Protein tyrosine phosphatase 1B (PTP1B) is implicated as a negative regulator of insulin receptor (IR) signaling and a potential drug target for the treatment of type 2 diabetes and other associated metabolic syndromes. To further define the role of PTP1B in insulin signaling and to test the hypothesis that blocking the activity of PTP1B would augment the action of insulin, we prepared several cell permeable, potent and selective, small molecule PTP1B inhibitors, and evaluated their biological effects in several insulin sensitive cell lines. Our data indicate that PTP1B inhibitors bind to and colocalize with PTP1B on the surface of the endoplasmic reticulum and PTP1B exerts its negative effect on insulin signaling upstream of phosphatidylinositol 3-kinase and MEK1. Treatment of cells with PTP1B inhibitors, both in the presence and in the absence of insulin, markedly enhances IRbeta and IRS-1 phosphorylation, Akt and ERK1/2 activation, Glut4 translocation, glucose uptake, and Elk1 transcriptional activation and cell proliferation. These results indicate that small molecule inhibitors targeted to PTP1B can act as both insulin mimetics and insulin sensitizers. Taken together, our findings combined with results from PTP1B knockout, antisense, and biochemical studies provide strong evidence that PTP1B negatively regulates insulin signaling and that small molecule PTP1B inhibitors have the ability to potentiate and augment the action of insulin.

Based on recent studies showing that phospholipase D (PLD)1 is associated with intracellular membranes and promotes membrane budding from the trans-Golgi, we tested its possible role in the membrane trafficking of GLUT4 glucose transporters. Using immunofluorescence confocal microscopy, expressed Myc epitopetagged PLD1 was found to associate with intracellular vesicular structures by a mechanism that requires its N-terminal pleckstrin homology domain. Partial co-localization with expressed GLUT4 fused to green fluorescent protein in both 3T3-L1 adipocytes and Chinese hamster ovary cells was evident. Furthermore, microinjection of purified PLD into cultured adipocytes markedly potentiated the effect of a submaximal concentration of insulin to stimulate GLUT4 translocation to cell surface membranes. Insulin stimulated PLD activity in cells expressing high levels of insulin receptors but no such insulin effect was detected in 3T3-L1 adipocytes. Taken together, these results are consistent with the hypothesis that PLD1 associated with GLUT4-containing membranes acts in a constitutive manner to promote the mechanism of GLUT4 translocation by insulin.Physiological glucose homeostasis in humans is largely dependent on the actions of the hormone insulin, particularly its ability to enhance glucose transport into fat and muscle. Insulin exerts this effect primarily through a process whereby sequestered, intracellular GLUT4 glucose transporter proteins are rapidly redistributed to cell surface membranes where they can catalyze glucose uptake into cells (1-3). In both the basal and insulin-stimulated states, GLUT4 proteins appear to cycle between intracellular membrane and plasma membrane locations, but exocytosis is relatively slow in the absence of insulin (4). The action of insulin to stimulate this process appears dependent on the activity of p85/p110-type phosphatidylinositol (PI) 1 3-kinase because it is blocked by inhibitors of this enzyme (5, 6) or by expression of dominant inhibitory constructs of the p85 regulatory subunit (7). Insulin receptor tyrosine kinase activity activates PI 3-kinase through recruitment of Src homology 2 domains of the p85 subunit to phosphotyrosine sites on insulin receptor substrate proteins (8). The concept that the 3Ј-polyphosphoinositide products of insulin-stimulated PI 3-kinase are key mediators of insulin action on GLUT4 translocation is consistent with extensive data supporting a major role of these lipids in membrane trafficking (9).Much effort has been expended attempting to identify downstream targets of the PI 3-kinase products, phosphatidylinositol 3-phosphate (PtdIns(3)P), PtdIns(3,4)P 2 , and PtdIns(3,4,5)P 3 , that might connect insulin receptor signaling to GLUT4 glucose transporter trafficking (1). Some evidence (10, 11) has implicated Akt/ protein kinase B, which is thought to be activated in the presence of 3Ј-polyphosphoinositide through the actions of PDK1 and another unknown protein kinase (12), in GLUT4 regulation. However, other data do not support this view but i...

Genetic variability has a profound effect on the development of cardiac hypertrophy in response to stress. Consequently, using a variety of inbred mouse strains with known genetic profiles may be powerful models for studying the response to cardiovascular stress. To explore this approach we looked at male C57BL/6J and 129/SvJ mice. Hemodynamic analyses of left ventricular pressures (LVPs) indicated significant differences in 129/SvJ and C57BL/6J mice that implied altered Ca2+ handling. Specifically, 129/SvJ mice demonstrated reduced rates of relaxation and insensitivity to dobutamine (Db). We hypothesized that altered expression of genes controlling the influx and efflux of Ca2+ from the sarcoplasmic reticulum (SR) was responsible and investigated the expression of several genes involved in maintaining the intracellular and sarcoluminal Ca2+ concentration using quantitative real-time PCR analyses (qRT-PCR). We observed significant differences in baseline gene expression as well as different responses in expression to isoproterenol (ISO) challenge. In untreated control animals, 129/SvJ mice expressed 1.68× more ryanodine receptor 2(Ryr2) mRNA than C57BL/6J mice but only 0.37× as much calsequestrin 2 (Casq2). After treatment with ISO, sarco(endo)plasmic reticulum Ca2+-ATPase(Serca2) expression was reduced nearly two-fold in 129/SvJ while expression in C57BL/6J was stable. Interestingly, β (1) adrenergic receptor(Adrb1) expression was lower in 129/SvJ compared to C57BL/6J at baseline and lower in both strains after treatment. Metabolically, the brain isoform of creatine kinase (Ckb) was up-regulated in response to ISO in C57BL/6J but not in 129/SvJ. These data suggest that the two strains of mice regulate Ca2+ homeostasis via different mechanisms and may be useful in developing personalized therapies in human patients.