Current Challenges and Future Directions in the Assessment of Glucocorticoid Status

Article type: Review Article

Keywords: adrenal, pituitary gland, Addison’s disease, steroids, glucocorticoid

Authors: Sophie A Clarke, Pei Chia Eng ⚬, Alexander N Comninos ⚬, Katharine Lazarus ⚬, Sirazum Choudhury ⚬, Christie Tsang, Karim Meeran ⚬, Tricia M Tan ⚬, Waljit S Dhillo ⚬, Ali Abbara ⚬

Affiliations: Section of Investigative Medicine, Imperial College London, London W12 ONN, UK; Department of Endocrinology, Imperial College Healthcare NHS Trust, London W6 8RF, UK; Department of Endocrinology, National University of Singapore, Singapore

License: © The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society. CC BY 4.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. See the journal About page for additional terms.

Article links: DOI: 10.1210/endrev/bnae016  |  PubMed: 38795365  |  PMC: PMC11581704

Relevance: Moderate: mentioned 3+ times in text

Glucocorticoid (GC) hormones are secreted in a circadian and ultradian pattern (ref. 1, ref. 2) and are critical for maintaining physiological homeostasis (ref. 2). Both excess and insufficient GC levels are associated with adverse health outcomes (ref. 3, ref. 4). However, symptoms and signs of GC dysfunction may only match GC status when perturbations are chronic and extreme, ie, markedly low or high cortisol levels.

Current assessment of GC status relies on clinical features in combination with measurement of cortisol levels, either in the basal state or stimulated/suppressed according to the GC disturbance being assessed. In addition, salivary and urinary cortisol may also be used. However, the accuracy of a single cortisol measure to reflect GC status is challenged by intra- and interindividual variation in cortisol concentrations, the pulsatile nature of cortisol secretion (ref. 5), variations in corticosteroid-binding globulin (CBG), and local tissue concentrations dependent on 11beta-hydroxysteroid dehydrogenase (11β-HSD) activity, as well as GC receptor sensitivity. Thus, several factors can influence individual sensitivity to GC (ref. 6).

Furthermore, GC status is not necessarily dichotomous (ie, either being unequivocally low or high), but rather there can exist a gradation in the degree of abnormality between insufficient and excess GC status, challenging confirmation of GC status. Moreover, GC status could be incongruent from measured cortisol levels or challenging in the setting of (1) concomitant use of exogenous steroid treatments (eg, inhalers or creams) that could suppress endogenous cortisol levels but still contribute to GC status; (2) assessment for Cushing’s syndrome (CS) with incongruent results of investigations (eg, due to factors such as altered renal function affecting reliability of 24-hour urine collections); (3) early presentation of adrenal insufficiency (AI) with residual adrenal function whereby cortisol levels are not absent and cortisol response to stimulation tests may still be normal; (4) suboptimal GC replacement regimens whereby signs and symptoms of GC deficiency/excess can be nuanced; (5) “non-neoplastic hypercortisolism” whereby abnormal investigations may be equivocal/misleading; (6) acute illness whereby cortisol metabolism/clearance and levels are altered; (7) concomitant use of medications that could alter liver metabolism of GC (eg, affecting the response to a dexamethasone suppression test); (8) serum cortisol levels that are falsely elevated (eg, due to increased CBG as a result of oral estrogen treatment).

Herein, we discuss the challenges in the diagnosis/management of GC deficiency/excess and aim to evaluate novel markers that could be used in the objective assessment of GC status in the future that do not rely solely on the measurement of circulating cortisol levels (Fig. 1).

The Hypothalamic-pituitary-adrenal Axis in Health

In healthy people who sleep at night, cortisol is released in both an ultradian (1 to 2 hourly cortisol pulses) (ref. 1) and circadian rhythm rising from ∼04:00 Am (ref. 7), peaking within 3 hours after waking (cortisol awakening response), before subsequently declining and reaching a nadir at around midnight (ref. 7). The frequency and amplitude of cortisol secretion are crucial to ensure synchronization of endogenous cellular rhythms to external cues (ref. 8). Clock genes in peripheral cells are responsible for circadian timekeeping with the activity of the suprachiasmatic nucleus and are modulated in the presence of external cues (eg, light, feeding, temperature) (ref. 8). Notably, acetylation of GC receptor by CLOCK/BMAL1 transcription factors is increased in the morning, leading to decreased cortisol tissue sensitivity (ref. 9).

The diurnal rhythm of circulating cortisol concentrations plays an important role in sleep timing and distribution of sleep stages throughout the night (ref. 10). However, the relationship is bidirectional with sleep onset and sleep disruption also influencing the release of cortisol (ref. 10). Sleep onset inhibits the hypothalamic-pituitary-adrenal (HPA) axis, typically within the first 20 minutes after slow-wave sleep with cortisol reaching a low level in the first half of the night (ref. 10). In the second half of the night, rapid eye movement sleep predominates, and inhibitory mechanisms are attenuated, which is associated by an increase in HPA activity (ref. 10). Nocturnal awakenings at the end of sleep are associated with transient release of cortisol, followed by temporary inhibition of cortisol secretion (ref. 10). Acute changes in the sleep-wake cycle (such as shiftwork or jetlag) lead to HPA axis activation and alter the normal circadian pattern of cortisol secretion (ref. 10). Some (ref. 11), but not all (ref. 12), chronic shift workers are reported to have a delay in the early morning cortisol rise. Prolonged exposure to high cortisol levels is associated with reduced glucose tolerance and insulin sensitivity (ref. 13).

Currently, disturbance of GC status is assessed using basal or stimulated/suppressed cortisol levels depending on the GC disturbance being assessed (Fig. 1). Moreover, ACTH is pulsatile and variable such that it may not be reliable as a marker of GC status in isolation especially if the disturbance in GC status is not at the extreme end of being very high or very low (ref. 2).

Factors Affecting Circulating Cortisol Concentration

Measurement of cortisol concentrations can be affected by several factors including differences in age, sex, time of collection, assay factors, and variation in binding protein levels.

Variation in Corticosteroid Binding Globulin

CBG is predominantly produced by the liver and has a high affinity for cortisol (ref. 14). Approximately 90% of serum cortisol is bound to CBG, 4% to 6% is bound to other binding proteins (eg, albumin or alpha-1-glycoprotein), and 4% to 6% circulates freely (ref. 14, ref. 15). Cortisol binding to CBG is a major determinant of bioavailability; thus, interindividual variability of binding to CBG may affect the measurement of total cortisol levels and dynamic tests (ref. 14, ref. 15), although salivary cortisol and urinary free cortisol are not affected by alterations in CBG (ref. 16). Synthetic GCs, including prednisolone and dexamethasone, bind CBG with less affinity than cortisol (ref. 15, ref. 17). Other factors affecting CBG include sex, temperature, glycosylation states, critical illness, genetic variants, medications, and insulin sensitivity (ref. 14, ref. 18).

Of clinical relevance, low albumin states (albumin < 25 g/L), as encountered in critical illness, are associated with lower total cortisol levels (435 vs 633 nmol/L) but similar active free cortisol levels (140 vs 143 nmol/L) (ref. 19). Likewise, nephrotic syndrome results in renal CBG loss and thus lower total cortisol levels and thus could lead to a false diagnosis of AI after a tetracosactide test (tetracosactide is amino acid 1-24 of the 36 amino acid structure of ACTH, commonly marketed as Synacthen®) (ref. 20). Conversely, exogenous oral estrogens (as well as high circulating levels of estrogens in pregnancy) increase CBG, resulting in a falsely elevated total cortisol (ref. 21).

Age and Cortisol Secretion

Age influences endogenous cortisol secretion and diurnal rhythmicity; increasing age leads to a reduction in the diurnal amplitude of cortisol secretion (ref. 22, ref. 23), an advancement of the early morning cortisol rise (ref. 24), a reduction in the amplitude of cortisol pulses, and an increase in serum cortisol nadir. However, age-specific cut-offs are typically not used in clinical practice.

Effect of Sex and the Menstrual Cycle

Cortisol levels vary between men and women, with additional differences due to the menstrual cycle and menopause (ref. 25). Men have higher cortisol output with higher mean peak, nadir, and cortisol awakening response than premenopausal women (ref. 26, ref. 27). The peak cortisol rise (acrophase) increases gradually with age in women but not in men, such that there is no longer a difference after menopause (ref. 25).

