Sparsely Granulated Corticotroph Pituitary Macroadenoma Presenting with Pituitary Apoplexy Resulting in Remission of Hypercortisolism

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Highlights

• We describe a rare case of a patient with a sparsely granulated corticotroph pituitary macroadenoma with pituitary apoplexy who underwent transsphenoidal resection resulting in remission of hypercortisolism.
• Corticotroph adenomas are divided into densely granulated, sparsely granulated and Crooke’s cell tumors.
• macroadenomas account for 7-23% of patients with pituitary corticotroph adenomas
• Sparsely granulated corticotroph tumors are associated with longer duration of Cushing disease prior to diagnosis, larger tumor size at diagnosis, decreased immediate remission rate, increased proliferative marker Ki-67 and increased recovery time of hypothalamic-pituitary-adrenal axis after surgery.
• Granulation pattern is an important clinicopathological distinction impacting the behavior and treatment outcomes of pituitary corticotroph adenomas

Abstract

Background

/Objective: Pituitary corticotroph macroadenomas, which account for 7% to 23% of corticotroph adenomas, rarely present with apoplexy. The objective of this report is to describe a patient with a sparsely granulated corticotroph tumor (SGCT) presenting with apoplexy and remission of hypercortisolism.

Case Report

A 33-year-old male presented via ambulance with sudden onset of severe headache and nausea/vomiting. Physical exam revealed bitemporal hemianopsia, diplopia from right-sided third cranial nerve palsy, abdominal striae, facial plethora, dorsal and supraclavicular fat pad. Magnetic resonance imaging (MRI) demonstrated a 3.2 cm mass arising from the sella turcica with hemorrhage compressing the optic chiasm, extension into the sphenoid sinus and cavernous sinus. Initial investigations revealed plasma cortisol of 64.08 mcg/dL (Reference Range (RR), 2.36 – 17.05). He underwent emergent transsphenoidal surgery. Pathology was diagnostic of SGCT. Post-operatively, cortisol was <1.8ug/dL (RR, 2.4 – 17), adrenocorticotropic hormone (ACTH) 36 pg/mL (RR, 0 – 81), thyroid stimulating hormone (TSH) 0.07 uIU/mL (RR, 0.36 – 3.74), free thyroxine 1 ng/dL (RR, 0.8 – 1.5), luteinizing hormone (LH) <1 mIU/mL (RR, 1 – 12), follicle stimulating hormone (FSH) 1 mIU/mL (RR, 1 – 12) and testosterone 28.8 ng/dL (RR, 219.2 – 905.6) with ongoing requirement for hydrocortisone, levothyroxine, testosterone replacement and continued follow-up.

Discussion

Corticotroph adenomas are divided into densely granulated, sparsely granulated and Crooke’s cell tumors. Sparsely granulated pattern is associated with larger tumor size and decreased remission rate after surgery.

Conclusion

This report illustrates a rare case of hypercortisolism remission due to apoplexy of a SGCT with subsequent central adrenal insufficiency, hypothyroidism and hypogonadism.

Keywords

pituitary apoplexy
pituitary macroadenoma
pituitary tumor
sparsely granulated corticotroph tumor
Cushing disease

Introduction

The incidence of Cushing Disease (CD) is estimated to be between 0.12 to 0.24 cases per 100,00 persons per year1,2. Of these, 7-23% are macroadenomas (>1 cm)345. Pituitary apoplexy is a potentially life-threatening endocrine and neurosurgical emergency which occurs due to infarction or hemorrhage in the pituitary gland. Apoplexy occurs most commonly in non-functioning macroadenomas with an estimated prevalence of 6.2 cases per 100,000 persons and incidence of 0.17 cases per 100,00 persons per year6. Corticotroph macroadenoma presenting with apoplexy is uncommon with only a handful of reports in the literature7. We present a case of a sparsely granulated corticotroph (SGCT) which presented with apoplexy leading to remission of hypercortisolism and subsequent central adrenal insufficiency.

Case Presentation

A 33-year-old male who was otherwise healthy and not on any medications presented to a community hospital with sudden and severe headache accompanied by hypotension, nausea, vomiting, bitemporal hemianopsia and diplopia. Computed Tomography (CT) scan of the brain demonstrated a hyperattenuating 2.0 cm x 2.8 cm x 1.5 cm mass at the sella turcica with extension into the right cavernous sinus and encasement of the right internal carotid arteries (Figure 1A). He was transferred to a tertiary care center for neurosurgical management with endocrinology consultation post-operatively.

