Activation of the hexosamine pathway leads to phosphorylation of insulin receptor substrate-1 on Ser307 and Ser612 and impairs the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin insulin biosynthetic pathway in RIN pancreatic beta-cells

Many adverse effects of glucose were attributed to its increased routing through the hexosamine pathway (HBP). There is evidence for an autocrine role of the insulin signaling in beta-cell function. We tested the hypothesis that activation of the HBP induces defects in insulin biosynthesis by affecting the insulin-mediated protein translation signaling. Exposure of human pancreatic islets and RIN beta-cells to glucosamine resulted in reduction in glucose- and insulin-stimulated insulin biosynthesis, which in RIN beta-cells was associated with impairment in insulin-stimulated insulin receptor substrate-1 (IRS-1) phosphorylation at Tyr(608) and Tyr(628), which are essential for engaging phosphatidylinositol 3-kinase (PI 3-kinase). These changes were accompanied by impaired activation of PI 3-kinase, and activation of Akt/mammalian target of rapamycin/phosphorylated heat- and acid-stable protein-1/p70S6 kinase pathway. RIN beta-cells exposed to high glucose exhibited increased c-Jun N-terminal kinase (JNK) and ERK1/2 activity, which was associated with increased IRS-1 phosphorylation at serine (Ser)(307) and Ser(612), respectively, that inhibits coupling of IRS-1 to the insulin receptor and is upstream of the inhibition of IRS-1 tyrosine phosphorylation. Azaserine reverted the stimulatory effects of high glucose on JNK and ERK1/2 activity and IRS-1 phosphorylation at Ser(307) and Ser(612). Glucosamine mimicked the stimulatory effects of high glucose on JNK and ERK1/2 activity and IRS-1 phosphorylation at Ser(307) and Ser(612). Inhibition of JNK and MAPK kinase-1 activity reverted the negative effects of glucosamine on insulin-mediated protein synthesis. These results suggest that activation of the HBP accounts, in part, for glucose-induced phosphorylation at Ser(307) and Ser(612) of IRS-1 mediated by JNK and ERK1/2, respectively. These changes result in impaired coupling of IRS-1 and PI 3-kinase, and activation of the Akt/mammalian target of rapamycin/phosphorylated heat- and acid-stable protein-1/p70S6 kinase pathway.     

Platelet-derived growth factor activates protein kinase C epsilon through redundant and independent signaling pathways involving phospholipase C gamma or phosphatidylinositol 3-kinase.

Protein kinase C (PKC), a major cellular receptor for tumor-promoting phorbol esters and diacylglycerols (DGs), appears to be involved in a variety of cellular functions, although its activation mechanism in vivo is not yet fully understood. To evaluate the signaling pathways involved in the activation of PKC epsilon upon stimulation by platelet-derived growth factor (PDGF) receptor (PDGFR), we used a series of PDGFR "add-back" mutants. Activation of a PDGFR mutant (Y40/51) that binds and activates phosphatidylinositol 3-kinase (PI 3-kinase) caused translocation of PKC epsilon from the cytosol to the membrane in response to PDGF. A PDGFR mutant (Y1021) that binds and activates phospholipase C gamma (PLC gamma), but not PI 3-kinase, also caused the PDGF-dependent translocation of PKC epsilon. The translocation of PKC epsilon upon stimulation of PDGFR (Y40/51) was inhibited by wortmannin, an inhibitor of PI 3-kinase. Activation of PKC epsilon was further confirmed in terms of PKC epsilon-dependent expression of a phorbol 12-tetradecanoate 13-acetate response element (TRE)-luciferase reporter. Further, purified PKC epsilon was activated in vitro by either DG or synthetic phosphatidylinositol 3,4,5-trisphosphate. These results clearly demonstrate that PKC epsilon is activated through redundant and independent signaling pathways which most likely involve PLC gamma or PI 3-kinase in vivo and that PKC epsilon is one of the downstream mediators of PI 3-kinase whose downstream targets remain to be identified.     

The inability of phosphatidylinositol 3-kinase activation to stimulate GLUT4 translocation indicates additional signaling pathways are required for insulin-stimulated glucose uptake.

