Involvement of TNF-alpha in abnormal adipocyte and muscle sortilin expression in obese mice and humans.

AIMS/HYPOTHESIS: Insulin resistance is caused by numerous factors including inflammation. It is characterised by defective insulin stimulation of adipocyte and muscle glucose transport, which requires the glucose transporter GLUT4 translocation towards the plasma membrane. Defects in insulin signalling can cause insulin resistance, but alterations in GLUT4 trafficking could also play a role. Our goal was to determine whether proteins controlling GLUT4 trafficking are altered in insulin resistance linked to obesity. METHODS: Using real-time RT-PCR, we searched for selected transcripts that were differentially expressed in adipose tissue and muscle in obese mice and humans. Using various adipocyte culture models and in vivo mice treatment, we searched for the involvement of TNF-alpha in these alterations in obesity. RESULTS: Sortilin mRNA and protein were downregulated in adipose tissue from obese db/db and ob/ob mice, and also in muscle. Importantly, sortilin mRNA was also decreased in morbidly obese human diabetic patients. Sortilin and TNF-alpha (also known as TNF) mRNA levels were inversely correlated in mice and human adipose tissues. TNF-alpha decreased sortilin mRNA and protein levels in cultured mouse and human adipocytes, an effect partly prevented by the peroxisome proliferator-activated receptor gamma activator rosiglitazone. TNF-alpha also inhibited adipocyte and muscle sortilin mRNA when injected to mice. CONCLUSIONS/INTERPRETATION: Sortilin, an essential player in adipocyte and muscle glucose metabolism through the control of GLUT4 localisation, is downregulated in obesity and TNF-alpha is likely to be involved in this defect. Chronic low-grade inflammation in obesity could thus contribute to insulin resistance by modulating proteins that control GLUT4 trafficking.

p38MAP Kinase activity is required for human primary adipocyte differentiation.

Little is known about the role of p38MAPK in human adipocyte differentiation. Here we showed that p38MAPK activity increases during human preadipocytes differentiation. Pharmacological inhibition of p38MAPK during adipocyte differentiation of primary human preadipocytes markedly reduced triglycerides accumulation and adipocyte markers expression. Cell cycle arrest or proliferation was not affected by p38MAPK inhibition. Although induction of C/EBPbeta was not altered by the p38MAPK inhibitor, its phosphorylation on Threonine(188) was decreased as well as PPARgamma expression. These results indicate that p38MAPK plays a positive role in human adipogenesis through regulation of C/EBPbeta and PPARgamma factors.

Potential role of Rab4 in the regulation of subcellular localization of Glut4 in adipocytes.

A role for Rab4 in the translocation of the glucose transporter Glut4 induced by insulin has been recently proposed. To study more directly the role of this small GTPase, freshly isolated adipocytes were transiently transfected with the cDNAs of both an epitope-tagged Glut4-myc and Rab4, a system which allows direct measurement of the concentration of Glut4 molecules at the cell surface. When cells were cotransfected with Glut4-myc and Rab4, the concentration of Glut4-myc at the cell surface decreased in parallel with the increased expression of Rab4, suggesting that Rab4 participates in the intracellular retention of Glut4. In parallel, the amount of Rab4 associated with the Glut4-containing vesicles increased. When Rab4 was moderately overexpressed, the number of Glut4-myc molecules recruited to the cell surface in response to insulin was similar to that observed in mock-transfected cells, and thus the insulin efficiency was increased. When Rab4 was expressed at a higher level, the amount of Glut4-myc present at the cell surface in response to insulin decreased. Since the overexpressed protein was predominantly cytosolic, this suggests that the cytosolic Rab4 might complex some factor(s) necessary for insulin action. This hypothesis was strengthened by the fact that Rab4 deltaCT, a Rab4 mutant lacking the geranylgeranylation sites, inhibited insulin-induced recruitement of Glut4-myc to the cell surface, even when moderately overexpressed. Rab3D was without effect on Glut4-myc subcellular distribution in basal or insulin-stimulated conditions. While two mutated proteins unable to bind GTP did not decrease the number of Glut4-myc molecules in basal or insulin-stimulated conditions at the plasma membrane, the behavior of a mutated Rab4 protein without GTPase activity was similar to that of the wild-type Rab4 protein, indicating that GTP binding but not its hydrolysis was required for the observed effects. Altogether, our results suggest that Rab4, but not Rab3D, participates in the molecular mechanism involved in the subcellular distribution of the Glut4 molecules both in basal and in insulin-stimulated conditions in adipocytes.

