Inhibition of insulin-induced GLUT4 translocation by Munc18c through interaction with syntaxin4 in 3T3-L1 adipocytes

Insulin induces the translocation of vesicles containing the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane in adipocytes. SNARE proteins have been implicated in the docking and fusion of these vesicles with the cell membrane. The role of Munc18c, previously identified as an n-Sec1/Munc18 homolog in 3T3-L1 adipocytes, in insulin-regulated GLUT4 trafficking has now been investigated in 3T3-L1 adipocytes. In these cells, Munc18c was predominantly associated with syntaxin4, although it bound both syntaxin2 and syntaxin4 to similar extents in vitro. In addition, SNAP-23, an adipocyte homolog of SNAP-25, associated with both syntaxins 2 and 4 in 3T3-L1 adipocytes. Overexpression of Munc18c in 3T3-L1 adipocytes by adenovirus-mediated gene transfer resulted in inhibition of insulin-stimulated glucose transport in a virus dose-dependent manner (maximal effect, approximately 50%) as well as in inhibition of sorbitol-induced glucose transport (by approximately 35%), which is mediated by a pathway different from that used by insulin. In contrast, Munc18b, which is also expressed in adipocytes but which did not bind to syntaxin4, had no effect on glucose transport. Furthermore, overexpression of Munc18c resulted in inhibition of insulin-induced translocation of GLUT4, but not of that of GLUT1, to the plasma membrane. These results suggest that Munc18c is involved in the insulin-dependent trafficking of GLUT4 from the intracellular storage compartment to the plasma membrane in 3T3-L1 adipocytes by modulating the formation of a SNARE complex that includes syntaxin4.     

The ras signaling pathway mimics insulin action on glucose transporter translocation

Recent observations suggest that insulin increases cellular levels of activated, GTP-bound Ras protein. We tested whether the acute actions of insulin on hexose uptake and glucose-transporter redistribution to the cell surface are mimicked by activated Ras. 3T3-L1 fibroblasts expressing an activated mutant (Lys-61) N-Ras protein exhibited a 3-fold increase in 2-deoxyglucose uptake rates compared with non-transfected cells. Insulin stimulated hexose uptake by approximately 2-fold in parental fibroblasts but did not stimulate hexose uptake in the N-Ras61K-expressing fibroblasts. Overexpression of N-Ras61K also mimicked the large effect of insulin on 2-deoxyglucose transport in 3T3-L1 adipocytes, and again the effects of the two agents were not additive. Total glucose transporter protein (GLUT) 1 was similar between parental and N-Ras61K-expressing 3T3-L1 fibroblasts or adipocytes, whereas total GLUT-4 protein was actually lower in the N-Ras61K-expressing compared with parental adipocytes. However, expression of N-Ras61K in 3T3-L1 adipocytes markedly elevated both GLUT-1 and GLUT-4 in plasma membranes relative to intracellular membranes, and insulin had no further effect. These modulations of glucose transporters by N-Ras61K expression are not due to upstream regulation of insulin receptors because receptor tyrosine phosphorylation and association of phosphatidylinositol 3-kinase with tyrosine-phosphorylated proteins were unaffected. These results show that activated Ras mimics the actions of insulin on membrane trafficking of glucose transporters, consistent with the concept that Ras proteins function as intermediates in this insulin signaling pathway.     

Requirement of atypical protein kinase clambda for insulin stimulation of glucose uptake but not for Akt activation in 3T3-L1 adipocytes

Phosphoinositide (PI) 3-kinase contributes to a wide variety of biological actions, including insulin stimulation of glucose transport in adipocytes. Both Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, and atypical isoforms of protein kinase C (PKCzeta and PKClambda) have been implicated as downstream effectors of PI 3-kinase. Endogenous or transfected PKClambda in 3T3-L1 adipocytes or CHO cells has now been shown to be activated by insulin in a manner sensitive to inhibitors of PI 3-kinase (wortmannin and a dominant negative mutant of PI 3-kinase). Overexpression of kinase-deficient mutants of PKClambda (lambdaKD or lambdaDeltaNKD), achieved with the use of adenovirus-mediated gene transfer, resulted in inhibition of insulin activation of PKClambda, indicating that these mutants exert dominant negative effects. Insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but not growth hormone- or hyperosmolarity-induced glucose uptake, were inhibited by lambdaKD or lambdaDeltaNKD in a dose-dependent manner. The maximal inhibition of insulin-induced glucose uptake achieved by the dominant negative mutants of PKClambda was approximately 50 to 60%. These mutants did not inhibit insulin-induced activation of Akt. A PKClambda mutant that lacks the pseudosubstrate domain (lambdaDeltaPD) exhibited markedly increased kinase activity relative to that of the wild-type enzyme, and expression of lambdaDeltaPD in quiescent 3T3-L1 adipocytes resulted in the stimulation of glucose uptake and translocation of GLUT4 but not in the activation of Akt. Furthermore, overexpression of an Akt mutant in which the phosphorylation sites targeted by growth factors are replaced by alanine resulted in inhibition of insulin-induced activation of Akt but not of PKClambda. These results suggest that insulin-elicited signals that pass through PI 3-kinase subsequently diverge into at least two independent pathways, an Akt pathway and a PKClambda pathway, and that the latter pathway contributes, at least in part, to insulin stimulation of glucose uptake in 3T3-L1 adipocytes.     

