Obesity-dependent metabolic signatures associated with nonalcoholic fatty liver disease progression.

Our understanding of the mechanisms by which nonalcoholic fatty liver disease (NAFLD) progresses from simple steatosis to steatohepatitis (NASH) is still very limited. Despite the growing number of studies linking the disease with altered serum metabolite levels, an obstacle to the development of metabolome-based NAFLD predictors has been the lack of large cohort data from biopsy-proven patients matched for key metabolic features such as obesity. We studied 467 biopsied individuals with normal liver histology (n=90) or diagnosed with NAFLD (steatosis, n=246; NASH, n=131), randomly divided into estimation (80% of all patients) and validation (20% of all patients) groups. Qualitative determinations of 540 serum metabolite variables were performed using ultraperformance liquid chromatography coupled to mass spectrometry (UPLC-MS). The metabolic profile was dependent on patient body-mass index (BMI), suggesting that the NAFLD pathogenesis mechanism may be quite different depending on an individual's level of obesity. A BMI-stratified multivariate model based on the NAFLD serum metabolic profile was used to separate patients with and without NASH. The area under the receiver operating characteristic curve was 0.87 in the estimation and 0.85 in the validation group. The cutoff (0.54) corresponding to maximum average diagnostic accuracy (0.82) predicted NASH with a sensitivity of 0.71 and a specificity of 0.92 (negative/positive predictive values=0.82/0.84). The present data, indicating that a BMI-dependent serum metabolic profile may be able to reliably distinguish NASH from steatosis patients, have significant implications for the development of NASH biomarkers and potential novel targets for therapeutic intervention.

Deficiency in the extracellular signal-regulated kinase 1 (ERK1) protects leptin-deficient mice from insulin resistance without affecting obesity.

AIMS/HYPOTHESIS: Extracellular signal-regulated kinase (ERK) activity is increased in adipose tissue in obesity and type 2 diabetes mellitus and strong evidences suggests that it is implicated in the downregulation of insulin signalling and action in the insulin-resistant state. To determine the role of ERK1 in obesity-associated insulin resistance in vivo, we inactivated Erk1 (also known as Mapk3) in obese leptin-deficient mice (ob/ob). METHODS: Mice of genotype ob/ob-Erk1⁻(/)⁻ were obtained by crossing Erk1⁻(/)⁻ mice with ob/ob mice. Glucose tolerance and insulin sensitivity were studied in 12-week-old mice. Tissue-specific insulin sensitivity, insulin signalling, liver steatosis and adipose tissue inflammation were determined. RESULTS: While ob/ob-Erk1⁻(/)⁻ and ob/ob mice exhibited comparable body weight and adiposity, ob/ob-Erk1⁻(/)⁻ mice did not develop hyperglycaemia and their glucose tolerance was improved. Hyperinsulinaemic-euglycaemic clamp studies demonstrated an increase in whole-body insulin sensitivity in the ob/ob-Erk1⁻(/)⁻ mice associated with an increase in both insulin-stimulated glucose disposal in skeletal muscles and adipose tissue insulin sensitivity. This occurred in parallel with improved insulin signalling in both tissues. The ob/ob-Erk1⁻(/)⁻ mice were also partially protected against hepatic steatosis with a strong reduction in acetyl-CoA carboxylase level. These metabolic improvements were associated with reduced expression of mRNA encoding inflammatory cytokine and T lymphocyte markers in the adipose tissue. CONCLUSIONS/INTERPRETATION: Our results demonstrate that the targeting of ERK1 could partially protect obese mice against insulin resistance and liver steatosis by decreasing adipose tissue inflammation and by increasing muscle glucose uptake. Our results indicate that deregulation of the ERK1 pathway could be an important component in obesity-associated metabolic disorders.

A new composite model including metabolic syndrome, alanine aminotransferase and cytokeratin-18 for the diagnosis of non-alcoholic steatohepatitis in morbidly obese patients.

