Glucocorticoid-induced Insulin Resistance Research Articles

Transcriptional coactivation by EHMT2 restricts glucocorticoid-induced insulin resistance in a study with male mice

The classical dogma of glucocorticoid-induced insulin resistance is that it is caused by the transcriptional activation of hepatic gluconeogenic and insulin resistance genes by the glucocorticoid receptor (GR). Here, we find that glucocorticoids also stimulate the expression of insulin-sensitizing genes, such as Irs2. The transcriptional coregulator EHMT2 can serve as a transcriptional coactivator or a corepressor. Using male mice that have a defective EHMT2 coactivation function specifically, we show that glucocorticoid-induced Irs2 transcription is dependent on liver EHMT2’s coactivation function and that IRS2 play a key role in mediating the limitation of glucocorticoid-induced insulin resistance by EHMT2’s coactivation. Overall, we propose a model in which glucocorticoid-regulated insulin sensitivity is determined by the balance between glucocorticoid-modulated insulin resistance and insulin sensitizing genes, in which EHMT2 coactivation is specifically involved in the latter process.

No effect of the turmeric root phenol curcumin on prednisolone-induced glucometabolic perturbations in men with overweight or obesity.

Preclinically, curcumin has been shown to protect against glucocorticoid-induced insulin resistance. We evaluated the effect of curcumin administered with prednisolone in healthy overweight or obese men. In a double-blind, parallel-group trial, 24 overweight/obese non-diabetic men were randomised to one of three intervention groups (A) prednisolone placebo+curcumin placebo, (B) prednisolone (50 mg/day)+curcumin placebo or (C) prednisolone and curcumin (400 mg/day). Curcumin or curcumin placebo treatment started 1 day prior to 10-day prednisolone or prednisolone placebo treatment. The primary endpoint was change in prednisolone-induced insulin resistance assessed by homeostatic model assessment of insulin resistance (HOMA2-IR). Other endpoints included anthropometric measurements, magnetic resonance spectroscopy-assessed hepatic fat content, blood pressure, circulating metabolic markers and continuous glucose monitoring measures. Baseline characteristics (mean ± s.d): age 44.2 ± 13.7 years, BMI 30.1 ± 3.5 kg/m2, HbAlc 33.3 ± 3.2 mmol/mol, HOMA2-IR 1.10 ± 0.45 and fasting plasma glucose 5.2 ± 0.4 mmol/L. Prednisolone significantly increased HOMA2-IR (estimated treatment difference 0.36 (95% CI 0.16; 0.57)). Co-treatment with curcumin had no effect on HOMA2-IR (estimated treatment difference 0.08 (95% CI -0.13; 0.39)). Prednisolone increased HbAlc, insulin, C-peptide, glucagon, blood pressure, mean interstitial glucose, time spent in hyperglycaemia and glucose variability, but no protective effect of curcumin on any of these measures was observed. In this double-blind, placebo-controlled parallel-group study involving 24 overweight or obese men randomised to one of three treatment arms, curcumin treatment had no protective effect on prednisolone-induced insulin resistance or other glucometabolic perturbations.

The effect of quinoa (Chenopodium quinoa) on apoptotic, autophagic, antioxidant and inflammation markers in glucocorticoid-induced insulin resistance in rats.

Insulin resistance plays an important role in predicting type 2 diabetes that may develops. This study was planned in order to investigate the beneficial effects of quinoa (Chenopodium quinoa) use in glucocorticoid induced-insulin resistance. Forty-two rats were used as the material (experimental) groups: the control group (C), the quinoa-administered group (Q), the insulin resistance-created group (IR), the IR + metformin group (IM), the IR + quinoa for treatment group (IQ) and the quinoa + IR for prophylaxis group (QI). Blood glucose, insulin levels and HOMA-IR were found to be highest (p < 0.05) in the IR group (p < 0.05). Glucose levels decreased significantly with the administration of quinoa and approached the levels of the control, but the insulin levels and the HOMA-IR did not significantly change. It was also observed that other biochemical parameters (ALT, AST, ALP, total cholesterol, total protein, urea and creatinine) changed significantly in the IR group and approached the levels of the control group with the administration of quinoa. Apoptotic (BCL2 5, BAX 9, CAS 3), autophagic (SQSTM1 7, ATG5) and inflammation (IL-1β, TNF-α) genes were upregulated by 5-11-fold in the IR group. In the groups in which quinoa was administered for treatment and protection, all these genes were found to be upregulated to a lower extent than the IR group. Antioxidant genes (GPX1, SOD1) increased by nine to tenfold in the quinoa groups. As a result, after administration of quinoa, it was determined that the glucose level increased due to experimental insulin resistance and the liver and kidney damage indicators decreased. It was determined that quinoa (Chenopodium quinoa) had significant beneficial effects on biochemical parameters and apoptotic, autophagic, antioxidant and inflammatory markers in experimental glucocorticoid-induced insulin resistance.