Antidiabetic thiazolidinediones block the inhibitory effect of tumor necrosis factor-alpha on differentiation, insulin-stimulated glucose uptake, and gene expression in 3T3-L1 cells

Tumor necrosis factor-alpha (TNF alpha) is a cytokine implicated in the development of septic shock, cachexia, and other pathological states. Recent studies indicated a direct role for adipose expression of TNF alpha in obesity-linked insulin resistance and diabetes. Pioglitazone, CP-86,325 (CP), AD-5075, CS-045, ciglitazone, and englitazone are members of a new class of insulin-sensitizing thiazolidinedione derivatives with in vivo antidiabetic activities. To test whether these agents antagonize the effect of TNF alpha, 3T3-L1 cells were induced to differentiate in the presence of TNF alpha with or without thiazolidinedione derivatives. Incubation of 3T3-L1 cells with TNF alpha alone completely inhibited adipocyte conversion and expression of fatty acid-binding protein messenger RNA (mRNA). However, coincubation of TNF alpha-treated cells with CP (1 microM), AD-5075 (1 microM), pioglitazone (10 microM), or CS-045 (10 microM) blocked these effects. Long term incubation of 3T3-L1 adipocytes with a low dose of TNF alpha (50 pM) significantly decreased the levels of the adipocyte/muscle-specific glucose transporter (GLUT4) and the CCAAT enhancer-binding protein mRNAs, but did not affect expression of the ubiquitously expressed glucose transporter (GLUT1) or lipoprotein lipase mRNAs. Incubation of 3T3-L1 adipocytes with TNF alpha also inhibited insulin-stimulated 2-deoxyglucose uptake as well as expression of GLUT4 protein. Furthermore, in 3T3-L1 adipocytes, incubation with TNF alpha attenuated the expression of fatty acid-binding protein mRNA in a time- and dose-dependent manner. These inhibitory effects were partially or completely blocked by coincubation of the cells with CP. These results implicate that the insulin-sensitizing agents may exert their antidiabetic activities by antagonizing the inhibitory effects of TNF alpha.     

Insulin-mediated targeting of phosphatidylinositol 3-kinase to GLUT4-containing vesicles

Phosphatidylinositol (PI) 3-kinase is hypothesized to be a signaling element in the acute redistribution of intracellular GLUT4 glucose transporters to the plasma membrane in response to insulin. However, some receptors activate PI 3-kinase without causing GLUT4 translocation, suggesting specific cellular localization may be critical to this PI 3-kinase function. Consistent with this idea, complexes containing PI 3-kinase bound to insulin receptor substrate 1 (IRS-1) in 3T3-L1 adipocytes are associated with intracellular membranes (Heller-Harrison, R., Morin, M. and Czech, M. (1995) J. Biol. Chem. 270, 24442-24450). We report here that in response to insulin, activated complexes of IRS-1.PI 3-kinase can be immunoprecipitated with anti-IRS-1 antibody from detergent extracts of immunoadsorbed GLUT4-containing vesicles prepared from 3T3-L1 adipocytes. The targeting of PI 3-kinase to rat adipocyte GLUT4-containing vesicles using vesicles prepared by sucrose velocity gradient ultracentrifugation was also demonstrated. Insulin treatment caused a 2.3-fold increase in immunoreactive p85 protein in these GLUT4-containing vesicles while anti-p85 immunoprecipitates of PI 3-kinase activity in GLUT4-containing vesicle extracts increased to a similar extent. HPLC analysis of the GLUT4 vesicle-associated PI 3-kinase activity showed insulin-mediated increases in PI 3-P, PI 3,4-P2, and PI 3,4,5-P3 when PI, PI 4-P, and PI 4,5-P2 were used as substrates. Our data demonstrate that insulin directs the association of PI 3-kinase with GLUT4-containing vesicles in 3T3-L1 and rat adipocytes, consistent with the hypothesis that PI 3-kinase is involved in the insulin-regulated movement of GLUT4 to the plasma membrane.     