BACKGROUND: Non-invasive approaches are useful to differentiate simple steatosis from non-alcoholic steatohepatitis (NASH) in obese and morbidly obese patients. AIM: To develop a new scoring system to diagnose definitive NASH. METHODS: Preoperative clinical and biological data including serum caspase 3-generated cytokeratin-18 fragments (CK18) and surgical liver biopsies were obtained from 464 morbidly obese patients who had undergone bariatric surgery. The cohort was divided into two groups: training group (n = 310) and validation group (n = 154). Definitive NASH was defined according to Kleiner's classification with a Non-alcoholic fatty liver disease Activity Score (NAS) ≥5. RESULTS: Alanine aminotransferase (ALT), CK18 fragments and the presence of metabolic syndrome were independent predictors for discriminating patients with NAS ≥5 in the training group. These three parameters were used to carry out a scoring system for the prediction of NAS ≥5. Whereas serum CK18 fragment alone had an area under the receiver operating characteristic (AUROC) curve = 0.74, AUROC curves of the scoring system were 0.88 and 0.83 in the training group and the validation group, respectively. CONCLUSION: A simple and non-invasive composite model (the Nice Model) including metabolic syndrome, ALT and CK18 fragments is able to predict accurately a non-alcoholic fatty liver disease activity score ≥5 in morbidly obese subjects.

Involvement of TNF-alpha in abnormal adipocyte and muscle sortilin expression in obese mice and humans.

AIMS/HYPOTHESIS: Insulin resistance is caused by numerous factors including inflammation. It is characterised by defective insulin stimulation of adipocyte and muscle glucose transport, which requires the glucose transporter GLUT4 translocation towards the plasma membrane. Defects in insulin signalling can cause insulin resistance, but alterations in GLUT4 trafficking could also play a role. Our goal was to determine whether proteins controlling GLUT4 trafficking are altered in insulin resistance linked to obesity. METHODS: Using real-time RT-PCR, we searched for selected transcripts that were differentially expressed in adipose tissue and muscle in obese mice and humans. Using various adipocyte culture models and in vivo mice treatment, we searched for the involvement of TNF-alpha in these alterations in obesity. RESULTS: Sortilin mRNA and protein were downregulated in adipose tissue from obese db/db and ob/ob mice, and also in muscle. Importantly, sortilin mRNA was also decreased in morbidly obese human diabetic patients. Sortilin and TNF-alpha (also known as TNF) mRNA levels were inversely correlated in mice and human adipose tissues. TNF-alpha decreased sortilin mRNA and protein levels in cultured mouse and human adipocytes, an effect partly prevented by the peroxisome proliferator-activated receptor gamma activator rosiglitazone. TNF-alpha also inhibited adipocyte and muscle sortilin mRNA when injected to mice. CONCLUSIONS/INTERPRETATION: Sortilin, an essential player in adipocyte and muscle glucose metabolism through the control of GLUT4 localisation, is downregulated in obesity and TNF-alpha is likely to be involved in this defect. Chronic low-grade inflammation in obesity could thus contribute to insulin resistance by modulating proteins that control GLUT4 trafficking.

The nitric oxide-donating derivative of acetylsalicylic acid, NCX 4016, stimulates glucose transport and glucose transporters translocation in 3T3-L1 adipocytes.

NCX 4016 is a nitric oxide (NO)-donating derivative of acetylsalicylic acid. NO and salicylate, in vivo metabolites of NCX 4016, were shown to be potential actors in controlling glucose homeostasis. In this study, we evaluated the action of NCX 4016 on the capacity of 3T3-L1 adipocytes to transport glucose in basal and insulin-stimulated conditions. NCX 4016 induced a twofold increase in glucose uptake in parallel with the translocation of the glucose transporters GLUT1 and GLUT4 to the plasma membrane, leaving unaffected their total expression levels. Importantly, NCX 4016 further increased glucose transport induced by a physiological concentration of insulin. The stimulatory effect of NCX 4016 on glucose uptake appears to be mediated by its NO moiety. Indeed, it is inhibited by a NO scavenger and treatment with acetylsalicylic or salicylic acid had no effect. Although NO is involved in the action of NCX 4016, it did not mainly depend on the soluble cGMP cyclase/protein kinase G pathway. Furthermore, NCX 4016-stimulated glucose transport did not involve the insulin-signaling cascade required to stimulate glucose transport. NCX 4016 induces a small activation of the mitogen-activated protein kinases p38 and c-Jun NH(2)-terminal kinase and no activation of other stress-activated signaling molecules, including extracellular signal-regulated kinase, inhibitory factor kappaB, or AMP-activated kinases. Interestingly, NCX 4016 modified the content of S-nitrosylated proteins in adipocytes. Taken together, our results indicate that NCX 4016 induced glucose transport in adipocytes through a novel mechanism possibly involving S-nitrosylation. NCX 4016 thus possesses interesting characteristics to be considered as a candidate molecule for the treatment of patients suffering from metabolic syndrome and type 2 diabetes.

Rab proteins in endocytosis and Glut4 trafficking.