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Figure 1. hyperattenuating 2.0 cm x 2.8 cm x 1.5 cm mass at the sella turcica on unenhanced CT (A); MRI demonstrated a 1.9 cm x 3.2 cm x 2.4 cm heterogeneous mass on T1 (B) and T2-weighted imaging (C) showing small hyperintense areas in solid part of the sella mass with flattening of the optic chiasm, remodeling/dehiscence of the floor of the sella and extending into the right cavernous sinus with at least partial encasement of the ICA

In retrospect, he reported a 3-year history of ongoing symptoms of hypercortisolism including increased central obesity, dorsal and supraclavicular fat pad, facial plethora, abdominal purple striae, easy bruising, fatigue, decreased libido and erectile dysfunction. Notably, at the time of presentation he did not have a history of diabetes, hypertension, osteoporosis, fragility fractures or proximal muscle weakness. He fathered 2 children previously. His physical examination was significant for Cushingoid facies, facial plethora, dorsal and supraclavicular fat pads and central obesity with significant axillary and abdominal wide purple striae (Figure 2). Neurological examination revealed bitemporal hemianopsia, right third cranial nerve palsy with ptosis and impaired extraocular movement. The fourth and sixth cranial nerves were intact as was the rest of his neurological exam. These findings were corroborated by Ophthalmology.

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Figure 2. Representative images illustrating facial plethora (A); abdominal striae (B, C); supraclavicular fat pad (D); dorsal fat pad (E)

Initial laboratory data at time of presentation to the hospital included elevated plasma cortisol of 64.08ug/dL (RR, 2.36 – 17.05), ACTH was not drawn at the time of presentation, normal TSH 0.89 mIU/L (RR, 0.36 – 3.74), free thyroxine 0.91ng/dL (RR, 0.76 – 1.46), evidence of central hypogonadism with low total testosterone 28.8 ng/dL (RR, 219.2 – 905.6) and inappropriately normal luteinizing hormone (LH) 1mIU/mL (RR, 1 – 12) and follicle stimulating hormone (FSH) 3mIU/mL (RR, 1 – 12), low prolactin <1 ng/mL (RR, 3 – 20), and normal insulin growth factor – 1 (IGF–1) 179ng/mL (RR, 82 – 242).

A pituitary gland dedicated MRI was performed to further characterize the mass, which re-demonstrated a 1.9 cm x 3.2 cm x 2.4 cm heterogenous mass at the sella turcica extending superiorly and flattening the optic chiasm, remodeling of the floor of the sella and bulging into the sphenoid sinus and extending laterally into the cavernous sinus with encasement of the right internal carotid artery (ICA). As per the radiologist’s diagnostic impression, this appearance was most in keeping with a pituitary macroadenoma with apoplexy (Figure 1B – C).

The patient underwent urgent TSS and decompression with no acute complications. Pathological examination of the pituitary adenoma showed features characteristic of sparsely granulated corticotroph pituitary neuroendocrine tumor (adenoma)8, with regional hemorrhage and tumor necrosis (apoplexy). The viable tumor exhibited a solid growth pattern (Figure 3A), t-box transcription factor (T-pit) nuclear immunolabeling (Figure 3B), diffuse cytoplasmic CAM5.2 (low molecular weight cytokeratin) immunolabeling (Figure 3C), and regional weak to moderate intense granular cytoplasmic ACTH immuno-staining (Figure 3D). The tumor was immuno-negative for: pituitary-specific positive transcription factor 1 (Pit-1) and steroidogenic factor 1 (SF-1) transcription factors, growth hormone, prolactin, TSH, FSH, LH, estrogen receptor-alpha, and alpha-subunit. Crooke hyalinization was not identified in an adjacent compressed fragment of non-adenomatous anterior pituitary tissue. Ki-67 immunolabeling showed a 1.5% proliferative index (11 of 726 nuclei).

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Figure 3. Hematoxylin phloxine saffron staining showing adenoma with solid growth pattern (A); immunohistochemical staining showing T-pit reactivity of tumor nuclei (B); diffuse cytoplasmic staining for cytokeratin CAM5.2 (C); and regional moderately intense granular cytoplasmic staining for ACTH (D). Scale bar = 20 μm

Post-operatively, he developed transient central diabetes insipidus requiring desmopressin but resolved on discharge. His postoperative cortisol was undetectable, ACTH 36 pg/mL (RR, 0 – 81), TSH 0.07 mIU/mL (RR, 0.36 – 3.74), free thyroxine 1 ng/dL (RR, 0.8 – 1.5), LH <1mIU/mL (RR, 1 – 12), FSH 1 mIU/mL (RR, 1 – 12) and testosterone 28.8 ng/dL (RR, 219.2 – 905.6) (Table 1 and Figure 4). One month later, he reported 15 pounds of weight loss and a 5-inch decrease in waist circumference. He also noted a reduction in the dorsal and supraclavicular fat pads, facial plethora, and Cushingoid facies as well as fading of the abdominal stretch marks. His visual field defects and right third cranial nerve palsy resolved on follow up with ophthalmology post-operatively. Repeat MRI six months post-operatively showed minor residual soft tissue along the floor of the sella. He is being followed by Neurosurgery, Ophthalmology, and Endocrinology for monitoring of disease recurrence, visual defects, and management of hypopituitarism.