Evidence for an insulin receptor substrate 1 independent insulin signaling pathway that mediates insulin-responsive glucose transporter (GLUT4) translocation

Interaction of the activated insulin receptor (IR) with its substrate, insulin receptor substrate 1 (IRS-1), via the phosphotyrosine binding domain of IRS-1 and the NPXY motif centered at phosphotyrosine 960 of the IR, is important for IRS-1 phosphorylation. We investigated the role of this interaction in the insulin signaling pathway that stimulates glucose transport. Utilizing microinjection of competitive inhibitory reagents in 3T3-L1 adipocytes, we have found that disruption of the IR/IRS-1 interaction has no effect upon translocation of the insulin-responsive glucose transporter (GLUT4). The activity of these reagents was demonstrated by their ability to block insulin stimulation of two distinct insulin bioeffects, membrane ruffling and mitogenesis, in 3T3-L1 adipocytes and insulin-responsive rat 1 fibroblasts. These data suggest that phosphorylated IRS-1 is not an essential component of the metabolic insulin signaling pathway that leads to GLUT4 translocation, yet it appears to be required for other insulin bioeffects.     

Association of the insulin receptor with phospholipase C-gamma (PLCgamma) in 3T3-L1 adipocytes suggests a role for PLCgamma in metabolic signaling by insulin

Phospholipase C-gamma (PLCgamma) is the isozyme of PLC phosphorylated by multiple tyrosine kinases including epidermal growth factor, platelet-derived growth factor, nerve growth factor receptors, and nonreceptor tyrosine kinases. In this paper, we present evidence for the association of the insulin receptor (IR) with PLCgamma. Precipitation of the IR with glutathione S-transferase fusion proteins derived from PLCgamma and coimmunoprecipitation of the IR and PLCgamma were observed in 3T3-L1 adipocytes. To determine the functional significance of the interaction of PLCgamma and the IR, we used a specific inhibitor of PLC, U73122, or microinjection of SH2 domain glutathione S-transferase fusion proteins derived from PLCgamma to block insulin-stimulated GLUT4 translocation. We demonstrate inhibition of 2-deoxyglucose uptake in isolated primary rat adipocytes and 3T3-L1 adipocytes pretreated with U73122. Antilipolytic effect of insulin in 3T3-L1 adipocytes is unaffected by U73122. U73122 selectively inhibits mitogen-activated protein kinase, leaving the Akt and p70 S6 kinase pathways unperturbed. We conclude that PLCgamma is an active participant in metabolic and perhaps mitogenic signaling by the insulin receptor in 3T3-L1 adipocytes.     

Syndet, an adipocyte target SNARE involved in the insulin-induced translocation of GLUT4 to the cell surface

In adipocytes, insulin stimulates the translocation of the glucose transporter, GLUT4, from an intracellular storage compartment to the cell surface. Substantial evidence exists to suggest that in the basal state GLUT4 resides in discrete storage vesicles. A direct interaction of GLUT4 storage vesicles with the plasma membrane has been implicated because the v-SNARE, vesicle-associated membrane protein-2 (VAMP2), appears to be a specific component of these vesicles. In the present study we sought to identify the cognate target SNAREs for VAMP2 in mouse 3T3-L1 adipocytes. Membrane fractions were isolated from adipocytes and probed by far Western blotting with the cytosolic portion of VAMP2 fused to glutathione S-transferase. Two plasma membrane-enriched proteins, p25 and p35, were specifically labeled with this probe. By using a combination of immunoblotting, detergent extraction, and anion exchange chromatography, we identified p35 as Syntaxin-4 and p25 as the recently identified murine SNAP-25 homologue, Syndet (mSNAP-23). By using surface plasmon resonance we show that VAMP2, Syntaxin-4, and Syndet form a ternary SDS-resistant SNARE complex. Microinjection of anti-Syndet antibodies into 3T3-L1 adipocytes, or incubation of permeabilized adipocytes with a synthetic peptide comprising the C-terminal 24 amino acids of Syndet, inhibited insulin-stimulated GLUT4 translocation to the cell surface by approximately 40%. GLUT1 trafficking remained unaffected by the presence of the peptide. Our data suggest that Syntaxin-4 and Syndet are important cell-surface target SNAREs within adipocytes that regulate docking and fusion of GLUT-4-containing vesicles with the plasma membrane in response to insulin.     

