Insulin resistance in fat cells from obese Zucker rats - Evidence for an impaired activation and translocation of protein kinase B and glucose transporter 4

The effect of insulin on glucose transport, glucose transporter 4 (Glut4) translocation, and intracellular signaling were measured in fat cells from lean and obese Zucker rats of different ages. Insulin-stimulated glucose transport was markedly reduced in adipocytes from old and obese animals. The protein content of Glut4 and insulin receptor substrates (IRS) 1 and 2 were also reduced while other proteins, including the p85 subunit of PI3-kinase, Shc and the MAP kinases (ERK1 and 2) were essentially unchanged. There was a marked impairment in the insulin stimulated tyrosine phosphorylation of IRS-1 and 2 as well as activation of PI3-kinase and PKB in cells from old and obese animals. Furthermore, insulin-stimulated translocation of both Glut4 and PKB to the plasma membrane was virtually abolished. The phosphotyrosine phosphatase inhibitor, vanadate, increased the insulin- stimulated upstream signaling including PI3-kinase and PKB activities as well as rate of glucose transport. Thus, the insulin resistance in cells from old and obese Zucker rats can be accounted for by an impaired translocation process, due to signaling defects leading to a reduced activation of PI3-kinase and PKB, as well as an attenuated Glut4 protein content.     

From insulin receptor signalling to Glut 4 translocation abnormalities in obesity and insulin resistance

Insulin resistance is commonly associated with obesity in rodents. Using mice made obese with goldthioglucose (GTG-obese mice), we have shown that insulin resistance results from defects at the level of the receptor and from intracellular alterations in insulin signalling pathway, without major alteration in the number of the Glut 4 glucose transporter. Activation of phosphatidylinositol 3-kinase (PI 3-kinase) was found to be profoundly affected in response to insulin. This defect appears very early in the development of obesity, together with a marked decrease in IRS 1 tyrosine phosphorylation. In order to better understand the abnormalities in glucose transport in insulin resistance, we have studied the pathway leading from the insulin receptor kinase stimulation to the translocation of the Glut 4 containing vesicles. This stimulation involves the activation of PI 3-kinase, which in turns activates protein kinase B. We have then focussed at the mechanism of vesicle exocytosis, and more specifically at the role of the small GTPase Rab4 in this process. We have shown that Rab4 participates, first in the intracellular retention of the Glut 4 containing vesicles, second in the insulin signalling pathway leading to glucose transporter translocation.     

From Insulin Receptor Signalling to Glut 4 Translocation Abnormalities in Obesity and Insulin Resistance

Abstract

Insulin resistance is commonly associated with obesity in rodents. Using mice made obese with goldthioglucose (GTG-obese mice), we have shown that insulin resistance results from defects at the level of the receptor and from intracellular alterations in insulin signalling pathway, without major alteration in the number of the Glut 4 glucose transporter. Activation of phosphatidylinositol 3-kinase (PI 3-kinase) was found to be profoundly affected in response to insulin. This defect appears very early in the development of obesity, together with a marked decrease in IRS 1 tyrosine phosphorylation. In order to better understand the abnormalities in glucose transport in insulin resistance, we have studied the pathway leading from the insulin receptor kinase stimulation to the translocation of the Glut 4 containing vesicles. This stimulation involves the activation of PI 3-kinase, which in turns activates protein kinase B. We have then focussed at the mechanism of vesicle exocytosis, and more specifically at the role of the small GTPase Rab4 in this process. We have shown that Rab4 participates, first in the intracellular retention of the Glut 4 containing vesicles, second in the insulin signalling pathway leading to glucose transporter translocation.     

Essential role of insulin receptor substrate-2 in insulin stimulation of Glut4 translocation and glucose uptake in brown adipocytes

