The P300 acetyltransferase inhibitor C646 promotes membrane translocation of insulin receptor protein substrate and interaction with the insulin receptor

Inhibition of P300 acetyltransferase activity by specific inhibitor C646 has been shown to improve insulin signaling. However, the underlying molecular mechanism of this improvement remains unclear. In this study, we analyzed P300 levels of obese patients and found that they were significantly increased in liver hepatocytes. In addition, large amounts of P300 appeared in the cytoplasm. Inhibition of P300 acetyltransferase activity by C646 drastically increased tyrosine phosphorylation of the insulin receptor protein substrates (IRS1/2) without affecting the tyrosine phosphorylation of the beta subunit of the insulin receptor (IRβ) in hepatocytes in the absence of insulin. Since IRS1/2 requires membrane translocation and binding to inositol compounds for normal functions, we also examined the role of acetylation on binding to phosphatidylinositol(4,5)P2 and found that IRS1/2 acetylation by P300 reduced this binding. In contrast, we show that inhibition of IRS1/2 acetylation by C646 facilitates IRS1/2 membrane translocation. Intriguingly, we demonstrate that C646 activates IRβ′s tyrosine kinase activity and directly promotes IRβ interaction with IRS1/2, leading to the tyrosine phosphorylation of IRS1/2 and subsequent activation of insulin signaling even in the absence of insulin. In conclusion, these data reveal the unique effects of C646 in activating insulin signaling in patients with obesity and diabetes.     

Activation and glucagon regulation of mitogen-activated protein kinases (MAPK) by insulin and epidermal growth factor in cultured rat and human hepatocytes

Many hepatocellular activities may be proximally regulated by intracellular signalling proteins including mitogen-activated protein kinases (MAPK). In this study, signalling events from epidermal growth factor (EGF) and insulin were examined in primary cultured human and rat hepatocytes. Using Western immunoblots, rat and human hepatocytes were found to produce a rapid tyrosine phosphorylation of the EGF receptor and MAPK following 0·5–1 min exposure to EGF. Phosphorylation of p42 and p44 MAPK was observed following 2·5 min exposure to EGF. Insulin treatment produced phosphorylation of the insulin receptor β subunit; shc phosphorylation was not observed. MAPK phosphorylation corresponded with a shift in molecular weight and an increase in kinase activity. Insulin-dependent activation of MAPK was unequivocally observed only in human hepatocytes, though a slight activation was detected in rat. Co-treatment with insulin and EGF produced phosphorylation and complete electrophoretic shift in molecular weight of MAPK, with an additive or synergistic increase in enzyme activity in rat but not human hepatocytes; human hepatocyte MAPK was maximally stimulated by EGF alone. Glucagon pretreatment blocked phosphorylation, gel mobility shift and kinase activity of MAPK induced by insulin but only partially blocked EGF-induced MAPK activation in human hepatocytes. Glucagon also reduced the activation of MAPK by EGF in rat hepatocytes. Pre-treatments with forskolin or cyclic AMP analogues diminished in the insulin-, EGF- and insulin plus EGF-dependent activation of MAPK in rat hepatocytes without effecting phosphorylation of receptors or MAPK. These results indicate that although EGF and insulin may both signal through the MAPK/ras/raf/MAPK pathway, the response for MAPK differs between these ligands and between species. Further, in both rat and human, glucagon exerts its effects through a cyclic AMP-dependent mechanism at a level in the insulin and EGF signal transduction pathways downstream of MAPK but promixal to MAPK. The partial inhibition of EGF-induced MAPK phosphorylation by glucagon in human hepatocytes provides further evidence for a raf-1-independent pathway for activation of MAPK. © 1998 John Wiley & Sons, Ltd.     

