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.     

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.     

Mechanism of insulin action--signal transductions after binding to insulin receptor

Insulin binds to the alpha subunit of the insulin receptor which activates the tyrosine kinase in the beta subunit and tyrosine-phosphorylates the insulin receptor substrates-1 (IRS-1). Insulin promotes the formation of a complex between tyrosine-phosphorylated IRS-1 and several proteins including phosphoinositide(PI) 3-kinase, a heterodimer consisting of regulatory 85-kDa (p85) and catalytic 110-kDa (p110) subunits, GRB2 and Syp via the Src homology region 2 (SH2) domains. Recently, it was suggested that GRB2-Sos complex binding to IRS-1 was linked to Ras activation and that PI 3-kinase binding to IRS-1 was linked to activation of glucose transport. Since the mechanism of insulin-stimulated glucose uptake is mainly due to translocation of glucose transporters from an intracellular vesicle pool to the plasma membrane, PI 3-kinase activity may be involved in vesicle transport in mammalian cells.     

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.     

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.     

Improved metabolic status and insulin sensitivity in obese fatty (fa/fa) Zucker rats and Zucker Diabetic Fatty (ZDF) rats treated with the thiazolidinedione, MCC-555

1. We examined the effect of chronic (21 days) oral treatment with the thiazolidinedione, MCC-555 ((+)-5-[[6-(2-fluorbenzyl)-oxy-2-naphy]methyl]-2,4-thiazo lid inedione) on metabolic status and insulin sensitivity in obese (fa/fa) Zucker rats and Zucker Diabetic Fatty (ZDF) rats which display an impaired glucose tolerance (IGT) or overt diabetic symptoms, respectively. 2. MCC-555 treatment to obese Zucker rats (10 and 30 mg kg(-1)) and diabetic ZDF rats (10 mg kg(-1)) reduced non-esterified fatty acid concentrations in both rat strains and reduced plasma glucose and triglyceride concentrations in the obese Zucker rats. Liver glycogen concentrations were significantly increased by chronic MCC-555 treatment in both obese Zucker rats (30 mg kg(-1) day(-1)) and diabetic ZDF rats (10 mg kg(-1) day(-1)), as compared with vehicle-treated lean and obese rats and there was a significant increase in hepatic glycogen synthase activity in MCC-555-treated diabetic ZDF rats as compared to vehicle-treated controls. 3. During a euglycaemic hyperinsulinaemic clamp, MCC-555-treated obese Zucker rats and diabetic ZDF rats required significantly higher glucose infusion rates to maintain stable glucose concentrations (2.01+/-0.19 mg min(-1) and 6.42+/-1.03 mg min(-1), respectively) than vehicle-treated obese controls (0.71+/-0.17 mg min(-1) and 2.09+/-0.71 mg min(-1); P<0.05), demonstrating improved insulin sensitivity in both Zucker and ZDF rats. MCC-555 treatment also enhanced insulin-induced suppression of hepatic glucose production in ZDF rats as measured using infusions of [6-3H]-glucose under clamp conditions. 4. In conclusion, we have demonstrated that MCC-555 improves metabolic status and insulin sensitivity in obese Zucker and diabetic ZDF rats. MCC-555 may prove a useful compound for alleviating the metabolic disturbances and IGT associated with insulin resistance in man.     

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.     

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.     

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.     