Studies evaluating cortisol responses at different stages of the menstrual cycle have shown discordant results. Some studies have not shown any difference in mean salivary cortisol levels across the menstrual cycle (ref. 28, ref. 29), while other studies have shown higher circulating cortisol levels in the follicular phase (ref. 30). A higher stimulated cortisol response following tetracosactide has been reported in the luteal phase (ref. 29, ref. 31). Such factors could cause a discrepancy between cortisol levels and GC action, and thus markers of GC action could more accurately reflect GC status in these situations.

Single Cortisol Value in Reflection of GC Status

The variation in plasma cortisol levels with time-of-day challenges interpretation of a single value taken at a random time. Early morning cortisol levels are generally high, and levels in patients who are asleep at midnight are usually very low. A major challenge in the interpretation of serum and stimulated cortisol levels is the lack of assay-specific normative data in specific population groups (ref. 32, ref. 33). Historically, serum cortisol was measured using a nonspecific fluorometric assay that measures both cortisol and corticosterone (ref. 34). Due to the poor specificity and low sample throughput of this method (ref. 35), cortisol measurement commonly is undertaken using automated cortisol immunoassays (ref. 36) with each clinical laboratory establishing its own performance characteristics and specificity for cortisol (ref. 33). Agreement between different immunoassays vary (ref. 37); according to some studies, levels can be comparable to those of mass spectrometry (ref. 38), while others have reported higher cortisol levels using immunoassay than mass spectrometry (ref. 39–41). A study comparing automated immunoassays [Advia Centaur (Siemens), Architect (Abbott), Modular Analytics E170 (Roche), Immulite 2000 (Siemens), and Access (Beckman)] with gas chromatography-mass spectrometry showed a bias of 1.08 to 1.36 in immunoassay-measured cortisol levels (ref. 32). In addition to the analytical bias among immunoassays, sex-specific normative cortisol responses in healthy individuals remain undefined (ref. 33). Direct cortisol immunoassays may be susceptible to cross-reactivity with other structurally similar compounds, in particular prednisolone and 11-deoxycortisol (ref. 42).

This difference among cortisol immunoassays ultimately affects the interpretation of GC status (ref. 42). The discrepancy between serum cortisol levels measurement between mass spectrometry and automated immunoassays (12.6% to 90.8%) can be greater in patients receiving treatment for CS e.g. metyrapone owing to the cross-reactivity of structural cortisol analogs in the steroidogenesis pathway (ref. 43). Mass spectrometry quantifies exogenous GC (ref. 44) and endogenous GC metabolites (ref. 45) simultaneously (ref. 43), and recent developments have improved sensitivity and reliability (ref. 46), allowing novel insights into GC processing to be investigated, although mass spectrometers are not ubiquitously available (ref. 47).

In view of the various factors that interfere with cortisol levels, measurement of serum-free cortisol (5% of total) was proposed to have better clinical utility in patients with total cortisol concentrations near the diagnostic threshold or in states of altered binding globulin (ref. 16). Free cortisol can be calculated after removing bound fractions via a suitable technique either by ultrafiltration, equilibrium dialysis, or gel-filtration chromatography (ref. 39, ref. 48). Although these techniques produce comparable interassay coefficients of variation < 10% (ref. 49), they are labor-intensive and require long incubation times. Surrogate methods (eg, Coolen’s equation) or the free cortisol index have been used to estimate free cortisol levels (ref. 50), but these equations are limited in their assumption of direct proportionality and can be inaccurate in patients with hypoalbuminemia or altered CBG capacity (ref. 51, ref. 52).

Thus, the validity of using cortisol response to tetracosactide test to diagnose AI can be influenced by assay performance and alterations in CBG (ref. 32, ref. 33). The widely used cortisol cut-offs of 500 (ref. 53) or 550 nmol/L (ref. 54) in response to a tetracosactide test could result in misclassification of AI (12%,19%, 4%, and 9%) for the Centaur, Architect, Immulite (2000), and Access assays, respectively, at 500 nM; 27%, 42%, 16%, and 21% for the respective assays at 550 nM (ref. 32), whereas patients with a morning (8:00 Am to 12:00 Pm) serum cortisol (using an ultrasensitive immunoassay on an Abbott Architect platform) of >275 nmol/L (96% sensitivity) are unlikely to have an insufficient response to tetracosactide (ref. 55).

Moreover, GC insufficiency can exist over a continuum between complete lack of cortisol and partial deficiency/inability to mount an adequate stress response, and conversely cortisol values marginally below the lower reference limit may not necessarily reflect GC inadequacy. Therefore, other parameters reflecting GC action could provide useful information in addition to clinical judgment in the assessment of GC status.

Current Assessment of Altered GC States

GC Insufficiency

AI is characterized by a failure to secrete sufficient GC to meet physiological demands. Primary adrenal insufficiency (PAI) accounts for 40% of AI (ref. 56), resulting from destruction of adrenal cortical cells (typically due to an autoimmune process or infection, eg, tuberculosis), or adrenal enzymatic failure of GC synthesis (eg, congenital adrenal hyperplasia) (ref. 4, ref. 57). Secondary adrenal insufficiency (SAI) occurs due to impaired ACTH production, eg, due to a pituitary adenoma or traumatic brain injury (ref. 56). Tertiary AI occurs due to impaired hypothalamic corticotrophin releasing hormone secretion, usually due to chronic exogenous GC use (ref. 4). In patients with AI, GC replacement is used to avoid the life-threatening complication of adrenal crisis and to optimize well-being (ref. 58). Patients with PAI also usually require additional mineralocorticoid therapy (ref. 59).

Clinical and biochemical assessment of GC insufficiency

Symptoms of AI depend on the degree of cortisol, mineralocorticoid, and adrenal androgen deficiency at the time of presentation and can be nonspecific, which can lead to delayed diagnosis (ref. 60). Clinical presentation and biochemical changes seen in patients with AI are presented in Table 1 and of GC excess in Table 2. Dynamic endocrine tests used in the diagnosis of both GC deficiency and GC excess and their associated challenges are summarized in Table 3.

Assessment of GC status in AI with replacement therapy

Patients with AI, including both primary and secondary, have increased mortality rates (ref. 96), attributed to excess GC exposure at nonphysiological times and adrenal crises (ref. 97). GC excess is associated with metabolic dysfunction, including hypertension, dyslipidemia, immune suppression (ref. 98), hyperglycemia, and osteoporosis (ref. 3). Optimizing replacement therapy for AI, using multidose oral regimens, therefore involves minimizing excess GC exposure and replicating the circadian rhythm (ref. 99); however, to date, there is a paucity of universally accepted methods to ascertain optimal GC replacement. In patients receiving multiple daily-dose hydrocortisone regimens, hydrocortisone day curves can be used predominantly to assess interindividual pharmacokinetic alterations in hydrocortisone metabolism (ref. 58). For those taking low-dose prednisolone (eg, 2-4 mg once daily), mass spectrometry can quantify prednisolone concentrations as low as 10 mg/L (ref. 100), enabling higher resolution day curves with the potential to individualize dosing regimens (ref. 101).

A study evaluating a novel GC replacement regimen highlighted potential measurable pharmacodynamic differences that could be monitored to assess GC status (ref. 99, ref. 102). Once-daily modified release hydrocortisone (dose 16 mg/m²) compared with standard hydrocortisone/cortisone acetate (dose 18 mg/m²) showed differences in body weight, immune profiles, and quality of life scores (ref. 101). Modified-release hydrocortisone was associated with more physiological immune profiles [reversal of the increased number of CD16+ natural killer (NK) cells at baseline] and reduced susceptibility to infections (ref. 101, ref. 103). Similarly, studies evaluating cortisol replacement via pulsatile administration pumps have used functional imaging, quality of life scores (SF-36), and behavioral and cognitive tests to assess GC effects on emotional processing (ref. 104).

GC Excess

Clinical and biochemical assessment of GC excess

Many symptoms of CS are common but nonspecific, while others are more specific but less common (ref. 68). This can be complicated by associated comorbidities such as obesity and hypertension that are commonly found in the general population, as well as sex, age (ref. 105), and the duration and extent of disease (ref. 106). Table 2 summarizes the clinical features and complications observed in CS. More discriminatory features relate to the catabolic state and increased protein breakdown observed secondary to GC excess (ref. 68). The use of facial recognition to diagnose CS is yet to be validated in a clinical setting, but face-classification software correctly classified 85% of patients and 95% of controls (ref. 68). Table 3 outlines 3 biochemical screening tests recommended by the Endocrine Society, where there is a strong pretest probability of a diagnosis of CS (ref. 89). However, the sensitivity and specificity of the tests will depend on the assay and threshold values used. There can also be intraindividual variability, and more than 1 measurement, eg, in the case of urinary free cortisol, is often required to avoid false-negative results (ref. 90). Moreover, in the setting of cyclical CS, results may be concordantly negative and require further evaluation and follow-up (ref. 89). Other methods of measuring cortisol levels include salivary cortisol, whereas hair and nail cortisol could be used to reflect longer term GC exposure.

