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Found 2 results

  1. Abstract Purpose: Literature regarding endogenous Cushing syndrome (CS) largely focuses on the challenges of diagnosis, subtyping, and treatment. The enigmatic phenomenon of glucocorticoid withdrawal syndrome (GWS), due to rapid reduction in cortisol exposure following treatment of CS, is less commonly discussed but also difficult to manage. We highlight the clinical approach to navigating patients from GWS and adrenal insufficiency to full hypothalamic-pituitary-adrenal (HPA) axis recovery. Methods: We review the literature on the pathogenesis of GWS and its clinical presentation. We provide strategies for glucocorticoid dosing and tapering, HPA axis testing, as well as pharmacotherapy and ancillary treatments for GWS symptom management. Results: GWS can be difficult to differentiate from adrenal insufficiency and CS recurrence, which complicates glucocorticoid dosing and tapering regimens. Monitoring for HPA axis recovery requires both clinical and biochemical assessments. The most important intervention is reassurance to patients that GWS symptoms portend a favorable prognosis of sustained remission from CS, and GWS typically resolves as the HPA axis recovers. GWS also occurs during medical management of CS, and gradual dose titration based primarily on symptoms is essential to maintain adherence and to eventually achieve disease control. Myopathy and neurocognitive dysfunction can be chronic complications of CS that do not completely recover. Conclusions: Due to limited data, no guidelines have been developed for management of GWS. Nevertheless, this article provides overarching themes derived from published literature plus expert opinion and experience. Future studies are needed to better understand the pathophysiology of GWS to guide more targeted and optimal treatments. Introduction Endogenous neoplastic hypercortisolism - Cushing syndrome (CS) - is one of the most challenging diagnostic and management problems in clinical endocrinology. CS may be due to either a pituitary tumor (Cushing disease, CD), or a non-pituitary (ectopic) tumor secreting ACTH. ACTH-independent hypercortisolism due to unilateral or bilateral adrenal nodular disease has been increasingly recognized as an important cause of CS. Regardless of the cause of CS, the clinical manifestations are protean and include a myriad of clinical, biochemical, neurocognitive, and neuropsychiatric abnormalities. The catabolic state of hypercortisolism causes signs and symptoms including skin fragility, bruising, delayed healing, violaceous striae, muscle weakness, and low bone mass with fragility fractures. Other clinical features include weight gain, fatigue, depression, difficulty concentrating, insomnia, facial plethora, and fat redistribution to the head and neck with resultant supraclavicular and dorsocervical fullness[1]. Metabolic consequences of hypercortisolism including hypertension, diabetes, and dyslipidemia are common. In addition, women often experience hirsutism and menstrual irregularity, while men may have hypogonadism. Management options of CS include surgery, medications, and radiation. The preferred first line treatment, regardless of source, is surgery, which offers the potential for remission[2,3,4]. The primary literature, reviews, and clinical practice guidelines for CS have traditionally focused on the diagnosis, subtyping, and surgical approach to CS. This bias derives first from the profound diagnostic challenge posed in the evaluation of cortisol production and dynamics, given that circulating cortisol follows a circadian rhythm, exhibits extensive protein binding and metabolism, and rises acutely with stress. CD and ectopic ACTH syndrome may be difficult to distinguish clinically and biochemically, and inferior petrosal sinus sampling is required in many patients to resolve this differential diagnosis. Ectopic ACTH-producing tumors can also be small, and these tumors can escape localization despite the best current methods. Although diagnosis and initial surgical remission can be achieved in the majority of patient with CS at experienced centers, up to 50% of patients with CD will require additional therapies after unsuccessful primary surgeries or recurrence up to many years later[5]. For patients who do not achieve surgical cure or who are not surgical candidates, several medical treatment options are now available. Pharmacotherapies directed at the pituitary include pasireotide[6, 7] (FDA approved) and cabergoline[8]. Adrenal steroidogenesis inhibitors such as osilodrostat[9] (FDA approved), metyrapone[10], levoketoconazole[11] (FDA approved) and ketoconazole[12], as well as the glucocorticoid antagonist, mifepristone[13] (FDA approved), are now widely used to treat CS. Pituitary radiotherapy is an additional treatment option for CD but can take months to years to lower cortisol production. Bilateral adrenalectomy (BLA) provides immediate, reliable correction of hypercortisolism but mandates life-long corticosteroid replacement therapy, and, in patients with CD, may be complicated by corticotroph tumor progression syndrome in 25–40% of patients[14]. After successful surgery for CS, the rapid onset of adrenal insufficiency (AI) is anticipated and usually portends a favorable prognosis [15,16,17,18]; however, despite the use of post-operative corticosteroid replacement, the rapid reduction in cortisol exposure often results in an enigmatic phenomenon referred to as the glucocorticoid withdrawal syndrome (GWS). This article addresses the clinical presentation and the pathogenesis of GWS, as well as its distinction from AI. When available, appropriate references are provided. Statements and guidance provided without references are derived from expert opinion and experience. Clinical Presentation and Pathogenesis of GWS GWS occurs following withdrawal of supraphysiologic exposure to either exogenous or endogenous glucocorticoids of at least several months duration[19]. After surgical cure of endogenous CS, GWS is usually characterized by biochemical evidence of hypothalamic-pituitary-adrenal (HPA) axis suppression with many signs and symptoms consistent with cortisol deficiency despite the use of supraphysiologic glucocorticoid replacement therapy. The degree of physical or psychologic glucocorticoid dependence experienced by patients may not correlate with the degree of HPA axis suppression[20, 21]. GWS symptom onset is typically 3–10 days postoperatively, often after the patient has been discharged from the hospital. The first symptoms of GWS vary but usually consist of myalgias, muscle weakness, fatigue, and hypersomnolence. Anorexia, nausea, and abdominal discomfort are common, but vomiting should raise concern for hyponatremia, cerebrospinal fluid leak, hydrocephalus, or other perioperative complications. Mood changes develop more gradually and range from mood swings to depression, and the fatigue with myalgias can exacerbate mood changes. An atypical depressive disorder has been described in many patients after CD surgery[22]. Weight loss should ensue in most patients but gradually and proportionate to the reduction in glucocorticoid exposure. It is important to complete a thorough symptom review and physical exam at postoperative visits, as the differentiation between GWS and bona fide AI – and even between GWS and recurrence of CS – can be challenging (Fig. 1). All three conditions are associated with symptoms of myalgias, weakness, and fatigue; however, rapid weight loss, hypoglycemia, and hypotension are suggestive of AI and the need for an increase in the glucocorticoid dose. In parallel, hypersomnia is more suggestive of GWS, while insomnia is more associated with recurrence of CS. Given the anticipation of GWS onset shortly after discharge and the potential for hyponatremia during this time, a widely employed strategy is a generous glucocorticoid dose for the first 2–3 weeks, at least until the first postoperative outpatient visit (Table 1). Fig. 1 Overlapping clinical features of Cushing syndrome (CS), glucocorticoid withdrawal syndrome (GWS), and adrenal insufficiency (AI) Full size image Table 1 Glucocorticoid Therapy Options After Surgery for CS Full size table The mechanisms responsible for the precipitation of the GWS after surgery for CS and the variability in its manifestations are not completely understood, yet alterations in the regulation of cortisol and cortisol-responsive genes appear to contribute. Down-regulation of