Reduction of insulin-stimulated glucose uptake by peroxynitrite is concurrent with tyrosine nitration of insulin receptor substrate-1.
Inducible nitric oxide synthetase plays an essential role in insulin resistance induced by a high-fat diet. The reaction of nitric oxide with superoxide leads to the formation of peroxynitrite (ONOO-), which can modify several proteins. In this study, we investigated whether peroxynitrite impairs insulin-signalling pathway. Our experiments showed that 3-(4-morpholinyl)sydnonimine hydrochloride (SIN-1), a constitutive producer of peroxynitrite, dose-dependently inhibited insulin-stimulated glucose uptake. While SIN-1 did not affect the insulin receptor protein level and tyrosine phosphorylation, it reduced the insulin receptor substrate-1 (IRS-1) protein level, and IRS-1 associated phosphatidylinositol-3 kinase (PI-3 kinase) activity. Although SIN-1 did not induce Ser307 phosphorylation of IRS-1, tyrosine nitration of IRS-1 was detected in SIN-1-treated-Rat1 fibroblasts expressing human insulin receptors. Mass spectrometry showed that peroxynitrite induced at least four nitrated tyrosine residues in rat IRS-1, including Tyr939, which is critical for association of IRS-1 with the p85 subunit of PI-3 kinase. Our results suggest that peroxynitrite reduces the IRS-1 protein level and decreases phosphorylation of IRS-1 concurrent with nitration of its tyrosine residues.
Nomiyama
Takashi
T
Department of Medicine, Metabolism and Endocrinology, Juntendo University, School of Medicine, Tokyo, Japan.
Igarashi
Yasuhiro
Y
Taka
Hikari
H
Mineki
Reiko
R
Uchida
Toyoyoshi
T
Ogihara
Takeshi
T
Choi
Jong Bock
JB
Uchino
Hiroshi
H
Tanaka
Yasushi
Y
Maegawa
Hiroshi
H
Kashiwagi
Atsunori
A
Murayama
Kimie
K
Kawamori
Ryuzo
R
Watada
Hirotaka
H
D041721:3T3-L1 Cells; D000818:Animals; D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D005947:Glucose / Q000493:pharmacokinetics*; D007328:Insulin / Q000378:metabolism*; D055504:Insulin Receptor Substrate Proteins; D051379:Mice; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D009566:Nitrates / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000737:chemistry* / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D014443:Tyrosine / Q000378:metabolism*
C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; C526296:Irs1 protein, rat; D009566:Nitrates; D010750:Phosphoproteins; D030421:Peroxynitrous Acid; D014443:Tyrosine; C002385:linsidomine; D008981:Molsidomine; D005947:Glucose