Anti-Diabetic and Hypolipidemic Effect of Coccinia Indica in Glucocorticoid Induced Insulin Resistance

This study explores the anti-diabetic, insulin sensitizing and hypolipidemic activity of Coccinia indica (C.indica) leaf extract (ethanolic) in glucocorticoid induced insulin resistance (IR). A 12 day study with 5 groups of 30 male Wistar albino rats, with 6 rats in each was conducted. The rats in all the groups except group 1 received dexamethasone (8mg/kg/i.p.) from 7th to 12th day to induce IR. The groups 1 and 2 received 2% gum acacia orally for 12 days whereas the groups 3 &amp; 4 received oral ethanolic extract of C.indica leaf in the dose of 1 and 2 gm/kg, respectively. The standard control (group 5) received metformin (1gm/kg) orally for 12 days. Fasting serum glucose, insulin and lipid levels were estimated at the beginning and end of the study. The insulin sensitivity indices (homeostatic model assessment of insulin resistance and sensitivity, fasting glucose to insulin ratio, hepatic &amp; atherogenic indices) were calculated. The body weight was monitored on alternate days. The liver weight, volume and histopathology were also done. Compared to group 2 rats, the group’s 3 &amp; 4 demonstrated significant(p&lt;0.05) dose dependent lowering of serum glucose, insulin and lipids as well as lowered IR, improved insulin sensitivity and reduced hepatic steatosis. Additionally, group 2 rats had low body weight and hepatomegaly. This extract demonstrated significant anti-diabetic, hypolipidemic and insulin sensitizing activity. Hence it can be used as an effective alternative for treating type2 diabetes mellitus.

Molecular Mechanisms of Glucocorticoid-Induced Insulin Resistance.

Glucocorticoids (GCs) are steroids secreted by the adrenal cortex under the hypothalamic-pituitary-adrenal axis control, one of the major neuro-endocrine systems of the organism. These hormones are involved in tissue repair, immune stability, and metabolic processes, such as the regulation of carbohydrate, lipid, and protein metabolism. Globally, GCs are presented as ‘flight and fight’ hormones and, in that purpose, they are catabolic hormones required to mobilize storage to provide energy for the organism. If acute GC secretion allows fast metabolic adaptations to respond to danger, stress, or metabolic imbalance, long-term GC exposure arising from treatment or Cushing’s syndrome, progressively leads to insulin resistance and, in fine, cardiometabolic disorders. In this review, we briefly summarize the pharmacological actions of GC and metabolic dysregulations observed in patients exposed to an excess of GCs. Next, we describe in detail the molecular mechanisms underlying GC-induced insulin resistance in adipose tissue, liver, muscle, and to a lesser extent in gut, bone, and brain, mainly identified by numerous studies performed in animal models. Finally, we present the paradoxical effects of GCs on beta cell mass and insulin secretion by the pancreas with a specific focus on the direct and indirect (through insulin-sensitive organs) effects of GCs. Overall, a better knowledge of the specific action of GCs on several organs and their molecular targets may help foster the understanding of GCs’ side effects and design new drugs that possess therapeutic benefits without metabolic adverse effects.