corticotropin-releasing hormone (CRH) and proopiomelanocortin (POMC) expression, combined with up-regulation of cytokines and prostaglandins are likely to be important components of GWS. Low CRH has been associated with atypical depression[23], and CRH levels in cerebrospinal fluid of patients with CD are significantly lower compared to healthy subjects[24]. CRH suppression gradually resolves after surgical cure over 12 months during glucocorticoid replacement[25], illustrative of the slow recovery process. The expression of POMC, the ACTH precursor molecule, is also suppressed with chronic glucocorticoid exposure[26], and the normalization of POMC-associated peptides mirrors HPA axis recovery[19]. In the acute phase of glucocorticoid withdrawal, interleukins IL-6 and IL-1β, as well as tumor-necrosis factor alpha (TNFα) have been observed to rise[27], suggesting that glucocorticoid-mediated suppression of cytokines and prostaglandins is then released in GWS, and these cytokines induce the associated flu-like symptoms. Glucocorticoid replacement with dexamethasone 0.5 mg/d reduced but did not normalize IL-6 after 4–5 days[27], consistent with resistance to suppression during GWS. Acute Care: Perioperative Planning, Coaching, and Management For patients with CD, transsphenoidal surgery performed by an experienced surgeon achieves remission in about 80% of pituitary microadenomas and 60% of macroadenomas[28,29,30,31]. Post-operative AI and GWS are some of the most challenging phases of management for endocrinologists and one of the most disheartening for CS patients. Many patients report feeling unprepared for the postsurgical recovery process[32]. For these reasons, it is important to prepare the patient prior to surgery for the difficult months ahead, and the same considerations apply to the commencement of medical therapies, as will be discussed later. On the one hand, more potent glucocorticoids and higher doses reliably mitigate symptoms, but on the other hand, substitution of exogenous for endogenous CS delays recovery of the HPA axis and perpetuates CS-related co-morbidities. Limited data that compare management strategies preclude evidence-based decisions, yet some themes can be derived from expert opinion and extensive experience from CS centers. In centers dedicated to the management of CS, surgeons and endocrinologists work closely together through all phases of the process. Although the goal of primary surgery for CD is adenoma resection, the tumor might not be found and/or removed completely after initial exploration. To prepare for this possibility, the surgeon should determine in advance with the patient and endocrinologist what to do next in this situation – dissect further, perform a hypophysectomy or hemi-hypophysectomy, or stop the operation. The plan for perioperative testing and glucocorticoid treatment varies widely among centers. The conundrum faced in the immediate perioperative period is that withholding glucocorticoids allows for rapid testing and demonstration of remission; however, complete resection of the causative tumor causes AI from prolonged suppression of the HPA axis and concerns for acute decompensation. Abundant evidence has shown that post-pituitary adenomectomy patients are not at risk for an adrenal crisis when monitored closely in an intensive care unit or equivalent setting[33]. Many studies have confirmed that post-operative AI almost always suggests a remission of CD[15,16,17,18, 34]. A standard protocol includes securing serum electrolytes and cortisol, plasma ACTH, capillary blood glucose, blood pressure, and urine specific gravity every 6 h for 24–48 h while withholding all glucocorticoids. Consecutive serum cortisol values less than 2–5 µg/dL (we use < 3 µg/dL) are sufficient to document successful tumor resection and to begin glucocorticoid therapy[35]. Post-operative signs and symptoms of AI including vomiting, hyponatremia, hypoglycemia, and hypotension should also mandate immediate glucocorticoid support. Although not clinically useful in the immediate post-operative period, some investigators have shown that low ACTH and DHEAS levels may be better predictors of long-term remission than serum cortisol[36]. A similar strategy for the management of possible post-operative AI/GWS following unilateral adrenalectomy for nodular adrenal disease has recently been reported. A post-operative day 1 basal cortisol and its response to cosyntropin stimulation can reliably segregate those patients with HPA axis suppression requiring cortisol replacement from those with an intact HPA axis who do not need to be discharged with glucocorticoid therapy[37]. Once remission is achieved, exogenous glucocorticoid replacement should be initiated and maintained during the months required for HPA axis recovery. Several glucocorticoids and dosing options are available (Table 1), and the initial dose is generally 3- to 4-fold higher than the physiologic range and graded based on age, comorbidities, and severity of disease. Fludrocortisone acetate should also be initiated following BLA for patients who receive glucocorticoids other than hydrocortisone, the only glucocorticoid with mineralocorticoid activity. By comparison, post-BLA patients receiving supraphysiologic hydrocortisone doses usually do not need mineralocorticoid support until their dose is tapered to near physiologic replacement. In the acute postoperative period, several medical comorbidities accompanying CS may reverse rapidly and require medication adjustments[35]. In particular, insulin and oral hypoglycemic drugs, potassium-sparing diuretics such as spironolactone, and other cardiovascular drugs are typically tapered or discontinued as glucose counter-regulation and electrolyte balance change rapidly upon cortisol reduction. Due to the high risk of postoperative venous thromboembolism[38,39,40], prophylaxis is frequently recommended and continued for several weeks after discharge. Posterior pituitary manipulation can disturb water balance and result in serum sodium alterations, including transient or permanent central diabetes insipidus, and in rare cases the triphasic response of diabetes insipidus, followed by syndrome of inappropriate secretion of antidiuretic hormone (SIADH), and finally permanent diabetes insipidus[41, 42]. In the first week or two after discharge, the most common cause for readmission is hyponatremia[43, 44], although the mechanisms responsible for this transient SIADH state are not known. For this reason, patients should be instructed to drink only when thirsty and not as an alternative to solid foods or for social reasons for 7–10 days after the surgery. Both diabetes insipidus and SIADH may not manifest for weeks after surgery; consequently, serum sodium should be monitored after hospital discharge as well [42]. Subacute Care: The GWS and HPA Axis Recovery When managing GWS symptoms, it is important to repeatedly emphasize to the patient that not only are GWS symptoms to be expected, but in fact these manifestations portend a favorable prognosis of sustained remission from CS. The most important treatment intervention is frequent reassurance to the patient that GWS typically resolves as the HPA axis recovers. Family members must be included in the conversation to help provide as much support as possible, as patients report that support from family and friends is the most helpful coping mechanism during the recovery process[32]. When appropriate, it may be necessary to provide the patient with temporary disability documentation, since GWS symptoms may be so severe to preclude gainful employment. The patient must know that the myalgias reflect the body’s attempts to repair the muscle damage, similar to the soreness experienced the day after resistance weight training, and these aches will eventually subside. Due to the challenges of differentiating between GWS and AI, a higher glucocorticoid dose can be briefly trialed to assess if this increased glucocorticoid exposure improves symptoms, but late-day dosing should be avoided to support recovery of the circadian rhythm. In parallel, the patient should be encouraged to adequately rest, particularly going to sleep early but limiting daytime sleep to short naps. Several other classes of medications can be trialed to target specific patient symptoms (Table 2). Antidepressants such as fluoxetine, sertraline, and trazodone might help to improve mood, sleep and appetite. A non-steroidal anti-inflammatory medication to address the musculoskeletal discomfort might