Recent experimental evidence has focused attention to the role of two molecules, insulin receptor substrate 1 (IRS-1) and phosphatidylinositol 3-kinase (PI3-kinase), in linking the insulin receptor to glucose uptake; IRS-1 knockout mice are insulin resistant, and pharmacological inhibitors of PI3-kinase block insulin-stimulated glucose uptake. To investigate the role of PI3-kinase and IRS-1 in insulin-stimulated glucose uptake we examined whether stimulation of insulin-sensitive cells with platelet-derived growth factor (PDGF) or with interleukin 4 (IL-4) stimulates glucose uptake; the activated PDGF receptor (PDGFR) directly binds and activates PI3-kinase, whereas the IL-4 receptor (IL-4R) activates PI3-kinase via IRS-1 or the IRS-1-related molecule 4PS. We found that stimulation of 3T3-L1 adipocytes with PDGF resulted in tyrosine phosphorylation of the PDGFR and activation of PI3-kinase in these cells. To examine whether IL-4 stimulates glucose uptake, L6 myoblasts were engineered to overexpress GLUT4 as well as both chains of the IL-4R (L6/IL-4R/GLUT4); when these L6/IL-4R/GLUT4 myoblasts were stimulated with IL-4, IRS-1 became tyrosine phosphorylated and associated with PI3-kinase. Although PDGF and IL-4 can activate PI3-kinase in the respective cell lines, they do not possess insulin's ability to stimulate glucose uptake and GLUT4 translocation to the plasma membrane. These findings indicate that activation of PI3-kinase is not sufficient to stimulate GLUT4 translocation to the plasma membrane. We postulate that activation of a second signaling pathway by insulin, distinct from PI3-kinase, is necessary for the stimulation of glucose uptake in insulin-sensitive cells.     

Phosphatidylinositol 3-kinase activation is required for sulfonylurea stimulation of glucose transport in rat skeletal muscle

Sulfonylureas are drugs widely used in the treatment of patients with type 2 diabetes mellitus. In addition to their pancreatic effect of stimulating insulin secretion, many studies suggest that sulfonylureas also have extrapancreatic actions. We have previously reported that gliclazide, a second-generation sulfonylurea, stimulates the glucose uptake by rat hindquarter skeletal muscle directly and immediately by promoting the translocation of glucose transporter 4 to the plasma membrane. The aim of our study was to approach the gliclazide intracellular signaling pathway. For this purpose, we incubated clamped and isolated soleus muscle from rat with gliclazide. The following results were obtained: 1) gliclazide stimulates insulin receptor substrate (IRS)-1-phosphatidylinositol 3 (PI3)-kinase-associated activity, and this activity is necessary for gliclazide-stimulated glucose transport; 2) gliclazide treatment produces a gradual translocation of the diacylglycerol (DAG)-dependent isoforms protein kinase C (PKC) alpha, theta, and epsilon from cytosolic to membrane fraction that is dependent on PI3-kinase and phospholipase C (PLC)-gamma activation; and 3) PKC and PLC-gamma activation is necessary for gliclazide-stimulated glucose transport. We propose a hypothetical signaling pathway by which gliclazide could stimulate IRS-1 that would allow its association with PI3-kinase, promoting its activation. PI3-kinase products could induce PLC-gamma activation, whose hydrolytic activity could activate the DAG-dependent isoforms PKC alpha, theta, and epsilon.     

Signaling pathways in ischemic preconditioning

Ischemic preconditioning renders the heart resistant to infarction from ischemia/reperfusion. Over the past two decades a great deal has been learned about preconditioning’s mechanism. Adenosine, bradykinin, and opioids act in parallel to trigger the preconditioned state and do so by activating PKC. While adenosine couples directly to PKC through the phospholipases, bradykinin and opioids do so through a complex pathway that includes in order: phosphatidylinositol 3-kinase (PI3-kinase), Akt, nitric oxide synthase, guanylyl cyclase, PKG, opening of mitochondrial K_ATP channels, and activation of PKC by redox signaling. There are even differences between the opioid and bradykinin coupling as the former activates PI3-kinase through transactivation of the epidermal growth factor receptor while the latter has an unknown coupling mechanism. Protection stems from inhibition of formation of mitochondrial permeability transition pores early in reperfusion through activation of the survival kinases, Akt and ERK. These kinases are activated as a result of PKC somehow promoting signaling from adenosine A₂ receptors early in reperfusion. The survival kinases are thought to inhibit pore formation by phosphorylating GSK-3β. The reperfused heart requires the support of the protective signals for only about an hour after which the ischemic injury is repaired and the signals are no longer needed.     

