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.     

Insulin-stimulated phosphatidylinositol 3-kinase. Association with a 185-kDa tyrosine-phosphorylated protein (IRS-1) and localization in a low density membrane vesicle

Insulin stimulates the appearance of anti-tyrosine(P)-immunoprecipitable phosphatidylinositol 3-kinase (PI 3-kinase) activity in adipocytes, predominantly in an intracellular membrane fraction (Kelly, K. L., Ruderman, N. B., and Chen, K. S. (1992) J. Biol. Chem. 267, 3423-3428). Neither the mechanism underlying this activation nor the precise subcellular compartment in which it occurs is known. To address these questions, studies were performed using isolated rat adipocytes and subcellular fractions of these cells. In intact cells, insulin stimulated the rapid appearance of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate in 32P-labeled adipocytes without changing the labeling of phosphatidylinositol 3-phosphate, phosphatidylinositol 4-phosphate, or phosphatidylinositol 4,5-bisphosphate. This effect was accompanied by the tyrosyl phosphorylation of a 185-kDa protein, tentatively identified as IRS-1, with which PI 3-kinase became associated. The majority of the p85, the regulatory subunit of PI 3-kinase, in untreated adipocytes was present in the cytosol; however, neither the activity of PI 3-kinase nor the total amount of p85 in this fraction was modified in response to insulin. In contrast, insulin increased the association of p85 with IRS-1, the tyrosyl phosphorylation of the IRS-1 associated with p85, and the total activity of PI 3-kinase in the plasma membranes and low density membranes. After insulin treatment, similar amounts of p85 were bound to IRS-1 in the low density and plasma membrane fractions; however, tyrosyl-phosphorylated IRS-1 and PI 3-kinase activity were an order of magnitude greater in the low density membranes. The complex of tyrosyl-phosphorylated IRS-1.p85 that formed in response to insulin was localized to a very low density vesicle subpopulation that could be distinguished from vesicles containing the GLUT-4 glucose transporter and the insulin receptor. These data suggest that the activation of PI 3-kinase by insulin in the adipocyte involves the formation of a complex between IRS-1 and PI 3-kinase in a very low density membrane fraction that is not enriched in GLUT-4 or insulin receptors. They also suggest that PI 3-kinase activation correlates more closely with the extent of tyrosyl phosphorylation of the IRS-1 complexed to PI 3-kinase than it does to the amount of p85 bound to IRS-1.     

Separation of IRS-1 and PI3-kinase from GLUT4 vesicles in rat skeletal muscle

In fat and muscle tissues, insulin stimulates cellular glucose uptake by initiating a phosphorylation cascade which ultimately results in the translocation of the GLUT4 glucose transporter isoform from an intracellular vesicular storage pool(s) to the plasma membrane in fat and to t-tubules in skeletal muscle. Insulin receptor substrate-1 (IRS-1) and phosphatidylinositol 3-kinase (PI3-kinase) are known to be involved in cellular responses to insulin such as GLUT4 translocation, but the biochemical mechanism(s) connecting IRS-1 and PI3-kinase to GLUT4-containing intracellular membranes remains unclear. Here, in control and insulin-stimulated rat skeletal muscle, the intracellular localization of these two proteins was compared to that of GLUT4 using subcellular fractionation by sucrose velocity gradients followed by immunoblotting. Our data show that insulin-sensitive GLUT4-containing vesicles are present in fractions 1 through 10, whereas IRS-1 and PI3-kinase are found in fractions 16 through 24. These results indicate that in intracellular fractions derived from skeletal muscle, IRS-1 and PI3-kinase are excluded from membranes harboring GLUT4.     

Different subcellular distribution and regulation of expression of insulin receptor substrate (IRS)-3 from those of IRS-1 and IRS-2

Adipocytes contain three major substrate proteins of the insulin receptor, termed IRS-1, IRS-2, and IRS-3. We demonstrated that IRS-1 and IRS-2 are located mainly in the low density microsome (LDM) fraction and are tyrosine phosphorylated in response to insulin stimulation, leading to phosphatidylinositol (PI) 3-kinase activation. In contrast, IRS-3 is located mainly in the plasma membrane (PM) fraction and contributes to PI 3-kinase activation in the PM fraction. The different cellular localizations of IRS proteins may account for the mechanism of insulin resistance induced by a high fat diet, considering that PI 3-kinase activation in the LDM fraction is reportedly essential for the translocation of GLUT4 in adipocytes. High fat feeding in rats increased both protein and mRNA levels of IRS-3 but decreased those of IRS-1 and IRS-2 in epididymal adipocytes. As a result, selective impairment of insulin-induced PI 3-kinase activation was observed in the LDM fraction, whereas PI 3-kinase activation was conserved in the PM fraction. This is the first report showing that different IRS proteins function in different subcellular compartments, which may contribute to determining the insulin sensitivity in adipocytes.     

