Differentiation-dependent suppression of platelet-derived growth factor signaling in cultured adipocytes.

A critical component of vertebrate cellular differentiation is the acquisition of sensitivity to a restricted subset of peptide hormones and growth factors. This accounts for the unique capability of insulin (and possibly insulin-like growth factor-1), but not other growth factors, to stimulate glucose uptake and anabolic metabolism in heart, skeletal muscle, and adipose tissue. This selectivity is faithfully recapitulated in the cultured adipocyte line, 3T3-L1, which responds to insulin, but not platelet-derived growth factor (PDGF), with increased hexose uptake. The serine/threonine protein kinases Akt1 and Akt2, which have been implicated as mediators of insulin-stimulated glucose uptake, as well as glycogen, lipid, and protein synthesis, were shown to mirror this selectivity in this tissue culture system. This was particularly apparent in 3T3-L1 adipocytes overexpressing an epitope-tagged form of Akt2 in which insulin activated Akt2 10-fold better than PDGF. Similarly, in 3T3-L1 adipocytes, only insulin stimulated phosphorylation of Akt's endogenous substrate, GSK-3beta. Other signaling molecules, including phosphatidylinositol 3-kinase, pp70 S6-kinase, mitogen-activated protein kinase, and PHAS-1/4EBP-1, did not demonstrate this selective responsiveness to insulin but were instead activated comparably by both insulin and PDGF. Moreover, concurrent treatment with PDGF and insulin did not diminish activation of phosphatidylinositol 3-kinase, Akt, or glucose transport, indicating that PDGF did not simultaneously activate an inhibitory mechanism. Interestingly, PDGF and insulin comparably stimulated both Akt isoforms, as well as numerous other signaling molecules, in undifferentiated 3T3-L1 preadipocytes. Collectively, these data suggest that differential activation of Akt in adipocytes may contribute to insulin's exclusive mediation of the metabolic events involved in glucose metabolism. Moreover, they suggest a novel mechanism by which differentiation-dependent hormone selectivity is conferred through the suppression of specific signaling pathways operational in undifferentiated cell types.     

Activation of SOCS-3 by resistin

Resistin is an adipocyte hormone that modulates glucose homeostasis. Here we show that in 3T3-L1 adipocytes, resistin attenuates multiple effects of insulin, including insulin receptor (IR) phosphorylation, IR substrate 1 (IRS-1) phosphorylation, phosphatidylinositol-3-kinase (PI3K) activation, phosphatidylinositol triphosphate production, and activation of protein kinase B/Akt. Remarkably, resistin treatment markedly induces the gene expression of suppressor of cytokine signaling 3 (SOCS-3), a known inhibitor of insulin signaling. The 50% effective dose for resistin induction of SOCS-3 is approximately 20 ng/ml, close to levels of resistin in serum. Association of SOCS-3 protein with the IR is also increased by resistin. Inhibition of SOCS function prevented resistin from antagonizing insulin action in adipocytes. SOCS-3 induction is the first cellular effect of resistin that is independent of insulin and is a likely mediator of resistin's inhibitory effect on insulin signaling in adipocytes.     

c-Jun N-terminal kinase (JNK) mediates feedback inhibition of the insulin signaling cascade

Activation of the c-Jun N-terminal kinase (JNK) by proinflammatory cytokines inhibits insulin signaling, at least in part, by stimulating phosphorylation of rat/mouse insulin receptor substrate 1 (Irs1) at Ser(307) (Ser(312) in human IRS1). Here we show that JNK mediated feedback inhibition of the insulin signal in mouse embryo fibroblasts, 3T3-L1 adipocytes, and 32D(IR) cells. Insulin stimulation of JNK activity required phosphatidylinositol 3-kinase and Grb2 signaling. Moreover, activation of JNK by insulin was inhibited by a cell-permeable peptide that disrupted the interaction of JNK with cellular proteins. However, the direct binding of JNK to Irs1 was not required for its activation by insulin, whereas direct binding was required for Ser(307) phosphorylation of Irs1. Insulin-stimulated Ser(307) phosphorylation was reduced 80% in cells lacking JNK1 and JNK2 or in cells expressing a mutant Irs1 protein lacking the JNK binding site. Reduced Ser(307) phosphorylation was directly related to increased insulin-stimulated tyrosine phosphorylation, Akt phosphorylation, and glucose uptake. These results support the hypothesis that JNK is a negative feedback regulator of insulin action by phosphorylating Ser(307) in Irs1.     