Mediation of beta-endorphin in exercise-induced improvement in insulin resistance in obese Zucker rats

BACKGROUND: Aerobic exercise including treadmill running has long been used to successfully treat and/or prevent insulin resistance and type-2 diabetes. Increase of plasma beta-endorphin is observed with exercise. The present study was designed to clarify the role of endogenous beta-endorphin in exercise-induced improvement in insulin resistance. METHODS: We used a moderate exercise program consisting of treadmill running at 20 m/min and 0% grade for 1 h/day, 7 days/week, for 8 weeks. Plasma glucose concentration was assessed by the glucose oxidase method. The enzyme-linked immunosorbent assay was performed to quantify the plasma level of beta-endorphin-like immunoreactivity (BER). The glucose disposal rate (GDR) was measured by the hyperinsulinemic euglycemic clamp technique. Changes of the insulin signaling in isolated soleus muscle were then detected by immunoprecipitation and immunoblotting. RESULTS: An increase of plasma BER in parallel with the reduction of plasma glucose was obtained in exercise-trained obese Zucker rats. Different from a marked reduction in sedentary obese rats, the value of insulin-stimulated GDR obtained from the exercised obese rats was reversed to near that of the sedentary lean group, eight weeks after the last period of exercise. This effect of exercise was inhibited by naloxone or naloxonazine at doses sufficient to block opioid micro-receptors. Signaling-related defects in the soleus muscle of sedentary obese Zucker rats, which impaired glucose transporter subtype 4 (GLUT 4), included decreased phosphorylation of insulin receptor substrate (IRS)-1, as well as an attenuated p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3 kinase) and Akt serine phosphorylation. In contrast, exercise training failed to modify the levels of insulin receptor (IR), IRS-1, and IR tyrosine autophosphorylation in obese Zucker rats. CONCLUSION: Enhanced insulin sensitivity via exercise training might be mediated by endogenous beta-endorphin through an increase of postreceptor insulin signaling related to the IRS-1-associated PI3-kinase step that leads to the enhancement of GLUT 4 translocation and improved glucose disposal in obese Zucker rats.     

Glucosamine enhances platelet-derived growth factor-induced DNA synthesis via phosphatidylinositol 3-kinase pathway in rat aortic smooth muscle cells

Vascular smooth muscle cells play a key role in the development of atherosclerosis. Culture of vascular smooth muscle A10 cells with high glucose for 4 weeks enhanced platelet-derived growth factor (PDGF)-induced BrdU incorporation. Since a long period of high glucose incubation was required for the effect, and it was inhibited by co-incubation with azaserine, the role of hexosamine biosynthesis in the development of atherosclerosis in diabetes was studied in A10 cells. Addition of glucosamine to the culture media enhanced PDGF-stimulated BrdU incorporation, and PDGF-induced tyrosine phosphorylation of the PDGF beta-receptor was increased by glucosamine treatment. Of the subsequent intracellular signaling pathways, PDGF-induced PDGF beta-receptor association with PLC gamma was not affected, whereas tyrosine phosphorylation of Shc, subsequent association of Shc with Grb2, and MAP kinase activation were relatively decreased. In contrast, PDGF-induced PDGF beta-receptor association with the p85 regulatory subunit of PI3-kinase and PI3-kinase activation were increased by 20% (P<0.01) and 36% (P<0.01), respectively. The intracellular signaling molecules responsible for the glucosamine effect were further examined using pharmacological inhibitors. Pretreatment with PLC inhibitor (U73122) had negligible effects, and MEK1 inhibitor (PD98059) showed only a slight inhibitory effect on the PDGF-induced BrdU incorporation. In contrast, pretreatment with PI3-kinase inhibitor (LY294002) significantly inhibited glucosamine enhancement of PDGF-induced BrdU incorporation. These findings suggest that glucosamine is involved in the development of atherosclerosis by enhancing PDGF-induced mitogenesis specifically via the PI3-kinase pathway.     

