GLUT4 Activity Research Articles

Antihyperglycemic Activity of Houttuynia cordata Thunb. in Streptozotocin-Induced Diabetic Rats.

Present study is an attempt to investigate plausible mechanism involved behind antidiabetic activity of standardized Houttuynia cordata Thunb. extract in streptozotocin-induced diabetic rats. The plant is used as a medicinal salad for lowering blood sugar level in North-Eastern parts of India. Oral administration of extract at 200 and 400 mg/kg dose level daily for 21 days showed a significant (P < 0.05) decrease in fasting plasma glucose and also elevated insulin level in streptozotocin-induced diabetic rats. It also significantly reversed all the alterations in biochemical parameters, that is, total lipid profile, blood urea, creatinine, protein, and antioxidant enzymes in liver, pancreas, and adipose tissue of diabetic rats. Furthermore, we have demonstrated that the extract significantly reversed the expression patterns of various glucose homeostatic enzyme genes like GLUT-2, GLUT-4, and caspase-3 levels but did not show any significant effect on PPAR-γ protein expressions. Additionally, the extract positively regulated mitochondrial membrane potential and succinate dehydrogenase (SDH) activity in diabetic rats. The findings justified the antidiabetic effect of H. cordata which is attributed to an upregulation of GLUT-4 and potential antioxidant activity, which may play beneficial role in resolving complication associated with diabetes.

PIP3 but not PIP2 increases GLUT4 surface expression and glucose metabolism mediated by AKT/PKCζ/λ phosphorylation in 3T3L1 adipocytes

Phosphatidylinositol-3,4,5-triphosphate (PIP3) and phosphatidylinositol-4,5-biphosphate (PIP2) are two well-known membrane bound polyphosphoinositides. Diabetes is associated with impaired glucose metabolism. Using a 3T3L1 adipocyte cell model, this study investigated the role of PIP3 and PIP2 on insulin stimulated glucose metabolism in high glucose (HG) treated cells. Exogenous PIP3 supplementation (1, 5, or 10 nM) increased the phosphorylation of AKT and PKCζ/λ, which in turn upregulated GLUT4 total protein expression as well as its surface expression, glucose uptake, and glucose utilization in cells exposed to HG (25 mM); however, PIP2 had no effect. Comparative signal silencing studies with antisense AKT2 and antisense PKCζ revealed that phosphorylation of PKCζ/λ is more effective in PIP3 mediated GLUT4 activation and glucose utilization than in AKT phosphorylation. Supplementation with PIP3 in combination with insulin enhanced glucose uptake and glucose utilization compared to PIP2 with insulin, or insulin alone, in HG-treated adipocytes. This suggests that a decrease in cellular PIP3 levels may cause impaired insulin sensitivity in diabetes. PIP3 supplementation also prevented HG-induced MCP-1 and resistin secretion and lowered adiponectin levels. This study for the first time demonstrates that PIP3 but not PIP2 plays an important role in GLUT4 upregulation and glucose metabolism mediated by AKT/PKCζ/λ phosphorylation. Whether PIP3 levels in blood can be used as a biomarker of insulin resistance in diabetes needs further investigation.

Expression and phosphorylation of the AS160_v2 splice variant supports GLUT4 activation and the Warburg effect in multiple myeloma