Salivary Cortisol and Cortisone

Unconjugated cortisol diffuses into the saliva and is converted to cortisone by the enzyme 11β-HSD type 2 (11β-HSD2) (ref. 107). Serum cortisone levels are 4-fold lower than serum cortisol levels, whereas salivary cortisone levels are 6-fold higher than salivary cortisol levels (ratio of salivary cortisone: salivary cortisol = 6:1) (ref. 108). Salivary cortisol and cortisone showed good correlation with serum free cortisol (ref. 108). However, in a study of women treated with estrogens and healthy controls (HCs), salivary cortisone correlated more strongly with serum cortisol levels (Spearman r = +0.95) than salivary cortisol (ref. 109). High levels of salivary cortisol were found for ∼45 minutes after oral dosing of hydrocortisone, likely due to detection of residual hydrocortisone in the oral cavity (ref. 109). However, as hydrocortisone pills do not contain cortisone, salivary cortisone measurement is not affected by oral hydrocortisone, and its level remains correlated with serum free cortisol (ρ = 0.91) in healthy male volunteers (n = 14) after both oral or intravenous hydrocortisone administration (ref. 110). Salivary cortisone is more readily measurable, remaining detectable across the whole 24-hour day, whereas salivary cortisol concentrations can fall below detection limits at low serum cortisol levels (ref. 110).

A recent prospective study demonstrated that waking salivary cortisone measured by liquid chromatography-tandem mass spectrometry could predict cortisol response >430 nmol/L at 30 minutes following tetracosactide (measured by immunoassay) with an area (AUC) of 0.95 [95% confidence interval (CI) 0.92-0.97] (ref. 111). This was greater than the performance of waking salivary cortisol (AUC 0.89, 95% CI 0.85-0.94), which is more susceptible to contamination from oral hydrocortisone from the previous day in a small number of patients (ref. 111). This approach could avoid the need for tetracosactide stimulation in 70% of patients (ref. 111).

Late-night salivary cortisol and cortisone measured by liquid chromatography-tandem mass spectrometry both have high specificity and sensitivity for identifying patients with endogenous CS (ref. 110). A threshold for late-night salivary cortisone of >14.5 nmol/L had a sensitivity of 95.2% and specificity of 100% for identification of CS (ref. 112). Although liquid chromatography-mass spectrometry offers better specificity and comparable sensitivity than antibody-based methods, it is not widely available (ref. 113).

Therefore, salivary cortisone can be used in the screening for AI to assess cortisol circadian rhythm in patients treated with oral hydrocortisone (ref. 110) or to evaluate hydrocortisone replacement in children where blood sampling is undesirable (ref. 114). Three 8-hourly salivary cortisone measurements provided a good estimate of AUC cortisol exposure in HCs (n = 28), patients with AI, or autonomous cortisol secretion (n = 8) and could be used to assess 24-hour cortisol secretion (ref. 115).

Saliva collection is easy for most adults but can be challenging for infants, elderly patients, patients with oral lesions, or patients with Sjogren’s syndrome (ref. 113). Immunoassays used to analyze salivary cortisol/cortisone are inexpensive to run, but there is a potential of cross-reactivity with other steroids, and they are generally not validated for the salivary matrix at the concentrations required (ref. 91, ref. 116, ref. 117).

Hair Cortisol

Hair cortisol analysis has been investigated in the research setting as a potential tool for the assessment of chronic GC excess (ref. 118). While spot cortisol levels in blood, saliva, and urine do not correlate with body mass index (BMI), a 9.8% increment in hair cortisol was associated with a higher BMI by 2.5 kg/m² (ref. 119). Higher levels of cortisol per 1 cm of scalp hair are found in patients with endogenous CS at 679 (range 279-2500) ng/g (n = 6) when compared to HCs of 116 (range 26-204) ng/g (n = 32) (ref. 120). Hair cortisol has also been used to assess the adequacy of GC replacement in patients treated with hydrocortisone for AI (ref. 6). Thus, it could have potential for diagnosis or for follow-up assessment of cyclical CS where a longer term view of GC status may be helpful (ref. 121-124); however, it remains to be validated outside a research setting and should be considered in light of confounding factors such as sex, ethnicity, physical activity, light exposure, and medications used (ref. 125).

Assessing GC Status: Tissue-specific GC Concentrations

Cortisol action in tissues is regulated by the enzyme 11β-HSD and the GC receptor (GR; the gene name is nuclear receptor subfamily 3, group C, member 1; NR3C1).

Role of 11β-HSD

11β-HSD is a bidirectional enzyme that interconverts active cortisol to inactive cortisone (ref. 126). Cortisone is not synthesized in the adrenal gland. However, cortisone is produced in the kidney and gut and is largely dependent on the activity of 11β-HSD2, which inactivates cortisol to cortisone (ref. 127, ref. 128). In metabolic tissues including liver, skeletal muscle, and adipose tissue, the reductase action of 11β-HSD type 1 (11β-HSD1) reactivates cortisol from cortisone, releasing ∼900 pmol of cortisol per 100 g of liver tissue per minute and ∼15 pmol of cortisol per 100 g of adipose tissue per minute, contributing to 30% to 40% of daily cortisol production (ref. 127, ref. 128). The prereceptor interconversion between bioactive cortisol and inactive cortisone regulates the concentration of GC available for binding to the GR (ref. 126) and alters feedback onto the HPA axis (ref. 129).

Overexpression of adipose HSD11B in mice recapitulates features of metabolic syndrome without changes in circulating GC levels (ref. 130). Conversely, knockout of HSD11B in mice improves glucose tolerance, reduces weight, and improves the lipid profile also without affecting circulating cortisol levels (ref. 131). Although 11β-HSD1 enzyme activity in adipose tissue has been associated with insulin resistance and leptin levels (ref. 132, ref. 133), HSD11B mRNA expression in subcutaneous or omental adipose biopsies was not associated with obesity (ref. 134). Genetic variants in the HSD11B gene have been associated with type 2 diabetes (ref. 135, ref. 136) but not with obesity (ref. 135). Patients with CS with a functional deficit in HSD11B are protected from the adverse effects of GC excess (ref. 137).

Clinical trials using nonselective 11β-HSD inhibition have shown improved insulin sensitivity in patients with type 2 diabetes (ref. 138). A selective 11β-HSD1 inhibitor, ICNB13739, administered to patients with type 2 diabetes significantly lowered body weight and plasma glucose levels (ref. 139). In a recent proof-of-concept study in 32 men, a potent competitive 11β-HSD1 inhibitor, AZD4017, mitigated the metabolic side effects of prednisolone treatment by preventing worsening of hepatic insulin sensitivity, nocturnal blood pressure elevation, lipid metabolism, and bone turnover (preventing the fall in osteocalcin and N-terminal propeptide of type I collagen with prednisolone) while maintaining its desired anti-inflammatory action (assessed using the OX40 assay) (ref. 140).

These demonstrate that variation in 11β-HSD1 activity could alter GC action without necessarily being reflected in circulating GC levels. Thus, manipulation of 11β-HSD1 offers an attractive prospect to modulate GC action; quantifying the extent to which local cortisol is lowered in different tissues for therapeutic benefit while avoiding insufficient GC action could benefit from novel markers reflecting GC action (ref. 141).

Variability in the Response of GC Receptor

The GR is comprised of 777 amino acids and is expressed in almost all human tissues and organs (ref. 142). Alternative splicing of NR3C1 precursor mRNA yields several GR splice elements that modulate GR function and affinity to GC, with the most abundant splice elements being GRα and GRβ (ref. 143). GC predominantly binds to GRα, whereas GRβ, being constitutively nuclear, can represses GRα-induced activity (ref. 143). Prior to ligand binding, GR predominantly resides in the cytoplasm and exists as a complex bound to 2 molecules of heat shock protein 90 (HSP90) and several other proteins (ref. 143). Upon binding to GC, GR undergoes conformational change, dissociates from HSP90, and is translocated to the nucleus (ref. 144) (Fig. 2). The ligand-bound GR dynamically interacts with GC responsive elements (GRE) or GRE half-sites in the promoter region of target genes or indirectly with other transcription factors to exert genomic effects (ref. 145). Variability in the GR response can therefore be influenced by GR expression level, the ligand-binding affinity of GR, posttranslational changes, the type of GREs, and the level of interaction with various coactivators or repressors (ref. 146).