Intracellular zinc fluctuations modulate protein tyrosine phosphatase activity in insulin/insulin-like growth factor-1 signaling

Zinc is an effector of insulin/IGF-1 signaling and has insulinomimetic effects, the molecular basis of which is not understood. The present study establishes the capacity of zinc to inhibit protein tyrosine phosphatases (PTPs) as a cause for these effects and, moreover, demonstrates modulation of the insulin response by changes in intracellular zinc. The inhibition of PTPs by zinc occurs at significantly lower concentrations than previously reported. In vitro, zinc inhibits PTPs 1B and SHP-1 with IC(50) values of 17 and 93 nM, respectively. A fluorescent probe with a similar binding constant [FluoZin-3, K(D)(Zn) = 15 nM] detects corresponding concentrations of zinc within cells. Increase of cellular zinc after incubation with both zinc and the ionophore pyrithione augments protein tyrosine phosphorylation, and in particular the phosphorylation of three activating tyrosine residues of the insulin/IGF-1 receptor. Vice versa, specific chelation of cellular zinc with the membrane-permeable N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine suppresses insulin- and IGF-1-stimulated phosphorylation. In the context of the emerging concept that intracellular zinc is tightly regulated and fluctuates dynamically, these results suggest that a pool of cellular zinc modulates phosphorylation signaling.     

The emergence of ErbB2 expression in cultured rat hepatocytes correlates with enhanced and diversified EGF-mediated signaling

The proliferative effects of EGF in liver have been extensively investigated in cultured hepatocytes. We studied the effects of EGF, insulin, and other growth regulators on the expression, interaction, and signaling of ErbB receptors in primary cultures of adult rat hepatocytes. Using immunological methods and ErbB tyrosine kinase inhibitors, we analyzed the expression and signaling patterns of the ErbB kinases over 120 h of culture. Basal and EGF-stimulated protein tyrosine phosphorylation increased as cells adapted in vitro. EGF receptor (EGFr) expression declined in the first 24 h, whereas ErbB3 expression rose. Although ErbB2 was not present in freshly isolated hepatocytes, EGF and insulin independently induced ErbB2 while suppressing ErbB3 expression. Low concentrations of EGF and insulin synergistically stimulated ErbB2 expression and DNA synthesis. The greatest increase in ErbB2, which is normally expressed by fetal and neonatal hepatocytes, occurred shortly before the onset of DNA synthesis (> 40 h). EGF promoted EGFr and ErbB2 coassociation, stimulating tyrosine phosphorylation of both proteins. In contrast, heregulin beta1 (HRG-beta1) did not promote ErbB2 and ErbB3 coassociation. A selective tyrphostin inhibitor of ErbB2 suppressed EGF-stimulated DNA synthesis, but maximum suppression required the blockade of the EGFr kinase as well. Maximal EGF stimulation of DNA synthesis in vitro depends on the induction of ErbB2 and involves an EGFr-ErbB2 heterodimer. The ability of insulin to induce ErbB2 suggests both a mechanism for the synergy between insulin and EGF and a possible metabolic control of ErbB2 in vivo.     

低频脉冲电场对胰岛素介导的细胞增殖影响及其作用机理研究

本文以胞外信号分子胰岛素为研究对象,从细胞信号系统与电磁场相互耦合角度,通过MTT比色法和间接免疫荧光技术研究脉冲电场(f=50Hz,τ=20μS,Epp=1V/m)对人肝细胞(L-02细胞株)的增殖能力以及胰岛素与细胞表面受体的专一结合特性的影响。MTT比色分析结果发现在脉冲电场对细胞增殖的直接作用中,处理5、10、20分钟对细胞增殖的抑制百分率分别为10.13％、18.10％和11.85％;脉冲电场对细胞增殖的间接作用中,胰岛素经脉冲电场处理20、40分钟,对细胞增殖的抑制百分率分别为10.31％和14.12％;流式细胞术检测结果表明细胞悬液和胰岛素共同经脉冲电场处理20分钟组其平均荧光强度降低22.74％,仅胰岛素受电场处理组和仅细胞受电场处理组的平均荧光强度分别下降12.96％和10.51％。本实验结果显示 脉冲电场对人肝细胞的作用体现在两个方面:一是电场直接作用于细胞膜上的胰岛素受体及其他膜成分;二是脉冲电场还可通过改变培养基中的胰岛素分子的构像,影响胰岛素与受体的结合能力,进而影响细胞的增殖等活动。     

Hepassocin activates the EGFR/ERK cascade and induces proliferation of L02 cells through the Src-dependent pathway