A molecular basis for insulin resistance. Elevated serine/threonine phosphorylation of IRS-1 and IRS-2 inhibits their binding to the juxtamembrane region of the insulin receptor and impairs their ability to undergo insulin-induced tyrosine phosphorylation

Tumor necrosis factor alpha (TNFalpha) or chronic hyperinsulinemia that induce insulin resistance trigger increased Ser/Thr phosphorylation of the insulin receptor (IR) and of its major insulin receptor substrates, IRS-1 and IRS-2. To unravel the molecular basis for this uncoupling in insulin signaling, we undertook to study the interaction of Ser/Thr-phosphorylated IRS-1 and IRS-2 with the insulin receptor. We could demonstrate that, similar to IRS-1, IRS-2 also interacts with the juxtamembrane (JM) domain (amino acids 943-984) but not with the carboxyl-terminal region (amino acids 1245-1331) of IR expressed in bacteria as His6 fusion peptides. Moreover, incubation of rat hepatoma Fao cells with TNFalpha, bacterial sphingomyelinase, or other Ser(P)/Thr(P)-elevating agents reduced insulin-induced Tyr phosphorylation of IRS-1 and IRS-2, markedly elevated their Ser(P)/Thr(P) levels, and significantly reduced their ability to interact with the JM region of IR. Withdrawal of TNFalpha for periods as short as 30 min reversed its inhibitory effects on IR-IRS interactions. Similar inhibitory effects were obtained when Fao cells were subjected to prolonged (20-60 min) pretreatment with insulin. Incubation of the cell extracts with alkaline phosphatase reversed the inhibitory effects of insulin. These findings suggest that insulin resistance is associated with enhanced Ser/Thr phosphorylation of IRS-1 and IRS-2, which impairs their interaction with the JM region of IR. Such impaired interactions abolish the ability of IRS-1 and IRS-2 to undergo insulin-induced Tyr phosphorylation and further propagate the insulin receptor signal. Moreover, the reversibility of the TNFalpha effects and the ability to mimic its action by exogenously added sphingomyelinase argue against the involvement of a proteolytic cascade in mediating the acute inhibitory effects of TNFalpha on insulin action.     

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.     

N-Methyl-D-aspartate inhibits apoptosis through activation of phosphatidylinositol 3-kinase in cerebellar granule neurons. A role for insulin receptor substrate-1 in the neurotrophic action of n-methyl-D-aspartate and its inhibition by ethanol

Primary cultured rat cerebellar granule neurons underwent apoptosis when switched from medium containing 25 mM K+ to one containing 5 mM K+. N-methyl-D-aspartate (NMDA) protected granule neurons from apoptosis in medium containing 5 mM K+. Inhibition of apoptosis by NMDA was blocked by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY294002, but it was unaffected by the mitogen-activated protein kinase kinase inhibitor PD 98059. The antiapoptotic action of NMDA was associated with an increase in the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), an increase in the binding of the regulatory subunit of PI 3-kinase to IRS-1, and a stimulation of PI 3-kinase activity. In the absence of extracellular Ca2+, NMDA was unable to prevent apoptosis or to phosphorylate IRS-1 and activate PI 3-kinase. Significant inhibition of NMDA-mediated neuronal survival by ethanol (10-15%) was observed at 1 mM, and inhibition was half-maximal at 45-50 mM. Inhibition of neuronal survival by ethanol corresponded with a marked reduction in the capacity of NMDA to increase the concentration of intracellular Ca2+, phosphorylate IRS-1, and activate PI 3-kinase. These data demonstrate that the neurotrophic action of NMDA and its inhibition by ethanol are mediated by alterations in the activity of a PI 3-kinase-dependent antiapoptotic signaling pathway.     

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.     

Intracellular localization of phosphatidylinositide 3-kinase and insulin receptor substrate-1 in adipocytes: potential involvement of a membrane skeleton