Protein kinase Cdelta mediates cyclic adenosine monophosphate-stimulated translocation of sodium taurocholate cotransporting polypeptide and multidrug resistant associated protein 2 in rat hepatocytes

Cyclic adenosine monophosphate (cAMP) stimulates translocation of Na(+)-taurocholate (TC) cotransporting polypeptide (Ntcp) and multidrug resistant associated protein 2 (Mrp2) to the plasma membrane. Because cAMP activates phosphoinositide-3-kinase (PI3K) and protein kinase C (PKC) activation is PI3K-dependent, the aim of the current study was to determine whether cAMP activates conventional and novel PKCs in hepatocytes and whether such activation plays a role in cAMP-stimulated Ntcp and Mrp2 translocation. The effect of cAMP on PKCs, TC uptake, and Ntcp and Mrp2 translocation was studied in isolated rat hepatocytes using a cell-permeable cAMP analog, CPT-cAMP. The activity of PKCs was assessed from membrane translocation of individual PKCs, and phospho-specific antibodies were used to determine PKCdelta phosphorylation. TC uptake was determined from time-dependent uptake of (14)C-TC, and a cell surface biotinylation method was used to determine Ntcp and Mrp2 translocation. CPT-cAMP stimulated nPKCdelta but not cPKCalpha or nPKCepsilon, and induced PI3K-dependent phosphorylation of nPKCdelta at Thr(505). Rottlerin, an inhibitor of nPKCdelta, inhibited cAMP-induced nPKCdelta translocation, TC uptake, and Ntcp and Mrp2 translocation. Bistratene A, an activator of nPKCdelta, stimulated nPKCdelta translocation, TC uptake, and Ntcp and Mrp2 translocation. The effects of cAMP and bistratene A on TC uptake and Ntcp and Mrp2 translocation were not additive. Conclusion: These results suggest that cAMP stimulates Ntcp and Mrp2 translocation, at least in part, by activating nPKCdelta via PI3K-dependent phosphorylation at Thr(505).     

Platelet-derived growth factor triggers translocation of the insulin-regulatable glucose transporter (type 4) predominantly through phosphatidylinositol 3-kinase binding sites on the receptor.

Insulin is the only known hormone which rapidly stimulates glucose uptake in target tissues, mainly by translocation to the cell surface of the intracellular insulin-regulatable glucose transporter (glucose transporter type 4, GLUT4). We have developed a cell line for direct, sensitive detection of GLUT4 on the cell surface. We have suggested that insulin-activated phosphatidylinositol (PI) 3-kinase may be involved in the signaling pathway of insulin-stimulated GLUT4 translocation. We report that platelet-derived growth factor (PDGF), which stimulates PI 3-kinase activity, triggers GLUT4 translocation in Chinese hamster ovary (CHO) cells stably overexpressing the PDGF receptor and in 3T3-L1 mouse adipocytes. Using mutant PDGF receptors that cannot bind to Ras-GTPase-activating protein, phospholipase C-gamma, and PI 3-kinase, respectively, we obtained evidence that PI 3-kinase binding sites play a key role in the signaling pathway of PDGF-stimulated GLUT4 translocation in the CHO cell system.     

Involvement of protein kinase C and phosphatidylinositol 3-kinase pathways in the survival of B-cell chronic lymphocytic leukemia cells

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived CD5(+) B lymphocytes. TPA (12-O-tetradecanoylphorbol 13- acetate) and interleukin-4 (IL-4) inhibit apoptosis of B-CLL lymphocytes ex vivo. We used specific inhibitors of protein kinase C (PKC), extracellular-regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3-kinase) to study their involvement in TPA- and IL-4-induced survival of B-CLL lymphocytes. BisI, a specific inhibitor of PKC, induced apoptosis and inhibited the antiapoptotic activity of TPA and IL-4. B-CLL cells have a basal PKC activity that was increased by TPA but not by IL-4. TPA, but not IL-4, induced ERK activation. However, the inhibition of ERK activation did not affect the viability of B-CLL lymphocytes, demonstrating that this pathway is not involved in their survival. Inhibition of PI3-kinase by LY294002 induced apoptosis of B-CLL cells and inhibited the survival effect of IL-4 and TPA. In addition, Akt, a downstream effector of PI3-kinase activity, was phosphorylated by TPA and IL-4 in B-CLL cells, though PI3-kinase had no effect on PKC-dependent phosphorylation of Akt. Furthermore, the inhibition of PKC or PI3-kinase increased dexamethasone- and fludarabine-induced apoptosis ex vivo in the presence of survival factors. These results demonstrate that PKC and PI3-kinase are involved in the survival of B-CLL cells and suggest that inhibitors of these pathways could be combined with the drugs used in the treatment of B-CLL.     