Defective Akt activation is associated with glucose- but not glucosamine-induced insulin resistance

3T3-L1 adipocytes develop insulin-resistant glucose transport upon preincubation with high glucose or glucosamine, provided insulin (0.6 nM) is present during preincubation. Insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol (PI) 3-kinase activity is unaffected (30). Total cellular IRS-1, PI 3-kinase, or Akt concentrations were unchanged. Akt activation in subcellular fractions was assessed by immunoblotting with two phospho-Akt-specific antibodies. Upon acute 100 nM insulin stimulation, plasma membrane (PM)-associated phospho-Akt was highest in cells preincubated in low glucose with no insulin, less in high glucose with no insulin, even less in low glucose+insulin, and lowest in high glucose+insulin. Only high glucose+insulin caused insulin-resistant glucose transport. Acute insulin stimulation increased total PM-Akt about twofold after preincubation without insulin in low or high glucose. Preincubation with 0.6 nM insulin decreased Akt PM translocation by approximately 25% in low and approximately 50% in high glucose. Preincubation with glucosamine did not affect Akt phosphorylation or translocation. Conclusions: chronic exposure to high glucose or insulin downregulates acute insulin-stimulated Akt activation, acting synergistically distal to PI 3-kinase. Maximal insulin activates more Akt than required for maximal glucose transport stimulation. Insulin resistance may ensue when PM-associated phospho-Akt decreases below a threshold. High glucose and glucosamine cause insulin resistance by different mechanisms in 3T3-L1 adipocytes.     

An inhibitor of p38 mitogen-activated protein kinase prevents insulin-stimulated glucose transport but not glucose transporter translocation in 3T3-L1 adipocytes and L6 myotubes

The precise mechanisms underlying insulin-stimulated glucose transport still require investigation. Here we assessed the effect of SB203580, an inhibitor of the p38 MAP kinase family, on insulin-stimulated glucose transport in 3T3-L1 adipocytes and L6 myotubes. We found that SB203580, but not its inactive analogue (SB202474), prevented insulin-stimulated glucose transport in both cell types with an IC50 similar to that for inhibition of p38 MAP kinase (0.6 microM). Basal glucose uptake was not affected. Moreover, SB203580 added only during the transport assay did not inhibit basal or insulin-stimulated transport. SB203580 did not inhibit insulin-stimulated translocation of the glucose transporters GLUT1 or GLUT4 in 3T3-L1 adipocytes as assessed by immunoblotting of subcellular fractions or by immunofluorescence of membrane lawns. L6 muscle cells expressing GLUT4 tagged on an extracellular domain with a Myc epitope (GLUT4myc) were used to assess the functional insertion of GLUT4 into the plasma membrane. SB203580 did not affect the insulin-induced gain in GLUT4myc exposure at the cell surface but largely reduced the stimulation of glucose uptake. SB203580 had no effect on insulin-dependent insulin receptor substrate-1 phosphorylation, association of the p85 subunit of phosphatidylinositol 3-kinase with insulin receptor substrate-1, nor on phosphatidylinositol 3-kinase, Akt1, Akt2, or Akt3 activities in 3T3-L1 adipocytes. In conclusion, in the presence of SB203580, insulin caused normal translocation and cell surface membrane insertion of glucose transporters without stimulating glucose transport. We propose that insulin stimulates two independent signals contributing to stimulation of glucose transport: phosphatidylinositol 3-kinase leads to glucose transporter translocation and a pathway involving p38 MAP kinase leads to activation of the recruited glucose transporter at the membrane.     