OR14-03 The Transcriptional Coactivation Function of EHMT2 Restricts Chronic Glucocorticoid Exposure Induced Insulin Resistance

Glucocorticoids are required for metabolic adaptations during times of stress. However, chronic glucocorticoid exposure is associated with metabolic disorders such as insulin resistance. Glucocorticoids mainly convey their signals through an intracellular glucocorticoid receptor (GR). GR is a transcription factor that requires interactions with transcriptional coregulators to modulate the transcription of GR primary target genes, which in turn regulate specific aspects of physiology. Euchromatic Histone Methyltransferase 2 (Ehmt2) is a transcriptional coregulator for GR that can act as a corepressor or a coactivator. We found that glucocorticoid-induced insulin resistance was exacerbated when Ehmt2 levels were reduced in the liver. Intriguingly, this phenotype resulted from the transactivation function of Ehmt2. This is because a mutation at the lysine 182 automethylation site, which is required for the coactivation but not the corepression function of Ehmt2, results in similar exacerbated GC-induced insulin resistance. These results suggest that Ehmt2 coactivation dependent GR primary target genes restrict the extent of glucocorticoid-induced insulin resistance. Gene expression analysis identified Dusp4 (a.k.a. Mkp-2) as an Ehmt2 coactivation dependent GR-activated gene, which when overexpressed in liver, attenuated glucocorticoid-induced insulin resistance. Thus, we have identified a novel GR-Ehmt2-Dusp4 axis that plays a key role in controlling the extent of the development of insulin resistance. Notably, the classical view of how GC induce hepatic insulin resistance is that GR activates genes that inhibit insulin signaling and enhance hepatic gluconeogenesis. Our study, however, provides a revolutionary concept in which the extent of GC-induced insulin resistance is controlled by the balance of GR-activated genes that promote insulin sensitivity or insulin resistance.

Androgens sensitise mice to glucocorticoid-induced insulin resistance and fat accumulation.

Chronic glucocorticoid therapy causes insulin resistance, dyslipidaemia, abnormal fat accumulation, loss of muscle mass and osteoporosis. Here we describe a hitherto unknown sexual dimorphism in the metabolic response to chronic glucocorticoid exposure in mice. This led us to investigate whether glucocorticoid-induced insulin resistance and obesity were dependent on sex hormones. Male and female CD1 mice were treated for 4weeks with supraphysiological doses (~250μg/day) of corticosterone, the main glucocorticoid in rodents, or equivalent volume of vehicle (drinking water without corticosterone). To investigate the effects of sex hormones, a separate group of mice were either orchidectomised or ovariectomised prior to corticosterone treatment, with or without dihydrotestosterone replacement. Body composition was determined before and after corticosterone treatment, and insulin tolerance was assessed after 7 and 28days of treatment. Adipocyte morphology was assessed in white and brown adipose tissues by immunohistochemistry, and fasting serum concentrations of NEFA, triacylglycerols, total cholesterol and free glycerol were measured using colorimetric assays. Obesity- and diabetes-related hormones were measured using multiplex assays, and RNA and protein expression in adipose tissues were measured by RT-PCR and immunoblotting, respectively. Chronic corticosterone treatment led to insulin resistance, fasting hyperinsulinaemia, increased adiposity and dyslipidaemia in male, but not female mice. In males, orchidectomy improved baseline insulin sensitivity and attenuated corticosterone-induced insulin resistance, but did not prevent fat accumulation. In androgen-deficient mice (orchidectomised males, and intact and ovariectomised females) treated with dihydrotestosterone, corticosterone treatment led to insulin resistance and dyslipidaemia. In brown adipose tissue, androgens were required for corticosterone-induced intracellular lipid accumulation ('whitening'), and dihydrotestosterone specifically exacerbated corticosterone-induced accumulation of white adipose tissue by increasing adipocyte hypertrophy. Androgens also suppressed circulating adiponectin concentrations, but corticosterone-induced insulin resistance did not involve additional suppression of adiponectin levels. In white adipose tissue, androgens were required for induction of the glucocorticoid target gene Gilz (also known as Tsc22d3) by corticosterone. In mice, androgens potentiate the development of insulin resistance, fat accumulation and brown adipose tissue whitening following chronic glucocorticoid treatment.

Estrogen interacts with glucocorticoids in the regulation of lipocalin 2 expression in human adipose tissue. Reciprocal roles of estrogen receptor α and β in insulin resistance?