Hepassocin (HPS) is a secreted protein with mitogenic activity on primary hepatocytes and protects hepatocytes from chemically-induced injury. Our previous studies showed that HPS stimulates proliferation of hepatocytes in an ERK pathway-dependent manner. However, the molecular mechanism of HPS-induced activation of the ERK pathway remains unclear. In this study, we found that HPS induced the phosphorylation of the epidermal growth factor receptor (EGFR) in the human L02 hepatocyte cell line, and this event was concomitant with the activation of the non-receptor tyrosine kinase Src. Specific inhibition of EGFR kinase activity by gefitinib or down-regulation of EGFR by specific EGFR siRNAs prevented HPS-induced activation of the ERK pathway and proliferation of L02 cells. Furthermore, inhibition of Src activity significantly blocked HPS-induced activation of the EGFR, which was suggestive of a ligand-independent transactivation mechanism of EGFR itself as well as ERK phosphorylation and proliferation of L02 cells. These results indicate that EGFR plays an important role in the mitogenic signaling induced by HPS in L02 cell lines and may further stimulate research on the role of HPS in hepatocytes within biological processes in human health and disease.     

Protein tyrosine phosphatase regulation in fibroblasts from patients with an insulin receptor gene mutation

Tyrosine phosphorylation of the insulin receptor is the initial event following receptor binding to insulin, and it induces further tyrosine phosphorylation of various intracellular molecules. This signaling is countered by protein tyrosine phosphatases (PTPases), which reportedly are associated with insulin resistance that can be reduced by regulation of PTPases. Protein tyrosine phosphatase 1B (PTP1B) and leukocyte antigen-related PTPase (LAR) are the PTPases implicated most frequently in insulin resistance and diabetes mellitus. Here, we show that PTP1B and LAR are expressed in human fibroblasts, and we examine the regulation of PTPase activity in fibroblasts from patients with an insulin receptor gene mutation as an in vitro model of insulin resistance. Total PTPase activity was significantly lower in the cytosolic and membrane fractions of fibroblasts with mutations compared with controls (p<0.05). Insulin stimulation of fibroblasts with mutations resulted in a significantly smaller increase in PTP1B activity compared with stimulation of wild-type fibroblasts (p<0.05). This indicates that insulin receptor gene mutations blunt increases in PTPase activity in response to insulin, possibly via a negative feedback mechanism. Our data suggest that the PTPase activity in patients with insulin receptor gene mutation and severe insulin resistance may differ from that in ordinary type 2 diabetes.     

Opposite Cross-Talk by Oleate and Palmitate on Insulin Signaling in Hepatocytes through Macrophage Activation

Chronic low grade inflammation in adipose tissue during obesity is associated with an impairment of the insulin signaling cascade. In this study, we have evaluated the impact of palmitate or oleate overload of macrophage/Kupffer cells in triggering stress-mediated signaling pathways, in lipoapoptosis, and in the cross-talk with insulin signaling in hepatocytes. RAW 264.7 macrophages or Kupffer cells were stimulated with oleate or palmitate, and levels of M1/M2 polarization markers and the lipidomic profile of eicosanoids were analyzed. Whereas proinflammatory cytokines and total eicosanoids were elevated in macrophages/Kupffer cells stimulated with palmitate, enhanced arginase 1 and lower leukotriene B₄ (LTB₄) levels were detected in macrophages stimulated with oleate. When hepatocytes were pretreated with conditioned medium (CM) from RAW 264.7 or Kupffer cells loaded with palmitate (CM-P), phosphorylation of stress kinases and endoplasmic reticulum stress signaling was increased, insulin signaling was impaired, and lipoapoptosis was detected. Conversely, enhanced insulin receptor-mediated signaling and reduced levels of the phosphatases protein tyrosine phosphatase 1B (PTP1B) and phosphatase and tensin homolog (PTEN) were found in hepatocytes treated with CM from macrophages stimulated with oleate (CM-O). Supplementation of CM-O with LTB₄ suppressed insulin sensitization and increased PTP1B and PTEN. Furthermore, LTB₄ decreased insulin receptor tyrosine phosphorylation in hepatocytes, activated the NFκB pathway, and up-regulated PTP1B and PTEN, these effects being mediated by LTB₄ receptor BTL1. In conclusion, oleate and palmitate elicit an opposite cross-talk between macrophages/Kupffer cells and hepatocytes. Whereas CM-P interferes at the early steps of insulin signaling, CM-O increases insulin sensitization, possibly by reducing LTB₄.     

Long-term interleukin-1alpha treatment inhibits insulin signaling via IL-6 production and SOCS3 expression in 3T3-L1 adipocytes.

