Journal List > J Korean Diabetes Assoc > v.30(1) > 1062385

Lee, Hong, Kwon, Moon, Chang, Son, Yoon, Cha, and Kang: The Long-Term Effect of Ramipril on Giα2-Protein and Protein Tyrosine Phosphatase 1B in an Animal Model of Type 2 Diabetes (OLETF rat)

Abstract

Background

The regulation of tyrosine phosphorylation/dephosphorylation is an important mechanism in various intracellular metabolism. Also impaired insulin signal transduction is important in pathogenesis of type 2 diabetes. It has been reported that PTP1B is a negative regulator of insulin action, and Giα2-protein is related to the regulation of PTP1B. Herein we investigated the long-term effects of ramipril on PTP1B/insulin signal protein interaction and the relation between Giα2 and PTP1B in animal model of type 2 diabetes (OLETF rat).

Methods

OLETF rats and age-matched LETO rats were divided into two groups. One group of rats received ramipril (10 mg/kg body weight) for 12 weeks, and another group did not. Finally, each group was divided into 2 subgroups, with or without insulin injection intravenously, before sacrifice. After sacrifice, tissues extracts of liver, hind limb muscle, and epididymal fat were obtained for quantification of PTP1B, Giα2, and several insulin signal proteins by western blotting.

Results

In liver and muscle, the levels of basal PTP1B and activated PTP1B of OLETF rats treated with ramipril and insulin were significantly decreased. The levels of Giα2, activated IRS-2, and activated p-85α were significantly increased in OLETF rats treated with ramipril and insulin. In adipose tissue, the levels of Giα2 and activated p-85α of OLETF rats treated with ramipril and insulin were slightly increased as in liver and muscle. But, the levels of basal PTP1B and activated PTP1B were significantly increased. And, the levels of activated IRS-1 and activated IRS-2 were decreased.

Conclusion

These results suggest that the improvement of insulin sensitivity by treatment with ramipril was related to the decreased level of activated PTP1B. Also, we could suggest that the changes of activated PTP1B level was related with the changes of Giα2-protein. However, the results of adipose tissue were different from those of liver and muscle. So it seemed likely that there would be various major modulators for regulation of insulin signal pathway according to tissue.

Figures and Tables

Fig. 1
The changes of body weight (A), and the differences of oral glucose tolerance test (B) of LETO and OLETF rats with or without ramipril treatment. Oral glucose tolerance tests were done at 12 weeks after ramipril treatment. Data were expressed as mean ± SE. *P < 0.05
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Fig. 2
Effects of ramipril on tyrosine phosphorylation of PTP1B in liver (A), muscle (B), and fat tissue (C) of OLETF rats with or without ramipril treatment. The proteins were isolated with extraction buffer as described in methods and kept on ice. After centrifugation, aliquots of the supernatant were immunoprecipitated (IP) with anti-IR- and immunoblotted (IB) with anti-PTP1B antibodies (left panel of A, B, C), IP with anti-phosphotyrosine antibodies and IB with anti-PTP1B antibodies (right panel of A, B, C). Data were expressed as mean ± SE and represented by fold increase comparing with the control protein (#) that were normalized to 1. *P < 0.05
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Fig. 3
Effects of ramipril on Giα2-protein in live r (A), muscle (B), and adipose tissue (C) of OLETF rats with or without ramipril treatment. The proteins were isolated with extraction buffer as described in methods and kept on ice. After centrifugation, aliquots of the supernatant were immunoblotted (IB) with anti-Giα2 antibodies (upper blot), and were immunoprecipitated (IP) with anti-phosphotyrosine antibodies and immunoblotted with anti-Giα2 antibodies (lower blot). Data were expressed as mean ± SE and represented by fold increase comparing with the control protein (#) that were normalized to 1. *P < 0.05
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Fig. 4
Effects of ramipril on insulin-induced tyrosine phosphorylation of IR-, IRS-1, IRS-2, and p-85 in liver of OLETF rats with or without ramipril treatment. The proteins were isolated with extraction buffer as described in methods and kept on ice. After centrifugation, aliquots of the supernatant were immunoprecipitated (IP) with anti-IR- and immunoblotted (IB) with anti-IR-antibodies (A, upper blot), IP with anti-IR- and IB with anti-phosphotyrosine antibodies (A, lower blot), IP with anti-phosphotyrosine and IB with anti-IRS1 antibodies (B), IP with anti-phosphotyrosine and IB with IRS-2 antibodies (C), IP with anti-phosphotyrosine and IB with anti- p-85 antibodies (D). Data were expressed as mean ± SE and represented by fold increase comparing with the control protein (#) that were normalized to 1. *P < 0.05
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Fig. 5
Effects of ramipril on insulin-induced tyrosine phosphorylation of IR-, IRS-2, and p-85 in muscle of OLETF rats with or without ramipril treatment. Other procedures were similar in figure 4. Data were expressed as mean S.E., and represented by fold increase comparing with the control protein(#) that were normalized to 1. *P < 0.05
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Fig. 6
Effects of ramipril on insulin-induced tyrosine phosphorylation of IR-, IRS-2, and p-85 in muscle of OLETF rats with or without ramipril treatment. Other procedures were similar in figure 4. Data were expressed as mean ± SE and represented by fold increase comparing with the control protein (#) that were normalized to 1. *P < 0.05
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Fig. 7
Schematic representation of molecular mechanisms for insulin resistance (A) and improvement of insulin sensitivity by ramipril treatment (B). IR: insulin receptor, PI3-K: phosphatidylinositide-3 kinase, PTP1B: protein tyrosine phosphatase, IRS-1/-2: insulin receptor substrate-1/-2, PKA: protein kinase A.
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