Phosphatidylinositol 3-kinase-dependent translocation of phospholipase Cgamma2 in mouse megakaryocytes is independent of Bruton tyrosine kinase translocation

Activation of the collagen receptor glycoprotein VI (GPVI) by a collagen-related peptide (CRP) induces stimulation of platelets and megakaryocytes through the phosphatidylinositol (PI) 3-kinase-dependent pathway leading to activation of Bruton tyrosine kinase (Btk) and phospholipase Cgamma2 (PLCgamma2). Here, we present evidence that both proteins undergo PI 3-kinase-dependent translocation to the plasma membrane on CRP stimulation that is markedly inhibited by wortmannin and LY294002. Translocation of PLCgamma2 but not Btk is also seen in megakaryocytes from X-linked immunodeficiency mice, which have a mutation that reduces the affinity of the pleckstrin homology (PH) domain of Btk for PI 3,4,5-trisphosphate (PI 3,4,5-P3). Activation of PC12 cells by epidermal growth factor (EGF) results in increased PI 3-kinase activity and high PI 3,4,5-P3 levels that trigger translocation of the green fluorescent protein (GFP)-labeled PH of Btk, but not the GFP-labeled PH and tandem Src homology 2 (SH2) domains of PLCgamma2. In contrast to the results with CRP, the G protein-coupled receptor agonist thrombin stimulates PI 3-kinase-independent translocation of Btk but not PLCgamma2. In conclusion, these results demonstrate that in mouse megakaryocytes, CRP leads to PI 3-kinase-dependent translocation of PLCgamma2 and Btk that are independent of one another, whereas thrombin only induces translocation of Btk through a pathway that is independent of PI 3-kinase activity.     

FLT3/ ITD regulates leukaemia cell adhesion through α4β1 integrin and Pyk2 signalling

Internal tandem duplication of FMS-like receptor tyrosine kinase 3 (FLT3/ITD) within its juxtamembrane domain is a frequent mutation in adult acute myeloid leukaemia (AML). This mutation causes constitutive activation of FLT3 and is associated with poor prognosis. The high relapse rate of FLT3/ITD-positive AML might be partly because of insufficient eradication of slow-cycling leukaemic stem cells in the bone marrow microenvironment. β1 integrin mediates haematopoietic stem and progenitor cell homing along with their retention in the bone marrow and also inhibits haematopoietic proliferation and differentiation. Here, we demonstrate that inhibition of FLT3/ITD kinase activity by a FLT3 selective inhibitor named FI-700 decreases affinity of α4β1 integrin to soluble VCAM-1. α4β1 integrin deactivation by FI-700 is independent of Rap1, which is the critical regulator of integrin inside-out signalling. In addition, selective inhibition of FLT3/ITD induces Pyk2 dephosphorylation together with the inhibition of phosphatidylinositol-3-kinase (PI3K)/Akt pathway. Both wild-type and ITD-FLT3 proteins co-immunoprecipitated with β1 integrin and Pyk2 indicating the signal crosstalk between FLT3, β1 integrin and Pyk2. These results collectively indicated that the inhibition of FLT3 kinase might contribute not only to the induction of apoptosis, but also to the leukaemia cell detachment from the bone marrow microenvironment in the treatment of AML.     

Overexpression of glutamine:fructose-6-phosphate amidotransferase in rat-1 fibroblasts enhances glucose-mediated glycogen accumulation via suppression of glycogen phosphorylase activity

The hexosamine biosynthesis pathway (HBP) mediates many of the adverse effects of excess glucose. We have shown previously that glucose down-regulates basal and insulin-stimulated glycogen synthase (GS) activity. Overexpression of the rate-limiting enzyme in the HBP, glutamine:fructose-6-phosphate amidotransferase (GFA), mimics these effects of high glucose and renders the cells more sensitive to glucose. Here we examine the role of the HBP in regulating cellular glycogen content. Glycogen content and glycogen phosphorylase (GP) activity were determined in Rat-1 fibroblasts that overexpress GFA. In both GFA and controls there was a dose-dependent increase in glycogen content (approximately 8-fold) in cells cultured in increasing glucose concentrations (1-20 mM). There was a shift to the left in the glucose dose-response curve for glycogen content in GFA cells (ED50 for glycogen content = 5.80+/-1.05 vs. 8.84+/-0.87 mM glucose, GFA vs. control). Inhibition of GFA reduced glycogen content by 28.4% in controls cultured in 20 mM glucose. In a dose-dependent manner, glucose resulted in a more than 35% decrease in GP activity in controls. GP activity in GFA cells was suppressed compared with that in controls, and there was no glucose-induced down-regulation of GP activity. Glucosamine and uridine mimicked the effects of glucose on glycogen content and GP activity. However, chronic overexpression of GFA is a unique model of hexosamine excess, as culturing control cells in low dose glucosamine (0.1-0.25 mM) did not suppress GP activity and did not eliminate the glucose-mediated down-regulation of GP activity. We conclude that increased flux through the HBP results in enhanced glycogen accumulation due to suppression of GP activity. These results demonstrate that the HBP is an important regulator of cellular glucose metabolism and supports its role as a cellular glucose/satiety sensor.     