Identification of G protein alpha-subunits in RINm5F cells and their selective interaction with galanin receptor.

Galanin, an inhibitor of insulin secretion in pancreatic beta-cells, exerts its multiple effects through mechanisms that are sensitive to pertussis toxin (PTX). G proteins have been characterized in RINm5F cells. By ADP ribosylation and immunoblotting, the alpha-subunits of Gi1, Gi2, Gi3, and two forms of Go were identified, Gi alpha 2 being predominant. As expected from a G protein-linked receptor, GTP and its nonhydrolyzable analogue GTP-gamma-S decreased tracer galanin binding to cell membranes. This resulted from a change in receptor affinity without any modification in the number of sites. Selective antibodies against the COOH-terminal decapeptide of the alpha-subunits of the Gi and Go proteins were used to block G protein interaction before we studied galanin binding. Antibody AS, which selectively recognizes Gi alpha 1 and Gi alpha 2, decreased tracer galanin binding to membranes at concentrations where there were no effects of other antibodies specifically directed against Gi alpha 3 or G alpha o. These data suggest that Gi1 and/or Gi2 interact with the galanin receptor and probably mediate the effects of galanin in pancreatic beta-cells.

Wortmannin inhibits the action of insulin but not that of okadaic acid in skeletal muscle: comparison with fat cells.

To look for the possible involvement of phosphatidylinositol-3-kinase (PI3-kinase) in insulin action in muscle, we have used wortmannin, described as a specific inhibitor of the enzyme, and compared its effect in muscle and in adipose cells. Both in intact mouse soleus muscle and in isolated rat adipocytes, wortmannin blocked insulin effect on glucose uptake, without markedly altering basal glucose uptake. In adipocyte, this effect results from a blockade of the translocation process because wortmannin inhibited the stimulatory action of insulin on both the Glut 4 movement from the internal compartment to the plasma membranes and the Rab4 departure from the microsomes. In a similar fashion, two other insulin effects, the activation of glycogen synthase and the stimulation of amino acid uptake, were blocked by wortmannin in skeletal muscle. Lipogenesis from acetate was also inhibited by wortmannin in adipocytes. By contrast, wortmannin did not affect muscle deoxglucose uptake when it was stimulated either by okadaic acid or by the protein kinase C activator tumor promoting agent. These results suggest that, in muscle and adipocyte, PI3-kinase inhibition causes a blockade of all insulin effects studied. By contrast, wortmannin did not affect the same responses elicited in muscle by okadaic acid or tumor promoting agent.

Insulin induces a change in Rab5 subcellular localization in adipocytes independently of phosphatidylinositol 3-kinase activation.

We investigated whether Rab5, a small guanosine triphosphatase that regulates early endocytic transport in different cell types is involved in the insulin-regulated endocytic pathways in adipocytes. Rab5 was detected in freshly isolated adipocytes and 3T3-L1 adipocytes, but its expression level was not markedly increased with adipocyte differentiation. After subcellular fractionation of adipocytes incubated in the absence of insulin, Rab5 was found to be abundant in plasma membrane and cytosol, but was also present in high and low density microsomes. This subcellular distribution was compatible with a role in early endocytosis. When cells were incubated with insulin, the concentration of Rab5 decreased by about 50% in the internal compartments. In contrast to Rab4, which also leaves the low density microsomes in response to insulin, Rab5 was not found in Glut4-containing vesicles purified by immunoadsorption on antibodies to Glut4. When adipocytes were treated with wortmannin, an inhibitor of phosphatidylinositol 3-kinase, the effect of insulin on Rab5 movement was not affected, whereas the insulin-induced movements of Rab4 and Glut4 were abolished. In parallel, wortmannin inhibited the increase in horseradish peroxidase uptake induced by insulin, an index of fluid phase endocytosis, but did not prevent the endocytosis of the glucose transporters. As a whole, our results suggest that Rab5 is not involved in insulin-stimulated Glut4 exocytosis. These results are compatible with the postulated role of Rab5 in the endocytotic pathway, at a step that does not require phosphatidyl-inositol 3-kinase activation.

