Insulin Signaling In Human Adipocytes Research Articles

Salt-inducible kinases are required for glucose uptake and insulin signaling in human adipocytes.

Salt-inducible kinase 2 (SIK2) is abundantly expressed in adipocytes and downregulated in adipose tissue from individuals with obesity or insulin resistance. The main aims of this work were to investigate the involvement of SIKs in the regulation of glucose uptake in primary mature human adipocytes and to identify mechanisms underlying this regulation. Primary mature adipocytes were isolated from human, rat, or mouse adipose tissue and treated with pan-SIK inhibitors. Adipocytes isolated from wild type, ob/ob, and SIK2 knockout mice were also used. Glucose uptake was examined by glucose tracer assay. The insulin signaling pathway was monitored by Western blotting, co-immunoprecipitation, and total internal reflection fluorescence microscopy. This study demonstrates that SIK2 is downregulated in obese ob/ob mice and that SIK activity is required for intact glucose uptake in primary human and mouse adipocytes. The underlying mechanism involves direct effects on the insulin signaling pathway, likely at the level of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) generation or breakdown. Moreover, lack of SIK2 alone is sufficient to attenuate glucose uptake in mouse adipocytes. SIK2 is required for insulin action in human adipocytes, and the mechanism includes direct effects on the insulin signaling pathway.

Development of a physiological insulin resistance model in human stem cell-derived adipocytes.

Adipocytes are key regulators of human metabolism, and their dysfunction in insulin signaling is central to metabolic diseases including type II diabetes mellitus (T2D). However, the progression of insulin resistance into T2D is still poorly understood. This limited understanding is due, in part, to the dearth of suitable models of insulin signaling in human adipocytes. Traditionally, adipocyte models fail to recapitulate in vivo insulin signaling, possibly due to exposure to supraphysiological nutrient and hormone conditions. We developed a protocol for human pluripotent stem cell–derived adipocytes that uses physiological nutrient conditions to produce a potent insulin response comparable to in vivo adipocytes. After systematic optimization, this protocol allows robust insulin-stimulated glucose uptake and transcriptional insulin response. Furthermore, exposure of sensitized adipocytes to physiological hyperinsulinemia dampens insulin-stimulated glucose uptake and dysregulates insulin-responsive transcription. Overall, our methodology provides a novel platform for the mechanistic study of insulin signaling and resistance using human pluripotent stem cell–derived adipocytes.

D-Chiro-Inositol Regulates Insulin Signaling in Human Adipocytes

D-Chiro-Inositol (D-Chiro-Ins) is a secondary messenger in the insulin signaling pathway. D-Chiro-Ins modulates insulin secretion, the mitochondrial respiratory chain, and glycogen storage. Due to these actions D-Chiro-Ins has been proposed to correct defective insulin function in a variety of conditions characterized by metabolic dysfunction, such as polycystic ovary syndrome (PCOS), obesity, gestational diabetes and fat accumulation at menopause. Since it is unclear whether D-Chiro-Ins directly acts on adipocytes, we aimed to study D-Chiro-Ins’s actions on adipocyte viability, proliferation, differentiation, and insulin-related protein expression using a human adipocyte cell line derived from Simpson–Golabi–Behmel Syndrome (SGBS) which fully differentiates to mature adipocytes. Throughout differentiation, cells were treated with D-Chiro-Ins, 17β-estradiol (E2) or Insulin. Cell viability and proliferation were not affected by D-Chiro-Ins, then D-Chiro-Ins promoted cell differentiation only during the final days of the process, while E2 enhanced it from the first phases. D-Chiro-Ins stimulated lipid storage and the production of big lipid droplets, thus reducing the content of free fatty acids. We also found that D-Chiro-Ins, either alone or in combination with insulin and E2 increased the expression and activation of insulin receptor substrate-1 (IRS1) and glucose transporter type 4 (GLUT4). In conclusion, this work shows that D-Chiro-Ins plays a direct role in the differentiation and in the function of human adipocytes, where it synergizes with insulin and estrogen through the recruitment of signal transduction pathways involved in lipid and glucose storage. These findings give clear insights to better understand the actions of D-Chiro-Ins on fat metabolism in women in physiology and in a variety of diseases.