The intracellular trafficking of numerous proteins requires a tight control to fulfil their physiological functions. It is the case of the adipocyte and muscle glucose transporter Glut4 that is retained intracellularly until insulin induces its recruitment to the plasma membrane. Rabs are evolutionarily conserved small GTPases that control intracellular traffic events from yeast to mammalian cells. In the past few decades, considerable progresses have been made in identifying the route of Glut4, the Rabs involved in controlling it, and more recently the connection between insulin signalling and Glut4 trafficking through Rab activity control.

The antidiabetic drug metformin exerts an antitumoral effect in vitro and in vivo through a decrease of cyclin D1 level.

Metformin is a widely used antidiabetic agent, which regulates glucose homeostasis through inhibition of liver glucose production and an increase in muscle glucose uptake. Recent studies suggest that metformin may reduce the risk of cancer, but its mode of action in cancer remains not elucidated. We investigated the effect of metformin on human prostate cancer cell proliferation in vitro and in vivo. Metformin inhibited the proliferation of DU145, PC-3 and LNCaP cancer cells with a 50% decrease of cell viability and had a modest effect on normal prostate epithelial cell line P69. Metformin did not induce apoptosis but blocked cell cycle in G(0)/G(1). This blockade was accompanied by a strong decrease of cyclin D1 protein level, pRb phosphorylation and an increase in p27(kip) protein expression. Metformin activated the AMP kinase pathway, a fuel sensor signaling pathway. However, inhibition of the AMPK pathway using siRNA against the two catalytic subunits of AMPK did not prevent the antiproliferative effect of metformin in prostate cancer cells. Importantly, oral and intraperitoneal treatment with metformin led to a 50 and 35% reduction of tumor growth, respectively, in mice bearing xenografts of LNCaP. Similar, to the in vitro study, metformin led to a strong reduction of cyclin D1 protein level in tumors providing evidence for a mechanism that may contribute to the antineoplastic effects of metformin suggested by recent epidemiological studies.

p38MAP Kinase activity is required for human primary adipocyte differentiation.

Little is known about the role of p38MAPK in human adipocyte differentiation. Here we showed that p38MAPK activity increases during human preadipocytes differentiation. Pharmacological inhibition of p38MAPK during adipocyte differentiation of primary human preadipocytes markedly reduced triglycerides accumulation and adipocyte markers expression. Cell cycle arrest or proliferation was not affected by p38MAPK inhibition. Although induction of C/EBPbeta was not altered by the p38MAPK inhibitor, its phosphorylation on Threonine(188) was decreased as well as PPARgamma expression. These results indicate that p38MAPK plays a positive role in human adipogenesis through regulation of C/EBPbeta and PPARgamma factors.

Differential effects of IRS1 phosphorylated on Ser307 or Ser632 in the induction of insulin resistance by oxidative stress.

AIMS/HYPOTHESIS: Induction of stress kinases leading to serine hyperphosphorylation of IRS1 may link oxidative stress to insulin resistance. The aim of this study was to investigate the roles of the phosphorylated serine residues Ser307 and Ser632, two sites implicated in the inhibition of IRS1 function in insulin signalling. MATERIALS AND METHODS: Fao hepatoma cells were exposed to an H(2)O(2)-generating system, and antibodies against the two phosphorylated serine residues were used for immunoprecipitation, immunoblot and immunofluorescence analyses. RESULTS: Exposure to approximately 50 mumol/l H(2)O(2) for 2 h resulted in IRS1 phosphorylation on both Ser307 and Ser632, concomitant with activation of inhibitor kappa kinase beta (IKKbeta) and c-Jun kinase (JNK). Immunoprecipitation studies revealed that the maximum overlap between phospho (p) Ser307-IRS1 and pSer632-IRS1 was 20%, and confocal microscopy suggested distinct localisations of IRS1 molecules phosphorylated on either site. Although pSer307-IRS1 showed decreased insulin-induced tyrosine phosphorylation and interaction with phosphatidylinositol 3-kinase (PI3K) in response to insulin, pSer632-IRS1 molecules were normally tyrosine-phosphorylated and exhibited typical associated PI3K activity. Salicylic acid and SP600125 partially inhibited IKKbeta and JNK, respectively, which indicated distinct roles for these two kinases in the phosphorylation of IRS1 at the two serine sites. Protection against oxidation-mediated impairment in insulin-induced phosphorylation of protein kinase B/Akt and in glycogen synthesis was achieved only by combining salicylic acid and SP600125. CONCLUSIONS/INTERPRETATION: These results suggest that pSer307-IRS1 and pSer632-IRS1 may define two minimally overlapping pools of IRS1 in response to oxidative stress, contributing differentially to insulin resistance. A combination of stress kinase inhibitors is required to protect against insulin resistance and IRS1 hyperphosphorylation induced by oxidative stress.