Insulin and insulin-like growth factor I signals are mediated via phosphorylation of a family of insulin receptor substrate (IRS) proteins, which may serve both complementary and overlapping functions in the cell. To study the metabolic effects of these proteins in more detail, we established brown adipocyte cell lines from wild type and various IRS knockout (KO) animals and characterized insulin action in these cells in vitro. Preadipocytes derived from both wild type and IRS-2 KO mice could be fully differentiated into mature brown adipocytes. In differentiated IRS-2 KO adipocytes, insulin-induced glucose uptake was decreased by 50% compared with their wild type counterparts. This was the result of a decrease in insulin-stimulated Glut4 translocation to the plasma membrane. This decrease in insulin-induced glucose uptake could be partially reconstituted in these cells by retrovirus-mediated re-expression of IRS-2, but not overexpression of IRS-1. Insulin signaling studies revealed a total loss of IRS-2-associated phosphatidylinositol (PI) 3-kinase activity and a reduction in phosphotyrosine-associated PI 3-kinase by 30% (p < 0.05) in the KO cells. The phosphorylation and activity of Akt, a major downstream effector of PI 3-kinase, as well as Akt-dependent phosphorylation of glycogen synthase kinase-3 and p70S6 kinase were not affected by the lack of IRS-2; however, there was a decrease in insulin stimulation of Akt associated with the plasma membrane. These results provide evidence for a critical role of IRS-2 as a mediator of insulin-stimulated Glut4 translocation and glucose uptake in adipocytes. This occurs without effects in differentiation, total activation of Akt and its downstream effectors, but may be caused by alterations in compartmentalization of these downstream signals.     

Requirements for pYXXM motifs in Cbl for binding to the p85 subunit of phosphatidylinositol 3-kinase and Crk, and activation of atypical protein kinase C and glucose transport during insulin action in 3T3/L1 adipocytes

Cbl is phosphorylated by the insulin receptor and reportedly functions within the flotillin/CAP/Cbl/Crk/C3G/TC10 complex during insulin-stimulated glucose transport in 3T3/L1 adipocytes. Cbl, via pYXXM motifs at tyrosine-371 and tyrosine-731, also activates phosphatidylinositol (PI) 3-kinase, which is required to activate atypical protein kinase C (aPKC) and glucose transport during thiazolidinedione action in 3T3/L1 and human adipocytes [Miura et al. (2003) Biochemistry 42, 14335-14341]. Presently, we have examined the importance of Cbl in activating PI 3-kinase and aPKC during insulin action in 3T3/L1 adipocytes by expressing Y371F and Y731F Cbl mutants, which nullify pYXXM binding of Cbl to SH2 domains of downstream effectors. Interestingly, these mutants inhibited insulin-induced increases in (a) binding of Cbl to both Crk and the p85 subunit of PI 3-kinase, (b) activation of Cbl-dependent PI 3-kinase, (c) activation and translocation of aPKC to the plasma membrane, (d) translocation of Glut4 to the plasma membrane, (e) and glucose transport. Importantly, coexpression of wild-type Cbl reversed the inhibitory effects of Cbl mutants. In contrast to Cbl-dependent PI 3-kinase, Cbl mutants did not significantly inhibit the activation of PI 3-kinase by IRS-1, which is also required during insulin action. Our findings suggest that (a) Cbl uses pYXXM motifs to simultaneously activate PI 3-kinase and Crk/C3G/TC10 pathways and (b) Cbl, along with IRS-1, functions upstream of PI 3-kinase and aPKCs during insulin-stimulated glucose transport in 3T3/L1 adipocytes.     

Cdc42 is a Rho GTPase family member that can mediate insulin signaling to glucose transport in 3T3-L1 adipocytes

We investigated the role of cdc42, a Rho GTPase family member, in insulin-induced glucose transport in 3T3-L1 adipocytes. Microinjection of anti-cdc42 antibody or cdc42 siRNA led to decreased insulin-induced and constitutively active G(q) (CA-G(q); Q209L)-induced GLUT4 translocation. Adenovirus-mediated expression of constitutively active cdc42 (CA-cdc42; V12) stimulated 2-deoxyglucose uptake to 56% of the maximal insulin response, and this was blocked by treatment with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, wortmannin, or LY294002. Both insulin and CA-G(q) expression caused an increase in cdc42 activity, showing that cdc42 is activated by insulin and is downstream of G alpha(q/11) in this activation pathway. Immunoprecipitation experiments showed that insulin enhanced a direct association of cdc42 and p85, and both insulin treatment and CA-cdc42 expression stimulated PI3-kinase activity in immunoprecipitates with anti-cdc42 antibody. Furthermore, the effects of insulin, CA-G(q), and CA-cdc42 on GLUT4 translocation or 2-deoxyglucose uptake were inhibited by microinjection of anti-protein kinase C lambda (PKC lambda) antibody or overexpression of a kinase-deficient PKC lambda construct. In summary, activated cdc42 can mediate 1) insulin-stimulated GLUT4 translocation and 2) glucose transport in a PI3-kinase-dependent manner. 3) Insulin treatment and constitutively active G(q) expression can enhance the cdc42 activity state as well as the association of cdc42 with activated PI3-kinase. 4) PKC lambda inhibition blocks CA-cdc42, CA-G(q), and insulin-stimulated GLUT4 translocation. Taken together, these data indicate that cdc42 can mediate insulin signaling to GLUT4 translocation and lies downstream of G alpha(q/11) and upstream of PI3-kinase and PKC lambda in this stimulatory pathway.     