Potential involvement of the carboxy-terminus of the Glut 1 transporter in glucose transport.

The role of the carboxy-terminal domain of the Glut 1 glucose transporter was investigated using an antipeptide antibody to the C-terminal part of the molecule. The study was performed in fibroblasts transfected with the cDNA coding for the human insulin receptor. These cells acutely respond to insulin for glucose transport. Using antipeptide antibodies to Glut 1 and Glut 4, we first established that these cells expressed only Glut 1. Then, to define the role of the C-terminal part of Glut 1 in glucose transport, the antibodies were loaded into the cells by electroporation. When anti-Glut 1 immunoglobulins were introduced into the cells, a 60% increase in basal deoxyglucose and 3-O-methylglucose transport was observed compared to that in cells electroporated with nonimmune immunoglobulins. The stimulatory action of the antipeptide was not due to an increase in the total amount of transporters. It was found only at low glucose concentrations, suggesting that the affinity of the transporter, rather than its maximal capacity, was changed. Finally, the effect of antibody was additive to that of insulin. The interaction between the anti-Glut 1 antibody and the carboxy-tail of the transporter seems to lead to an increase in the intrinsic activity of the transporter, suggesting that this part of the molecule could be implicated in the regulation of glucose uptake.

Subcellular distribution of low molecular weight guanosine triphosphate-binding proteins in adipocytes: colocalization with the glucose transporter Glut 4.

Insulin stimulation of glucose transport involves the translocation of vesicles containing the glucose transporter Glut 4 to the plasma membrane. Since low mol wt GTP-binding proteins (LMW-GTP-binding proteins) have been implicated in the regulation of vesicular trafficking, we have analyzed these proteins in adipocytes. Isolated adipocytes were incubated in the absence or presence of insulin before separation of plasma membranes (PM) and low density microsomes (LDM). [alpha-32P]GTP binding to proteins transferred to nitrocellulose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed specific and distinct subsets of proteins in the PM and LDM; those proteins were more abundant in PM than in LDM. [alpha-32P]GTP binding to these proteins was specific for the guanylnucleotides, since it was competed for by GTP and guanosine 5'-O-(3-thiotriphosphate), but not by ATP or adenosine 5'-O-(3-thiotriphosphate). The LMW-GTP-binding proteins were tightly associated with the membranes, as treatment with 1.5 M KCl did not modify this association. The distribution of the LMW-GTP-binding proteins in the fractions and their affinity for guanylnucleotides were the same in control and insulin-treated adipocytes. When the presence of Gi alpha subunits was looked for with a specific antibody, Gi alpha 1 and Gi alpha 2 were found almost exclusively in PM. By contrast, the same antibody revealed the presence of a 100 kDa band in the LDM. Insulin treatment of adipocytes did not modify the amounts of those G-proteins in PM or LDM fractions, although it promoted the translocation of Glut 4 proteins from LDM to PM. LDM fractions contain a specific subset of vesicles markedly enriched in Glut 4 molecules. When those vesicles were isolated from the total LDM fraction by immunoadsorption on highly specific antibodies to Glut 4 protein, LMW-GTP-binding proteins were found in the immune pellet. Those proteins were absent when immunoprecipitation was performed after solubilization of the vesicles with 1% Triton X-100. Our results strongly suggest that the vesicles containing the Glut 4 protein also contained LMW-GTP-binding proteins and indicate that these GTP-binding proteins could play a role in the exocytosis of the Glut 4-containing vesicles.