Maresin 1 regulates insulin signaling in human adipocytes as well as in adipose tissue and muscle of lean and obese mice.

Maresin 1 (MaR1) is a DHA-derived pro-resolving lipid mediator. The present study aimed to characterize the ability of MaR1 to prevent the alterations induced by TNF-α on insulin actions in glucose uptake and Akt phosphorylation in cultured human adipocytes from overweight/obese subjects, as well as to investigate the effects of MaR1 acute and chronic administration on Akt phosphorylation in absence/presence of insulin in white adipose tissue (WAT) and skeletal muscle from lean and diet-induced obese (DIO) mice. MaR1 (0.1nM) prevented the inhibitory effect of TNF-α on insulin-stimulated 2-Deoxy-D-glucose uptake and Akt phosphorylation in human adipocytes. Acute treatment with MaR1 (50μg/kg, 3h, i.p.) induced Akt phosphorylation in WAT and skeletal muscle of lean mice. However, MaR1 did not further increase the stimulatory effect of insulin on Akt activation. Interestingly, intragastric chronic treatment with MaR1 (50μg/kg, 10days) in DIO mice reduced the hyperglycemia induced by the high fat diet (HFD) and improved systemic insulin sensitivity. In parallel, MaR1 partially restored the impaired insulin response in skeletal muscle of DIO mice and reversed HFD-induced lower Akt phosphorylation in WAT in non-insulin-stimulated DIO mice while did not restore the defective Akt activation in response to acute insulin observed in DIO mice. Our results suggest that MaR1 attenuates the impaired insulin signaling and glucose uptake induced by proinflammatory cytokines. Moreover, the current data support that MaR1 treatment could be useful to reduce the hyperglycemia and the insulin resistance associated to obesity, at least in part by improving Akt signaling.

Effect of protocatechuic acid on insulin responsiveness and inflammation in visceral adipose tissue from obese individuals: possible role for PTP1B

Background/ObjectivesThe occurrence of chronic inflammation in visceral adipose tissue (VAT) in obese subjects precipitates the development of insulin resistance and type 2 diabetes (T2D). Anthocyanins and their main metabolite protocatechuic acid (PCA) have been demonstrated to stimulate insulin signaling in human adipocytes. The aim of this study was to investigate whether PCA is able to modulate insulin responsiveness and inflammation in VAT from obese (OB) and normal weight (NW) subjects.Subjects/MethodsVATs obtained from NW and OB subjects were incubated or not (control) with 100 μM PCA for 24 h. After incubation, tissues untreated and treated with PCA were acutely stimulated with insulin (20 nM, 20 min). PTP1B, p65 NF-κB, phospho-p65 NF-κB, IRS-1, IRβ, Akt, GLUT4 as well as basal and insulin-stimulated Tyr-IRS-1 and Ser-Akt phosphorylations were assessed by Western blotting in NW- and OB-VAT. Samples were assessed for PTP1B activity and adipocytokine secretion.ResultsPCA restored insulin-induced phosphorylation in OB-VAT by increasing phospho-Tyr-IRS-1 and phospho-Ser-Akt after insulin stimulation as observed in NW-VAT (p < 0.05). PTP1B activity was lower in OB-VAT treated with PCA with respect to untreated (p < 0.05). Compared to non-treated tissues, PCA reduced phospho-p65 NF-κB and IL-6 in OB-VAT, and IL-1β in NW-VAT (p < 0.05); and increased adiponectin secretion in NW-VAT (p < 0.05).ConclusionPCA restores the insulin responsiveness of OB-VAT by increasing IRS-1 and Akt phosphorylation which could be related with the lower PTP1B activity found in PCA-treated OB-VAT. Furthermore, PCA diminishes inflammation in VAT. These results support the beneficial role of an anthocyanin-rich diet against inflammation and insulin resistance in obesity.

Zinc-α2-Glycoprotein Modulates AKT-Dependent Insulin Signaling in Human Adipocytes by Activation of the PP2A Phosphatase.