PTEN, but not SHIP2, suppresses insulin signaling through the phosphatidylinositol 3-kinase/Akt pathway in 3T3-L1 adipocytes

Glucose homeostasis is controlled by insulin in part through the stimulation of glucose transport in muscle and fat cells. This insulin signaling pathway requires phosphatidylinositol (PI) 3-kinase-mediated 3'-polyphosphoinositide generation and activation of Akt/protein kinase B. Previous experiments using dominant negative constructs and gene ablation in mice suggested that two phosphoinositide phosphatases, SH2 domain-containing inositol 5'-phosphatase 2 (SHIP2) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) negatively regulate this insulin signaling pathway. Here we directly tested this hypothesis by selectively inhibiting the expression of SHIP2 or PTEN in intact cultured 3T3-L1 adipocytes through the use of short interfering RNA (siRNA). Attenuation of PTEN expression by RNAi markedly enhanced insulin-stimulated Akt and glycogen synthase kinase 3alpha (GSK-3alpha) phosphorylation, as well as deoxyglucose transport in 3T3-L1 adipocytes. In contrast, depletion of SHIP2 protein by about 90% surprisingly failed to modulate these insulin-regulated events under identical assay conditions. In control studies, no diminution of insulin signaling to the mitogen-activated protein kinases Erk1 and Erk2 was observed when either PTEN or SHIP2 were depleted. Taken together, these results demonstrate that endogenous PTEN functions as a suppressor of insulin signaling to glucose transport through the PI 3-kinase pathway in cultured 3T3-L1 adipocytes.     

The mitogenic and antiapoptotic actions of ghrelin in 3T3-L1 adipocytes

Ghrelin, a stomach-derived hormone, induces adiposity when administered to rodents. Because ghrelin receptor is abundantly expressed in adipose tissue, we investigated the role of ghrelin in adipocyte biology. We observed ghrelin receptor expression in 3T3-L1 preadipocytes and adipocytes. Treatment of preadipocytes with ghrelin induced cellular proliferation and differentiation to mature adipocytes, as well as basal and insulin-stimulated glucose transport, but it inhibited adipocyte apoptosis induced by serum deprivation. Exposure of 3T3-L1 cells to ghrelin caused a rapid activation of MAPKs, especially ERK1/2. Chemical inhibition of MAPK blocked the mitogenic and antiapoptotic effects of ghrelin. Ghrelin also stimulated the insulin receptor substrate-associated phosphatidylinositol 3-kinase/Akt pathway in 3T3-L1 preadipocytes and adipocytes, whereas inhibition of this pathway blocked the effects of ghrelin on cell proliferation, antiapoptosis and glucose uptake. These findings suggest that the direct effects of ghrelin on proliferation, differentiation, and apoptosis in adipocytes may play a role in regulating fat cell number. These effects may be mediated via activation of the MAPK and phosphatidylinositol 3-kinase/Akt pathways.     

Requirement for Activation of the Serine-Threonine Kinase Akt (Protein Kinase B) in Insulin Stimulation of Protein Synthesis but Not of Glucose Transport

