Maria J. Pereira

mTOR inhibition with rapamycin causes impaired insulin signalling and glucose uptake in human subcutaneous and omental adipocytes

Rapamycin is an immunosuppressive agent used after organ transplantation, but its molecular effects on glucose metabolism needs further evaluation. We explored rapamycin effects on glucose uptake and insulin signalling proteins in adipocytes obtained via subcutaneous (n=62) and omental (n=10) fat biopsies in human donors. At therapeutic concentration (0.01 μM) rapamycin reduced basal and insulin-stimulated glucose uptake by 20-30%, after short-term (15 min) or long-term (20 h) culture of subcutaneous (n=23 and n=10) and omental adipocytes (n=6 and n=7). Rapamycin reduced PKB Ser473 and AS160 Thr642 phosphorylation, and IRS2 protein levels in subcutaneous adipocytes. Additionally, it reduced mTOR-raptor, mTOR-rictor and mTOR-Sin1 interactions, suggesting decreased mTORC1 and mTORC2 formation. Rapamycin also reduced IR Tyr1146 and IRS1 Ser307/Ser616/Ser636 phosphorylation, whereas no effects were observed on the insulin stimulated IRS1-Tyr and TSC2 Thr1462 phosphorylation. This is the first study to show that rapamycin reduces glucose uptake in human adipocytes through impaired insulin signalling and this may contribute to the development of insulin resistance associated with rapamycin therapy.

Effects of decavanadate and insulin enhancing vanadium compounds on glucose uptake in isolated rat adipocytes.

The effects of different vanadium compounds namely pyridine-2,6-dicarboxylatedioxovanadium(V) (V5-dipic), bis(maltolato) oxovanadium(IV) (BMOV) and amavadine, and oligovanadates namely metavanadate and decavanadate were analysed on basal and insulin stimulated glucose uptake in rat adipocytes. Decavanadate (50 microM), manifest a higher increases (6-fold) on glucose uptake compared with basal, followed by BMOV (1 mM) and metavanadate (1 mM) solutions (3-fold) whereas V5 dipic and amavadine had no effect. Decavanadate (100 microM) also shows the highest insulin like activity when compared with the others compounds studied. In the presence of insulin (10 nM), only decavanadate increases (50%) the glucose uptake when compared with insulin stimulated glucose uptake whereas BMOV and metavanadate, had no effect and V5 dipic and amavadine prevent the stimulation to about half of the basal value. Decavanadate is also able to reduce or eradicate the suppressor effect caused by dexamethasone on glucose uptake at the level of the adipocytes. Altogether, vanadium compounds and oligovanadates with several structures and coordination spheres reveal different effects on glucose uptake in rat primary adipocytes.

The immunosuppressive agents rapamycin, cyclosporin A and tacrolimus increase lipolysis, inhibit lipid storage and alter expression of genes involved in lipid metabolism in human adipose tissue

Cyclosporin A (CsA), tacrolimus and rapamycin are immunosuppressive agents (IAs) associated with insulin resistance and dyslipidemia, although their molecular effects on lipid metabolism in adipose tissue are unknown. We explored IAs effects on lipolysis, lipid storage and expression of genes involved on lipid metabolism in isolated human adipocytes and/or adipose tissue obtained via subcutaneous and omental fat biopsies. CsA, tacrolimus and rapamycin increased isoproterenol-stimulated lipolysis and inhibited lipid storage by 20-35% and enhanced isoproterenol-stimulated hormone-sensitive lipase Ser552 phosphorylation. Rapamycin also increased basal lipolysis (~20%) and impaired insulins antilipolytic effect. Rapamycin, down-regulated the gene expression of perilipin, sterol regulatory element-binding protein 1 (SREBP1) and lipin 1, while tacrolimus down-regulated CD36 and aP2 gene expression. All three IAs increased IL-6 gene expression and secretion, but not expression and secretion of TNF-α or adiponectin. These findings suggest that CsA, tacrolimus and rapamycin enhance lipolysis, inhibit lipid storage and expression of lipogenic genes in adipose tissue, which may contribute to the development of dyslipidemia and insulin resistance associated with immunosuppressive therapy.

