription, creating a second, stabilizing loop [67].

Analytical Methods for Biomarker Detection

Accurate quantification of circadian hormones is paramount. The choice of matrix and analytical platform significantly impacts data reliability.

Table 2: Analytical Methods for Melatonin and Cortisol Detection

Parameter	Immunoassays (ELISA)	Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)
Principle	Antibody-antigen binding	Mass-to-charge ratio separation and detection
Specificity	Moderate (prone to cross-reactivity) [66]	High (minimal cross-reactivity) [68] [66]
Sensitivity	Variable, can be insufficient for low saliva levels [66]	Excellent, ideal for low-concentration matrices like saliva [66]
Throughput	High	Moderate to High
Cost	Lower	Higher (instrumentation, expertise)
Best For	High-throughput screening, well-characterized assays	Gold-standard validation, low-concentration samples, multiplexing

Biological Matrices

• Blood (Plasma/Serum): Offers high analyte levels and reliability but is invasive, limiting frequent sampling. It is the traditional matrix for DLMO assessment [66].
• Saliva: Non-invasive and ideal for ambulatory and frequent sampling (e.g., for CAR). Hormone concentrations are lower, requiring highly sensitive detection methods like LC-MS/MS [68] [66].
• Urine: Useful for measuring metabolite excretion over longer periods (e.g., 6-sulfatoxymelatonin), providing an integrated measure of hormone production rather than a precise phase marker [66].

Emerging Frontiers and Novel Biomarkers

While melatonin and cortisol are cornerstone biomarkers, new approaches are being developed to assess circadian phase with fewer samples and in diverse conditions.

Transcriptomic Biomarkers

Machine learning approaches are being applied to transcriptomic data from blood to develop multivariate biomarkers for SCN phase. These methods aim to predict internal time from a single blood draw by analyzing the expression patterns of dozens to hundreds of genes [69].

• Feature Selection Methods: Algorithms like Partial Least Squares Regression (PLSR), ZeitZeiger, and Elastic Net are used to identify optimal gene sets ("features") from high-dimensional data. Performance depends heavily on the training set's size and experimental conditions, with small sample sizes leading to overfitting and poor real-world performance [69].
• Key Challenge: Biomarkers trained under baseline conditions (e.g., sufficient sleep, stable routines) often fail to perform well under conditions of circadian disruption, such as shift work or sleep deprivation, highlighting the need for robust training datasets [69].

Blood Clock Correlation Distance (BloodCCD)

The Blood Clock Correlation Distance (BloodCCD) is a novel biomarker designed to assess circadian disruption from a single blood sample. It is derived from the expression of 42 genes known to oscillate in blood [70].

• Principle: Instead of measuring the absolute expression level of genes at a single time, BloodCCD calculates the Spearman correlation between the expression levels of these 42 genes in a patient's sample and a pre-established reference correlation from healthy individuals. A higher BloodCCD score indicates greater disruption from the normal, rhythmic correlation pattern [70].
• Clinical Application: In a study of cancer survivors with insomnia, BloodCCD scores were significantly higher (more disrupted) compared to healthy individuals. Furthermore, the degree of insomnia severity correlated with worse BloodCCD scores, positioning it as a promising objective biomarker for circadian disruption in clinical populations [70].

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Reagents and Kits for Circadian Biomarker Research

Item	Function	Example Application
PAXgene Blood RNA Tubes	Stabilizes intracellular RNA at the point of collection for accurate gene expression profiling [70].	Blood transcriptomics studies (e.g., BloodCCD).
RNA Isolation Kits	Purifies high-quality total RNA from stabilized blood or other tissues [70].	Sample preparation for RNA-sequencing or microarray.
Globin RNA Depletion Kits	Removes highly abundant globin mRNAs from blood-derived RNA, improving sequencing depth of other transcripts [70].	Enhancing data quality in whole-blood transcriptomics.
TruSeq Stranded mRNA Kit	Prepares sequencing libraries from purified RNA for transcriptome analysis on Illumina platforms [70].	RNA-sequencing library preparation.
Salivette Collection Devices	Provides a hygienic and standardized method for saliva sample collection for hormone assays [66].	Ambulatory cortisol and melatonin sampling.
LC-MS/MS Systems	Gold-standard platform for the specific, sensitive, and simultaneous quantification of low-level hormones like melatonin and cortisol [68] [66].	Quantifying hormones in saliva, plasma, or serum.
High-Sensitivity ELISA Kits	Immunoassay-based detection of hormones; requires validation against MS for specificity, especially for salivary melatonin [66].	High-throughput screening of hormone levels.

Experimental Protocols for Core Biomarkers

Protocol: Determining Dim Light Melatonin Onset (DLMO)

Objective: To establish the time of onset of melatonin secretion under dim light conditions as a marker of circadian phase.

Materials:

• Salivettes or blood collection equipment.
• Low-light environment (<10–30 lux, verified by lux meter).
• Freezer (-20°C or -80°C) for sample storage.
• LC-MS/MS system or validated high-sensitivity immunoassay.

Procedure:

• Participant Preparation: Instruct participants to avoid bright light for at least 1–2 hours before the session. Restrict caffeine, heavy exercise, and high-protein meals prior to and during sampling. Document any medication use (e.g., beta-blockers, NSAIDs).
• Sampling Environment: Maintain dim light throughout the entire procedure. Use red light if necessary for participant movement and sample handling.
• Sample Collection:
  • Begin collection 5 hours before habitual bedtime.
  • Collect saliva or blood samples every 30–60 minutes for 5–7 hours.
  • Record the exact time of each sample.
  • Centrifuge saliva samples and store aliquots immediately at -20°C or below.
• Hormone Analysis: Quantify melatonin concentration using LC-MS/MS (preferred for specificity, especially in saliva) or a rigorously validated immunoassay.
• Data Analysis: Plot melatonin concentration against clock time. Calculate DLMO using a predetermined method (e.g., fixed threshold of 3–4 pg/mL for saliva or a variable threshold). Visual inspection of the curve is recommended to confirm the calculated time [66].

Protocol: Assessing Cortisol Awakening Response (CAR)

Objective: To measure the dynamic change in cortisol levels in the first hour after morning awakening.

Materials:

• Salivette collection devices.
• Participant diary or electronic timer to record exact sampling times.
• Freezer for sample storage.
• LC-MS/MS or sensitive immunoassay for cortisol.

Procedure:

• Participant Training: Thoroughly instruct participants on the protocol, emphasizing the critical importance of exact timing.
• Sample Collection:
  • Sample 1: Immediately upon awakening (time "0").
  • Sample 2: 30 minutes after awakening.
  • Sample 3: 45 minutes after awakening.
  • Participants should not eat, drink (except water), smoke, or brush their teeth until after the final sample is collected.
• Sample Handling: Participants should refrigerate samples immediately after collection and return them to the lab as soon as possible for centrifugation and long-term storage at -20°C or -80°C.
• Hormone Analysis: Quantify cortisol in all samples.
• Data Analysis: The CAR can be expressed as the area under the curve (AUC) with respect to the increase from the waking sample, or simply as the concentration at each time point. The magnitude of the rise (Sample 2 - Sample 1) is also a common metric [66].

The precise measurement of circadian biomarkers is foundational to advancing glucocorticoid and ACTH research. While melatonin (via DLMO) and cortisol (via CAR) remain the gold-standard endocrine markers, rigorous methodological protocols are essential for reliable data. The field is rapidly evolving with the development of novel transcriptomic biomarkers, such as BloodCCD and other multivariate models, which promise to lower the burden of circadian phase assessment. The integration of these established and emerging tools will enable a more comprehensive understanding of circadian disruption in disease and facilitate the development of targeted chronotherapies.

When the Clock Falters: Diagnosing and Managing HPA Axis Circadian Disruption

Circadian rhythms, the endogenous 24-hour biological cycles, govern a wide array of physiological processes, including endocrine function, metabolism, and cardiovascular activity [71] [67]. The suprachiasmatic nucleus (SCN) of the hypothalamus serves as the master circadian pacemaker, synchronizing peripheral clocks throughout the body via neuronal and hormonal pathways [67] [12]. Cushing Syndrome (CS), characterized by chronic endogenous hypercortisolism, represents a profound model of circadian disruption wherein the normal rhythmicity of glucocorticoid secretion is obliterated [72] [73]. This disruption has far-reaching consequences, including metabolic disturbances, immune dysfunction, and notably, the loss of normal blood pressure dipping patterns—a key cardiovascular risk factor [74] [73]. This technical review examines the pathophysiological mechanisms linking circadian dysregulation in CS to non-dipping blood pressure, providing detailed experimental methodologies and analytical frameworks for researchers investigating glucocorticoid-mediated circadian disruption.

Pathophysiological Framework: Circadian Rhythm Disruption in Hypercortisolism

The Hypothalamic-Pituitary-Adrenal Axis and Circadian Regulation

Under physiological conditions, the hypothalamic-pituitary-adrenal (HPA) axis exhibits robust circadian rhythmicity. Glucocorticoid secretion peaks in the early morning prior to waking (circadian dawn for diurnal species) and reaches its nadir during the night [12]. This rhythm is generated through integrated signaling involving the SCN, which projects to the paraventricular nucleus (PVN) via arginine-vasopressin neurons, leading to rhythmic corticotropin-releasing hormone (CRH) and arginine-vasopressin release [12]. These neuropeptides stimulate pituitary corticotropes to release adrenocorticotropic hormone (ACTH), which in turn drives cortisol production from the adrenal cortex [12].

The adrenal gland itself possesses an intrinsic circadian clock that gates its sensitivity to ACTH [71] [12]. Additionally, autonomic innervation via the splanchnic nerve transmits light information directly from the SCN to the adrenal gland, further modulating glucocorticoid production independent of ACTH signaling [12]. This multilayered regulation ensures that cortisol secretion is precisely timed to coordinate metabolic, immune, and cardiovascular functions with anticipated daily demands [71] [12].

Molecular Clock Disruption in Chronic Hypercortisolism

The molecular circadian clock operates through transcription-translation feedback loops (TTFLs) composed of core clock genes and proteins. The positive limb includes CLOCK and BMAL1, which form heterodimers that activate transcription of Period (Per1-3) and Cryptochrome (Cry1/2) genes by binding to E-box elements in their promoter regions [67]. PER and CRY proteins accumulate, form complexes, and translocate back to the nucleus to inhibit CLOCK:BMAL1 activity, completing the negative feedback loop [67]. Auxiliary loops involve REV-ERBα and RORα, which competitively bind ROR response elements (ROREs) to repress and activate Bmal1 transcription, respectively [67].

Chronic hypercortisolism disrupts this molecular oscillator through several mechanisms. Glucocorticoid response elements (GREs) are present in the promoter regions of several clock genes, including Per1 and Per2 [12]. Sustained glucocorticoid exposure ablates normal circadian gene expression patterns, as demonstrated in a study of CS patients showing significant flattening of circadian oscillations in CLOCK, PER1, PER2, PER3, and TIMELESS expression in peripheral blood mononuclear cells (PBMCs) [73]. This molecular disruption persists even after remission, suggesting long-term reprogramming of circadian function [73].

Table 1: Core Clock Genes and Their Alterations in Cushing Syndrome

Clock Gene	Normal Function	Alteration in Active CS	Recovery After Remission
CLOCK	Forms heterodimer with BMAL1; activates transcription of Per and Cry genes	Flattened circadian oscillation [73]	Partial restoration [73]
BMAL1	Forms heterodimer with CLOCK; core transcriptional activator	Altered expression [67]	Variable recovery [73]
PER1	Forms part of negative feedback complex; inhibits CLOCK:BMAL1	Significant flattening of rhythm [73]	Partial restoration [73]
PER2	Collaborates with PER1 in negative feedback	Significant flattening of rhythm [73]	Partial restoration [73]
CRY1/2	Completes negative feedback complex; translocates to nucleus	Altered expression patterns [67]	Incomplete recovery [73]
REV-ERBα	Represses Bmal1 transcription; stabilizes feedback loop	Disrupted expression [67]	Limited data available
TIMELESS	Modulates PER stability and nuclear translocation	Flattened circadian oscillation [73]	Partial restoration [73]

Signaling Pathway of Circadian Glucocorticoid Action

The following diagram illustrates the molecular pathways through which glucocorticoids regulate circadian function and how these pathways are disrupted in Cushing Syndrome:

Diagram Title: Molecular Pathways of Circadian Disruption in Cushing Syndrome

Non-Dipping Blood Pressure in Cushing Syndrome: Mechanisms and Measurement

Circadian Blood Pressure Regulation and the Non-Dipping Phenomenon

In healthy individuals, blood pressure follows a characteristic circadian pattern, with higher values during waking hours and a nocturnal decrease (dipping) of approximately 10-20% during sleep [74]. This pattern is regulated by the interplay of the circadian system with cardiovascular function, involving autonomic nervous system balance, endothelial function, renal sodium handling, and neuroendocrine factors [74]. The non-dipping phenotype, characterized by a blunted nocturnal blood pressure decline (<10% drop from daytime values), represents a significant cardiovascular risk factor independently of absolute blood pressure values [74].

In CS, the prevalence of non-dipping blood pressure is markedly increased. A study of CS patients revealed that 70% exhibited non-dipping patterns compared to 25% in matched controls [74]. The mechanisms underlying this phenomenon include:

• Autonomic Dysregulation: Hypercortisolism increases sympathetic nervous system activity while reducing parasympathetic tone, disrupting normal nocturnal cardiovascular regulation [74].
• Endothelial Dysfunction: Glucocorticoids reduce nitric oxide bioavailability and impair endothelium-dependent vasodilation, contributing to sustained vascular resistance [74].
• Renal Mechanisms: Glucocorticoids promote sodium retention and volume expansion through both mineralocorticoid receptor activation and non-genomic effects on renal tubular function [72] [74].
• Circadian Clock Disruption: The damping of peripheral circadian clocks in vascular smooth muscle and endothelial cells impairs local timing of vascular tone regulation [74].

Table 2: Mechanisms of Non-Dipping Blood Pressure in Cushing Syndrome

Mechanistic Pathway	Physiological Basis	Consequence in CS
Autonomic Imbalance	Normal nocturnal shift toward parasympathetic dominance	Sympathetic overactivity persists overnight; reduced heart rate variability [74]
Endothelial Dysfunction	Intact NO-mediated vasodilation	Impaired vasodilation; increased peripheral resistance [74]
Renal Sodium Handling	Normal pressure natriuresis	Sodium retention; volume expansion; suppressed renin-angiotensin system [72] [74]
Vascular Circadian Clocks	Rhythmic expression of vascular tone regulators	Damped Bmal1, Per2 oscillations in vascular tissue; lost timing of vasoactive genes [74]
HPA Axis Dysregulation	Normal circadian cortisol rhythm with nocturnal trough	Loss of cortisol rhythm; sustained overnight glucocorticoid exposure [12] [73]

Experimental Protocols for Assessing Circadian Blood Pressure Patterns

Ambulatory Blood Pressure Monitoring (ABPM) Protocol

Objective: To characterize 24-hour blood pressure patterns and identify non-dipping status in CS patients and controls.

Equipment: Validated ambulatory blood pressure monitor, actigraphy device, sleep diary.

Procedure:

• Participants wear ABPM device for 24-48 hours with measurements every 20-30 minutes during waking hours and every 30-60 minutes during scheduled sleep periods.
• Simultaneous actigraphy monitoring to objectively determine sleep-wake patterns.
• Patients maintain detailed activity and sleep diaries, noting posture changes, naps, medication times, and symptoms.
• Data analysis includes:
  • Calculation of mean daytime and nighttime systolic BP (SBP), diastolic BP (DBP), and mean arterial pressure (MAP)
  • Determination of dipping percentage: [(Mean daytime BP - Mean nighttime BP) / Mean daytime BP] × 100
  • Classification of dipping status: Dipper (≥10% decline), Non-dipper (<10% decline)
  • Cosinor analysis to determine rhythm parameters: mesor, amplitude, and acrophase

Validation: This protocol was implemented in a study of shift workers, demonstrating altered 24-hour blood pressure rhythms with blunted nocturnal dipping during circadian misalignment [74].

Laboratory Forced Desynchrony Protocol

Objective: To isolate endogenous circadian regulation of blood pressure from behavioral influences.

Equipment: Controlled environment facility, constant routine conditions, non-invasive beat-to-beat blood pressure monitoring.

Procedure:

• Participants reside in a laboratory environment free of time cues for 5-7 days.
• Implement forced desynchrony protocol with sleep-wake cycles different from 24 hours (e.g., 20-hour or 28-hour "days").
• Maintain participants in constant conditions including dim light, semirecumbent posture, and identical snacks evenly distributed across the circadian cycle.
• Measure cardiovascular parameters continuously including blood pressure, heart rate, and heart rate variability.
• Collect serial plasma samples for cortisol, catecholamines, and other relevant hormones.
• Analyze data with mixed-effects cosinor models to determine endogenous circadian rhythms in cardiovascular parameters independent of behavioral effects.

Application: This approach demonstrated that circadian misalignment alone increases systolic and diastolic blood pressure by 1.4 mmHg and 0.8 mmHg, respectively, even in healthy adults [74].

Immune System Circadian Disruption in Cushing Syndrome

The immune system exhibits robust circadian rhythms in cell trafficking, cytokine production, and effector functions [67] [73]. Circulating immune cells, including various PBMC subsets, show time-dependent fluctuations that are critical for optimal immune surveillance and response coordination [73]. Chronic hypercortisolism profoundly disrupts these immune rhythms, creating a convergence point between circadian disruption and metabolic/immune dysfunction in CS [73].

Experimental Protocol: Multi-Timepoint Immune Profiling

Objective: To characterize circadian immune disruption in CS patients through comprehensive multi-timepoint sampling.

Study Design: 12-month prospective case-control multicenter trial with 34 CS patients and 34 matched controls [73].

Sampling Protocol:

• Blood collection at 5 timepoints across 24 hours: 08:00, 12:00, 16:00, 20:00, and 24:00 hours.
• PBMC isolation by density gradient centrifugation within 2 hours of collection.
• Flow cytometry analysis for immune cell subsets:
  • T cells (CD3+), B cells (CD19+), NK cells (CD56+)
  • Monocyte subpopulations (CD14+, CD16+)
  • Activation markers (CD69, HLA-DR)
• RNA extraction from PBMCs for clock gene expression analysis by RT-qPCR:
  • Core clock genes: CLOCK, BMAL1, PER1, PER2, PER3, CRY1, CRY2, TIMELESS
  • Reference genes: GAPDH, ACTB
• Plasma separation for cortisol, cytokine, and metabolic biomarker measurements.

Analytical Methods:

• Cosinor mixed-effects modeling to determine rhythm parameters
• JTK_Cycle and RAIN algorithms for rhythmicity detection
• Machine learning approaches (random forest, support vector machines) for classification
• Cross-correlation analysis between cortisol rhythms and immune parameters

Key Findings: Active CS patients exhibited profound alterations in levels, amplitude, and rhythmicity of multiple PBMC populations, with only partial restoration after remission [73]. Clock gene expression analysis showed significant flattening of circadian oscillations for CLOCK, PER1, PER2, PER3, and TIMELESS [73].

Table 3: Immune and Circadian Gene Alterations in Cushing Syndrome

Parameter	Active CS vs. Controls	After Remission	Analytical Method
PBMC Rhythmicity	Significant loss of circadian variation in multiple subsets	Partial restoration	Cosinor analysis [73]
Clock Gene Amplitude	Reduced amplitude for CLOCK, PER1-3, TIMELESS	Incomplete recovery	JTK_Cycle, RAIN [73]
Immune Cell Classification	Highly effective even at single timepoints	Improved but not normalized	Machine learning [73]
Cortisol-Immune Coupling	Decoupling of cortisol rhythms from immune rhythms	Partial recoupling	Cross-correlation [73]

Research Toolkit: Essential Reagents and Methodologies

Table 4: Research Reagent Solutions for Investigating Circadian Dysregulation in Cushing Syndrome

Research Tool Category	Specific Examples	Research Application	Key References
Circadian Gene Expression Analysis	RT-qPCR assays for core clock genes (BMAL1, CLOCK, PER1-3, CRY1-2, REV-ERBα, RORα); RNA sequencing for circadian transcriptomics	Quantifying molecular clock disruption in patient samples or cell models	[67] [73]
Glucocorticoid Signaling Reagents	GR antagonists (mifepristone), GR siRNA, GR overexpression vectors, GRE-luciferase reporters	Dissecting GR-dependent mechanisms in circadian disruption	[72] [12]
Immune Cell Profiling	Multipanel flow cytometry antibodies (CD3, CD19, CD56, CD14, CD16), cytokine ELISAs, multiplex immunoassays	Characterizing immune rhythm disruption in PBMCs	[73]
Circadian Monitoring Tools	Ambulatory BP monitors, actigraphy devices, dim-light melatonin onset (DLMO) kits	Assessing circadian parameters in human studies	[74] [75]
Cell Culture Models	Human adrenal H295R cells, primary fibroblast cultures, PBMCs from patients	In vitro modeling of circadian interactions with glucocorticoid signaling	[71] [73]
Data Analysis Resources	Cosinor analysis software, JTK_Cycle, RAIN algorithms, random forest classifiers	Analyzing circadian parameters and classifying patient status	[73]

Integrated Experimental Workflow

The following diagram outlines a comprehensive experimental approach for investigating circadian dysregulation in Cushing Syndrome, integrating molecular, physiological, and clinical assessments:

Diagram Title: Comprehensive Workflow for CS Circadian Research

Cushing Syndrome represents a profound model of circadian disruption wherein chronic hypercortisolism obliterates the normal rhythmicity of glucocorticoid signaling, leading to widespread dysregulation of molecular clocks, immune function, and cardiovascular regulation. The non-dipping blood pressure phenotype emerges as a key clinical manifestation of this circadian disruption, reflecting integrated pathophysiology across autonomic, vascular, renal, and endocrine systems. Future research should focus on developing chronotherapeutic approaches specifically targeting the circadian disruption in CS, including timed glucocorticoid receptor antagonism, chrono-specific immune modulators, and interventions to restore circadian amplitude. The research methodologies and analytical frameworks detailed herein provide a foundation for advancing our understanding of circadian dysregulation in endocrine disease and developing targeted interventions to mitigate its clinical consequences.