Domain Swapping Used To Investigate the Mechanism of Protein Kinase B Regulation by 3-Phosphoinositide-Dependent Protein Kinase 1 and Ser473 Kinase

Protein kinase B (PKB or Akt), a downstream effector of phosphoinositide 3-kinase (PI 3-kinase), has been implicated in insulin signaling and cell survival. PKB is regulated by phosphorylation on Thr308 by 3-phosphoinositide-dependent protein kinase 1 (PDK1) and on Ser473 by an unidentified kinase. We have used chimeric molecules of PKB to define different steps in the activation mechanism. A chimera which allows inducible membrane translocation by lipid second messengers that activate in vivo protein kinase C and not PKB was created. Following membrane attachment, the PKB fusion protein was rapidly activated and phosphorylated at the two key regulatory sites, Ser473 and Thr308, in the absence of further cell stimulation. This finding indicated that both PDK1 and the Ser473 kinase may be localized at the membrane of unstimulated cells, which was confirmed for PDK1 by immunofluorescence studies. Significantly, PI 3-kinase inhibitors prevent the phosphorylation of both regulatory sites of the membrane-targeted PKB chimera. Furthermore, we show that PKB activated at the membrane was rapidly dephosphorylated following inhibition of PI 3-kinase, with Ser473 being a better substrate for protein phosphatase. Overall, the results demonstrate that PKB is stringently regulated by signaling pathways that control both phosphorylation/activation and dephosphorylation/inactivation of this pivotal protein kinase.     

G alpha-q/11 protein plays a key role in insulin-induced glucose transport in 3T3-L1 adipocytes.

We evaluated the role of the G alpha-q (Galphaq) subunit of heterotrimeric G proteins in the insulin signaling pathway leading to GLUT4 translocation. We inhibited endogenous Galphaq function by single cell microinjection of anti-Galphaq/11 antibody or RGS2 protein (a GAP protein for Galphaq), followed by immunostaining to assess GLUT4 translocation in 3T3-L1 adipocytes. Galphaq/11 antibody and RGS2 inhibited insulin-induced GLUT4 translocation by 60 or 75%, respectively, indicating that activated Galphaq is important for insulin-induced glucose transport. We then assessed the effect of overexpressing wild-type Galphaq (WT-Galphaq) or a constitutively active Galphaq mutant (Q209L-Galphaq) by using an adenovirus expression vector. In the basal state, Q209L-Galphaq expression stimulated 2-deoxy-D-glucose uptake and GLUT4 translocation to 70% of the maximal insulin effect. This effect of Q209L-Galphaq was inhibited by wortmannin, suggesting that it is phosphatidylinositol 3-kinase (PI3-kinase) dependent. We further show that Q209L-Galphaq stimulates PI3-kinase activity in p110alpha and p110gamma immunoprecipitates by 3- and 8-fold, respectively, whereas insulin stimulates this activity mostly in p110alpha by 10-fold. Nevertheless, only microinjection of anti-p110alpha (and not p110gamma) antibody inhibited both insulin- and Q209L-Galphaq-induced GLUT4 translocation, suggesting that the metabolic effects induced by Q209L-Galphaq are dependent on the p110alpha subunit of PI3-kinase. In summary, (i) Galphaq appears to play a necessary role in insulin-stimulated glucose transport, (ii) Galphaq action in the insulin signaling pathway is upstream of and dependent upon PI3-kinase, and (iii) Galphaq can transmit signals from the insulin receptor to the p110alpha subunit of PI3-kinase, which leads to GLUT4 translocation.     