BackgroundMultiple myeloma (MM) is a fatal plasma cell malignancy exhibiting enhanced glucose consumption associated with an aerobic glycolytic phenotype (i.e., the Warburg effect). We have previously demonstrated that myeloma cells exhibit constitutive plasma membrane (PM) localization of GLUT4, consistent with the dependence of MM cells on this transporter for maintenance of glucose consumption rates, proliferative capacity, and viability. The purpose of this study was to investigate the molecular basis of constitutive GLUT4 plasma membrane localization in MM cells.FindingsWe have elucidated a novel mechanism through which myeloma cells achieve constitutive GLUT4 activation involving elevated expression of the Rab-GTPase activating protein AS160_v2 splice variant to promote the Warburg effect. AS160_v2-positive MM cell lines display constitutive Thr642 phosphorylation, known to be required for inactivation of AS160 Rab-GAP activity. Importantly, we show that enforced expression of AS160_v2 is required for GLUT4 PM translocation and activation in these select MM lines. Furthermore, we demonstrate that ectopic expression of a full-length, phospho-deficient AS160 mutant is sufficient to impair constitutive GLUT4 cell surface residence, which is characteristic of MM cells.ConclusionsThis is the first study to tie AS160 de-regulation to increased glucose consumption rates and the Warburg effect in cancer. Future studies investigating connections between the insulin/IGF-1/AS160_v2/GLUT4 axis and FDG-PET positivity in myeloma patients are warranted and could provide rationale for therapeutically targeting this pathway in MM patients with advanced disease.

Effects of Amyloid Precursor Protein 17 Peptide on the Protection of Diabetic Encephalopathy and Improvement of Glycol Metabolism in the Diabetic Rat

Researchers have proposed that amyloid precursor protein 17 peptide (APP17 peptide), an active fragment of amyloid precursor protein (APP) in the nervous system, has therapeutic effects on neurodegeneration. Diabetic encephalopathy (DE) is a neurological disease caused by diabetes. Here we use multiple experimental approaches to investigate the effect of APP17 peptide on changes in learning behavior and glycol metabolism in rats. It was found that rats with DE treated by APP17 peptide showed reversed behavioral alternation. The [18F]-FDG-PET images and other results all showed that the APP17 peptide could promote glucose metabolism in the brain of the DE rat model. Meanwhile, the insulin signaling was markedly increased as shown by increased phosphorylation of Akt and enhanced GLUT4 activation. Compared with the DE group, the activities of SOD, GSH-Px, and CAT in the rat hippocampal gyrus were increased, while MDA decreased markedly in the DE + APP17 peptide group. No amyloid plaques in the cortex and the hippocampus were detected in either group, indicating that the experimental animals in the current study were not suffering from Alzheimer's disease. These results indicate that APP17 peptide could be used to treat DE effectively.

Insulin sensitization via partial agonism of PPARγ and glucose uptake through translocation and activation of GLUT4 in PI3K/p-Akt signaling pathway by embelin in type 2 diabetic rats

BackgroundThe present study was aimed at isolating an antidiabetic molecule from a herbal source and assessing its mechanism of action. MethodsEmbelin, isolated from Embelia ribes Burm. (Myrsinaceae) fruit, was evaluated for its potential to regulate insulin resistance, alter β-cell dysfunction and modulate key markers involved in insulin sensitivity and glucose transport using high-fat diet (HFD) fed-streptozotocin (STZ) (40mg/kg)-induced type 2 diabetic rats. Molecular-dockings were performed to investigate the binding modes of embelin into PPARγ, PI3K, p-Akt and GLUT4 active sites. ResultsEmbelin (50mg/kg b wt.) reduced body weight gain, blood glucose and plasma insulin in treated diabetic rats. It further modulated the altered lipid profiles and antioxidant enzymes with cytoprotective action on β-cell. Embelin significantly increased the PPARγ expression in epididymal adipose tissue compared to diabetic control group; it also inhibited adipogenic activity; it mildly activated PPARγ levels in the liver and skeletal muscle. It also regulated insulin mediated glucose uptake in epididymal adipose tissue through translocation and activation of GLUT4 in PI3K/p-Akt signaling cascade. Embelin bound to PPARγ; it disclosed stable binding affinities to the active sites of PI3K, p-Akt and GLUT4. ConclusionsThese findings show that embelin could improve adipose tissue insulin sensitivity without increasing weight gain, enhance glycemic control, protect β-cell from damage and maintain glucose homeostasis in adipose tissue. General significanceEmbelin can be used in the prevention and treatment of type 2 diabetes mellitus caused due to obesity.