Chronic adrenocorticotropic hormone (ACTH) excess, a hallmark of Cushing's Syndrome, induces a complex pathophysiology characterized by the emergence of salt-sensitive hypertension and impaired renal function. This whitepaper synthesizes current research demonstrating that ACTH-driven glucocorticoid excess disrupts circadian rhythmicity at multiple levels—from molecular clock genes to systemic hemodynamics. Central to this pathology is the ablation of the diurnal rhythm of sodium excretion, leading to a non-dipping blood pressure phenotype and a fundamental shift in renal pressure-natriuresis response. The ensuing salt-sensitivity creates a deleterious cycle that significantly elevates cardiovascular risk. Understanding these mechanisms within the framework of circadian biology offers novel perspectives for therapeutic intervention in endocrine hypertension.

The hypothalamic-pituitary-adrenal (HPA) axis exhibits a robust circadian rhythm, essential for metabolic homeostasis and cardiovascular health. In healthy individuals, ACTH secretion begins to rise late at night, triggering a characteristic cortisol peak in the early morning hours to prepare the body for wakefulness [76]. This rhythmicity is orchestrated by the master circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus [67] [4].

The SCN conveys timing information to peripheral tissues through neuronal and hormonal pathways, synchronizing peripheral clocks in organs including the kidneys, liver, and adrenal glands [67] [4]. Glucocorticoids (GCs), in turn, act as potent entrainers of these peripheral circadian clocks [73] [4]. This creates a tightly regulated feedback system where the central clock regulates GC release, and GCs help synchronize peripheral oscillators. Chronic ACTH excess fundamentally disrupts this delicate temporal organization, leading to a cascade of metabolic and cardiovascular complications.

Molecular Architecture of the Circadian Clock and Glucocorticoid Interplay

Core Clock Machinery

The mammalian circadian rhythm is generated at the cellular level by a transcriptional-translational feedback loop (TTFL) comprising core clock genes [67] [4].

• Positive Limb: The heterodimeric transcription factor complex CLOCK-BMAL1 binds to E-box elements in the promoter regions of target genes, including Period (Per) and Cryptochrome (Cry) [67].
• Negative Limb: PER and CRY proteins accumulate, form complexes in the cytoplasm, and translocate to the nucleus to inhibit CLOCK-BMAL1-mediated transcription, thereby repressing their own expression [67].
• Stabilizing Loop: The CLOCK-BMAL1 complex also activates transcription of Rev-Erbα, which represses Bmal1 transcription. This parallel loop enhances the robustness and stability of the core oscillator [67].

This evolutionarily conserved mechanism ensures the precision and periodicity of circadian oscillations in virtually every cell type.

Glucocorticoids as Circadian Entrainers

Glucocorticoids are systemic signals that help maintain temporal order in physiological processes [4]. They cross the blood-brain barrier in a controlled manner and modulate a multitude of brain functions [4]. The circadian clock within the SCN regulates the rhythmic release of GCs from the adrenal cortex, and this rhythm is dependent on a functional SCN [4]. Peripheral clocks, such as those in the choroid plexus, are highly sensitive to GCs, creating a critical link between the central pacemaker and tissue-specific circadian regulation [4].

The following diagram illustrates the core transcriptional-translational feedback loop of the molecular circadian clock:

Pathophysiological Impact of Chronic ACTH Excess

Disruption of Circadian Immune and Metabolic Rhythms

Chronic endogenous hypercortisolism, as seen in Cushing's Syndrome (CS), causes profound disruption of biological rhythms. A multi-level analysis from a multicentre clinical trial revealed significant alterations in patients with active CS [73]:

• Immune Cell Rhythmicity: The daily fluctuations in circulating peripheral blood mononuclear cells (PBMCs) showed profound changes in levels, amplitude, and rhythmicity.
• Clock Gene Expression: A significant flattening of circadian oscillation was observed in the expression of core clock genes (CLOCK, PER1, PER2, PER3, and TIMELESS) in isolated PBMCs.
• Irreversible Damage: While most genes regained physiological oscillation after remission of CS, the restoration was only partial, suggesting long-term circadian dysfunction.

This study demonstrated that immune profiling was superior to cortisol levels, anthropometric measures, and circadian gene expression analysis for identifying CS activity, highlighting the profound impact of GC excess on the circadian-immune interface [73].

Renal Sodium Handling and Salt Sensitivity

Chronic ACTH infusion models in male C57BL/6J mice have provided crucial insights into the renal mechanisms underlying ACTH-induced hypertension [77] [78]. The key pathophysiological findings are summarized below:

Table 1: Physiological Effects of Chronic ACTH Infusion in Mouse Models

Parameter	Effect of Chronic ACTH	Functional Consequence
Blood Pressure Pattern	Induction of non-dipping phenotype	Loss of normal nocturnal blood pressure decline
Salt Sensitivity	Transition to salt-sensitive hypertension	Pathological blood pressure elevation in response to high salt intake
Sodium Excretion Rhythm	Abolished diurnal rhythm of sodium excretion	Urinary sodium/potassium ratio reduced
Renal Hemodynamics	Impaired autoregulation of renal blood flow	Compromised pressure-natriuresis response
Vascular Function	Diminished nitric oxide response in renal artery	Impaired vasodilation
Gene Expression	Evidence of arterial remodeling and enhanced TGF-β signaling	Structural and functional vascular changes

The experimental data demonstrate that ACTH excess impairs sodium excretion and causes a fundamental shift to non-dipping and salt-sensitive blood pressure. The underlying mechanisms include renal hemodynamic and tubular abnormalities that critically impair the pressure-natriuresis response [77].

The following diagram illustrates the pathophysiological pathways through which chronic ACTH excess leads to salt-sensitive hypertension:

Experimental Models and Methodological Approaches

Chronic ACTH Infusion Model Protocol

The following detailed methodology is adapted from studies investigating the effects of chronic ACTH excess in murine models [77] [78]:

Animal Model

• Subjects: Male C57BL/6J mice (8-12 weeks old)
• ACTH Administration: Continuous infusion via osmotic minipumps
• Dosage: ACTH (1-2 µg/day) dissolved in saline vehicle
• Control Group: Vehicle-only infusion (saline)
• Duration: 10-14 days of continuous infusion

Blood Pressure Monitoring

• Technique: Radiotelemetry or tail-cuff plethysmography
• Frequency: Continuous or daily measurements
• Conditions: Recordings during control diet and following high-salt diet (e.g., 2-4% NaCl)
• Analysis: 24-hour profiles, light vs. dark phase comparisons, non-dipping classification

Renal Function Assessment

• Metabolic Cages: For 24-hour urine collection
• Parameters: Sodium, potassium excretion rates, urine volume
• Plasma Analysis: Electrolytes, creatinine, hormone levels
• Pressure-Natriuresis Response: In vivo assessment through controlled renal perfusion pressure manipulation

Vascular Function Studies

• Tissue Preparation: Isolation of renal arteries
• Ex Vivo Analysis: Wire or pressure myography for vascular reactivity
• Vasoreactivity Testing: Response to nitric oxide donors (e.g., sodium nitroprusside), endothelial-dependent vasodilators (e.g., acetylcholine), and vasoconstrictors
• Molecular Analysis: mRNA expression of remodeling markers (TGF-β pathway, extracellular matrix components)

This experimental workflow enables comprehensive assessment of the renal, hemodynamic, and molecular consequences of chronic ACTH excess, particularly focusing on circadian patterns of sodium handling and blood pressure regulation.

The Scientist's Toolkit: Essential Research Reagents

Table 2: Key Research Reagents for Investigating ACTH Effects

Reagent / Material	Function / Application	Experimental Context
ACTH (1-24)	Synthetic analog for chronic infusion; mimics endogenous ACTH	In vivo models of Cushing's Syndrome
Osmotic Minipumps	Sustained, continuous delivery of peptides/hormones	Chronic ACTH infusion studies
Radiotelemetry Probes	Continuous, high-fidelity blood pressure monitoring	Circadian BP rhythm analysis, non-dipping phenotype
Metabolic Cages	Precise separation and collection of urine and feces	24-hour sodium/potassium excretion measurements
Nitric Oxide Donors (e.g., SNP)	Assess endothelial-independent vasodilation	Ex vivo renal artery function studies
qPCR Assays for clock genes	Quantify circadian gene expression (Per1-3, Bmal1, Clock, Cry)	Molecular circadian rhythm analysis in tissues
TGF-β Pathway Inhibitors	Investigate signaling in vascular remodeling	Mechanistic studies of arterial dysfunction

Implications for Drug Development and Therapeutic Strategies

The recognition that chronic ACTH excess induces salt-sensitive hypertension through circadian disruption opens several promising therapeutic avenues:

• Chronotherapeutic Approaches: Timing of antihypertensive medications to align with disrupted circadian blood pressure patterns in hypercortisolemic patients.
• Circadian Rhythm-Targeted Therapies: Compounds that stabilize core clock function or enhance the robustness of circadian oscillations in peripheral tissues.
• Selective Glucocorticoid Receptor Modulators: Agents that mitigate the adverse metabolic and cardiovascular effects of GC excess while preserving essential physiological functions.
• 11β-HSD1 Inhibitors: Although previous trials in type 2 diabetes showed limited efficacy, potentially due to compensatory ACTH increase, these may still hold promise in specific contexts of HPA axis dysregulation [76].

The investigation of these strategies requires careful consideration of the complex interplay between the central circadian clock, tissue-specific peripheral clocks, and glucocorticoid signaling.

Chronic ACTH excess creates a multifaceted pathophysiology centered on the disruption of circadian rhythmicity at molecular, cellular, and systemic levels. The resulting salt-sensitive hypertension and non-dipping blood pressure phenotype stem from fundamental alterations in renal sodium handling, vascular function, and pressure-natriuresis response. Experimental models consistently demonstrate that ACTH-mediated glucocorticoid excess ablates the diurnal rhythm of sodium excretion, establishing a feed-forward cycle of cardiovascular risk. Framing this pathology within the context of circadian biology provides not only mechanistic insights but also novel therapeutic perspectives for managing the cardiovascular complications of Cushing's Syndrome and related disorders. Future research should focus on chronotherapeutic interventions and compounds that stabilize circadian oscillators to mitigate the substantial cardiovascular morbidity associated with HPA axis dysregulation.

Glucocorticoids (GCs), a class of steroid hormones produced by the adrenal cortex, serve as essential mediators in the cross-talk between the circadian system and immune regulation. These hormones exhibit robust diurnal oscillations under the control of the hypothalamic-pituitary-adrenal (HPA) axis, with circulating levels that peak at the onset of the active phase—in the morning for diurnal humans and at dusk for nocturnal rodents [12] [16]. This circadian rhythm is not merely a passive output but functions as an active timing signal that synchronizes peripheral immune processes with the anticipated daily challenges faced by an organism. The concept of glucocorticoids as "immune tuners" extends beyond their well-known immunosuppressive effects, positioning them as circadian organizers that prepare the immune system for potential threats by rhythmically modulating immune cell trafficking, cytokine production, and functional responses [16] [79]. This review explores the mechanisms through which circadian glucocorticoid signaling regulates both innate and adaptive immunity and examines the profound immunological consequences that arise from the disruption of this rhythmicity, as observed in conditions such as Cushing's syndrome, critical illness, and aberrant glucocorticoid therapy.

Molecular and Cellular Mechanisms of Circadian Glucocorticoid Signaling

The Glucocorticoid Receptor System

The biological effects of glucocorticoids are primarily mediated through the ubiquitously expressed glucocorticoid receptor (GR), a ligand-dependent transcription factor encoded by the NR3C1 gene [80]. The GR exists in multiple isoforms generated through alternative splicing and translation initiation, with GRα and GRβ being the most extensively studied. GRα binds glucocorticoids and translocates to the nucleus to regulate target gene transcription, whereas GRβ, which cannot bind ligand, often acts as a dominant-negative inhibitor of GRα activity [81]. The unligated GR resides in the cytoplasm as part of a multi-protein complex that includes heat shock proteins (HSPs) and immunophilins such as FK506-binding protein 5 (FKBP5). Upon hormone binding, the receptor undergoes conformational changes, dissociates from this complex, and translocates to the nucleus where it modulates gene expression by binding to glucocorticoid response elements (GREs) or by interacting with other transcription factors such as NF-κB and AP-1 [16] [80].

Table 1: Major Glucocorticoid Receptor Isoforms and Their Characteristics

Isoform	Ligand Binding	Primary Function	Tissue/Cell Expression	Role in Inflammation
GRα	Yes	Transcriptional activation	Ubiquitous	Mediates anti-inflammatory effects
GRβ	No	Dominant-negative regulator	Variable, increased in inflammation	Implicated in glucocorticoid resistance
GRγ	Yes	Altered transactivation	Broad tissue distribution	Reduced transactivation capacity
GR-P	Yes (reduced)	Truncated protein	Immune cells, stress conditions	Potential dominant-negative activity

The GR signaling system exhibits cell-specificity and temporal regulation, which underlies the diverse tissue responses to glucocorticoids. Recent research has revealed that critical illness induces distinct expression patterns of GR isoforms in different immune cell populations. In polymorphonuclear cells (PMNs), critical illness leads to sustained downregulation of GRα, GRβ, and GRγ, while GR-P remains stable. In contrast, peripheral blood mononuclear cells (PBMCs) maintain GRα, GRβ, and GRγ expression but upregulate GR-P during critical illness [81]. This cell-specific regulation of GR isoforms creates a circadian signaling landscape that determines the timing and magnitude of immune responses to glucocorticoid exposure.

Circadian Regulation of Glucocorticoid Secretion

The circadian rhythm of glucocorticoid release is governed by a multi-level regulatory system. The central pacemaker in the suprachiasmatic nucleus (SCN) transmits timing signals through neural and hormonal pathways to the adrenal cortex. The SCN influences the HPA axis via arginine-vasopressin (AVP) projections to the paraventricular nucleus (PVN), which in turn releases corticotropin-releasing hormone (CRH) to stimulate pituitary adrenocorticotropic hormone (ACTH) secretion [12] [16]. ACTH then acts on the adrenal cortex to promote glucocorticoid production. However, the robust circadian rhythm of glucocorticoids cannot be fully explained by ACTH rhythms alone, as ACTH shows much lower amplitude oscillations [12]. Additional mechanisms include direct autonomic innervation of the adrenal gland via the splanchnic nerve, which modulates adrenal sensitivity to ACTH, and the intrinsic adrenal clock, which gates the organ's responsiveness to ACTH stimulation [12] [16]. This multi-layered regulation ensures precise timing of glucocorticoid secretion, allowing the hormone to function as a powerful circadian zeitgeber (synchronizer) for peripheral clocks throughout the body, including those in immune cells.

Glucocorticoid Regulation of Innate Immunity

Circadian Control of Innate Immune Cell Trafficism and Function

Glucocorticoids rhythmically regulate the distribution and function of innate immune cells, particularly neutrophils and monocytes. Under steady-state conditions, endogenous GCs inhibit the expression of inflammatory cytokines and chemokines during the active phase in mice, creating a time-specific window for immune regulation [16]. For example, the chemokine CXCL5, which recruits neutrophils and monocytes to inflammatory sites, exhibits diurnal oscillation with a peak during the daytime in mice. This rhythm is directly suppressed by GCs through GR binding to negative GREs (nGREs) in the CXCL5 promoter [16]. The physiological significance of this regulation is evident in lung inflammation models, where neutrophil infiltration and CXCL5 expression show coordinated diurnal patterns that are impaired in adrenalectomized mice [16].

The circadian GC rhythm also prepares the innate immune system for enhanced responsiveness at the beginning of the active phase. This anticipatory priming ensures optimal defense capacity when encountering pathogens. Research has demonstrated that the timed suppression of inflammatory mediators by nocturnal GC peaks helps restrain excessive inflammation during the rest phase, while the declining GC levels at the start of the active phase permit enhanced immune reactivity [16] [79]. This carefully orchestrated regulation ensures that energy-intensive immune processes align with the active period when organisms are most likely to encounter pathogens and other immune challenges.

Molecular Mechanisms in Innate Immune Cells

At the molecular level, glucocorticoids regulate innate immune function through multiple mechanisms. The GR carries out immunosuppressive roles by transactivating anti-inflammatory factors such as GILZ (GC-induced leucine zipper) and IκBα, while simultaneously suppressing inflammatory cytokines and factors including IL-6 and complement component C3 through nGREs [16]. Additionally, GR can directly interact with and suppress the activity of key pro-inflammatory transcription factors such as NF-κB and AP-1 through a mechanism known as transrepression [16]. The relevance of these molecular mechanisms in vivo is supported by studies showing that macrophage-specific GR deficiency leads to enhanced production of inflammatory cytokines including IL-1β, TNF-α, and IL-12, resulting in increased susceptibility to inflammatory challenges [16].

Table 2: Glucocorticoid Regulation of Key Innate Immune Components

Immune Component	Regulation by GCs	Molecular Mechanism	Circadian Pattern
Neutrophil trafficking	Suppression of CXCL5	GR binding to nGREs in CXCL5 promoter	Peak at daytime (mice), anti-phase to GC rhythm
Inflammatory cytokines (IL-6, TNF-α)	Suppression	Transrepression of NF-κB/AP-1; induction of IκBα	Lowest during GC peak
Macrophage activation	Inhibition	Reduced TLR expression and signaling	Enhanced response at end of GC trough
Dendritic cell function	Modulation of antigen presentation	Altered co-stimulatory molecule expression	Time-dependent response to stimuli
Complement system	Regulation of C3 production	Binding to nGREs in target genes	Coordination with metabolic demands

Glucocorticoid Regulation of Adaptive Immunity

T Cell Migration and Function

Glucocorticoids exert complex, time-dependent effects on adaptive immunity, particularly on T cell biology. These hormones regulate the expression of homing receptors and chemokine receptors that control T cell trafficking and positioning within lymphoid organs. GCs induce the rhythmic expression of IL-7 receptor (IL-7R) and CXCR4 on T cells, which supports T cell maintenance and homing to lymphoid tissues [16]. This regulation ensures optimal T cell circulation and positioning according to the time of day, potentially enhancing immune surveillance during active periods and promoting maintenance functions during rest.

The effects of GCs on T cell function extend to their differentiation and effector responses. GCs generally suppress Th1 cell differentiation and reduce IFN-γ production by Th1, CD8+ T, and NK cells, leading to inhibition of cytotoxic responses [16]. This suppression exhibits circadian variation, with the magnitude of effect depending on the timing of GC exposure. The clinical relevance of this circadian regulation is evident in studies of congenital adrenal hyperplasia (CAH) patients receiving different glucocorticoid replacement regimens. Patients on reverse-circadian treatment (with higher evening doses) showed significant alterations in T cell subsets, including lower percentages of CD4+CD25+ T cells and Th17 cells compared to those on conventional circadian treatment [82]. These findings highlight how therapeutic manipulation of GC rhythms can substantially impact adaptive immune profiles.

B Cell and Antibody Responses

While less extensively characterized, emerging evidence suggests that B cell function and antibody responses are also subject to circadian regulation by glucocorticoids. The rhythmic distribution and function of B cells within different immunological compartments appears to be influenced by the HPA axis, though the specific mechanisms remain an active area of investigation. Studies in patients with GC rhythm disruptions have noted alterations in B cell populations, suggesting that proper timing of GC signaling is important for optimal B cell function [82]. The interplay between circadian GC signals and B cell responses represents a promising frontier for understanding how humoral immunity is coordinated with daily physiological cycles.

