Roles for insulin receptor, PI3-kinase, and Akt in insulin-signaling pathways related to production of nitric oxide in human vascular endothelial cells.

BACKGROUND: Previously, we demonstrated that insulin stimulates production of nitric oxide (NO) in endothelial cells. However, specific insulin-signaling pathways mediating production of NO have not been elucidated. METHODS AND RESULTS: We developed methods for transfection of human umbilical vein endothelial cells (HUVECs) and direct measurement of NO to begin defining insulin-signaling pathways related to NO production. HUVECs were cotransfected with enhanced Green Fluorescent Protein (eGFP) and another gene of interest. Transfection efficiencies >95% were obtained by selecting cells expressing eGFP. Overexpression of insulin receptors in HUVECs resulted in an approximately 3-fold increase in production of NO in response to insulin. In contrast, HUVECs overexpressing a tyrosine kinase-deficient mutant insulin receptor had a dose-response curve similar to that of control cells. Overexpression of inhibitory mutants of either phosphatidylinositol 3-kinase (PI3K) or Akt resulted in nearly complete inhibition of insulin-stimulated production of NO. Overexpression of an inhibitory mutant of Ras had a much smaller effect. CONCLUSIONS: Receptor kinase activity is necessary to mediate production of NO through the insulin receptor. Both PI3K and Akt contribute importantly to this process, whereas the contribution of Ras is small.

Phosphorylation of PTP1B at Ser(50) by Akt impairs its ability to dephosphorylate the insulin receptor.

PTP1B is a protein tyrosine phosphatase that negatively regulates insulin sensitivity by dephosphorylating the insulin receptor. Akt is a ser/thr kinase effector of insulin signaling that phosphorylates substrates at the consensus motif RXRXXS/T. Interestingly, PTP1B contains this motif (RYRDVS(50)), and wild-type PTP1B (but not mutants with substitutions for Ser(50)) was significantly phosphorylated by Akt in vitro. To determine whether PTP1B is a substrate for Akt in intact cells, NIH-3T3(IR) cells transfected with either wild-type PTP1B or PTP1B-S50A were labeled with [(32)P]-orthophosphate. Insulin stimulation caused a significant increase in phosphorylation of wild-type PTP1B that could be blocked by pretreatment of cells with wortmannin or cotransfection of a dominant inhibitory Akt mutant. Similar results were observed with endogenous PTP1B in untransfected HepG2 cells. Cotransfection of constitutively active Akt caused robust phosphorylation of wild-type PTP1B both in the absence and presence of insulin. By contrast, PTP1B-S50A did not undergo phosphorylation in response to insulin. We tested the functional significance of phosphorylation at Ser(50) by evaluating insulin receptor autophosphorylation in transfected Cos-7 cells. Insulin treatment caused robust receptor autophosphorylation that could be substantially reduced by coexpression of wild-type PTP1B. Similar results were obtained with coexpression of PTP1B-S50A. However, under the same conditions, PTP1B-S50D had an impaired ability to dephosphorylate the insulin receptor. Moreover, cotransfection of constitutively active Akt significantly inhibited the ability of wild-type PTP1B, but not PTP1B-S50A, to dephosphorylate the insulin receptor. We conclude that PTP1B is a novel substrate for Akt and that phosphorylation of PTP1B by Akt at Ser(50) may negatively modulate its phosphatase activity creating a positive feedback mechanism for insulin signaling.

Up-regulation of endothelial nitric oxide synthase expression by cyclic guanosine 3',5'-monophosphate.

In this study, the role of cyclic GMP, the end product of the NO-cyclic GMP signalling pathway, in the regulation of ecNOS was investigated. Bovine pulmonary endothelial cells were exposed to 8-bromo-cyclic GMP and its effect on NO production, ecNOS protein, and mRNA levels was analyzed. Endothelial cells on exposure to 8-bromo-cyclic GMP produced significantly increased amounts of NO, detected as increased cyclic GMP in cocultures with vascular smooth muscle cells both under basal conditions and with agonist stimulation. 8-Bromo-cyclic GMP significantly increased the ecNOS protein and mRNA levels as detected on Western and Northern blots respectively. This 8-bromo-cyclic GMP mediated increase of NO production, ecNOS protein and mRNA levels suggests that cyclic GMP up-regulates the expression of ecNOS. Thus, there may be an intercellular feedback mechanism involved at the molecular level in the expression of the NO-cyclic GMP signalling pathway in blood vessels.