Proinflammatory cytokines are well-known to inhibit insulin signaling to result in insulin resistance. IL-1alpha is also one of the proinflammatory cytokines, but the mechanism of how IL-1alpha induces insulin resistance remains unclear. We have now examined the effects of IL-1alpha on insulin signaling in 3T3-L1 adipocytes. Prolonged IL-1alpha treatment for 12 to 24 hours partially decreased the protein levels as well as the insulin-stimulated tyrosine phosphorylation of IRS-1 and Akt phosphorylation. mRNA for SOCS3, an endogenous inhibitor of insulin signaling, was dramatically augmented 4 hours after IL-1alpha treatment. Concomitantly, the level of IL-6 in the medium and STAT3 phosphorylation were increased by the prolonged IL-1alpha treatment. Addition of anti-IL-6 neutralizing antibody to the medium or overexpression of dominant-negative STAT3 decreased the IL-1alpha-stimulated STAT3 activation and SOCS3 induction, and ameliorated insulin signaling. These results suggest that the IL-1alpha-mediated deterioration of insulin signaling is largely due to the IL-6 production and SOCS3 induction in 3T3-L1 adipocytes.     

The tyrosine kinase activity of the chicken insulin receptor is similar to that of the human insulin receptor

The tyrosine kinase activity of a chimeric insulin receptor composed of the extracellular domain of the human insulin receptor (IR) and the intracellular domain of the chicken IR was compared with wild-type human IR. The degrees of autophosphorylation, phosphorylation of IRS-1, and in vitro phosphorylation of an exogenous substrate after stimulation by human insulin were similar to that seen with the human IR. We conclude that the insulin resistance of chickens is not attributable to a lower level of intrinsic tyrosine kinase activity of IR.     

Functional trans-inactivation of insulin receptor kinase by growth-inhibitory angiotensin II AT2 receptor

The present study demonstrates negative intracellular cross-talk between angiotensin II type 2 (AT2) and insulin receptors. AT2 receptor stimulation leads to inhibition of insulin-induced extracellular signal-regulated protein kinase (ERK2) activity and cell proliferation in transfected Chinese hamster ovary (CHO-hAT2) cells. We show that AT2 receptor interferes at the initial step of insulin signaling cascade, by impairing tyrosine phosphorylation of the insulin receptor (IR) beta-chain. AT2-mediated inhibition of IR phosphorylation is insensitive to pertussis toxin and is also detected in neuroblastoma N1E-115 and pancreatic acinar AR42J cells that express endogenous receptors. We present evidence that AT2 receptor inhibits the autophosphorylating tyrosine kinase activity of IR, with no significant effect on insulin binding properties. AT2-mediated inactivation of IR does not mainly involve tyrosine dephosphorylation by vanadate-sensitive tyrosine phosphatases nor serine/threonine phosphorylation by protein kinase C. As a consequence of IR inactivation, AT2 receptor inhibits tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and signal-regulatory protein (SIRPalpha1) and prevents subsequent association of both IRS-1 and SIRPalpha1 with Src homology 2 (SH2)-containing tyrosine phosphatase SHP-2. Our results thus demonstrate functional trans-inactivation of IR kinase by G protein-coupled AT2 receptor, illustrating a novel mode of negative communication between two families of membrane receptors.     

Dual action of eicosapentaenoic acid in hepatoma cells: up-regulation of metabolic action of insulin and inhibition of cell proliferation

Exogenous administration of eicosapentaenoic acid (EPA) improves insulin sensitivity, but its precise mechanism remains unknown. Here we show that EPA stimulates the intracellular insulin signaling pathway in hepatoma cells. Exposure of these cells to EPA caused up-regulation of several insulin-induced activities including tyrosine phosphorylation of insulin receptor substrate-1, insulin receptor substrate-1-associated phosphatidylinositol 3-kinase, and its downstream target Akt kinase activity as well as down-regulation of gluconeogenesis. In contrast, EPA decreased mitogen-activated protein kinase activity and inhibited cell proliferation. These findings raise the possibility that EPA up-regulates metabolic action of insulin and inhibits cell growth in humans.     