FM19G11 Favors Spinal Cord Injury Regeneration and Stem Cell Self-Renewal by Mitochondrial Uncoupling and Glucose Metabolism Induction

Spinal cord injury is a major cause of paralysis with no currently effective therapies. Induction of self-renewal and proliferation of endogenous regenerative machinery with noninvasive and nontoxic therapies could constitute a real hope and an alternative to cell transplantation for spinal cord injury patients. We previously showed that FM19G11 promotes differentiation of adult spinal cord-derived ependymal stem cells under hypoxia. Interestingly, FM19G11 induces self-renewal of these ependymal stem cells grown under normoxia. The analysis of the mechanism of action revealed an early increment of mitochondrial uncoupling protein 1 and 2 with an early drop of ATP, followed by a subsequent compensatory recovery with activated mitochondrial metabolism and the induction of glucose uptake by upregulation of the glucose transporter GLUT-4. Here we show that phosphorylation of AKT and AMP-activated kinase (AMPK) is involved in FM19G11-dependent activation of GLUT-4, glucose influx, and consequently in stem cell self-renewal. Small interfering RNA of uncoupling protein 1/2, GLUT-4 and pharmacological inhibitors of AKT, mTOR and AMPK signaling blocked the FM19G11-dependent induction of the self-renewal-related markers Sox2, Oct4, and Notch1. Importantly, FM19G11-treated animals showed accelerated locomotor recovery. In vivo intrathecal sustained administration of FM19G11 in rats after spinal cord injury showed more neurofilament TUJ1-positive fibers crossing the injured area surrounded by an increase of neural precursor Vimentin-positive cells. Overall, FM19G11 exerts an important influence on the self-renewal of ependymal stem progenitor cells with a plausible neuroprotective role, providing functional benefits for spinal cord injury treatment.

Comment on: Gorboulev et al. Na+-D-glucose Cotransporter SGLT1 Is Pivotal for Intestinal Glucose Absorption and Glucose-Dependent Incretin Secretion. Diabetes 2012;61:187–196

The elegant paper from Koepsell and colleagues (1) demonstrates the apical GLUT2 mechanism, but appears at first sight to question its significance. However, when set in a physiological context, the results are those expected from the three roles of SGLT1 and the differences between fed rat and starved mouse. SGLT1 acts as 1 ) a transporter and 2 ) a powerful scavenger. In 2000, my laboratory proposed that SGLT1 also has 3 ) a pivotal regulatory role, controlling the rapid insertion and activation of GLUT2 at the enterocyte apical membrane so that absorptive capacity matches glucose load. …

Transcription Factor GATA-6 Recruits PPARα to Cooperatively Activate Glut4 Gene Expression

Peroxisome proliferator-activated receptor α (PPARα) is a nuclear hormone receptor that regulates energy metabolism, but its precise mechanisms remain unknown. Here, we demonstrate that the PPARα agonist fenofibrate activated expression of the glucose transporter Glut4. Moreover, PPARα was associated with the Glut4 promoter through GATA sites upon fenofibrate stimulation in cardiomyocytes. This occupancy is achieved through an interaction between amino acids 1–136 of PPARα with amino acids 276–443 of the cardiac transcription factor GATA-6. In addition, the interaction of PPARα with GATA-6 activated Glut4 gene expression, improved glucose consumption, and enhanced activity of mitochondrial citrate synthase in C2C12 myoblasts; both mutants of PPARα (1–101 aa) and GATA-6 (227–331 aa) were unable to cooperate in Glut4 activation. Thus, GATA-6 is an important component of the transcription network required for energy metabolism mediated by PPARα, and these findings provide a molecular basis for understanding the role of GATA-6 proteins in muscle development and disease.