The adipokine lipocalin 2 (LCN2) is linked to insulin resistance. Its expression in human adipose tissue (AT) can be regulated in a sex-specific manner by a synthetic glucocorticoid, dexamethasone, suggesting an underlying role of sex steroids. We show that 17-β-estradiol (E2) dose-dependently increased LCN2 gene expression in subcutaneous AT from postmenopausal women. This was also seen in the presence of estrogen receptor (ER) α antagonist alone but not with ERβ antagonist, suggesting that E2 effects on LCN2 are mediated via ERβ pathway. Dexamethasone alone or E2+dexamethasone had no significant effect on LCN2. However, E2+dexamethasone increased LCN2 expression with ERα-blockade. Dexamethasone reduced ERα but increased ERβ expression. Dexamethasone can regulate LCN2 expression via inhibition of ERα and stimulation of ERβ and may contribute to the development of glucocorticoid-induced insulin resistance in human AT. In conclusion, ERβ and ERα pathways have opposite effects on LCN2 expression and they interact with glucocorticoid action.

Knockout of glucocorticoid receptor on AgRP/NPY/GABA (ANG) neurons identifies a potential role for this neuronal population in mediating glucocorticoid-induced insulin resistance in female mice

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Adaptive β-Cell Neogenesis in the Adult Mouse in Response to Glucocorticoid-Induced Insulin Resistance.

Both type 1 and type 2 diabetes are characterized by deficient insulin secretion and decreased β-cell mass. Thus, regenerative strategies to increase β-cell mass need to be developed. To characterize mechanisms of β-cell plasticity, we studied a model of severe insulin resistance in the adult mouse and defined how β-cells adapt. Chronic corticosterone (CORT) treatment was given to adult mice and led to rapid insulin resistance and adaptive increased insulin secretion. Adaptive and massive increase of β-cell mass was observed during treatment up to 8 weeks. β-Cell mass increase was partially reversible upon treatment cessation and reinduced upon subsequent treatment. β-Cell neogenesis was suggested by an increased number of islets, mainly close to ducts, and increased Sox9 and Ngn3 mRNA levels in islets, but lineage-tracing experiments revealed that neoformed β-cells did not derive from Sox9- or Ngn3-expressing cells. CORT treatment after β-cell depletion partially restored β-cells. Finally, β-cell neogenesis was shown to be indirectly stimulated by CORT because serum from CORT-treated mice increased β-cell differentiation in in vitro cultures of pancreatic buds. Altogether, the results present a novel model of β-cell neogenesis in the adult mouse and identify the presence of neogenic factors in the serum of CORT-treated mice.

Glucocorticoid-Induced Insulin Resistance in Men Is Associated With Suppressed Undercarboxylated Osteocalcin.

In mice, glucocorticoid-induced insulin resistance occurs largely through impaired osteoblast function and decreased circulating undercarboxylated osteocalcin (ucOC). Whether these mechanisms contribute to glucocorticoid-induced insulin resistance in humans has yet to be established. In addition, the effects of glucocorticoids on the exercise-induced increase in circulating ucOC and insulin sensitivity are also unknown. We hypothesized that acute glucocorticoid treatment would lead to basal and postexercise insulin resistance in part through decreased circulating ucOC and ucOC-mediated skeletal muscle protein signaling. Nine healthy men completed two separate cycling sessions 12 hours after ingesting either glucocorticoid (20 mg prednisolone) or placebo (20 mg Avicel). The homeostatic model assessment was used to assess basal insulin sensitivity and a 2-hour euglycemic-hyperinsulinemic clamp was commenced 3 hours after exercise to assess postexercise insulin sensitivity. Serum ucOC and skeletal muscle protein signaling were measured. Single-dose glucocorticoid ingestion increased fasting glucose (27%, p < 0.01) and insulin (83%, p < 0.01), and decreased basal insulin sensitivity (-47%, p < 0.01). Glucocorticoids reduced insulin sensitivity after cycling exercise (-34%, p < 0.01), reduced muscle GPRC6A protein content (16%, p < 0.05), and attenuated protein phosphorylation of mTORSer2481 , AktSer374 , and AS160Thr642 (59%, 61%, and 50%, respectively; all ps < 0.05). Serum ucOC decreased (-24%, p < 0.01) which correlated with lower basal insulin sensitivity (r = 0.54, p = 0.02), lower insulin sensitivity after exercise (r = 0.72, p < 0.05), and attenuated muscle protein signaling (r = 0.48-0.71, p < 0.05). Glucocorticoid-induced basal and postexercise insulin resistance in humans is associated with the suppression of circulating ucOC and ucOC-linked protein signaling in skeletal muscle. Whether ucOC treatment can offset glucocorticoid-induced insulin resistance in human subjects requires further investigation. © 2018 American Society for Bone and Mineral Research.