Insulin-stimulated GLUT4 translocation is relevant to the phosphorylation of IRS-1 and the activity of PI3-kinase

We examined the role of 185-kDa insulin receptor substrate-1 (IRS-1) and phosphatidylinositol 3-kinase (PI3-kinase) in the signaling pathway of insulin-stimulated GLUT4 translocation. We had already developed a novel cell line to detect GLUT4 on the cell surface, directly and sensitively (Kanai, F., Nishioka, Y., Hayashi, H., Kamohara, S., Todaka, M., and Ebina, Y. (1993) J. Biol. Chem. 268, 14523-14526). We stably expressed a mutant insulin receptor in which Tyr972 was replaced with phenylalanine. Insulin-stimulated tyrosyl phosphorylation of IRS-1 and GLUT4 translocation were decreased in cells expressing the mutant receptor, as compared to findings in cells expressing the normal receptor. Wortmannin, an inhibitor of PI3-kinase, inhibits the insulin-stimulated PI3-kinase activity and GLUT4 translocation at 50 nM, but not the NaF-stimulated GLUT4 translocation. These results suggest that the tyrosine phosphorylation of IRS-1 and activation of PI3-kinase may be involved in the signaling pathway of the insulin-stimulated GLUT4 translocation.     

Tumor necrosis factor-alpha causes insulin receptor substrate-2-mediated insulin resistance and inhibits insulin-induced adipogenesis in fetal brown adipocytes

Treatment of fetal brown adipocytes with 0.6 nM tumor necrosis factor (TNF)-alpha for 24 h resulted in a partial impairment in the expression of fatty acid synthase, glycerol-3-phosphate dehydrogenase, and glucose transporter (GLUT)-4 messenger RNAs (mRNAs), as well as in the enhancement in the cytoplasmic lipid content in response to insulin. However, the expression of the tissue-specific gene, uncoupling protein 1, is increased by the presence of TNF-alpha. The antiadipogenic effect of TNF-alpha was accompanied by a down-regulation of CCAAT/enhancer-binding protein-alpha and beta mRNAs and up-regulation of CCAAT/enhancer-binding protein-delta, with the expression of peroxisome proliferator-activated receptor-gamma remaining essentially unmodified. Moreover, TNF-alpha caused an insulin resistance on the insulin-induced glucose uptake in brown adipocytes. Pretreatment with TNF-alpha resulted in hypophosphorylation of the insulin receptor in response to insulin, without affecting the number of insulin receptors per cell or its molecular mass. However, insulin receptor substrate (IRS)-1 and IRS-2 signaling in response to insulin showed functional differences. Thus, TNF-alpha pretreatment induced a hypophosphorylation of IRS-2 but not of IRS-1. This effect leads to an impairment in the IRS-2-associated phosphatidylinositol (PI) 3-kinase activation due to a decreased association of alpha-p85 regulatory subunit of PI 3-kinase with IRS-2 but not in the IRS-1-associated PI 3-kinase activation in response to insulin. Our results indicate that TNF-alpha induced an IRS-2- but not IRS-1-mediated insulin resistance on glucose transport and lipid synthesis in fetal brown adipocytes.     

Stimulation of IRS-1-associated phosphatidylinositol 3-kinase and Akt/protein kinase B but not glucose transport by beta1-integrin signaling in rat adipocytes