Role of MAPKs in development and differentiation: lessons from knockout mice.

The ERK, p38MAPK, JNK mitogen-activated protein kinases (MAPKs) are intracellular signaling pathways that play a pivotal role in many essential cellular processes such as proliferation and differentiation. These cascades are activated by a large variety of stimuli and display a high degree of homology. So far, seven MAPK isoforms have been invalidated in mice leading to the discovery of their important functions in development and differentiation. As we could expect because of their multiple and specific properties in vitro, knockout (KO) of MAPK pathways leads to distinct phenotypes in mice. Surprisingly, into a given cascade, KOs of the various isoforms assign specific non-redundant biological functions to each isoform, without compensation by the others. These results emphasize the notion that, although initiated by the same external stimuli, these intracellular cascades activate kinase isoforms each with its own specific role.

The formation of an intrachain disulfide bond in the leptin protein is necessary for efficient leptin secretion.

Leptin is a cytokine secreted by the adipose tissue that is involved in the control of body weight. We previously showed that a point mutation (R105W) in leptin results in leptin deficiency, marked obesity and hypogonadism in humans adults. Expression in COS1 cells showed impaired secretion and intracellular accumulation of the mutated protein. However, impaired secretion of the mutant leptin had not been demonstrated in adipose cells. In this work, we demonstrate that secretion of R105W mutant is impaired in rat and human adipocytes. We also show that R105W mutant expressed in COS1 cells and in PAZ6 adipocytes forms large molecular aggregates that cannot cross a filtration membrane with a cut-off of 100 kDa. Moreover, we have engineered, by site directed mutagenesis, the cDNAs coding for leptin in which either Cys 117, Cys 167, or both, were replaced by a serine. When expressed in COS1 cells or PAZ6 adipocytes, cysteine mutants also show impaired secretion and formation of large molecular aggregates. Therefore, our work indicates that the formation of an intramolecular disulfide bridge is necessary for normal processing and secretion of leptin. Moreover, the similarity of the behavior of R105W mutant and cystein mutants suggests that the lack of secretion observed with the naturally occurring mutant could result from impaired disulfide bond formation.

Alteration in insulin action: role of IRS-1 serine phosphorylation in the retroregulation of insulin signalling.

Insulin resistance, when combined with impaired insulin secretion, contributes to the development of type 2 diabetes. Insulin resistance is characterised by a decrease in insulin effect on glucose transport in muscle and adipose tIssue. Tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) and its binding to phosphatidylinositol 3-kinase (PI 3-kinase) are critical events in the insulin signalling cascade leading to insulin-stimulated glucose transport. Modification of IRS-1 by serine phosphorylation could be one of the mechanisms leading to a decrease in IRS-1 tyrosine phosphorylation, PI 3-kinase activity and glucose transport. Recent findings demonstrate that "diabetogenic" factors such as FFA, TNFalpha, hyperinsulinemia and cellular stress, increase the serine phosphorylation of IRS-1 and identified Ser307/612/632 as phosphorylated sites. Moreover, several kinases able to phosphorylate these serine residues have been identified. These exciting results suggest that serine phosphorylation of IRS-1 is a possible hallmark of insulin resistance in biologically insulin responsive cells or tIssues. Identifying the pathways by which "diabetogenic" factors activate IRS-1 kinases and defining the precise role of serine phosphorylation events in IRS-1 regulation represent important goals. Such studies may enable rational drug design to selectively inhibit the activity of the relevant enzymes and generate a novel class of therapeutic agents for type 2 diabetes.

Fatty acid-induced insulin resistance: role of insulin receptor substrate 1 serine phosphorylation in the retroregulation of insulin signalling.