Glucocorticoid-induced insulin resistance is related to macrophage visceral adipose tissue infiltration

Insulin resistance is frequently present in patients with glucocorticoid (GC) excess (Cushing’s syndrome) or treated with high doses of GCs. Furthermore, others similarities between metabolic syndrome (visceral obesity, elevated blood glucose levels, dyslipidemia) and Cushing’s syndrome suggest that GCs could play a role in obesity-linked complications. Here we reported that long-term corticosterone (CORT) exposure in mice induced weight gain, dyslipidemia as well as hyperglycaemia and systemic insulin resistance. CORT-treated mice exhibited an increased 11β-Hsd1 expression and corticosterone levels in fat depots but a specific upregulation of glucocorticoid receptor (Gr) and hexose-6-phosphate dehydrogenase only in gonadal adipose tissue, suggesting that GC could act differentially on various fat depots. Despite fat accumulation in all depots, an increased expression of adipogenic (Pparγ, C/ebpα) and lipogenic (Acc, Fas) key genes was restricted to gonadal adipose tissue. Hypertrophied adipocytes observed in both visceral and subcutaneous depots also resulted from reduced lipolytic activity due to CORT treatment. Surprisingly, GC treatment promoted macrophage infiltration (F4/80, Cd68) within all adipose tissues along with predominant M2-like macrophage phenotype, and can directly act on macrophages to induce this phenotype. Moreover, macrophage infiltration preceded mass gain and adipocyte hypertrophy. Of note, specific macrophage depletion in gonadal fat preferentially reduced the M2-like macrophage content, and partially restored insulin sensitivity in mice with GC-induced obesity and insulin resistance. These data provide evidence that GCs act on adipose tissue in a depot-dependent manner and that gonadal adipose macrophages are key effectors of GC-associated insulin resistance.

FKBP5 expression in human adipose tissue: potential role in glucose and lipid metabolism, adipogenesis and type 2 diabetes

PurposeHere, we explore the involvement of FKBP51 in glucocorticoid-induced insulin resistance (IR) in human subcutaneous adipose tissue (SAT), including its potential role in type 2 diabetes (T2D). Moreover, we assess the metabolic effects of reducing the activity of FKBP51 using the specific inhibitor SAFit1.MethodsHuman SAT was obtained by needle biopsies of the lower abdominal region. FKBP5 gene expression was assessed in fresh SAT explants from a cohort of 20 T2D subjects group-wise matched by gender, age and BMI to 20 non-diabetic subjects. In addition, human SAT was obtained from non-diabetic volunteers (20F/9M). SAT was incubated for 24 h with or without the synthetic glucocorticoid dexamethasone and SAFit1. Incubated SAT was used to measure the glucose uptake rate in isolated adipocytes.ResultsFKBP5 gene expression levels in SAT positively correlated with several indices of IR as well as glucose area under the curve during oral glucose tolerance test (r = 0.33, p < 0.05). FKBP5 gene expression levels tended to be higher in T2D subjects compared to non-diabetic subjects (p = 0.088). Moreover, FKBP5 gene expression levels were found to inversely correlate with lipolytic, lipogenic and adipogenic genes. SAFit1 partly prevented the inhibitory effects of dexamethasone on glucose uptake.ConclusionsFKBP5 gene expression in human SAT tends to be increased in T2D subjects and is related to elevated glucose levels. Moreover, FKBP5 gene expression is inversely associated with the expression of lipolytic, lipogenic and adipogenic genes. SAFit1 can partly prevent glucose uptake impairment by glucocorticoids, suggesting that FKBP51 might be a key factor in glucocorticoid-induced IR.

Glucocorticoid-Induced Metabolic Disturbances Are Exacerbated in Obese Male Mice.

The purpose of this study was to determine the effects of glucocorticoid-induced metabolic dysfunction in the presence of diet-induced obesity. C57BL/6J adult male lean and diet-induced obese mice were given dexamethasone, and levels of hepatic steatosis, insulin resistance, and lipolysis were determined. Obese mice given dexamethasone had significant, synergistic effects on fasting glucose, insulin resistance, and markers of lipolysis, as well as hepatic steatosis. This was associated with synergistic transactivation of the lipolytic enzyme adipose triglyceride lipase. The combination of chronically elevated glucocorticoids and obesity leads to exacerbations in metabolic dysfunction. Our findings suggest lipolysis may be a key player in glucocorticoid-induced insulin resistance and fatty liver in individuals with obesity.