be used early in the GWS, with the cyclooxygenase type 2 (COX-2) inhibitor celecoxib (100–200 mg once or twice daily) preferred when several weeks of daily treatment is needed, generally not more than 3 months. With anorexia and reduced food intake, adequate protein intake is necessary to allow muscle recovery. Egg whites, nuts, and lean meats are nutritionally dense and generally easy to tolerate despite poor appetite. Table 2 Pharmacotherapy and Ancillary Treatment Options for GWS Symptoms Full size table Following surgical remission, the duration of glucocorticoid taper can vary from 6 to 12 months or more, depending on age, severity of disease, and duration of disease [45, 46]. Monitoring for HPA axis recovery involves both clinical and biochemical assessments. Since the HPA axis is likely to remain suppressed with prolonged supraphysiologic glucocorticoid replacement, the first goal is to shift from all-day dosing to a circadian schedule as soon as possible, such as hydrocortisone 20 mg on rising and 10 mg in the early afternoon by 2–6 weeks after surgery. The advantages of hydrocortisone include rapid absorption for symptom mitigation, the ability to measure serum cortisol as a measure of drug exposure when helpful, and the relatively short half-life [47], which ensures a glucocorticoid-free period in the early morning when it is most critical to avoid prolonged HPA axis suppression and to enhance recovery. The second goal, which should not be attempted until GWS symptoms – particularly the anorexia and myalgias – are considerably improved, is to limit replacement to a single morning dose. Biochemical assessment should begin once patients are taking a physiologic dose of glucocorticoid replacement (total daily dose of hydrocortisone 15 to 20 mg per day) and clinically feel well enough to begin the final stage to discontinuation of glucocorticoid replacement (Fig. 2). Biochemical evaluation begins with basal testing, and dynamic assessment of adrenal function might be necessary to confirm completion of recovery. For basal testing, patients should not take their afternoon hydrocortisone dose (if prescribed) the day before testing and then have a blood draw by 0830 prior to the morning hydrocortisone dose on the day of testing. While a serum cortisol alone is adequate to taper hydrocortisone, a simultaneous plasma ACTH assists in gauging the state of HPA axis recovery. Often the ACTH and cortisol rise gradually in parallel, but sometimes the ACTH rises above the normal range despite a low cortisol, which indicates recovery of the hypothalamus (CRH neuron) and pituitary corticotrophs in advance of adrenal function. Serum DHEAS can remain suppressed for months to years after cortisol normalization, and a low DHEAS does not indicate continued HPA axis suppression. A rapid rise in DHEAS, in contrast, is concerning for disease recurrence, but a slow drift to a measurable amount in parallel with the cortisol rise is consistent with HPA axis recovery. Periodic assessment of electrolytes is prudent to screen for hyponatremia and hypo- or hyperkalemia as medications are changed, particularly diuretics. Hypercalcemia that is parathyroid-hormone independent might be observed during the recovery phase, probably related to the rise in cytokines that accompany resolution of hypercortisolemia[48, 49]. Fig. 2 Glucocorticoid withdrawal algorithm. TDD, total daily dose Full size image Basal testing is performed at 4- to 6-week intervals during glucocorticoid replacement. A rule of thumb is that the AM cortisol in µg/dL plus the morning dose of hydrocortisone in milligrams should sum to 15–20. Thus, once endogenous cortisol production is measurable, the hydrocortisone dose should be not more than 20 mg on arising. Once the AM cortisol rises to near 5 and then 10 µg/dL, the AM hydrocortisone dose is dropped to 15 and then 10 mg, respectively. Once the AM cortisol is 12–14 µg/dL, recovery is essentially complete, and the morning hydrocortisone dose is dropped to 5 mg for 4–6 weeks and then stopped or held for dynamic testing (Fig. 2). A clinical pearl related to HPA axis recovery is that patients who state that they are finally feeling better and getting over the GWS usually have started to make some endogenous cortisol, yet not enough to stop glucocorticoid tapering. Nevertheless, a smidgeon of endogenous cortisol production with the waning of GWS symptoms is a harbinger that HPA axis recovery is imminent. If basal testing is equivocal, dynamic testing might be necessary. The gold standard testing for central AI is the insulin tolerance test, which is rarely used, and metyrapone testing might be employed once the basal cortisol is > 10 µg/dL. Although designed to test for primary adrenal insufficiency, the cosyntropin stimulation test is often employed in this setting due to greater availability, simplicity, and safety than insulin or metyrapone testing. The duration of full HPA axis recovery can be highly variable depending on the individual and postoperative glucocorticoid dosing[50]. GWS During Medical Management of CS Patients who are not surgical candidates or do not have successful remission of CS following surgery may be offered medical treatment or BLA. After BLA, the GWS will ensue without eventual recovery of the HPA axis, so glucocorticoids are tapered until a chronic physiologic replacement dose is reached as described previously. With medical management, patients might also experience GWS, particularly at the onset of treatment. Therefore, patients must be counseled that the typical symptoms of fatigue, myalgias, and anorexia are not only possible but indeed expected, rather than “side effects” of the medication, with two caveats. First, as described for glucocorticoid replacement following surgical remission, the endocrinologist must distinguish GWS from AI due to over-treatment of CS. The same parameters of vomiting, hypotension, and hypoglycemia favor inadequate cortisol exposure and the need for dose reduction or treatment pause and/or supplementation with a potent glucocorticoid such as dexamethasone to reverse an acute event. Second, known adverse effects of the specific drug in use should be considered and excluded. The quandary of distinguishing GWS from over-treatment raises an important principle of medical management: under-dose initially and gauge primarily the severity of GWS symptoms in the first several days. The initial goal of medical therapy is not to rapidly achieve normal cortisol milieu, but rather to “dial in” just enough inhibition of cortisol production or receptor antagonism to precipitate mild to moderate GWS symptoms. Once GWS symptoms appear and/or a typical dose of the medication is achieved, further assessments, including glucose, serum cortisol and/or UFC (except when treated with mifepristone), clinical appearance, and body weight are conducted while the dose is maintained constant until GWS symptoms begin to dissipate. If the patient is not experiencing adequate clinical and/or biochemical benefit from the medication in the absence of GWS symptoms, the dose is gradually raised incrementally. This iterative process might require periodic dose reduction or perhaps even temporarily discontinuing the medication if the patient’s daily living activities are affected at any point in the process. For several medications, a block-and-replacement strategy is an option[3], particularly for very compliant patients for whom a priority is placed on avoidance of over-treatment. This strategy resembles thionamide-plus-levothyroxine therapy for the treatment of Graves disease. The patient is given both a generous dose of medication to completely block endogenous glucocorticoid production, plus simultaneous exogenous glucocorticoid therapy, titrated to replacement dose or greater. This approach allows for greater control over glucocorticoid exposure and low risk of AI, as long as the patient always takes both medications each day. Long-acting pasireotide, for example, would not be an appropriate drug for the block-and-replace strategy. Based on the drug mechanism of action, this block-and-replace strategy is feasible with ketoconazole or levoketoconazole, the 11β-hydroxylase inhibitors osilodrostat and metyrapone, and the adrenolytic agent mitotane (the latter three are off-label uses). Alternatively, the patient might be given a double replacement dose of glucocorticoid to take only if symptoms concerning for over-treatment occur, and the medical therapy for hypercortisolemia is then interrupted until