Do adipocytes contain high affinity sulfonylurea receptors

Sulfonylureas interact with specific, high affinity receptors on the pancreatic beta-cell to close ATP-sensitive K+ channels, depolarize the cell, activate Ca2+ influx through voltage-dependent Ca2+ channels, and trigger insulin secretion. We tested the hypothesis that sulfonylureas promote glucose uptake into 3T3-L1 cells or isolated rat adipocytes by similar mechanisms. Using 125I-labeled 5-iodo-2-hydroxyglyburide and either equilibrium binding or photoaffinity labeling, a high affinity sulfonylurea receptor was not found on plasma membranes of either the 3T3-L1 cells or rat adipocytes. Furthermore, glyburide did not inhibit 86Rb+ efflux (a marker for ATP-sensitive K+ channel conductance), increase free cytosolic calcium in adipocytes or 3T3-L1 cells, or increase basal or insulin-stimulated glucose uptake into 3T3-L1 cells or rat adipocytes. Parallel studies using a hamster insulin-secreting tumor cell line (HIT cells) easily demonstrated both the receptor and biological effects of glyburide on free cytosolic calcium and insulin secretion. Thus, rat adipocytes and 3T3-L1 cells do not possess the high affinity sulfonylurea receptor or respond to glyburide alone. We conclude that the antidiabetogenic effects of sulfonylureas are not mediated by a direct action of sulfonylureas to increase glucose uptake into adipose tissue and suggest that the major locus for sulfonylurea action is the beta-cell.     

Fructose uptake in rat adipocytes: GLUT5 expression and the effects of streptozotocin-induced diabetes

Previous studies have shown that rat adipocytes possess the capacity to take up fructose by a mechanism that is distinct from that involved in the transport of glucose. In this investigation we report that rat adipocytes express the GLUT5 fructose transporter and that it is responsible for mediating a substantial component (approximately 80%) of the total cellular fructose uptake. This proposition is based on the finding that only 21% of the total fructose uptake was cytochalasin B (CB) sensitive which most likely reflects transport via GLUT1 and/or GLUT4. Consistent with this suggestion we found (i) that insulin caused a small, but significant stimulation in fructose uptake (approximately 35%) which was abolished in the presence of CB and (ii) that 3-O-methyl glucose inhibited fructose uptake to a level comparable with that observed in the presence of CB. GLUT5 was found to be localised only in the adipocyte plasma membrane and, unlike GLUT4 or GLUT1, its cell surface abundance was not modulated by insulin. GLUT5 expression fell substantially (by approximately 75%) in adipocytes of streptozotocin-diabetic rats and was accompanied by a reduction in fructose uptake by approximately 50%. Treatment of streptozotocin-diabetic rats with sodium orthovanadate for a period of 3 days led to a significant reduction in blood glycaemia by approximately 40% and a partial restoration in both GLUT5 expression and adipocyte fructose uptake. We suggest that fructose uptake in rat adipocytes is principally mediated by GLUT5 in an insulin- and CB-insensitive manner and that expression of GLUT5 in rat adipocytes may be regulated by changes in blood glycaemia.     

Transgenic mice expressing the human GLUT4/muscle-fat facilitative glucose transporter protein exhibit efficient glycemic control

To examine the physiological role of the GLUT4/muscle-fat specific facilitative glucose transporter in regulating glucose homeostasis, we have generated transgenic mice expressing high levels of this protein in an appropriate tissue-specific manner. Examination of two independent founder lines demonstrated that high-level expression of GLUT4 protein resulted in a marked reduction of fasting glucose levels (approximately 70 mg/dl) compared to wild-type mice (approximately 130 mg/dl). Surprisingly, 30 min following an oral glucose challenge the GLUT4 transgenic mice had only a slight elevation in plasma glucose levels (approximately 90 mg/dl), whereas wild-type mice displayed a typical 2- to 3-fold increase (approximately 250-300 mg/dl). In parallel to the changes in plasma glucose, insulin levels were approximately 2-fold lower in the transgenic mice compared to the wild-type mice. Furthermore, isolated adipocytes from the GLUT4 transgenic mice had increased basal glucose uptake and subcellular fractionation indicated elevated levels of cell surface-associated GLUT4 protein. Consistent with these results, in situ immunocytochemical localization of GLUT4 protein in adipocytes and cardiac myocytes indicated a marked increase in plasma membrane-associated GLUT4 protein in the basal state. Taken together these data demonstrate that increased expression of the human GLUT4 gene in vivo results in a constitutively high level of cell surface GLUT4 protein expression and more efficient metabolic control over fluctuations in plasma glucose concentrations.     