Differential effects of insulin and exercise on Rab4 distribution in rat skeletal muscle.

Insulin and exercise cause the translocation of GLUT4 from an intracellular location to the plasma membrane in skeletal muscle. The purpose of this study was to determine if Rab4, a small GTP binding protein that has been implicated in the insulin-stimulated translocation of GLUT4 in adipose cells, is involved in the regulation of transporter translocation in skeletal muscle. Male rats were injected with insulin (20 U i.p.) or exercised on a treadmill(1 h, 20 m/min, 10% grade). Rats were killed 30 min after insulin injection or immediately after exercise, and the hind limb muscles dissected. Plasma membrane and intracellular microsomal membrane fractions were prepared, and the distribution of GLUT4 and Rab4 was determined by immunoblotting. Both insulin and exercise caused GLUT4 translocation as demonstrated by a decrease in microsomal membrane GLUT4 and an increase in plasma membrane GLUT4. In contrast, only insulin caused a decrease in Rab4 in the microsomal membrane. Rab4 was associated with GLUT4-containing vesicles isolated by immunoprecipitation. Rab4 was not detected in plasma membrane under any condition. These data demonstrate that insulin modulates the subcellular distribution of both GLUT4 and Rab4 in rats skeletal muscle, suggesting that Rab4 may play a role in the insulin-stimulated movement of GLUT4-containing vesicles. Although both insulin and exercise increase skeletal muscle glucose uptake by the translocation of GLUT4, the regulation of translocation may occur by different mechanisms.

The small guanosine triphosphate-binding protein Rab4 is involved in insulin-induced GLUT4 translocation and actin filament rearrangement in 3T3-L1 cells.

Insulin's stimulation of glucose transport involves the translocation of vesicles containing the glucose transporter GLUT4 to the plasma membrane. Small GTP-binding proteins have been implicated in the regulation of vesicular traffic. We studied the effects of microinjection of wild-type Rab4 glutathione S-transferase fusion protein (WT Rab4), a GTP-binding defective mutant (Rab4 N121I), a guanosine triphosphatase-defective mutant (Rab4 Q67L), and a Rab4 antibody on insulin-induced GLUT4 translocation in 3T3-L1 adipocytes. Microinjection of Rab4 N121I and Rab4 antibodies had no effect on basal GLUT4 staining, but inhibited insulin-induced GLUT4 translocation by 50% compared with that in control IgG-injected cells. WT Rab4 and Rab4 Q67L microinjection had no effect on either basal or insulin-induced GLUT4 translocation. Premixing and coinjection of the Rab4 antibody with WT Rab4 almost completely abolished its inhibitory effect on insulin-induced GLUT4 translocation. In contrast, microinjection of an antibody directed against the highly conserved region of Rab3 proteins had no effect on insulin-induced GLUT4. These results point to a direct role of Rab4 in insulin-induced GLUT4 translocation, and that this effect is dependent on nucleotide binding to the protein. We also studied the effect of microinjection of the same proteins on insulin-induced actin filament rearrangement (membrane ruffling) in the same cell line. Microinjection of Rab4 N121I and Rab4 antibodies inhibited insulin-induced membrane ruffling by 40%, whereas WT Rab4 or a Rab3 antibody injection had no effect on cytoskeletal rearrangement. In summary, 1) Rab4 is a necessary component of the insulin/GLUT4 translocation signaling pathway; 2) the function of Rab4 in this pathway requires GTP binding; 3) Rab4 also participates in the process of insulin-induced membrane ruffling; and 4) Rab3 proteins do not seem to be involved in these processes.

Parallel changes in Glut 4 and Rab4 movements in two insulin-resistant states.

