Insulin-stimulated NADH/NAD+ redox state increases NAD(P)H oxidase activity in cultured rat vascular smooth muscle cells.
BACKGROUND
We reported that insulin modestly stimulates NAD(P)H oxidase activity in cultured rat vascular smooth muscle cells (VSMC) and synergistically stimulates enzyme activity with angiotensin II (Ang II), leading to synergistic stimulation of VSMC migration. The aim of this study was to determine the mechanism of insulin-stimulated NAD(P)H oxidase activity.


METHODS
Cultured rat VSMC O(2)(-) and H(2)O(2) production was measured by lucigenin and luminol luminescence, respectively, and lactate and pyruvate content of cell lysates determined by measuring increase or loss, respectively, of NADH in the presence of lactate dehydrogenase. Migration of VSMC was determined by a wound closure method.


RESULTS
Administration of 1 nmol/L insulin increased the lactate/pyruvate ratio (LPR), and hence the NADH/NAD(+) ratio by 122 +/- 16% (P < .05). Exogenous lactate (5 mmol/L), which increased the LPR similarly to insulin, increased O(2)(-) production by 123% +/- 32% (P < .05); Ang II (50 nmol/L) increased it by 60% +/- 9% (P < .05); but together these agents synergistically increased O(2)(-) production to 343% +/- 72% above the control value (P < .05 v the sum of the lactate-and Ang II-stimulated values), all in a diphenyleneiodonium-sensitive or apocynin-sensitive manner. Blocking the increase of insulin in the LPR with exogenous pyruvate or oxaloacetate blocked insulin's stimulation and insulin's plus Ang II's synergistic stimulation of O(2)(-)production as well as insulin's stimulation of migration of Ang II-treated VSMC. Insulin plus Ang II also stimulated H(2)O(2) production. Neither wortmannin nor pertussis toxin, which inhibit insulin-stimulated NAD(P)H oxidase activity in other cell types, affected insulin or insulin plus Ang II-stimulated O(2)(-)production.


CONCLUSIONS
Insulin stimulates NAD(P)H oxidase activity and with Ang II synergistically stimulates it in cultured rat VSMC by increasing the NADH/NAD(+) redox potential, but not by phosphatidylinositol 3-kinase or heterotrimeric G(iota) protein-dependent pathways.
Yang
Ming
M
Division of Renal Diseases and Hypertension, Department of Internal Medicine, The University of Texas Health Science Center, 6431 Fannin Street, Houston, TX 77030, USA.
Kahn
Andrew M
AM
D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D004357:Drug Synergism; D004789:Enzyme Activation / Q000187:drug effects; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D019344:Lactic Acid / Q000378:metabolism; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology*; D009243:NAD / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism*; D010084:Oxidation-Reduction; D019289:Pyruvic Acid / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014662:Vasoconstrictor Agents / Q000494:pharmacology
D007004:Hypoglycemic Agents; D007328:Insulin; D014662:Vasoconstrictor Agents; D009243:NAD; D000804:Angiotensin II; D019344:Lactic Acid; D019289:Pyruvic Acid; D019255:NADPH Oxidases