PKB inhibition prevents the stimulatory effect of insulin on glucose transport and protein translocation but not the antilipolytic effect in rat adipocytes

We identified 1-(5 chloronaphthalenesulfonyl)-1H-hexahydro-1, 4-diazepine, also known as ML-9, as a powerful inhibitor of PKB activity in different cells as well as of recombinant PKB. It also inhibits other downstream serine/threonine kinases, such as PKA and p90 S6 kinase, but not upstream tyrosine phosphorylation or PI3-kinase activation in response to insulin. We compared the effects of ML-9 and wortmannin on several insulin-stimulated effects in isolated rat fat cells. Both ML-9 and wortmannin inhibited glucose transport and GLUT4/IGF II receptor translocation to the plasma membrane. In contrast, only wortmannin inhibited the antilipolytic effect and PDE3B activation by insulin. Thus, ML-9 inhibits PKB but not PI3-kinase activation in response to insulin and is useful to differentiate between these effects. Both PI3-kinase and PKB are important for glucose transport and intracellular protein translocation while PKB does not appear to play an important role for the antilipolytic effect or activation of PDE3B in response to insulin.     

Overexpression of SH2-containing inositol phosphatase 2 results in negative regulation of insulin-induced metabolic actions in 3T3-L1 adipocytes via its 5'-phosphatase catalytic activity

Phosphatidylinositol (PI) 3-kinase plays an important role in various metabolic actions of insulin including glucose uptake and glycogen synthesis. Although PI 3-kinase primarily functions as a lipid kinase which preferentially phosphorylates the D-3 position of phospholipids, the effect of hydrolysis of the key PI 3-kinase product PI 3,4,5-triphosphate [PI(3,4,5)P3] on these biological responses is unknown. We recently cloned rat SH2-containing inositol phosphatase 2 (SHIP2) cDNA which possesses the 5'-phosphatase activity to hydrolyze PI(3,4,5)P3 to PI 3,4-bisphosphate [PI(3,4)P2] and which is mainly expressed in the target tissues of insulin. To study the role of SHIP2 in insulin signaling, wild-type SHIP2 (WT-SHIP2) and 5'-phosphatase-defective SHIP2 (Delta IP-SHIP2) were overexpressed in 3T3-L1 adipocytes by means of adenovirus-mediated gene transfer. Early events of insulin signaling including insulin-induced tyrosine phosphorylation of the insulin receptor beta subunit and IRS-1, IRS-1 association with the p85 subunit, and PI 3-kinase activity were not affected by expression of either WT-SHIP2 or Delta IP-SHIP2. Because WT-SHIP2 possesses the 5'-phosphatase catalytic region, its overexpression marked by decreased insulin-induced PI(3,4,5)P3 production, as expected. In contrast, the amount of PI(3,4,5)P3 was increased by the expression of Delta IP-SHIP2, indicating that Delta IP-SHIP2 functions in a dominant-negative manner in 3T3-L1 adipocytes. Both PI(3,4,5)P3 and PI(3,4)P2 were known to possibly activate downstream targets Akt and protein kinase C lambda in vitro. Importantly, expression of WT-SHIP2 inhibited insulin-induced activation of Akt and protein kinase C lambda, whereas these activations were increased by expression of Delta IP-SHIP2 in vivo. Consistent with the regulation of downstream molecules of PI 3-kinase, insulin-induced 2-deoxyglucose uptake and Glut4 translocation were decreased by expression of WT-SHIP2 and increased by expression of Delta IP-SHIP2. In addition, insulin-induced phosphorylation of GSK-3beta and activation of PP1 followed by activation of glycogen synthase and glycogen synthesis were decreased by expression of WT-SHIP2 and increased by the expression of Delta IP-SHIP2. These results indicate that SHIP2 negatively regulates metabolic signaling of insulin via the 5'-phosphatase activity and that PI(3,4,5)P3 rather than PI(3,4)P2 is important for in vivo regulation of insulin-induced activation of downstream molecules of PI 3-kinase leading to glucose uptake and glycogen synthesis.     