A wide variety of biological activities including the major metabolic actions of insulin is regulated by phosphatidylinositol (PI) 3-kinase. However, the downstream effectors of the various signaling pathways that emanate from PI 3-kinase remain unclear. Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, is thought to be one such downstream effector. A mutant Akt (Akt-AA) in which the phosphorylation sites (Thr³⁰⁸ and Ser⁴⁷³) targeted by growth factors are replaced by alanine has now been shown to lack protein kinase activity and, when overexpressed in CHO cells or 3T3-L1 adipocytes with the use of an adenovirus vector, to inhibit insulin-induced activation of endogenous Akt. Akt-AA thus acts in a dominant negative manner in intact cells. Insulin-stimulated protein synthesis, which is sensitive to wortmannin, a pharmacological inhibitor of PI 3-kinase, was abolished by overexpression of Akt-AA without an effect on amino acid transport into the cells, suggesting that Akt is required for insulin-stimulated protein synthesis. Insulin activation of p70 S6 kinase was inhibited by ~75% in CHO cells and ~30% in 3T3-L1 adipocytes, whereas insulin-induced activation of endogenous Akt was inhibited by 80 to 95%, by expression of Akt-AA. Thus, Akt activity appears to be required, at least in part, for insulin stimulation of p70 S6 kinase. However, insulin-stimulated glucose uptake in both CHO cells and 3T3-L1 adipocytes was not affected by overexpression of Akt-AA, suggesting that Akt is not required for this effect of insulin. These data indicate that Akt acts as a downstream effector in some, but not all, of the signaling pathways downstream of PI 3-kinase.     

Functional interactions of phosphatidylinositol 3-kinase with GTPase-activating protein in 3T3-L1 adipocytes.

The role of phosphatidylinositol (PI) 3-kinase in specific aspects of insulin signaling was explored in 3T3-L1 adipocytes. Inhibition of PI 3-kinase activity by LY294002 or wortmannin significantly enhanced basal and insulin-stimulated GTPase-activating protein (GAP) activity in 3T3-L1 adipocytes. Furthermore, removal of the inhibitory influence of PI 3-kinase on GAP resulted in dose-dependent decreases in the ability of insulin to stimulate p21ras. This effect was specific to adipocytes, as inhibition of PI 3-kinase did not influence GAP in either 3T3-L1 fibroblasts, Rat-1 fibroblasts, or CHO cells. Immunodepletion of either of the two subunits of the PI 3-kinase (p85 or p110) yielded similar activation of GAP, suggesting that catalytic activity of p110 plays an important role in controlling GAP activity in 3T3-L1 adipocytes. Inhibition of PI 3-kinase activity in 3T3-L1 adipocytes resulted in abrogation of insulin-stimulated glucose uptake and thymidine incorporation. In contrast, effects of insulin on glycogen synthase and mitogen-activated protein kinase activity were inhibited only at higher concentrations of LY294002. It appears that in adipocytes, P1 3-kinase prevents activation of GAP. Inhibition of PI 3-kinase activity or immunodepletion of either one of its subunits results in activation of GAP and decreases in GTP loading of p21ras.     

Dissecting the Mechanism of Insulin Resistance Using a Novel Heterodimerization Strategy to Activate Akt

Insulin resistance can occur in response to many different external insults, including chronic exposure to insulin itself as well as other agonists such as dexamethasone. It is generally thought that such defects arise due to a defect(s) at an early stage in the insulin signaling cascade. One model suggests that this involves activation of the mammalian target of rapamycin/S6 kinase pathway, which inactivates insulin receptor substrate via Ser/Thr phosphorylation. However, we have recently shown that insulin receptor substrate is not a major node for insulin resistance defects. To explore the mechanism of insulin resistance, we have developed a novel system to activate Akt independently of its upstream effectors as well as other insulin-responsive pathways such as mitogen-activated protein kinase. 3T3-L1 adipocytes were rendered insulin-resistant either with chronic insulin or dexamethasone treatment, but conditional activation of Akt2 stimulated hemagglutinin-tagged glucose transporter 4 translocation to the same extent in these insulin-resistant and control cells. However, addition of insulin to cells in which Akt was conditionally activated resulted in a reversion to the insulin-resistant state, indicating a feedforward inhibitory mechanism activated by insulin itself. This effect was overcome with wortmannin, implicating a role for phosphatidylinositol 3-kinase in this inhibitory process. We conclude that in chronic insulin- and dexamethasone-treated cells, acute activation with insulin itself is required to activate a feedforward inhibitory pathway likely emanating from phosphatidylinositol 3-kinase that converges on a target downstream of Akt to cause insulin resistance.     