Insulin resistance, when combined with impaired insulin secretion, contributes to the development of type 2 diabetes. Insulin resistance is characterized by a decrease in the insulin effect on glucose transport in muscle and adipose tissue. Tyrosine phosphorylation of IRS-1 (insulin receptor substrate 1) and its binding to PI 3-kinase (phosphoinositide 3-kinase) are critical events in the insulin signalling cascade leading to insulin-stimulated glucose transport. Various studies have implicated lipids as a cause of insulin resistance in muscle. Elevated plasma fatty acid concentrations are associated with reduced insulin-stimulated glucose transport activity as a consequence of altered insulin signalling through PI 3-kinase. Modification of IRS-1 by serine phosphorylation could be one of the mechanisms leading to a decrease in IRS-1 tyrosine phosphorylation, PI 3-kinase activity and glucose transport. Recent findings demonstrate that non-esterified fatty acids, as well as other factors such as tumour necrosis factor alpha, hyperinsulinaemia and cellular stress, increase the serine phosphorylation of IRS-1 and identified Ser(307) as one of the phosphorylated sites. Moreover, several kinases able to phosphorylate this serine residue have been identified. These exciting results suggest that Ser(307) phosphorylation is a possible hallmark of insulin resistance in biologically insulin-responsive cells or tissues. Identification of IRS-1 kinases could enable rational drug design in order to selectively inhibit the activity of the relevant enzymes and generate a novel class of therapeutic agents for type 2 diabetes.

MAP kinases and mTOR mediate insulin-induced phosphorylation of insulin receptor substrate-1 on serine residues 307, 612 and 632.

AIM/HYPOTHESIS: Insulin-induced IRS-1 serine phosphorylation could be physiologically important to regulate insulin action. In a hyperinsulinaemic state such as obesity or Type 2 diabetes, this phosphorylation could be modified and exacerbate insulin resistance. We aimed at identifying serine residues in IRS-1 phosphorylated in response to insulin stimulation and at determining the involved kinases. METHODS: 3T3-L1 adipocytes, muscle and adipose tissue of mice were subjected to Western Blot analysis with phosphospecific antibodies to identify phosphorylation sites in IRS-1 following insulin treatment. Pharmacological inhibitors were used to determine the serine kinases involved in this phosphorylation. RESULTS: In 3T3-L1 adipocytes, insulin promoted the phosphorylation of serine 307, 612 and 632 with Serine(612/632) more rapidly phosphorylated than Serine(307). Insulin-induced phosphorylation of Serine(307) was dependent on the activation of a PI 3-kinase/mTOR pathway. The phosphorylation of Serine(612/632) required the activation of the MAP kinase pathway following short-term insulin stimulation and activation of the PI 3-kinase/mTOR pathway following prolonged insulin stimulation. Phosphorylation of Serine(307) and Serine(632) occurred in vivo in skeletal muscle and white adipose tissue of mice injected with insulin and was dependent on the activation of mTOR. Moreover, inhibition of mTOR led to a persistent PI 3-kinase activation by insulin. CONCLUSION/INTERPRETATION: Insulin-induced IRS-1 serine phosphorylation is a complex process involving different sites and kinases. This complexity could be physiologically important to accurately regulate insulin signalling. Abnormal phosphorylation of these serine residues in hyperinsulinaemic state could participate in the down-regulation of insulin signalling.

Positive and negative regulation of glucose uptake by hyperosmotic stress.

This review will provide insight on the current understanding of the intracellular signaling mechanisms by which hyperosmolarity mimics insulin responses such as Glut 4 translocation and glucose transport but also antagonizes insulin effects. Glucose uptake induced by insulin is largely dependent on the PI 3-kinase/PKB pathway. In both adipocyte and muscle cells, hyperosmolarity promotes glucose uptake by multiple mechanisms which do not require PI 3-kinase/PKB pathway but are dependent on the cell type. In muscle, osmotic stress induces glucose uptake by stimulation of AMP-Kinase and/or inhibition of Glut 4 endocytosis. In adipocytes, activation of Gab1-dependent signaling pathway plays an important role in osmotic stress-mediated glucose uptake. Apart of its insulin-like effects, hyperosmolarity can lead to cellular insulin resistance mediated by both prevention of PKB activation and inhibition of the Insulin Receptor Substrate-1 (IRS1) function. Serine phosphorylation and degradation of IRS1 negatively regulate its functions. Understanding how osmotic stress induces glucose transport or mediates insulin resistance may provide novel targets for strategies to enhance glucose transport or to prevent insulin resistance.

The role of small G-proteins in the regulation of glucose transport (review).