Hepatic very low density lipoprotein-ApoB overproduction is associated with attenuated hepatic insulin signaling and overexpression of protein-tyrosine phosphatase 1B in a fructose-fed hamster model of insulin resistance

A fructose-fed hamster model of insulin resistance was previously documented to exhibit marked hepatic very low density lipoprotein (VLDL) overproduction. Here, we investigated whether VLDL overproduction was associated with down-regulation of hepatic insulin signaling and insulin resistance. Hepatocytes isolated from fructose-fed hamsters exhibited significantly reduced tyrosine phosphorylation of the insulin receptor and insulin receptor substrates 1 and 2. Phosphatidylinositol 3-kinase activity as well as insulin-stimulated Akt-Ser473 and Akt-Thr308 phosphorylation were also significantly reduced with fructose feeding. Interestingly, the protein mass and activity of protein-tyrosine phosphatase-1B (PTP-1B) were significantly higher in fructose-fed hamster hepatocytes. Chronic ex vivo exposure of control hamster hepatocytes to high insulin also appeared to attenuate insulin signaling and increase PTP-1B. Elevation in PTP-1B coincided with marked suppression of ER-60, a cysteine protease postulated to play a role in intracellular apoB degradation, and an increase in the synthesis and secretion of apoB. Sodium orthovanadate, a general phosphatase inhibitor, partially restored insulin receptor phosphorylation and significantly reduced apoB secretion. In summary, we hypothesize that fructose feeding induces hepatic insulin resistance at least in part via an increase in expression of PTP-1B. Induction of hepatic insulin resistance may then contribute to reduced apoB degradation and enhanced VLDL particle assembly and secretion.     

Role of the pleckstrin homology domain of PLCgamma1 in its interaction with the insulin receptor

A thiol-reactive membrane-associated protein (TRAP) binds covalently to the cytoplasmic domain of the human insulin receptor (IR) beta-subunit when cells are treated with the homobifunctional cross-linker reagent 1,6-bismaleimidohexane. Here, TRAP was found to be phospholipase C gamma1 (PLCgamma1) by mass spectrometry analysis. PLCgamma1 associated with the IR both in cultured cell lines and in a primary culture of rat hepatocytes. Insulin increased PLCgamma1 tyrosine phosphorylation at Tyr-783 and its colocalization with the IR in punctated structures enriched in cortical actin at the dorsal plasma membrane. This association was found to be independent of PLCgamma1 Src homology 2 domains, and instead required the pleckstrin homology (PH)-EF-hand domain. Expression of the PH-EF construct blocked endogenous PLCgamma1 binding to the IR and inhibited insulin-dependent phosphorylation of mitogen-activated protein kinase (MAPK), but not AKT. Silencing PLCgamma1 expression using small interfering RNA markedly reduced insulin-dependent MAPK regulation in HepG2 cells. Conversely, reconstitution of PLCgamma1 in PLCgamma1-/- fibroblasts improved MAPK activation by insulin. Our results show that PLCgamma1 is a thiol-reactive protein whose association with the IR could contribute to the activation of MAPK signaling by insulin.     

The docking protein FRS2alpha controls a MAP kinase-mediated negative feedback mechanism for signaling by FGF receptors

The docking protein FRS2alpha functions as a major mediator of signaling by FGF and NGF receptors. Here we demonstrate that, in addition to tyrosine phosphorylation, FRS2alpha is phosphorylated by MAP kinase on multiple threonine residues in response to FGF stimulation or by insulin, EGF, and PDGF, extracellular stimuli that do not induce tyrosine phosphorylation of FRS2alpha. Prevention of FRS2alpha threonine phosphorylation results in constitutive tyrosine phosphorylation of FRS2alpha in unstimulated cells and enhanced tyrosine phosphorylation of FRS2alpha, MAPK stimulation, cell migration, and proliferation in FGF-stimulated cells. Expression of an FRS2alpha mutant deficient in MAPK phosphorylation sites induces anchorage-independent cell growth and colony formation in soft agar. These experiments reveal a novel MAPK-mediated, negative feedback mechanism for control of signaling pathways that are dependent on FRS2 and a mechanism for heterologous control of signaling via FGF receptors.     