the patient communicates with the endocrinologist. Treatment monitoring with medical management includes biochemical and symptom assessment. For all medications other than mifepristone, normalization of 24-hour UFC is the minimal goal [2]. Basal morning cortisol and late-night salivary cortisol may be more challenging to interpret in the setting of diurnal rhythm loss characteristic of CS. Because mifepristone blocks glucocorticoid receptors, ACTH and cortisol increase with treatment for most forms of CS; dose titration therefore relies on assessment of clinical features, glycemia, body weight, and other metabolic parameters [2]. For occult tumors, periodic imaging to screen for a surgical target and/or tumor regrowth is prudent, and a pause in treatment for repeat surgery might be indicated. The End Game: Comprehensive Recovery for the Patient with CS Besides navigating the GWS and shepherding recovery of the HPA axis, recovery from co-morbidities of CS must be addressed to the extent possible. Hypertension, hyperglycemia, hypokalemia, and dyslipidemia often improve substantially but do not always resolve. Insomnia, skin thinning and bruising, and risk of thrombosis also generally resolve, and associated treatments might be discontinued. Although there is usually an improvement in bone density and decreased fracture risk following correction of CS, anabolic and/or anti-resorptive therapies may be warranted in some patients. The deformities of vertebral compression fractures may be permanent, and some authors have recommended the use of vertebroplasty for symptom relief[51]. Violaceous striae and chronic skin tears might heal with hyperpigmentation, leaving “the scars of Cushing’s,” which can persist for a lifetime. These milestones or minor victories can be used as evidence of healing and encouragement for the patient during the dark days of the GWS, and these changes herald further improvements. Fat redistribution and significant weight loss take some weeks to manifest and usually follow next. The myopathy from CS is an example of a co-morbidity that rarely improves without targeted treatment, and the German Cushing’s Registry has provided evidence for chronic muscle dysfunction following cure of CS[52]. Recent data indicate that a low IGF-1 after curative surgery is associated with long-term myopathy [53]. This persistent myopathy is a common source of chronic fatigue following HPA axis recovery, which is unresponsive to glucocorticoids. For these reasons, an important ancillary modality is physical therapy, and an ideal time to initiate this treatment is at the first signs of HPA axis recovery when the GWS symptoms have subsided. A complete evaluation from an experienced physical therapist should focus on core and proximal muscle strength, balance, and other factors that limit function. Exercises targeting these factors (stand on one foot, sit-to-stand, straight-arm raises with 1- to 5-pound weights) rather than traditional gym exercises (arm curls, bench press, treadmill) are necessary to restore functional status and avoid frustration and injury when the patient is not yet prepared for the latter stages of recovery. Professional supervision of this initial phase is a critical component of the recovery process, and failure to attend to musculoskeletal rehabilitation – as would be routine following survival of a critical illness – risks long-term morbidities from a curable disease. Patients with CS often complain of cognitive defects, which usually improve but may not completely recover following treatment[54, 55]. Glucocorticoids are toxic to the hippocampus, and both rats treated with high-dose corticosterone and patients with CD experience reductions in hippocampal volume, which does not completely return to normal even with correction of hypercortisolemia[56, 57]. Because the hippocampus is an important brain region for memory, the main complaint is impaired formation of new memories and recall of recent events. When significant cognitive dysfunction persists, a formal neuropsychologic testing session is prudent, both to screen for additional sources of memory loss (degenerative brain diseases) and to identify aspects that might be amenable to functional management approaches. Cognitive therapy can be effective for mental health and overall disease coping strategies as well. Finally, for patients undergoing transsphenoidal surgery for CD, complications associated with pituitary surgeries in general should also be considered. Anterior pituitary hormone axes should be assessed biochemically and symptomatically for hypothyroidism and hypogonadism, as hypopituitarism is an independent predictor of decreased quality of life after surgical cure [58]. Hypopituitarism can not only complicate the assessment of GWS with overlapping symptoms such as fatigue, but treatment of hypopituitarism can also be important for GWS recovery. Prior to initiating physical therapy, testosterone replacement in male patients with hypogonadism should be optimized. Hypothyroidism can contribute to hyponatremia and can also slow the metabolism of glucocorticoids. Therefore, optimizing the treatment of hypothyroidism and hypogonadism prior to completing glucocorticoid taper is prudent. Growth hormone deficiency may also be evaluated in symptomatic patients in the setting of other anterior pituitary hormone deficiencies, although formal evaluation is best delayed for at least 6–12 months when HPA axis recovery has occurred or at least the glucocorticoid dose is reduced to a physiologic range [2]. Summary and Final Thoughts After a diagnosis of CS has been well established, a multidisciplinary team of endocrinologists and surgeons must design the best treatment strategy for the patient. Expectations and possible adverse side effects of surgery or pharmacotherapy should be reviewed with the patient. The GWS is a very difficult concept for patients to understand. It seems inconceivable to them that they could possibly feel worse (and that this is a good omen) six weeks after resolution of their hypercortisolism than they do pre-operatively; however, there are no studies that address whether comprehensive pre-operative patient education regarding GWS has any impact on the patient’s post-operative perception and outcome after successful surgery. An addiction metaphor is sometimes helpful: the patient’s body and brain has become addicted to steroids (cortisol) and after steroids are abruptly reduced, their body and brain are dysphoric — much like removal of any other addictive substance (e.g., opioids, alcohol, nicotine). The patient and their care team need to know that this treatment odyssey will be a marathon, not a sprint. It may take as long as 12–18 months for patients to have full HPA axis recovery, regression of GWS, and, most importantly, resolution of the devastating effects of chronic excessive glucocorticoid exposure. Conclusions GWS following surgery or during medical treatment of CS can be challenging to manage. There are currently no standard guidelines for management of GWS, but various available medical and ancillary therapies are discussed here. Studies are needed to better understand the pathophysiology of GWS to guide more targeted treatments. There may be yet unrecognized steroids produced by the adrenal glands, the withdrawal of which contributes to GWS symptoms[59]. Future observational and interventional studies would be beneficial for identifying optimal management options. References Carroll TB, Findling JW (2010) The diagnosis of Cushing’s syndrome. 