Gq-coupled receptors transmit the signal for GLUT4 translocation via an insulin-independent pathway

Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) induces the translocation of glucose transporter type 4 (GLUT4) from an intracellular pool to the cell surface and increases glucose uptake in adipocytes. The GTP-binding protein(s) responsible for the translocation has remained to be identified. Using a sensitive and quantitative method to assess the translocation of c-MYC epitope-tagged GLUT4, we obtained evidence that the activation of receptor-coupled Gq (neither Gi nor Gs) triggered GLUT4 translocation in cells, independently of insulin signaling pathway(s). Platelet-activating factor (PAF) induced GLUT4 translocation in the cells expressing the Gi- and Gq-coupled PAF receptor, but the translocation was induced even after pretreatment with wortmannin, an islet-activating protein and phorbol 12, 13-dibutyrate. Norepinephrine triggered GLUT4 translocation in cells expressing the Gq-coupled alpha1-adrenergic receptor, but prostaglandin E2 did not cause GLUT4 translocation in cells expressing the Gs-coupled EP4 receptor or the Gi-coupled EP3alpha receptor. The norepinephrine-stimulated GLUT4 translocation and glucose uptake via Gq may possibly contribute to the fuel supply required for thermogenesis in brown adipocytes and for the enhanced contractility in cardiomyocytes, both of which have an abundant endogenous GLUT4.     

An annotated bibliography on autologous transfusion: second edition

JTT-501 is an insulin-sensitising compound with an isoxazolidinedione rather than a thiazolidionedione structure. Sprague-Dawley rats fed a high fat diet for 2 weeks were used as an animal model of insulin resistance, and JTT-501 was administered for the final week of the diet. An euglycaemic glucose clamp study showed that the glucose infusion rate (GIR) required to maintain euglycaemia was 57% lower in rats fed a high fat diet than in control rats, and that JTT-501 treatment restored the reduction in GIR produced by the high fat diet. To explain the mechanisms underlying the effects of a high fat diet and JTT-501 treatment, epididymal fat pads were excised and used in the analysis of insulin action. The high fat diet caused: (1) a 58% decrease in insulin receptor substrate-1 (IRS-1) content with a 58% decrease in IRS-1 tyrosine phosphorylation; (2) reductions of 56% and 73% respectively in insulin-induced maximal PI 3-kinase activation in anti-phosphotyrosine and anti-IRS-1 antibody immunoprecipitates; (3) a 46% reduction in the glucose transporter protein, GLUT4 content and, consequently, (4) severely impaired insulin-induced GLUT4 translocation to the plasma membrane and glucose uptake in adipocytes. JTT-501 treatment restored appreciably the protein content and tyrosine phosphorylation level of IRS-1. Insulin-stimulated PI 3-kinase activation was also restored in anti-phosphotyrosine and anti-IRS-1 antibody immunoprecipitates. As reflected by these improvements in insulin signalling, JTT-501 treatment improved considerably insulin-induced GLUT4 translocation to the plasma membrane as well as insulin-induced glucose uptake. However, JTT-501 had no effect on the decrease in GLUT4 content produced by the high fat diet. These observations suggest that JTT-501 enhances insulin signalling and may be effective in reducing insulin resistance.     

"Cross talk" between the bioactive glycerolipids and sphingolipids in signal transduction