Protein phosphatase-2C alpha as a positive regulator of insulin sensitivity through direct activation of phosphatidylinositol 3-kinase in 3T3-L1 adipocytes

During differentiation, expression of protein phosphatase-2Calpha (PP2Calpha) is increased in 3T3-L1 adipocytes. To elucidate the role of PP2Calpha in insulin signaling, we overexpressed wild-type (WT) PP2Calpha by adenovirus-mediated gene transfer in 3T3-L1 adipocytes. Overexpression of PP2Calpha-WT enhanced the insulin sensitivity of glucose uptake without any changes in the early steps of insulin signaling. Infection with adenovirus 5 expressing PP2Calpha-WT increased phosphatidylinositol 3-kinase (PI3K) activities in the immunoprecipitate using antibody against the p85 or p110 subunit under both basal and insulin-stimulated conditions, followed by activation of downstream steps in the PI3K pathway, such as phosphorylation of Akt, glycogen synthase kinase-3, and atypical protein kinase C. In contrast, overexpression of the phosphatase-defective mutant PP2Calpha(R174G) did not produce such effects. Furthermore, overexpression of PP2Calpha-WT (but not PP2Calpha(R174G)) decreased the (32)P-labeled phosphorylation state as well as the gel mobility shift of the p85 subunit, suggesting that dephosphorylation of the p85 subunit by PP2Calpha activation might stimulate PI3K catalytic activity. Moreover, knockdown of PP2Calpha by transfection of small interfering RNA led to a significant decrease in Akt phosphorylation. In addition, microinjection of anti-PP2Calpha antibody or PP2Calpha small interfering RNA led to decreased insulin-stimulated GLUT4 translocation. In conclusion, PP2Calpha is a new positive regulator of insulin sensitivity that acts through a direct activation of PI3K in 3T3-L1 adipocytes.     

Tumor necrosis factor alpha produces insulin resistance in skeletal muscle by activation of inhibitor kappaB kinase in a p38 MAPK-dependent manner

Insulin stimulation produced a reliable 3-fold increase in glucose uptake in primary neonatal rat myotubes, which was accompanied by a similar effect on GLUT4 translocation to plasma membrane. Tumor necrosis factor (TNF)-alpha caused insulin resistance on glucose uptake and GLUT4 translocation by impairing insulin stimulation of insulin receptor (IR) and IR substrate (IRS)-1 and IRS-2 tyrosine phosphorylation, IRS-associated phosphatidylinositol 3-kinase activation, and Akt phosphorylation. Because this cytokine produced sustained activation of stress and proinflammatory kinases, we have explored the hypothesis that insulin resistance by TNF-alpha could be mediated by these pathways. In this study we demonstrate that pretreatment with PD169316 or SB203580, inhibitors of p38 MAPK, restored insulin signaling and normalized insulin-induced glucose uptake in the presence of TNF-alpha. However, in the presence of PD98059 or SP600125, inhibitors of p42/p44 MAPK or JNK, respectively, insulin resistance by TNF-alpha was still produced. Moreover, TNF-alpha produced inhibitor kappaB kinase (IKK)-beta activation and inhibitor kappaB-beta and -alpha degradation in a p38 MAPK-dependent manner, and treatment with salicylate (an inhibitor of IKK) completely restored insulin signaling. Furthermore, TNF-alpha produced serine phosphorylation of IR and IRS-1 (total and on Ser(307) residue), and these effects were completely precluded by pretreatment with either PD169316 or salicylate. Consequently, TNF-alpha, through activation of p38 MAPK and IKK, produces serine phosphorylation of IR and IRS-1, impairing its tyrosine phosphorylation by insulin and the corresponding activation of phosphatidylinositol 3-kinase and Akt, leading to insulin resistance on glucose uptake and GLUT4 translocation.     

