Insulin-mediated Signal Transduction Research Articles

Inhibition of the insulin receptor kinase phosphorylation by nitric oxide: functional and structural aspects.

Previous studies on cultured skeletal muscle cells have indicated that the insulin-induced expression of GLUT4 transporter protein is inhibited by nitric oxide (NO). Therefore, we determined the effect of NO on the insulin-induced autophosphorylation of the insulin receptor kinase (IRK), i.e., the first step in the insulin-mediated signal transduction pathway. The experiments showed that the insulin-induced autophosphorylation of the insulin receptor beta-chain is strongly inhibited by the NO donors 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA-NO) or S-nitroso-N-acetylpenicillamine (SNAP). The inhibitory effect was ameliorated in cells depleted of glutathione (GSH), suggesting the possibility that S-nitroso-glutathione may operate as an intermediate NO donor. Complementary experiments with different Cys --> Ala mutant proteins showed, surprisingly, that all mutant proteins were inhibited by DEA-NO. Three-dimensional models of the nonphosphorylated IR beta-chain nitrosylated at the accessible cysteine residues 1056, 1138, 1234, or 1245 revealed that derivatization of any of these four cysteine residues leads essentially to the same structural changes of the IRK domain. These changes involve a movement of the amino-terminal lobe against the carboxy-terminal lobe in a direction opposite to the direction of the "lobe closure" that was previously proposed to facilitate the accessibility for ATP and the expression of catalytic activity. Our findings suggest that the occurrence of several functionally relevant cysteine residues in distinct regions of the IRK protein increases the probability of regulatory redox interactions and thus the redox sensitivity of the IRK.

Ethanol inhibits insulin receptor substrate-1 tyrosine phosphorylation and insulin-stimulated neuronal thread protein gene expression.

Neuronal thread proteins (NTPs) are molecules that accumulate in the brains of patients with Alzheimer's disease, and may play a key role in both normal and neurodegenerative neuritic sprouting. In this investigation we determined whether NTP expression is up-regulated by insulin, an important neurotrophic factor that stimulates differentiation-associated neurite outgrowth, and studied the effects of ethanol, a known inhibitor of growth factor receptor tyrosine phosphorylation, on NTP expression and insulin-mediated signal transduction cascade in neuronal [primitive neuroectodermal tumour cell line 2; (PNET2)] cells. PNET2 cells were treated with 50 m-units/ml insulin in the presence or absence of 100 mM ethanol for 0.2-96 h, and cell proliferation and expression of NTP molecules were investigated by metabolic labelling, immunoprecipitation and immunohistochemical staining. Insulin stimulation resulted in an immediate increase in the levels of three (38, 18 and 15 kDa) of five NTP species (the others were of 26 and 21 kDa), followed by a decline in expression within 120 min; however, studies performed up to 96 h of culture demonstrated up-regulation by insulin of all five NTP species. Ethanol either abolished or severely muted the short- and long-term insulin-mediated upregulation of NTP expression, and substantially reduced insulin-mediated neuronal differentiation. The effects of ethanol on NTP gene expression were associated with impaired insulin-mediated tyrosine phosphorylation of both the insulin receptor beta subunit and the insulin receptor substrate-1 (IRS-1), resulting in decreased association of phosphatidylinositol 3-kinase with IRS-1. The findings suggest that ethanol may inhibit NTP expression associated with central nervous system neuronal differentiation by uncoupling the IRS-1-mediated insulin signal transduction pathway.

Insulin-induced differentiation and modulation of neuronal thread protein expression in primitive neuroectodermal tumor cells is linked to phosphorylation of insulin receptor substrate-1.

Neuronal thread proteins (NTPs) are a family of developmentally regulated molecules expressed in central nervous system (CNS) neurons and primitive neuroectodermal tumor (PNET) cell lines. NTP gene expression is modulated with DNA synthesis, neuritic sprouting, and neuronal differentiation. The present study explores the mechanism of insulin modulation of NTP gene expression during neuronal differentiation using PNET cell lines of CNS origin. PNET2 cells underwent neuronal differentiation with neurite outgrowth coupled with transient up-regulation of several species of NTP. In contrast, PNET1 cells failed to differentiate in response to insulin stimulation, although insulin receptors were more abundant than in PNET2 cells. Analysis of the insulin-mediated signal transduction pathway demonstrated that the lack of insulin responsiveness in PNET1 cells was primarily caused by impaired insulin-mediated tyrosyl phosphorylation of the insulin receptor substrate-1 (IRS-1). Correspondingly, the association between phosphatidyl-inositol 3 (PI3) kinase and phosphorylated IRS-1 was reduced in PNET1 compared with PNET2 cells. In contrast, the levels of IRS-1 protein were similar in PNET1 and PNET2 cells, and expression of the insulin receptor beta subunit (Ir beta) and insulin-mediated tyrosyl phosphorylation of the Ir beta were greater in PNET1 than PNET2 cells. The findings suggest that insulin effected neuronal differentiation and modulation of NTP gene expression in PNET cells utilizes a signal transduction cascade that requires tyrosyl phosphorylation of IRS-1.

Insulin receptor substrate 1 is required for insulin-mediated mitogenic signal transduction.

Insulin treatment of mammalian cells immediately stimulates the tyrosine phosphorylation of a cellular protein of 185 kDa referred to as pp185 or IRS-1 (insulin receptor substrate 1). The potential role of the IRS-1 protein in insulin signaling has been examined by microinjecting affinity-purified antibodies into living cells. Stably transfected Rat-1 fibroblasts, which overexpress the human insulin receptor, were microinjected and subsequently stimulated with insulin or other growth factors. Progression through the cell cycle was monitored by using a single-cell assay, which employs bromodeoxyuridine labeling of DNA and analysis with immunofluorescence microscopy. Microinjection of anti-IRS-1 antibody completely inhibited incorporation of bromodeoxyuridine into the nuclei of cells stimulated with insulin or insulin-like growth factor I but did not affect cells stimulated with serum or a variety of purified growth factors. These studies indicate that IRS-1 is a critical component of the insulin and insulin-like growth factor I signaling pathways, which lead to DNA synthesis and cell growth.