FKBP5 expression in human adipose tissue increases following dexamethasone exposure and is associated with insulin resistance

OBJECTIVE To study effects of dexamethasone on gene expression in human adipose tissue aiming to identify potential novel mechanisms for glucocorticoid-induced insulin resistance. MATERIALS/METHODS Subcutaneous and omental adipose tissue, obtained from non-diabetic donors (10 M/15 F; age: 28-60 years; BMI: 20.7-30.6 kg/m²), was incubated with or without dexamethasone (0.003-3 μmol/L) for 24 h. Gene expression was assessed by microarray and real time-PCR and protein expression by immunoblotting. RESULTS FKBP5 (FK506-binding protein 5) and CNR1 (cannabinoid receptor 1) were the most responsive genes to dexamethasone in both subcutaneous and omental adipose tissue (~7-fold). Dexamethasone increased FKBP5 gene and protein expression in a dose-dependent manner in both depots. The gene product, FKBP51 protein, was 10-fold higher in the omental than in the subcutaneous depot, whereas the mRNA levels were similar. Higher FKBP5 gene expression in omental adipose tissue was associated with reduced insulin effects on glucose uptake in both depots. Furthermore, FKBP5 gene expression in subcutaneous adipose tissue was positively correlated with serum insulin, HOMA-IR and subcutaneous adipocyte diameter and negatively with plasma HDL-cholesterol. FKBP5 SNPs were found to be associated with type 2 diabetes and diabetes-related phenotypes in large population-based samples. CONCLUSIONS Dexamethasone exposure promotes expression of FKBP5 in adipose tissue, a gene that may be implicated in glucocorticoid-induced insulin resistance.

Cyclosporine A and Tacrolimus Reduce the Amount of GLUT4 at the Cell Surface in Human Adipocytes: Increased Endocytosis as a Potential Mechanism for the Diabetogenic Effects of Immunosuppressive Agents

CONTEXT Immunosuppressive agents are associated with profound metabolic side effects including new-onset diabetes and dyslipidemia after organ transplantation. OBJECTIVE To investigate the effects of cyclosporine A (CsA) and tacrolimus on glucose uptake and insulin signaling in human adipocytes and their impact on the regulation of cellular trafficking of the glucose transporter 4 (GLUT4). DESIGN Isolated human adipocytes were incubated with therapeutic concentrations of either CsA or tacrolimus, and glucose uptake and expression of insulin signaling proteins were assessed. Furthermore, we studied effects of CsA and tacrolimus on the regulation of cellular trafficking of GLUT4 in differentiated human preadipocytes and L6 cells. RESULTS CsA and tacrolimus had a concentration-dependent inhibitory effect on basal and insulin-stimulated (14)C-glucose uptake in adipocytes. Although phosphorylation at Tyr1146 of the insulin receptor was inhibited by tacrolimus, the phosphorylation and/or protein levels of the insulin signaling proteins IRS1/2, p85-PI3K, PKB, AS160, and mTORC1, as well as GLUT4 and GLUT1, were unchanged by CsA or tacrolimus. Furthermore, CsA and tacrolimus reduced the GLUT4 amount localized at the cell surface of differentiated human preadipocytes and L6 cells in the presence of insulin. This occurred by an increased rate of GLUT4 endocytosis, with no change in the exocytosis rate. CONCLUSIONS These results suggest that therapeutic concentrations of CsA and tacrolimus can inhibit glucose uptake independent of insulin signaling by removing GLUT4 from the cell surface via an increased rate of endocytosis. This mechanism can contribute to the development of insulin resistance and diabetes associated with immunosuppressive therapy. In addition, it may provide novel pharmacological approaches for the treatment of diabetes.

Molecular mechanisms underlying the effects of cyclosporin A and sirolimus on glucose and lipid metabolism in liver, skeletal muscle and adipose tissue in an in vivo rat model.