Table 1. Pre- and post-operative hormonal panel

POD -1 POD 0 POD1 POD2 POD3 POD16 6 -9 months Comments
Cortisol(2.4 – 17 ug/dL) 64↓ 32↓ 11↓ <1.8↓ <1.8↓ 1.8↓ HC started POD3 post bloodwork
ACTH(0 – 81 pg/mL) 41↓ 36↓ 28↓ 13↓
TSH(0.36 – 3.74 uIU/mL) 0.89 0.43 0.12↓ 0.07↓ 0.05↓ 0.73
Thyroxine, free(0.8 – 1.5 ng/dL) 0.9 0.9 1.1 1 2.1↑ 1 Levothyroxine started POD4
LH(1 – 12 miU/mL) 1↓ <1↓ 1↓ 3
FSH(1 – 12 mIU/mL) 3↓ 1↓ 1↓ 3
Testosterone(219.2 – 905.6 ng/dL) 28.8↓ <20↓ 175.9↓ Testosterone replacement started as outpatient
Testosterone, free(160 – 699 pmol/L) <5.8↓ 137↓
IGF-1(82 – 242 ng/mL) 179 79
GH(fasting < 6 mIU/L) 4.5 <0.3
Prolactin(3 – 20 ng/mL) <1↓ <1↓

POD, postoperative day; HC, hydrocortisone; ACTH, adrenocorticotropic hormone; TSH, thyroid stimulating hormone; LH, luteinizing Hormone; FSH, follicle stimulating hormone; IGF-1, insulin like growth factor – 1; GH, growth hormone

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Figure 4. Trend of select pituitary hormonal panel with key clinical events denoted by black arrows.

Discussion

Microadenomas account for the majority of corticotroph tumors, but 7% – 23% of patients are diagnosed with a macroadenoma345. It is even rarer for a corticotroph macroadenoma to present with apoplexy with only a handful of case reports or series in the literature7. Due to its rarity, appropriate biochemical workup on presentation, such as including an ACTH with the blood work, may be omitted especially if the patient is going for emergent surgery. In this case, the undetectable prolactin can reflect loss of anterior pituitary function and also suggest a functioning corticotroph adenoma due to the inhibitory effect of long term serum glucocorticoids on prolactin secretion9. After undergoing TSS, the patient developed central adrenal insufficiency, hypothyroidism and hypogonadism requiring hormone replacement. Presumably, the development of adrenal insufficiency demonstrated the remission of hypercortisolism as a result of apoplexy and/or TSS. The ACTH remains detectable likely representing residual tumor that was not obliterated by apoplexy nor excised by TSS given it location near the carotid artery and cavernous sinus. The presence of adrenal insufficiency in the setting of detectable ACTH is not contradictory as the physiological hypothalamic-pituitary-adrenal axis has been suppressed by the long-term pathological production of ACTH. IGF-1 and prolactin also failed to recover post-operatively. In CD where the production of IGF-1 and prolactin are attenuated by elevated cortisol, it would then be expected that IGF-1 and prolactin recover after hypercortisolism remission. However, the absence of this observation in our case is likely a sequalae of the apoplexy and extensive surgery leading to pituitary hypofunction.

We also want to highlight features of the pre-operative radiographical findings which can provide valuable insight into the subsequent histology. Previous literature has shown that, on T2-weight MRI, silent corticotroph adenomas are strongly correlated with characteristic a multimicrocystic appearance while nonfunctional gonadotroph macroadenomas are not correlated with this MRI finding10. The multimicrocystic appearance is described as small hyperintense areas with hyperintense striae in the solid part of the tumor (Figure 1C)10. This is an useful predictive tool for silent corticotroph adenomas with a sensitivity of 76%, specificity of 95% and a likelihood ratio of 15.310.