Assay and screening methods for bioactive substances based on cellular signaling pathways

Assay and screening methods for bioactive substances based on cellular signaling pathways are presented. Examples include: (1) intracellular protein phosphorylation and protein-protein interaction, (1-i) a new assay method for evaluating chemical selectivity of agonists for insulin signaling pathways based on agonist-induced phosphorylation of a target peptide, (1-ii) an SPR-based screening method for agonist selectivity for insulin signaling pathways based on the binding of phosphotyrosine to its specific binding protein, (1-iii) a fluorescent indicator for tyrosine phosphorylation-based insulin signaling pathways, and (1-iv) split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing; (2) a screening method for antigen-specific IgE using mast cells based on intracellular calcium signaling; (3) a screening method for substrates of multidrug resistance-associated protein (MRP); and (4) fluorescent indicators for cyclic GMP based on cyclic GMP-dependent protein kinase Ialpha and green fluorescent proteins.     

Modification of Akt2 by 4-hydroxynonenal inhibits insulin-dependent Akt signaling in HepG2 cells

The production of reactive aldehydes such as 4-hydroxy-2-nonenal (4-HNE) is a key component of the pathogenesis in a spectrum of hepatic diseases involving oxidative stress such as alcoholic liver disease (ALD). One consequence of ALD is increased insulin resistance in hepatocytes. To understand the effects of 4-HNE on insulin signaling in liver cells, we employed a model using hepatocellular carcinoma cell line HepG2. Previously, we have demonstrated an increase in the level of Akt phosphorylation is mediated by 4-HNE inhibition of PTEN, a direct regulator of Akt. In this work, we evaluated the effects of 4-HNE on insulin-dependent stimulation of the Akt2 pathway. We demonstrate that 4-HNE selectively leads to phosphorylation of Akt2. Although Akt2 is phosphorylated following 4-HNE treatment, the level of downstream phosphorylation of Akt substrates such as GSK3β and MDM2 is significantly decreased. Pretreatment with 4-HNE prevented insulin-dependent Akt signaling and decreased intracellular Akt activity by 87%. Using biotin hydrazide capture, it was confirmed that 4-HNE treatment of cells resulted in carbonylation of Akt2, which was not observed in untreated control cells. Using a synthetic GSK3α/β peptide as a substrate, treatment of recombinant human myristoylated Akt2 (rAkt2) with 20 or 40 μM 4-HNE inhibited rAkt2 activity by 30 or 85%, respectively. Matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF) identified Michael addition adducts of 4-HNE with His196, His267, and Cys311 of rAkt2. Computation-based molecular modeling analysis of 4-HNE adducted to His196 and Cys311 of Akt2 suggests inhibition of GSK3β peptide binding by 4-HNE in the Akt2 substrate binding pocket. The inhibition of Akt by 4-HNE provides a novel mechanism for increased insulin resistance in ALD. These data provide a potential mechanism of dysregulation of Akt2 during events associated with sustained hepatocellular oxidative stress.     

Low proliferation capacity of lymphocytes from alloxan-diabetic rats: involvement of high glucose and tyrosine phosphorylation of Shc and IRS-1

The proliferation capacity of lymphocytes obtained from mesenteric lymph nodes of control and alloxan-diabetic (40 mg/kg) rats in response to concanavalin A (ConA) and lipopolysaccharide (LPS) stimuli was examined. Proliferation response of lymphocytes from diabetic rats was significantly reduced under Con A (43%) and LPS (46%) stimulation as compared with the control group. Insulin (166 microM) promoted a marked increase of lymphocyte proliferation (7.5-fold) in the control group and this response was much lower (2.6-fold) in lymphocyte from diabetic rats. Cells were also cultured in medium containing glucose at 5, 10 or 20 mM. High glucose concentration (20 mM) caused a marked inhibition of lymphocyte proliferation reaching the values of the diabetic group. In lymphocytes from control rats, the degree of Shc tyrosine phosphorylation was gradually increased, whereas that of cells from diabetic rats was much lower in response to insulin. In lymphocytes obtained from control rats, the tyrosine phosphorylation of IRS-1 was time-dependent on insulin. In cells from diabetic rats, the basal tyrosine phosphorylation of IRS-1 was higher than that of control rats, however, there was no further phosphorylation after insulin addition. We conclude that the response of lymphocyte proliferation from diabetic rats to Con A and LPS stimuli is decreased but insulin was able to promote a significant proliferative effect on these cells. Also, high glycemia in addition to the lack of insulin participates in the reduced proliferation capacity of lymphocytes from diabetic rats.