Hydrogen Sulfide and l-Cysteine Increase Phosphatidylinositol 3,4,5-Trisphosphate (PIP3) and Glucose Utilization by Inhibiting Phosphatase and Tensin Homolog (PTEN) Protein and Activating Phosphoinositide 3-Kinase (PI3K)/Serine/Threonine Protein Kinase (AKT)/Protein Kinase Cζ/λ (PKCζ/λ) in 3T3l1 Adipocytes

This work examined the novel hypothesis that reduced levels of H(2)S or L-cysteine (LC) play a role in the impaired glucose metabolism seen in diabetes. 3T3L1 adipocytes were treated with high glucose (HG, 25 mM) in the presence or absence of LC or H(2)S. Both LC and H(2)S treatments caused an increase in phosphatidylinositol-3,4,5 trisphosphate (PIP3), AKT phosphorylation, and glucose utilization in HG-treated cells. The effect of LC on PIP3 and glucose utilization was prevented by propargylglycine, an inhibitor of cystathionine γ-lyase that catalyzes H(2)S formation from LC. This demonstrates that H(2)S mediates the effect of LC on increased PIP3 and glucose utilization. H(2)S and LC caused phosphatidylinositol 3-kinase activation and PTEN inhibition. Treatment with LC, H(2)S, or PIP3 increased the phosphorylation of IRS1, AKT, and PKCζ/λ as well as GLUT4 activation and glucose utilization in HG-treated cells. This provides evidence that PIP3 is involved in the increased glucose utilization observed in cells supplemented with LC or H(2)S. Comparative signal silencing studies with siAKT2 or siPKCζ revealed that PKCζ phosphorylation is more effective for the GLUT4 activation and glucose utilization in LC-, H(2)S-, or PIP3-treated cells exposed to HG. This is the first report to demonstrate that H(2)S or LC can increase cellular levels of PIP3, a positive regulator of glucose metabolism. The PIP3 increase is mediated by PI3K activation and inhibition of PTEN but not of SHIP2. This study provides evidence for a molecular mechanism by which H(2)S or LC can up-regulate the insulin-signaling pathways essential for maintenance of glucose metabolism.

Epigenetic Regulation of Glucose Transporter 4 by Estrogen Receptor β

Glucose transporter 4 (Glut4) is an important regulator of cellular glucose uptake in adipose tissue and skeletal muscle. The estrogen receptors α and β (ERα and ERβ) have been shown to regulate Glut4. However, the regulatory mechanisms are unclear, and there are conflicting results about the effects of the two ER isoforms on Glut4 activity. In this study we investigated how the lack of either ER isoform affects Glut4 expression in differentiated mouse embryonic fibroblasts. Our results demonstrate that Glut4 transcription is markedly reduced in cells lacking ERβ, both basally and upon induction by liver X receptor. These changes in Glut4 expression could not be explained by the lack of ERβ as ligand-activated transcription factor. They were rather brought about by hypermethylation of one single CpG in the Glut4 promoter in the ERβ-deficient cells. This CpG is part of an Sp1-binding site, and Sp1 binding was reduced by its methylation. Treatment with Sp1 inhibitor diminished Glut4 expression in wild-type, but not in ERβ-deficient cells, suggesting that reduced recruitment of Sp1 to the Glut4 promoter is responsible for the differences in Glut4 expression. Reintroduction of ERβ into ERβ-deficient cells partly restored Glut4 transcription and stabilized low DNA methylation after treatment with the DNA demethylating agent 5-Aza-2'-deoxycytidine. Our findings demonstrate the involvement of DNA methylation in Glut4 regulation and imply a novel function for ERβ in mediating epigenetic events and thereby regulating gene expression.

Human subcutaneous adipose tissue Glut 4 mRNA expression in obesity and type 2 diabetes

Cellular resistance to insulin caused by reduced glucose transport and metabolism is a primary defect leading to the development of metabolic disease. While the etiology of insulin resistance is multifactorial, reduced insulin action is associated with impaired activity of the glucose transporter GLUT4 in insulin-sensitive tissues. Yet, the role of adipose tissue GLUT4 deregulation in the pathogenesis of insulin resistance, obesity, and diabetes is still unclear. In this study, we assessed the relative GLUT4 level in human subcutaneous adipose tissue from obese, diabetic, and diabetic obese versus control subjects, using a real-time PCR method. GLUT4 mRNA levels were considerably decreased among type 2 diabetic patients compared with those of the controls (P<0.01), whereas no such difference was found between obese and normal-weight controls. Multiple linear regressions analysis in both diabetic non-obese and diabetic obese groups showed a negative correlation between GLUT4 mRNA expression and both markers of obesity or insulin resistance (P<0.01). However, in obese group, GLUT4 was inversely associated only with HOMA-IR (P<0.01). Our findings showed that adipose GLUT4 gene expression changes were more related to insulin resistance and type 2 diabetes rather than to obesity.