Consequences of Circadian Disruption

Experimental Models of Circadian Disruption

Animal models of circadian disruption have been instrumental in elucidating the immunological consequences of disturbed glucocorticoid rhythms. Chronic jet-lag protocols and altered feeding-fasting rhythms that disrupt the normal phase relationship between the central clock and peripheral oscillators lead to significant immune dysregulation [83]. These models demonstrate that chronic circadian disruption increases the risk of metabolic syndrome, type-2 diabetes, obesity, chronic inflammation, cancer, and impaired cognitive function [83]. The common feature in these models is the desynchronization of the carefully coordinated timing between the SCN and peripheral oscillators, which includes disruption of the normal glucocorticoid rhythm that serves as a key synchronizing signal for immune tissues.

Clinical Evidence from Cushing's Syndrome and Critical Illness

Clinical studies in patients with Cushing's Syndrome (CS) provide compelling evidence for the immunological impact of glucocorticoid rhythm disruption. CS is characterized by chronic endogenous hypercortisolism that obliterates the normal circadian rhythm of glucocorticoid secretion. A comprehensive multi-level analysis from a multicentre clinical trial revealed that active CS causes profound alterations in the levels, amplitude, and rhythmicity of several PBMC populations [73]. These changes included a significant flattening of circadian oscillations in the expression of core clock genes (CLOCK, PER1, PER2, PER3, and TIMELESS) in PBMCs from CS patients compared to matched controls [73]. Importantly, these immune and molecular rhythm disruptions were only partially restored after the remission of hypercortisolism, suggesting that prolonged GC rhythm disturbance may cause persistent immunological alterations.

Critical illness represents another condition characterized by profound disruption of glucocorticoid circadian rhythms. Studies in critically ill patients have revealed cell-specific changes in GR isoform expression and signaling over time. In PMNs, critical illness leads to sustained downregulation of GRα, GRβ, and GRγ, while GILZ expression is preserved. In contrast, PBMCs maintain GRα, GRβ, and GRγ expression but upregulate GR-P and DUSP1 [81]. These divergent responses highlight the cell-specific nature of GC signaling disruption in critical illness and may contribute to the simultaneous presence of hyperinflammatory and immunosuppressive features in these patients.

Table 3: Immune Consequences of Glucocorticoid Rhythm Disruption in Different Conditions

Condition	GC Rhythm Alteration	Innate Immune Effects	Adaptive Immune Effects
Cushing's Syndrome	Loss of rhythm, sustained high levels	Flattened circadian oscillation of PBMCs; altered clock gene expression in immune cells	Reduced T cell populations; altered homing receptor expression
Critical Illness	Loss of rhythm, elevated baseline	Cell-specific GR isoform changes; simultaneous inflammation and immunosuppression	Impaired T cell function; increased infection risk
Reverse-Circadian Treatment	Inverted therapeutic rhythm	Altered monocyte subsets	Reduced CD4+CD25+ T cells; lower Th17 cells; impaired NK cytotoxicity
Chronic Jet-Lag	Misaligned GC rhythm	Increased inflammatory cytokines; enhanced tissue inflammation	Autoimmunity predisposition; impaired immune memory

Experimental Approaches and Methodologies

Assessing Circadian Immune Parameters

Investigating the interplay between glucocorticoid rhythms and immune function requires specialized methodological approaches that account for temporal dynamics. A critical consideration is the implementation of dense sampling protocols across the 24-hour cycle to accurately capture circadian variations. The multicentre study on Cushing's Syndrome employed 5-point daily sampling to comprehensively characterize daily fluctuations in PBMC populations and clock gene expression [73]. Such high-resolution temporal profiling is essential for distinguishing true circadian oscillations from random fluctuations and for accurately determining rhythm parameters including acrophase (peak time), amplitude (rhythm strength), and mesor (mean level).

For immune cell profiling, flow cytometry with extensive antibody panels enables detailed characterization of immune cell populations and their activation states. In studies of GC replacement regimens in CAH patients, flow cytometric analysis included T cell subsets (CD4+, CD8+, Th1, Th2, Th9, Th17, Th22, Treg), B cells, NK cells, and monocyte subsets (classical, intermediate, non-classical) [82]. Functional assessments such as NK cell cytotoxicity (measured by CD107 expression) and receptor expression (NKG2D, NKp30) provide additional layers of information beyond mere cell counts [82].

Molecular Analyses of GC Signaling

At the molecular level, quantitative assessment of GR isoforms and downstream targets requires sensitive and specific techniques. Reverse transcription quantitative PCR (RT-qPCR) is commonly used to measure the expression of different GR variants (GRα, GRβ, GRγ, GR-P) and GC-responsive genes such as GILZ and DUSP1 [81]. Proper normalization using reference genes (e.g., CYPA and GAPDH) is essential for accurate quantification. For protein-level analyses, western blotting and immunofluorescence can determine GR expression, localization, and post-translational modifications.

The assessment of glucocorticoid sensitivity involves both in vitro and in vivo approaches. Dexamethasone suppression tests evaluate the functional response of the HPA axis to synthetic GCs, while cell-based assays can measure the suppression of cytokine production (e.g., LPS-induced TNF-α) by GCs. More recently, transcriptomic approaches including RNA sequencing have been employed to develop GC response signatures that provide comprehensive assessments of GC sensitivity in health and disease [80] [81].

Statistical Analyses for Circadian Data

Specialized statistical methods are required for analyzing circadian data. Cosinor analysis uses regression techniques to fit cosine curves to time-series data and test for the presence of significant rhythms. More advanced methods include mixed-effects models that account for within-subject correlations in longitudinal studies, and machine learning approaches that can identify complex patterns in high-dimensional circadian data [73]. These analytical techniques enable researchers to rigorously test hypotheses about glucocorticoid-immune interactions while accounting for the temporal structure of the data.

Visualization of Core Concepts

Circadian Glucocorticoid Signaling in Immune Cells

Diagram Title: Circadian GC Signaling in Immune Cells

This diagram illustrates the multi-level regulation of immune function by circadian glucocorticoid signaling. The pathway begins with the central clock in the suprachiasmatic nucleus (SCN) coordinating HPA axis activity through neural and hormonal signals. The resulting circadian cortisol rhythm transmits timing information to immune cells by activating glucocorticoid receptors (GRs), which translocate to the nucleus and regulate gene expression through GREs and nGREs. This includes both direct immune effects and feedback regulation of cellular clock genes, creating an integrated time-keeping system that coordinates immune function with daily physiological cycles.

Experimental Workflow for Assessing GC-Immune Interactions

Diagram Title: GC-Immune Rhythm Study Design

This workflow outlines the key methodological steps for investigating glucocorticoid-immune interactions, emphasizing the importance of dense temporal sampling across the 24-hour cycle. The approach integrates immune phenotyping, molecular analyses of GR signaling, and hormonal assessments to provide a comprehensive view of how glucocorticoid rhythms regulate immune function. Specialized statistical methods for circadian data are then applied to characterize rhythm parameters and test hypotheses about rhythm disruption in various physiological and pathological conditions.

The Scientist's Toolkit: Essential Research Reagents and Methods

Table 4: Key Research Reagents and Methods for Studying GC-Immune Interactions

Category	Specific Reagents/Methods	Key Applications	Considerations
Cell Isolation	Ficoll gradient centrifugation, Magnetic bead separation (CD15+ PMNs, PBMCs)	Obtain pure immune cell populations for cell-specific analyses	Maintain circadian timing during isolation; process immediately
Flow Cytometry	Antibodies to CD3, CD4, CD8, CD19, CD56, CD14, CD15, CD25, chemokine receptors	Immune cell phenotyping, trafficking receptor expression	Multi-color panels for comprehensive profiling; fixation for temporal batches
Molecular Biology	RT-qPCR primers for GR isoforms (GRα, GRβ, GRγ, GR-P), clock genes (PER, CRY, BMAL1), GILZ, DUSP1	Gene expression rhythm analysis, GR signaling assessment	Normalize to multiple reference genes; parallel protein validation
Hormonal Assays	CLIA for cortisol, ICMA for ACTH, salivary cortisol kits	HPA axis rhythm characterization, free vs. total cortisol	Strict timing for circadian assessment; consider pulsatility
GC Sensitivity Assays	Dexamethasone suppression, LPS-induced cytokine production with GC co-treatment	Functional assessment of GC responsiveness in immune cells	Dose-response curves; compare across time points
Circadian Tools	Cosinor analysis, JTK_CYCLE, mixed-effects models	Rhythm detection, parameter estimation in longitudinal data	Sufficient sampling density; account for individual differences

Therapeutic Implications and Future Directions

The recognition of glucocorticoids as circadian immune tuners has profound implications for clinical practice and therapeutic development. The timing of glucocorticoid administration significantly impacts treatment efficacy and side effect profiles. This is exemplified in studies of congenital adrenal hyperplasia, where reverse-circadian treatment (higher evening doses) resulted in distinct immunological alterations compared to conventional circadian treatment, including lower percentages of CD4+CD25+ T cells and reduced NK cell cytotoxicity [82]. These findings highlight the importance of chronotherapeutic approaches that align glucocorticoid treatment with physiological rhythms to optimize immune function while minimizing adverse effects.

Future research should focus on several key areas. First, a deeper understanding of how cell-specific GR isoform expression contributes to differential immune responses across circadian time is needed. Second, the development of modified-release glucocorticoid preparations that better mimic the physiological cortisol rhythm may improve immunological outcomes in conditions requiring replacement therapy [82]. Third, exploring how circadian GC signaling interacts with other rhythmic processes, such as metabolism and the microbiome, will provide a more integrated understanding of immune timing. Finally, translating these chronobiological insights into personalized timing regimens for glucocorticoid therapy in inflammatory and autoimmune conditions represents a promising frontier for improving patient care.

The emerging paradigm of glucocorticoids as circadian immune tuners underscores the fundamental importance of biological timing in immune regulation. Maintaining proper glucocorticoid rhythm is essential for coordinated innate and adaptive immune function, while disruption of this rhythm contributes to immunological dysregulation in various disease states. Incorporating circadian principles into both basic research and clinical practice will advance our understanding of immune function and open new avenues for therapeutic innovation.

Iatrogenic Cushing's syndrome represents the most common form of hypercortisolism in clinical practice, resulting from prolonged exposure to exogenous glucocorticoids administered for therapeutic purposes [84]. This condition provides a unique model for understanding the systemic consequences of glucocorticoid excess, particularly in the context of disrupted circadian rhythmicity. Under physiological conditions, cortisol secretion follows a robust circadian rhythm with peak levels in the early morning and a nadir around midnight in diurnal animals like humans [85] [6]. This rhythm is orchestrated by the hypothalamic-pituitary-adrenal (HPA) axis, which is precisely regulated by the central circadian clock in the suprachiasmatic nucleus (SCN) [6]. Exogenous glucocorticoid administration disrupts this finely tuned system, leading to the characteristic clinical manifestations of Cushing's syndrome while providing insights into the critical importance of circadian hormone patterning.

The significance of iatrogenic Cushing's syndrome extends beyond its clinical presentation to inform fundamental aspects of glucocorticoid biology. Circadian rhythms represent endogenously regulated cycles controlled by molecular clocks that synchronize with the 24-hour light-dark cycle [85]. The HPA axis forms a crucial neuroendocrine pathway through which the central clock regulates peripheral physiology, with neuronal activity from the SCN inducing corticotropin-releasing hormone (CRH) secretion from the hypothalamus, which subsequently stimulates pituitary adrenocorticotropic hormone (ACTH) release, ultimately driving cortisol production from the adrenal cortex [85]. When therapeutic glucocorticoids bypass this regulatory system, they not only produce clinical hypercortisolism but also disrupt the temporal organization of glucocorticoid signaling, creating a valuable model for investigating the relationship between hormone excess and circadian disruption in human disease.

Epidemiology and Clinical Presentation

Prevalence and Risk Factors

Iatrogenic Cushing's syndrome is considerably more common than endogenous forms, with prevalence dependent on the frequency and spectrum of medical conditions requiring glucocorticoid treatment within a population [84]. An estimated 2% to 3% of the population is currently prescribed systemic or topical glucocorticoid treatments, establishing a substantial at-risk population [86]. The clinical manifestations vary based on treatment parameters including glucocorticoid potency, dosage, duration of therapy, and route of administration [84]. Notably, even non-systemic administration routes such as inhalational or intranasal corticosteroids can precipitate iatrogenic Cushing's syndrome, particularly when concomitant administration of cytochrome P450 (CYP450) inhibitors occurs, as these interactions reduce glucocorticoid metabolism and increase systemic exposure [87].

Clinical Features and Morbidity

The clinical presentation of iatrogenic Cushing's syndrome mirrors that of endogenous Cushing's syndrome, though some distinguishing features exist. Common manifestations include rapid weight gain with central obesity, moon facies, buffalo hump, facial plethora, wide violaceous striae, easy bruising, proximal muscle weakness, and psychological disturbances [84] [88]. Compared to spontaneous Cushing's syndrome, the iatrogenic form demonstrates different complication profiles, with higher frequencies of osteoporosis, increased intraocular pressure, benign intracranial hypertension, aseptic necrosis of the femoral head, and pancreatitis, while hypertension, hirsutism, and menstrual irregularities are less commonly observed [87].

Table 1: Comparative Glucocorticoid Potencies and Pharmacologic Properties

Glucocorticoid	Equivalent Dose (mg)	Relative Glucocorticoid Potency	Relative Mineralocorticoid Potency	Biologic Half-Life (hours)
Cortisol	20	1.0	2	8-12
Hydrocortisone	25	0.8	2	8-12
Prednisone	5	4	1	18-36
Methylprednisolone	4	5	0	18-36
Dexamethasone	0.5	25-50	0	36-54

Untreated Cushing's syndrome carries a five-fold increased mortality risk over the general population, with a five-year mortality rate of 50% attributable to cardiovascular complications, thromboembolic events, opportunistic infections, and suicide [84]. The morbidity profile reflects the diverse physiological systems affected by glucocorticoid excess, including metabolic, cardiovascular, musculoskeletal, immune, and neuropsychiatric systems [86] [88]. This multi-system involvement underscores the critical importance of recognizing iatrogenic Cushing's syndrome early and implementing appropriate management strategies.

Pathophysiology: Circadian Disruption and Molecular Mechanisms

Circadian Rhythm Disruption in Iatrogenic Cushing's Syndrome

The pathophysiology of iatrogenic Cushing's syndrome extends beyond simple receptor activation to encompass profound disruption of circadian biology. Under physiological conditions, the molecular clock regulates circadian rhythm through a transcription-translation feedback loop involving core clock components including BMAL1, CLOCK, PER, CRY, REV-ERB, and ROR [85]. This system generates 24-hour oscillations in gene expression that synchronize physiological processes with environmental cycles. Glucocorticoids interact intimately with this system, as glucocorticoid response elements (GREs) are present in the gene loci of core clock components including Per1, Per2, and Nfil3 [85]. The normal circadian rhythm of cortisol features a characteristic diurnal pattern that is abolished in iatrogenic Cushing's syndrome, resulting in non-physiological, sustained glucocorticoid exposure without the protective trough periods.

The molecular interplay between glucocorticoid signaling and circadian regulation occurs at multiple levels. The glucocorticoid receptor (GR) functions as a ligand-dependent transcription factor that regulates target gene expression through multiple mechanisms. Upon binding glucocorticoids, GR dimerizes, translocates to the nucleus, and binds to specific DNA sequences known as glucocorticoid response elements (GREs) to transactivate or transrepress target genes [85]. Additionally, GR can directly interact with other transcription factors including NF-κB and AP-1 to modulate their activity, a mechanism known as transrepression [85]. These interactions disrupt the normal circadian regulation of immune and metabolic processes, contributing to the clinical features of iatrogenic Cushing's syndrome.

Diagram 1: Molecular Pathophysiology of Iatrogenic Cushing's Syndrome. This diagram illustrates how exogenous glucocorticoids disrupt normal HPA axis function and circadian regulation, leading to altered gene transcription and clinical manifestations. Key disrupted pathways are highlighted in red.

Tissue-Specific Mechanisms and Systemic Consequences

The clinical manifestations of iatrogenic Cushing's syndrome reflect the pleiotropic effects of glucocorticoid excess across multiple organ systems. In the immune system, glucocorticoids exert potent anti-inflammatory and immunosuppressive effects through multiple mechanisms, including transactivation of anti-inflammatory mediators like GILZ and IκBα, and transrepression of inflammatory cytokines including IL-1, IL-2, IL-6, TNF-α, and IFN-γ [85]. These immunomodulatory effects follow circadian patterns under physiological conditions but become dysregulated in iatrogenic Cushing's syndrome. For instance, glucocorticoids regulate the circadian expression of chemokines such as CXCL5, which demonstrates diurnal oscillation that is impaired by exogenous glucocorticoid administration [85].

In metabolic tissues, glucocorticoid excess promotes hyperglycemia through multiple mechanisms including increased hepatic gluconeogenesis via induction of phosphoenolpyruvate carboxykinase (PCK1) and glucose-6-phosphatase (G6PC), and induction of insulin resistance in peripheral tissues [85] [86]. Glucocorticoids also enhance the effects of adrenergic signaling, known as permissive effects, further amplifying catecholamine-induced lipolysis and gluconeogenesis [85]. These metabolic disturbances are compounded by glucocorticoid effects on adipose tissue distribution, leading to characteristic central fat deposition. In bone, glucocorticoids promote osteoporosis through dual mechanisms: inhibiting bone formation by suppressing osteoblast differentiation and function, while simultaneously increasing bone resorption through altered RANKL/OPG signaling [86] [88].

Diagnostic Approaches: Assessment and Circadian Evaluation

Diagnostic Criteria and Testing Protocols

The diagnosis of iatrogenic Cushing's syndrome requires demonstration of inappropriately high cortisol levels in the context of exogenous glucocorticoid exposure [84]. Several biochemical tests confirm hypercortisolism, though interpretation must account for the unique characteristics of iatrogenic cases, which typically show HPA axis suppression rather than the autonomy seen in endogenous Cushing's syndrome.

Table 2: Diagnostic Tests for Cushing's Syndrome

Test	Procedure	Interpretation	Sensitivity	Specificity	Circadian Considerations
Late-night Salivary Cortisol	Saliva sample collected between 11 PM - 12 AM	Cortisol >1.8 mcg/dL suggests CS	95-98%	95-98%	Directly assesses circadian rhythm disruption
24-hour Urinary Free Cortisol	24-hour urine collection	Results >3x normal suggest CS	Varies	81%	Measures integrated cortisol exposure
Overnight 1mg DST	1mg dexamethasone at 11 PM, measure cortisol at 8 AM	Cortisol >1.8 mcg/dL suggests CS	High	88%	Tests HPA axis suppressibility
Low-dose DST (48-hour)	0.5mg dexamethasone q6h for 48h, measure cortisol	Cortisol >1.8 mcg/dL suggests CS	100%	88%	Extended assessment of HPA axis

The low-dose dexamethasone suppression test (DST) serves as a standard screening tool, with failure to suppress cortisol levels below 1.8 mcg/dL indicating hypercortisolism [89] [84]. The late-night salivary cortisol test has gained prominence due to its non-invasive nature and direct assessment of circadian rhythm disruption, as loss of the normal circadian cortisol nadir is one of the earliest biochemical abnormalities in Cushing's syndrome [89] [88]. For patients with confirmed hypercortisolism, measurement of ACTH levels helps distinguish between ACTH-dependent and ACTH-independent causes, though in iatrogenic Cushing's syndrome, ACTH is typically suppressed due to negative feedback on the HPA axis [84].

Circadian-Specific Diagnostic Considerations

The assessment of circadian glucocorticoid rhythm provides crucial diagnostic information in iatrogenic Cushing's syndrome. The midnight cortisol measurement, whether in serum or saliva, directly evaluates the integrity of the circadian rhythm, which is characteristically flattened in Cushing's syndrome [84] [88]. This circadian disruption has implications beyond diagnosis, as research indicates that circadian rhythm abnormalities contribute to the metabolic and cardiovascular complications associated with glucocorticoid excess [85] [6]. Special consideration must be given to patients on exogenous glucocorticoids, as the timing of administration can influence test interpretation. For patients receiving long-acting glucocorticoid preparations like dexamethasone, the suppressive effects on the HPA axis may persist for extended periods, complicating biochemical assessment.

In iatrogenic Cushing's syndrome, the primary diagnostic challenge often lies in recognizing the condition, as the relationship between glucocorticoid therapy and symptom development may be overlooked, particularly with non-oral administration routes or intermittent dosing regimens [87]. A high index of suspicion is warranted in patients receiving any form of glucocorticoid therapy who develop characteristic signs or symptoms. Once identified, the diagnostic evaluation should include assessment of HPA axis suppression to guide safe glucocorticoid tapering and prevent adrenal insufficiency.

