Najma Begum

Regulation of Mitogen-activated Protein Kinase Phosphatase-1 Induction by Insulin in Vascular Smooth Muscle Cells EVALUATION OF THE ROLE OF THE NITRIC OXIDE SIGNALING PATHWAY AND POTENTIAL DEFECTS IN HYPERTENSION

In this study, we examined the regulation of mitogen-activated protein kinase phosphatase (MKP-1) expression by insulin in primary vascular smooth muscle cell cultures. Insulin caused a rapid time- and dose-dependent induction of MKP-1 mRNA and protein expression. Blockade of nitric-oxide synthase (NOS) withN G-monomethyl-l-arginine acetate, and cGMP with RpcGMP, completely inhibited MKP-1 expression. Insulin-mediated MKP-1 expression was preceded by inducible NOS (iNOS) induction and cGMP production. Blockade of phosphatidylinositol 3-kinase (PI3-kinase) signaling with wortmannin inhibited insulin-mediated iNOS protein induction, cGMP production, and MKP-1 expression. To evaluate potential interactions between NOS and the mitogen-activated protein kinase (MAPK) signaling pathways, we employed PD98059 and SB203580, two specific inhibitors of ERKs and p38 MAPK. These inhibitors abolished the effect of insulin on MKP-1 expression. Only PD98059 inhibited insulin-mediated iNOS protein induction. Vascular smooth muscle cells from spontaneous hypertensive rats exhibited a marked decrease in MKP-1 induction due to defects in insulin-induced iNOS expression because of reductions in PI3-kinase activity. Treatment with sodium nitroprusside and 8-bromo-cGMP restored MKP-1 mRNA expression to levels comparable with controls. We conclude that insulin-induced MKP-1 expression is mediated by PI3-kinase-initiated signals, leading to the induction of iNOS and elevated cGMP levels that stimulates MKP-1 expression.

cAMP Counter-regulates Insulin-mediated Protein Phosphatase-2A Inactivation in Rat Skeletal Muscle Cells

In this study, we examined the mechanism of recently reported inactivation of protein phosphatase-2A (PP-2A) by insulin (Srinivasan, M., and Begum, N. (1994) J. Biol. Chem. 269, 12514-12520) and its counter-regulation by cAMP agonists. Exposure of L6 myotubes to insulin resulted in a rapid inhibition of PP-2A that was accompanied by a 3-fold increase in the phosphotyrosine content of the immunoprecipitated PP-2A catalytic subunit. Pretreatment with (Sp)-cAMP, a cAMP agonist, completely blocked insulin-mediated inhibition of PP-2A activity and decreased the tyrosine phosphorylation of PP-2A catalytic subunit to control levels. To understand the mechanism of counter-regulation of PP-2A by (Sp)-cAMP, cells were pretreated with sodium orthovanadate, an inhibitor of phosphotyrosine phosphatases. Vanadate prevented the effect of (Sp)-cAMP on PP-2A activity and increased the phosphorylation status of PP-2A catalytic subunit to the level observed with insulin. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, and rapamycin, an inhibitor of 70-kDa S6 kinase activation, prevented insulin-mediated inactivation of PP-2A, suggesting that these pathways may participate in insulin-mediated phosphorylation and inactivation of PP-2A. These results show that insulin signaling results in a rapid inactivation of PP-2A by increased tyrosine phosphorylation and cAMP agonists counter-regulate insulins effect on PP-2A by decreasing phosphorylation, presumably via an activated phosphatase.

Protein Phosphatase-1 and insulin action

Protein Phosphatase-1 (PP-1) appears to be the key component of the insulin signalling pathway which is responsible for bridging the initial insulin-simulated phosphorylation cascade with the ultimate dephosphorylation of insulin sensitive substrates. Dephosphorylations catalyzed by PP-1 activate glycogen synthase (GS) and simultaneously inactivate phosphorylase a and phosphorylase kinase promoting glycogen synthesis. Our in vivo studies using L6 rat skeletal muscle cells and freshly isolated adipocytes indicate that insulin stimulates PP-1 by increasing the phosphorylation status of its regulatory subunit (PP-1G). PP-1 activation is accompanied by an inactivation of Protein Phosphatase-2A (PP-2A) activity. To gain insight into the upstream kinases that mediate insulin-stimulated PP-1G phosphorylation, we employed inhibitors of the ras/MAPK, PI3-kinase, and PKC signalling pathways. These inhibitor studies suggest that PP-1G phosphorylation is mediated via a complex, cell type specific mechanism involving PI3-kinase/PKC/PKB and/or the ras/MAP kinase/Rsk kinase cascade. cAMP agonists such as SpcAMP (via PKA) and TNF-α (recently identified as endogenous inhibitor of insulin action via ceramide) block insulin-stimulated PP-1G phosphorylation with a parallel decrease of PP-1 activity, presumably due to the dissociation of the PP-1 catalytic subunit from the regulatory G-subunit. It appears that any agent or condition which interferes with the insulin-induced phosphorylation and activation of PP-1, will decrease the magnitude of insulins effect on downstream metabolic processes. Therefore, regulation of the PP-1G subunit by site-specific phosphorylation plays an important role in insulin signal transduction in target cells. Mechanistic and functional studies with cell lines expressing PP-1G subunit site-specific mutations will help clarify the exact role and regulation of PP-1G site-specific phosphorylations on PP-1 catalytic function.

