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 agentes are associated with profound metabolic side effects including new-onset diabetes and dyslipidemia after organ transplantation. Objective: Toi nvestigated the effects of the cyclosporine A(CsA)or tacrolimus ong lucose uptake and insulin signalling in human adipocytes and their impact on the regulation of celular trafficking of the glucose transporter 4 (GLUT4). Design:Human isolated 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 celular trafficking of the GLUT4 in differentiated human pre-adipocytes and L6 cells. Results:CsA and tacrolimus had a concentration-dependent inhibitory effect on basal and insulin stimulated 14C-glucose uptake in adipocytes. Although phosphorylation at Tyr1146 of insulin receptor (IR) was inhibited by tacrolimus, the phosphorylation and/or protein levels of the insulin signalling proteinsIRS1/2,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 surfasse of differentiated human pre-adipocytes and L6 cells in the presence of insulin.This occurred by na 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 independente of insulin signalling by removing GLUT4 from the cell surface via na increased rate of endocytosis.Such mechanisms can contribute to the development of insulin resistance and diabetes associated with immunosuppressive therapy.In addition,they may provide novel pharmacological approaches for treatment of diabetes

Human adipose tissue. Genes predominantly expressed in the visceral depot and in hypertrophic adipocytes

The obesity prevalence is increasing worldwide and obesity is closely linked to type 2 diabetes and cardiovascular disease. Both visceral fat accumulation and enlarged adipocyte size are risk factors for obesity related metabolic disorders. The aim of this thesis was therefore to detect genes, with higher expression in visceral versus subcutaneous adipose tissue, and in large versus small adipocytes, that potentially may contribute to metabolic disease related to obesity.DNA microarray was used to identify genes differentially expressed in human adipose tissue depots of obese subjects, and real-time PCR was used for verification and further investigation of selected genes. Complement (C2, C3, C4, C7, factor B) and immunoglobulin (Ig) (IGHG3, IGK@, IGL@) genes were higher expressed in omental compared to subcutaneous adipose tissue. Serum C3 and C4 levels correlated to measures of adiposity and analysis of tissue distribution showed that, in comparison with the omentum, only liver had higher expression of the classical complement components C2, C3 and C4. These results suggest that the complement system may be involved in metabolic complications associated with increased visceral fat mass. The density of Ig producing plasma cells was twice as high in omental compared to subcutaneous adipose tissue analyzed by immunohistochemistry. Fc receptors (FcgammaRII) were present on adipocyte membranes and the Fc moiety of IgG reduced adipocyte interleukin expression and stimulated lipogenesis in vitro, indicating that Ig have anti-inflammatory and lipid accumulating effects in human adipose tissue.Serum amyloid A (SAA) and NAD(P)H:quinone oxidoreductase 1 (NQO1) were identified as predominantly expressed in omental adipocytes when compared to expression profiles from 32 other human tissues and cell types. Adipose tissue expression and serum levels of the acute-phase protein SAA were down regulated in obese subjects during diet induced weight loss, and serum levels of SAA correlated to measures of adiposity and insulin levels during the diet. Liver displayed the second highest SAA expression indicating that adipose tissue is a major expression site for SAA during nonacute-phase. The NQO1 expression also decreased during weight loss and correlated to measures of adiposity, insulin, AST and ALT.A technique to separate human adipocytes from an adipose tissue sample into populations of small and large cells was developed and the expression profiles of the populations were compared. Fourteen genes, of which five immune-related (E-selectin, IL-8, SAA, C1q receptor 1, MIP-2), were markedly higher expressed in large versus small cells. SAA was 19-fold higher expressed in large adipocytes and the expression correlated to adipocyte size.Many immune-related genes (complement, Ig, SAA) and NQO1, implicated in oxidative stress, are highly expressed in human visceral adipose tissue and large subcutaneous adipocytes. These genes may link visceral and hypertrophic obesity to metabolic disorders. The results support the suggested connection between obesity and inflammation

Plasma cells and Fc receptors in human adipose tissue--lipogenic and anti-inflammatory effects of immunoglobulins on adipocytes

We have previously reported high immunoglobulin expression in human omental adipose tissue. The aim of this work was to investigate plasma cell density and Fc receptor (FcR) expression in human adipose tissue depots and in vitro effects of immunoglobulins on adipocyte function. Plasma cell density was higher in the visceral compared to the subcutaneous depot (10.0+/-1.56% and 5.2+/-0.98%, respectively, n=20,

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 insulin’s 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-a 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

mTOR inhibition with rapamycin cause 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 lM) 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

Characterization of brown adipose tissue in the human perirenal depot.