The signal transduction pathway by which insulin stimulates glucose transport is not understood, but a role for complexes of insulin receptor substrate (IRS) proteins and phosphatidylinositol (PI) 3-kinase as well as for Akt/protein kinase B (PKB) has been proposed. Here, we present evidence suggesting that formation of IRS-1/PI 3-kinase complexes and Akt/PKB activation are insufficient to stimulate glucose transport in rat adipocytes. Cross-linking of beta1-integrin on the surface of rat adipocytes by anti-beta1-integrin antibody and fibronectin was found to cause greater IRS-1 tyrosine phosphorylation, IRS-1-associated PI 3-kinase activity, and Akt/PKB activation, detected by anti-serine 473 antibody, than did 1 nM insulin. Clustering of beta1-integrin also significantly potentiated stimulation of insulin receptor and IRS-1 tyrosine phosphorylation, IRS-associated PI 3-kinase activity, and Akt/PKB activation caused by submaximal concentrations of insulin. In contrast, beta1-integrin clustering caused neither a change in deoxyglucose transport nor an effect on the ability of insulin to stimulate deoxyglucose uptake at any concentration along the entire dose-response relationship range. The data suggest that (i) beta1-integrins can engage tyrosine kinase signaling pathways in isolated fat cells, potentially regulating fat cell functions and (ii) either formation of IRS-1/PI 3-kinase complexes and Akt/PKB activation is not necessary for regulation of glucose transport in fat cells or an additional signaling pathway is required.     

Suppression of insulin-stimulated phosphatidylinositol 3-kinase activity by the beta3-adrenoceptor agonist CL316243 in rat adipocytes

Insulin increased 2-deoxyglucose (2-DG) uptake via the translocation of glucose transporter (GLUT) 4 to the plasma membrane fraction in rat adipocytes. The stimulatory actions of insulin were accompanied by both an increase in the immunoreactive p85 subunit of phosphatidylinositol (PI) 3-kinase in the plasma membrane fractions and PI 3-kinase activation by tyrosine phosphorylation of the p85 subunit. The beta3-adrenoceptor agonist CL316243 (CL) suppressed all the insulin actions in adenosine deaminase (ADA)-treated cells, but was without effect in non-ADA-treated cells. The inhibitory effects of CL on GLUT 4 translocation and PI 3-kinase activation were abolished by the addition of N6-phenylisopropyl adenosine. Cholera toxin treatment, which markedly increased intracellular cAMP levels, suppressed increases in the levels of GLUT 4 and PI 3-kinase in the plasma membrane fractions in response to insulin. In addition, dibutyryl (Bt2) cAMP also impaired the activation of PI 3-kinase by insulin. These results indicated that CL suppressed insulin-stimulated glucose transport under conditions where cAMP levels were markedly increased (approximately 12-fold). The inhibitory actions of PI 3-kinase activation by insulin were exerted even when cAMP, 8-bromo-cAMP, or Bt2 cAMP was added to immunoprecipitates of the p85 subunit of PI 3-kinase, after treating the cells with insulin. These results suggest that CL suppressed insulin-stimulated PI 3-kinase activity via a cAMP-dependent mechanism, at least in part, direct cAMP action in ADA-treated adipocytes, by which PI 3-kinase activation was inhibited, resulting in the decrease in GLUT 4 translocation and subsequent 2-DG uptake in response to insulin.     

Urocortin, a newly identified corticotropin-releasing factor-related mammalian peptide, stimulates atrial natriuretic peptide and brain natriuretic peptide secretions from neonatal rat cardiomyocytes

Vanadate stimulates adipocyte 2-deoxyglucose transport and GLUT-4 translocation to the membrane through an insulin receptor-independent but wortmannin-inhibitable pathway. Vanadate stimulates PI 3-kinase in anti-IRS-1 immunoprecipitates and the binding between IRS-1 and the p85alpha subunit of PI 3-kinase. In insulin-resistant adipocytes from old rats vanadate fully stimulates IRS-1-associated PI 3-kinase, but partially activates glucose uptake. We conclude that: (a) vanadate stimulates 2-deoxyglucose uptake using a pathway that converges with that of insulin at the level of PI 3-kinase; and (b) adipocytes from old rats are defective in the insulin pathway at steps located both upstream and downstream of PI 3-kinase.     

Root resorption of dental and traumatic origin: classification based on etiology

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.     