Therapeutic Strategies and Circadian-Rhythm Based Approaches

Management of Established Iatrogenic Cushing's Syndrome

The cornerstone of management for iatrogenic Cushing's syndrome is gradual glucocorticoid withdrawal to allow recovery of the suppressed HPA axis [84]. The tapering schedule must be individualized based on the glucocorticoid potency, duration of therapy, and clinical context, with careful attention to preventing adrenal insufficiency. For patients requiring ongoing anti-inflammatory therapy, several strategies can minimize glucocorticoid exposure, including transition to non-glucocorticoid alternatives, use of local rather than systemic administration, and selection of glucocorticoids with favorable pharmacokinetic profiles [86] [87].

Novel therapeutic approaches focus on minimizing off-target effects while preserving therapeutic efficacy. Selective glucocorticoid receptor agonists and modulators (SEGRAs and SEGRMs) represent an emerging class of compounds designed to dissociate transrepressive (anti-inflammatory) from transactivative (metabolic) effects [86]. Additionally, strategies exploiting tissue-specific glucocorticoid metabolism through isoforms of 11β-hydroxysteroid dehydrogenase show early potential for mitigating adverse effects [86]. These mechanism-based approaches hold promise for maintaining therapeutic benefits while reducing the risk of iatrogenic Cushing's syndrome.

Circadian-Optimized Therapeutic Approaches

Emerging understanding of glucocorticoid circadian biology supports the development of chronotherapeutic approaches to minimize iatrogenic complications. Circadian-informed treatment strategies include timed administration to align with physiological cortisol rhythms and the development of modified-release formulations that mimic the circadian cortisol profile [6]. These approaches aim to preserve the beneficial anti-inflammatory effects of glucocorticoids while reducing disruption to endogenous circadian systems.

For patients who must continue glucocorticoid therapy, management of complications follows established protocols for endogenous Cushing's syndrome, including blood pressure control, glycemic management, and osteoporosis prevention/treatment [86] [88]. The accompanying research toolkit provides essential resources for investigating circadian aspects of iatrogenic Cushing's syndrome and developing improved therapeutic strategies.

Table 3: Research Reagent Solutions for Investigating Glucocorticoid Circadian Biology

Research Tool Category	Specific Examples	Research Applications	Key Insights Generated
GR Signaling Modulators	Mifepristone (GR antagonist), Dexamethasone (synthetic glucocorticoid)	GR pathway dissection, HPA axis feedback studies	Mechanisms of GR transactivation vs transrepression [90] [91]
Enzyme Inhibitors	Ketoconazole, Osilodrostat (CYP11B1 inhibitors), Etomidate	Cortisol synthesis inhibition studies	Steroidogenic pathway regulation [90] [91]
Circadian Biology Tools	PER2::LUCIFERASE reporters, Clock gene mutants	Circadian rhythm disruption assessment	GC regulation of peripheral clocks [85] [6]
Cell Type-Specific Models	CD11c-Cre GR knockout (DCs), Adrenal-specific clock mutants	Cell-type specific GC action studies	Tissue-specific GC effects [85]
HPA Axis Assessment	CRH, ACTH measurements, DEX suppression tests	HPA axis function evaluation	HPA axis suppression kinetics [89] [84]

Iatrogenic Cushing's syndrome represents both a clinical challenge and a valuable model for understanding the systemic consequences of glucocorticoid excess and circadian disruption. The condition highlights the critical importance of the precise circadian regulation of glucocorticoid signaling for maintaining metabolic, immune, and cardiovascular homeostasis. Future research directions should focus on elucidating the molecular mechanisms connecting circadian disruption to specific disease manifestations, developing circadian-optimized glucocorticoid therapies that minimize HPA axis suppression and metabolic complications, and identifying biomarkers that predict individual susceptibility to glucocorticoid-related adverse effects.

Advancements in our understanding of glucocorticoid circadian biology hold promise for transforming the management of conditions requiring therapeutic glucocorticoids. By applying principles of chronotherapy and developing targeted glucocorticoid receptor modulators that preserve circadian rhythmicity, it may be possible to maintain the considerable therapeutic benefits of glucocorticoids while mitigating the risk of iatrogenic Cushing's syndrome. This integrated approach, recognizing the fundamental interconnection between glucocorticoid signaling and circadian biology, represents the future of glucocorticoid therapy and the prevention of its most significant adverse effect.

The hypothalamic-pituitary-adrenal (HPA) axis represents a critical neuroendocrine system that mediates the body's stress response and exhibits a robust circadian rhythm, with glucocorticoids as its final effector hormones. Recent research has fundamentally revised the understanding of glucocorticoid-circadian interactions, revealing that the suprachiasmatic nucleus (SCN) possesses functional glucocorticoid receptors (GR) in adulthood, contrary to long-standing dogma. This discovery, coupled with the established role of glucocorticoids in synchronizing peripheral clocks, opens novel therapeutic avenues for circadian re-alignment. This technical guide synthesizes current mechanistic understanding and emerging strategies for targeting the HPA axis to correct circadian rhythm disorders, framed within the context of advancing glucocorticoid and ACTH research. We provide detailed experimental methodologies, pathway visualizations, and reagent solutions to facilitate research and drug development in this evolving field.

The HPA axis functions as a sophisticated communication network between the hypothalamus, pituitary gland, and adrenal glands, serving as the body's primary stress response system while simultaneously maintaining circadian homeostasis [92]. This axis operates through a sequential hormone cascade: in response to stressors, the hypothalamus releases corticotropin-releasing hormone (CRH), which stimulates the anterior pituitary to secrete adrenocorticotropic hormone (ACTH), ultimately triggering the adrenal cortex to release glucocorticoids (cortisol in humans, corticosterone in rodents) [93] [92].

The HPA axis demonstrates complex temporal organization, operating across ultradian (~90 minute pulses), circadian (~24 hour), and stress-responsive timeframes [12] [4]. The central circadian pacemaker in the SCN regulates the HPA axis through multiple pathways: neuronal projections to the paraventricular nucleus (PVN) using arginine-vasopressin (AVP), autonomic nervous system signals to the adrenal gland that gate its sensitivity to ACTH, and by maintaining a circadian clock within the adrenal cortex itself [12] [4]. This multi-layered regulation generates a robust circadian glucocorticoid rhythm that peaks immediately before the active phase (morning in humans, evening in nocturnal rodents) to support anticipated metabolic and behavioral demands [12].

The relationship between the circadian system and glucocorticoids is fundamentally reciprocal. While the SCN regulates glucocorticoid rhythm, glucocorticoids in turn synchronize peripheral clocks throughout the body by binding to glucocorticoid response elements (GREs) in promoter regions of clock genes such as Per1 and Per2 [93] [12]. This feedback mechanism allows glucocorticoids to function as potent zeitgebers for peripheral tissues, though the SCN itself exhibits remarkable resistance to glucocorticoid-mediated phase resetting in adulthood—a property previously attributed to absence of GR but now understood to involve complex intercellular coupling mechanisms [94].

Table 1: Core Components of the HPA Axis and Their Circadian Functions

Component	Location	Core Function in HPA Axis	Circadian Role
Hypothalamus (PVN)	Brain	Releases CRH in response to stress	Receives circadian input from SCN via AVP projections
Anterior Pituitary	Base of brain	Releases ACTH in response to CRH	Conveys hypothalamic signals to adrenals
Adrenal Cortex	Top of kidneys	Produces/releases glucocorticoids	Contains peripheral clock; gates glucocorticoid release via neural input from SCN
Suprachiasmatic Nucleus (SCN)	Hypothalamus	Not part of HPA axis proper	Master circadian pacemaker; regulates HPA axis timing

Molecular Mechanisms and Novel Insights

Glucocorticoid Signaling and Clock Gene Regulation

Glucocorticoids exert their effects primarily through the ubiquitously expressed glucocorticoid receptor (GR), encoded by the NR3C1 gene [93]. This receptor exists in multiple isoforms generated through alternative splicing and translation initiation, with GRα being the classic ligand-activated transcription factor [93]. Upon glucocorticoid binding, GR translocates to the nucleus and homodimerizes, binding to GREs in target gene promoters to regulate transcription [93]. Approximately 15-20% of the human leukocyte transcriptome is influenced by glucocorticoids, with about two-thirds of targets being induced and one-third suppressed [93].

The molecular integration between glucocorticoid signaling and the circadian clock occurs at multiple levels. First, several core clock genes, including Per1, Per2, and Nr1d1, contain GREs in their promoter regions, allowing direct glucocorticoid regulation of their transcription [94] [12]. Second, the CLOCK protein, a core component of the molecular clock, physically interacts with the ligand-binding domain of GR and acetylates specific lysine residues within its hinge region, thereby repressing GR's transcriptional activity [93]. This acetylation reduces GR binding to GREs and represents a fundamental mechanism of circadian gating of glucocorticoid sensitivity [93].

Paradigm Shift: Glucocorticoid Receptors in the Adult SCN

A pivotal recent discovery has challenged the long-standing paradigm that the adult SCN lacks glucocorticoid receptors and is therefore immune to glucocorticoid influence. Research demonstrates that GR mRNA (Nr3c1) expression actually increases from the fetal stage to adulthood (postnatal day 28), with GR immunoreactivity present in both neurons and glia of the adult SCN [94]. Furthermore, these receptors are functionally competent, as dexamethasone (DEX) administration acutely increases expression of downstream target genes Gilz and Sgk1 in the adult SCN [94].

The resistance of the adult SCN clock to glucocorticoid-mediated phase shifting is not due to receptor absence but rather emerges postnatally through development of network-level properties. While DEX efficiently resets the fetal SCN clock, this effect disappears shortly after birth [94]. Mechanistically, this acquisition of resistance depends on neuronal coupling, as tetrodotoxin (TTX) treatment sensitizes the adult SCN to DEX and induces phase shifts comparable to those observed in fetal stages [94]. In contrast, inhibition of glial metabolism with fluorocitrate does not affect DEX resistance, indicating the primacy of neuronal communication in this protective mechanism [94].

Therapeutic Targeting Strategies

Therapeutic targeting of the HPA axis for circadian re-alignment encompasses pharmacological interventions aimed at specific regulatory nodes within the axis. The strategic approaches are summarized in the table below.

Table 2: Therapeutic Strategies for HPA Axis Targeting in Circadian Re-alignment

Therapeutic Approach	Molecular Targets	Representative Agents	Circadian Effect	Development Status
Glucocorticoid Signaling Modulation	GR agonists/antagonists	Dexamethasone, Mifepristone	Phase resetting of peripheral clocks	Established corticosteroids; investigational for circadian applications
Melatonin-Based Therapies	MT1/MT2 receptors	Melatonin, Ramelteon, Tasimelteon	SCN phase resetting; enhances rhythm amplitude	FDA-approved for Non-24-hour sleep-wake disorder
HPA Axis Feedback Enhancement	CRH, ACTH signaling	CRH antagonists, ACTH analogs	Normalizes hyperactive HPA axis; restores rhythm	Primarily investigational

Glucocorticoid-Based Interventions

Synthetic glucocorticoids like dexamethasone can be strategically timed to phase-shift peripheral clocks, leveraging the widespread expression of GREs in clock gene promoters [93] [12]. This approach is particularly relevant for shift workers and jet lag management, where peripheral clocks become desynchronized from the central SCN pacemaker. The critical consideration is that while glucocorticoids effectively synchronize peripheral clocks, they have minimal direct phase-resetting effects on the adult SCN clock due to its network-based resistance mechanism [94].

Emerging approaches seek to modulate GR activity more subtly than full agonists or antagonists. Selective GR modulators (SEGRMs) that exhibit tissue-specific actions represent a promising direction, potentially allowing separation of circadian benefits from adverse metabolic effects [94] [12].

Adjunctive Chronobiological Interventions

Melatonin receptor agonists constitute another key therapeutic strategy, working through distinct but complementary mechanisms to glucocorticoid approaches. Melatonin acts directly on the SCN, primarily through MT1 and MT2 receptors, to reinforce circadian phase and enhance rhythm amplitude [12]. Unlike glucocorticoids, melatonin can directly phase-shift the SCN clock, making it particularly valuable for conditions like Delayed Sleep Phase Disorder (DSPD) and Non-24-Hour Sleep-Wake Disorder [12] [95]. Tasimelteon, an FDA-approved melatonin receptor agonist, is specifically indicated for Non-24-hour sleep-wake disorder in blind individuals, representing a targeted HPA-axis-independent circadian therapy [95].

The combination of strategically timed melatonin and low-dose glucocorticoid regimens represents an innovative approach currently under investigation, potentially leveraging both SCN and peripheral clock resetting mechanisms for more comprehensive circadian re-alignment.

Experimental Approaches and Methodologies

Assessing Circadian Glucocorticoid Effects In Vitro

The organotypic SCN explant culture from mPer2^Luc knock-in mice provides a robust model for investigating direct glucocorticoid effects on the central circadian clock. This system enables real-time monitoring of PER2 protein expression rhythms through bioluminescence recording [94].

Protocol: SCN Explant Preparation and Drug Treatment

• Tissue Preparation: Sacrifice mice at desired developmental stage (E17 to adult). Rapidly dissect brains in cold Earle's Balanced Salt Solution (EBSS). Section coronally (250-300 μm) using vibratome (adults) or tissue chopper (neonates).
• Explant Culture: Place medial SCN sections on Millicell culture inserts in 35 mm Petri dishes with recording medium (DMEM supplemented with 2% B27, 0.1 mM D-Luciferin, antibiotics).
• Bioluminescence Monitoring: Transfer cultures to LumiCycle apparatus for continuous bioluminescence recording at 37°C.
• Drug Application: At stable rhythm amplitude (3-5 days in vitro), apply treatments: 100 nM dexamethasone (1 μL of 0.1 mM stock in 1% ethanol/mL medium) or vehicle control.
• Network Disruption Studies: For neuronal uncoupling, add 1 μM tetrodotoxin (TTX) to recording medium 24h prior to dexamethasone treatment. For glial inhibition, apply 7 μL of 7 mM fluorocitrate (FLC) stock/mL medium 48h before treatment.
• Phase Shift Analysis: Determine phase shifts by comparing treatment cycles with baseline pre-treatment cycles and vehicle-controlled cycles.

Evaluating HPA Axis Function In Vivo

Comprehensive assessment of HPA axis circadian function requires integrated measurements across multiple regulatory levels.

Protocol: Circadian HPA Axis Profiling in Rodents

• Animal Housing: Maintain under standardized LD12:12 conditions (lights on 06:00, ZT0) with minimal disturbance for at least two weeks prior to experiments.
• Circadian Sampling: For 24h rhythm characterization, sacrifice groups of animals (n=5-8/time point) every 4h across the circadian cycle. Collect trunk blood for hormone measurement, rapidly dissect tissues (SCN, PVN, pituitary, adrenal), and freeze.
• Laser Microdissection: Cryosection brains (20 μm), stain with cresyl violet, and laser microdissect SCN and PVN for region-specific RNA/protein analysis.
• Hormone Assays: Measure plasma corticosterone/cortisol (ELISA/RIA) and ACTH (ELISA) levels. Include corticosterone awakening response assessment by frequent sampling around light-dark transitions.
• Gene Expression Analysis: Extract RNA from microdissected tissues, synthesize cDNA, and perform RT-qPCR for key targets (Nr3c1, Per1, Per2, Bmal1, Gilz, Sgk1, Crh, Avp).
• Drug Challenges: For axis reactivity tests, inject CRH (1 μg/kg i.v.) or dexamethasone (0.1-1 mg/kg i.p.) at different circadian times, sampling blood at 0, 30, 60, 120 min post-injection.

Research Reagent Solutions

Table 3: Essential Research Reagents for HPA-Circadian Investigations

Reagent/Category	Specific Examples	Research Application	Key Function
Circadian Reporter Models	mPer2Luc knock-in mice	Real-time clock function monitoring	PER2 protein fusion with luciferase enables bioluminescence recording
GR Ligands	Dexamethasone, Corticosterone, Mifepristone (RU486)	GR pathway manipulation	Receptor agonists/antagonists for probing GR function
Ion Channel Modulators	Tetrodotoxin (TTX), Fluorocitrate (FLC)	Network disruption studies	Inhibits neuronal activity (TTX) or glial metabolism (FLC)
Molecular Analysis Tools	Laser microdissection systems, RT-qPCR assays	Tissue-specific gene expression	Enables analysis of specific nuclei (SCN, PVN)
Hormone Assays	Corticosterone/Cortisol ELISA, ACTH ELISA	HPA axis output measurement	Quantifies endocrine endpoints in plasma/tissue
Circadian Monitoring	LumiCycle, intravital microscopy	Rhythm recording in tissues	Long-term bioluminescence/fluorescence monitoring

Signaling Pathways and Experimental Workflows

Core HPA Axis Circadian Signaling Circuitry

Diagram 1: HPA axis circadian circuitry and SCN resistance.

Molecular Integration: Clock Genes and Glucocorticoid Signaling

Diagram 2: Molecular integration of glucocorticoid signaling and clock genes.

Experimental Workflow for Assessing Circadian Glucocorticoid Effects

Diagram 3: Experimental workflow for circadian glucocorticoid studies.

Therapeutic targeting of the HPA axis for circadian re-alignment represents a promising approach with evolving mechanistic understanding. The paradigm-shifting recognition that the adult SCN contains functional GRs but resets glucocorticoid influence through neuronal coupling opens new avenues for selective circadian interventions. Future directions should focus on developing compounds that can modulate this coupling mechanism, potentially allowing controlled access to the master clock while preserving its stability against random stressors.

Emerging research connecting HPA axis function to glymphatic clearance and neurodegenerative processes highlights the broader implications of circadian glucocorticoid signaling for brain health [4]. The development of tissue-specific GR modulators that can disentangle desirable circadian effects from adverse metabolic consequences represents a key challenge for the field. Furthermore, integrating HPA-targeted approaches with emerging digital therapeutics and personalized chronotyping promises more comprehensive management of circadian rhythm disorders.

As our understanding of the intricate reciprocity between the HPA axis and circadian system deepens, particularly through single-cell resolution studies and advanced in vivo monitoring techniques, increasingly sophisticated therapeutic strategies will emerge. These advances hold significant promise for addressing the growing burden of circadian rhythm disorders in our modern, 24/7 society.

Validating Mechanisms and Comparative Pathophysiology: Insights from Models and Clinical Data

The circadian rhythm of glucocorticoid (GC) secretion is a fundamental physiological process, orchestrating daily fluctuations in metabolism, immune function, and cardiovascular activity. This rhythm is primarily governed by the hypothalamus-pituitary-adrenal (HPA) axis, where corticotropin-releasing hormone (CRH) from the hypothalamus stimulates pituitary secretion of adrenocorticotropic hormone (ACTH), which in turn drives GC production from the adrenal cortex [6]. The suprachiasmatic nucleus (SCN), the master circadian clock in the hypothalamus, entrains this entire system to the light-dark cycle [1] [96]. In undisturbed states, circulating GC levels exhibit a robust diurnal variation, with a peak around the onset of the active period—morning in diurnal humans and night in nocturnal rodents [85] [6]. Research using animal models, particularly through chronic ACTH infusion and genetic manipulation of clock components, has been instrumental in elucidating the complex regulatory mechanisms that underlie this rhythm and the profound health consequences when it is disrupted. This whitepaper synthesizes key insights from these experimental approaches for the research community.

The Chronic ACTH Infusion Model: A Tool for Dissecting Glucocorticoid Excess

The chronic infusion of adrenocorticotropic hormone (ACTH) in animals is a well-established model for studying conditions of glucocorticoid excess, such as Cushing's syndrome, and for investigating the circadian control of the HPA axis.

Experimental Protocol and Methodology

A standard protocol, as demonstrated in a 2025 study using male C57BL/6J mice, involves the continuous subcutaneous infusion of ACTH via osmotic minipumps [77]. Key methodological steps include:

• Animal Model: Male C57BL/6J mice are commonly used.
• ACTH Administration: ACTH (1-24) is typically dissolved in a vehicle like gelatin and administered via osmotic minipumps implanted subcutaneously. A common dosage is 0.5 μg/hr, delivered continuously for a period such as two weeks.
• Control Groups: Control animals undergo the same surgical procedure and receive minipumps infusing only the vehicle solution.
• Dietary Manipulation: To probe salt-sensitivity, blood pressure is measured first on a control diet and then following a switch to a high-salt diet (e.g., 6% NaCl).
• Blood Pressure Monitoring: Blood pressure is measured longitudinally using non-invasive tail-cuff plethysmography or telemetric methods, allowing for the assessment of diurnal dipping patterns.
• Endpoint Analyses: At the end of the infusion period, animals are euthanized for tissue collection. Analyses can include:
  • Renal Function: Assessment of salt excretion rhythms and the pressure-natriuresis response.
  • Vascular Reactivity: Ex vivo wire or pressure myography on isolated renal arteries to test responses to vasoactive substances like nitric oxide.
  • Molecular Profiling: Quantitative PCR (qPCR) or RNA sequencing to evaluate gene expression changes related to signaling pathways like TGF-β in vascular tissues [77].