Insulin-induced Glut 4 and Rab4 movements were studied in two insulin-resistant states. In adipocytes from streptozotocin diabetic rats, the amount of Glut 4 was decreased by 60%. The remaining Glut 4 molecules were translocated in response to insulin, and in parallel, Rab4 left the intracellular compartment. In contrast, in 3T3-L1 adipocytes rendered insulin-resistant by a prolonged insulin treatment, both Rab4 and Glut 4 remained in the intracellular compartment following an acute insulin stimulation. Those results illustrate a similar behavior of Glut 4 and Rab4 in two situations where insulin resistance results from different mechanisms, and add further support for a role of Rab4 in Glut 4 translocation.

Expression of guanine-nucleotide-binding proteins in lean and obese insulin-resistant mice.

To examine whether G protein were affected in the obese insulin-resistant state, the level of various G proteins (alpha i1, alpha i2, alpha i3, alpha o and alpha s) was assessed by immunodetection in lean and experimentally induced obese mice. Crude membranes were prepared from adipose tissues, muscle, liver, kidney and brain. G alpha-subunits were similar in lean and obese animals in brain, kidney, skeletal or heart muscle. Hepatic G alpha s, G alpha i2 and G alpha i3 subunits were markedly elevated in obese mice. When total tissue contents were considered, interscapular brown adipose tissue and epididymal fat pads from obese animals contained more alpha i2 than the lean tissues, while alpha i1, alpha i3 and alpha s were similar in both groups. However, when expressed per mg of membrane protein, alpha i1, alpha i3 and alpha s were decreased and alpha i2 was normal in white adipose tissue of obese animals. Thus the expression of the G protein alpha-subunits seems to be regulated by tissue-specific factors rather than by circulating factors.

Essential role of microphthalmia transcription factor for DNA replication, mitosis and genomic stability in melanoma.

Malignant melanoma is an aggressive cancer known for its notorious resistance to most current therapies. The basic helix-loop-helix microphthalmia transcription factor (MITF) is the master regulator determining the identity and properties of the melanocyte lineage, and is regarded as a lineage-specific 'oncogene' that has a critical role in the pathogenesis of melanoma. MITF promotes melanoma cell proliferation, whereas sustained supression of MITF expression leads to senescence. By combining chromatin immunoprecipitation coupled to high throughput sequencing (ChIP-seq) and RNA sequencing analyses, we show that MITF directly regulates a set of genes required for DNA replication, repair and mitosis. Our results reveal how loss of MITF regulates mitotic fidelity, and through defective replication and repair induces DNA damage, ultimately ending in cellular senescence. These findings reveal a lineage-specific control of DNA replication and mitosis by MITF, providing new avenues for therapeutic intervention in melanoma. The identification of MITF-binding sites and gene-regulatory networks establish a framework for understanding oncogenic basic helix-loop-helix factors such as N-myc or TFE3 in other cancers.

Rab proteins in endocytosis and Glut4 trafficking.

The intracellular trafficking of numerous proteins requires a tight control to fulfil their physiological functions. It is the case of the adipocyte and muscle glucose transporter Glut4 that is retained intracellularly until insulin induces its recruitment to the plasma membrane. Rabs are evolutionarily conserved small GTPases that control intracellular traffic events from yeast to mammalian cells. In the past few decades, considerable progresses have been made in identifying the route of Glut4, the Rabs involved in controlling it, and more recently the connection between insulin signalling and Glut4 trafficking through Rab activity control.

The nitric oxide-donating derivative of acetylsalicylic acid, NCX 4016, stimulates glucose transport and glucose transporters translocation in 3T3-L1 adipocytes.