Shikonin stimulates glucose uptake in 3T3-L1 adipocytes via an insulin-independent tyrosine kinase pathway

Type 2 diabetes is due to defects in both insulin action and secretion. In an attempt to discover small molecules that stimulate glucose uptake, similar to insulin, a cell-based glucose uptake screening assay was performed using 3T3-L1 adipocytes. Shikonin, a substance originally isolated from the root of the Chinese plant that has been used as an ointment for wound healing, was thus identified. Shikonin stimulated glucose uptake and potentiated insulin-stimulated glucose uptake in a concentration-dependent manner in 3T3-L1 adipocytes. Stimulation of glucose uptake was also observed in rat primary adipocytes and cardiomyocytes. Like insulin, shikonin-stimulated glucose uptake was inhibited by genistein, a tyrosine kinase inhibitor, and enhanced by vanadate, a tyrosine phosphatase inhibitor. However, in contrast to insulin, shikonin-stimulated glucose uptake was not strongly inhibited by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K). In vitro phosphorylation analyses revealed that shikonin did not induce tyrosine phosphorylation of the insulin receptor, but significantly induced both Thr-308 and Ser-473 phosphorylation of Akt. Our results suggest that in 3T3-L1 adipocytes, shikonin action is not mediated primarily via the insulin receptor/PI3K pathway, but rather via another distinct tyrosine kinase-dependent pathway leading to glucose uptake involving Akt phosphorylation.     

Interleukin-4 Restores Insulin Sensitivity in Lipid-Induced Insulin-Resistant Adipocytes

Obesity and latent inflammation in adipose tissue significantly contribute to the development of insulin resistance (IR) and type 2 diabetes. Here we studied whether the antiinflammatory interleukin-4 (IL-4) can restore insulin sensitivity in cultured 3T3-L1 adipocytes. The activity of key components of the insulin signaling cascade was assessed by immunoblotting using phospho-specific antibodies to insulin receptor substrate IRS1 (Tyr612), Akt (Thr308 and Ser473), and AS160 (Ser318) protein that regulates translocation of the GLUT4 glucose transporter to the plasma membrane. IR was induced in mature adipocytes with albumin-conjugated palmitate. IR significantly reduced phosphorylation levels of all the above-mentioned proteins. Addition of IL-4 to the culturing medium during IR induction led to a dose-dependent stimulation of the insulin-promoted phosphorylation of IRS1, Akt, and AS160. At the optimal concentration of 50 ng/ml, IL-4 fully restored activation of the insulin cascade in IR cells, but it did not affect insulin signaling activation in the control cells. IL- 4 neither upregulated expression of key adipogenesis markers GLUT4 and PPARγ nor caused lipid accumulation in the adipocytes. These results demonstrate that IL-4 can restore insulin sensitivity in adipocytes via mechanisms not associated with induced adipogenesis or de novo formation of lipid depots.     

Infusion of beta-endorphin improves insulin resistance in fructose-fed rats.

In an attempt to probe the effect of beta-endorphin on insulin resistance, we used Wistar rats that were fed fructose-rich chow to induce insulin resistance. Insulin action on glucose disposal rate (GDR) was measured using the hyperinsulinemic euglycemic clamp technique, in which glucose (variable), insulin (40 mU/kg/min), and beta-endorphin (6 ng/kg/min) or vehicle were initiated simultaneously and continued for 120 min. A marked reduction in insulin-stimulated GDR was observed in fructose-fed rats compared to normal control rats. Infusion of beta-endorphin reversed the value of GDR, which was inhibited by naloxone and naloxonazine each at doses sufficient to block opioid mu-receptors. Opioid mu-receptors may therefore be activated by beta-endorphin to improve insulin resistance. Next, soleus muscle was isolated to investigate the effect of beta-endorphin on insulin signals. Insulin resistance in rats induced by excess fructose was associated with the impaired insulin receptor (IR), tyrosine autophosphorylation, and insulin receptor substrate (IRS)-1 protein content in addition to the significant decrease in IRS-1 tyrosine phosphorylation in soleus muscle. This impaired glucose transportation was also due to signaling defects that included an attenuated p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase) and Akt serine phosphorylation. However, IR protein levels were not markedly changed in rats with insulin resistance. beta-endorphin infusion reversed the fructose-induced decrement in the insulin-signaling cascade with increased GDR. Apart from IR protein levels, infusion of beta-endorphin reversed the decrease in protein expression for the IRS-1, p85 regulatory subunit of PI3-kinase, and Akt serine phosphorylation in soleus muscle in fructose-fed rats. The decrease in insulin-stimulated protein expression of glucose transporter subtype 4 (GLUT 4) in fructose-fed rats returned to near-normal levels after beta-endorphin infusion. Infusion of beta-endorphin may improve insulin resistance by modulating the insulin-signaling pathway to reverse insulin responsiveness.     