The ability to distinguish between silent corticotroph macroadenoma and other macroadenomas is important for assessing rate of remission and recurrence risk. In 2017, the WHO published updated classification for pituitary tumors. In this new classification, corticotroph adenomas are further divided into densely granulated, sparsely granulated and Crooke’s cell tumors11. DGCT are intensely Periodic Acid Schiff (PAS) stain positive and exhibit strong diffuse pattern of ACTH immunoreactivity, whereas SGCT exhibit faintly positive PAS alongside weak focal ACTH immunoreactivity4,12. Crooke’s cell tumors are characterized by Crooke’s hyaline changes in more than 50% of the tumor cells4. In the literature, SGCT account for an estimated 19-29% of corticotroph adenomas131415. The clinicopathological relevance of granulation pattern in corticotroph tumors was unclear until recently.

In multiple studies examining granulation pattern and tumor size, SGCT were statistically larger13,15,16. Hence, we suspect that many of the previously labelled silent corticotroph macroadenomas in the literature were SGCT. The traditional teaching of CD has been “small tumor, big Cushing and big tumor, small Cushing” which reflects the inverse relationship between tumor size and symptomatology17. This observation appears to hold true as Doğanşen et al. found a trend towards longer duration of CD in SGCT of 34 months compared to 26 months in DGCT based on patient history13,17. It has been postulated that the underlying mechanism of the inverse relationship between tumor size and symptomatology is impaired processing of proopiomelanocortin resulting in less effective secretion of ACTH in corticotroph macroadenomas3. Doğanşen et al. also found that the recurrence rate was doubled for SGCT, while Witek et al. showed that SGCT were less likely to achieve remission postoperatively13,16.

Similar to other cases of SGCT, the diagnosis was only arrived retrospective after pathological confirmation10. Interestingly, the characteristic Crooke’s hyaline change of surrounding non-adenomatous pituitary tissue was not observed as one would expect in a state of prolonged glucocorticoid excess in this case. Although classically described, the absence of this finding does not rule out CD. As evident in a recent retrospective study where 10 out of 144 patients with CD did not have Crooke’s hyaline change18. In patients without Crooke’s hyaline change, the authors found a lower remission rate of 44.4% compared to 73.5% in patients with Crooke’s hyaline change. Together with the detectable post-operative ACTH, sparsely granulated pattern and absence of Crooke’s hyaline change in surrounding pituitary tissue, the risk of recurrence is increased. These risk factors emphasize the importance of close monitoring to ensure early detection of recurrence.

Declaration of Interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Conclusion

We present a case of a sparsely granulated corticotroph macroadenoma presenting with apoplexy leading to remission of hypercortisolism and development of central adrenal insufficiency, hypothyroidism and hypogonadism requiring hormone replacement.

References

Novel Application of Amniotic Membrane Saves Adrenal Tissue in Patients Undergoing Adrenal Surgery

The Carling Adrenal Center, a worldwide destination for the surgical treatment of adrenal tumors, becomes the first center to offer the use of amniotic membrane during adrenal surgery which saves functional adrenal tissue in patients undergoing adrenal surgery. This novel technique enables more patients to have a partial adrenalectomy thereby preserving some normal adrenal physiology, potentially eliminating life-long adrenal hormone replacement.

Preliminary clinical data from the Carling Adrenal Center suggest that the use of a human amniotic membrane allograph on the adrenal gland remnant following partial adrenal surgery leads to faster recovery of normal adrenal gland function. Rather than removing the entire adrenal gland—which has been standard of care for decades—a portion of the adrenal gland is able to be salvaged with amniotic membrane placed upon the remnant as a biologic covering.

Preliminary clinical data from the Carling Adrenal Center suggest that the use of a human amniotic membrane allograph on the adrenal gland remnant following partial adrenal surgery leads to faster recovery of normal adrenal gland function. Rather than removing the entire adrenal gland—which has been standard of care for decades—a portion of the adrenal gland is able to be salvaged with amniotic membrane placed upon the remnant as a biologic covering. The preliminary data from an ongoing clinical trial shows this technique translates into fewer patients needing steroid hormone replacement following adrenal surgery, and if they do, it is for a significantly shorter period of time.

“Sometimes it is possible, and preferable, to remove the adrenal tumor without removing the entire adrenal gland. This is called partial adrenal surgery and our study shows this technique is more successful when amniotic membrane is used,” said Dr. Carling. He further stresses that “removing only part of the adrenal gland is a more advanced operation and is typically only performed by expert adrenal surgeons. The goal is to leave some normal adrenal tissue so that the patient can avoid adrenal insufficiency which requires a daily dose of several adrenal hormones and steroids. Partial adrenal surgery is especially beneficial for patients with pheochromocytoma, as well as Conn’s and Cushing’s syndrome. Avoiding daily steroids is life-changing for these patients so this is a major breakthrough.”