NCX 4016 is a nitric oxide (NO)-donating derivative of acetylsalicylic acid. NO and salicylate, in vivo metabolites of NCX 4016, were shown to be potential actors in controlling glucose homeostasis. In this study, we evaluated the action of NCX 4016 on the capacity of 3T3-L1 adipocytes to transport glucose in basal and insulin-stimulated conditions. NCX 4016 induced a twofold increase in glucose uptake in parallel with the translocation of the glucose transporters GLUT1 and GLUT4 to the plasma membrane, leaving unaffected their total expression levels. Importantly, NCX 4016 further increased glucose transport induced by a physiological concentration of insulin. The stimulatory effect of NCX 4016 on glucose uptake appears to be mediated by its NO moiety. Indeed, it is inhibited by a NO scavenger and treatment with acetylsalicylic or salicylic acid had no effect. Although NO is involved in the action of NCX 4016, it did not mainly depend on the soluble cGMP cyclase/protein kinase G pathway. Furthermore, NCX 4016-stimulated glucose transport did not involve the insulin-signaling cascade required to stimulate glucose transport. NCX 4016 induces a small activation of the mitogen-activated protein kinases p38 and c-Jun NH(2)-terminal kinase and no activation of other stress-activated signaling molecules, including extracellular signal-regulated kinase, inhibitory factor kappaB, or AMP-activated kinases. Interestingly, NCX 4016 modified the content of S-nitrosylated proteins in adipocytes. Taken together, our results indicate that NCX 4016 induced glucose transport in adipocytes through a novel mechanism possibly involving S-nitrosylation. NCX 4016 thus possesses interesting characteristics to be considered as a candidate molecule for the treatment of patients suffering from metabolic syndrome and type 2 diabetes.

The role of small G-proteins in the regulation of glucose transport (review).

Insulin increases the rate of glucose transport into fat and muscle cells by stimulating the translocation of intracellular Glut 4-containing vesicles to the plasma membrane. This results in a marked increase in the amount of the facilitative glucose transporter Glut 4 at the cell surface, allowing for an enhanced glucose uptake. This process requires a continuous cycling through the early endosomes, a Glut 4 specific storage compartment and the plasma membrane. The main effect of insulin is to increase the rate of Glut 4 trafficking from its specific storage compartment to the plasma membrane. The whole phenomenon involves signal transduction from the insulin receptor, vesicle trafficking (sorting and fusion processes) and actin cytoskeleton modifications, which are all supposed to require small GTPases. This review describes the potential role of the various members of the Ras, Rad, Rho, Arf and Rab families in the traffic of the Glut 4-containing vesicles.

Okadaic acid stimulates IGF-II receptor translocation and inhibits insulin action in adipocytes.

Okadaic acid, an inhibitor of protein phosphatases 2A and 1, stimulates glucose transport in muscle and fat cells, suggesting that serine/threonine phosphorylation steps are involved in the translocation of glucose transporters. Here we have investigated whether such phosphorylation events could also participate in another membrane-associated insulin-stimulated process: insulin-like growth factor II (IGF-II) receptor translocation in adipocytes. Maximally effective concentrations of insulin and okadaic acid stimulated deoxyglucose uptake by 5.5- and 2.5-fold, respectively, whereas IGF-II binding was increased 3.5-fold and 1.5-fold. Subcellular fractionation indicated that the okadaic acid-induced stimulation of IGF-II binding resulted from an increase in the number of IGF-II receptors in the plasma membrane with a concomitant disappearance from the low-density microsomal fraction. These changes occurred in parallel to those observed for the glucose transporter GLUT-4. Both insulin-stimulated glucose transport and IGF-II binding were prevented when cells were pretreated with okadaic acid. To understand the mechanism of this inhibitory effect, insulin receptor autophosphorylation and the tyrosine phosphorylation of endogenous proteins were studied. Insulin induced the tyrosine phosphorylation of its receptor beta-subunit and of proteins at 120 and 185 kDa, whereas okadaic acid alone had no effect. When okadaic acid and insulin were added together, the beta-subunit autophosphorylation was similar to that observed with insulin alone, but the tyrosine phosphorylation of substrates was prevented. Taken together, our data suggest that, in adipocytes, serine/threonine phosphorylation events mimicked by okadaic acid are required for the translocation of IGF-II receptors and glucose transporters.(ABSTRACT TRUNCATED AT 250 WORDS)

Polymyxin B inhibits insulin-induced glucose transporter and IGF II receptor translocation in isolated adipocytes.