Berberine inhibits PTP1B activity and mimics insulin action

Type 2 diabetes patients show defects in insulin signal transduction that include lack of insulin receptor, decrease in insulin stimulated receptor tyrosine kinase activity and receptor-mediated phosphorylation of insulin receptor substrates (IRSs). A small molecule that could target insulin signaling would be of significant advantage in the treatment of diabetes. Berberine (BBR) has recently been shown to lower blood glucose levels and to improve insulin resistance in db/db mice partly through the activation of AMP-activated protein kinase (AMPK) signaling and induction of phosphorylation of insulin receptor (IR). However, the underlying mechanism remains largely unknown. Here we report that BBR mimics insulin action by increasing glucose uptake ability by 3T3-L1 adipocytes and L6 myocytes in an insulin-independent manner, inhibiting phosphatase activity of protein tyrosine phosphatase 1B (PTP1B), and increasing phosphorylation of IR, IRS1 and Akt in 3T3-L1 adipocytes. In diabetic mice, BBR lowers hyperglycemia and improves impaired glucose tolerance, but does not increase insulin release and synthesis. The results suggest that BBR represents a different class of anti-hyperglycemic agents.     

Growth factor receptor-binding protein 10 (Grb10) as a partner of phosphatidylinositol 3-kinase in metabolic insulin action

The regulation of the metabolic insulin response by mouse growth factor receptor-binding protein 10 (Grb10) has been addressed in this report. We find mouse Grb10 to be a critical component of the insulin receptor (IR) signaling complex that provides a functional link between IR and p85 phosphatidylinositol (PI) 3-kinase and regulates PI 3-kinase activity. This regulatory mechanism parallels the established link between IR and p85 via insulin receptor substrate (IRS) proteins. A direct association was demonstrated between Grb10 and p85 but was not observed between Grb10 and IRS proteins. In addition, no effect of mouse Grb10 was observed on the association between IRS-1 and p85, on IRS-1-associated PI 3-kinase activity, or on insulin-mediated activation of IR or IRS proteins. A critical role of mouse Grb10 was observed in the regulation of PI 3-kinase activity and the resulting metabolic insulin response. Dominant-negative Grb10 domains, in particular the SH2 domain, eliminated the metabolic response to insulin in differentiated 3T3-L1 adipocytes. This was consistently observed for glycogen synthesis, glucose and amino acid transport, and lipogenesis. In parallel, the same metabolic responses were substantially elevated by increased levels of Grb10. A similar role of Grb10 was confirmed in mouse L6 cells. In addition to the SH2 domain, the Pro-rich amino-terminal region of Grb10 was implicated in the regulation of PI 3-kinase catalytic activity. These regulatory roles of Grb10 were extended to specific insulin mediators downstream of PI 3-kinase including PKB/Akt, glycogen synthase kinase, and glycogen synthase. In contrast, a regulatory role of Grb10 in parallel insulin response pathways including p70 S6 kinase, ubiquitin ligase Cbl, or mitogen-activated protein kinase p38 was not observed. The dissection of the interaction of mouse Grb10 with p85 and the resulting regulation of PI 3-kinase activity should help elucidate the complexity of the IR signaling mechanism.     

Transient effect of platelet-derived growth factor on GLUT4 translocation in 3T3-L1 adipocytes.