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Funding XH is supported by grant T32DK07245 from the National Institutes of Diabetes and Digestive and Kidney Diseases. Author information Affiliations Department of Internal Medicine, Division of Metabolism, Endocrinology and Diabetes, University of Michigan Medical School, Ann Arbor, MI, USA Xin He & Richard J. Auchus Department of Medicine, Division of Endocrinology and Molecular Medicine, Medical College of Wisconsin, Milwaukee, WI, USA James W. Findling Endocrinology Center and Clinics, Medical College of Wisconsin, Milwaukee, WI, USA James W. Findling Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI, USA Richard J. Auchus Lieutenant Colonel Charles S. Kettles Veterans Affairs Ann Arbor Healthcare System, Ann Arbor, MI, USA Richard J. Auchus Contributions All authors contributed to the manuscript conception, design, and content. All authors read, edited, and approved the final manuscript. Corresponding author Correspondence to Richard J. Auchus. Ethics declarations Financial Interests Dr. Auchus has received research support from Novartis Pharmaceuticals, Corcept Therapeutics, Spruce Biosciences, and Neurocrine Biosciences and has served as a consultant for Corcept Therapeutics, Janssen Pharmaceuticals, Novartis Pharmaceuticals, Quest Diagnostics, Adrenas Therapeutics, Crinetics Pharmaceuticals, PhaseBio Pharmaceuticals, OMass Therapeutics, Recordati Rare Diseases, Strongbridge Biopharma, and H Lundbeck A/S. Dr. Findling has received research support from Novartis Pharmaceuticals and has served as a consultant for Corcept Therapeutics and Recordati Rare Diseases. Human Subjects and Animals No human subjects or animals were used to collect data for this manuscript. Additional information Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Electronic Supplementary Material Below is the link to the electronic supplementary material. Supplementary Material 1 Rights and permissions Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Reprints and Permissions Cite this article He, X., Findling, J.W. & Auchus, R.J. Glucocorticoid Withdrawal Syndrome following treatment of endogenous Cushing Syndrome. Pituitary (2022). https://doi.org/10.1007/s11102-022-01218-y Download citation Accepted15 March 2022 Published26 April 2022 DOIhttps://doi.org/10.1007/s11102-022-01218-y From https://link.springer.com/article/10.1007/s11102-022-01218-y
  2. Background: In Cushing’s syndrome (CS), chronic glucocorticoid excess (GC) and disrupted circadian rhythm lead to insulin resistance (IR), diabetes mellitus, dyslipidaemia and cardiovascular comorbidities. As undifferentiated, self-renewing progenitors of adipocytes, mesenchymal stem cells (MSCs) may display the detrimental effects of excess GC, thus revealing a promising model to study the molecular mechanisms underlying the metabolic complications of CS. Methods: MSCs isolated from the abdominal skin of healthy subjects were treated thrice daily with GCs according to two different regimens: lower, circadian-decreasing (Lower, Decreasing Exposure, LDE) versus persistently higher doses (Higher, Constant Exposure, HCE), aimed at mimicking either the physiological condition or CS, respectively. Subsequently, MSCs were stimulated with insulin and glucose thrice daily, resembling food uptake and both glucose uptake/GLUT-4 translocation and the expression of LIPE, ATGL, IL-6 and TNF-α genes were analyzed at predefined timepoints over three days. Results: LDE to GCs did not impair glucose uptake by MSCs, whereas HCE significantly decreased glucose uptake by MSCs only when prolonged. Persistent signs of IR occurred after 30 hours of HCE to GCs. Compared to LDE, MSCs experiencing HCE to GCs showed a downregulation of lipolysis-related genes in the acute period, followed by overexpression once IR was established. Conclusions: Preserving circadian GC rhythmicity is crucial to prevent the occurrence of metabolic alterations. Similar to mature adipocytes, MSCs suffer from IR and impaired lipolysis due to chronic GC excess: MSCs could represent a reliable model to track the mechanisms involved in GC-induced IR throughout cellular differentiation. Introduction Glucocorticoids (GCs) regulate a variety of physiological processes, such as metabolism, immune response, cardiovascular activity and brain function (1, 2). Chronic excess and dysregulation of GCs induces Cushing’s syndrome (CS), a complex clinical condition characterized by multisystem morbidities such as central obesity, hypertension, type 2 diabetes mellitus, insulin resistance (IR), dyslipidaemia, fatty liver, hypercoagulability, myopathy and osteoporosis (3–5). In patients with CS, GC secretion does not follow the circadian rhythm and consistently high serum GC levels are observed throughout the day (6, 7). IR, defined as the reduced ability of insulin to control the breakdown of glucose in target organs, represents the common thread among obesity, metabolic syndrome and type 2 diabetes mellitus (8). GCs induce IR, but the mechanisms are complex and not completely understood. Under physiological conditions, the binding of insulin to its receptor on the cell surface induces the autophosphorylation of tyrosine in the insulin receptor substrate (IRS)-1 subunit with a consequent complex cascade of intracellular signals that leads to the inhibition of glycogen synthase kinase 3, the inhibition of apoptosis and the translocation of glucose transporter 4 (GLUT4) to the cell membrane with consequent glucose uptake (9, 10). Several studies have shown how chronic exposure to high levels of GCs reduces IRS-1 phosphorylation and protein expression, resulting in a lack of GLUT4 translocation and a reduction in glucose uptake in adipose tissue (11). In addition, the chronic excess of GCs increases lipoprotein activity and expression with subsequent release of circulating fatty acids, which, in turn, induce the phosphorylation of serine in IRS-1, thus compromising the mechanisms that lead to glucose transport into the cell (12). In recent years, the involvement of mesenchymal stem cells (MSCs) in the onset of different pathologies has been addressed, and for some of them, MSCs have been identified as the real target for lasting therapeutic approaches (13, 14). MSCs are undifferentiated cells inside many tissues that are able to self-renew and differentiate into adipocytes, osteocytes and chondrocytes (15). Adipose tissue, muscle tissue and bone are compromised in CS, so the involvement of MSCs in CS complications has been hypothesized; this was confirmed by our previous work reporting that MSCs isolated from the skin of patients affected by CS showed an altered wound healing process that is recognized as a clinical manifestation of CS (16). In this scenario, it is tempting to speculate that the detrimental effects of excess GC could also affect MSCs, which may represent a promising cellular model to study the mechanisms leading to IR. The choice to use MSCs as a model is particularly interesting, since MSCs are the progenitors of mature adipocytes that may inherit and spread dysregulated mechanisms already present in MSCs. Here, MSCs isolated from the abdominal skin of healthy subjects were treated in vitro with two different GC regimens, mimicking circadian cortisol rhythm and chronic hypercortisolism. Subsequently, cells were stimulated with insulin and glucose three times/day, resembling the normal uptake of food, and both glucose uptake and the expression of selected genes were analyzed to clarify the mechanisms underlying the development of IR and the occurrence of altered carbohydrate and lipid metabolism under chronic exposure to high levels of GCs. Materials and Methods Sample Collection Seven abdominal skin samples were collected from healthy subjects (four males and three females age matched 42.3 ± 3.4) undergoing abdominoplasty at the Clinic of Plastic and Reconstructive Surgery, Università Politecnica delle Marche. Patients gave their informed consent; the study was approved by the Università Politecnica delle Marche Ethical Committee and conducted in accordance with the Declaration of Helsinki. The main demographical and clinical characteristics of enrolled patients are summarized in Table 1. TABLE 1 Table 1 Demographical and functional characteristics of enrolled patients. Isolation and Characterization of MSCs Cells were isolated from abdominal skin and then cultured with a Mesenchymal Stem Cell Growth Medium bullet kit (MSCGM, Lonza Group® Ltd) as previously described (16) and characterized according to the criteria by Dominici (15). Plastic adherence, immunophenotype and multipotency were tested as already described (17–19). After the Oil Red staining, a semiquantitative analysis was carried out by dissolving the staining with 100% isopropanol and the absorbance was measured at 510nm in a microplate reader (Thermo Scientific Multiskan GO Microplate Spectrophotometer, Milano, Italy). In addition, the expression of PPAR-γ (peroxisome proliferator-activated receptor gamma) and C/EBP-α (CCAAT/enhancer-binding protein alpha) was tested by Real time PCR to confirm the adipocytes differentiation. Undifferentiated MSCs were used as control (C-MSCs). Briefly, after 21 days of culture in adipocytes differentiation medium, 2.5x105 cells from the 7 patients were collected; cDNA synthesis and qRT–PCR were carried out as previously described (20). The primer sequences are summarized in Table 2. mRNA expression was calculated by the 2−ΔΔCt method (21), where ΔCt=Ct (gene of interest)—Ct (control gene) and Δ (ΔCt)=ΔCt (differentiated MSCs)—ΔCt (undifferentiated MSCs). Genes were amplified in triplicate with the housekeeping genes RPLP0 (Ribosomal Protein Lateral Stalk Subunit P0) and GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) for data normalization. Of the two, GAPDH was the most stable and was used for subsequent normalization. The values of the relative expression of the genes are mean ± SD of three independent experiments. TABLE 2 Table 2 Primer sequences. Experimental Design: In Vitro Reproduction of Both Circadian Rhythm and Chronic Excess GCs and Food Uptake Cells were treated with two different GC regimens: some were given lower, circadian-decreasing GC doses (Lower and Decreasing Exposure, LDE), some were exposed to persistently higher GC doses (Higher and Constant Exposure, HCE), to mimic in vitro either the preserved circadian rhythm or its pathologic abolishment in CS, as shown in Figure 1A and described in detail below. LDE cells were first exposed (8:00 a.m.