So how does it work? The increased viability of the adrenal gland remnant is presumed to be related to the release of growth factors known to be present in amniotic tissue which is in direct contact with the adrenal gland remnant as a covering. The results are improved rates of viable adrenal cortical tissues with faster regeneration and recovery to normal endocrine physiology by the adrenal cortical cells.

These findings come during Adrenal Disease Awareness Month. Adrenal gland diseases cause many debilitating symptoms like chronic headaches, anxiety, depression, fatigue, brain fog, memory loss, dangerously high blood pressure, heart arrythmia, weight gain, tremors, and more, yet they are often misdiagnosed or improperly treated. Since many doctors are inexperienced in the workup of adrenal hormone problems and only see a handful of adrenal tumors during their careers, it is important for patients to know about the symptoms of adrenal tumor disease and request their doctor measure adrenal hormones.

Adrenal.com is the leading resource for adrenal gland function, tumors and cancers, and an award-winning resource for adrenal gland surgery. The diagnosis and surgical treatment of all types of adrenal tumor types are discussed. Adrenal.com is edited by Dr. Tobias Carling who has performed more adrenal surgery than any other surgeon and has published some of the most important scientific studies of adrenal disease and adrenal surgery including the understanding of the pathogenesis of pheochromocytoma and adrenal tumors causing Conn’s and Cushing’s syndrome.

Established by Dr. Tobias Carling in 2020, the Carling Adrenal Center located at the Hospital for Endocrine Surgery in Tampa FL, is the highest volume adrenal surgical center in the world. The Center now averages nearly 20 adrenal tumor patients every week. Dr Carling was the Director of Endocrine Surgery at Yale University prior to opening the Center in Tampa. At the new Hospital for Endocrine Surgery, Dr Carling joins the Norman Parathyroid Center, the Clayman Thyroid Center and the Scarless Thyroid Surgery Center as the highest volume endocrine surgery center in the world.

About the Carling Adrenal Center: Founded by Dr. Tobias Carling, one of the world’s leading experts in adrenal gland surgery, the Carling Adrenal Center is a worldwide destination for the surgical treatment of adrenal tumors. Dr. Carling spent nearly 20 years at Yale University, including 7 as the Chief of Endocrine Surgery before leaving in 2020 to open to Carling Adrenal Center, which performs more adrenal operations than any other hospital in the world. (813) 972-0000. More about partial adrenalectomy for adrenal tumors can be found at the Center’s website www.adrenal.com.

From https://www.streetinsider.com/PRNewswire/Novel+application+of+amniotic+membrane+saves+adrenal+tissue+in+patients+undergoing+adrenal+surgery/19915274.html

Osilodrostat Normalizes Urinary Free Cortisol in Most Adults with Cushing’s Disease

More than three-quarters of adults with Cushing’s disease assigned osilodrostat had a normalized mean urinary free cortisol level at 12 weeks and maintained a normal level at 36 weeks, according to data from the LINC 4 phase 3 trial.

In findings published in The Journal of Clinical Endocrinology & Metabolism, 77% of adults with Cushing’s disease randomly assigned to osilodrostat (Isturisa, Recordati) had mean urinary free cortisol (UFC) levels reduced to below the upper limit of normal at 12 weeks compared with 8% of adults assigned to placebo.

Osilodrostat normalizes UFC in most people with Cushing's disease at 12 weeks
Most adults with Cushing’s disease taking 2 mg twice daily osilodrostat had normalized mean UFC levels at 12 weeks compared with placebo. Data were derived from Gadelha M, et al. J Clin Endocrinol Metab. 2022;doi:10.1210/clinem/dgac178.

Osilodrostat is a highly effective treatment for Cushing’s disease, normalizing urinary free cortisol excretion in 77% of patients after 12 weeks’ treatment,” Mônica Gadelha, MD, professor of endocrinology at The Federal University of Rio de Janeiro, and colleagues wrote. “Cortisol reductions were maintained throughout 48 weeks of treatment and were accompanied by improvements in clinical signs of hypercortisolism and quality of life.”