Collagen levels in isoproterenol induced myocardial infarction in rats.

Myocardial infarction was induced in albino rats by the subcutaneous administration of isoproterenol. A significant was observed increase in weight of the myocardium, total protein, DNA levels, neutral salt soluble collagen content, serum and urinary hydroxyproline levels at peak infarction stage in treated rats while the body weight, insoluble and total collagen content of heart were found to be decreased. These results show an early degradation of collagen immediately after myocardial infarction and enhanced synthesis during the different stages of recovery. Histological studies confirmed the incidence of peak infarction after two doses of isoproterenol treatment and recovery to normalcy from the third day onwards.

In vivo labeling studies on the biosynthesis and degradation of collagen in experimental myocardial infarction.

The biosynthesis and degradation of myocardial collagen was studied in myocardium infarcted rats after a single intraperitoneal injection of 3H-proline. The incorporation of tritiated proline into collagen as 3H-hydroxyproline was regarded as a measure of collagen synthesis. The total content as well as total activity of hydroxyproline were measured in the whole homogenate, neutral salt soluble fraction, insoluble fraction and in urine collected at different time intervals and specific activities were calculated. Both collagen anabolism and catabolism were found to be affected in infarcted rat hearts. Degradation of existing collagen at the earlier stages of myocardial infarction and a simultaneous new collagen synthesis and deposition as insoluble form later took place.

Direct and reversible inhibition of endothelial nitric oxide synthase by nitric oxide.

The objective of this study was to investigate the regulation of endothelial nitric oxide (NO) synthase by NO. Partially purified endothelial NO synthase was exposed to authentic NO (10-200 microM) and to the nitrovasodilators sodium nitroprusside (SNP; 10-1,000 microM) and S-nitroso-N-acetylpenicillamine (SNAP; 100-1,000 microM), and enzyme activity was assayed by measuring the conversion of L-[3H]arginine to L-[3H]citrulline in the presence of added cofactors. NO, SNP, and SNAP inhibited NO synthase activity in a dose-dependent manner, NO being the most potent inhibitor. The Michaelis constant for L-arginine was not altered (4.87 microM) by NO (50 microM), whereas the maximal velocity of the enzyme decreased from 784 to 633 pmol.mg-1.min-1. Oxyhemoglobin (10 microM) partially prevented the inhibition of NO synthase by NO (50 microM). The data also suggest that NO inhibits endothelial NO synthase activity by directly interacting with the NO synthase and not by an indirect mechanism such as limitation of cofactor or oxygen availability. Dialysis of NO synthase treated with NO (50 microM) partially restored the enzyme activity. This study demonstrates a direct and reversible inhibition of NO synthase by NO, suggesting a feedback mechanism in vivo.

Influence of isoproterenol-induced myocardial infarction on certain glycohydrolases and cathepsins in rats.

The changes in the activities of certain lysosomal hydrolases, viz., beta-glucuronidase, beta-N-acetylglucosaminidase, beta-galactosidase, beta-glucosidase, alpha-glucosidase, alpha-galactosidase, alpha-mannosidase, cathepsin B, cathepsin D, and collagenolytic cathepsin, in serum and heart of rats subject to myocardial infarction with isoproterenol, were studied during the periods of peak infarction and recovery. The activities of all the enzymes assayed exhibited a significant increase both in serum and in heart at peak infarction stage and these levels returned to normal during the stage of recovery and repair. The infiltration of inflammatory cells at the infarct regions and the altered lysosomal fragility are probably responsible for the increased activity of the enzymes studied. This may also bring about the catabolism of connective tissue constituents as reported in literature.

Alterations in the heart lysosomal stability in isoproterenol induced myocardial infarction in rats.

The alterations in the heart lysosomal stability following isoproterenol induced myocardial infarction were studied in albino rats. The rate of release of beta-glucuronidase at various time intervals at 37 degrees C from lysosome rich fraction was taken as a measure of lysosomal stability. As compared to the control day one, three and five samples exhibited a significant increase in beta-glucuronidase activity at all the time intervals. The subcellular distribution of beta-glucuronidase was also studied and the soluble and total activities exhibited an increase at peak infarction stage and returned to normal during the recovery. The decrease in the lysosomal stability might be attributed to the increased beta-glucuronidase activity observed following myocardial infarction.