Comparative effects of GTPgammaS and insulin on the activation of Rho, phosphatidylinositol 3-kinase, and protein kinase N in rat adipocytes. Relationship to glucose transport

Electroporation of rat adipocytes with guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) elicited sizable insulin-like increases in glucose transport and GLUT4 translocation. Like insulin, GTPgammaS activated membrane phosphatidylinositol (PI) 3-kinase in rat adipocytes, but, unlike insulin, this activation was blocked by Clostridium botulinum C3 transferase, suggesting a requirement for the small G-protein, RhoA. Also suggesting that Rho may operate upstream of PI 3-kinase during GTPgammaS action, the stable overexpression of Rho in 3T3/L1 adipocytes provoked increases in membrane PI 3-kinase activity. As with insulin treatment, GTPgammaS stimulation of glucose transport in rat adipocytes was blocked by C3 transferase, wortmannin, LY294002, and RO 31-8220; accordingly, the activation of glucose transport by GTPgammaS, as well as insulin, appeared to require Rho, PI 3-kinase, and another downstream kinase, e.g. protein kinase C-zeta (PKC-zeta) and/or protein kinase N (PKN). Whereas insulin activated both PKN and PKC-zeta, GTPgammaS activated PKN but not PKC-zeta. In transfection studies in 3T3/L1 cells, stable expression of wild-type Rho and PKN activated glucose transport, and dominant-negative forms of Rho and PKN inhibited insulin-stimulated glucose transport. In transfection studies in rat adipocytes, transient expression of wild-type and constitutive Rho and wild-type PKN provoked increases in the translocation of hemagglutinin (HA)-tagged GLUT4 to the plasma membrane; in contrast, transient expression of dominant-negative forms of Rho and PKN inhibited the effects of both insulin and GTPgammaS on HA-GLUT4 translocation. Our findings suggest that (a) GTPgammaS and insulin activate Rho, PI 3-kinase, and PKN, albeit by different mechanisms; (b) each of these signaling substances appears to be required for, and may contribute to, increases in glucose transport; and (c) PKC-zeta may contribute to increases in glucose transport during insulin, but not GTPgammaS, action.     

Overexpression of a constitutively active form of phosphatidylinositol 3-kinase is sufficient to promote Glut 4 translocation in adipocytes

Insulin stimulates glucose transport in its target cells by recruiting the glucose transporter Glut 4 from an intracellular compartment to the cell surface. Previous studies have indicated that phosphatidylinositol 3-kinase (PI 3-kinase) is a necessary step in this insulin action. We have investigated whether PI 3-kinase activation is sufficient to promote Glut 4 translocation in transiently transfected adipocytes. Rat adipose cells were cotransfected with expression vectors that allowed transient expression of epitope-tagged Glut 4 and a constitutively active form of PI 3-kinase (p110*). The expression of p110* induced the appearance of epitope-tagged Glut 4 at the cell surface at a level similar to that obtained after insulin treatment, whereas a kinase-dead version of p110* had no effect. The p110* effect was observed over a wide range of the transfected cDNA. When subcellular fractionation of adipocytes was performed, p110* was found, similar to the endogenous PI 3-kinase, enriched in the low density microsomal compartment, which also contains the Glut 4 vesicles. This could suggest that a specific localization of PI 3-kinase in this compartment is required for the action on Glut 4. The observations made with PI 3-kinase are in contrast with those seen with the MAP kinase cascade. Indeed, a constitutively active form of MAP kinase kinase had no effect on Glut 4 translocation in basal conditions. At the highest degree of expression, the constitutively active form of MAP kinase kinase slightly inhibited the insulin stimulation of Glut 4 translocation. Taken together, our results indicate that Glut 4 translocation can be efficiently promoted by an active form of PI 3-kinase but not by the activation of the MAP kinase pathway.     

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.     

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.     

Inhibition of phosphatidylinositol 3-kinase activity by adenovirus-mediated gene transfer and its effect on insulin action