Phosphodiesterase 3A (PDE3A) Deletion Suppresses Proliferation of Cultured Murine Vascular Smooth Muscle Cells (VSMCs) via Inhibition of Mitogen-activated Protein Kinase (MAPK) Signaling and Alterations in Critical Cell Cycle Regulatory Proteins

Cyclic nucleotide phosphodiesterase 3 (PDE3) is an important regulator of cyclic adenosine monophosphate (cAMP) signaling within the cardiovascular system. In this study, we examined the role of PDE3A and PDE3B isoforms in regulation of growth of cultured vascular smooth muscle cells (VSMCs) and the mechanisms by which they may affect signaling pathways that mediate mitogen-induced VSMC proliferation. Serum- and PDGF-induced DNA synthesis in VSMCs grown from aortas of PDE3A-deficient (3A-KO) mice was markedly less than that in VSMCs from PDE3A wild type (3A-WT) and PDE3B-deficient (3B-KO) mice. The reduced growth response was accompanied by significantly less phosphorylation of extracellular signal-regulated kinase (ERK) in 3A-KO VSMCs, most likely due to a combination of greater site-specific inhibitory phosphorylation of Raf-1Ser-259 by protein kinase A (PKA) and enhanced dephosphorylation of ERKs due to elevated mitogen-activated protein kinase phosphatase 1 (MKP-1). Furthermore, 3A-KO VSMCs, compared with 3A-WT, exhibited higher basal PKA activity and cAMP response element-binding protein (CREB) phosphorylation, higher levels of p53 and p53 phosphorylation, and elevated p21 protein together with lower levels of Cyclin-D1 and retinoblastoma (Rb) protein and Rb phosphorylation. Adenoviral overexpression of inactive CREB partially restored growth effects of serum in 3A-KO VSMCs. In contrast, exposure of 3A-WT VSMCs to VP16 CREB (active CREB) was associated with inhibition of serum-induced DNA synthesis similar to that in untreated 3A-KO VSMCs. Transfection of 3A-KO VSMCs with p53 siRNA reduced p21 and MKP-1 levels and completely restored growth without affecting amounts of Cyclin-D1 and Rb phosphorylation. We conclude that PDE3A regulates VSMC growth via two complementary pathways, i.e. PKA-catalyzed inhibitory phosphorylation of Raf-1 with resulting inhibition of MAPK signaling and PKA/CREB-mediated induction of p21, leading to G0/G1 cell cycle arrest, as well as by increased accumulation of p53, which induces MKP-1, p21, and WIP1, leading to inhibition of G1 to S cell cycle progression.

Studies on the Possible Involvement of Prostaglandins in Insulin Generation of Pyruvate Dehydrogenase Activator

Insulin-exposed liver particulate fraction supernatants from control rats stimulated mitochondrial pyruvate dehydrogenase (PDH) activity by 26%, while the stimulation by similar preparations from indomethacin-injected rats (5 mg/kg twice daily, i.p., for 2 days) was 4%. In vitro addition of indomethacin to the particulate fraction during insulin exposure also inhibited stimulation of PDH by insulin. This inhibitory effect of indomethacin was completely overcome by the in vitro addition of prostaglandin E2 (PGE2) to the liver particulate incubation mixture. Intact adipocytes showed a similar (62%) decrease in insulin activation of PDH in the presence of indomethacin. In a cell-free adipocyte system (co-incubation of mitochondria and plasma membrane), indomethacin addition resulted in 90% decrease in insulin-stimulated PDH response. PGE2 addition completely reversed this inhibition. In contrast to its effects on PDH activation, indomethacin had no effect on insulin-stimulated glucose oxidation. In vitro incubation of fat cells with dexamethasone (1 μLM) also resulted in decreased insulin activation of PDH. Inclusion of arachidonic acid during dexamethasone exposure of fat cells resulted in partial restoration of the insulin effect on PDH in fat cells and in cell-free preparations. However, addition of PGE2 during insulin exposure of plasma membranes from dexamethasonetreated preparations showed no significant restoration of the insulin effect on PDH. These studies suggest that: (1) PG metabolism is involved in insulins generation of the second messenger, and (2) the mechanism of dexamethasone-induced inhibition of insulin effect on PDH is a complex phenomenon involving the synthesis and action of eicosanoids.

