J Korean Diabetes Assoc.  2007 Jan;31(1):22-32. 10.4093/jkda.2007.31.1.22.

Oxidative Stress Causes Vascular Insulin Resistance in OLETF Rat Through Increased IRS-1 Degradation

• Park JL
• Lee YS
• Kim BH
• Kang YH
• Kim IJ
• Kim YK
• Son SM

• KMID: 2008093
• DOI: http://doi.org/10.4093/jkda.2007.31.1.22

Abstract

BACKGROUND: Insulin resistance and oxidative stress have been reported to play essential pathophysiological roles in diabetic cardiovascular complication. The relationship between insulin resistance and oxidative stress in vasculature remains unclear. The study was conducted to assess whether oxidative stress induce vascular insulin resistance in OLETF rat, a model of type 2 diabetes
METHODS
We used OLETF rats (20/30/40 weeks, n = 5/5/5), as models of type 2 DM, and LETO rats (20/30/40 weeks, n = 5/5/5) as controls. Aortas of each rats were extracted. Superoxide anion production was detected by NBT assay and lucigenin assay. 8-hydroxyguanosine (OHdG) and nitrotyrosine were detected as markers of oxidative stress in 20 and 40 weeks groups. The glucose uptake of aortas was measured by detecting 2-deoxyglucose uptake in both groups. The expression of IR, IRS-1, PI3-K and Akt/PKB were detected by immuno precipitation and immunoblotting in 20, 30 and 40 weeks groups
RESULTS
Superoxide anion production and markers of oxidative stress (8-OHdG, nitrotyrosine) were significantly increased in aortas of OLETF rats compared with controls. Aortas of OLETF rats exhibited decreased IRS-1 content and increased phosphorylation of IRS-1 at Ser307 compared with LETO rats. There were no significant differences in expressions of IR, PI3-K and Akt/PKB between two groups
CONCLUSION
These results suggest that oxidative stress induces insulin resistance in vasculature of OLETF rat specifically through increasing serine phosphorylation of IRS-1 and its degradation by a proteasome-dependent pathway, providing an alternative mechanism that may explain the association with insulin resistance and diabetic vascular complications.

Keyword

Aorta; Insulin resistance; IRS-1; Oxidative stress

MeSH Terms

Animals
Aorta
Deoxyglucose
Diabetic Angiopathies
Glucose
Immunoblotting
Insulin Resistance*
Insulin*
Oxidative Stress*
Phosphorylation
Rats
Rats, Inbred OLETF*
Serine
Superoxides
Deoxyglucose
Glucose
Insulin
Serine
Superoxides

Figure

• 

  Fig. 1 Vascular superoxide production in the aortic segments from LETO and OLETF rats was assessed by lucigenin chemiluminescence assay. Data were expressed as nmol/min/mg of dry weight of vessels. Bars represent mean ± SEM of 4 experiments in each group. *P < 0.01 compared with LETO rats at 40 weeks.

• 

  Fig. 2 NAD(P)H oxidase-stimulated superoxide production in aortic segments by NBT reduction. Bars represent mean ± SEM of 4 experiments in each group. *P < 0.05 compared with LETO rats at 20 weeks. **P <0.01 compared with LETO rats at 40 weeks.

• 

  Fig. 3 Localization of superoxide anion production in aortic rings from LETO and OLETF rats by NBT reduction. Cross-sections of a rat thoracic aorta stained with NBT in LETO (A) and OLETF (B) rats were embedded in paraffin, and photographed.

• 

  Fig. 4 8-OHdG content in the aortic tissue DNA (A) and nitrotyrosine content in the aortic tissue lysate (B). Bars represent mean ± SEM of 4 experiments in each group. *P < 0.01 compared with LETO rats at 40 weeks. **P < 0.01 compared with LETO rats at 40 weeks.

• 

  Fig. 5 Aortic tissues were exposed to 100 nmol/L insulin for 120 minutes. Uptake of 2-deoxy-D-[3H]glucose was measured. Bar graph represents means ± SEM (n = 4) of percent change in 2-deoxy-D-[3H]glucose uptake. *P < 0.05 compared with LETO rats at 20 weeks. **P < 0.01 compared with LETO rats at 40 weeks.

• 

  Fig. 6 IR (A) and IRS-1 (B) expressions on aortas of LETO and OLETF rats. Western blotting with anti-IR or anti-IRS-1 antibody was performed. Bars represent mean ± SEM of 4 experiments in each group. *P < 0.05 compared with LETO rats at 30 weeks. **P < 0.01 compared with LETO rats at 40 weeks.

• 

  Fig. 7 Phosphorylation of IRS-1 on aorta of LETO and OLETF rats. Aortic tissues were lysed and immunoprecipitated with anti-IRS-1 antibody. Western analysis was performed using phospho-tyrosine-IRS-1, phospho-Ser307-IRS-1 or phospho-Ser612-IRS-1 antibody. Bars represent mean ± SEM of 4 experiments in each group. *P < 0.01 compared with LETO rats at 40 weeks.

• 

  Fig. 8 PI3-K expression (A) and phosphorylation of Akt/PKB (B) on aortas of LETO and OLETF rats. Aortic tissues were lysed and immunoblotted with PI3-K, phospho-Ser473-Akt/PKB, or phospho-Thr308-Akt/PKB antibody. Bars represent mean ± SEM of 4 experiments in each group.

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