Phosphatidylinositol 3-kinase (PI 3-K) is implicated in cellular events including glucose transport, glycogen synthesis, and protein synthesis. It is activated in insulin-stimulated cells by binding of the Src homology 2 (SH2) domains in its 85-kDa regulatory subunit to insulin receptor substrate-1 (IRS-1), and, others. We have previously shown that IRS-1-associated PI 3-kinase activity is not essential for insulin-stimulated glucose transport in 3T3-L1 adipocytes, and that alternate pathways exist in these cells. We now show that adenovirus-mediated overexpression of the p85N-SH2 domain in these cells behaves in a dominant-negative manner, interfering with complex formation between endogenous PI 3-K and its SH2 binding targets. This not only inhibited insulin-stimulated IRS-1-associated PI 3-kinase activity, but also completely blocked anti-phosphotyrosine-associated PI 3-kinase activity, which would include the non-IRS-1-associated activity. This resulted in inhibition of insulin-stimulated glucose transport, glycogen synthase activity and DNA synthesis. Further, Ser/Thr phosphorylation of downstream molecules Akt and p70 S6 kinase was inhibited. However, co-expression of a membrane-targeted p110(C) with the p85N-SH2 protein rescued glucose transport, supporting our argument that the p85N-SH2 protein specifically blocks insulin-mediated PI 3-kinase activity, and, that the signaling pathways downstream of PI 3-kinase are intact. Unexpectedly, GTP-bound Ras was elevated in the basal state. Since p85 is known to interact with GTPase-activating protein in 3T3-L1 adipocytes, the overexpressed p85N-SH2 peptide could titrate out cellular GTPase-activating protein by direct association, such that it is unavailable to hydrolyze GTP-bound Ras. However, insulin-induced mitogen-activated protein kinase phosphorylation was inhibited. Thus, PI 3-kinase may be required for this action at a step independent of and downstream of Ras. We conclude that, in 3T3-L1 adipocytes, non-IRS-1-associated PI 3-kinase activity is crucial for insulin's metabolic signaling, and that overexpressed p85N-SH2 protein inhibits a variety of insulin's ultimate biological effects.     

Glucosamine-induced insulin resistance in 3T3-L1 adipocytes is caused by depletion of intracellular ATP

Glucosamine, which enters the hexosamine pathway downstream of the rate-limiting step, has been routinely used to mimic the insulin resistance caused by high glucose and insulin. We investigated the effect of glucosamine on insulin-stimulated glucose transport in 3T3-L1 adipocytes. The Delta-insulin (insulin-stimulated minus basal) value for 2-deoxyglucose uptake was dramatically inhibited with increasing concentrations of glucosamine with an ED50 of 1.95 mM. Subcellular fractionation experiments demonstrated that reduction in insulin-stimulated 2-deoxyglucose uptake by glucosamine was due to an inhibition of translocation of both Glut 1 and Glut 4 from the low density microsomes (LDM) to the plasma membrane. Analysis of the insulin signaling cascade revealed that glucosamine impaired insulin receptor autophosphorylation, insulin receptor substrate (IRS-1) phosphorylation, IRS-1-associated PI 3-kinase activity in the LDM, and AKT-1 activation by insulin. Measurement of intracellular ATP demonstrated that the effects of glucosamine were highly correlated with its ability to reduce ATP levels. Reduction of intracellular ATP using azide inhibited Glut 1 and Glut 4 translocation from the LDM to the plasma membrane, insulin receptor autophosphorylation, and IRS-1 tyrosine phosphorylation. Additionally, both the reduction in intracellular ATP and the effects on insulin action caused by glucosamine could be prevented by the addition of inosine, which served as an alternative energy source in the medium. We conclude that direct administration of glucosamine can rapidly lower cellular ATP levels and affect insulin action in fat cells by mechanisms independent of increased intracellular UDP-N-acetylhexosamines and that increased metabolism of glucose via the hexosamine pathway may not represent the mechanism of glucose toxicity in fat cells.     

Enhanced insulin-stimulated activation of phosphatidylinositol 3-kinase in the liver of high-fat-fed rats

Insulin receptor substrate (IRS)-1 and IRS-2, which mediate phosphatidylinositol (PI) 3-kinase activation, play essential roles in insulin-induced translocation of GLUT4 and in glycogen synthesis. In this study, we investigated the process of PI 3-kinase activation via binding with IRS-1 and -2 in liver, muscle, and fat of high-fat-fed rats, a model of insulin-resistant diabetes. In the liver of high-fat-fed rats, insulin increased the PI 3-kinase regulatory subunit p85alpha and the PI 3-kinase activities associated with IRS-1 3.6- and 2.4-fold, and with IRS-2, 4.7- and 3.0-fold, respectively, compared with those in control rats. The tyrosine phosphorylation levels of IRS-1 and IRS-2 were not significantly altered, however. In contrast with the liver, tyrosine phosphorylation levels and associated PI 3-kinase proteins and activities were decreased in the muscle and adipose tissue of high-fat-fed rats. Thus, high-fat feeding appears to cause insulin resistance in the liver by a mechanism different from the impaired PI 3-kinase activation observed in muscle and adipose tissue. Taking into consideration that hepatic PI 3-kinase activation is severely impaired in obese diabetic models such as Zucker fatty rats, it is possible that the mechanism by which a high-fat diet causes insulin resistance is quite different from that associated with obesity and overeating due to abnormality in the leptin system. This is the first report to show increased PI 3-kinase activation by insulin in an insulin-resistant diabetic animal model. These findings may be important for understanding the mechanism of insulin resistance in human NIDDM, since a high-fat diet is considered to be one of the major factors exacerbating insulin insensitivity in humans.     