Key Quantitative Findings from Chronic ACTH Models

Chronic ACTH infusion produces a reproducible phenotype characterized by hypertension, abolished blood pressure dipping, and altered renal sodium handling. The table below summarizes core quantitative findings from a recent murine study [77].

Table 1: Key Physiological and Molecular Changes Induced by Chronic ACTH Infusion in Mice

Parameter	Findings in ACTH-Infused Mice vs. Controls
Blood Pressure	Induced hypertension and a transition to a "nondipping" phenotype.
Salt Sensitivity	Developed salt-sensitive blood pressure following high-salt intake.
Sodium Excretion	Abolished the diurnal rhythm of sodium excretion; reduced urine sodium/potassium ratio.
Renal Hemodynamics	Impaired autoregulation of renal blood flow and a blunted pressure-natriuresis response.
Vascular Function	Diminished vasorelaxant response to nitric oxide in isolated renal arteries.
Gene Expression	Evidence of arterial remodeling and enhanced TGF-β signaling at the mRNA level.

Underlying Mechanisms and Pathways

The findings from the chronic ACTH infusion model point to several interconnected mechanisms that disrupt circadian cardiovascular and metabolic rhythms:

• Disruption of Renal Clocks and Sodium Handling: The model demonstrates that GC excess directly interferes with the kidney's intrinsic circadian clock, which normally regulates the diurnal rhythm of sodium excretion. This leads to salt retention and a blunted pressure-natriuresis response, fundamentally shifting the system toward salt-sensitivity and hypertension [77].
• Impairment of Vascular Function: GC excess induced by ACTH leads to endothelial dysfunction, specifically a reduced vasodilatory response to nitric oxide in resistance arteries such as the renal artery. This contributes to increased peripheral resistance and pressure nondipping [77].
• Systemic Circadian Misalignment: By overdriving the adrenal output, chronic ACTH disrupts the tightly coordinated temporal signals between the central SCN clock and peripheral clocks in the kidney, heart, and vasculature. This systemic misalignment is a key pathophysiological contributor to the observed metabolic and cardiovascular deficits [96].

Genetic Manipulation of Circadian Clocks

Complementing the ACTH infusion model, genetic manipulations of core clock components have been essential for elucidating the molecular machinery governing circadian rhythms and its role in HPA axis function.

Experimental Models and Protocols

Research employs both systemic and tissue-specific knockout models to dissect the function of specific clock genes. Common approaches include:

• Global Knockout Models: Generation of mice with constitutive knockout of core clock genes such as Per2 (e.g., Per2 mutant mice) [85].
• Conditional Knockout Models: Use of the Cre-lox system to delete clock genes in a tissue-specific manner (e.g., cardiomyocyte-specific deletion of BMAL1 or CLOCK) [96] [97].
• Molecular Profiling: Transcriptomic analyses (e.g., RNA-seq) of tissues from genetically modified mice to identify rhythmically expressed genes and pathways under circadian control.
• Behavioral and Physiological Phenotyping: Comprehensive assessment of circadian behaviors (e.g., wheel-running activity), metabolic parameters, cardiovascular function, and immune responses under controlled light-dark cycles and in free-running conditions (constant darkness) [85] [96].

Key Findings from Genetic Clock Manipulations

Table 2: Phenotypes Associated with Genetic Manipulation of Circadian Clock Components

Genetic Model	Key Findings Related to HPA Axis, Immunity, and Physiology
Per2 Mutant Mice	Altered circadian response to endotoxin; disrupted oscillation of inflammatory cytokines; increased corticosterone concentration after LPS challenge due to upregulation of CLOCK, BMAL1, and the steroidogenic enzyme StAR [85].
Cardiomyocyte-specific BMAL1 or CLOCK knockout	Impaired cardiac contractility, mitochondrial dysfunction, and heightened sensitivity to ischemic injury [96] [97].
Systemic Clock Gene Disruption	Leads to altered neurogenesis, increased neuroinflammation, cognitive decline, and is linked to neurological and psychiatric conditions in both animal models and human studies [96].

Underlying Mechanisms and Pathways

Genetic models reveal that the molecular clock is a non-negligible regulator of the HPA axis and peripheral physiology:

• Core Clock Feedback Loops: The molecular clock operates via transcriptional-translational feedback loops (TTFLs). The core components BMAL1 and CLOCK form a heterodimer that activates transcription of Per, Cry, Rev-erbα, and Ror genes. The PER/CRY complex then represses BMAL1/CLOCK activity, creating a ~24-hour oscillation [85] [96].
• Clock-Immune System Crosstalk: Clock genes directly regulate the circadian rhythm of immune responses. For instance, the BMAL1/CLOCK complex can bind to the promoter of the Star gene, a rate-limiting enzyme in GC synthesis, thereby linking the core clock machinery directly to the adrenal production of GCs [85].
• Integration of Central and Peripheral Timing: The SCN synchronizes peripheral clocks via neural, hormonal, and behavioral signals. Glucocorticoids themselves act as potent zeitgebers (time-giving signals) for peripheral clocks in organs like the liver, heart, and immune cells. Genetic disruption of this hierarchical network leads to local and systemic circadian misalignment, predisposing to disease [96].

Visualization of Key Pathways and Workflows

The following diagrams illustrate the core molecular clock mechanism and the experimental workflow for the chronic ACTH infusion model.

Molecular Circadian Clock and HPA Axis

Diagram 1: Circadian Clock and HPA Axis Interaction. This figure illustrates the hierarchical regulation of the HPA axis by the central SCN clock and the molecular TTFL of peripheral clocks. Core clock components BMAL1 and CLOCK drive the expression of period (PER) and cryptochrome (CRY) proteins. The PER/CRY complex then inhibits BMAL1/CLOCK activity, creating a self-sustaining ~24-hour cycle. Glucocorticoids (GCs), whose secretion is ultimately driven by the SCN via CRH and ACTH, in turn act as zeitgebers to synchronize peripheral clocks [85] [6] [96].

Chronic ACTH Infusion Experimental Workflow

Diagram 2: ACTH Infusion Experimental Workflow. This diagram outlines the key steps in a standard protocol for modeling glucocorticoid excess and circadian disruption via chronic ACTH infusion in mice, culminating in comprehensive physiological and molecular analyses [77].

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Reagents and Models for Circadian ACTH/GC Research

Reagent / Model	Function and Application in Research
ACTH (1-24)	A synthetic bioactive fragment of ACTH used in chronic infusion models to stimulate the adrenal cortex and induce a state of glucocorticoid excess, mimicking conditions like Cushing's syndrome [77].
Osmotic Minipumps	Subcutaneously or intraperitoneally implanted devices for the continuous and sustained delivery of compounds like ACTH over days or weeks, crucial for chronic disease modeling [77].
C57BL/6J Mouse Strain	A widely used inbred mouse strain that serves as the genetic background for most transgenic and knockout models in circadian and metabolic research [77].
Clock Gene KO Mice (e.g., Per2m, BMAL1⁻/⁻)	Genetically modified mice with disruptions in core clock genes. These models are essential for dissecting the specific roles of molecular clock components in regulating the HPA axis and peripheral physiology [85] [96].
CRF1 Receptor Antagonists (e.g., Crinecerfont)	Investigational oral, nonpeptide compounds that block the corticotropin-releasing factor type 1 receptor. Used in clinical and preclinical research to suppress ACTH secretion and study HPA axis regulation [98] [32].
MC2R Antagonists (e.g., Atumelnant)	First-in-class, investigational oral antagonists of the melanocortin type 2 receptor (ACTH receptor). These tools directly block ACTH action at the adrenal gland, offering a novel approach to treat conditions of ACTH excess [32].

Research utilizing chronic ACTH infusion and genetic clock manipulations has unequivocally established that the precise circadian rhythm of glucocorticoid secretion is critical for systemic health. These models demonstrate that disruption of this rhythm—whether by hormonal excess or genetic alteration of the core clockwork—promotes a pathological state characterized by cardiovascular dysfunction, metabolic dysregulation, and immune imbalance. The interplay between the central SCN clock, the HPA axis, and peripheral tissue clocks creates a complex, integrated timing system that is highly vulnerable to misalignment.

The insights gleaned from these animal models are directly informing the development of novel chronotherapeutic strategies. These include timing drug administration to align with circadian biology (e.g., antihypertensives) and developing new classes of drugs that target the HPA axis with greater precision, such as oral CRF1 and MC2R antagonists [98] [32] [97]. Future research should focus on deepening our understanding of the communication mechanisms between different organ clocks and on translating the robust findings from animal models into personalized diagnostic and therapeutic approaches for patients with circadian rhythm disorders.

The hypothalamic-pituitary-adrenal (HPA) axis represents a fundamental neuroendocrine system regulating physiological and psychological stress adaptation through glucocorticoid secretion. While the ACTH stimulation test remains a cornerstone for assessing adrenal function, its interpretation is complicated by the dynamic, adaptive nature of the HPA axis under chronic stress exposure. This technical review explores a mechanistic modeling framework that integrates hormonal kinetics, feedback inhibition, and functional mass adaptation of corticotroph and adrenal compartments. By simulating HPA axis dynamics across 180 days encompassing baseline, chronic stress, and recovery phases, this framework demonstrates how glandular remodeling, glucocorticoid receptor (GR) resistance, and delayed feedback recovery alter test outcomes without indicating primary adrenal failure. Our analysis specifically frames these findings within the context of circadian glucocorticoid and ACTH research, highlighting implications for drug development and diagnostic innovation in adrenal disorders.

The hypothalamic-pituitary-adrenal (HPA) axis is a complex neuroendocrine system that coordinates endocrine responses to physiological and psychological stress, serving as a vital regulator of homeostasis [99] [100]. This system operates through a cascade of hormonal signals: the hypothalamus secretes corticotropin-releasing hormone (CRH), which stimulates the pituitary gland to release adrenocorticotropic hormone (ACTH), which in turn prompts the adrenal cortex to produce glucocorticoids (primarily cortisol in humans) [100] [101]. The HPA axis is characterized by its robust circadian rhythmicity, with ACTH and cortisol levels typically peaking in the early morning and reaching their nadir late in the evening [67] [102] [103]. This circadian regulation is orchestrated by the suprachiasmatic nucleus (SCN) of the hypothalamus, which serves as the body's master circadian pacemaker [67].

The HPA axis stress response is driven primarily by neural mechanisms, invoking CRH release from hypothalamic paraventricular nucleus (PVN) neurons [100]. Pathways activating CRH release are stressor-dependent: reactive responses to homeostatic disruption frequently involve direct noradrenergic or peptidergic drive of PVN neurons by sensory relays, whereas anticipatory responses use oligosynaptic pathways originating in upstream limbic structures [100]. Stress responses are inhibited by negative feedback mechanisms, whereby glucocorticoids act to diminish drive and promote trans-synaptic inhibition [100]. The context in which stressors occur determines whether an individual's acute or chronic stress responses are adaptive or maladaptive (pathological) [100].

Table 1: Core Components of the HPA Axis

Component	Key Structures	Secreted Factors	Primary Functions
Hypothalamus	Paraventricular Nucleus (PVN)	Corticotropin-releasing Hormone (CRH)	Integration of stress signals; initiation of HPA axis response
Pituitary	Anterior Pituitary	Adrenocorticotropic Hormone (ACTH)	Stimulation of adrenal glucocorticoid production
Adrenal	Adrenal Cortex	Glucocorticoids (Cortisol)	Metabolic regulation; immune modulation; stress adaptation
Regulatory Centers	Suprachiasmatic Nucleus (SCN)	Vasoactive intestinal peptide	Master circadian pacemaker; entrainment of peripheral rhythms

Circadian Biology of Glucocorticoid Signaling

Circadian rhythms are intrinsic 24-hour biological cycles that govern various physiological processes, with the hypothalamic suprachiasmatic nucleus (SCN) serving as the primary circadian pacemaker [67]. The SCN communicates its rhythmic signals to the entire body through neural and endocrine pathways, synchronizing biological clocks in peripheral tissues and organs [67]. It governs the rhythmic secretion of key hormones, including cortisol and melatonin, establishing itself as a critical hub linking central rhythms with peripheral metabolism [67]. The secretion rhythm of cortisol peaks in the early morning to awaken the body and falls to a nadir at night, forming a fundamental circadian pattern that influences virtually every organ system [67].

At the molecular level, the circadian rhythm is controlled by a core group of regulatory clock genes, including BMAL1, CLOCK, PER, CRY, REV-ERB, and ROR [67] [104]. These genes form interlocking transcription-translation feedback loops that generate approximately 24-hour molecular oscillations. The core negative feedback loop establishes the fundamental framework: BMAL1 and CLOCK form a heterodimer that activates transcription of PER and CRY genes, whose protein products then inhibit their own transcription, creating a self-sustaining oscillator [67]. A parallel auxiliary loop provides sophisticated dynamic regulation through the rhythmic expression of the BMAL1 gene itself, where REV-ERB and ROR competitively bind to the ROR response element (RORE) on the BMAL1 promoter, with REV-ERB repressing and ROR activating transcription [67].

Recent research has revealed that glucocorticoids play a significant role in synchronizing peripheral circadian clocks. In glioblastoma models, daily glucocorticoids synchronize circadian rhythms in tumor cells through glucocorticoid receptor signaling, regulating clock gene expression in a time-of-day dependent manner [104]. This synchronization has functional consequences, as blocking circadian signals like vasoactive intestinal peptide or glucocorticoids dramatically slows tumor growth and disease progression [104]. These findings highlight the bidirectional relationship between glucocorticoid signaling and circadian biology, with implications for both physiology and disease pathogenesis.

The ACTH Stimulation Test: Clinical Applications and Limitations

The ACTH stimulation test, also known as a cosyntropin stimulation test, is a dynamic endocrine test that assesses adrenal gland responsiveness to adrenocorticotropic hormone [39]. This test serves as the primary medical assessment for diagnosing adrenal insufficiency—whether primary, secondary, or tertiary [39]. The standard protocol involves administering synthetic ACTH (cosyntropin) via intramuscular injection and measuring cortisol levels at baseline and specific intervals post-stimulation (typically 30 and 60 minutes) [39].

In clinical practice, a normal response is characterized by a post-stimulation cortisol level higher than 12.6 μg/dL, indicating adequate adrenal reserve [39]. Abnormal responses help differentiate among various adrenal disorders: in primary adrenal insufficiency (Addison's disease), cortisol levels show little or no increase despite ACTH stimulation due to adrenal gland damage; in secondary adrenal insufficiency, blunted responses result from pituitary dysfunction and subsequent adrenal atrophy; and in Cushing syndrome, interpretation depends on both cortisol and ACTH levels [39] [101] [103].

Table 2: Interpretation of ACTH Stimulation Test Results

Condition	Baseline Cortisol	Post-ACTH Cortisol	Baseline ACTH	Physiological Basis
Normal Adrenal Function	Normal circadian variation	>12.6 μg/dL	Normal (7.2-63.3 pg/mL) [102]	Intact HPA axis with proper adrenal reserve
Primary Adrenal Insufficiency	Low	Minimal or no increase	High	Adrenal gland destruction; loss of cortisol production
Secondary Adrenal Insufficiency	Low	Blunted response	Low or normal	Pituitary dysfunction; inadequate ACTH production
Cushing Disease (Pituitary)	High	High	High	ACTH-producing pituitary tumor
Adrenal Tumor	High	High	Low	Autonomous cortisol production independent of ACTH

The standard ACTH test has significant limitations in its ability to detect subtle HPA axis adaptations. The test employs supraphysiological ACTH doses (250μg) that may mask partial adrenal dysfunction, as this pharmacological stimulus can overcome mild to moderate impairments in adrenal responsiveness [99]. Additionally, the test fails to account for the dynamic and adaptive nature of the HPA axis, particularly under conditions of chronic stress where glandular remodeling, glucocorticoid receptor resistance, and altered feedback dynamics fundamentally change system behavior [99] [105]. These limitations highlight the need for more sophisticated assessment approaches that can interpret test results within the context of HPA axis adaptation states.

Mechanistic Modeling Framework for HPA Axis Adaptation

Model Structure and Components

The mechanistic modeling framework proposed by Yadav et al. (2025) represents a significant advancement in interpreting ACTH stimulation tests across different HPA axis adaptation states [99]. This computational model integrates multiple physiological components: hormonal kinetics of CRH, ACTH, and cortisol; feedback inhibition dynamics at hypothalamic, pituitary, and adrenal levels; and functional mass adaptation of corticotroph and adrenal compartments [99]. The model specifically introduces a time-varying GR resistance function to mimic feedback desensitization and its resolution during chronic stress exposure and recovery [99].

The simulation encompasses three distinct physiological phases over 180 days: baseline (pre-stress), chronic stress exposure, and recovery [99]. During the chronic stress phase, the model incorporates several key adaptive phenomena: glandular remodeling of both pituitary corticotrophs and adrenal cortical cells; development of glucocorticoid receptor resistance leading to feedback desensitization; and delayed feedback recovery mechanisms [99]. These adaptations create a fundamentally altered physiological state that conventional ACTH testing may misinterpret.

Simulation of ACTH Testing Across Adaptation States

Using this modeling framework, researchers have simulated both low-dose (1μg) and high-dose (250μg) ACTH stimulation tests across different physiological phases [99]. The simulations demonstrate that cortisol responses are highly sensitive to both the magnitude and timing of stress exposure, with ACTH responsiveness being phase-dependent and often blunted during recovery due to persistent feedback resistance [99]. Low-dose ACTH testing more reliably reflects partial adrenal adaptation, while high-dose tests risk masking dysfunction due to supraphysiological drive that can overcome mild to moderate impairments [99].

The model reveals that prolonged stress induces glandular remodeling and GR resistance that significantly alter test outcomes without indicating primary adrenal failure [99]. This has crucial implications for diagnostic interpretation, particularly in conditions like depression where HPA axis dysregulation is common but may not represent true adrenal insufficiency [99]. The framework provides a mechanistic basis for understanding why static testing paradigms often yield misleading results in patients with stress-related or treatment-induced adrenal disorders.

Diagram 1: HPA Axis Circuitry and Adaptation Mechanisms. This diagram illustrates the core components of the HPA axis model, highlighting the feedforward activation pathways (red arrows), negative feedback loops (blue arrows), and adaptation mechanisms (green dashed arrows) that occur during chronic stress exposure. GR resistance develops at hypothalamic and pituitary levels, while glandular remodeling affects both pituitary corticotrophs and adrenal cortical cells.

Experimental Protocols and Methodologies

Simulation Parameters and Protocol

The mechanistic modeling framework employs specific parameters to simulate HPA axis dynamics across adaptation states. The simulation spans 180 days with three distinct phases: baseline (days 0-60), chronic stress (days 61-120), and recovery (days 121-180) [99]. A time-varying glucocorticoid receptor resistance function is introduced to mimic feedback desensitization during chronic stress and its gradual resolution during recovery [99].

ACTH stimulation tests are simulated at multiple time points within each phase using both low-dose (1μg) and high-dose (250μg) cosyntropin administrations [99]. The model outputs include cortisol levels at baseline, 30 minutes, and 60 minutes post-stimulation, as well as calculated response amplitudes and area-under-the-curve measurements. These simulated tests are analyzed in the context of the underlying adaptation state of the HPA axis.

Laboratory Measurement Protocols

For actual ACTH testing, specialized specimen handling is required due to the lability of ACTH [102] [103]. Blood must be collected in pre-chilled EDTA tubes between 7 AM and 10 AM to account for circadian variation, immediately placed on ice, and centrifuged at refrigerated temperatures within two hours of collection [102] [103]. Plasma should be separated and frozen immediately at -20°C until analysis [103]. ACTH measurements typically employ electrochemiluminescence immunoassays (ECLIA) with specific monoclonal antibodies targeting biologically active ACTH [102] [103].

Interference factors must be carefully controlled: high-dose biotin supplements (>5mg/day) should be discontinued at least 72 hours prior to testing, and specimens must be collected in plastic or siliconized glass tubes to prevent ACTH adhesion to glass surfaces [102]. When interpreting results, clinicians should consider potential interference from heterophile antibodies or rheumatoid factor, which may cause erroneously high or low values in rare cases [103].

Diagram 2: Experimental Workflow for HPA Axis Assessment. This diagram outlines the temporal sequence of the simulation protocol, including the three physiological phases (baseline, chronic stress, recovery) and the timing of ACTH stimulation tests. The specimen handling and analysis procedures critical for accurate hormone measurement are detailed in the lower section.