Polymorphisms in the GR gene result in impaired function of GR as a transcriptional activator or repressor. Indeed, there is interindividual variation suggesting that there is variation in the set-point of the HPA axis. Genetic polymorphisms, such as N363S (rs6195), are associated with increased GC action, increased BMI, and reduced bone mineral density (ref. 148). Similarly, individuals with BclI polymorphism (rs41423247) also exhibit increased GC sensitivity, characterized by increased central obesity, insulin resistance, hypertension, and depression (ref. 149). By contrast, carriers of ER22/23EK (rs6189 and rs6190) polymorphism had reduced GC action and lower BMI and cardiovascular risk (ref. 149). Individuals who were homozygous in the intronic region of PDGFD locus (rs591118) are more likely to develop adrenal suppression following treatment with GC compared to those who are homozygous wild-type (ref. 150). Thus, variability in the action of the GR can affect the GC action beyond alterations in circulating GC levels, and in vivo and in vitro assessment of GC sensitivity has been proposed (ref. 6).

Body Weight/Appetite

Changes in body weight can reflect alterations in GC action. GC deficiency is associated with weight loss due to anorexia as well as salt and water depletion (ref. 64). Adrenalectomy in rodents is associated with decreased food intake, which is reversed with GC replacement, mediated through an effect on appetite-controlling neuropeptides in the hypothalamus, including insulin (ref. 151), oxytocin, and corticotrophin-releasing hormone (ref. 152). Additionally, patients with PAI have delayed gastric emptying, which may further contribute to anorexia and weight loss (ref. 153).

By contrast, GC excess may increase oral intake and cause weight gain (ref. 154). In vitro and in vivo studies also showed that GCs can increase hypothalamic endocannabinoids, which have been shown to increase hypothalamic AMP-activated protein kinase (AMPK) activity resulting in increased appetite (ref. 13, ref. 155). In a randomized controlled trial of pulsed high-dose dexamethasone vs standard prednisolone therapy, 17 of 40 (42.5%) reported some increase in appetite and 19 of 40 (47.5%) gained at least 1 kg of body weight in both groups over 24 weeks (ref. 156). In healthy men, energy intake was increased compared to baseline intake after 4 days in those receiving methylprednisolone (40 mg per day, n = 10) by 4554 kcal per day compared to 2867 kcal per day in those receiving placebo (n = 10) (ref. 157).

In a bidirectional Mendelian randomization study, single nucleotide variants independently associated with morning plasma cortisol were obtained from the CORtisol NETwork consortium. Of note, the CORtisol NETwork consortium undertook genome-wide association studies for plasma cortisol and identified that <1% of plasma cortisol variance is accounted for by variation in chromosome 14. The locus identified spans SERPINA6 encoding cortisol-binding globulin and SERPINA1 encoding α1-antitrypsin (which inhibits cleavage of the reactive center loop that releases cortisol from CBG) (ref. 158). Surprisingly, genetically predicted cortisol levels were negatively associated with BMI class and vice versa, with genetically predicted blunted morning cortisol secretion being associated with obesity (ref. 159). Given that the morning cortisol rise is an important feature of the physiological HPA axis, this suggests that disruption to the circadian rhythm of cortisol secretion could contribute to the development of obesity (ref. 160).

Blood Pressure

Changes in blood pressure occur in both AI (including SAI where mineralocorticoid production is preserved) (ref. 161) and GC excess (ref. 3). In patients with PAI, 88% have hypotension and 12% have symptomatic postural dizziness, although mineralocorticoid deficiency leading to salt and water loss is a dominant factor in PAI (ref. 162). GCs also contribute to blood pressure via the transcriptional expression of the enzyme phenylethanolamine-N-methyltransferase, which is responsible for the conversion of norepinephrine to epinephrine in the adrenal medulla (ref. 163). GCs also contribute to catecholamine-induced vasoconstriction (ref. 164). Therefore, GC deficiency results in a relative decrease in epinephrine (reduced vasoconstriction and systolic blood pressure) and an increase in norepinephrine (increased heart rate) (ref. 165).

Hypertension is observed in 80% of patients with CS (ref. 165) through a multifactorial mechanism, including a reduction in vasodilation via inhibition of nitric oxide synthase expression (ref. 166) and modulation of catecholamine synthesis (ref. 165). Cortisol and aldosterone have the same affinity for the mineralocorticoid receptor, but 11β-HSD2 inactivates cortisol under basal conditions. However, the enzyme can become saturated at high cortisol levels, eg, in ectopic-ACTH CS, resulting in excess mineralocorticoid action and hypertension.

GC Status and Muscle Function

Up to 50% of patients with PAI experience generalized weakness, muscle cramps, and fatigue (ref. 167, ref. 168), whereas around 40% to 60% of patients with CS present with muscle weakness (ref. 169-171). Mechanisms contributing to muscle weakness in GC deficiency include impaired muscle carbohydrate metabolism, depleted muscle glycogen stores, and suppression of adrenaline-induced activation of hepatic and skeletal muscle glycogen phosphorylase (ref. 168). By contrast, GC excess induces myopathy via activation of major cellular proteolytic systems [eg, ubiquitin-proteasome system, lysosomal system (cathepsins), and calcium-dependent system (calpains)], inhibition of protein synthesis (ref. 172), alteration of growth factors (eg, IGF-1, myostatin), and testosterone levels (ref. 172). Patients with CS tend to have predominant postural and proximal muscle weakness due to preferential atrophy of fast-twitch glycolytic (type IIb) muscle fibers (ref. 173, ref. 174).

Biomarkers for the Assessment of Muscle Status

GC-induced muscle atrophy is associated with changes in the muscle secretome. SerpinA3N is a secretory serine protease inhibitor that has a wide range of effects including suppression of osteoblasts, as well as being a myokine (secreted into the blood in response to muscle contraction) that is released in association with muscle atrophy. Expression of skeletal muscle SerpinA3N is increased in vitro from skeletal muscle (ref. 175); circulating levels of serpina3n were increased by 2.5-fold in 9 patients with CS (ref. 175), suggesting that it could represent a potential biomarker of GC-induced muscle atrophy pending larger studies (ref. 175).

Myostatin is also a protein produced in muscle tissue that negatively regulates muscle mass. Myostatin administration induced a 35% to 50% decrease in muscle mass (ref. 176), whereas an antimyostatin antibody attenuated chronic kidney disease associated muscle proteolysis in murine models (ref. 177). In in vitro studies, dexamethasone treatment increased myostatin mRNA levels at 24 hours, but the rise in myostatin protein levels was only observed at 36 hours (ref. 178). This discrepancy in mRNA and protein levels suggests that myostatin may be upregulated transiently at the beginning of GC exposure, but it may not be the main pathway that mediates the protein catabolic process (ref. 178). In animal studies, daily intraperitoneal administration of dexamethasone at 60, 600, and 1200 μg/kg body weight for 5 days resulted in a dose-dependent reduction in weight and muscle atrophy, and this decrease was associated with a marked upregulation of myostatin mRNA by 66%, 450%, and 527.6% compared to pair-fed control rats (ref. 179). However, the expression of myostatin mRNA and protein levels was not sustained, and levels returned to basal levels when dexamethasone treatment was extended to 10 days (ref. 179). Another research group reported similar findings in that in vivo myostatin expression was time-dependent with a transient elevation at day 5 during the 10-day dexamethasone treatment (ref. 180). A case-control study showed that serum levels of myostatin did not significantly differ in 30 patients with CS from HCs (ref. 181). Inder et al also showed that short-term oral dexamethasone treatment (4 mg for 4 days) induced relative changes in plasma testosterone (male: 15.0 vs 11.3 nmol/L; female: 1.8 vs 0.5 nmol/L) but skeletal muscle androgen receptor expression and skeletal muscle myostatin mRNA levels were unchanged (ref. 182). Overall, these studies suggest that muscle loss associated with GC is mediated in part by transient upregulation of myostatin at the beginning of GC exposure, but this may not be the main pathway mediating GC-induced muscle atrophy.