Abstract 1268: Aberrant plasma membrane localization of GLUT4 sustains the glycolytic phenotype in multiple myeloma

Abstract The incurable plasma cell malignancy multiple myeloma (MM) is characterized by the progressive development of chemoresistance, thus warranting the design of new therapeutic strategies tailored to unique molecular mechanisms driving tumor propagation. One aspect of myeloma pathogenesis which has not been exploited therapeutically is an abnormal avidity for glucose. Inhibition of glucose metabolism has broad applicability in cancer and has demonstrated in vitro potency; unfortunately, previous attempts focusing on hexokinase inhibition have been largely unsuccessful. Targeting the upstream process of glucose transport has not been a major area of investigation due to the common association between GLUT1 and cancer and the importance of GLUT1 in many normal tissues. However, the specific glucose transporters responsible for maintaining the glycolytic phenotype in myeloma have not been identified, leaving open the possibility that family members other than GLUT1 play important roles in this cellular context. Therefore, we undertook an unbiased real time PCR-based screen of GLUT gene expression profiles in myeloma cell lines and control B lymphocytes. GLUTs 4, 8, and 11 exhibit widespread overexpression in MM cells with no significant difference noted in GLUT1 levels. Examination of microarray studies of MM patient samples and western blot analyses of MM cell lines and normal B cells confirm the upregulation of GLUT8 and GLUT11 in clinical specimens and at the protein level. However, we did not find further evidence for GLUT4 overexpression. After evaluating the subcellular localization of GLUT4 through microscopy, we determined that a substantial fraction of GLUT4 protein is constitutively mislocalized to the plasma membrane in myeloma cells, an event normally regulated by insulin in non-malignant tissues. Indeed, cell fractionation studies demonstrate a dramatic increase in plasma membrane GLUT4 content in MM cell lines relative to PBMC. Lentiviral transduction of GLUT4-targeted shRNA verifies the functional relevance of GLUT4 activity, as MM cells with reduced GLUT4 expression exhibit dramatic deficiencies in glucose transport and lactate production rates, which leads to growth inhibition and significant cell death. Similar studies with a GLUT1-targeted shRNA reveal comparable trends but less potent effects. Next, we investigated a class of HIV protease inhibitors which elicit off-target inhibitory effects on GLUT4 in vivo. Treatment with these compounds recapitulates the effects of GLUT4 knockdown, thus supporting the relevance of GLUT4 in myeloma. These studies represent the first time a prominent, functional role for GLUT4 has been demonstrated in any cancer and highlight a therapeutic strategy involving the repositioning of an FDA-approved class of drugs to target myeloma cell metabolism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1268. doi:10.1158/1538-7445.AM2011-1268

The Activation of GLUT1, AMPK alpha and PPAR gamma by Tinospora crispa in L6 Myotubes

ABSTRACT AIM: Tinospora crispa (T. crispa, TC) has long been used in traditional medicine in South East Asia for the treatment of Type 2 diabetes mellitus. In the present study, it was aim to investigate the effect of water-ethanol extract of T. crispa (WETC) on glucose transporter (GLUT1 and GLUT4), 5\\\\\\\\\\\\\\\'AMP-activated protein kinase (AMPK alpha1) and peroxisome proliferator-activated receptors (PPAR gamma) expression. METHODS: The activation of GLUT1, GLUT4, AMPK alpha1 and PPAR gamma by T. crispa were assessed in vitro by the treatment of WETC to L6 myotubes followed by determined these genes expression using reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: WETC at 400 µg/ml significantly increased glucose uptake at 24 hours (214.49 +/- 9.00%, p