International audienceTo characterize brown adipose tissue (BAT) in the human perirenal adipose tissue depot. Perirenal adipose tissue biopsies were obtained from 55 healthy kidney donors. Expression analysis was performed using microarray, real-time PCR, immunoblotting and immunohistochemistry. Additional studies using human stem cells were performed. UCP1 gene expression analysis revealed a large intra-individual variation in the perirenal adipose tissue biopsies. Both multi- and unilocular UCP1-positive adipocytes were detected in several of the adipose tissue samples analyzed by immunohistochemical staining. Microarray analysis identified 54 genes that were overexpressed in UCP1-positive perirenal adipose tissue. Real-time PCR analysis of BAT candidate genes revealed a set of genes that were highly correlated to UCP1 and a set of three transcription factor genes (PRDM16, PGC1α, and RXRγ) that were highly correlated to each other. RXRγ displayed nuclear immunoreactivity in brown adipocytes and an increased gene expression during brown adipogenesis in human stem cells. Our data provides the first molecular characterization of BAT in the perirenal adipose tissue depot. Furthermore, it highlights the transcription factor RXRγ as a new player in BAT development

Detection of genetic hypopituitarism in an adult population of idiopathic pituitary insufficiency patients with growth hormone deficiency.

International audienceIdiopathic pituitary insufficiency (IPI) is diagnosed in 10% of all hypopituitary patients. There are several known and unknown aetiologies within the IPI group. The aim of this study was to investigate an adult IPI population for genetic cause according a screening schedule. From files of 373 GH deficient (GHD) patients on GH replacement 50 cases with IPI were identified. Of the 39 patients that approved to the study, 25 patients were selected for genetic investigation according to phenotype and 14 patients were not further tested, as sporadic isolated GHD (n = 9) and GHD with diabetes insipidus (n = 5) have low probability for a known genetic cause. Genotyping of all coding exons of HESX1, LHX4, PROP1, POU1F1 and GH1 genes were performed according to a diagnostic algorithm based on clinical, hormonal and neuroradiological phenotype. Among the 25 patients, an overall rate of 8% of mutations was found, and a 50% rate in familial cases. Among two sibling pairs, one pair that presented with complete anterior pituitary insufficiency, had a compound heterozygous PROP1 gene mutation (codons 117 and 120: exon 3 p Phe 117 Ile (c349 T>A) and p Arg 120 Cys (c358 C>T)) with a phenotype of very late onset ACTH-insufficiency. In the other sibling pair and in the sporadic cases no mutation was identified. This study suggests that currently known genetic causes are rare in sporadic adult IPI patients, and that systematic genetic screening is not needed in adult-onset sporadic cases of IPI. Conversely, familial cases are highly suspect for genetic causes

Separation of human adipocytes by size: hypertrophic fat cells display distinct gene expression

Enlarged adipocytes are associated with insulin resistance and are an independent predictor of type 2 diabetes. To understand the molecular link between these diseases and adipocyte hypertrophy, we developed a technique to separate human adipocytes from an adipose tissue sample into populations of small cells (mean 57.6+-3.54 um) and large cells (mean 100.1+-3.94 um). Microarray analysis of the cell populations separated from adipose tissue from three subjects identified 14 genes, of which five immune-related, with more than fourfold higher expression in large cells than small cells. Two of these genes were serum amyloid A (SAA) and transmembrane 4 L six family member 1 (TM4SF1). Real-time RT-PCR analysis of SAA and TM4SF1 expression in adipocytes from seven subjects revealed 19-fold and 22-fold higher expression in the large cells, respectively, and a correlation between adipocyte size and both SAA and TM4SF1 expression. The results were verified using immunohistochemistry. In comparison with 17 other human tissues and cell types by microarray, large adipocytes displayed by far the highest SAA and TM4SF1 expression. Thus, we have identified genes with markedly higher expression in large, compared with small, human adipocytes. These genes may link hypertrophic obesity to insulin resistance/type 2 diabetes

High expression of complement components in omental adipose tissue in obese men.

OBJECTIVE: Accumulation of visceral fat is recognized as a predictor of obesity-related metabolic disturbances. Factors that are predominantly expressed in this depot could mediate the link between visceral obesity and associated diseases. RESEARCH METHODS AND PROCEDURES: Paired subcutaneous and omental adipose tissue biopsies were obtained from 10 obese men. Gene expression was analyzed by DNA microarrays in triplicate and by real-time polymerase chain reaction. Serum C3 and C4 were analyzed by radial immunodiffusion assays in 91 subjects representing a cross section of the general population. Body composition was measured by computerized tomography. RESULTS: Complement components C2, C3, C4, C7, and Factor B had higher expression in omental compared with subcutaneous adipose tissue ( approximately 2-, 4-, 17-, 10-, and 7-fold, respectively). In addition, adipsin, which belongs to the alternative pathway, and the classical pathway components C1QB, C1R, and C1S were expressed in both depots. Analysis of tissue distribution showed high expression of C2, C3, and C4 in omental adipose tissue, and only liver had higher expression of these genes. Serum C3 levels correlated with both visceral and subcutaneous adipose tissue in both men (r = 0.65 and