GC can also cause muscle atrophy by altering the production of growth factors (eg, IGF-1) that control muscle mass development. IGF-1 stimulates protein synthesis and differentiation of muscle satellite cells (ref. 183) and downregulates the muscle proteolytic systems by inhibiting the expression of atrogenes that cause muscle atrophy, such as atrogin-1, MuRF-1, and cathepsin L, which are induced by GC (ref. 184, ref. 185). Muscle IGF-1 overexpression prevents muscle atrophy in GC-treated animal models. However, although IGF-1 appears to have a dominant effect in reversing GC-induced muscle atrophy, systemic administration of IGF-1 could be limited by its hypoglycaemic (ref. 186) and cardiac hypertrophic actions (ref. 187). In human studies, short-term administration of GC increases serum IGF-1 levels (ref. 182, ref. 188, ref. 189), but levels of IGF-1 in tissue fluid remain unchanged (ref. 189). Skeletal muscle expression of IGF1 mRNA was significantly downregulated, suggesting a distinct tissue-specific effect on IGF1 action that could be partly mediated by GC-induced changes in plasma testosterone levels (ref. 182). Therefore, although IGF-1 appears to have a beneficial effect in reversing GC-induced muscle catabolism, further studies are needed to determine its independent effect.

Imaging in Assessment of Muscle Status

Dual-energy X-ray absorptiometry and bioelectrical impedance analysis can be used in the assessment of muscle mass, but due to the nonuniform nature of muscle atrophy in CS (ref. 190) and the reliance on a constant for hydration of fat free mass (ref. 191), dual-energy X-ray absorptiometry and bioelectrical impedance analysis could give inaccurate results.

Computed tomography (CT) and magnetic resonance imaging are considered gold standards for assessing muscle density and muscle composition as both modalities quantify intramuscular fat content based on attenuation characteristics to discern between fat and muscle tissue (ref. 192). Muscle density has been shown to better reflect handgrip strength and functional outcomes compared to muscle mass (ref. 193). In patients with CS, psoas muscle density measured by CT negatively correlated with urinary free cortisol (r = −0.30) (ref. 194), whereas treatment of GC excess increased CT-measured cross-sectional area of thigh and calf muscle (ref. 195). The use of CT or magnetic resonance imaging is limited in practice due to cost, radiation, and operational complexity. Thus, ultrasound has emerged as a reliable alternative to measure muscle mass (ie, the thickness) and structure (ie, the echo density), which comprise muscle composition (ref. 196). Muscle thickness was similar, but ultrasound-determined echo intensity of vastus lateralis, tibialis anterior, and medial gastrocnemius were significantly higher in patients with active CS compared to those in remission, indicating that muscle structure recovery precedes muscle mass recovery following resolution of hypercortisolemia (ref. 196). Needle biopsy and surface electromyography showed atrophy of type II fibers and slowing of conduction in patients with CS (ref. 197). Atrophy of muscles reduce muscle fiber length and cause architectural changes that contribute to reduced force production capacity (ref. 197). Slowing of muscle fiber conduction velocity could be related to the suppressive effect of GC on sacrolemmal excitability. The preferential GC impairment of fast fibers translates to a greater difference in muscle fiber contraction velocity of vastus lateralis and vastus medialis muscles (∼26%) compared to that of tibialis anterior muscle (∼11%) between patients with CS and age-sex matched HCs (ref. 197).

However, these changes are also observed in other conditions such as aging and neuromuscular diseases and thus lack specificity for the diagnosis of CS (ref. 198, ref. 199). Thus, it is possible that imaging can be used as an adjunct to provide objective evidence for changes in GC status; however, specificity may be lacking for diagnosis. Although emerging technologies provide a means of assessment of muscle quality changes, there is currently no standardized imaging modality that is preferred for use as a diagnostic tool for assessing changes in muscle mass.

Immunomodulatory Effects of GC

GCs have a broad range of immuno-modulatory and immune-suppressive actions on both innate and adaptive immunity, including inhibiting lymphocyte activation, promoting lymphocyte and dendritic cell apoptosis, and downregulation of inflammatory cytokines. It is worth noting that many studies have used much higher doses of GCs than are used clinically for GC replacement (ref. 152, ref. 200).

In patients with PAI, population studies have demonstrated an increased risk of infection, which contributes to an increased mortality rate (ref. 201, ref. 202). This may be explained by impaired NK-induced cellular toxicity. NK cells in patients with PAI have a reduced expression of the activating receptors NKG2D and NK-46 (ref. 186), which may explain the propensity for increased severe bacterial infections. Similarly, peripheral blood mononuclear cells (PBMC) from patients with PAI have a deficient chemokine response to interferons reflective of the adaptive immunity and the ability to defend against viral infections (ref. 203).

Whether these findings are secondary to imperfect GC replacement therapy or a feature of AI is difficult to tease apart. One study showed a normalization of immune cells implicated in innate bacterial defense and viral immunity in response to changing to a modified-release hydrocortisone preparation, linking this to a reduction in viral illnesses (ref. 101). However, it is not clear how a mild excess of GC replacement could affect immune cell function (ref. 204). Further work is needed to assess the effects of different replacement regimens on different immune cell populations.

In CS, there are a variety of changes within the innate and adaptive immune system that increase the risk of severe and opportunistic infections and contribute to a low-grade inflammatory state (ref. 205). These include an increase in neutrophils [due to increased release of polymorphonuclear leucocytes (PMNs) from the bone marrow and reduced apoptosis and extravasation of PMNs leading to an increased PMN half-life in the blood stream]. GC excess also reduces NK cell activity and macrophage phagocytic activity due to a reduction in circulating monocytes. Adaptive changes include a relative reduction in CD4+ T cells, a relative increase in CD8+ T cells, and a reduced B lymphocyte count (ref. 190), increasing susceptibility to intracellular and opportunistic infections.

Increased proinflammatory adipokines, altered tumor necrosis factor α regulation, IL-6, and C-reactive protein also contribute to the chronic, inflammatory state that is thought to contribute to the pathogenesis of the other clinical complications, including diabetes, atherosclerosis, and osteoporosis, with an increased risk of cardiovascular disease that persists after remission (ref. 206).

It has also been observed that CS patients experience rebound autoimmune diseases during remission. This may be due to overactivity of previously suppressed immune function. The extensive effects of GCs on the immune system are beyond the remit of this review; further in-depth discussions on the immune system changes can be found in a recent comprehensive review (ref. 205). A better understanding of changes in the immune system could offer further biomarkers that could be used to assess GC status.

GC Effects on Glycemia

The mechanisms by which GCs exert a diabetogenic effect are complex and are context dependent, but proposed effects include increased insulin resistance, both increased or decreased insulin secretion, and stimulation of hepatic gluconeogenesis. Additional interorgan signaling and tissue-specific effects in vivo further contribute and amplify GC’s diabetogenic effects. The reader is directed to a recent comprehensive review focusing on glucocorticoid effects on glycemia (ref. 207).

Insulin resistance occurs both peripherally and in the liver. As a result of hepatic insulin resistance, there is removal of insulin-mediated repression of hepatic gluconeogenesis. Peripherally, GCs directly disrupt insulin signaling through the downregulation and phosphorylation of IRS1, PI3K, and PKB-AKT (ref. 208-210). This inhibits insulin-induced recruitment of the glucose transporter 4 in skeletal muscle, reducing insulin sensitivity and glucose uptake (ref. 211). Additional effects on protein degradation increase serum amino acids, providing substrates for gluconeogenesis (ref. 212, ref. 213).

De novo glucose production predominantly occurs via hepatic gluconeogenesis via upregulation of the enzymes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase. The action of GCs on other tissues, such as skeletal muscle (via protein catabolism) and lipolysis in white adipose tissue, increase gluconeogenic precursors that are shuttled to the liver (ref. 172, ref. 214), further driving hepatic gluconeogenesis. Understanding the effects of GCs on insulin secretion in vivo is challenging due to the duration of GC exposure and compensatory effects that occur with increased exposure duration, which is difficult to elucidate in in vitro and ex vivo studies. Preclinical studies have demonstrated an increase in beta cell mass following GC exposure in a dose-dependent manner (ref. 215); however, this has not been demonstrated in humans where exposure to GC resulted in minimal changes to beta cell mass (ref. 216). In humans, an acute single oral GC dose has been shown to acutely impair the initial beta cell response to hyperglycemia with increased AUC_glucose and reduced AUC_c-peptide, but this demonstrated acute recovery with subsequent increased insulin secretion (both fasting and in response to a mixed meal test) 24 hours later. In individuals exposed to 15 days of treatment, there was evidence of increased c-peptide secretion but increased levels of fasting and postprandial AUC_glucose, suggesting that the increase in insulin is insufficient to maintain euglycemia (ref. 217).

In addition, studies have also demonstrated increased basal and stimulated glucagon levels in participants following GC administration (ref. 217, ref. 218), further contributing to hyperglycemia. However, the literature is not consistent, with other studies failing to demonstrate this (ref. 219). This may be explained by differences in the insulin sensitivity in the individuals studied. It has been suggested that additional alpha cell disturbances may be present in those with reduced insulin sensitivity (ref. 219).