H-ras Induces Glucose Uptake in Brown Adipocytes in an Insulin- and Phosphatidylinositol 3-Kinase-Independent Manner

Fetal brown adipocytes (parental cells) expressed mainly Glut4 mRNA glucose transporter, the expression of Glut1 mRNA being much lower. At physiological doses, insulin stimulation for 15 min increased 3-fold glucose uptake and doubled the amount of Glut4 protein located at the plasma membrane. Moreover, phosphatidylinositol (PI) 3-kinase activity was induced by the presence of insulin in those cells, glucose uptake being precluded by PI 3-kinase inhibitors such as wortmannin or LY294002. H-ras^lys12-transformed brown adipocytes showed a 10-fold higher expression of Glut1 mRNA and protein than parental cells, Glut4 gene expression being completely down-regulated. Glucose uptake increased by 10-fold in transformed cells compared to parental cells; this uptake was unaltered in the presence of insulin and/or wortmannin. Transient transfection of parental cells with a dominant form of active Ras increased basal glucose uptake by 5-fold, no further effects being observed in the presence of insulin. However, PI 3-kinase activity (immunoprecipitated with anti-αp85 subunit of PI 3-kinase) remained unaltered in H-ras permanent and transient transfectants. Our results indicate that activated Ras induces brown adipocyte glucose transport in an insulin-independent manner, this induction not involving PI 3-kinase activation.     

Regulation of protein kinase B

Protein kinase B (PKB) is a member of the second-messenger regulated subfamily of protein kinases implicated in signalling downstream of growth factor and insulin receptor tyrosine kinases and phosphatidylinositol 3-kinase (PI 3-kinase). PKB is activated by phosphorylation in response to mitogens and survival factors. Membrane recruitment driven by lipid second-messengers derived from PI 3-kinase leads to PKB phosphorylation and activation by upstream kinases (PDK1 and an as yet identified protein kinase). Prolonged stimulation with growth factors results in nuclear translocation, providing evidence that PKB activation at the plasma membrane precedes its nuclear translocation and supporting a role for PKB in signalling from receptor tyrosine kinases to the nucleus.     

Roles of 1-phosphatidylinositol 3-kinase and ras in regulating translocation of GLUT4 in transfected rat adipose cells.

Insulin stimulates glucose transport in insulin target tissues by recruiting glucose transporters (primarily GLUT4) from an intracellular compartment to the cell surface. Previous studies have demonstrated that insulin receptor tyrosine kinase activity and subsequent phosphorylation of insulin receptor substrate 1 (IRS-1) contribute to mediating the effect of insulin on glucose transport. We have now investigated the roles of 1-phosphatidylinositol 3-kinase (PI 3-kinase) and ras, two signaling proteins located downstream from tyrosine phosphorylation. Rat adipose cells were cotransfected with expression vectors that allowed transient expression of epitope-tagged GLUT4 and the other genes of interest. Overexpression of a mutant p85 regulatory subunit of PI 3-kinase lacking the ability to bind and activate the p110 catalytic subunit exerted a dominant negative effect to inhibit insulin-stimulated translocation of epitope-tagged GLUT4 to the cell surface. In addition, treatment of control cells with wortmannin (an inhibitor of PI 3-kinase) abolished the ability of insulin to recruit epitope-tagged GLUT4 to the cell surface. Thus, our data suggest that PI 3-kinase plays an essential role in insulin-stimulated GLUT4 recruitment in insulin target tissues. In contrast, over-expression of a constitutively active mutant of ras (L61-ras) resulted in high levels of cell surface GLUT4 in the absence of insulin that were comparable to levels seen in control cells treated with a maximally stimulating dose of insulin. However, wortmannin treatment of cells overexpressing L61-ras resulted in only a small decrease in the amount of cell surface GLUT4 compared with that of the same cells in the absence of wortmannin. Therefore, while activated ras is sufficient to recruit GLUT4 to the cell surface, it does so by a different mechanism that is probably not involved in the mechanism by which insulin stimulates GLUT4 translocation in physiological target tissues.     

Calmodulin antagonists inhibit insulin-stimulated GLUT4 (glucose transporter 4) translocation by preventing the formation of phosphatidylinositol 3,4,5-trisphosphate in 3T3L1 adipocytes