In isolated adipocytes, polymyxin B inhibited insulin-induced glucose incorporation into lipids in a dose-dependent manner, while polymyxin E, a structurally related antibiotic, was ineffective. To approach the mechanism of this effect, the subcellular distribution of the glucose transporter Glut 4 was investigated. Adipocytes were pretreated without or with polymyxin B before insulin stimulation, subcellular fractionation was performed and Glut 4 was detected by immunodetection. Incubation of adipocytes with polymyxin B prevented the insulin-induced appearance of Glut 4 in the plasma membranes, but did not prevent their decrease from the low-density microsomal fraction. A lower purity of the plasma membrane fractions, a detergent effect of polymyxin B on the membranes or an interference of the substance with the immunodetection of the Glut 4 molecules were excluded. These results suggest that polymyxin B was interfering with the Glut 4 translocation process stimulated by insulin in adipocytes. In a similar fashion, polymyxin B inhibited the insulin-induced increase in IGF II binding to adipocytes. This resulted from a blockade of the appearance of IGF II receptors in the plasma membranes. Since low-molecular-mass GTP-binding proteins have been implicated in the regulation of vesicular trafficking, we have used [alpha-32P]GTP binding to analyze such proteins in adipocyte fractions, after SDS/PAGE and transfer to nitrocellulose. Specific and distinct subsets of GTP-binding proteins were revealed in plasma membrane and low-density microsomal fractions of control adipocytes, whether they were stimulated or not with insulin. Polymyxin B treatment of adipocytes markedly modified the profile of the low-molecular-mass GTP-binding proteins in plasma membranes, but not in low-density microsomal fractions. Our results suggest that polymyxin B was interfering with the exocytotic process of the Glut 4 and IGF II receptor-containing vesicles, perhaps at the fusion step between vesicles and plasma membranes.

Insulin stimulates phosphatidylinositol-3-kinase activity in rat adipocytes.

Phosphatidylinositol (PtdIns) 3-kinase is thought to participate in the signal transduction pathways initiated by the activation of receptor tyrosine kinases including the insulin receptor. To approach the physiological relevance of this enzyme in insulin signaling, we studied the activation of PtdIns-3-kinase in adipocytes, a major insulin target tissue for glucose transport and utilisation. To analyze possible interactions of the enzyme with cellular proteins, immunoprecipitations with the following antibodies were performed: (a) anti-phosphotyrosine antibodies, (b) two antibodies to the 85-kDa subunit of PtdIns-3-kinase (p85) and (c) an antibody to the 185-kDa major insulin receptor substrate (p185). We show that in cell extracts from adipocytes exposed to insulin, and after immunoprecipitation with an anti-phosphotyrosine antibody and an antibody to p85, we are able to detect a PtdIns-3-kinase activity stimulated by the hormone. Similarly, after immunoprecipitation with an antibody to p185, an increase in the PtdIns-3-kinase activity could be demonstrated. Taken together these results suggest that, upon insulin stimulation of fat cells, PtdIns-3-kinase itself is tyrosine phosphorylated and/or associated with an insulin receptor substrate, such as p185, which could function as a link between the insulin receptor and PtdIns-3-kinase. The PtdIns-3-kinase was activated within 1 min of exposure to insulin, and the half-maximal effect was reached at the same concentration, i.e. 3 nM, as for stimulation of the insulin receptor kinase. Subcellular fractionation showed that PtdIns-3-kinase activity was found both in the membranes and in the cytosol. Further, immunoprecipitation with an antibody to p85, which possesses the capacity to activate PtdIns-3-kinase, suggests that the presence of the enzyme in the membrane may be due to an insulin-induced recruitment of the PtdIns-3-kinase from the cytosol to the membrane. Finally, we used isoproterenol, which exerts antagonistic effects on insulin action. This drug was found to inhibit both the PtdIns-3-kinase and the insulin receptor activation by insulin, suggesting that the activation of the PtdIns-3-kinase was closely regulated by the insulin receptor tyrosine kinase. The occurrence of an insulin-stimulated PtdIns-3-kinase in adipocytes leads us to propose that this enzyme might be implicated in the generation of metabolic responses induced by insulin.