Effects of the HIV Protease Inhibitor Ritonavir on GLUT4 Knock-out Mice

HIV protease inhibitors acutely block glucose transporters (GLUTs) in vitro, and this may contribute to altered glucose homeostasis in vivo. However, several GLUT-independent mechanisms have been postulated. To determine the contribution of GLUT blockade to protease inhibitor-mediated glucose dysregulation, the effects of ritonavir were investigated in mice lacking the insulin-sensitive glucose transporter GLUT4 (G4KO). G4KO and control C57BL/6J mice were administered ritonavir or vehicle at the start of an intraperitoneal glucose tolerance test and during hyperinsulinemic-euglycemic clamps. G4KO mice exhibited elevated fasting blood glucose compared with C57BL/6J mice. Ritonavir impaired glucose tolerance in control mice but did not exacerbate glucose intolerance in G4KO mice. Similarly, ritonavir reduced peripheral insulin sensitivity in control mice but not in G4KO mice. Serum insulin levels were reduced in vivo in ritonavir-treated mice. Ritonavir reduced serum leptin levels in C57BL/6J mice but had no effect on serum adiponectin. No change in these adipokines was observed following ritonavir treatment of G4KO mice. These data confirm that a primary effect of ritonavir on peripheral glucose disposal is mediated through direct inhibition of GLUT4 activity in vivo. The ability of GLUT4 blockade to contribute to derangements in the other molecular pathways that influence insulin sensitivity remains to be determined.

Insulin resistance for glucose uptake and Akt2 phosphorylation in the soleus, but not epitrochlearis, muscles of old vs. adult rats

The slow-twitch soleus, but not fast-twitch muscle, of old vs. adult rats has previously been demonstrated to become insulin resistant for in vivo glucose uptake. We probed cellular mechanisms for the age effect by assessing whether insulin resistance for glucose uptake was an intrinsic characteristic of the muscle ex vivo and by analyzing key insulin signaling steps. We hypothesized that isolated soleus and epitrochlearis (fast-twitch) muscles from old (25 mo) vs. adult (9 mo) male Fisher-344 x Brown Norway rats would have insulin resistance for Akt2 Thr308 phosphorylation (pAkt2Thr308), AS160 phosphorylation Thr642 (pAS160Thr642), and atypical PKC (aPKCzeta/lambda) activity corresponding in magnitude to the extent of insulin resistance for [3H]-2-deoxyglucose (2-DG) uptake. Epitrochlearis insulin-stimulated 2-DG uptake above basal values was unaltered by age, and epitrochlearis pAkt2Thr308, pAS160Thr642, and aPKCzeta/lambda activity were not significantly different in adult vs. old rats. Conversely, insulin-stimulated 2-DG uptake by the soleus of old vs. adult rats was reduced with 1.2 nM (42%) and 30 nM (28%) insulin concomitant with an age-related decline in pAkt2Thr308 of the insulin-stimulated soleus. There were no age effects on pAS160Thr642 or aPKCzeta/lambda activity or abundance of Akt2, AS160, GLUT4 or Appl1 protein in either muscle. The results suggest the possibility that an age-related decline in pAkt2Thr308, acting by a mechanism other than reduced pAS160Thr642, may play a role in the insulin resistance in the soleus of old rats. Skeletal muscle insulin resistance in old age is distinctive compared with other insulin-resistant rodent models that are not selective for greater insulin resistance in the soleus vs. the epitrochlearis.