Gadelha and colleagues enrolled 73 adults aged 18 to 75 years with Cushing’s disease from 40 centers in 14 countries into the LINC 4 phase 3 trial. Participants were randomly assigned to 2 mg osilodrostat twice daily (n = 48) or placebo (n = 25) for 12 weeks. Urinary samples were collected at weeks 2, 5 and 8 to measure mean UFC, and dosage was adjusted based on efficacy and tolerability. After 12 weeks, participants from both groups received osilodrostat in a 36-week open-label treatment period. All participants restarted the open-label portion of the trial at 2 mg osilodrostat unless they were on a lower dose at week 12. Dose adjustments in the open-label phase were made using the same guidelines in the randomized, double-blind, placebo-controlled trial. The primary endpoint was the efficacy of osilodrostat at achieving a mean UFC below the upper limit of normal of 138 nmol per 24 hours at 12 weeks vs. placebo; the key secondary endpoint was the percentage of participants achieving a normal mean UFC at 36 weeks.

At 12 weeks, the percentage of adults with a normalized mean UFC level was higher in the osilodrostat group compared with placebo (77.1% vs. 8%; P < .0001).

At 36 weeks, 80.8% of all participants had a normal mean UFC level. The overall response rate was 79.5% at 48 weeks.

Median time to first controlled mean UFC response was 35 days for those randomly assigned to osilodrostat as well as those randomly assigned to placebo who crossed over to osilodrostat for the open-label phase. At 48 weeks, 84% of participants were receiving 10 mg or less of osilodrostat per day, including 56% receiving 4 mg or less daily.

At 12 weeks, the osilodrostat group had several cardiovascular and metabolic-related improvements, including systolic and diastolic blood pressure, HbA1c, HDL cholesterol, body weight and waist circumference. No changes were observed in the placebo group.

“The improvements in cardiovascular and metabolic parameters were sustained throughout osilodrostat treatment and have the potential to alleviate the burden of comorbidities in many patients with Cushing’s disease,” the researchers wrote.

At 12 weeks, 52.5% of those receiving osilodrostat had a reduction in supraclavicular fat pad and 50% had a reduction in dorsal fat pad. At least 25% of participants also had improvements in facial redness, striae, proximal muscle atrophy and central obesity. Improvements were sustained through week 48.

During the placebo-controlled trial, grade 3 and 4 adverse events occurred for about 20% of participants in both groups. For the entire study, 38.4% of adults reported grade 3 and 4 adverse events, with the most common being hypertension. Eight participants discontinued the study due to adverse events.

From https://www.healio.com/news/endocrinology/20220408/osilodrostat-normalizes-urinary-free-cortisol-in-most-adults-with-cushings-disease

A Promising In Vitro Model to Study Cushing’s Syndrome

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 LIPEATGLIL-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 (12). 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 (35). In patients with CS, GC secretion does not follow the circadian rhythm and consistently high serum GC levels are observed throughout the day (67).

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 (910). 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 (1314). 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

www.frontiersin.orgTable 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 (1719). 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, 2.5×105 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

www.frontiersin.orgTable 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

www.frontiersin.orgFigure 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 3×103 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, 3×103 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, 1.5×104 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 LIPEATGLIL-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 (2528). A total of 2.5×105 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

www.frontiersin.orgFigure 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

www.frontiersin.orgFigure 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

www.frontiersin.orgFigure 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

www.frontiersin.orgFigure 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

www.frontiersin.orgFigure 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

www.frontiersin.orgFigure 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 (3032) 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 (3334), 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 (3638), 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 (2540). 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.

Publisher’s Note

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.

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Keywords: glucocorticoids, MSCs, lipolysis, glucose uptake, insulin resistance

Citation: Di Vincenzo M, Martino M, Lariccia V, Giancola G, Licini C, Di Benedetto G, Arnaldi G and Orciani M (2022) Mesenchymal Stem Cells Exposed to Persistently High Glucocorticoid Levels Develop Insulin-Resistance and Altered Lipolysis: A Promising In Vitro Model to Study Cushing’s Syndrome. 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, Canada
Małgorzata 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

Recordati Rare Diseases Announce Publication in the Journal of Clinical Endocrinology & Metabolism of the Phase III LINC 4 Study Confirming the Efficacy and Safety of Isturisa® (Osilodrostat) in Patients With Cushing’s Disease

The LINC 4 study demonstrated superiority of Isturisa® (osilodrostat) over placebo in achieving cortisol normalisation during the 12-week, double-blind, randomised phase (77% vs 8%, P<0.0001).

Isturisa provided rapid and sustained control of cortisol secretion in the majority of patients throughout the 48-week core phase of the study.