Mechanisms of the fasting-induced dissociation of insulin binding from its action in isolated rat hepatocytes

SummaryFasting leads to an increase in insulin binding to isolated rat hepatocytes from 12 to 17%. This increase was accounted for by changes in the affinity of insulin receptors without alteration in their number. In contrast, the responsiveness of hepatocytes to insulin was markedly diminished in fasted rats. Both basal and insulin-stimulated rates of 14C-glucose incorporation into glycogen were significantly decreased in fasted animals. When insulin-induced 14C-glucose incorporation into glycogen was expressed as a percent above the basal rate, hepatocytes isolated both from control and fasted animals showed the same magnitude of maximal response (66 ± 13% in fed and 59 ± 12% in fasted animals, respectively). However, more insulin must be bound to hepatocytes isolated from fasted animals in order to elicit the same percent of insulins maximal effect.Incubation of ‘fed’ hepatocytes in the serum obtained from fasted rats significantly diminished their responsiveness to insulin. An addition of insulin (100 ng/ml), glucose (10 mM) and antibodies to glucagon (1:100) eliminated the inhibitory effect of ‘fasted’ serum on ‘fed’ hepatocytes.A 48-hour fast increased significantly the microviscosity (decreased fluidity) of hepatocyte plasma membranes and altered membrane phospholipid composition. These changes correlated with enhanced insulin binding to isolated membranes. Moreover, in response to insulin, plasma membranes isolated from ‘fasted’ hepatocytes generated only one half the amount of the second messenger (PDH activator) observed in membranes of fed animals. The amount of PDH activator generated by incubation of plasma membranes with insulin correlated inversely with both insulin binding and membrane microviscosity.We conclude that 1) fasting induces both coupling defect and post-receptor changes in insulins action; 2) both extracellular and intracellular factors contribute to fasting-induced dissociation of insulin binding from insulin action; 3) insulin/ glucagon ratio may influence hepatocyte responsiveness to insulin; 4) alterations in plasma membrane fluidity and phospholipid composition may alter insulin binding and contribute to its dissociation from the subsequent action; 5) membranes isolated from ‘fasted’ hepatocytes generate less mediator of insulin action than do membranes isolated from ‘fed’ hepatocytes.

Effect of Acute Exercise on Insulin Generation of Pyruvate Dehydrogenase Activator by Rat Liver and Adipocyte Plasma Membranes

Groups of young adult rats with body weights of 125–135 g (group A) or 300–400 g (group B) were subjected to one bout of prolonged exercise to exhaustion on a treadmill and were studied 2 h postexercise. Liver glycogen levels were markedly depleted in the exercised rats. Adipocytes from group A exercised rats showed a significantly greater increase in pyruvate dehydrogenase (PDH) activity in response to insulin than those from sedentary controls. Incubation with insulin of liver part iculate fractions from exercised group A rats resulted in an increased production of a mitochondrial PDH activator compared with preparations from sedentary controls. The tissues of both exercised and sedentary group B rats were less responsive to insulin than those of the smaller rats. A significant effect of exercise on increased production of a PDH activator in response to insulin was found only in experiments in which adipocyte plasma membranes were coincubated with mitochondria and insulin. For group B rats exercise provided no significant enhancement of insulin activation of intact adipocyte PDH or stimulation of the production of a PDH activator by liver paniculate preparations. Insulin binding to fat cells was not affected by exercise. Group A rats made insulin resistant by a high-fat diet did not respond to exercise by significantly increasing the insulin stimulation of PDH activator by liver membranes. The enhancing effect of a single bout of exercise on insulin response was not readily demonstrable in rats resistant to insulin either in association with age and weight or with a high-fat diet. In insulin-sensitive, young adult rats a single bout of exercise to exhaustion increases the effect of insulin on adipocyte PDH activity and on the generation of PDH-stimulating activity by liver and fat cell membranes. A postbinding site is probably responsible for the exercise effect.

Insulin-induced Internalization and Replacement of Insulin Receptors in Adipocytes of Rats Adapted to Fat Feeding

To elucidate the mechanisms of the previously observed decrease in adipocyte surface insulin binding in fat diet-induced insulin resistance (Ip et al., J. Lipid Res. 1976; 17:588–99), we performed studies on (1) the distribution of insulin receptors between the cell surface and cell interior, and (2) the extent of internalization and recycling of insulin receptors in adipocytes obtained from fat-fed and glucose-fed rats. Intact cell binding and binding to solubilized cells (total) was decreased by 50% in rats fed fat for 7 days when compared with glucose-fed rats. Incubation of adipocytes with insulin in Tris buffer (100 ng/ml) resulted in a 40–60% decrease in cell surface insulin binding capacity. In two separate experiments, it was found that this insulin-induced receptor loss (%) in fat-fed rat adipocyte preparations was either comparable to that of glucosefed rats or somewhat decreased. The degradation of the receptors was not affected, as seen by the lack of difference in the chloroquine effect between the two groups. Incubation of fat cells with insulin in tissue culture medium promoted complete reinsertion of receptors into the cell membrane in glucose-fed rat adipocytes, while fat-fed rat preparations demonstrated a significant decrease (37%) in the extent of reinsertion of insulin receptors. Thus, the decrease in cell surface insulin binding and receptor number in fat-fed rat adipocytes is related to an adaptive decrease in the total receptor content coupled with an impairment in the ability to reinsert insulin receptors from the cell interior after insulin-induced internalization.