-9:50 a.m.) to 500 nM hydrocortisone (MedChemExpress, MCE, Monmouth Junction, NJ, USA) and then to decreasing concentrations by replacing the medium with a fresh medium containing 250 nM hydrocortisone (9:50 a.m.-01:50 p.m.) and 100 nM (01:50 p.m.-05:50 p.m. and 05:50 p.m.-08:00 a.m.) of hydrocortisone (22). To mimic CS, HCE cells were exposed to 500 nM hydrocortisone for 24/24 hours. The 500 nM hydrocortisone medium was replaced with fresh medium at the same time as the physiological condition medium was changed. FIGURE 1 Figure 1 (A) In vitro reproduction of preserved versus abolished GC circadian rhythm. (B). Daily experimental design. Cells were starved and exposed three times/day to 10 mM glucose with or without prestimulation with 1 μM insulin (Sigma–Aldrich, Milano, Italy) to resemble daily food uptake. Protocol is resumed in Figure 1B. Cells derived from each single patient were divided into six experimental groups (Exp): 1) Exp 1, GLU: Cells exposed to glucose; 2) Exp 2, INS+GLU: Cells stimulated with insulin before glucose exposure; 3) Exp 3, LDE+GLU: LDE cells treated with glucose; 4) Exp 4, HCE+GLU: HCE cells treated with glucose; 5) Exp 5, LDE+INS+GLU: LDE cells stimulated with insulin before glucose exposure; 6) Exp 6, HCE+INS+GLU: HCE cells stimulated with insulin before glucose exposure. In detail, cells were seeded in DMEM/F-12+10% FBS (Corning, NY, USA). After 24 hours, the medium was changed, and the cells were starved overnight with Advanced DMEM/F-12 w/o glucose (Lonza) with 0.5% FBS. At 8:00 a.m., starvation medium was replaced with a new medium containing hydrocortisone 500 nM for 30 minutes in groups exposed to GCs. After washing, the cells were glucose starved with KRPH buffer (20 mM HEPES, 5 mM KH2PO4, 1 mM MgSO4, 1 mM CaCl2, 136 mM NaCl and 4.7 mM KCl, pH 7.4) containing 2% BSA (Sigma–Aldrich) and hydrocortisone for 40 minutes. Cells from Exp 2, 5 and 6 were then stimulated with 1 μM insulin (Sigma–Aldrich) for 20 minutes. Finally, 10 mM glucose was added, and the time sampling was after 20 minutes. The same protocol starting with starvation for 2 hours in DMEM/F-12 w/o glucose was repeated two times during the day, and the hydrocortisone concentration in the medium of LDE and HCE cells varied accordingly. To evaluate the long-term impact on metabolism and IR, the experiment was performed for three days with repeated sampling times after glucose administration: T1, T2 and T3 at 9:50 a.m., 1:50 p.m., 5:50 p.m. of the first day; T4, T5 and T6 at 9:50 a.m., 1:50 p.m., 5:50 p.m. of the second day; T7 at 1:50 p.m. of the third day (Figure 1A). The entire experiment (Exp 1-6, from T1 to T7) was repeated thrice, and data are reported as mean± standard deviation (SD) over the three independent experiments. XTT Assay To evaluate whether repeated starvation steps and treatments would affect cell viability and consequently influence the measurement of glucose uptake, an XTT assay (Sigma–Aldrich) was initially performed. A total of 3x103 cells/well belonging to Exp 1, 2, 4 and 6 derived from the 7 patients were plated in a 96-well plate and treated as previously described. Another experimental group was included as a control, consisting of cells continuously cultured in starvation medium (STARVED CTRL). The XTT assay was performed at the end of each day (T3, T6 and T7 sampling times) following the manufacturer’s instructions. The experiment was repeated thrice, and data are reported as mean ± SD over the three independent experiments. MSCs Responsiveness to Insulin To evaluate whether MSCs were responsive to insulin, glucose uptake and the cellular localization of GLUT4 were first evaluated in MSCs not treated with GCs (Exp 1 and 2) from T1 to T6. For the glucose uptake assay, 3x103 cells/well were plated in a 96-well plate and treated according to the above protocol; after insulin stimulation, 10 mM of 2-deoxyglucose (2-DG) was added for 20 minutes, and a colorimetric assay was performed following the manufacturer’s instructions. The readings were at 420 nm in a microplate reader (Thermo Scientific Multiskan GO Microplate Spectrophotometer, Milano, Italy). For the cellular distribution of GLUT4, 1.5x104 cells (Exp 1 and 2 derived from the 7 patients) were seeded in triplicate on coverslips and treated as indicated before until T5 sampling time. Cells were then washed, fixed with 4% PFA and permeabilized for 30 min. Subsequently, cells were incubated with anti-GLUT4 antibody (Santa Cruz Biotechnology, USA) followed by treatment for 30 min with a goat anti-mouse FITC-conjugated antibody (23). Finally, coverslips were mounted on glass slides in Vectashield (Vectorlabs, CA, USA), and confocal imaging was performed using a Zeiss LSM510/Axiovert 200 M microscope with an objective lens at 20× magnification (24). Line scans were acquired excluding nuclear regions, and GLUT4 immunofluorescence was analyzed as described elsewhere. Effects of Different GC Regimens on Glucose Uptake and GLUT4 Translocation After having proven that MSCs could function as a cellular model, since they were responsive to insulin, the potential effects of both GC regimens on glucose uptake were evaluated. Glucose uptake was measured in the experimental groups treated with GCs (Exp 3, 4, 5 and 6 derived from the 7 patients), and GLUT4 translocation was evaluated in cells from Exp 4 and 6 as described above. Expression of Genes Involved in the Development of IR The expression of selected genes, such as LIPE, ATGL, IL-6 and TNF-α (coding for hormone-sensitive Lipase E, Adipose TriGlyceride Lipase, InterLeukin-6 and Tumour Necrosis Factor-α, respectively), was evaluated to clarify the mechanisms involved in the development of IR in MSCs (25–28). A total of 2.5x105 cells/well belonging to Exp 5 and 6 from the 7 patients were seeded in triplicates in a 6-well plate and treated following the experimental design. Pellets were collected at T2 and T7, which were chosen as sampling times representing acute and chronic exposure to GCs. RNA extraction, cDNA synthesis and qRT–PCR were carried out as previously described (20). The primer sequences are summarized in Table 2. mRNA expression was calculated by the 2−ΔΔCt method (21), where ΔCt=Ct (gene of interest)—Ct (control gene) and Δ (ΔCt)=ΔCt (HCE+INS+GLU)—ΔCt (LDE+INS+GLU). All selected genes were amplified in triplicate with the housekeeping genes RPLP0 and GAPDH for data normalization. Of the two, GAPDH was the most stable and was used for subsequent normalization. The values of the relative expression of the genes are mean ± SD of three independent experiments. Statistical Analysis For statistical analysis, GraphPad Prism 6 Software was used. All data are expressed as the mean ± standard deviation (SD). For parametric analysis all groups were first tested for normal distribution by the Shapiro–Wilk test (29) and comparison between 2 groups were performed by unpaired Student’s t test. For two-sample comparisons, significance was calculated by unpaired t-Student’s test while the ordinary one-way ANOVA test was used for multiple comparison (Tukey’s multiple comparisons test). Significance was set at p value < 0.05. Results MSCs Isolation and Characterization From Abdominal Skin MSCs isolated from abdominal skin appeared homogeneous with a fibroblastoid morphology and showed adherence to plastic. According to Dominici’s criteria (17), cells were positive for CD73, CD90 and CD105, and negative for HLA-DR, CD14, CD19, CD34 and CD45. Cells were also able to differentiate