ObjectiveEvidence from mouse models suggests that zinc-α2-glycoprotein (ZAG) is a novel anti-obesity adipokine. In humans, however, data are controversial and its physiological role in adipose tissue (AT) remains unknown. Here we explored the molecular mechanisms by which ZAG regulates carbohydrate metabolism in human adipocytes.MethodsZAG action on glucose uptake and insulin action was analyzed. β1 and β2-adrenoreceptor (AR) antagonists and siRNA targeting PP2A phosphatase were used to examine the mechanisms by which ZAG modulates insulin sensitivity. Plasma levels of ZAG were measured in a lean patient cohort stratified for HOMA-IR.ResultsZAG treatment increased basal glucose uptake, correlating with an increase in GLUT expression, but induced insulin resistance in adipocytes. Pretreatment of adipocytes with propranolol and a specific β1-AR antagonist demonstrated that ZAG effects on basal glucose uptake and GLUT4 expression are mediated via β1-AR, whereas inhibition of insulin action is dependent on β2-AR activation. ZAG treatment correlated with an increase in PP2A activity. Silencing of the PP2A catalytic subunit abrogated the negative effect of ZAG on insulin-stimulated AKT phosphorylation and glucose uptake but not on GLUT4 expression and basal glucose uptake. ZAG circulating levels were unchanged in a lean patient cohort stratified for HOMA-IR. Neither glucose nor insulin was associated with plasma ZAG.ConclusionsZAG inhibits insulin-induced glucose uptake in human adipocytes by impairing insulin signaling at the level of AKT in a β2-AR- and PP2A-dependent manner.

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.

Immunosuppressive agents are associated with profound metabolic side effects including new-onset diabetes and dyslipidemia after organ transplantation. 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). 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. 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. 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.

Fat-specific Protein 27 (FSP27) Interacts with Adipose Triglyceride Lipase (ATGL) to Regulate Lipolysis and Insulin Sensitivity in Human Adipocytes

In adipocytes, lipolysis is a highly regulated process involving hormonal signals, lipid droplet-associated proteins, and lipases. The discovery of new lipid droplet-associated proteins added complexity to the current model of lipolysis. In this study, we used cultured human adipocytes to demonstrate that fat-specific protein 27 (FSP27), an abundantly expressed protein in adipocytes, regulates both basal and stimulated lipolysis by interacting with adipose triglyceride lipase (ATGL, also called desnutrin or PNPLA2). We identified a core domain of FSP27, amino acids 120-220, that interacts with ATGL to inhibit its lipolytic function and promote triglyceride storage. We also defined the role of FSP27 in free fatty acid-induced insulin resistance in adipocytes. FSP27 depletion in human adipocytes increased lipolysis and inhibited insulin signaling by decreasing AKT phosphorylation. However, reducing lipolysis by either depletion of ATGL or expression of exogenous full-length FSP27 or amino acids 120-220 protected human adipocytes against the adverse effects of free fatty acids on insulin signaling. In embryonic fibroblasts derived from ATGL KO mice, exogenous free fatty acids did not affect insulin sensitivity. Our results demonstrate a crucial role for FSP27-ATGL interactions in regulating lipolysis, triglyceride accumulation, and insulin signaling in human adipocytes.

Disruption of GIP/GIPR Axis in Human Adipose Tissue Is Linked to Obesity and Insulin Resistance

Glucose-dependent insulinotropic peptide (GIP) has a central role in glucose homeostasis through its amplification of insulin secretion; however, its physiological role in adipose tissue is unclear. Our objective was to define the function of GIP in human adipose tissue in relation to obesity and insulin resistance. GIP receptor (GIPR) expression was analyzed in human sc adipose tissue (SAT) and visceral adipose (VAT) from lean and obese subjects in 3 independent cohorts. GIPR expression was associated with anthropometric and biochemical variables. GIP responsiveness on insulin sensitivity was analyzed in human adipocyte cell lines in normoxic and hypoxic environments as well as in adipose-derived stem cells obtained from lean and obese patients. GIPR expression was downregulated in SAT from obese patients and correlated negatively with body mass index, waist circumference, systolic blood pressure, and glucose and triglyceride levels. Furthermore, homeostasis model assessment of insulin resistance, glucose, and G protein-coupled receptor kinase 2 (GRK2) emerged as variables strongly associated with GIPR expression in SAT. Glucose uptake studies and insulin signaling in human adipocytes revealed GIP as an insulin-sensitizer incretin. Immunoprecipitation experiments suggested that GIP promotes the interaction of GRK2 with GIPR and decreases the association of GRK2 to insulin receptor substrate 1. These effects of GIP observed under normoxia were lost in human fat cells cultured in hypoxia. In support of this, GIP increased insulin sensitivity in human adipose-derived stem cells from lean patients. GIP also induced GIPR expression, which was concomitant with a downregulation of the incretin-degrading enzyme dipeptidyl peptidase 4. None of the physiological effects of GIP were detected in human fat cells obtained from an obese environment with reduced levels of GIPR. GIP/GIPR signaling is disrupted in insulin-resistant states, such as obesity, and normalizing this function might represent a potential therapy in the treatment of obesity-associated metabolic disorders.