The new antidiabetic drug MCC-555 acutely sensitizes insulin signaling in isolated cardiomyocytes

Freshly isolated adult rat ventricular cardiomyocytes have been used to characterize the action profile of the new thiazolidinedione antidiabetic drug MCC-555. Preincubation of cells with the compound (100 microM for 30 min or 10 microM for 2 h) did not modify basal 3-O-methylglucose transport, but produced a marked sensitizing effect (2- to 3-fold increase in insulin action at 3 x 10(-11) M insulin) and a further enhancement of maximum insulin action (1.8-fold). MCC-555 did not modulate autophosphorylation of the insulin receptor and tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). However, insulin action (10(-10) and 10(-7) M) on IRS-1-associated phosphatidylinositol (PI) 3-kinase activity was enhanced 2-fold in the presence of MCC-555. Association of the p85 adapter subunit of PI 3-kinase to IRS-1 was not modified by the drug. Immunoblotting experiments demonstrated expression of the peroxisomal proliferator-activated receptor-gamma in cardiomyocytes reaching about 30% of the abundance observed in adipocytes. The insulin-sensitizing effect of MCC-555 was lost after inhibition of protein synthesis by preincubation of the cells with cycloheximide (1 mM; 30 min). Cardiomyocytes from obese Zucker rats exhibited a completely blunted response of glucose transport at 3 x 10(-11) M insulin. MCC-555 ameliorates this insulin resistance, producing a 2-fold stimulation of glucose transport, with maximum insulin action being 1.6-fold higher than that in control cells. This drug effect was paralleled by a significant dephosphorylation of IRS-1 on Ser/Thr. In conclusion, MCC-555 rapidly sensitizes insulin-stimulated cardiac glucose uptake by enhancing insulin signaling resulting from increased intrinsic activity of PI 3-kinase. Acute activation of protein expression leading to a modulation of the Ser/Thr phosphorylation state of signaling proteins such as IRS-1 may be underlying this process. It is suggested that MCC-555 may provide a causal therapy of insulin resistance by targeted action on the defective site in the insulin signaling cascade.     

The pleckstrin homology and phosphotyrosine binding domains of insulin receptor substrate 1 mediate inhibition of apoptosis by insulin

Insulin and insulin-like growth factor 1 (IGF-1) evoke diverse biological effects through receptor-mediated tyrosine phosphorylation of insulin receptor substrate (IRS) proteins. We investigated the elements of IRS-1 signaling that inhibit apoptosis of interleukin 3 (IL-3)-deprived 32D myeloid progenitor cells. 32D cells have few insulin receptors and no IRS proteins; therefore, insulin failed to inhibit apoptosis during IL-3 withdrawal. Insulin stimulated mitogen-activated protein kinase in 32D cells expressing insulin receptors (32DIR) but failed to activate the phosphatidylinositol 3 (PI 3)-kinase cascade or to inhibit apoptosis. By contrast, insulin stimulated the PI 3-kinase cascade, inhibited apoptosis, and promoted replication of 32DIR cells expressing IRS-1. As expected, insulin did not stimulate PI 3-kinase in 32DIR cells, which expressed a truncated IRS-1 protein lacking the tail of tyrosine phosphorylation sites. However, this truncated IRS-1 protein, which retained the NH2-terminal pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains, mediated phosphorylation of PKB/akt, inhibition of apoptosis, and replication of 32DIR cells during insulin stimulation. These results suggest that a phosphotyrosine-independent mechanism mediated by the PH and PTB domains promoted antiapoptotic and growth actions of insulin. Although PI 3-kinase was not activated, its phospholipid products were required, since LY294002 inhibited these responses. Without IRS-1, a chimeric insulin receptor containing a tail of tyrosine phosphorylation sites derived from IRS-1 activated the PI 3-kinase cascade but failed to inhibit apoptosis. Thus, phosphotyrosine-independent IRS-1-linked pathways may be critical for survival and growth of IL-3-deprived 32D cells during insulin stimulation.