Defective Insulin and Acetylcholine Induction of Endothelial Cell–Nitric Oxide Synthase Through Insulin Receptor Substrate/Akt Signaling Pathway in Aorta of Obese Rats

Abstract

The actions of acetylcholine (ACh) on endothelium mainly are mediated through muscarinic receptors, which are members of the G protein-coupled receptor family. In the present study, we show that ACh induces rapid tyrosine phosphorylation and activation of Janus kinase 2 (JAK2) in rat aorta. Upon JAK2 activation, tyrosine phosphorylation of insulin receptor substrate (IRS)-1 is detected. In addition, ACh induces JAK2/IRS-1 and IRS-1/phosphatidylinositol (PI) 3-kinase associations, downstream activation of Akt/protein kinase B, endothelial cell-nitric oxide synthase (eNOS), and extracellular signal-regulated kinase (ERK)-1/2. The pharmacological blockade of JAK2 or PI 3-kinase reduced ACh-stimulated eNOS phosphorylation, NOS activity, and aorta relaxation. These data indicate a new signal transduction pathway for IRS-1/PI 3-kinase/Akt/ eNOS activation and ERK1/2 by means of JAK2 tyrosine phosphorylation stimulated by ACh in vessels. Moreover, we demonstrate that in aorta of obese rats (high-fat diet), there is an impairment in the insulin-and ACh-stimulated IRS-1/PI 3-kinase pathway, leading to reduced activation with lower protein levels of eNOS associated with a hyperactivated ERK/mitogen-activated protein kinase pathway. These results suggest that in aorta of obese rats, there not only is insulin resistance but also ACh resistance, probably mediated by a common signaling pathway that controls the activity and the protein levels of eNOS. Diabetes 56: 1014 -1024, 2007 U nder physiological conditions, insulin regulates many vascular functions (1,2), including the release of nitric oxide (NO) (3) and the regulation of mRNA expression of matrix proteins (4,5) and constitutive endothelial cell-nitric oxide synthase (eNOS) (6,7). At the molecular level, binding of insulin to its cognate receptor (insulin receptor) results in activation of the insulin receptor's tyrosine kinase activity, which, in turn, phosphorylates tyrosine residues of insulin receptor substrates (IRSs). IRSs are adaptor proteins that transduce signals from the insulin receptor to downstream signaling cascades, including the phosphatidylinositol (PI) 3-kinase/Akt pathway (8,9). In the vasculature, the activation of PI 3-kinase increases serine phosphorylation of Akt, which, in turn, directly phosphorylates eNOS on ser1177 and activates the enzyme, leading to increased NO production (10,11). In addition, a second postreceptor insulin signaling pathway involves the activation of Ras, Raf, mitogen-activated protein and extracellular signalregulated kinase kinase (MEK), and mitogen-activated protein kinase (MAPK) (extracellular signal-regulated kinase [ERK]-1/2), which is related to cellular growth (12,13). Recent findings (14,15) suggest that impaired IRS-1/PI 3-kinase/Akt/eNOS signal transduction may play a mechanistic role in endothelial dysfunction and in the development of cardiovascular diseases in situations of insulin resistance.Although insulin is well known in inducing vascular relaxation, the physiological significance of circulating insulin ...

Functional and Morphological Evaluation of the Mesenteric Artery, Kidney and Liver from Obese Rats: Impact of a High Fat Diet plus Fructose

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NO/beta-catenin crosstalk modulates primitive streak formation prior to embryonic stem cell osteogenic differentiation

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