PUTEAUX, France, March 29, 2022–(BUSINESS WIRE)–Recordati Rare Diseases announce today the publication of positive results from the Phase III LINC 4 study of Isturisa in The Journal of Clinical Endocrinology & Metabolism.1 These data reinforce Isturisa as an effective and well-tolerated oral therapy for patients with Cushing’s disease. Isturisa is indicated in the EU for the treatment of adult patients with endogenous Cushing’s syndrome,2 a rare and debilitating condition of hypercortisolism that is most commonly caused by a pituitary adenoma (Cushing’s disease).3

The LINC 4 study augments the efficacy and safety data for Isturisa in patients with Cushing’s disease, confirming the results from the Phase III LINC 3 study. This study in 73 adults is the first Phase III study of a medical treatment in patients with Cushing’s disease to include an upfront, randomised, double-blind, placebo-controlled period during which 48 patients received Isturisa and 25 received placebo for the first 12 weeks, followed by an open-label period during which all patients received Isturisa until week 48; thereafter, patients could enter an optional extension phase.

Key findings published in the manuscript entitled ‘Randomised trial of osilodrostat for the treatment of Cushing’s disease’ include:1

  • LINC 4 met the primary endpoint: Isturisa was significantly superior to placebo at normalising mUFC at the end of a 12-week randomised, double-blind period (77% vs 8%; P<0.0001).
  • Effects of Isturisa were rapid. Over one-quarter of patients randomised to Isturisa achieved normal mUFC as early week 2 and 58% achieved control by week 5.
  • The key secondary endpoint was also met, with 81% of all patients in the study having normal mUFC at week 36.
  • Improvements in cardiovascular and metabolic parameters of Cushing’s disease, including blood pressure and blood glucose metabolism, were seen by week 12 and were maintained throughout the study.
  • Physical features of hypercortisolism improved during Isturisa treatment, including fat pads, facial rubor, striae, and muscle wasting. Improvements were observed by week 12, with continued improvement throughout the study to week 48.
  • Patient-reported QoL scores (CushingQoL and Beck Depression Inventory) also improved during Isturisa treatment.
  • Isturisa was well tolerated in the majority of patients, with no unexpected adverse events (AEs). The most common AEs overall were decreased appetite, arthralgia, fatigue and nausea.

“These results show convincingly that osilodrostat is an effective treatment for Cushing’s disease,” said Peter J. Snyder MD, Professor of Medicine at the University of Pennsylvania. “Osilodrostat rapidly lowered cortisol excretion to normal in most patients with Cushing’s disease and maintained normal levels throughout the core phase of the study. Importantly, this normalisation was accompanied by improvements in cardiovascular and metabolic parameters, which increase morbidity and mortality in Cushing’s disease.”

“These compelling data build on the positive Phase III LINC 3 study, published in The Lancet Diabetes & Endocrinology in 2020,4 demonstrating that Isturisa enables most patients with Cushing’s disease to gain rapid control of their cortisol levels, which in turn provides relief from a host of undesirable symptoms,” said Alberto Pedroncelli, Clinical Development & Medical Affairs Lead, Global Endocrinology, Recordati AG. “Recordati Rare Diseases is committed to improving the lives of patients with this rare, debilitating and life-threatening condition. I would like to thank everyone who has contributed to LINC 4 and the LINC clinical programme.”

“I had Cushing’s disease for 8 years without being diagnosed,” said Thérèse Fournier from L’association “Surrénales”. “I was trapped in a vicious circle of missed diagnoses and worsening physical and psychological symptoms that became life-threatening. I lost everything – my job, my house, my partner, my friends – I was isolated. When I finally received my diagnosis, I was relieved because I knew the truth. Since my surgery, I have been learning to live again, enjoying the moments that make a life. I am still on the path to remission, but I feel deeply happy, even if I carry this journey that nobody can understand.”

About Cushing’s syndrome
Cushing’s syndrome is a rare disorder caused by chronic exposure to excess levels of cortisol from either an exogenous (eg medication) or an endogenous source.5 Cushing’s disease is the most common cause of endogenous Cushing’s syndrome and arises as a result of excess secretion of adrenocorticotropic hormone from a pituitary adenoma, a tumour of the pituitary gland.5,6 There is often a delay in diagnosing Cushing’s syndrome, which consequently leads to a delay in treating patients.7 Patients who are exposed to excess levels of cortisol for a prolonged period have increased comorbidities associated with the cardiovascular and metabolic systems, which consequently reduce QoL and increase the risk of mortality.3,6 To alleviate the clinical signs associated with excess cortisol exposure, the primary treatment goal in Cushing’s syndrome is to reduce cortisol levels to normal.8