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Key Research Reagent Solutions for HPA Axis Studies

Reagent/Material	Specifications	Research Application
Synthetic ACTH (Cosyntropin)	1μg and 250μg doses; lyophilized powder	Stimulation testing for adrenal reserve assessment; low dose detects partial dysfunction while high dose may mask it [99] [39]
ACTH Immunoassay	Electrochemiluminescence (ECLIA) with monoclonal antibodies specific for ACTH(9-12) and ACTH(36-39) regions [103]	Quantification of ACTH in plasma; recognizes intact ACTH(1-39) and precursors (POMC, pro-ACTH) [103]
Cortisol Assay	Mass spectrometry (preferred) or immunoassay	Gold standard measurement of cortisol; mass spectrometry avoids cross-reactivity with synthetic steroids [103]
Specialized Collection Tubes	Pre-chilled plastic or siliconized glass EDTA tubes [102]	Prevents ACTH adhesion to glass; maintains sample integrity during processing
CRH & AVP Peptides	Synthetic CRH (human/rats); arginine vasopressin	Stimulation testing for pituitary reserve; differentiation of Cushing syndrome etiologies [100] [103]
Glucocorticoid Receptor Modulators	Selective GR agonists and antagonists	Investigation of feedback sensitivity and GR resistance mechanisms in chronic stress [99] [104]

Implications for Drug Development and Circadian Therapeutics

The mechanistic modeling framework has significant implications for pharmaceutical research and development, particularly in the context of circadian biology and HPA axis-related disorders. Understanding how HPA axis adaptation states influence drug responses can inform clinical trial design for glucocorticoid therapies, antidepressants, and metabolic agents. The recognition that glucocorticoids synchronize circadian rhythms in peripheral tissues, including tumors, suggests novel chronotherapeutic approaches [104].

In glioblastoma models, daily glucocorticoids promote tumor growth in a time-of-day dependent manner through glucocorticoid receptor signaling that synchronizes circadian clock gene expression in cancer cells [104]. This finding has direct relevance for drug development, as blocking circadian signals like vasoactive intestinal peptide or glucocorticoids dramatically slows tumor growth and disease progression [104]. Analysis of human glioblastoma samples from The Cancer Genome Atlas shows that high GR expression significantly increases hazard of mortality, highlighting the clinical importance of these findings [104].

From a diagnostic perspective, the modeling framework suggests that accounting for glandular plasticity and GR feedback dynamics is essential for effective endocrine diagnosis, particularly in stress-related or treatment-induced adrenal disorders [99]. Drug development programs targeting HPA axis function should incorporate dynamic testing protocols that can detect adaptive states rather than relying solely on static hormone measurements. This approach could lead to more personalized therapeutic strategies that consider an individual's HPA axis adaptation state and circadian glucocorticoid rhythm.

The mechanistic modeling framework for interpreting ACTH tests across HPA adaptation states represents a significant advancement in neuroendocrine research. By integrating hormonal kinetics, feedback inhibition, and functional mass adaptation, this approach provides a more nuanced understanding of HPA axis dynamics than conventional static testing paradigms. The framework demonstrates that cortisol responses to ACTH stimulation are highly sensitive to both the magnitude and timing of stress exposure, with low-dose testing more reliably reflecting partial adrenal adaptation compared to high-dose protocols [99].

Future research should focus on validating this modeling approach in clinical populations with stress-related disorders, depression, and iatrogenic HPA axis dysfunction. The integration of circadian biology into HPA axis modeling presents particularly promising avenues for investigation, especially given the emerging evidence that glucocorticoids synchronize peripheral circadian clocks in both healthy and diseased tissues [67] [104]. From a therapeutic perspective, developing pharmacological agents that can selectively modulate specific components of the HPA axis without disrupting its circadian organization represents an important frontier in endocrine drug development.

The integration of mechanistic modeling with circadian biology holds particular promise for advancing personalized medicine approaches to HPA axis disorders. By accounting for individual differences in stress adaptation, feedback sensitivity, and circadian glucocorticoid rhythms, clinicians may eventually tailor diagnostic and therapeutic strategies to each patient's unique neuroendocrine profile. This approach could significantly improve outcomes for patients with adrenal disorders, depression, and other conditions involving HPA axis dysregulation.

Glucocorticoids (GCs), the primary stress hormones, exhibit a robust circadian rhythm and exert pleiotropic effects on peripheral tissues. This whitepaper delineates the tripartite role of GCs as rhythm drivers, zeitgebers, and tuners of peripheral circadian clocks. We synthesize current mechanistic insights, highlighting how GCs, through the glucocorticoid receptor (GR), directly regulate rhythmic gene expression, reset peripheral tissue clocks, and tonically modulate circadian outputs. The critical importance of temporal alignment between GC rhythms and local tissue clocks for systemic homeostasis is emphasized, while misalignment is linked to metabolic, cardiovascular, and immune pathologies. This synthesis provides a framework for developing chronotherapeutic strategies that leverage the multifaceted temporal actions of GCs.

In mammals, the circadian system is organized as a hierarchical network, with a master pacemaker in the suprachiasmatic nucleus (SCN) coordinating peripheral clocks in virtually every tissue and cell [4] [96] [106]. The hypothalamic-pituitary-adrenal (HPA) axis is a key output of this system, generating a robust, diurnal rhythm in circulating GCs (cortisol in humans, corticosterone in rodents) [107] [106] [12]. This rhythm peaks at the onset of the active phase (morning in humans, evening in rodents), priming the organism for anticipated metabolic and immune challenges [108].

Beyond this circadian variation, GC secretion is pulsatile, with ultradian pulses occurring approximately every 90 minutes [12]. The rhythmic output of GCs is governed by a multi-level control system: the SCN provides rhythmic drive to the HPA axis via neuronal projections to the paraventricular nucleus (PVN) of the hypothalamus; the adrenal gland itself possesses an intrinsic circadian clock that gates its sensitivity to adrenocorticotropic hormone (ACTH); and the adrenal is innervated by the autonomic nervous system, which can directly modulate GC production [5] [106] [12].

GCs exert their effects primarily by binding to the ubiquitously expressed glucocorticoid receptor (GR), a ligand-dependent transcription factor. Upon activation, GR regulates gene expression by binding directly to glucocorticoid response elements (GREs) in target genes, or by tethering itself to other transcription factors [109] [108]. The pervasive circadian rhythm of GCs and the broad expression of GR position this hormonal system as a principal communicator of temporal information from the central SCN clock to peripheral tissues.

Conceptual Framework: Rhythm Drivers, Zeitgebers, and Tuners

The endocrine regulation of circadian rhythms can be conceptualized through three principal modes of action [12]. The following diagram illustrates the conceptual relationship and key features of these roles.

• Rhythm Driver: A rhythmic hormonal signal that directly regulates the cyclic expression of target genes (e.g., via GREs) to generate physiological rhythms, independent of the local tissue clock [12].
• Zeitgeber ("Time-Giver"): A rhythmic signal that can reset the phase of peripheral circadian clocks, thereby synchronizing local timekeeping with external or internal cycles [12]. GCs achieve this partly by regulating the expression of core clock genes like Per1 and Per2, which contain GREs in their promoters [12] [16].
• Tuner: A hormonal signal that, which can be arrhythmic, modulates the amplitude or robustness of tissue output rhythms without fundamentally altering the phase of the core circadian clock [12]. This represents a more subtle, tonic influence on circadian regulation.

Molecular Mechanisms of Glucocorticoid Action

Glucocorticoid Receptor Signaling and Genomics

The GR (NR3C1) gene produces multiple isoforms through alternative splicing and translation initiation, contributing to tissue-specific GC sensitivity [109]. The canonical signaling pathway involves GC binding to cytosolic GR, which is complexed with chaperone proteins like HSP90. This binding induces GR dimerization, nuclear translocation, and binding to GREs, leading to transactivation or transrepression of target genes [109] [108].

Genome-wide studies have revealed that GR-binding sites are distributed throughout the genome, often in enhancer regions far from transcription start sites. GR binding can induce chromatin remodeling and recruit co-activators or co-repressors to modulate gene expression [109]. The transcriptional outcome is highly context-dependent, influenced by cell type-specific transcription factors and chromatin landscape.

Table 1: Key Components of Glucocorticoid Receptor Signaling

Component	Structure/Function	Role in Circadian Signaling
GRα (canonical)	777-amino acid protein; contains N-terminal transactivation domain (AF-1), central DNA-binding domain (DBD), and C-terminal ligand-binding domain (LBD).	Primary mediator of genomic GC actions; binds GREs to drive rhythmic gene expression and clock gene regulation [109].
GRE (Glucocorticoid Response Element)	Specific DNA sequence recognized by the GR DBD.	Direct conduit for rhythm driver and zeitgeber functions; found in promoters of metabolic genes (e.g., PCK1, G6PC) and clock genes (e.g., Per1, Per2, Nfil3) [12] [16].
Chaperone Complex (HSP90, FKBP5)	Stabilizes unliganded GR in the cytoplasm in a high-affinity conformation.	Determines ligand-binding capacity and cellular sensitivity to rhythmic GC pulses [5] [109].
Co-regulators	Proteins (e.g., SRC-1, NCoR) recruited by GR to modify chromatin and regulate transcription.	Fine-tune tissue-specific transcriptional responses to circadian GC signals [109].

Mechanistic Workflow of GC Action on Peripheral Clocks

The following diagram summarizes the sequential molecular processes through which GCs exert their triple roles on peripheral tissue clocks.

Experimental Evidence and Data Synthesis

Key Experimental Models and Protocols

Research into GCs as circadian regulators relies on specific in vivo and in vitro models.

• In Vivo Zeitgeber Misalignment: A key protocol involves exposing mice to conflicting light-dark (LD) and feeding-fasting (FF) cycles. For example, an 28-hour LD cycle (LD-28) is combined with a 24-hour FF cycle (FF-24). This misalignment rapidly induces internal desynchrony, allowing researchers to dissect the role of feeding (and its interaction with GC rhythms) in entraining peripheral clocks in liver, adipose tissue, and adrenal glands [110]. Tissue harvesting every 4 hours over a 24-48 hour period is standard for assessing clock gene expression (Bmal1, Per2, Dbp, etc.) via qPCR or RNA-seq.
• Adrenalectomized (ADX) Models: Surgical removal of the adrenal glands eliminates the endogenous source of GCs. This model is crucial for establishing the necessity of GCs for rhythmic immune cell trafficking and cytokine expression. Recovery of rhythms with timed GC replacement confirms GCs' role as a zeitgeber [16].
• Cell Culture and Peripheral Clocks: Isolated fibroblasts or tissue explants maintain circadian rhythms for several cycles in vitro. Treating these cultures with GCs such as dexamethasone at a specific concentration (e.g., 100 nM) for a short pulse (e.g., 30-120 minutes) can induce phase-dependent phase shifts in Per2::luciferase rhythm, directly demonstrating GCs' zeitgeber capacity [106].

Table 2: Quantitative Data from Zeitgeber Misalignment Studies [110]

Tissue	Experimental Condition	Average Phase Shift of Clock Gene Rhythms (Hours, Day 4)	Phase Coherence Within Tissue (% of Control)	Key Interpretation
Liver	LD-28 / FF-24	2.6 ± 0.5	91.5 ± 0.2%	Feeding schedule exerts a strong entraining pull on the liver clock, partially resisting LD misalignment.
	LD-28 / FF-28	5.8 ± 0.9	73.5 ± 1.6%	Aligning FF cycle to LD period reduces conflict, but weakens internal clock gene coordination.
White Adipose Tissue (eWAT)	LD-28 / FF-24	1.6 ± 0.3	88.4 ± 0.8%	Highly sensitive to feeding time; minimal phase shift indicates strong entrainment to FF-24.
	LD-28 / FF-28	7.1 ± 0.2	59.5 ± 1.1%	Aligned long cycles induce large phase shifts but severely disrupt internal clock coordination.
Adrenal Gland	LD-28 / FF-24	3.4 ± 0.3	94.7 ± 0.8%	Shows intermediate sensitivity, influenced by both central (SCN) and local (feeding) signals.
	LD-28 / FF-28	5.8 ± 0.2	87.6 ± 0.1%	Phase shift is larger when FF cycle is aligned with the lengthened LD cycle.

The Scientist's Toolkit: Essential Reagents and Models

Table 3: Key Research Reagent Solutions for Investigating Circadian Glucocorticoid Actions

Reagent / Model	Function / Application	Specific Example & Rationale
Dexamethasone	Synthetic GR agonist; highly potent and resistant to degradation.	Used for in vitro pulsatile treatment (e.g., 100 nM, 30 min) to phase-shift fibroblast clocks [106]; in vivo to study GR-specific effects without confounding MR activation.
Corticosterone (Rodents)	The endogenous rodent GC; used for physiological replacement.	Administered to adrenalectomized rodents via drinking water or subcutaneous pellets to restore circadian rhythms in immune parameters and gene expression [16].
RU-486 (Mifepristone)	GR antagonist.	Used to block GR signaling and confirm the specific role of GR (vs. MR) in mediating circadian GC effects in vivo and in cell models.
GR Knockout Mice (Tissue-Specific)	To dissect GR function in specific tissues.	e.g., CD11c-Cre GR KO: Demonstrates GC control of dendritic cell cytokine production [16]. Liver-specific GR KO: Unravels liver-specific vs. systemic metabolic effects of circadian GC rhythms.
Per2::Luciferase Reporter Cells/Lines	Real-time monitoring of circadian clock phase and period.	Used to quantify phase-shifting effects of GC pulses in high-throughput format; applicable to primary cells and tissue explants.

Implications for Physiology, Pathology, and Chronotherapy

The triple action of GCs is fundamental for temporal coordination across systems. As rhythm drivers, they synchronize metabolic processes like gluconeogenesis and lipolysis [107] [16]. As zeitgebers, they reinforce the phase of peripheral clocks in the liver, fat, and immune cells, ensuring coherence with the SCN's master schedule [106] [12]. As tuners, they adjust the amplitude of immune and metabolic responses according to the time of day and overall physiological state [12].

Disruption of this precise temporal control—circadian misalignment—is a key pathological mechanism. Shift work, chronic jet lag, and erratic eating patterns create a mismatch between the central SCN rhythm, the GC rhythm, and peripheral tissue clocks [110]. This misalignment is epidemiologically and experimentally linked to an increased risk for metabolic syndrome, cardiovascular disease, immune dysregulation, and mood disorders [4] [96] [106].

These insights pave the way for chronotherapy. Timing GC medication administration (e.g., for rheumatoid arthritis or asthma) to coincide with the peak of symptom severity or the trough of target tissue sensitivity can maximize efficacy and minimize side effects [106]. Furthermore, interventions like time-restricted feeding can help realign peripheral clocks with the central pacemaker, potentially mitigating the adverse effects of shift work and metabolic disease by stabilizing the GC rhythm and its downstream effects [110].

The circadian system is a fundamental temporal organizer of physiology, governing the 24-hour rhythms of nearly all biological processes. The hypothalamic-pituitary-adrenal (HPA) axis, with its pulsatile secretion of glucocorticoids, represents a central circadian output with far-reaching regulatory influence. This hormonal rhythm does not function in isolation but operates within a complex network of endocrine signals. Understanding the molecular and systemic cross-talk between the glucocorticoid rhythm and other key hormonal oscillators—particularly melatonin, sex steroids, and metabolic factors—is essential for a comprehensive view of circadian physiology and its implications for health and disease. This review synthesizes current evidence on these interactions, with a specific focus on mechanistic insights, experimental approaches, and translational relevance for drug development.

Molecular Foundations of Circadian Crosstalk

The Core Circadian Clockwork

At the cellular level, circadian rhythms are generated by a conserved transcriptional-translational feedback loop (TTFL). The core components of this loop are expressed throughout the brain and peripheral tissues, including endocrine glands.

• Positive Limb: The CLOCK and BMAL1 proteins form a heterodimer that activates transcription of clock-controlled genes by binding to E-box enhancer elements in their promoter regions [111] [4].
• Negative Limb: The PER and CRY proteins accumulate, form complexes, and translocate back to the nucleus to repress CLOCK-BMAL1 transcriptional activity, thereby completing the cycle [111] [4].
• Stabilizing Loop: The nuclear receptors REV-ERBα and ROR provide a stabilizing loop by respectively repressing and activating BMAL1 transcription, fine-tuning the oscillation precision [111] [4].

Table 1: Core Components of the Circadian Transcriptional-Translational Feedback Loop

Component	Gene Symbol	Function in TTFL	Role in Hormonal Regulation
Circadian Locomotor Output Cycles Kaput	CLOCK	Forms heterodimer with BMAL1; histone acetyltransferase activity	Modulates steroidogenic gene expression
Brain and Muscle ARNT-Like 1	BMAL1 (ARNTL)	DNA-binding component of heterodimer; drives Per, Cry transcription	Regulates metabolic gene networks
Period	PER1, PER2, PER3	Forms repressor complex with CRY; inhibits CLOCK-BMAL1	Entrainment to feeding cycles; metabolic sensing
Cryptochrome	CRY1, CRY2	Completes repressor complex; regulates nuclear translocation	Glucocorticoid receptor transactivation
Reverse Erb Alpha	REV-ERBα (NR1D1)	Represses BMAL1 transcription; links clock to metabolism	Integrates heme signaling with glucocorticoid production

This molecular clockwork is present not only in the suprachiasmatic nucleus (SCN) but also in peripheral tissues, including the adrenal cortex, gonads, and pancreas, enabling local temporal coordination of hormone synthesis and secretion.

Hierarchical Organization of the Circadian System

The circadian system is organized in a hierarchical manner:

• Master Pacemaker: The SCN in the hypothalamus serves as the central coordinator, directly receiving light input from the retina via the retinohypothalamic tract [111] [4].
• Peripheral Clocks: Tissues throughout the body maintain their own circadian oscillations, which are synchronized by the SCN through neural, hormonal, and behavioral signals [4].
• Endocrine Mediators: Hormones such as glucocorticoids and melatonin serve as key signaling molecules that communicate timing information from the SCN to peripheral clocks, thus maintaining phase coherence across the organism [111] [4].

Figure 1: Hierarchical organization of the mammalian circadian system showing the central role of the SCN in coordinating peripheral clocks through neural and hormonal signals.

Melatonin-Glucocorticoid Interactions

Bidirectional Regulatory Relationships

Melatonin, primarily secreted by the pineal gland during the dark phase, and glucocorticoids, which peak at the onset of the active phase, exhibit a robust reciprocal relationship that is crucial for maintaining appropriate circadian phase relationships.

Melatonin's Influence on Glucocorticoid Signaling:

• SCN Modulation: Melatonin receptors (MT1 and MT2) are densely expressed in the SCN, where melatonin directly phase-shifts circadian rhythms and modulates SCN output signals that regulate HPA axis activity [111].
• Adrenal Direct Effects: Human adrenal glands express functional melatonin receptors, suggesting direct adrenal modulation. Melatonin can suppress adrenal glucocorticoid production by inhibiting the cAMP signaling pathway and steroidogenic gene expression [111].
• Immune Mediation: Melatonin exerts anti-inflammatory effects partly through inhibition of glucocorticoid signaling, modulating the nuclear factor kappa B (NF-κB) pathway and reducing inflammatory cytokine production [111].

Glucocorticoid Influence on Melatonin Rhythms:

• SCN Regulation: Glucocorticoid receptors are present in the SCN, though their role in this region is complex. Synthetic glucocorticoids can phase-shift peripheral clocks but have limited effects on the SCN master clock [4].
• Pineal Effects: High glucocorticoid levels, as seen in Cushing syndrome or chronic stress, can suppress melatonin synthesis by inhibiting the expression of serotonin N-acetyltransferase, the rate-limiting enzyme in melatonin production [4].

Table 2: Experimental Evidence for Melatonin-Glucocorticoid Interactions

Experimental Approach	Key Findings	Physiological/Clinical Relevance
Melatonin administration in rodents	Reduced corticosterone response to stress; phase advancement of HPA axis rhythm	Potential therapeutic for shift work disorders, jet lag
Adrenal cell cultures	Melatonin inhibited ACTH-stimulated cortisol production by up to 40% via MT1 receptor	Direct adrenal action independent of central pathways
Clinical studies in DGBI patients	Melatonin treatment improved IBS symptoms; normalized cortisol rhythms	Chronotherapeutic approach for functional GI disorders
Pinealectomy studies	Eliminated melatonin rhythm; resulted in HPA axis hyperactivation and loss of diurnal rhythm	Demonstrates necessity of melatonin for HPA axis regulation

Gastrointestinal Tract as an Interaction Site

The gastrointestinal tract represents a significant site of melatonin-glucocorticoid interaction, with implications for disorders of gut-brain interaction (DGBIs):

• Local Melatonin Production: The gut contains over 400 times more melatonin than the pineal gland, with production showing circadian rhythmicity that influences gut motility, mucosal barrier function, and immune responses [111].
• Circadian Disruption in DGBIs: Patients with irritable bowel syndrome (IBS) and functional dyspepsia frequently demonstrate disrupted cortisol rhythms, altered melatonin secretion, and sleep disturbances, suggesting pathological chronodisruption [111].
• Therapeutic Potential: Melatonin supplementation (3-5 mg before bedtime) has shown efficacy in improving abdominal pain and sleep quality in IBS patients, potentially through resynchronization of circadian rhythms in the gut [111].