AMPK is a key regulatory enzyme of appetite, lipid, and carbohydrate metabolism and is believed to mediate some of the deleterious metabolic effects of excess GC (ref. 220). AMPK activity in visceral adipose tissue was inversely associated with 9:00 Am cortisol levels and urinary free cortisol (ref. 220). The effects of AMPK inhibition have demonstrated impaired insulin-mediated glucose uptake by skeletal muscle and adipose, increased hepatic glucose output, reduced beta-cell glucose-stimulated insulin secretion, and increased lipogenesis and fat storage (ref. 221). Activation of AMPK has been shown to reverse GC-induced hepatic steatosis and effects on glucose metabolism (ref. 222). In nondiabetic patients established on GC for inflammatory disease, treatment with metformin (an activator of AMPK) for 12 weeks reduced truncal subcutaneous fat (−3835 mm²), improved insulin resistance and glucose concentrations, and reduced inflammation and progression of carotid intima thickness (ref. 223), suggesting that concomitant use of metformin can mitigate the deleterious effects of GC excess (ref. 223).

In summary, there is evidence that, through a variety of mechanisms, GC deficiency may result in hypoglycemia, especially in children (ref. 66), whereas chronic GC excess can result in hyperinsulinemia and, in those at risk, hyperglycemia (ref. 3).

GC Effects on Lipid Profiles

The effects of GC on lipid dysregulation are multifactorial and involve both direct and indirect pathways. GC promotes lipolysis of mature adipocytes (ref. 214, ref. 224) but stimulates lipogenesis on preadipocyte cells (ref. 224). Low to moderate concentrations of GC (1-10 µM) stimulate lipolysis of mouse adipocyte 3T3-L1 cells, but high doses of GC suppress lipolytic rate (ref. 224). Lipolysis is likely to be mediated via increased intracellular cAMP levels and protein kinase A activity, and this effect could be blunted by downregulation of cyclic-nucleotide phosphodiesterase 3B, a major enzyme responsible for cAMP hydrolysis (ref. 214, ref. 224). GC-induced lipolysis may also in part be mediated via a direct upregulation of GR target such as angiopoietin-like 4, an adipokine that inhibits lipoprotein lipase activity that is involved in lipolysis (ref. 225). In vivo studies reported a 60% rise in fatty acid palmitate level when circulating cortisol concentrations increased from 245 ± 12 nmol/L to 888 ± 7 nmol/L during a 6-hour hydrocortisone infusion (2 μg/kg/min) in the presence of a pancreatic pituitary infusion clamp (ref. 226).

Notably, in an individual, prevailing levels of insulin, somatostatin, and adrenaline concentrations could modulate the lipolytic effects of GC. Healthy volunteers (n = 15) who underwent a paired 2-step hyperinsulinaemic euglycemic clamp with a concurrent overnight HC (0.2 mg/kg/h) or saline infusion (at least 2 weeks apart, in random order) along with adipose tissue microdialysis had lower circulating free fatty acid levels during the high insulin concentration phase compared to low insulin level period compared to placebo (low insulin with HC vs placebo: 564 ± 40 vs 403 ± 38, P < .01 μmol/L·h; high insulin with HC vs placebo: 712 ± 35 vs 443 ± 38 μmol/L·h, P < .005) (ref. 227). During the clamp studies, glucose infusion rates decreased in response to insulin infusion in the HC group compared to placebo, suggesting a reduced glucose disposal rate, representing an increase in systemic insulin resistance. Endogenous glucose production did not differ between the HC or saline group, but the glucose production rate in the presence of a high insulin level was lower in the HC arm compared to saline, suggesting hepatic insulin resistance (ref. 227). Insulin also suppresses the magnitude of GC-induced lipolysis potentiated by adrenaline and stimulates lipoprotein lipase activity (ref. 226). As a result, despite a possible increase in lipolysis, an overall increase in lipogenesis (ref. 224) and hepatic and systemic insulin resistance occurs (ref. 224) and, in turn, promotes hepatic gluconeogenesis (via induction of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase) (ref. 228). Adipogenesis promotes free fatty acid release into the circulation and ectopic storage of lipids in liver and skeletal muscle (ref. 229). Effects of GC-induced adipogenesis are more notably observed in the visceral adipose tissue, which has a relatively higher number of GC receptors (ref. 230). Consequently, expansion of visceral adipose tissue and reduction of subcutaneous adipose tissue further impair liver and muscle sensitivity, leading to cardiometabolic disease (ref. 231). In summary, GC hypercortisolism promotes lipolysis but hyperinsulinemia above a certain threshold suppresses GC lipolysis and lipogenesis and insulin resistance predominates (ref. 232).

Dsylipidemia is a well-recognized feature in patients with hypercortisolism. Patients with active CS exhibit increased very low density lipoprotein, low-density lipoprotein, and triglycerides, but no change in the lipid profile was observed on remission of CS (ref. 233). GC replacement in patients with SAI reduced plasma cholesteryl ester transfer protein activity, resulting in increased high-density lipoprotein (HDL) cholesterol levels (ref. 234) and HDL size (ref. 235), but triglyceride or low-density lipoprotein cholesterol levels were unchanged (ref. 234). Consistent with this, daily administration of low-dose dexamethasone to healthy people for 3 weeks increased HDL cholesterol (ref. 236). By contrast, a large study of 2424 patients with hypopituitarism showed no significant changes in lipid levels between patients treated with GC replacement and those who were GC sufficient (ref. 237). Further clinical data are needed prior to consideration of lipid as a potential marker of GC status.

Adiponectin and Leptin

In humans, treatment with 0.5 mg of dexamethasone 4 times daily for 2 to 5 days reduced insulin sensitivity, but plasma levels of adiponectin, resistin, and leptin remained unchanged (ref. 238). Administration of hydrocortisone (25 mg) reduced adiponectin levels at 30 and 60 minutes both in patients with and without obesity (ref. 239). Adiponectin levels are lower in nonobese patients with CS (20.9 mcg/mL) than in HCs (30.9 mcg/mL), but levels of adiponectin did not differ between patients with CS and obesity compared to those with obesity alone (20.1-22.1 mcg/mL) (ref. 239). Thus, adiponectin could be useful in identifying GC excess but only in the absence of obesity.

Leptin levels were increased dose-dependently after 1 day of a 4-day course of oral hydrocortisone (at either 40 or 160 mg daily) by more than double after the higher dose in women; however, levels returned to baseline level by the fourth day and were less increased in men (ref. 240). Diurnal secretion of leptin is preserved in patients with CS (ref. 241). In patients without obesity, fasting leptin levels are 2-fold higher in patients with CS compared to nonobese controls (33.5 vs 14.2 ng/mL) (ref. 241). In contrast, a smaller difference in leptin levels was observed between patients with obesity compared to BMI-matched HCs (55.0 vs 41.7 ng/mL) (ref. 241). Leptin levels remained stable during the immediate postoperative period (10 days), despite a concurrent decrease in cortisol and ACTH levels after surgical cure of CS (ref. 241, ref. 242). This lack of change in leptin levels after successful surgery could be more associated with the lack of change in BMI and adiposity in the immediate postoperative period (ref. 241) as others have reported a reduction in leptin concentration and BMI in patients with CS 3 to 6 months after surgery (ref. 243). In summary, hypercortisolaemia may indirectly induce body fat changes and hyperinsulinemia, which in turn could influence leptin concentrations (ref. 241, ref. 243). Overall, there are several markers that may be used to assess GC status; however, given the tissue-specific responsiveness to GC that exists (ref. 244), there is no single marker that accurately reflects GC status in isolation.

Novel Markers That Could Be Used in the Future to Quantify GC Status

Please see Table 4.

Metabolomic Assessment

To date, there have been relatively few studies evaluating whether there is a metabolomic signature reflective of healthy GC status and how it may be altered after a changed GC status. Recently, Kachroo et al used metabolomic assessment to identify 17 steroid metabolites that were altered in patients treated with inhaled corticosteroid for asthma (ref. 245). They highlight that genetic variation can underpin the heterogeneity in treatment response and adrenal suppression with inhaled steroids (ref. 245). A significant proportion of patients on inhaled steroids were found to have low cortisol levels, although they may yet have increased GC status due to inhaled steroid use, highlighting a clinical example where GC levels may not reflect GC status (ref. 245).