Myosin IIA participates in docking of Glut4 storage vesicles with the plasma membrane in 3T3-L1 adipocytes

In adipocytes and myocytes, insulin stimulation translocates glucose transporter 4 (Glut4) storage vesicles (GSVs) from their intracellular storage sites to the plasma membrane (PM) where they dock with the PM. Then, Glut4 is inserted into the PM and initiates glucose uptake into these cells. Previous studies using chemical inhibitors demonstrated that myosin II participates in fusion of GSVs and the PM and increase in the intrinsic activity of Glut4. In this study, the effect of myosin IIA on GSV trafficking was examined by knocking down myosin IIA expression. Myosin IIA knockdown decreased both glucose uptake and exposures of myc-tagged Glut4 to the cell surface in insulin-stimulated cells, but did not affect insulin signal transduction. Interestingly, myosin IIA knockdown failed to decrease insulin-dependent trafficking of Glut4 to the PM. Moreover, in myosin IIA knockdown cells, insulin-stimulated binding of GSV SNARE protein, vesicle-associated membrane protein 2 (VAMP2) to PM SNARE protein, syntaxin 4 was inhibited. These data suggest that myosin IIA plays a role in insulin-stimulated docking of GSVs to the PM in 3T3-L1 adipocytes through SNARE complex formation.

Cinnamic acid, from the bark of Cinnamomum cassia, regulates glucose transport via activation of GLUT4 on L6 myotubes in a phosphatidylinositol 3‐kinase‐independent manner

Cinnamomum cassia (Family: Lauraceae) is an Ayurvedic medicinal plant used traditionally for the treatment of a number of diseases, including diabetes. The hypoglycemic effect of this plant has been established in vivo. However, the effects of cinnamic acid, isolated from C. cassia, on the insulin signaling cascade in an in vitro model have not been elucidated. Hence, the aim of the present study was to evaluate the anti-diabetic effect of cinnamic acid on glucose transport by L6 myotubes. The mechanism of action of cinnamic acid was determined using specific targets in the insulin signaling pathway, including protein tyrosine phosphatase (PTP) 1B, phosphatidylinositol 3-kinase (PI3-K) and the glucose transporter GLUT4. After differentiation of myoblast to myotubes, the cells were serum deprived for 5 h and then treated with 1 ng/mL cinnamic acid and 50 μmol/L rosiglitazone for 18 h and 100 nmol/L insulin for 20 min for gene expression studies. Expression of GLUT4 mRNA was increased following treatment of L6 myotubes with 1 ng/mL cinnamic acid. Furthermore, cinnamic acid inhibited PTP1B activity (by 96.5%), but had no significant effect on PI3-K activity. On the basis of the results of the present study, we postulate that cinnamic acid isolated from the hydro-alcoholic extract of Cinnamomum cassia activates glucose transport by a PI3-K-independent pathway. However, the detailed mechanism of action requires further analysis.

Insulin stimulates GLUT4 translocation in the semitendinosus muscle of Shetland ponies

Glucose homeostasis depends on insulin-regulated glucose uptake in the skeletal muscles and fat tissues via glucose transporter (GLUT) 4 translocation into cellular plasma membranes. The present study sought to elucidate GLUT4 expression, GLUT1 and GLUT4 translocation and glucose uptake in the skeletal muscles of Shetland ponies. Semitendinosus muscle explants were removed by open muscle biopsy from six Shetland pony geldings under general anaesthesia. The expression of GLUT4 was analysed by measuring muscle crude membrane (CM) GLUT4 protein contents. To determine the insulin-stimulated GLUT translocation, GLUT1 and GLUT4 concentrations were measured in partially purified plasma membranes (PM) and cytoplasmic vesicles (CV). GLUT contents were determined semi-quantitatively by Western blotting. Insulin-stimulated glucose uptake was analysed using 3-O-d-methyl[(3)H]glucose uptake. Incubation of semitendinosus muscle strips with 0.1 and 20mIU/mL insulin significantly increased GLUT4 translocation (PM GLUT4 contents), but had no significant effect on GLUT4 expression (CM GLUT4 concentrations) or PM GLUT1. The uptake of myocyte 3-O-Methylglucose was not significantly increased following insulin stimulation. The sub-cellular fractionation technique proved to be an appropriate tool for determining insulin-stimulated GLUT4 translocation in equine skeletal muscle. GLUT4 translocation in equines is insulin-dependent, as has been described in rodents and farm animals, but insulin-stimulated GLUT4 activation in ponies is lower than reported for pigs and cows under the same experimental conditions. Poor insulin-activated GLUT4 translocation may account for insulin resistance in ponies in previous euglycaemic, hyperinsulinaemic clamp tests.