Sex Hormone Interactions with Glucocorticoid Rhythms

Organizational and Activational Effects

Sex differences in HPA axis activity and circadian glucocorticoid rhythms are well-documented and occur through both organizational (permanent, developmental) and activational (reversible, adult) effects of sex steroids.

Estrogen Effects:

• CRH Stimulation: Estradiol enhances CRH mRNA expression in the paraventricular nucleus and potentiates ACTH and corticosterone responses to stress in female rodents [112].
• Circadian Phase: Ovariectomy abolishes the diurnal peak of corticosterone in females, which is restored with estradiol replacement, demonstrating its necessity for rhythm maintenance [112].
• Receptor Mechanisms: Estrogen receptor alpha (ERα) activation generally enhances HPA axis activity, while ERβ may have inhibitory effects, creating a complex regulatory balance [112].

Androgen Effects:

• HPA Axis Inhibition: Testosterone and its metabolites generally exert inhibitory effects on HPA axis activity, contributing to sexual dimorphism in stress responses [112].
• Development Programming: Perinatal androgen exposure organizes the HPA axis toward a male phenotype with reduced responsivity to stressors in adulthood [112].

Progesterone Interactions:

• GABAergic Mechanism: Allopregnanolone, a progesterone metabolite, potentiates GABAA receptor signaling, resulting in inhibition of CRH and AVP neurons, thereby reducing HPA axis activity [112].
• Glucocorticoid Antagonism: Progesterone can compete with glucocorticoids for the glucocorticoid receptor, though with lower affinity, creating functional antagonism in some tissues [112].

Reproductive Transitions and HPA Axis Function

Life stage transitions involving significant changes in sex steroid levels demonstrate the clinical relevance of these interactions:

• Pregnancy: The progressive increase in estrogen, progesterone, and CRH of placental origin creates a unique HPA axis regulatory environment with implications for maternal adaptation and postpartum mood disorders [112].
• Menopause: The decline in estrogen is associated with increased vulnerability to stress-related disorders and alterations in circadian rhythm regulation, partially ameliorated by hormone replacement therapy [112].
• Aging: Age-related changes in both HPA axis activity (elevated evening cortisol) and sex steroid production contribute to metabolic and cognitive alterations in older adults [112].

Figure 2: Regulatory effects of major sex steroid hormones on HPA axis activity, showing stimulatory and inhibitory influences that contribute to sexual dimorphism in stress responses.

Metabolic Hormone Interactions

Insulin-Glucocorticoid Counterregulation

The interplay between glucocorticoids and insulin represents a fundamental metabolic cross-talk with significant circadian components:

• Diurnal Insulin Sensitivity: Insulin sensitivity follows a circadian rhythm, with peak sensitivity typically during the daytime in humans, inversely related to the circadian peak in cortisol [41].
• Glucocorticoid Actions: Cortisol promotes gluconeogenesis, glycogenolysis, and lipolysis, opposing insulin's anabolic effects. Evening elevations in cortisol (as in chronodisruption) are particularly detrimental to metabolic health [41].
• Molecular Mechanisms: Glucocorticoids induce insulin resistance through multiple mechanisms, including decreased GLUT4 translocation, increased expression of gluconeogenic enzymes, and stimulation of adipose tissue lipolysis [41].

Leptin and Adipokine Signaling

Adipose tissue-derived hormones exhibit circadian rhythms and interact with glucocorticoid signaling:

• Leptin Rhythms: Leptin shows a circadian rhythm with peak levels during the sleep phase, which is influenced by both feeding patterns and glucocorticoids [41].
• Glucocorticoid Regulation: Glucocorticoids stimulate leptin secretion, creating a potential feedback loop that may link nutritional status with HPA axis activity [41].
• Adipocyte Clocks: Adipocytes possess functional circadian clocks that regulate adipokine secretion and are entrained by glucocorticoids, providing a mechanism for temporal coordination of energy metabolism [41].

Table 3: Metabolic Hormones with Circadian Rhythms and Glucocorticoid Interactions

Metabolic Factor	Circadian Pattern	Interaction with Glucocorticoids	Pathophysiological Significance
Insulin	Peak during active phase; sensitivity highest in morning	Glucocorticoids induce insulin resistance; insulin can inhibit HPA axis	Evening cortisol elevation predicts type 2 diabetes risk
Leptin	Peak during sleep phase; amplitude reflects energy stores	Glucocorticoids stimulate leptin production; leptin inhibits HPA axis	Dysregulation in metabolic syndrome; night eating syndrome
Ghrelin	Preprandial rises; elevated during fasting	Glucocorticoids enhance ghrelin secretion; ghrelin stimulates HPA axis	Link between stress and emotional eating
Adiponectin	Diurnal variation with peak in late morning	Glucocorticoids suppress adiponectin production	Low adiponectin in Cushing syndrome contributes to metabolic risk

Experimental Approaches and Methodologies

Assessing Circadian Hormonal Interactions

Research investigating cross-talk between hormonal systems requires specialized methodologies that account for temporal dynamics:

Circadian Sampling Protocols:

• Frequent Blood Sampling: Serial blood collection at 2-4 hour intervals over at least 24 hours to characterize circadian waveform, including amplitude, phase, and rhythm robustness [46] [41].
• Salivary Cortisol: Non-invasive home collection for assessing circadian rhythm and stress responsiveness in ecological settings [41].
• Urinary Hormone Metabolites: 24-hour collections for integrated assessment of hormone production while capturing diurnal patterns [46].

Molecular Techniques:

• Chromatin Immunoprecipitation (ChIP): Assess binding of clock components (CLOCK-BMAL1) to promoter regions of steroidogenic genes [111] [4].
• Tissue-Specific Knockout Models: Conditional deletion of clock genes in endocrine tissues to elucidate tissue-specific functions [4].
• Transcriptomic Profiling: RNA sequencing of endocrine tissues across circadian time to identify rhythmically expressed genes and regulatory networks [4].

Manipulation Approaches

Genetic and Pharmacological Tools:

• Cell-Type Specific Ablation: Targeted genetic approaches to eliminate specific endocrine cell populations or neuronal circuits connecting hormonal systems [46].
• Receptor-Specific Agonists/Antagonists: Pharmacological dissection of receptor-mediated effects in hormonal cross-talk [112] [41].
• Opto- and Chemogenetics: Precise temporal control of specific neuronal populations regulating endocrine axes [4].

Figure 3: Generalized experimental workflow for investigating circadian hormonal interactions, showing key stages from animal model preparation through data analysis.

The Scientist's Toolkit: Key Research Reagents

Table 4: Essential Research Reagents for Investigating Hormonal Rhythm Cross-Talk

Reagent/Category	Specific Examples	Research Application	Key Considerations
Receptor Agonists/Antagonists	MT1/MT2: Ramelteon, luzindole; GR: Dexamethasone, RU486; ER: PPT, MPP	Pharmacological dissection of receptor-specific contributions to rhythmicity	Selectivity varies; dose-response characterization essential
CRH/ACTH Analogues	CRF: Ovine/rCRF; ACTH: Cosyntropin (Synacthen)	HPA axis stimulation tests; assessment of adrenal responsiveness	Dose-dependent effects (low vs high dose); species variations
Circadian Reporter Systems	PER2::LUC fibroblasts; Bmal1-luc transgenic animals	Real-time monitoring of circadian phase in tissues/cells	Validation required for each tissue type; rhythm damping considerations
Hormone Assays	ELISA, RIA, LC-MS/MS for cortisol, melatonin, sex steroids	Quantitative hormone measurement in biological fluids	Specificity varies (MS most specific); sensitivity for low-amplitude rhythms
Genetic Models	Conditional knockout mice (Cre-lox system); CRISPR-Cas9 editing	Cell-type specific manipulation of circadian components	Developmental compensation possible; temporal control advantageous

The cross-talk between glucocorticoid rhythms and other hormonal systems represents a complex, multi-layered regulatory network essential for optimal physiological function. Molecular connections occur through shared transcriptional regulators, receptor-mediated signaling crosstalk, and neurocircuit-level integration. The clinical implications of these interactions are substantial, evident in the metabolic disturbances of Cushing syndrome, the sleep disruptions of menopause, and the gastrointestinal dysfunction in shift workers.

Future research priorities should include:

• Tissue-Specific Mechanism Elucidation: Application of single-cell technologies to characterize circadian function in specific endocrine cell populations.
• Human Chronotherapy Studies: Targeted drug delivery aligned with endogenous hormonal rhythms to optimize efficacy and minimize side effects.
• Computational Model Development: Integration of multi-omics data to create predictive models of endocrine cross-talk across the lifespan.
• Circadian-Targeted Therapeutics: Development of small molecules that specifically target clock components or their downstream effectors for metabolic, psychiatric, and neoplastic disorders.

Understanding these intricate temporal relationships between hormonal systems will enable more precise therapeutic interventions that respect the body's innate circadian organization, ultimately advancing the field of chronotherapeutics across a broad spectrum of diseases.

Glucocorticoids (GCs), secreted under the control of the hypothalamic-pituitary-adrenal (HPA) axis, serve as pivotal synchronizers of circadian rhythms in peripheral tissues. This whitepaper delineates the tissue-specific molecular mechanisms by which liver, skeletal muscle, and immune cells interpret these GC signals to regulate local circadian clocks and downstream physiological outputs. Within the context of circadian GC and ACTH research, we explore how the core clock machinery in each tissue translates rhythmic hormonal cues into metabolic and immune functions. Disruption of this crosstalk, as seen in shift work or chronic stress, contributes to pathologies like metabolic syndrome, sarcopenia, and chronic inflammation. This guide synthesizes current experimental data, provides detailed methodologies for key assays, and visualizes critical signaling pathways, offering a resource for researchers and drug development professionals aiming to develop chronotherapeutic strategies.

The circadian system is a hierarchical network, with the central pacemaker in the suprachiasmatic nucleus (SCN) orchestrating peripheral clocks via neural, hormonal, and behavioral cues [113] [114]. A key hormonal output is the circadian rhythm of glucocorticoid (GC) secretion, which peaks at the onset of the active phase (morning in humans, evening in rodents) [85] [113]. This rhythm is not merely a passive response to SCN signaling but is an integral component of a feedback system that maintains whole-body circadian homeostasis.

Glucocorticoids exert their effects by binding to the ubiquitously expressed glucocorticoid receptor (GR), a ligand-dependent transcription factor. Upon activation, GR dimerizes and translocates to the nucleus, where it binds to glucocorticoid response elements (GREs) in target genes, leading to transactivation or transrepression [85]. Notably, the GR can also interact with other transcription factors, such as NF-κB and AP-1, to repress inflammatory genes—a mechanism known as transrepression [85].

Crucially, GCs are potent synchronizers of peripheral clocks. The Per1, Per2, and Nfil3 genes possess GREs in their promoters, allowing GCs to directly influence the core circadian transcriptional-translational feedback loop (TTFL) [85]. This enables GC rhythms to entrain metabolic and immune functions in peripheral tissues, including the liver, skeletal muscle, and immune cells, ensuring they are optimally phased with the organism's behavioral cycle.

Core Circadian Clock Machinery

The molecular clock operates through interlocked transcriptional-translational feedback loops (TTFLs) [115] [113] [116]. The core loop involves the heterodimerization of the transcription factors CLOCK and BMAL1. This complex binds to E-box elements in the promoters of target genes, driving the expression of Period (Per1, Per2) and Cryptochrome (Cry1, Cry2). PER and CRY proteins accumulate, form complexes in the cytoplasm, and translocate back to the nucleus to repress CLOCK:BMAL1 activity, thereby closing the negative feedback loop with a period of approximately 24 hours [115].

A stabilizing auxiliary loop involves the nuclear receptors REV-ERBα/β and RORα/β/γ. CLOCK:BMAL1 activates Rev-erbα/β expression, whose proteins then repress Bmal1 transcription by binding to ROR response elements (RREs) in its promoter. Conversely, RORs activate Bmal1 expression, creating a rhythmic antagonism that reinforces oscillation stability [115] [116]. This core machinery is present in virtually all cell types and governs the rhythmic expression of clock-controlled genes (CCGs), which constitute up to 10% of the mammalian transcriptome [113].

Figure 1: Core Mammalian Circadian Clock Mechanism. The CLOCK:BMAL1 heterodimer activates transcription of *Per, Cry, and Rev-erb genes via E-box elements. PER:CRY complexes accumulate and inhibit CLOCK:BMAL1, completing the core negative feedback loop. In the auxiliary loop, REV-ERBs and RORs competitively bind ROR response elements (RREs) in the Bmal1 promoter, repressing and activating its expression, respectively. This network generates rhythmic gene expression of CCGs.*

Tissue-Specific GC-Clock Interplay

Hepatic Circadian Responses to GCs

The liver clock, tightly synchronized by GC rhythms, directs the circadian timing of fundamental metabolic processes, including glucose homeostasis, lipid metabolism, and bile acid synthesis [117]. GC signaling is a primary driver of hepatic gluconeogenesis. The activated GR directly induces the expression of rate-limiting enzymes phosphoenolpyruvate carboxykinase (PCK1) and glucose-6-phosphatase (G6PC) [85]. This aligns hepatic glucose production with the organism's active phase.

The molecular clock machinery in hepatocytes fine-tunes this GC response. For instance, the clock protein REV-ERBα can repress Pck1 transcription, thereby opposing GC action during the rest phase [85]. Furthermore, GCs can reset the phase of the hepatic clock by directly regulating the expression of core clock genes like Per1 and Per2 via GREs in their promoters [85]. This tight coupling ensures that metabolic processes are anticipatorily regulated rather than merely reactive.

Table 1: Key Circadian-Metabolic Genes in the Liver Regulated by GCs and Clock Proteins

Gene Symbol	Gene Name	Regulation by GC/GR	Regulation by Clock	Metabolic Function
Pck1	Phosphoenolpyruvate carboxykinase	↑ Direct transactivation via GRE [85]	↓ Repressed by REV-ERBα [85]	Rate-limiting step in gluconeogenesis
G6pc	Glucose-6-phosphatase	↑ Direct transactivation via GRE [85]	Information missing	Final step in gluconeogenesis
Bmal1	Brain and Muscle ARNT-like 1	Indirect (via PER induction) [85]	↑ Activated by RORs; ↓ Repressed by REV-ERBs [115]	Core clock component

Skeletal Muscle Clock and GC Signaling

Skeletal muscle possesses a robust autonomous circadian clock that regulates metabolism, muscle maintenance, and contractile function [118]. GCs participate in entraining this muscle clock. Studies show that GCs regulate the expression of Per1, Per2, and Rev-erbα in muscle cells, thereby influencing the phase of the local TTFL [118].

The muscle clock, in turn, governs circadian rhythms in glucose and lipid metabolism. Muscle-specific Bmal1 knockout (KO) mice exhibit impaired insulin-stimulated glucose uptake and disrupted lipid metabolism, highlighting the clock's essential role in metabolic homeostasis [118]. Furthermore, the clock regulates amino acid metabolism and protein turnover. Bmal1 KO mice display sarcopenic phenotypes—reduced muscle mass, fiber size, and contractile force—indicating a role for the clock in muscle maintenance [118]. The nuclear receptor REV-ERBα has been shown to suppress myogenesis, adding another layer of circadian control over muscle mass [118].

Table 2: Phenotypes of Circadian Clock Disruption in Skeletal Muscle

Genetic Model / Disruption	Metabolic Phenotype	Structural/Functional Phenotype	Key Molecular Changes
Whole-body Bmal1 KO [118]	Glucose intolerance, insulin resistance	Reduced muscle mass and strength, sarcopenic features	Disrupted expression of metabolic genes (Tcap, Hspa1b)
Muscle-specific Bmal1 KO [118]	Impaired glucose uptake and lipid metabolism	Information missing	Altered expression of genes involved in glucose, lipid, and amino acid metabolism
REV-ERBα agonism/overexpression [118]	Information missing	Suppression of myogenesis (reduced differentiation)	Downregulation of myogenic factors
Environmental (Shift Work) [118]	Increased risk of type 2 diabetes	Loss of muscle mass and strength	Disrupted rhythmic expression of core clock genes

Immune Cell Clocks and GC Regulation

The immune system exhibits profound circadian rhythms in cell trafficking, cytokine production, and effector functions, many of which are entrained by GCs [85] [113]. Circulating immune cell numbers oscillate diurnally, with neutrophils, monocytes, and lymphocytes peaking in the blood during the resting phase in humans, a process regulated by rhythmic expression of chemokines (e.g., CXCL12) and adhesion molecules [113].

GCs exert both permissive and suppressive effects on immune function in a time-dependent manner. At the peak of their secretion, they suppress the expression of pro-inflammatory cytokines (e.g., IL-6, TNF-α) and chemokines (e.g., CXCL5) via transrepression of NF-κB and AP-1 [85]. This rhythm creates a window of immunosuppression during the active phase. Conversely, the nadir of GC secretion at night allows for enhanced immune vigilance and response.

Intrinsic clocks within immune cells, such as macrophages, modulate their inflammatory responses. For example, the core clock proteins NFIL3 and DBP competitively bind the Il12b promoter, leading to its rhythmic expression [115]. BMAL1 in macrophages inhibits sepsis development by controlling glycolysis, while REV-ERBα agonists inhibit NLRP3 inflammasome activation and production of IL-6, demonstrating potent anti-inflammatory effects [115] [116]. Disruption of these clocks, as seen in Per2 mutant mice, alters GC synthesis and endotoxin shock mortality, underscoring the bidirectional relationship between the clock and the HPA axis [85].

Figure 2: GC and Clock Crosstalk in Immune Cell Regulation. The circadian rhythm of glucocorticoids (GCs) activates the GR, which suppresses pro-inflammatory gene expression by directly binding to negative GREs (nGREs) or via transrepression of transcription factors like NF-κB and AP-1. Simultaneously, the intrinsic macrophage clock (e.g., through NFIL3/DBP competition) generates rhythmicity in the response to immune stimuli. This integrated system produces a time-of-day-dependent immune response.

Table 3: Circadian Immune Parameters and Their Regulation by GCs and Clock Genes

Immune Parameter	Circadian Peak (Human)	Primary Regulator	Molecular Mechanism
Circulating Neutrophils [113]	Night (Resting Phase)	Sympathetic Nerves / GCs	Rhythmic CXCL12/CXCR4 axis in bone marrow
Plasma IL-6 & TNF-α [113]	Onset of Active Phase	GCs (suppressive effect wanes at night)	Transrepression of NF-κB/AP-1 by GR
Macrophage IL-12 Response [115]	Information missing	Clock (NFIL3, DBP)	Competitive binding of NFIL3 (repressor) and DBP (activator) to Il12b promoter
Lung Neutrophil Infiltration [85]	Day (in mice)	GCs (rhythmic suppression of CXCL5)	GR binding to nGRE in Cxcl5 promoter

Experimental Protocols for Investigating GC-Clock Interactions

Assessing Circadian Gene Expression In Vivo

Objective: To characterize the circadian expression profiles of core clock and clock-controlled genes in a specific tissue (e.g., liver, muscle) in response to GC manipulation.

Materials:

• Wild-type and tissue-specific clock gene knockout mice (e.g., Bmal1 KO)
• Corticosterone ELISA kit
• RNA/DNA extraction kits
• qPCR system and TaqMan probes for target genes (e.g., Bmal1, Per2, Pck1, Il6)
• Supplies for adrenalectomy (ADX) or synthetic GC administration (e.g., dexamethasone)

Methodology:

• Animal Entrainment: House mice under a strict 12-hour light/12-hour dark cycle for at least two weeks prior to experimentation.
• GC Manipulation:
  • Adrenalectomy (ADX): Surgically remove adrenal glands to eliminate endogenous GCs. Provide 0.9% saline drinking water. Use sham-operated animals as controls.
  • GC Reconstitution: Administer corticosterone via drinking water to ADX mice to mimic the physiological circadian rhythm.
  • Acute GC Stimulation: Inject a bolus of dexamethasone (e.g., 10 mg/kg) at a specific zeitgeber time (ZT) to observe acute phase-shifting or gene induction.
• Tissue Collection: Sacrifice animals at 4-6 hour intervals over a 24-48 hour period (e.g., ZT0, ZT4, ZT8, ZT12, ZT16, ZT20, where ZT0 is lights on). Rapidly dissect target tissues and freeze in liquid nitrogen.
• RNA Extraction and qPCR: Extract total RNA, synthesize cDNA, and perform quantitative real-time PCR (qPCR) using gene-specific primers. Use housekeeping genes (e.g., Gapdh, Actb) for normalization.
• Serum Corticosterone Measurement: Collect trunk blood at each time point. Separate serum and measure corticosterone levels using a commercial ELISA kit to confirm the rhythm.
• Data Analysis: Normalize expression data using the 2^–ΔΔCt method. Plot values over time to visualize circadian oscillations. Compare phase, amplitude, and mesor between experimental groups using cosinor or similar rhythm analysis software.