Human adipocyte extracellular vesicles in reciprocal signaling between adipocytes and macrophages

Extracellular vesicles (EVs) released by human adipocytes or adipose tissue (AT)-explants play a role in the paracrine interaction between adipocytes and macrophages, a key mechanism in AT inflammation, leading to metabolic complications like insulin resistance (IR) were determined. EVs released from in vitro differentiated adipocytes and AT-explants ex vivo were characterized by electron microscopy, Western blot, multiplex adipokine-profiling, and quantified by flow cytometry. Primary monocytes were stimulated with EVs from adipocytes, subcutaneous (SCAT) or omental-derived AT (OAT), and phenotyped. Macrophage supernatant was subsequently used to assess the effect on insulin signaling in adipocytes. Adipocyte and AT-derived EVs differentiated monocytes into macrophages characteristic of human adipose tissue macrophages (ATM), defined by release of both pro- and anti-inflammatory cytokines. The adiponectin-positive subset of AT-derived EVs, presumably representing adipocyte-derived EVs, induced a more pronounced ATM-phenotype than the adiponectin-negative AT-EVs. This effect was more evident for OAT-EVs versus SCAT-EVs. Furthermore, supernatant of macrophages pre-stimulated with AT-EVs interfered with insulin signaling in human adipocytes. Finally, the number of OAT-derived EVs correlated positively with patients HOMA-IR. A possible role for human AT-EVs in a reciprocal pro-inflammatory loop between adipocytes and macrophages, with the potential to aggravate local and systemic IR was demonstrated.

Tumor necrosis factor-alpha acutely inhibits insulin signaling in human adipocytes: implication of the p80 tumor necrosis factor receptor.

Tumor necrosis factor (TNF)-alpha is postulated to play a major role in the pathogenesis of obesity-linked insulin resistance, probably resulting from an interaction with insulin signaling pathways. This cross talk has now been investigated in human adipocytes at the level of phosphatidylinositol (PI) 3-kinase, and the TNF receptors (TNFRs) mediating these processes have been identified. Equilibrium binding studies using human adipocytes from mammary tissue indicated the presence of two populations of TNFR with apparent affinity constants of 13 pmol/l and 1.6 nmol/l, respectively. Interaction of TNF-alpha with insulin signaling was determined by quantification of insulin receptor substrate (IRS)-1-associated PI 3-kinase activity. Under control conditions, PI 3-kinase was activated about 10-fold in response to insulin (10[-7] mol/l, 5 min). Preincubation of adipocytes with 5 nmol/l TNF-alpha for 15 min resulted in a 60-70% reduction of insulin action, reaching a stable inhibition (40%) after longer incubation with the cytokine. The inhibitory action of TNF-alpha was dose-dependent, already detectable at 10 pmol/l, and was correlated to inhibition of tyrosine phosphorylation of IRS-1 with an unaltered autophosphorylation of the insulin receptor beta-subunit. The modulation of insulin signaling by TNF-alpha was found to be paralleled by a comparable inhibition of insulin-stimulated glucose transport. An agonistic TNFR1 antibody completely mimicked the inhibitory action of TNF-alpha on insulin signaling, whereas at 100 pmol/l TNF-alpha, a nonagonistic p80 TNFR antibody, was shown to ameliorate the inhibitory action of the cytokine. These findings indicate that in human adipocytes, low concentrations of TNF-alpha induce a rapid inhibition of insulin signaling at the level of PI 3-kinase. We suggest that under these conditions, the p80 TNFR is essential for initiating the intracellular cross talk that involves signaling by the p60 TNFR.