It has been previously reported that calmodulin plays a regulatory role in the insulin stimulation of glucose transport. To examine the basis for this observation, we examined the effect of a panel of calmodulin antagonists that demonstrated a specific inhibition of insulin-stimulated glucose transporter 4 (GLUT4) but not insulin- or platelet-derived growth factor (PDGF)-stimulated GLUT1 translocation in 3T3L1 adipocytes. These treatments had no effect on insulin receptor autophosphorylation or tyrosine phosphorylation of insulin receptor substrate 1 (IRS1). Furthermore, IRS1 or phosphotyrosine antibody immunoprecipitation of phosphatidylinositol (PI) 3-kinase activity was not affected. Despite the marked insulin and PDGF stimulation of PI 3-kinase activity, there was a near complete inhibition of protein kinase B activation. Using a fusion protein of the Grp1 pleckstrin homology (PH) domain with the enhanced green fluorescent protein, we found that the calmodulin antagonists prevented the insulin stimulation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] formation in vivo. Similarly, although PDGF stimulation increased PI 3-kinase activity in in vitro immunoprecipitation assays, there was also no significant formation of PI(3,4,5)P3 in vivo. These data demonstrate that calmodulin antagonists prevent insulin-stimulated GLUT4 translocation by inhibiting the in vivo production of PI(3,4,5)P3 without directly affecting IRS1- or phosphotyrosine-associated PI 3-kinase activity. This phenomenon is similar to that observed for the PDGF stimulation of 3T3L1 adipocytes.     

Activation of glycogen synthase by insulin in 3T3-L1 adipocytes involves c-Cbl-associating protein (CAP)-dependent and CAP-independent signaling pathways

In adipose and muscle, insulin stimulates glucose uptake and glycogen synthase activity. Phosphatidylinositol 3-kinase (PI3K) activation is necessary but not sufficient for these metabolic actions of insulin. The insulin-stimulated translocation of phospho-c-Cbl to lipid rafts, via its association with CAP, comprises a second pathway regulating GLUT4 translocation. In 3T3-L1 adipocytes, overexpression of a dominant negative CAP mutant (CAP Delta SH3) completely blocked the insulin-stimulated glucose transport and glycogen synthesis but only partially inhibited glycogen synthase activation. In contrast, CAP Delta SH3 expression did not affect glycogen synthase activation by insulin in the absence of extracellular glucose. Moreover, CAP Delta SH3 has no effect on the PI3K-dependent activation of protein phosphatase-1 or phosphorylation of glycogen synthase kinase-3. These results indicate blockade of the c-Cbl/CAP pathway directly inhibits insulin-stimulated glucose uptake, which results in secondary inhibition of glycogen synthase activation and glycogen synthesis.     

Requirement of Atypical Protein Kinase Cλ for Insulin Stimulation of Glucose Uptake but Not for Akt Activation in 3T3-L1 Adipocytes

Phosphoinositide (PI) 3-kinase contributes to a wide variety of biological actions, including insulin stimulation of glucose transport in adipocytes. Both Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, and atypical isoforms of protein kinase C (PKCζ and PKCλ) have been implicated as downstream effectors of PI 3-kinase. Endogenous or transfected PKCλ in 3T3-L1 adipocytes or CHO cells has now been shown to be activated by insulin in a manner sensitive to inhibitors of PI 3-kinase (wortmannin and a dominant negative mutant of PI 3-kinase). Overexpression of kinase-deficient mutants of PKCλ (λKD or λΔNKD), achieved with the use of adenovirus-mediated gene transfer, resulted in inhibition of insulin activation of PKCλ, indicating that these mutants exert dominant negative effects. Insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but not growth hormone- or hyperosmolarity-induced glucose uptake, were inhibited by λKD or λΔNKD in a dose-dependent manner. The maximal inhibition of insulin-induced glucose uptake achieved by the dominant negative mutants of PKCλ was ~50 to 60%. These mutants did not inhibit insulin-induced activation of Akt. A PKCλ mutant that lacks the pseudosubstrate domain (λΔPD) exhibited markedly increased kinase activity relative to that of the wild-type enzyme, and expression of λΔPD in quiescent 3T3-L1 adipocytes resulted in the stimulation of glucose uptake and translocation of GLUT4 but not in the activation of Akt. Furthermore, overexpression of an Akt mutant in which the phosphorylation sites targeted by growth factors are replaced by alanine resulted in inhibition of insulin-induced activation of Akt but not of PKCλ. These results suggest that insulin-elicited signals that pass through PI 3-kinase subsequently diverge into at least two independent pathways, an Akt pathway and a PKCλ pathway, and that the latter pathway contributes, at least in part, to insulin stimulation of glucose uptake in 3T3-L1 adipocytes.     