Phosphatidylinositide (PI) 3-kinase binds to tyrosyl-phosphorylated insulin receptor substrate-1 (IRS-1) in insulin-treated adipocytes, and this step plays a central role in the regulated movement of the glucose transporter, GLUT4, from intracellular vesicles to the cell surface. PDGF, which also activates PI 3-kinase in adipocytes, has no significant effect on GLUT4 trafficking in these cells. We propose that this specificity may be mediated by differential localization of PI 3-kinase in response to insulin versus PDGF activation. Using subcellular fractionation in 3T3-L1 adipocytes, we show that insulin- and PDGF-stimulated PI 3-kinase activities are located in an intracellular high speed pellet (HSP) and in the plasma membrane (PM), respectively. The HSP is also enriched in IRS-1, insulin-stimulated tyrosyl-phosphorylated IRS-1 and intracellular GLUT4-containing vesicles. Using sucrose density gradient sedimentation, we have been able to segregate the HSP into two separate subfractions: one enriched in IRS-1, tyrosyl-phosphorylated IRS-1, PI 3-kinase as well as cytoskeletal elements, and another enriched in membranes, including intracellular GLUT4 vesicles. Treatment of the HSP with nonionic detergent, liberates all membrane constituents, whereas IRS-1 and PI 3-kinase remain insoluble. Conversely, at high ionic strength, membranes remain intact, whereas IRS-1 and PI 3-kinase become freely soluble. We further show that this IRS-1-PI 3-kinase complex exists in CHO cells overexpressing IRS-1 and, in these cells, the cytosolic pool of IRS-1 and PI 3-kinase is released subsequent to permeabilization with Streptolysin-O, whereas the particulate fraction of these proteins is retained. These data suggest that IRS-1, PI 3-kinase, as well as other signaling intermediates, may form preassembled complexes that may be associated with the actin cytoskeleton. This complex must be in close apposition to the cell surface, enabling access to the insulin receptor and presumably other signaling molecules that somehow confer the absolute specificity of insulin signaling in these cells.     

Characterization of apolipoprotein E7 (Glu(244)-->Lys, Glu(245)--->Lys), a mutant apolipoprotein E associated with hyperlipidemia and atherosclerosis

To examine the mechanism by which free fatty acids (FFA) induce insulin resistance in human skeletal muscle, glycogen, glucose-6-phosphate, and intracellular glucose concentrations were measured using carbon-13 and phosphorous-31 nuclear magnetic resonance spectroscopy in seven healthy subjects before and after a hyperinsulinemic-euglycemic clamp following a five-hour infusion of either lipid/heparin or glycerol/heparin. IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity was also measured in muscle biopsy samples obtained from seven additional subjects before and after an identical protocol. Rates of insulin stimulated whole-body glucose uptake. Glucose oxidation and muscle glycogen synthesis were 50%-60% lower following the lipid infusion compared with the glycerol infusion and were associated with a approximately 90% decrease in the increment in intramuscular glucose-6-phosphate concentration, implying diminished glucose transport or phosphorylation activity. To distinguish between these two possibilities, intracellular glucose concentration was measured and found to be significantly lower in the lipid infusion studies, implying that glucose transport is the rate-controlling step. Insulin stimulation, during the glycerol infusion, resulted in a fourfold increase in PI 3-kinase activity over basal that was abolished during the lipid infusion. Taken together, these data suggest that increased concentrations of plasma FFA induce insulin resistance in humans through inhibition of glucose transport activity; this may be a consequence of decreased IRS-1-associated PI 3-kinase activity.     

Differential signaling by insulin receptor substrate 1 (IRS-1) and IRS-2 in IRS-1-deficient cells