Patients with adrenal tumous, including those with nonfunctionating adrenal tumors or those with mild autonomous cortisol secretion (MACS) or adrenal CS, were assessed by multisteroid profiling of 24-hour urine by tandem mass spectrometry (ref. 271). Those with MACS or adrenal CS had a decrease in 24-hour urinary excretion of androgen metabolites (androsterone, etiocholanolone, dehydroepiandrosterone) and of pregnenetriol (metabolite of 17-hydroxypregnenolone) and an increase in the 24-hour urinary excretion of cortisol and tetrahydro-11-deoxycortisol (metabolite of 11-deoxycortisol) (ref. 271). Notably, there was a step-wise change in the pattern of urinary steroids from those with nonfunctionating adrenal tumors to MACS to CS, suggesting that such an approach could be used to aid in the objective assessment of GC status (ref. 271). Similarly, in patients with AI (n = 50), dual-release hydrocortisone compared with thrice-daily hydrocortisone decreased cortisol exposure and shifted the urinary steroid metabolome toward that of HCs and thus could potentially be used as a tool to optimize GC replacement (ref. 247).

Growth Differentiating Factor 15

Growth differentiating factor 15 (GDF15) (previously called MIC-1) is a member of the transforming growth factor β family, expressed by cells in response to stress (ref. 250, ref. 251). As a marker of stress, it can be elevated in both physiological and pathological states, including pregnancy, exercise, chronic inflammatory disease, renal failure, and cardiac failure (ref. 250). In vivo, GCs repress transcription of GDF15 mRNA, suggesting that GDF15 could potentially be used as a downstream marker of GC signaling in the endoplasmic reticulum stress pathway (ref. 272).

Utility of GDF15 has been explored in patients with PAI (ref. 251). In a proof-of-concept study, patients with PAI (n = 10) without GC replacement for 39 hours (cortisol levels: PAI 47 ± 17 nmol/L, controls 371 ± 72 nmol/L) had higher circulating GDF15 levels (PAI: 739 ± 226 pg/mL) compared to HCs (498 ± 168 pg/mL) (ref. 251). However, these elevated levels in patients with PAI were reduced to 559 ± 120 pg/mL after a 22-hour hydrocortisone infusion (ref. 251). Additionally, there was a dose-dependent decrease in GDF15 levels (mean ± SD) in patients on low- (491.0 ± 157.7 pg/mL), medium- (427.0 ± 152.1 pg/mL), or high- (360 ± 143.1 pg/mL) dose oral GC replacement (ref. 251). GDF15 levels were also significantly lower in patients with congenital adrenal hyperplasia after a 300-minute hydrocortisone infusion (hydrocortisone 547.0 pg/mL vs placebo 606.0 pg/mL) (ref. 251). Likewise, a 22-hour hydrocortisone infusion (0.008-0.024 mg/kg/hr) reduced GDF15 levels in a dose-dependent manner in patients with PAI (baseline 754 vs 610 pg/mL at 22 hours) (ref. 251). Although this is early data, GDF15 could have a potential utility to contribute as a marker of insufficient GC status pending further study in larger cohorts (ref. 251).

MicroRNAs—miR-122-5p

MicroRNAs (miRNAs) are a group of small noncoding RNAs that regulate gene expression. Given their presence in many extracellular compartments such as plasma, serum, urine, and saliva, miRNAs hold potential as noninvasive biomarkers (ref. 273). In particular, miR-122-5p has been identified as having a key role in regulating gene expression in several cancers, including breast cancer and pancreatic ductal adenocarcinoma (ref. 274). GC exposure was associated with a 2-fold reduction in levels of miR-122-5p in circulating peripheral blood mononuclear cells (P = .009) (ref. 248). By contrast, circulating miR-182-5p was upregulated in Cushing’s disease in comparison to controls, with an area under the receiver operating characteristic (AuROC) curve of 0.89 (ref. 249). After 1 mg dexamethasone treatment, miR-96-5p was upregulated and miR-185-5p was downregulated (ref. 249) in postdexamethasone samples. Thus, while currently only used in the sphere of investigative research, miRNA biomarkers could in the future aid in the assessment of GC status.

GREs

A given GC dose can have variable efficacy and side-effect profile in different individuals. At present, no clinically useful biomarker of GC activity has been established for diagnosis or for use in therapeutic monitoring.

Thrombospondin-1 (TSP1) is a matricellular protein that is widely expressed in endothelial cells, monocytes, macrophages, and adipocytes (ref. 252, ref. 253). It has a plasma half-life of 9 hours and is unaffected by age, sex, BMI, or commonly administered medications for hypertension, hyperlipidemia, or diabetes (ref. 275, ref. 276). TSP1 mRNA was increased by dexamethasone in different cell types (eg, osteoblast, endometrial, or peripheral blood mononuclear cells) in vitro (ref. 277), and the antiangiogenic effect of cortisol on endometrial endothelial cells was abolished by small interfering RNA against TSP1 mRNA, indicating that TSP1 may be a downstream GC effector (ref. 278). A single 4-mg dose of dexamethasone increased plasma TSP1 mRNA in peripheral blood mononuclear cells in healthy individuals at 12 hours (ref. 254). Median (interquartile range) morning plasma TSP1 was significantly higher in patients with CS (n = 8) compared to healthy volunteers (n = 20) [CS: 638 (535-756) ng/mL vs HCs: 272 (237-336) ng/mL] (ref. 254). A threshold of 400 ng/mL identified CS with 100% sensitivity and 85% specificity (AuROC 0.94) (ref. 254). Likewise, morning plasma TSP1 levels were increased in patients with AI (n = 16) when the maintenance dose of HC was increased from 20 to 30 mg within a week from a baseline level of 139 (86-199) ng/mL to 256 (133-516 ng/mL) (ref. 254). These studies suggest that TSP1 is a GC-responsive protein in humans that could be used to reflect GC action. To improve diagnostic sensitivity and specificity, the ratio of TSP1 to osteocalcin taken from a morning blood sample was used in a study of 20 healthy volunteers and 19 people with CS (ref. 256). A TSP1:osteocalcin ratio of >73 reliably identified individuals with CS with an AuROC of 0.997 (ref. 256), highlighting it as a promising potential biomarker of GC status. Proteomic analysis of the secretome from PBMC cells isolated from healthy volunteers exposed to 100 ng/mL of dexamethasone yielded 3 additional novel candidate GC-responsive proteins that altered with GC exposure (eg, lysozyme C, high-mobility group protein 2, and nucleophosmin-1), which could be further explored in future studies (ref. 255).

FKBP5 Expression

FKBP5, a protein encoded by the FKBP5 gene, is a potential biomarker of cortisol activity at the GR (ref. 257, ref. 258). FKBP51 is a cochaperone of HSP90, which together form part of the GR chaperone complex (ref. 279). When bound to the complex, FKBP51 decreases GR activity by reducing its cortisol-binding affinity and preventing its translocation to the nucleus (ref. 259). Thus, FKBP51 interacts with the GR to maintain it in an unbound inactive state (ref. 259). Upon GC binding to the GR, FKBP51 dissociates from the complex and is replaced by FKBP52 enabling nuclear translocation of GR (ref. 258, ref. 279). Once migrated into the nucleus, the GR binds to GRE to affect gene expression, including of FKBP5 (ref. 258, ref. 279). This leads to an increase in the amount of FKBP5 mRNA and FKBP51 protein, which in turn restricts GR nuclear translocation, thus completing an intracellular negative feedback loop for GR activity (ref. 258, ref. 279). Thus, FKBP5 could be used as a biomarker to reflect GC receptor action.

FKBP5 expression levels positively correlate with serum cortisol (r = 0.419) in patients with PAI (ref. 260). Furthermore, an infusion of 100 mg hydrocortisone in patients with PAI off medications induced a 1.35-fold increase in FKBP5 (ref. 260). However, at very low cortisol levels, no correlation was found between FKBP5 expression and serum cortisol levels, although it negatively correlated with plasma ACTH (r = −0.399) (ref. 260).

Oral administration of a single dose of 25 mg of the synthetic GC prednisone increased FKBP5 mRNA expression by 16-fold within 4 hours, and levels returned to baseline within 24 hours of prednisone discontinuation (ref. 261). A single dose of mifepristone was equally effective in reducing prednisone-induced FKBP5 mRNA levels back to unstimulated levels within 4 hours (ref. 261). Likewise, repeated administration of the GC antagonist once daily for 14 days completely ablated prednisolone-induced FKBP5 (ref. 261).

