Development of insulin resistance and obesity in mice overexpressing cellular glutathione peroxidase

Abstract

Insulin resistance, a hallmark of type 2 diabetes, is associated with oxidative stress. However, the role of reactive oxygen species or specific antioxidant enzymes in its development has not been tested under physiological conditions. The objective of our study was to investigate the impact of overexpression of glutathione peroxidase 1 (GPX1), an intracellular selenoprotein that reduces hydrogen peroxide (H(2)O(2)) in vivo, on glucose metabolism and insulin function. The GPX1-overexpressing (OE) and WT male mice (n = 80) were fed a selenium-adequate diet (0.4 mg/kg) from 8 to 24 weeks of age. Compared with the WT, the OE mice developed (P < 0.05) hyperglycemia (117 vs. 149 mg/dl), hyperinsulinemia (419 vs. 1,350 pg/ml), and elevated plasma leptin (5 vs. 16 ng/ml) at 24 weeks of age. Meanwhile, these mice were heavier (37 vs. 27 g, P < 0.001) and fatter (37% vs. 17% fat, P < 0.01) than the WT mice. At 30-60 min after an insulin challenge, the OE mice had 25% less (P < 0.05) of a decrease in blood glucose than the WT mice. Their insulin resistance was associated with a 30-70% reduction (P < 0.05) in the insulin-stimulated phosphorylations of insulin receptor (beta-subunit) in liver and Akt (Ser(473) and Thr(308)) in liver and soleus muscle. Here we report the development of insulin resistance in mammals with elevated expression of an antioxidant enzyme and suggest that increased GPX1 activity may interfere with insulin function by overquenching intracellular reactive oxygen species required for insulin sensitizing.

Fig. 1.

Effect of GPX1 overexpression on GPX1 activity in liver and muscle of mice (24 weeks of age). Values are means ± SE (n = 5), and the asterisk indicates a difference (P < 0.05) between the two genotypes.

Fig. 2.

Effect of GPX1 overexpression on whole-blood glucose (A), body weight (B), and body fat (C) of mice. Whole-blood glucose was measured in mice fasted overnight by using tail blood. Body fat accretion in mice (24 weeks of age) was determined by dual-energy x-ray absorptiometry. Values are means ± SE (n = 4–6 per genotype), and the asterisks indicate differences (P < 0.05) between the two genotypes.

Fig. 3.

Effect of GPX1 overexpression on plasma insulin (A) and leptin concentrations (B) of mice at 8 and 24 weeks of age. Both hormones were measured by ELISA in mice fasted overnight. Values are means ± SE (n = 4–6 per genotype), and the asterisks indicate differences (P < 0.05) between the two genotypes at a given time point.

Fig. 4.

Effect of GPX1 overexpression on insulin sensitivity of mice at 8 (A) and 24 (B) weeks of age. After fasted mice (n = 8–13 per genotype) were given an injection (i.p.) of insulin (0.25 unit/kg), whole-blood glucose was measured at the indicated time points by using the Glucometer (Bayer). Values are expressed as the percentage of the initial glucose levels, and the asterisks indicate differences (P < 0.05) between genotypes within a given time point.

Fig. 5.

Effect of GPX1 overexpression on hepatic insulin receptor (β-subunit) (IRβ) protein (A), the insulin-stimulated phosphorylation of hepatic insulin receptor (β-subunit) (P-IRβ)(B), and the relative quantification of phosphorylated hepatic insulin receptor (β-subunit) (C). Fasted mice (24 weeks of age) were given an injection of saline (S) or insulin (I; 5.0 units/kg of body weight), and liver samples were collected 3 min after the injection. The Western blot analysis of insulin receptor protein was performed by separating freshly prepared tissue homogenates (50 μg of protein per lane) on an SDS/PAGE gel (10%) and probing with an antibody against insulin receptor (β-subunit, C-19; Santa Cruz Biotechnology). Phosphorylation of hepatic insulin receptor was detected by SDS/PAGE (10% gel) using the 4G10 antibody (Upstate Biotechnology) after the immunoprecipitation of tissue homogenate (200 μg protein per sample) with the above-described antibody against insulin receptor. The blots are representative of three independent experiments. The relative density values of phosphorylated hepatic insulin receptor (β-subunit) are means of six mice for each genotype (n = 6) from the three experiments, and the asterisk indicates a significant genotype difference (P < 0.05).

Fig. 6.

Effect of GPX1 overexpression on Akt protein (AKT), phospho-Akt (Thr³⁰⁸) (P-308), and phospho-Akt (Ser⁴⁷³) (P-473) in liver (A) and soleus muscle (B) and the relative quantification of P-308 and P-473 in both tissues (C). Fasted mice (24 weeks of age) were given an injection of saline (S) or insulin (I; 10 units/kg of body weight), and tissue samples were collected 8 min after the injection. The Western blot analyses were performed by separating freshly prepared tissue homogenates (30 μg of protein per lane) on an SDS/PAGE gel (10%) and probing with appropriate antibodies (Cell Signaling Technology). The blots are representative of three independent experiments. The relative density values of phosphorylated Akt are means of six to eight mice for each genotype (n = 6–8) from the three experiments, and the asterisk indicates a significant genotype difference (P < 0.05).