Protein kinase C-zeta phosphorylates insulin receptor substrate-1 and impairs its ability to activate phosphatidylinositol 3-kinase in response to insulin.

Protein kinase C-zeta (PKC-zeta) is a serine/threonine kinase downstream from phosphatidylinositol 3-kinase in insulin signaling pathways. However, specific substrates for PKC-zeta that participate in the biological actions of insulin have not been reported. In the present study, we identified insulin receptor substrate-1 (IRS-1) as a novel substrate for PKC-zeta. Under in vitro conditions, wild-type PKC-zeta (but not kinase-deficient mutant PKC-zeta) significantly phosphorylated IRS-1. This phosphorylation was reversed by treatment with the serine-specific phosphatase, protein phosphatase 2A. In addition, the overexpression of PKC-zeta in NIH-3T3(IR) cells caused significant phosphorylation of cotransfected IRS-1 as demonstrated by [(32)P]orthophosphate labeling experiments. In rat adipose cells, endogenous IRS-1 coimmunoprecipitated with endogenous PKC-zeta, and this association was increased 2-fold upon insulin stimulation. Furthermore, the overexpression of PKC-zeta in NIH-3T3(IR) cells significantly impaired insulin-stimulated tyrosine phosphorylation of cotransfected IRS-1. Importantly, this was accompanied by impaired IRS-1-associated phosphatidylinositol 3-kinase activity. Taken together, our results raise the possibility that IRS-1 is a novel physiological substrate for PKC-zeta. Because PKC-zeta is located downstream from IRS-1 and phosphatidylinositol 3-kinase in established insulin signaling pathways, PKC-zeta may participate in negative feedback pathways to IRS-1 similar to those described previously for Akt and GSK-3.

Inhalational anesthetics do not alter nitric oxide synthase activity.

Inhalational anesthetics inhibit the nitric oxide (NO)-soluble guanylate cyclase signaling pathway in vascular and neuronal tissues and it has been proposed that this inhibition is due to several mechanisms, which include a direct inhibition of NO synthase. To determine the direct interaction of anesthetics with NO synthase, the effects of halothane, isoflurane and enflurane on NO synthase activity of bovine and rat brains and cultured bovine aortic endothelial cells were investigated. Halothane and enflurane at 1% to 3% concentrations produced no significant effect on crude bovine brain NO synthase activity, as measured by the conversion of L-[3H]arginine to L-[3H]citrulline. Similarly, crude rat brain NO synthase activity was not affected by exposure to 1% to 4% halothane or isoflurane. The effects of inhalational anesthetics on the crude bovine brain NO synthase activity were not altered when assayed at two different temperatures (22 degrees C and 37 degrees C). Halothane and isoflurane produced no significant effects on the activity of partially purified rat brain NO synthase at different concentrations of L-[3H]arginine in the reaction mixture. Partially purified endothelial NO synthase, when equilibrated with halothane or isoflurane (0.5-2%), exhibited no significant alteration in enzyme activity. This study suggests that the effects of inhalational anesthetics on NO synthesis in rat and bovine brains and in vascular endothelial cells are not due to their direct interaction with NO synthase.

Purification and characterization of collagens from rat fibrosarcoma induced by 3-methylcholanthrene.

The distribution of various collagen types was studied in rat fibrosarcoma. Collagens extracted from fibrosarcoma tissue were characterized by the criteria of solubility in NaCl, SDS-PAGE, ion exchange chromatography, CNBr peptide mapping and amino acid analysis. Fibrosarcoma was found to produce excess amount of type V, type I trimer and type III collagens; comparatively, type I collagen and total collagen content were noticed to decrease in fibrosarcoma. We observe that the increase in type V collagen content in fibrosarcoma might be due to the enhanced transcription of type V collagen gene. Increased type I trimer collagen in fibrosarcoma might be attributed to the differential expression of alpha 1(I) and alpha 2(I) gene and might also be due to the expression of a different gene for type I trimer collagen.