We earlier developed a novel method to detect translocation of the glucose transporter (GLUT) directly and simply using c-MYC epitope-tagged GLUT (GLUTMYC). To define the effect of platelet-derived growth factor (PDGF) on glucose transport in 3T3-L1 adipocytes, we investigated the PDGF- and insulin-induced glucose uptake, translocation of glucose transporters, and phosphatidylinositol (PI) 3-kinase activity in 3T3-L1, 3T3-L1GLUT4MYC, and 3T3-L1GLUT1MYC adipocytes. Insulin and PDGF stimulated glucose uptake by 9-10- and 5.5-6.5-fold, respectively, in both 3T3-L1 and 3T3-L1GLUT4MYC adipocytes. Exogenous GLUT4MYC expression led to enhanced PDGF-induced glucose transport. In 3T3-L1GLUT4MYC adipocytes, insulin and PDGF induced an 8- and 5-fold increase in GLUT4MYC translocation, respectively, determined in a cell-surface anti-c-MYC antibody binding assay. This PDGF-induced GLUT4MYC translocation was further demonstrated with fluorescent detection. In contrast, PDGF stimulated a 2-fold increase of GLUT1MYC translocation and 2.5-fold increase of glucose uptake in 3T3-L1GLUT1MYC adipocytes. The PDGF-induced GLUT4MYC translocation, glucose uptake, and PI 3-kinase activity were maximal (100%) at 5-10 min and thereafter rapidly declined to 40, 30, and 12%, respectively, within 60 min, a time when effects of insulin were maximal. Wortmannin (0.1 microM) abolished PDGF-induced GLUT4MYC translocation and glucose uptake in 3T3-L1GLUT4MYC adipocytes. These results suggest that PDGF can transiently trigger the translocation of GLUT4 and stimulate glucose uptake by translocation of both GLUT4 and GLUT1 in a PI 3-kinase-dependent signaling pathway in 3T3-L1 adipocytes.     

Mechanisms of high-glucose/insulin-mediated desensitization of acute insulin-stimulated glucose transport and Akt activation

High-glucose/low-dose insulin-mediated insulin resistance of glucose transport was studied in 3T3-L1 adipocytes. In this model, proximal insulin signaling, including insulin receptor substrate (IRS)-1-bound phosphatidylinositol 3-kinase (PI 3-kinase) activation, is preserved, but insulin-stimulated protein kinase B (Akt) activation is markedly impaired. To assess a difference in acute insulin-stimulated production of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3], cells were labeled with [32P]orthophosphate, and glycerophosphoinositides were quantified by HPLC. Although basal PtdIns(3,4,5)P3 was similar, insulin stimulated its production 33.6% more in controls (P < 0.03) than in insulin-resistant cells. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein, a lipid phosphatase that dephosphorylates PtdIns(3,4,5)P3 in the 3-position, was significantly and specifically increased in insulin-resistant cells. Treatment with rapamycin [a specific inhibitor of mammalian target of rapamycin complex 1 (mTORC1)] inhibited the increased PTEN expression and partially restored insulin-stimulated glucose transport and Akt activation to insulin-resistant cells. Acute insulin markedly stimulated Ser(636/639) phosphorylation of IRS-1; this was rapamycin inhibited but was significantly decreased in cells that had been preexposed to insulin, whereas total IRS-1 was unaffected. These findings were essentially paralleled by changes in the activation of p70 S6 kinase and S6-ribosomal protein. Overexpression of uncoupling protein-1 or manganese superoxide dismutase did not prevent the development of insulin-resistant glucose transport and impaired Akt activation in high-glucose/low-insulin-pretreated cells. The insulin resistance associated with glucotoxicity in our model reflects in part decreased availability of PtdIns(3,4,5)P3, which correlates with increased PTEN protein expression. Chronic activation of mTORC1 plays a role in stimulating PTEN expression and possibly in activation or induction of a phosphoprotein phosphatase. No evidence was found for a role for increased mitochondrial superoxide production in this model.