Influence of prednisolone on parameters of de novo lipogenesis and indices for stearoyl-CoA- and Δ6- desaturase activity in healthy males: A Post-hoc analysis of a randomized, placebo-controlled, double-blind trial

Glucocorticoid treatment decreases liver insulin sensitivity and may modify fatty acid metabolism. We investigated the influence of oral prednisolone on indices for de novo lipogenesis (DNLi), stearoyl-CoA desaturase (SCDi) and Δ6-desaturase (D6Di) activity in healthy males. In addition, we explored whether the changes may be associated with prednisolone-induced changes in glucose and lipid metabolism and insulin sensitivity.Thirty-two healthy young males (mean ± SD age 22 ± 3 years, BMI 22.4 ± 1.7 kg/m2) were allocated to receive prednisolone 7.5 mg/day (PRED7.5; n = 12), prednisolone 30 mg/day (PRED30; n = 12), or placebo (n = 8) in a randomized double-blind fashion for 2 weeks. Fatty acid compositions of plasma cholesteryl esters (CE), phospholipids (PL) and triglycerides (TG) were measured at baseline and on day 14. DNLi, SCDi and D6Di were estimated from product/precursor ratios in CE, with DNLi primary deriving from 16:1ω7/18:2ω6, SCDi from 16:1ω7/16:0 and D6Di from 22:6ω3/20:5ω3. Ratios were also assessed in PL and TG.In CE, PRED30 increased DNLi by 51.2 [95%CI 14.8; 87.6]%, increased SCDi by 48.6 [18.7; 78.5]%, and decreased D6Di by 57.7 [-91.8; -23.5]% (p ≤ 0.01 for all, compared to placebo). The prednisolone-induced increases in DNLi and SCDi were positively correlated with insulin sensitivity (r = 0.35 and 0.50, respectively). Similar results were found in PL and TG.Prednisolone dose-dependently increases DNLi and SCDi and decreases D6Di in plasma CE, PL and TG in healthy males after 2 weeks. The observed unfavorable effects on fatty acid metabolism were related to the induction of glucocorticoid-induced insulin resistance.

11β-Hydroxysteroid Dehydrogenase Type 1(11β-HSD1) mediates insulin resistance through JNK activation in adipocytes.

Glucocorticoids are used to treat a number of human diseases but often lead to insulin resistance and metabolic syndrome. 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a key enzyme that catalyzes the intracellular conversion of cortisone to physiologically active cortisol. Despite the known role of 11β-HSD1 and active glucocorticoid in causing insulin resistance, the molecular mechanisms by which insulin resistance is induced remain elusive. The aim of this study is to identify these mechanisms in high fat diet (HFD) experimental models. Mice on a HFD were treated with 11β-HSD1 inhibitor as well as a JNK inhibitor. We then treated 3T3-L1-derived adipocytes with prednisone, a synthetic glucocorticoid, and cells with 11β-HSD1 overexpression to study insulin resistance. Our results show that 11β-HSD1 and JNK inhibition mitigated insulin resistance in HFD mice. Prednisone stimulation or overexpression of 11β-HSD1 also caused JNK activation in cultured adipocytes. Inhibition of 11β-HSD1 blocked the activation of JNK in adipose tissue of HFD mice as well as in cultured adipocytes. Furthermore, prednisone significantly impaired the insulin signaling pathway, and these effects were reversed by 11β-HSD1 and JNK inhibition. Our study demonstrates that glucocorticoid-induced insulin resistance was dependent on 11β-HSD1, resulting in the critical activation of JNK signaling in adipocytes.

Glucocorticoids Inhibit Basal and Hormone-Induced Serotonin Synthesis in Pancreatic Beta Cells.