Median (range) FKBP5 mRNA levels were higher in patients with ACTH-dependent CS (n = 24) than in HCs [2357.3 (1071.45, 17215.80) vs 918.7 (661.68, 1735.91)] (ref. 257). FKBP5 mRNA strongly correlated with cortisol levels both before and after surgery (r = 0.72-0.85) (ref. 257). Notably, after successful surgical treatment of CS, FKBP5 mRNA expression decreased to levels similar to those of HCs (CS postsurgery 1148.7 ± 372.8, HCs 918.7 ± 311.1) but remained elevated in patients with failed surgical treatment (2505.3 ± 1843.4) (ref. 257). Overall, the change in FKBP5 mRNA expression with GC exposure shows promise as an in vivo transcriptional biomarker of GC receptor activity that could contribute to assessment of GC status.

FKBP5 Methylation

Epigenetic changes in gene expression due to environmental influence cause alterations in gene activity rather than in gene sequence (ref. 280). The most extensively investigated epigenetic modification is DNA methylation (DNAm), which acts to repress transcription of the gene and can result in gene silencing via changes in chromatin structure (ref. 281).

Changes in chromatin structure involve intron 7 of the GREs, which is thought to contain a transcription enhancer (ref. 282). Less DNA methylation of GREs at intron 7 is associated with higher induction of FKBP5 by GC receptor activation and thus can result in differential expression and responsiveness of FKBP5 to GR (ref. 282).

Several studies have begun to examine the role of DNAm in relation to GC status, in particular, FKBP5 promoter methylation levels (ref. 262). In healthy volunteers, DNAm of the FKBP5 gene in leukocytes was measured before and after ACTH stimulation (ref. 262). FKBP5 methylation was negatively correlated with basal serum cortisol (r = −0.439) (ref. 262). The change in FKBP5 methylation at 24 hours after ACTH negatively correlated with the increase in cortisol levels at 1 hour after ACTH (r = −0.537) (ref. 262).

Despite no significant changes in the FKBP5 methylation status at 24 hours, in vitro studies have suggested changes in methylation of FKBP5 following longer term GC exposure for up to 4 weeks (ref. 283, ref. 284). In fact, patients with CS were found to have reduced FKBP5 methylation of intron 7 compared to HCs, although there was no difference between cured vs active CS patients (cured CS 77.1%, active CS 73.7%, and HCs 79.7%) (ref. 285).

Several studies have explored the potential of FKBP5 methylation as a biomarker of chronic GC load. FKBP5 methylation levels after 4 weeks of corticosterone exposure in mice significantly correlated with average corticosterone load (ref. 284). Overall, this supports the notion that FKBP5 expression levels reflect short-term GC exposure, whereas FKBP5 methylation reflects longer term GC exposure.

REDD1

Regulated in development and DNA damage response 1 (REDD1) is a hypoxia and stress-response gene that inhibits mammalian target of rapamycin complex-1 activity (ref. 286). It has a low level of expression in basal conditions and is induced in response to hypoxia, stress, DNA damage, or transcription factor activation (ref. 286).

Recent studies have identified the hypoxia and stress-response gene REDD1 as a molecular target for GCs that mediates cutaneous and skeletal muscle atrophy in skin (ref. 264). In REDD1 knockout mice, skin and muscle atrophy are averted (ref. 263, ref. 264). REDD1 is upregulated in mouse and human skin and in skeletal muscles following topical or oral GC administration. In mice, the expression of REDD1 is increased between 4 to 24 hours after treatment with topical GC (ref. 263) or after oral dexamethasone at 1 mg/kg (ref. 264). High levels of REDD1 correlate with reduced phosphorylation of downstream mammalian target of rapamycin complex-1 targets (Akt signaling pathway proteins S6 Ser^240/244; 4E-BP1 Thr^37/46, and ULK1 Ser⁷⁵⁷) resulting in reduction of protein synthesis (ref. 263, ref. 264). In contrast, REDD1 knockout mice did not demonstrate such downstream changes, and gene activation by GCs are altered in such a way that the therapeutic anti-inflammatory effects of GC were preserved. Thus, REDD1 expression could represent a surrogate biomarker for GC signaling in skin or skeletal muscle; however, data is limited to only preclinical models receiving supraphysiological GC doses to date (ref. 240, ref. 242).

Bone Turnover Makers

The incidence of osteoporotic fractures may be as high as 20% in endogenous hypercortisolism and 30% to 50% in patients with long-term GC use (ref. 287). Prolonged exposure to GCs induces insulin resistance in bone cells resulting in increased bone marrow adiposity and bone fragility (ref. 288). Dose and duration of GC use influences fracture risk (ref. 289). Individuals receiving higher doses of oral prednisolone (≥7.5 mg per day) had a 2- to 2.5-fold increased fracture risk compared to those on lower doses (<2.5 mg per day) (ref. 289). Cumulative GC doses of >1 g over a year result in significant bone loss compared to those receiving lower cumulative GC doses (ref. 290). Interestingly, bone loss is more rapid in new GC users during the first year with bone density of up to 12% followed by a slower decline of 2% to 3% per year with persistent GC use (ref. 291).

GCs inhibit osteocalcin, which is a marker of osteoblast activity (ref. 266). In healthy subjects, 1 mg oral dexamethasone reduced osteocalcin levels from 28.3 ng/mL to 21.8 ng/mL at 8 hours (ref. 267). Likewise, intramuscular administration of 1 mg of ACTH at 2 Pm in healthy individuals (which induced a rise in cortisol to 58.3 mcg/dL, ie, 1608 nmol/L) reduced osteocalcin levels from 28.3 ng/mL to 12.5 ng/mL at 8 Am the following morning (ref. 267). In fact, GC administered in both oral (prednisolone 5 mg) or inhaled forms depresses osteocalcin levels acutely at 3 to 4 hours postdose with levels returning to pretreatment levels on tapering of GC doses (ref. 266, ref. 268). A randomized cross-over study showed that healthy subjects (n = 37) receiving 7 days of treatment with prednisone at doses of 2.5 to 60 mg had a dose-dependent decrease in plasma osteocalcin levels as early as 4 hours postdose, which remained suppressed throughout the week (ref. 292).

The utility of serum osteocalcin as a biomarker has been explored in patients with CS. Morning serum osteocalcin levels were 21.5 ng/mL in healthy individuals and 8.35 ng/mL in overt CS, which corresponds to an AuROC of 0.923 in identifying individuals with CS (ref. 269). There was a negative correlation between osteocalcin levels and serum cortisol levels in patients with active CS in the morning (r = −0.39), at midnight (r = −0.54), and after low-dose dexamethasone suppression test (LDDST) (r = −0.53) (ref. 269). An increase in osteocalcin levels was also observed as early as 1 month after transsphenoidal surgery or adrenalectomy in patients with CS (ref. 270). While osteocalcin is helpful in validating the diagnosis of overt CS, its utility in identifying individuals with milder degrees of cortisol excess is yet to be evaluated. A recent retrospective study suggests osteocalcin could be a promising tool for identifying CS in postmenopausal women (age > 45 years) with osteoporosis (from those without CS) [AuROC: 0.960 (95% CI 0.892-1.000)] (ref. 293).

Other markers of bone formation, such as total and bone-specific alkaline phosphatase and procollagen type I carboxy-propeptide, are less sensitive to GC effects and display a milder and delayed decrease under GC therapy (ref. 268).

Bone resorption increases during GC treatment (ref. 294); however, data are inconsistent. C-terminal telopeptide of type 1 collagen (CTX) is a marker of osteoclastic activity and changes with cortisol levels but less sensitively than osteocalcin. Serum CTX levels correlate with morning (r +0.76) and postdexamethasone cortisol levels (r +0.43). CTX did not change in patients with CS; however, it was elevated in patients with ectopic ACTH (1.0 vs 0.4 ng/mL) who have higher cortisol levels (50 mcg/dL in ectopic ACTH CS vs 21 mcg/dL in other CS) (ref. 270). Overall, osteocalcin could present a relevant marker of GC status pending further study.

Conclusion

GCs are essential hormones for the normal functioning of most organ systems in the body with both GC excess and GC deficiency leading to adverse health consequences. However, symptoms can be nonspecific, and reliance on cortisol levels alone as a readout of GC status can be unreliable. Dynamic tests used to make diagnoses are based on historical cut-off values and aim to detect more profound abnormalities in GC status. Moreover, cortisol values can be challenged due to a number of factors, including the pulsatile nature of cortisol secretion, interindividual variation in cortisol binding proteins, local tissue availability dictated by 11 β-HSD activity, and GC receptor sensitivity.

Downstream markers of GC action may provide an objective method to assess GC status. Such markers include those reflecting metabolomic and epigenetic changes. A comprehensive multiomic approach, applied in concert with serum GC levels, assessment of clinical symptoms and signs such as weight change and metabolic parameters, along with other markers of cortisol action, could enable a comprehensive and objective assessment of GC status in the future.

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