High glucose increases phosphocofilin via phosphorylation of LIM kinase due to Rho/Rho kinase activation in cultured pig proximal tubular epithelial cells

In proximal tubular epithelial cells (PTECs), depolymerization of actin by cofilin plays a crucial role in maintaining polarity and function. Cofilin is inactivated when phosphorylated by p-Lin-11/Isl-1/Mec-3 kinase (LIMK) to give p-cofilin. LIMK is phosphorylated by phosphorylated p21-activated kinase (PAK), a downstream signal of phosphoinositide 3-kinase (PI3K), or by Rho kinase (ROCK), and is dephosphorylated by slingshot (SSH). However, in PTECs the signaling pathways regulating phosphorylation and dephosphorylation of cofilin, and the influence of high glucose (HG) on these pathways remain to be elucidated. Here, we show that HG in cultured porcine PTECs (LLC-PK1) increases p-cofilin and p-LIMK1 beyond 6h and that the simultaneous presence of phlorizin reverses the increase. HG did not influence the levels of PI3K-p85, downstream signals to SSH1 and p-PAK1, and mRNA of cofilin, LIMK1 and SSH1. On the other hand, wortmannin and LY294002 markedly increased p-cofilin and p-LIMK1 without influencing on the level of SSH1 protein. HG-activated RhoA and ROCK2 beyond 3h, and phlorizin attenuated this activation. GF109203X inhibited HG-induced increase in membranous RhoA and ROCK2, and phorbol ester increased these proteins. Y27632 (a ROCK inhibitor) reversed HG-induced increases of p-cofilin and p-LIMK1. We conclude that HG increases p-cofilin by phosphorylating LIMK1 through activation of Rho/Rho kinase, probably due to diacylglycerol-sensitive PKC activation resulting from increased glucose influx. HG did not alter PI3K or its downstream signals, even though PI3K has a physiological role in maintaining the cofilin level by activating SSH1.     

Leptin stimulates glucose transport and glycogen synthesis in C2C12 myotubes: evidence for a PI3-kinase mediated effect

Summary It was recently shown that leptin impairs insulin signalling, i. e. insulin receptor autophosphorylation and insulin-receptor substrate (IRS)-1 phosphorylation in rat-1 fibroblasts, NIH3T3 cells and HepG2 cells. To evaluate whether leptin might impair the effects of insulin in muscle tissue we studied the interaction of insulin and leptin in a muscle cell system, i. e. C₂C₁₂ myotubes. Preincubation of C₂C₁₂ cells with leptin (1–500 ng/ml) did not significantly affect insulin stimulated glucose transport and glycogen synthesis (1.8 to 2 fold stimulation); however, leptin by itself (1 ng/ml) was able to mimic approximately 80–90 % of the insulin effect on glucose transport and glycogen synthesis. Both glucose transport as well as glycogen synthesis were inhibited by the phosphatidylinositol-3 (PI3)-kinase inhibitor wortmannin and the protein kinase C inhibitor H7 while no effect was observed with the S6-kinase inhibitor rapamycin. We determined whether the effect of leptin occurs through activation of IRS-1 and PI3-kinase. Leptin did not stimulate PI3-kinase activity in IRS-1 immunoprecipitates; however, PI3-kinase activation could be demonstrated in p85α immunoprecipitates (3.04 ± 1.5 fold of basal). In summary the data provide the first evidence for a positive crosstalk between the signalling chain of the insulin receptor and the leptin receptor. Leptin mimics in C₂C₁₂ myotubes insulin effects on glucose transport and glycogen synthesis most likely through activation of PI3-kinase. This effect of leptin occurs independently of IRS-1 activation in C₂C₁₂ cells. [Diabetologia (1997) 40: 606–609] Received: 24 January 1997 and in revised form: 3 March 1997