Diabetes is a major complication of chronic Glucocorticoids (GCs) treatment. GCs induce insulin resistance and also inhibit insulin secretion from pancreatic beta cells. Yet, a full understanding of this negative regulation remains to be deciphered. In the present study, we investigated whether GCs could inhibit serotonin synthesis in beta cell since this neurotransmitter has been shown to be involved in the regulation of insulin secretion. To this aim, serotonin synthesis was evaluated in vitro after treatment with GCs of either islets from CD1 mice or MIN6 cells, a beta-cell line. We also explored the effect of GCs on the stimulation of serotonin synthesis by several hormones such as prolactin and GLP 1. We finally studied this regulation in islet in two in vivo models: mice treated with GCs and with liraglutide, a GLP1 analog, and mice deleted for the glucocorticoid receptor in the pancreas. We showed in isolated islets and MIN6 cells that GCs decreased expression and activity of the two key enzymes of serotonin synthesis, Tryptophan Hydroxylase 1 (Tph1) and 2 (Tph2), leading to reduced serotonin contents. GCs also blocked the induction of serotonin synthesis by prolactin or by a previously unknown serotonin activator, the GLP-1 analog exendin-4. In vivo, activation of the Glucagon-like-Peptide-1 receptor with liraglutide during 4 weeks increased islet serotonin contents and GCs treatment prevented this increase. Finally, islets from mice deleted for the GR in the pancreas displayed an increased expression of Tph1 and Tph2 and a strong increased serotonin content per islet. In conclusion, our results demonstrate an original inhibition of serotonin synthesis by GCs, both in basal condition and after stimulation by prolactin or activators of the GLP-1 receptor. This regulation may contribute to the deleterious effects of GCs on beta cells.

T25: Selective glucocorticoid receptor agonists as novel anti-cancer agents

Glucocorticoids (GCs) are widely used in treatment of many cancer types due to its ability to induce apoptosis in malignant cells in blood cancer therapy, and to prevent nausea, emesis and chemotherapy-associated hepatotoxicity in case of solid tumors. However, severe dose-limiting side effects occur, including osteoporosis, diabetes and other metabolic complications. Moreover, in therapy of solid tumors GCs strongly affect microenvironment which could be associated with poor prognosis, risk of metastasis and high frequency of relapses. Biological response to GCs is mediated by glucocorticoid receptor (GR), a well-characterized transcription factor. GR controls gene expression via (1) transactivation, which requires binding of GR homodimers to glucocorticoid-responsive elements (GRE) in gene promoters and enhancers, and (2) dimerization-independent transrepression mediated via negative interaction between GR and other transcription factors including major effectors of inflammation and proliferation. Transrepression plays an important role in anti-inflammatory and anti-cancer effects of GR, including normalizing influence on microenvironment, while side effects are associated with GR transactivation. In particular, GCs induce insulin resistance in adipocytes, a major component of the mammary microenvironment, which secrete pro-inflammatory cytokines and growth factors, implicated in tumor progression. Selective GR agonists (SEGRA) that preferentially activate GR transrepression could be a better option for treatment of cancer. Dozens of candidate SEGRAs were identified, synthesized and tested by industry and academia, with some having reached clinical trials. One of the novel GR modulators is 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium-chloride, or CpdA, synthetic analogue of aziridine precursor isolated from Namibian shrub Salsola tuberculatiformis Botschantzev. It was shown that CpdA acts as “dissociated” GR ligand: it competes with GCs for GR binding and efficiently induces GR transrepression but not transactivation. We and other authors reported recently that CpdA inhibits survival of prostate cancer cells as well as blood cancer cells in GR-dependent fashion. Furthermore, primary leukemia cells from T-ALL patients appeared to be equally sensitive to GCs and CpdA. Our further studies were concentrated on three directions: (1) GC/SEGRA-based chemotherapy. We screened biological effect of CpdA in combination with traditional agents (doxorubicin, vincristine) and newer therapeutics (Bortezomib, Carfilzomib, MLN-4924, Rapamycin). Pretreatment of lymphoma cells with proteasome inhibitor Bortezomib resulted in GR accumulation and enhanced ligand properties of CpdA. We also revealed remarkable GR-dependent cooperation between CpdA and Bortezomib in suppressing survival of lymphoma cells in vitro and in vivo. Also surprising findings were substantial cooperation in anti-cancer effect of immunosuppressant Rapamycin and CpdA in vitro, and unexpected “dissociated” effect of Rapamycin on GR signaling realized through down -regulation of REDD1, mTORC1 inhibitor. These data suggested high clinical potential of Rapamycin/GC combination in cancer treatment. (2) SEGRA list extention We used two approaches to extend SEGRA list: (1) synthesis of CpdA enantiomers and (2) its chemical derivatives. Chemical analogues of CpdA were designed by appending of bulky substituent into benzene ring, alkylation of carbon atom adjacent to chlorine atom or appending of substituents to nitrogen atom. Evaluation of biological properties of enantiomers revealed higher GR-dependent anti-cancer potential of S-CpdA. Cytotoxic and proapoptotic effects of CpdA analogues were comparable with precursor. (3) Selection of tumor types acceptable for SEGRA treatment. CpdA was selected for NCI-60 in Vitro Cell Line Screening Project providing direct support to anticancer drug discovery program. It was shown that CpdA affect viability of some adherent cancer cell lines. We demonstrated that CpdA unlike GCs did not modify microenvironment and disintegrate tight junctions between cells decreasing risk of metastasis in case of solid tumors. It demonstrates reasonability of further investigations. Overall, our data provide the rationale for novel therapy of cancer based on combination of non-steroidal GR modulators with classic and modern chemotherapeutics. Approaches to obtain more SEGRAs were elaborated.