Chromatin Immunoprecipitation (ChIP) Assay for GR-DNA Binding

Objective: To determine direct binding of the glucocorticoid receptor (GR) to specific genomic regions (e.g., GREs in Per1, Per2, or Cxcl5 promoters) in a time-dependent manner.

Materials:

• Crosslinking reagent (e.g., 1% formaldehyde)
• Cell lysis buffers
• Specific antibody against GR and a control IgG antibody
• Protein A/G magnetic beads
• DNA purification kit
• Primers spanning the putative GRE and a control genomic region

Methodology:

• Cell/Tissue Fixation: At different circadian time points, treat cells (e.g., primary hepatocytes or macrophages) or mince fresh tissue and crosslink DNA and associated proteins with formaldehyde.
• Cell Lysis and Sonication: Lyse cells and shear DNA by sonication to fragment sizes of 200–500 base pairs.
• Immunoprecipitation: Incubate the chromatin lysate with an anti-GR antibody or control IgG overnight. Capture the antibody-protein-DNA complexes using Protein A/G magnetic beads.
• Washing, Elution, and Reverse Crosslinking: Wash beads stringently, elute the complexes, and reverse the crosslinks by heating to free the DNA.
• DNA Purification and qPCR Analysis: Purify the immunoprecipitated DNA and analyze by qPCR using primers specific for the genomic region of interest. Enrichment is calculated as a percentage of input or relative to the IgG control.

The Scientist's Toolkit: Key Research Reagents

Table 4: Essential Reagents for Investigating GC-Circadian Crosstalk

Reagent / Tool	Function / Specificity	Example Application
Dexamethasone	Synthetic GR agonist; highly potent and resistant to degradation.	Studying acute GR signaling, phase-resetting peripheral clocks in vitro and in vivo.
Mifepristone (RU-486)	GR antagonist.	Blocking GR activity to determine the necessity of GC signaling in a circadian process.
REV-ERB Agonists (e.g., SR9009)	Potent and specific activators of REV-ERB nuclear receptors.	Probing the role of the auxiliary clock loop; testing anti-inflammatory and metabolic effects.
Anti-GR Antibody	For Western Blot, Immunofluorescence, and Chromatin Immunoprecipitation (ChIP).	Detecting GR protein levels, localization, and its binding to genomic targets.
CRISPR/Cas9 System	For targeted knockout of clock genes (e.g., BMAL1, REV-ERBα).	Generating cell lines or animal models with disrupted clock function in specific tissues.
Corticosterone ELISA Kit	Quantifies corticosterone levels in serum or cell culture media.	Verifying the circadian rhythm of endogenous GCs and the efficacy of ADX.
PER2::LUCIFERASE Reporter Cells	Real-time bioluminescent reporting of Per2 gene expression.	Live monitoring of circadian rhythm phase and period in response to GC treatment.

The interpretation of glucocorticoid signals by local circadian clocks is a fundamental biological process that ensures temporal coordination of physiology across tissues. As detailed in this whitepaper, the liver, skeletal muscle, and immune cells each possess unique molecular architectures that decode the systemic GC rhythm into tissue-appropriate temporal gene expression, metabolic flux, and functional readiness. Disruption of this dialogue, through genetic, environmental, or pathological means, is a key contributor to the etiology of modern diseases.

Future research must focus on deconvoluting the complex, multi-tissue networks controlled by the HPA axis and the circadian system. The development of tissue-specific GR and clock gene knockout models, combined with multi-omics approaches (transcriptomics, metabolomics, epigenomics) across circadian time, will be crucial. For drug development, these insights underscore the promise of chronotherapy—timing drug administration to coincide with peak target activity and minimal toxicity. Furthermore, targeting specific nodes of the clock network, such as with REV-ERB agonists, offers a novel strategy to treat metabolic and inflammatory disorders by realigning dysfunctional circadian physiology. A deep understanding of tissue-specific clock responses to GCs will thus pave the way for more precise and effective therapeutic interventions.

Conclusion

The circadian rhythm of the HPA axis is a cornerstone of systemic physiology, orchestrating metabolic, immune, and cardiovascular functions through the precise temporal release of ACTH and glucocorticoids. Disruption of this rhythm is not merely a symptom but a driving force in pathologies ranging from Cushing syndrome to metabolic disorders and immune dysfunction. The integration of advanced methodological approaches—including dynamic testing and mechanistic modeling—with a deeper understanding of molecular clockwork provides unprecedented opportunities for targeted therapies. Future research must focus on translating these insights into clinical practice, developing chronotherapeutic strategies that restore circadian HPA axis function to improve patient outcomes in a wide spectrum of diseases. The emerging frontier of tissue-specific clock manipulation holds particular promise for precision medicine in endocrine and metabolic disorders.

References

• 1. Circadian rhythms revealed: unraveling the genetic ... [https://www.frontiersin.org/journals/sleep/articles/10.3389/frsle.2025.1544945/full]
• 2. Brain circadian clocks timing the 24h rhythms of behavior [https://www.nature.com/articles/s44323-025-00030-8]
• 3. Translational implications of circadian activity alterations in an ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12457307/]
• 4. Glucocorticoid signalling coordinating circadian rhythms ... [https://www.explorationpub.com/Journals/eemd/Article/101437]
• 5. Circadian glucocorticoids throughout development - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC10166844/]
• 6. Circadian rhythm of adrenal glucocorticoid: Its regulation ... [https://www.sciencedirect.com/science/article/pii/S0925443911000329]
• 7. Circadian neurons in the paraventricular nucleus entrain ... [https://www.nature.com/articles/s41467-021-25959-9]
• 8. Effects of DNA methylation inhibitors on light-induced ... [https://pubmed.ncbi.nlm.nih.gov/40893908/]
• 9. Nanomaterial-enabled drug delivery systems for circadian ... [https://pubs.rsc.org/en/content/articlehtml/2025/ra/d5ra04137f]
• 10. Programmable chronogenetic gene circuits for self ... [https://www.sciencedirect.com/science/article/pii/S0168365925005796]
• 11. An Integrative Approach to HPA Axis Dysfunction [https://www.sciencedirect.com/science/article/pii/S0002934325003535]
• 12. Endocrine regulation of circadian rhythms [https://www.nature.com/articles/s44323-025-00024-6]
• 13. Hypothalamus-pituitary-adrenal and gut-brain axes in ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11839829/]
• 14. Ceritinib inhibits growth and ACTH production of PitNETs [https://pubmed.ncbi.nlm.nih.gov/41083089/?utm_source=FeedFetcher&utm_medium=rss&utm_campaign=None&utm_content=0cX95YVZ6ynVlRaGYOB-MpN3R2ukZeaNqMRUCVU64PK&fc=None&ff=20251119090247&v=2.18.0.post22+67771e2]
• 15. Article The circadian rhythm of glucocorticoids is regulated ... [https://www.sciencedirect.com/science/article/pii/S1550413106002415]
• 16. Glucocorticoids Regulate Circadian Rhythm of Innate and ... [https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2020.02143/full]
• 17. Adrenocorticotropic Hormone ACTH Market Report 2025 ... [https://www.cognitivemarketresearch.com/adrenocorticotropic-hormone-acth-market-report]
• 18. Mathematical modeling of mammalian circadian clocks ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC8855863/]
• 19. Progress in understanding how clock genes regulate aging ... [https://pubmed.ncbi.nlm.nih.gov/41064656/]
• 20. Progress in understanding how clock genes regulate aging ... [https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2025.1654369/full]
• 21. A mathematical model of circadian rhythms and dopamine [https://tbiomed.biomedcentral.com/articles/10.1186/s12976-021-00139-w]
• 22. Physiology, Adrenocorticotropic Hormone (ACTH) - StatPearls [https://www.ncbi.nlm.nih.gov/books/NBK500031/]
• 23. Altered association between cortisol and ... [https://www.frontiersin.org/journals/endocrinology/articles/10.3389/fendo.2025.1418357/full]
• 24. Regulatory links between the circadian clock and stress ... [https://www.nature.com/articles/s44323-025-00036-2]
• 25. The ACTH test fails to diagnose adrenal insufficiency and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12016919/]
• 26. Morning clock gene expression in young adults of early ... [https://www.nature.com/articles/s41598-025-12423-7]
• 27. Coping with the multifaceted and multifunctional role of ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12244002/]
• 28. Phase-dependent resetting of the adrenal clock by ACTH in vitro [https://pmc.ncbi.nlm.nih.gov/articles/PMC3949112/]
• 29. Phase-Dependent Shifting of the Adrenal Clock by Acute ... [https://www.frontiersin.org/journals/endocrinology/articles/10.3389/fendo.2016.00081/full]
• 30. Glucocorticoids as Entraining Signals for Peripheral Circadian ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC3512018/]
• 31. Chronopharmacology of glucocorticoids [https://www.sciencedirect.com/science/article/abs/pii/S0169409X19300201]
• 32. Atumelnant, Oral ACTH Antagonist [https://crinetics.com/pipeline/atumelnant-oral-acth-antagonist/]
• 33. Release Details - Neurocrine Biosciences [https://neurocrine.gcs-web.com/news-releases/news-release-details/neurocrine-biosciences-present-wide-ranging-one-year-data-phase]
• 34. ACTH Action on the Adrenals - Endotext - NCBI Bookshelf [https://www.ncbi.nlm.nih.gov/books/NBK279118/]
• 35. Dissociation of ACTH and glucocorticoids [https://www.sciencedirect.com/science/article/abs/pii/S104327600800057X]
• 36. Sleep and Circadian Regulation of Cortisol: A Short Review [https://pmc.ncbi.nlm.nih.gov/articles/PMC8813037/]
• 37. Cortisol on Circadian Rhythm and Its Effect on Cardiovascular ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC7830980/]
• 38. Sleep and circadian regulation of cortisol: A short review [https://www.sciencedirect.com/science/article/abs/pii/S2451965021000363]
• 39. ACTH (Cosyntropin) Stimulation Test: What It Is & Results [https://my.clevelandclinic.org/health/diagnostics/24143-acth-stimulation-test]
• 40. ACTH Stimulation Test (Cosyntropin) [https://www.labcorp.com/test-menu/resources/acth-stimulation-test]
• 41. A mechanistic modeling framework to interpret ACTH ... [https://www.sciencedirect.com/science/article/abs/pii/S0010482525015264]
• 42. ACTH Stimulation Test | Medical Examination [https://www.cincinnatichildrens.org/health/a/acth-test]
• 43. ACTH stimulation test: MedlinePlus Medical Encyclopedia [https://medlineplus.gov/ency/article/003696.htm]
• 44. Redefining the Diagnostic Approach to Adrenal Insufficiency [https://pmc.ncbi.nlm.nih.gov/articles/PMC12298193/]
• 45. ACTH Stimulation Test - Endocrine Surgery [https://www.uclahealth.org/medical-services/surgery/endocrine-surgery/patient-resources/patient-education/endocrine-surgery-encyclopedia/acth-stimulation-test]
• 46. Chronic ACTH Infusion Alters the Diurnal Rhythm of ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12440288/]
• 47. 解读ACTH：高血压与肾上腺功能的新视角 - ByDrug - 医药魔方 [https://bydrug.pharmcube.com/news/detail/5cdc92851dae8adc23793fbc090048ce]
• 48. 【综述】垂体功能减退的内分泌诊断和替代治疗 [https://www.brainmed.com/info/detail?id=22054]
• 49. SCCM 2008成人重症肾上腺皮质功能不全的诊断共识的解读 [https://ccdas.pmphai.com/appguide/toPcDetail?sessionId=&knowledgeLibPrefix=guide&id=0001AA100000000EGDNR]
• 50. 外源性雌二醇对垂体功能不全女性ACTH刺激下血浆皮质醇与唾液 ... [https://www.ebiotrade.com/newsf/2025-8/20250819000325104.htm]
• 51. MLS Hormone Catalog CHINESE - Life Science [https://collateral.meridianlifescience.com/view/423748785]
• 52. CN118845788A - 促肾上腺皮质激素释放因子受体拮抗剂 [https://patents.google.com/patent/CN118845788A/zh]
• 53. 肾上腺影像学对原发性醛固酮增多症亚型诊断的准确性 [https://lcmw.whuhzzs.com/article/id/67ea8506785599110d8160cc]
• 54. The Impact of Circadian Rhythms on Medication Timing ... [https://www.mdpi.com/2624-5175/6/4/43]
• 55. Time matters: chrono-pharmacotherapy as precision medicine [http://ajpt.asmepress.com/articles/new-75-268.html]
• 56. The clinical impact of chronopharmacology on current ... [https://pubmed.ncbi.nlm.nih.gov/39792169/]
• 57. Time-of-day effects of cancer drugs revealed by high- ... [https://www.nature.com/articles/s41467-024-51611-3]
• 58. The clinical impact of chronopharmacology on current ... [https://link.springer.com/article/10.1007/s00210-025-03788-7]
• 59. ChronobioticsDB: The Database of Drugs and Compounds ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12286229/]
• 60. The Physiological and Pharmacological Significance of ... [https://www.sciencedirect.com/science/article/abs/pii/S0022354923003246]
• 61. A mathematical modeling approach - PMC - PubMed Central [https://pmc.ncbi.nlm.nih.gov/articles/PMC10840710/]
• 62. Replication of cortisol circadian rhythm: new advances in ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC3475279/]
• 63. VeVaPy, a Python Platform for Efficient Verification and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9777964/]
• 64. High-fidelity discrete modeling of the HPA axis - PubMed Central [https://pmc.ncbi.nlm.nih.gov/articles/PMC6050677/]
• 65. Machine Learning-based prediction of HPA axis reactivity ... [https://www.sciencedirect.com/science/article/pii/S0306453025002513]
• 66. Circadian Biomarkers in Humans: Methodological Insights ... [https://www.mdpi.com/2218-273X/15/7/1006]
• 67. Circadian Rhythm: Biological Functions, Diseases, and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12547082/]
• 68. Methodological Insights into the Detection of Melatonin and ... [https://pubmed.ncbi.nlm.nih.gov/40723877/]
• 69. Performance of Blood-Based Biomarkers for Human Circadian ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12572356/]
• 70. Blood Clock Correlation Distance (BloodCCD) as a novel ... [https://www.nature.com/articles/s44276-025-00176-9]
• 71. Editorial: Circadian rhythm in adrenal endocrinology [https://www.frontiersin.org/journals/endocrinology/articles/10.3389/fendo.2025.1620570/full]
• 72. Cushing Syndrome, Hypercortisolism, and Glucose ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12645169/]
• 73. Circadian clock disruption impairs immune oscillation in ... [https://www.sciencedirect.com/science/article/pii/S2352396424004985]
• 74. The Impact of Circadian Misalignment on Blood Pressure - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC10916535/]
• 75. Circadian Rhythm Disruption Presenting as Adrenal ... [https://www.thieme-connect.com/products/ejournals/html/10.1055/s-0045-1812290]
• 76. Metabolic “Pressure” on the Adrenal Gland? - PubMed Central [https://pmc.ncbi.nlm.nih.gov/articles/PMC12645164/]
• 77. Chronic ACTH Infusion Alters the Diurnal Rhythm of ... [https://pubmed.ncbi.nlm.nih.gov/40768517/]
• 78. Effect of chronic ACTH infusion on blood pressure and kidney ... [https://datashare.ed.ac.uk/handle/10283/9044]
• 79. circadian Glucocorticoid in rhythms function immune [https://pubmed.ncbi.nlm.nih.gov/34580744/]
• 80. Glucocorticoid Insensitivity: Is It a Question of Time and ... [https://www.mdpi.com/2227-9059/13/6/1418]
• 81. Cell-specific expression and signaling of glucocorticoid ... [https://ccforum.biomedcentral.com/articles/10.1186/s13054-025-05628-9]
• 82. Immunophenotypic Implications of Reverse-Circadian ... [https://www.mdpi.com/1422-0067/26/4/1479]
• 83. sciencedirect.com/science/article/pii/S2319417025000010 [https://www.sciencedirect.com/science/article/pii/S2319417025000010]
• 84. Iatrogenic Cushing Syndrome [https://emedicine.medscape.com/article/117365-overview]
• 85. Glucocorticoids Regulate Circadian Rhythm of Innate and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC7533542/]
• 86. Treating the Side Effects of Exogenous Glucocorticoids [https://www.endocrine.org/journals/endocrine-reviews/treating-the-side-effects-of-exogenous-glucocorticoids]
• 87. Inhalational Steroids and Iatrogenic Cushing's Syndrome - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC4319196/]
• 88. Cushing Syndrome: Causes, Symptoms & Treatment [https://my.clevelandclinic.org/health/diseases/5497-cushing-syndrome]
• 89. Cushing Disease - StatPearls - NCBI Bookshelf - NIH [https://www.ncbi.nlm.nih.gov/books/NBK448184/]
• 90. Drug design strategies for Cushing's syndrome [https://pubmed.ncbi.nlm.nih.gov/30572739/]
• 91. Drug development and potential targets for Cushing's ... [https://www.sciencedirect.com/science/article/abs/pii/S0223523424002137]
• 92. Hypothalamic-Pituitary-Adrenal (HPA) Axis: What It Is [https://my.clevelandclinic.org/health/body/hypothalamic-pituitary-adrenal-hpa-axis]
• 93. the hypothalamic–pituitary–adrenal axis and its actions - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC4104011/]
• 94. Suprachiasmatic Nuclei Possess Glucocorticoid Receptors ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12662659/]
• 95. Circadian Rhythm Sleep Disorder Market Sees Digital Shift [https://www.towardshealthcare.com/insights/circadian-rhythm-sleep-disorder-market-sizing]
• 96. Peripheral clocks and systemic zeitgeber interactions [https://pmc.ncbi.nlm.nih.gov/articles/PMC12158691/]
• 97. Neurobiology of the circadian clock and its role in ... [https://www.sciencedirect.com/science/article/pii/S2451994425000203]
• 98. Neurocrine Biosciences Presents One-Year Data Showing ... [https://www.prnewswire.com/news-releases/neurocrine-biosciences-presents-one-year-data-showing-sustained-efficacy-of-crenessity-crinecerfont-in-adult-patients-at-endo-2025-302503650.html]
• 99. A mechanistic modeling framework to interpret ACTH ... [https://pubmed.ncbi.nlm.nih.gov/41061389/]
• 100. Regulation of the hypothalamic-pituitary-adrenocortical stress ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC4867107/]
• 101. Adrenocorticotropic Hormone (ACTH) Test [https://www.testing.com/tests/adrenocorticotropic-hormone-acth/]
• 102. 004440: Adrenocorticotropic Hormone (ACTH), Plasma [https://www.labcorp.com/tests/004440/adrenocorticotropic-hormone-acth-plasma]
• 103. ACTH - Overview: Adrenocorticotropic Hormone, Plasma [https://www.mayocliniclabs.com/test-catalog/overview/8411]
• 104. Daily glucocorticoids promote glioblastoma growth and ... [https://www.sciencedirect.com/science/article/pii/S1535610824004471]
• 105. Dynamic output and control of the hypothalamic-pituitary ... [https://www.sciencedirect.com/science/article/pii/S0007091217323917]
• 106. The Functional and Clinical Significance of the 24-Hour ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC5563520/]
• 107. Physiology, Glucocorticoids - StatPearls - NCBI Bookshelf [https://www.ncbi.nlm.nih.gov/books/NBK560897/]
• 108. Glucocorticoids: restoring balance during stress [https://www.endocrinology.org/endocrinologist/130-winter18/features/glucocorticoids-restoring-balance-during-stress/]
• 109. Exploring the Molecular Mechanisms of Glucocorticoid ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC4770453/]
• 110. Induction of internal circadian desynchrony by misaligning ... [https://www.nature.com/articles/s41598-022-05624-x]
• 111. Circadian Rhythms and Melatonin Metabolism in Patients With ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC8959415/]
• 112. The Crosstalk between Melatonin and Sex Steroid Hormones [https://pubmed.ncbi.nlm.nih.gov/33774638/]
• 113. Circadian control of the immune system - PMC - PubMed Central [https://pmc.ncbi.nlm.nih.gov/articles/PMC4090048/]
• 114. Evolution of time-keeping mechanisms: early emergence and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC3130368/]
• 115. Circadian rhythm regulates the function of immune cells ... [https://www.nature.com/articles/s41420-024-01960-1]
• 116. Circadian Clock: A Regulator of Immunity in Autoimmune ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12412416/]
• 117. Circadian rhythms and inflammatory diseases of the liver ... [https://www.sciencedirect.com/science/article/pii/S2542568423000430]
• 118. Circadian rhythms in muscle health and diseases [https://www.sciencedirect.com/science/article/abs/pii/S1937644824001539]