Growth Hormone Inhibition of Glucose Uptake in Adipocytes Occurs without Affecting GLUT4 Translocation through an Insulin Receptor Substrate-2-Phosphatidylinositol 3-Kinase-dependent Pathway

Growth hormone (GH) pretreatment of 3T3-L1 adipocytes resulted in a concentration- and time-dependent inhibition of insulin-stimulated glucose uptake. Surprisingly, this occurred without significant effect on insulin-stimulated glucose transporter (GLUT) 4 translocation or fusion with the plasma membrane. In parallel, the inhibitory actions of chronic GH pretreatment also impaired insulin-dependent activation of phosphatidylinositol (PI) 3-kinase bound to insulin receptor substrate (IRS)-2 but not to IRS-1. In addition, insulin-stimulated Akt phosphorylation was inhibited by GH pretreatment. In contrast, overexpression of IRS-2 or expression of a constitutively active Akt mutant prevented the GH-induced insulin resistance of glucose uptake. Moreover, small interfering RNA-mediated IRS-2 knockdown also inhibited insulin-stimulated Akt activation and glucose uptake without affecting GLUT4 translocation and plasma membrane fusion. Together, these data support a model in which chronic GH stimulation inhibits insulin-dependent activation of phosphatidylinositol 3-kinase through a specific interaction of phosphatidylinositol 3-kinase bound to IRS-2. This inhibition leads to suppression of Akt activation coupled to glucose transport activity but not translocation or plasma membrane fusion of GLUT4.

Abstract 1361: Leukocyte Antigen-Related Phosphatase Regulates Insulin Sensitivity by Interaction with Snapin

Several protein tyrosine phosphatases including leukocyte antigen-related (LAR) phosphatase have been implicated in insulin resistance, which is a risk factor for atherosclerosis. We showed previously that LAR negatively regulates insulin-like growth factor-1 (IGF1) signaling in vascular smooth muscle cells (VSMC) leading to increased proliferation and migration. Absence of LAR also enhanced neointima formation in response to arterial injury in mice. However, the role of LAR-modulated signaling in the development of insulin resistance has not been elucidated. Here, we investigated the function of LAR in regulating glucose uptake and insulin sensitivity. We identified snapin, a SNARE-associated protein involved in glucose transporter Glut4 vesicle fusion with plasma membrane, as a LAR-interacting protein using a yeast two-hybrid screen. IGF1-induced serine phosphorylation of snapin, its translocation to membrane and association with SNARE complex were enhanced in VSMC lacking LAR. Similarly, PI3K-PDK1-PKCζ signaling pathway was more active in LAR-/- cells after IGF1 treatment. This resulted in enhanced Glut4 activation, its membrane translocation and association with snapin. Glut4 membrane translocation and association with snapin after IGF1 treatment were impaired in snapin+/− VSMC. IGF1 treatment also increased serine phosphorylation of GSK3 β in LAR−/− VSMC leading to increased activation of glycogen synthase. Consistent with this, enhanced glucose uptake was observed in LAR−/− VSMC compared to wild-type cells after IGF1 treatment. Basal and IGF1-induced glucose uptake were significantly lower in snapin+/− VSMC than in wild-type cells. Snapin+/− mice had higher levels of blood glucose, lower quantitative insulin sensitivity check index (QUICKI) and impaired response to insulin in insulin tolerance test (ITT) compared to wild-type mice. Decrease of QUICKI and impairment of IIT were more pronounced in snapin+/− mice fed a high-fat diet. In addition, Doppler ultrasonography indicated increased arterial stiffness in snapin+/− mice. Together, these data indicate that LAR negatively regulates snapin phosphorylation which in turn affects glucose uptake leading to the development of insulin resistance and vascular pathology.