Search for Compounds with Hypoglycemic Activity in the Series of 1-[2-(1H-Tetrazol-5-yl)-R(1)-phenyl]-3-R(2)-phenyl(ethyl)ureas and R(1)-Tetrazolo[1,5-c]quinazolin-5(6H)-ones.

Methods of 1-[2-(1H-tetrazol-5-yl)-R1-phenyl]-3-R2-phenyl(ethyl)ureas and R1-tetrazolo[1,5-c]quinazolin-5(6H)-ones synthesis were designed. IR, LC-MS, 1H NMR, and elemental analysis data evaluated the structure and purity of the obtained compounds. Different products, depending on the reaction conditions, were distinguished and discussed. The preliminary hypoglycemic activity of 36 synthesized compounds was revealed. Docking studies to 11β-hydroxysteroid dehydrogenase 1, γ-peroxisome proliferator-activated receptor, and dipeptidyl peptidase-4 were conducted. Eight of these substances were further tested on glucocorticoid-induced insulin resistance models, namely glucose tolerance, oral rapid insulin, and adrenalin tests. One of the most active compounds turned out to be tetrazolo[1,5-c]quinazolin-5(6H)-one 3.1, exceeding the reference drugs Metformin (50 and 200 mg/kg) and Gliclazide (50 mg/kg).

Important role of 5-hydroxytryptamine in glucocorticoid-induced insulin resistance in liver and intra-abdominal adipose tissue of rats.

Both glucocorticoids and 5-hydroxytryptamine (5-HT) have been shown to induce insulin resistance (IR) in hepatocytes and adipocytes. Here, we explore whether there is a correlation between them. Except for the control group, male rats were exposed to dexamethasone treated with or without para-chlorophenylalanine (pCPA), or carbidopa for 20 days. Except for the control group, buffalo rat liver 3A (BRL-3A) cells were exposed to dexamethasone for 24 h, treated with or without pCPA, carbidopa, or clorgiline for 48 h, or exposed to 5-HT treated with or without fluoxetine for 48 h. Whole-body IR was determined by both glucose tolerance test and measurement of fasting blood glucose and insulin, whereas hepatocytes or adipocytes IR was determined by examining either hepatic gluconeogenesis, steatosis and glucose transporter 2 expression or lipolysis. Dexamethasone-induced whole-body IR, liver and intraabdominal adipose IR were accompanied by upregulated expressions of tryptophan hydroxylase-1 and aromatic amino acid decarboxylase with increased 5-HT level in both tissues, which were attenuated significantly by pCPA, inhibiting tryptophan hydroxylase-1, or carbidopa, inhibiting aromatic amino acid decarboxylase. [Correction added on 22 September 2015, after first online publication: 'inhibiting aromatic amino acid decarboxylase' was duplicated and has been replaced by 'tryptophan hydroxylase-1'.] In the BRL-3A cells, dexamethasone-induced IR was also accompanied by upregulated 5-HT synthesis in dose- and time-dependent manners, and was attenuated by pCPA or carbidopa, but exacerbated by clorgiline, inhibiting monoamine oxidase-A to further increase 5-HT level. Dexamethasone also enhanced 5-HT 2A and 2B receptor expressions in both tissues and BRL-3A cells. Additionally, blocking 5-HT transporter with fluoxetine significantly suppressed 5-HT-induced IR in BRL-3A cells. Enhancement of 5-HT synthesis in liver and intra-abdominal adipose is an important reason for glucocorticoids-induced IR.