Mice made insulin receptor substrate 1 (IRS-1) deficient by targeted gene knockout exhibit growth retardation and abnormal glucose metabolism due to resistance to the actions of insulin-like growth factor 1 (IGF-1) and insulin (E. Araki et al., Nature 372:186-190, 1994; H. Tamemoto et al., Nature 372:182-186, 1994). Embryonic fibroblasts and 3T3 cell lines derived from IRS-1-deficient embryos exhibit no IGF-1-stimulated IRS-1 phosphorylation or IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity but exhibit normal phosphorylation of IRS-2 and Shc and normal IRS-2-associated PI 3-kinase activity. IRS-1 deficiency results in a 70 to 80% reduction in IGF-1-stimulated cell growth and parallel decreases in IGF-1-stimulated S-phase entry, PI 3-kinase activity, and induction of the immediate-early genes c-fos and egr-1 but unaltered activation of the mitogen-activated protein kinases ERK 1 and ERK 2. Expression of IRS-1 in IRS-1-deficient cells by retroviral gene transduction restores IGF-1-stimulated mitogenesis, PI 3-kinase activation, and c-fos and egr-1 induction in proportion to the level of reconstitution. Increasing the level of IRS-2 in these cells by using a retrovirus reconstitutes IGF-1 activation of PI 3-kinase and immediate-early gene expression to the same degree as expression of IRS-1; however, IRS-2 overexpression has only a minor effect on IGF-1 stimulation of cell cycle progression. These results indicate that IRS-1 is not necessary for activation of ERK 1 and ERK 2 and that activation of ERK 1 and ERK 2 is not sufficient for IGF-1-stimulated activation of c-fos and egr-1. These data also provide evidence that IRS-1 and IRS-2 are not functionally interchangeable signaling intermediates for stimulation of mitogenesis despite their highly conserved structure and many common functions such as activating PI 3-kinase and early gene expression.     

Assignment of the gene encoding the limbic system-associated membrane protein (LAMP) to mouse chromosome 16B5 and human chromosome 3q13.2-q21

Both insulin and muscle contraction stimulate glucose transport activity. However, contraction stimulation does not involve the insulin signalling intermediate phosphatidylinositol 3-kinase (PI 3-kinase). Protein kinase B (PKB) has recently been identified as a direct downstream target of PI 3-kinase in the insulin signalling pathway. We have examined here whether the two stimuli share PKB as a convergent step in separate signalling pathways. Insulin stimulates both glucose transport, GLUT4 cell-surface content and PKB activity (by 4-6-fold above basal) in a wortmannin-sensitive manner in in vitro incubated rat soleus muscles. By contrast, muscle contraction, which stimulates glucose transport and the cell surface content of GLUT4 by 3-fold above basal levels, had no effect on PKB activity. These data demonstrate that PKB is not a mediator of contraction-induced glucose transport and GLUT4 translocation.     

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.     

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.     

The development of overt diabetes in young Zucker Diabetic Fatty (ZDF) rats and the effects of chronic MCC-555 treatment

1. Young (6-week-old) pre-diabetic Zucker Diabetic Fatty (ZDF) rats displaying impaired glucose tolerance (IGT), moderate hyperglycaemia and hyperinsulinaemia were treated with the novel thiazolidinedione, MCC-555, for 28 days, during which time beta-cell failure and progression to overt diabetes occurs. 2. Treated ZDF rats exhibited consistently lower blood glucose levels than vehicle-treated diabetic controls, with a delayed rise and lower plateau levels. MCC-555 maintained plasma insulin levels throughout the treatment period, whereas these fell by 40% in untreated ZDF rats. 3. The rise in body weight was maintained in MCC-555-treated rats, whereas vehicle-treated rats exhibited blunted body weight gain after 8 weeks of age. Daily food intake was higher in diabetic, as compared to non-diabetic rats, but treatment did not modify food intake in diabetic rats. Water intake was lower in treated ZDF rats, concomitant with lowering of blood glucose. 4. The hyperinsulinaemic-euglycaemic clamp technique was applied to all rats after treatment to examine the effects of MCC-555 on insulin sensitivity. The glucose infusion rate to maintain normoglycaemia was lower in diabetic than in non-diabetic rats, demonstrating reduced glucose entry into insulin-sensitive tissues in diabetic rats. Increased glucose infusion rates were required to maintain euglycaemia in treated diabetic rats, demonstrating increased insulin sensitivity in these animals. 5. In conclusion, chronic MCC-555 treatment of young ZDF rats displaying IGT attenuates the development of overt diabetes through improved insulin sensitivity and maintenance of beta-cell function. MCC-555 may thus be beneficial in humans with IGT, to prevent or delay the progression of diabetes.