Abstract
Insulin stimulates the tyrosine kinase activity of its receptor resulting in the tyrosine phosphorylation of pp185, which contains insulin receptor substrates IRS-1 and IRS-2. These early steps in insulin action are essential for the metabolic effects of insulin. Feeding animals a high-fructose diet results in insulin resistance. However, the exact molecular mechanism underlying this effect is unknown. In the present study, we determined the levels and phosphorylation status of the insulin receptor and pp185 (IRS-(1/2)) in liver and muscle of rats submitted to a high-fructose diet evaluated by immunoblotting with specific antibodies. Feeding fructose (28 days) induced a discrete insulin resistance, as demonstrated by the insulin tolerance test. Plasma glucose and serum insulin and cholesterol levels of the two groups of rats, fructose-fed and control, were similar, whereas plasma triacylglycerol concentration was significantly increased in the rats submitted to the fructose diet (P<0.05). There were no changes in insulin receptor concentration in the liver or muscle of either group. However, insulin-stimulated receptor autophosphorylation was reduced to 72 +/- 4% (P<0.05) in the liver of high-fructose rats. The IRS-1 protein levels were similar in both liver and muscle of the two groups of rats. In contrast, there was a significant decrease in insulin-induced pp185 (IRS-(1/2)) phosphorylation, to 83 +/- 5% (P<0.05) in liver and to 77 +/- 4% (P<0.05) in muscle of the high-fructose rats. These data suggest that changes in the early steps of insulin signal transduction may have an important role in the insulin resistance induced by high-fructose feeding.
Highlights
22oC with antiphosphotyrosine antibody, anti-insulin receptor antibody or anti-insulin receptor substrate 1 (IRS-1) antibody diluted in blocking buffer, pH 7.4 (3% nonfat dry milk, 10 mmol/l Tris, 150 mmol/l NaCl, and 0.02% Tween 20)
At the end of the 4-week experimental period there were no significant differences between control and fructose-fed rats in total body weight, or in the basal serum glucose, cholesterol and insulin concentration
After stimulation with insulin, in samples previously immunoprecipitated with anti-IR antibody and immunoblotted with antiphosphotyrosine antibody there was a clear decrease in insulin-stimulated receptor autophosphorylation to 72 ± 4% in fructose-fed animals compared to controls (P
Summary
22oC with antiphosphotyrosine antibody, anti-insulin receptor antibody (anti-IR) or anti-IRS-1 antibody diluted in blocking buffer, pH 7.4 (3% nonfat dry milk, 10 mmol/l Tris, 150 mmol/l NaCl, and 0.02% Tween 20). At the end of the 4-week experimental period there were no significant differences between control and fructose-fed rats in total body weight, or in the basal serum glucose, cholesterol and insulin concentration. Insulin receptor and IRS-1 protein levels (Figure 1a and b) did not change in the liver of fructose-fed rats compared to controls, as determined by immunoblotting with anti-IR and anti-IRS-1 antibodies.
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