Cyclosporin A (CsA) and sirolimus (SRL) are immunosuppressive agents (IAs) associated with dyslipidemia, insulin resistance and new onset diabetes after transplantation (NODAT). However, the molecular mechanisms involved are not fully understood. We investigated the effects of six-week treatment of either CsA or SRL on glucose and lipid metabolism in Wistar rats. The results show that, compared with vehicle-treated rats, SRL-treated rats were significantly lighter starting at week 5. CsA or SRL caused glucose intolerance, increased storage of lipids in the liver and skeletal muscle, and decreased the insulin-stimulated glucose uptake in isolated adipocytes. Furthermore, these agents significantly decreased genes involved in insulin action and glucose uptake, such as, IRS-1, Glut4 and Glut1, and increased genes and/or proteins involved in hepatic lipogenesis and gluconeogenesis, while decreasing them in adipose tissue. After either treatment PGC1α gene expression was down regulated in skeletal muscle, an important player in fatty acid oxidation. Moreover, there was an increase in IL-6 gene expression in adipose tissue in the SRL-treated rats, suggesting stimulation of lipolysis. The results of the present study suggest that CsA and SRL lead to metabolic alterations in liver, muscle and adipose tissue, which may contribute to the development of dyslipidemia and insulin resistance associated with immunosuppressive therapy.

Dapagliflozin once-daily and exenatide once-weekly dual therapy: A 24-week randomized, placebo-controlled, phase II study examining effects on body weight and prediabetes in obese adults without diabetes.

To explore the effects of dual therapy with dapagliflozin and exenatide on body weight, body composition, glycaemic variables and systolic blood pressure (SBP) in obese adults without diabetes.

The TGR5 gene is expressed in human subcutaneous adipose tissue and is associated with obesity, weight loss and resting metabolic rate

Highlights • Human adipose tissue (AT) expresses the bile acid receptor TGR5.• Human AT TGR5 expression is linked to obesity.• Resting metabolic rate and AT TGR5 expression is positively correlated.• TGR5 expression is not higher in brown compared to white human AT.

Impaired adipose tissue lipid storage, but not altered lipolysis, contributes to elevated levels of NEFA in type 2 diabetes. Degree of hyperglycemia and adiposity are important factors

BACKGROUND Elevated levels of circulating non-esterified fatty acids (NEFA) mediate many adverse metabolic effects. In this work we aim to determine the impact of type 2 diabetes (T2D), glycemic control and obesity on lipolysis regulation. DESIGN AND PARTICIPANTS 20 control and 20 metformin-treated T2D subjects were matched for sex (10M/10 F), age (58±11 vs 58±9 y) and BMI (30.8±4.6 vs 30.7±4.9kg/m2). In vivo lipolysis was assessed during a 3h-OGTT with plasma glycerol and NEFA levels. Subcutaneous adipose tissue (SAT) biopsies were obtained to measure mRNA and metabolite levels of factors related to lipolysis and lipid storage and to assess in vitro lipolysis in isolated subcutaneous adipocytes. RESULTS Plasma NEFA AUC during the OGTT where higher 30% (P=0.005) in T2D than in control subjects, but plasma glycerol AUC and subcutaneous adipocyte lipolysis in vitro were similar, suggesting that adipose tissue lipolysis is not altered. Expression in SAT of genes involved in lipid storage (FABP4, DGAT1, FASN) were reduced in T2D subjects compared with controls, but no differences were seen for genes involved in lipolysis. T2D subjects had elevated markers of beta-oxidation, α-hydroxybutyrate (1.4-fold, P<0.01) and β-hydroxybutyrate (1.7-fold, P<0.05) in plasma. In multivariate analysis, HbA1c, visceral adipose tissue volume and sex (male) were significantly associated with NEFA AUC in T2D subjects. CONCLUSIONS In T2D subjects, NEFA turnover is impaired, but not due to defects in lipolysis or lipid beta-oxidation. Impaired adipose NEFA re-esterification or de novo lipogenesis is likely to contribute to higher NEFA plasma levels in T2D. The data suggest that hyperglycemia and adiposity are important contributing factors for the regulation of plasma NEFA concentrations.

Characterization of brown adipose tissue in the human perirenal depot.

To characterize brown adipose tissue (BAT) in the human perirenal adipose tissue depot.