Hydrolysis of phosphatidylcholine via receptor-mediated stimulation of phospholipase D produces phosphatidate that can be converted to lysophosphatidate and diacylglycerol. Diacylglycerol is an activator of protein kinase C, whereas phosphatidate and lysophosphatidate stimulate tyrosine kinases and activate the Ras-Raf-mitogen-activated protein kinase pathway. These three lipids can stimulate cell division. Conversely, activation of sphingomyelinase by agonists (e.g., tumor necrosis factor-alpha) causes ceramide production that inhibits cell division and produces apoptosis. If ceramides are metabolized to sphingosine and sphingosine 1-phosphate, then these lipids can stimulate phospholipase D and are also mitogenic. By contrast, ceramides inhibit the activation of phospholipase D by decreasing its interaction with the G-proteins, ARF and Rho, which are necessary for its activation. In whole cells, ceramides also stimulate the degradation of phosphatidate, lysophosphatidate, ceramide 1-phosphate, and sphingosine 1-phosphate through a multifunctional phosphohydrolase (the Mg(2+)-independent phosphatidate phosphohydrolase), whereas sphingosine inhibits phosphatidate phosphohydrolase. Tumor necrosis factor-alpha causes insulin resistance, which may be partly explained by ceramide production. Cell-permeable ceramides decrease insulin-stimulated glucose uptake in 3T3-L1 adipocytes after 2-24 h, whereas they stimulate basal glucose uptake. These effects do not depend on decreased tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 or the interaction of insulin receptor substrate-1 with phosphatidylinositol 3-kinase. They appear to rely on the differential effects of ceramides on the translocation of GLUT1-and GLUT4-containing vesicles. It is concluded that there is a significant interaction and "cross-talk" between the sphingolipid and glycerolipid pathways that modifies signal transduction to control vesicle movement, cell division, and cell death.     

A novel, multifuntional c-Cbl binding protein in insulin receptor signaling in 3T3-L1 adipocytes

The protein product of the c-Cbl proto-oncogene is prominently tyrosine phosphorylated in response to insulin in 3T3-L1 adipocytes and not in 3T3-L1 fibroblasts. After insulin-dependent tyrosine phosphorylation, c-Cbl specifically associates with endogenous c-Crk and Fyn. These results suggest a role for tyrosine-phosphorylated c-Cbl in 3T3-L1 adipocyte activation by insulin. A yeast two-hybrid cDNA library prepared from fully differentiated 3T3-L1 adipocytes was screened with full-length c-Cbl as the target protein in an attempt to identify adipose-specific signaling proteins that interact with c-Cbl and potentially are involved in its tyrosine phosphorylation in 3T3-L1 adipocytes. Here we describe the isolation and the characterization of a novel protein that we termed CAP for c-Cbl-associated protein. CAP contains a unique structure with three adjacent Src homology 3 (SH3) domains in the C terminus and a region showing significant sequence similarity with the peptide hormone sorbin. Both CAP mRNA and proteins are expressed predominately in 3T3-L1 adipocytes and not in 3T3-L1 fibroblasts. CAP associates with c-Cbl in 3T3-L1 adipocytes independently of insulin stimulation in vivo and in vitro in an SH3-domain-mediated manner. Furthermore, we detected the association of CAP with the insulin receptor. Insulin stimulation resulted in the dissociation of CAP from the insulin receptor. Taken together, these data suggest that CAP represents a novel c-Cbl binding protein in 3T3-L1 adipocytes likely to participate in insulin signaling.     

Regional variation in adipose tissue insulin action and GLUT4 glucose transporter expression in severely obese premenopausal women

Insulin action and GLUT4 expression were examined in adipose tissue of severely obese premenopausal women undergoing gastrointestinal surgery. Fat samples were taken from three different anatomical regions: the subcutaneous abdominal site, the round ligament (deep abdominal properitoneal fat), and the greater omentum (deep abdominal intraperitoneal fat). The stimulatory effect of insulin on glucose transport and the ability of the hormone to inhibit lipolysis were determined in adipocytes isolated from these three adipose depots. Insulin stimulated glucose transport 2-3 times over basal rates in all adipocytes. However, round ligament adipose cells showed a significantly greater responsiveness to insulin when compared to subcutaneous and omental adipocytes. Round ligament fat cells also displayed the greatest sensitivity and maximal antilipolytic response to insulin. We also investigated whether regional differences in fat cell insulin-stimulated glucose transport were linked to a differential expression of the GLUT4 glucose transporter. GLUT4 protein content in total membranes was 5 and 2.2 times greater in round ligament adipose tissue than in subcutaneous and omental fat depots, respectively. Moreover, GLUT4 mRNA levels were 2.1 and 3 times higher in round ligament than in subcutaneous or omental adipose tissues, respectively. Adipose tissue GLUT4 protein content was strongly and negatively associated (r = -0.79 to -0.89, p < 0.01) with the waist-to-hip ratio but not with total adiposity. In conclusion, these results demonstrate the existence of site differences in adipose tissue insulin action in morbidly obese women. The greater insulin effect on glucose transport in round ligament adipocytes was associated with a higher expression of GLUT4 when compared to subcutaneous abdominal and omental fat cells. Moreover, despite the regional variation in GLUT4 expression, an increased proportion of abdominal fat was found to be associated with lower levels of GLUT4 in all adipose regions investigated.