Hepatocyte growth factor induces glucose uptake in 3T3-L1 adipocytes through A Gab1/phosphatidylinositol 3-kinase/Glut4 pathway

Adipose tissue is a source of hepatocyte growth factor (HGF), and circulating HGF levels have been associated with elevated body mass index in human. However, the effects of HGF on adipocyte functions have not yet been investigated. We show here that in 3T3-L1 adipocytes HGF stimulates the phosphatidylinositol (PI) 3-kinase-dependent protein kinase B (PKB) activity, AS160 phosphorylation, Glut4 translocation, and consequently, glucose uptake. The initial steps involved in HGF- and insulin-induced glucose uptake are different. HGF enhanced the tyrosine phosphorylation of Gab1, leading to the recruitment of the p85-regulated subunit of PI 3-kinase, whereas p85 was exclusively recruited by IRS1 in response to insulin. In adipocytes rendered insulin-resistant by a long-lasting tumor necrosis factor alpha treatment, the protein level of Gab1 was strongly decreased, and HGF-stimulated PKB activation and glucose uptake were also altered. Moreover, treatment of 3T3-L1 adipocytes with thiazolidinedione, an anti-diabetic drug, enhanced the expression of both HGF and its receptor. These data provide the first evidence that in vitro HGF promotes glucose uptake through a Gab1/PI 3-kinase/PKB/AS160 pathway which was altered in tumor necrosis factor alpha-treated adipocytes.     

Inhibition of insulin-induced activation of Akt by a kinase-deficient mutant of the epsilon isozyme of protein kinase C

Akt, also known as protein kinase B, is a protein-serine/threonine kinase that is activated by growth factors in a phosphoinositide (PI) 3-kinase-dependent manner. Although Akt mediates a variety of biological activities, the mechanisms by which its activity is regulated remain unclear. The potential role of the epsilon isozyme of protein kinase C (PKC) in the activation of Akt induced by insulin has now been examined. Expression of a kinase-deficient mutant of PKCepsilon (epsilonKD), but not that of wild-type PKCepsilon or of kinase-deficient mutants of PKCalpha or PKClambda, with the use of adenovirus-mediated gene transfer inhibited the phosphorylation and activation of Akt induced by insulin in Chinese hamster ovary cells or L6 myotubes. Whereas the epsilonKD mutant did not affect insulin stimulation of PI 3-kinase activity, the phosphorylation and activation of Akt induced by a constitutively active mutant of PI 3-kinase were inhibited by epsilonKD, suggesting that epsilonKD affects insulin signaling downstream of PI 3-kinase. PDK1 (3'-phosphoinositide-dependent kinase 1) is thought to participate in Akt activation. Overexpression of PDK1 with the use of an adenovirus vector induced the phosphorylation and activation of Akt; epsilonKD inhibited, whereas wild-type PKCepsilon had no effect on, these actions of PDK1. These results suggest that epsilonKD inhibits the insulin-induced phosphorylation and activation of Akt by interfering with the ability of PDK1 to phosphorylate Akt.     

Resistin inhibits glucose uptake in L6 cells independently of changes in insulin signaling and GLUT4 translocation

Elevated levels of resistin have been proposed to cause insulin resistance and therefore may serve as a link between obesity and type 2 diabetes. However, its role in skeletal muscle metabolism is unknown. In this study, we examined the effect of resistin on insulin-stimulated glucose uptake and the upstream insulin-signaling components in L6 rat skeletal muscle cells that were either incubated with recombinant resistin or stably transfected with a vector containing the myc-tagged mouse resistin gene. Transfected clones expressed intracellular resistin, which was released in the medium. Incubation with recombinant resistin resulted in a dose-dependent inhibition of insulin-stimulated 2-deoxyglucose (2-DG) uptake. The inhibitory effect of resistin on insulin-stimulated 2-DG uptake was not the result of impaired GLUT4 translocation to the plasma membrane. Furthermore, resistin did not alter the insulin receptor (IR) content and its phosphorylation, nor did it affect insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation, its association with the p85 subunit of phosphatidylinositol (PI) 3-kinase, or IRS-1-associated PI 3-kinase enzymatic activity. Insulin-stimulated phosphorylation of Akt/protein kinase B-alpha, one of the downstream targets of PI 3-kinase and p38 MAPK phosphorylation, was also not affected by resistin. Expression of resistin also inhibited insulin-stimulated 2-DG uptake when compared with cells expressing the empty vector (L6Neo) without affecting GLUT4 translocation, GLUT1 content, and IRS-1/PI 3-kinase signaling. We conclude that resistin does not alter IR signaling but does affect insulin-stimulated glucose uptake, presumably by decreasing the intrinsic activity of cell surface glucose transporters.