About LINC 4
LINC 4 is a multicentre, randomised, double-blind, 48-week study with an initial 12-week placebo-controlled period to evaluate the safety and efficacy of Isturisa® in patients with Cushing’s disease. The LINC 4 study enrolled patients with persistent or recurrent Cushing’s disease or those with de novo disease who were ineligible for surgery; 73 randomised patients were treated with Isturisa® (n=48) or placebo (n=25).1 The primary endpoint of the study is the proportion of randomised patients with a complete response (mUFC ≤ULN) at the end of the placebo-controlled period (week 12). The key secondary endpoint is the proportion of patients with an mUFC ≤ULN at week 36.1,9

About Isturisa®
Isturisa® is an oral inhibitor of 11β-hydroxylase (CYP11B1), which catalyses the final step of cortisol synthesis in the adrenal glands.2 Isturisa® is available as 1 mg, 5 mg and 10 mg film-coated tablets.2 Isturisa® is approved for the treatment of adult patients with endogenous Cushing’s syndrome in the EU and is now available in France, Germany, Greece and Austria.2

Isturisa® was granted marketing authorisation by the European Commission on 9 January 2020. For detailed recommendations on the appropriate use of this product, please consult the summary of product characteristics.2

References

1. Gadelha M, Bex M, Feelders RA et al. Randomised trial of osilodrostat for the treatment of Cushing’s disease. J Clin Endocrinol Metab 2022; dgac178, https://doi.org/10.1210/clinem/dgac178.
2. Isturisa® summary of product characteristics. May 2020.
3. Ferriere A, Tabarin A. Cushing’s syndrome: Treatment and new therapeutic approaches. Best Pract Res Clin Endocrinol Metab 2020;34:101381.
4. Pivonello R, Fleseriu M, Newell-Price J et al. Efficacy and safety of osilodrostat in patients with Cushing’s disease (LINC 3): a multicentre phase III study with a double-blind, randomised withdrawal phase. Lancet Diabetes Endocrinol 2020;8:748-61.
5. Lacroix A, Feelders RA, Stratakis CA et al. Cushing’s syndrome. Lancet 2015;386:913-27.
6. Pivonello R, Isidori AM, De Martino MC et al. Complications of Cushing’s syndrome: state of the art. Lancet Diabetes Endocrinol 2016;4:611-29.
7. Rubinstein G, Osswald A, Hoster E et al. Time to diagnosis in Cushing’s syndrome: A meta-analysis based on 5367 patients. J Clin Endocrinol Metab 2020;105:dgz136.
8. Nieman LK, Biller BM, Findling JW et al. Treatment of Cushing’s syndrome: An Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 2015;100:2807-31.
9. ClinicalTrials.gov. NCT02697734; available at https://clinicaltrials.gov/ct2/show/NCT02697734 (accessed March 2021).

Recordati Rare Diseases, the company’s EMEA headquarters are located in Puteaux, France, with global headquarter offices in Milan, Italy.

For a full list of products, please click here: www.recordatirarediseases.com/products.

Recordati, established in 1926, is an international pharmaceutical group, listed on the Italian Stock Exchange (Reuters RECI.MI, Bloomberg REC IM, ISIN IT 0003828271), with a total staff of more than 4,300, dedicated to the research, development, manufacturing and marketing of pharmaceuticals. Headquartered in Milan, Italy, Recordati has operations in Europe, Russia and the other C.I.S. countries, Ukraine, Turkey, North Africa, the United States of America, Canada, Mexico, some South American countries, Japan and Australia. An efficient field force of medical representatives promotes a wide range of innovative pharmaceuticals, both proprietary and under license, in several therapeutic areas including a specialized business dedicated to treatments for rare diseases. Recordati is a partner of choice for new product licenses for its territories. Recordati is committed to the research and development of new specialties with a focus on treatments for rare diseases. Consolidated revenue for 2021 was € 1,580.1 million, operating income was € 490.2 million and net income was € 386.0 million.

For further information:

Recordati website: www.recordatirarediseases.com

This document contains forward-looking statements relating to future events and future operating, economic and financial results of the Recordati group. By their nature, forward-looking statements involve risk and uncertainty because they depend on the occurrence of future events and circumstances. Actual results may therefore differ materially from those forecast as a result of a variety of reasons, most of which are beyond the Recordati group’s control. The information on the pharmaceutical specialties and other products of the Recordati group contained in this document is intended solely as information on the Recordati group’s activities and therefore, as such, it is not intended as medical scientific indication or recommendation, nor as advertising.

View source version on businesswire.com: https://www.businesswire.com/news/home/20220325005169/en/

Contacts

Celine Plisson, MD
Medical Affairs Director
Telephone: +33(0)147739463
Email: PLISSON.C@recordati.com

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