towards osteogenic, chondrogenic and adipogenic lineages. After 7 days of osteogenic differentiation, cells showed alkaline phosphatase activity (Figure 2A), and after 14 days, cells were strongly positive for alizarin red staining (Figure 2B). Chondrogenic differentiation was achieved after 30 days, as shown by safranin-O staining (Figure 2C). MSCs differentiation into adipocytes occurred after 21 days, as evidenced by the presence of lipid vacuoles after oil red staining (Figure 2D). Its quantification confirmed as the amount of lipid vacuoles was higher in differentiated cells than in control cells (C-MSCs; Figure 2E). The expression of PPAR-γ and C/EBP-α was tested after 21 days of culture in differentiating medium and it was higher in differentiated than in undifferentiated MSCs (Figures 2F, G). FIGURE 2 Figure 2 Multilineage differentiation of MSCs from abdominal skin. Representative images of MSCs derived from the seven patients and differentiated towards osteogenic lineage as assessed by ALP reaction (A, Scale bar 100μm) and Alizarin red staining (B, Scale bar 100μm); chondrogenic lineage as indicated by Safranin-O staining (C, Scale bar 100 μm); adipocyte lineage as confirmed by Oil red staining (D, Scale bar 100μm); (E) Oil Red staining quantification. Data are expressed as mean ± SD of the absorbance read for undifferentiated and differentiated cells (C-MSCs and DIFF-MSCs respectively). (F, G) Expression of PPAR-γ and C/EBP-α by RT-PCR in differentiated vs undifferentiated MSCs towards adipogenic lineage. Data are expressed as mean ± SD (over three independent experiments) of the X-fold (2−ΔΔCt method) of differentiated MSCs compared to undifferentiated MSCs, arbitrarily expressed as 1, where ΔCt=Ct (gene of interest)—Ct (control gene) and Δ (ΔCt)=ΔCt (DIFF-MSCs)—ΔCt (C-MSCs). Unpaired t-Student’s test; ***p<0.001, ****p<0.0001. Cell Viability by XTT Assay Figure 3 shows that the viability of the STARVED CTRL (cells continuously cultured in starvation medium) was significantly increased compared to that of the HCE cells at T3 but not thereafter. Although repeated interventions caused a proliferation block earlier than starvation alone, the different treatments did not interfere with vitality, and further analyses on glucose uptake were unaffected by different cell mortality during the experiment. FIGURE 3 Figure 3 XTT test. The bars indicate cells’ viability at T3, T6 and T7 sampling times. One-way ANOVA; **p < 0.01 vs STARVED CTRL inside each time sampling. STARVED CTRL: cells continuously cultured in starvation medium; GLU: Cells exposed to glucose; INS+GLU: Cells stimulated with insulin before glucose exposure; HCE+GLU: HCE (Higher and Constant Exposure) cells treated with glucose; HCE+INS+GLU: HCE cells stimulated with insulin before glucose exposure. Data are expressed as mean ± SD of the absorbance read for MSCs derived from each single patient over three independent experiments. MSCs Responsiveness to Insulin As shown in Figure 4, stimulation with insulin significantly increased glucose uptake at T1, T2, T4 and T5, whereas at T3 and T6, the level of glucose uptake did not differ significantly between insulin-treated (Exp2, INS+GLU) and nontreated (Exp1, GLU) cells. FIGURE 4 Figure 4 Responsiveness of MSCs to insulin. The bars show the glucose uptake expressed in pM at T1, T2, T3, T4, T5 and T6 in insulin-stimulated or non-stimulated MSCs. Unpaired t-Student’s test; *p < 0.05, **p < 0.01. GLU: Cells exposed to glucose; INS+GLU: Cells stimulated with insulin before glucose exposure. Data are expressed as mean ± SD of the readings for MSCs derived from each single patient over three independent experiments. Notably, in the absence of insulin, GLUT4 was more localized in the perinuclear area of the cells (Figures 5A, E). Insulin stimulation enhanced GLUT4 translocation towards the plasma membrane (Figures 5B, F). FIGURE 5 Figure 5 GLUT4 translocation. Representative confocal images of GLUT4 in MSCs derived from the seven patients and stimulated (B, D) or not (A, C) with insulin and exposed to 500nM of GCs (C, D). The graphs (E–H) show the fluorescence ratio between the edge and the centre of the cell; yellow arrows indicate the portion of cell subjected to analysis. GLU: Cells exposed to glucose; INS+GLU: Cells stimulated with insulin before glucose exposure; HCE+GLU: HCE (Higher and Constant Exposure) cells treated with glucose; HCE+INS+GLU: HCE cells stimulated with insulin before glucose exposure. Effects of LDE and HCE on GCs on Glucose Uptake and GLUT4 Translocation In LDE cells, insulin induced a significant increase in glucose uptake at all sampling times (Figure 6). Conversely, GC administration did not interfere with glucose uptake by HCE cells in the acute period (T1, T2) but led to a significant decrease in glucose uptake when prolonged (T3, T5, T6, T7). Accordingly, GLUT4 translocation was inhibited irrespective of insulin stimulation (Figures 5C, G and D, H) in HCE cells. FIGURE 6 Figure 6 Glucose uptake in MSCs undergoing a LDE or a HCE to GCs. The bars represent the glucose uptake expressed in pM at T1 (9:50 a.m. first day, A), T2 (1:50 p.m. first day, B), T3 (5:50 p.m. first day, C), T4 (9:50 a.m. second day, D), T5 (1:50 p.m. second day, E), T6 (5:50 p.m. second day, F) and T7(1:50 p.m. third day, G) in MSCs undergoing a LDE or a HCE to GCs and stimulated or not with insulin. One-way ANOVA; *p < 0.05,**p < 0,01,***p < 0,001. LDE+GLU: LDE (Lower and Decreasing Exposure) cells treated with glucose; HCE+GLU: HCE (higher and Constant Exposure) cells treated with glucose; LDE+INS+GLU: LDE cells stimulated with insulin before glucose exposure; HCE+INS+GLU: HCE cells stimulated with insulin before glucose exposure. Data are expressed as mean ± SD of the readings for MSCs derived from each single patient over three independent experiments. Effect on Lipolysis and Development of IR: Gene Expression A downregulation of both genes involved in the breakdown of triglycerides to fatty acids (LIPE and ATGL) was found at T2, whereas at T7, their expression was significantly increased in HCE cells compared to LDE cells. At T7, HCE cells showed a significant increase in the expression of both IL-6 and TNF-α genes, whereas at T2, only the expression of TNF-α was lower than that of LDE cells (Figure 7). FIGURE 7 Figure 7 Gene expression in MSCs undergoing a LDE or a HCE to GCs. The bars display the expression of genes referred specifically to the development of IR: (A): LIPE, (B): ATGL, (C): IL-6 and (D): TNF-α at T2 and T7 sampling times. LDE+GLU+INS: LDE (Lower and Decreasing Exposure) cells stimulated with insulin before glucose exposure; HCE+GLU +INS: HCE (higher and Constant Exposure) cells stimulated with insulin before glucose exposure. Data are expressed as mean ± SD (over three independent experiments) of the X-fold (2−ΔΔCt method) of HCE+INS+GLU compared to LDE+INS+GLU arbitrarily expressed as 1, where ΔCt=Ct (gene of interest)—Ct (control gene) and Δ (ΔCt)=ΔCt (HCE+INS+GLU)—ΔCt (LDE+INS+GLU). Unpaired t-Student’s test; *p < 0.05,**p < 0.01,***p < 0.001;****p < 0.0001. Discussion The clinical presentation of CS is well established, but the mechanisms underlying the onset of some of its complications, IR above all, have not yet been fully understood and may involve tissue-specific players. As progenitors of specialized cellular lines that are directly implicated in the progression of chronic GC excess-induced damage (such as adipocytes, skeletal muscle cells and osteocytes), MSCs are of particular interest: in a previous study, we showed that MSCs derived from the skin of patients with CS displayed dysregulated inflammatory markers and altered expression of genes related to wound healing, demonstrating not only how they could be a useful cellular model to study this event but also their potential contribution to the development of CS manifestations (16). With this premise, we hypothesized that MSCs exposed to excess GC encounter altered glucose uptake mechanisms, which are then inherited and consolidated by their derived, specialized cells. Our work aimed to explore and compare the effects of two different GC regimens (LDE- Lower and Decreasing Exposure- and HCE- Higher and Constant Exposure) on glucose and lipid metabolism in MSCs. First, MSCs were isolated from abdominal skin and characterized by confirming their undifferentiated state (15). To faithfully reproduce the circadian variations in GC concentrations and food intake, cells were treated by following an articulated protocol (Figure 1). It is well established that insulin stimulation promotes glucose uptake via GLUT4 translocation (30–32) in adipocytes and skeletal muscle cells, but the same mechanism has not yet been demonstrated for MSCs. Therefore, the responsiveness of MSCs to insulin, as well as the involvement of GLUT4 in glucose uptake, were addressed before evaluating the effects of GCs. We demonstrated that the exposure of MCSs to insulin increased their glucose uptake and insulin-induced GLUT4 translocation with mechanisms that are similar to those described for adipocytes and muscle cells by confocal imaging. In contrast to what was previously reported for adipocytes (33, 34), GLUT4 expression before insulin stimulation occurred in the cytoplasmic, perinuclear and nuclear compartments in a nonvacuolized pattern. The same localization was observed by Tonack et al. in mouse embryonic stem cells (35). As in adipocytes, the protein translocated on the cell surface, favoring glucose uptake after insulin stimulation. These results opened the second part of the research aimed at evaluating the IR-inducing effects of GCs on MSCs. MSCs were exposed to two different GC regimens: in LDE cells, insulin stimulation always caused an increase in glucose uptake, confirming that insulin sensitivity of MSCs is not altered when cortisol circadian rhythm is preserved; conversely, in HCE cells, an impaired response to insulin was observed, as demonstrated by their decreased glucose uptake. These observations were also confirmed by confocal data, showing how excess GC blocked the insulin-induced translocation of GLUT4 from the intracellular compartment to the cell surface. Of note, a reduction in glucose uptake was not detected in earlier sampling times (T1, T2) but later (T3, T5, T6, T7). These results, taken together with the lack of GLUT4 translocation, suggest that IR develops over time. The development of IR following chronic exposure to GCs has been widely demonstrated in differentiated cells such as adipocytes, hepatocytes, muscle and endothelial cells (36–38), but to our knowledge, this has never been observed in human stem cells before. Our results are in line with those by Gathercole et al. (12), who reported increased insulin-stimulated glucose uptake in a human immortalized subcutaneous adipocyte line (Chub-S7) after acute exposure to dexamethasone, as well as to hydrocortisone (up to 48 hours, in a dose- and time-dependent manner for the latter), thus proposing that the development of GC-induced obesity was promoted by enhanced adipocyte differentiation. However, it must be noted that although Chub-S7 are not fully differentiated adipocytes, they cannot be considered MSCs. In our study, MSCs showed transient signs of IR at T3. In our opinion, this finding represents a physiologic phenomenon and is in line with previous findings in healthy volunteers who were administered hydrocortisone at two different time points and whose endogenous cortisol production was suppressed by metyrapone and nutrient intake was controlled by means of a continuous glucose infusion (39😞 subjects receiving hydrocortisone in the evening showed a more pronounced delayed hyperglycaemic effect than those taking hydrocortisone in the morning (39). Persistent signs of IR in our MSCs appeared even earlier (from T5, after 30 hours of HCE to GCs) than Gathercole’s Chub-S7 (12😞 the ability of MSCs to develop early documentable and conceptually plausible alterations, which can be tracked even once differentiated, further confirms that they are a reliable model for physiopathology studies. The relationship between insulin and lipolysis is bidirectional: inhibition of lipolysis is mainly due to insulin (24), but different mechanisms have been identified where increased lipolysis is involved in the impairment of insulin sensitivity (25, 40). Boden et al. (41) reported that increasing circulating nonesterified fatty acid (NEFA) levels by lipid infusion induced transient IR. To obtain a clearer picture of the possible mechanisms involved in the development of IR in MSCs, we analyzed the expression of LIPE and ATGL genes at different timepoints. We found that HCE cells showed an initial reduction (T2), followed by a significant increase (T7), in the expression of LIPE and ATGL genes compared to LDE cells. The results from previous works on this topic are partially conflicting: Slavin (42) and Villena (43) found upregulated expression of the LIPE and ATGL genes, respectively, after a short treatment with GCs, but studies examining the effects of prolonged GC administration suggested that the acute induction of systemic lipolysis by GCs was not sustained over time (44). However, in these in vitro studies, cells were never treated with insulin, whose counterregulatory effect on lipolysis could not be highlighted. Notably, diabetic patients with CS show an increased activation of lipolysis due to IR (44). Our results fully reflect this scenario, showing that the lipolytic effects are even more marked once insulin levels fail to compensate for associated IR. LIPE and ATGL gene expression was downregulated at T2, when IR had not yet been reached; at T7, when chronic exposure to high GC levels compromised insulin sensitivity, both lipolysis-related enzymes were overexpressed. Of note, increased expression of LIPE and ATGL genes in the presence of IR was also reported by Sumuano et al. in mature adipocytes (37). Given its ability to decrease the tyrosine kinase activity of the insulin receptor, TNF-α is an important mediator of IR in obesity and type 2 diabetes mellitus (26). IL-6 is notably associated with IR by both sustaining low-grade chronic inflammation (45) and impairing the phosphorylation of insulin receptor and IRS-1 (27). In agreement with these statements, TNF-α and IL-6 expression was lower before IR induction (T2) and higher after prolonged exposure (T7) in HCE cells than in LDE cells, further confirming the importance of preserved circadian GC rhythmicity to prevent the occurrence of metabolic alterations. Conclusions MSCs derived from skin could be a good human model for studying the toxic effects of GCs. Like mature adipocytes, they are responsive to insulin stimulation that promotes glucose uptake via GLUT4 translocation, and their chronic exposure to excessive levels of GCs induces the development of IR. For differentiated cells, impaired lipolysis is observed in MSCs once IR has arisen. Furthermore, MSCs could be a promising model to track the mechanisms involved in GC-induced IR throughout cellular differentiation. Functional analyses will be necessary to elucidate the mechanisms behind these first descriptive results and overcame the actual weakness of this research. In addition, co-cultures with MSCs and mature adipocytes will be performed to investigate the crosstalk between these two cell types. Data Availability Statement The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. Ethics Statement The studies involving human participants were reviewed and approved by Università Politecnica delle Marche Ethical Committee. The patients/participants provided their written informed consent to participate in this study. Author Contributions Conceptualization, MO and GA. Methodology, MDV and MM. Formal analysis, MDV, VL, and CL. Data curation, GDB and GG. Writing—original draft preparation, MO and MDV. Writing—review and editing, MO, GA, and MM. Supervision, MO and GA. All authors have read and agreed to the published version of the manuscript. Funding This work was supported by 2017HRTZYA_005 project grant. Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 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Front. Endocrinol. 13:816229. doi: 10.3389/fendo.2022.816229 Received: 16 November 2021; Accepted: 20 January 2022;Published: 24 February 2022. Edited by: Pierre De Meyts, Université Catholique de Louvain, Belgium Reviewed by: Jacqueline Beaudry, University of Toronto, CanadaMałgorzata Małodobra-Mazur, Wroclaw Medical University, Poland Copyright © 2022 Di Vincenzo, Martino, Lariccia, Giancola, Licini, Di Benedetto, Arnaldi and Orciani. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Giorgio Arnaldi, g.arnaldi@univpm.it †These authors have contributed equally to this work and share first authorship ‡These authors have contributed equally to this work and share last authorship Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. From https://www.frontiersin.org/articles/10.3389/fendo.2022.816229/full
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