Insulin increases the rate of glucose transport into fat and muscle cells by stimulating the translocation of intracellular Glut 4-containing vesicles to the plasma membrane. This results in a marked increase in the amount of the facilitative glucose transporter Glut 4 at the cell surface, allowing for an enhanced glucose uptake. This process requires a continuous cycling through the early endosomes, a Glut 4 specific storage compartment and the plasma membrane. The main effect of insulin is to increase the rate of Glut 4 trafficking from its specific storage compartment to the plasma membrane. The whole phenomenon involves signal transduction from the insulin receptor, vesicle trafficking (sorting and fusion processes) and actin cytoskeleton modifications, which are all supposed to require small GTPases. This review describes the potential role of the various members of the Ras, Rad, Rho, Arf and Rab families in the traffic of the Glut 4-containing vesicles.

Role of the FYVE finger and the RUN domain for the subcellular localization of Rabip4.

Rabip4 is a Rab4 effector, which possesses a RUN domain, two coiled-coil domains, and a FYVE finger. It is associated with the early endosomes and leads, in concert with Rab4, to the enlargement of endosomes, resulting in the fusion of sorting and recycling endosomes. Our goal was to characterize the role of these various domains in Rabip4 subcellular localization and their function in Chinese hamster ovary cells. Although the FYVE finger domain specifically bound phosphatidylinositol 3-phosphate and was necessary for the function of Rabip4, it was not sufficient for the protein association with membranes. Indeed a protein containing the FYVE finger and the Rab4-binding site was cytosolic, whereas the total protein was mostly associated to the membrane fraction, whether or not cells were pretreated with wortmannin. By contrast, a construct corresponding to the N-terminal end, Rabip4-(1-212), and containing the RUN domain was membrane-associated. The complete protein partitioned between the Triton X-100-insoluble and -soluble fractions and a wortmannin treatment increased the amount of the protein in the Triton X-100 fraction. Rabip4-(1-212) was totally Triton X-100-insoluble, and confocal microscopic examination showed that it labeled not only the endosomes, positive for Rabip4, but also a filamentous network with a honeycomb appearance. The Triton X-100-insoluble fraction that contains Rabip4 did not correspond to the caveolin or glycosylphosphatidylinositol-enriched lipid rafts. Rabip4 did not appear directly linked to actin but seemed associated to the actin network. We propose that the subcellular localization of the protein is primarily driven by the RUN domain to endosomal microdomains characterized by Triton X-100 insolubility and that the FYVE domain and the Rab4-binding domain then allow for the recruitment of the protein to lipophilic microdomains enriched in phosphatidylinositol 3-phosphate.

The lipid phosphatase SHIP2 controls insulin sensitivity.

Insulin is the primary hormone involved in glucose homeostasis, and impairment of insulin action and/or secretion has a critical role in the pathogenesis of diabetes mellitus. Type-II SH2-domain-containing inositol 5-phosphatase, or 'SHIP2', is a member of the inositol polyphosphate 5-phosphatase family. In vitro studies have shown that SHIP2, in response to stimulation by numerous growth factors and insulin, is closely linked to signalling events mediated by both phosphoinositide-3-OH kinase and Ras/mitogen-activated protein kinase. Here we report the generation of mice lacking the SHIP2 gene. Loss of SHIP2 leads to increased sensitivity to insulin, which is characterized by severe neonatal hypoglycaemia, deregulated expression of the genes involved in gluconeogenesis, and perinatal death. Adult mice that are heterozygous for the SHIP2 mutation have increased glucose tolerance and insulin sensitivity associated with an increased recruitment of the GLUT4 glucose transporter and increased glycogen synthesis in skeletal muscles. Our results show that SHIP2 is a potent negative regulator of insulin signalling and insulin sensitivity in vivo.

Expression of a prenylation-deficient Rab4 inhibits the GLUT4 translocation induced by active phosphatidylinositol 3-kinase and protein kinase B.

The small GTPase Rab4 has been shown to participate in the subcellular distribution of GLUT4 under both basal and insulin-stimulated conditions in adipocytes. In the present work, we have characterized the effect of Rab4 DeltaCT, a prenylation-deficient and thus cytosolic form of Rab4, in this process. We show that the expression of Rab4 DeltaCT in freshly isolated adipocytes inhibits insulin-induced GLUT4 translocation, but only when this protein is in its GTP-bound active form. Further, it not only blocks the effect of insulin, but also that of a hyperosmotic shock, but does not interfere with the effect of zinc ions on GLUT4 translocation. Rab4 DeltaCT was then shown to prevent GLUT4 translocation induced by the expression of an active form of phosphatidylinositol 3-kinase or of protein kinase B, without altering the activities of the enzymes. Our results are consistent with a role of Rab4 DeltaCT acting as a dominant negative protein towards Rab4, possibly by binding to Rab4 effectors.