Overexpression of Rad in muscle worsens diet-induced insulin resistance and glucose intolerance and lowers plasma triglyceride level

Abstract

Rad is a low molecular weight GTPase that is overexpressed in skeletal muscle of some patients with type 2 diabetes mellitus and/or obesity. Overexpression of Rad in adipocytes and muscle cells in culture results in diminished insulin-stimulated glucose uptake. To further elucidate the potential role of Rad in vivo, we have generated transgenic (tg) mice that overexpress Rad in muscle using the muscle creatine kinase (MCK) promoter-enhancer. Rad tg mice have a 6- to 12-fold increase in Rad expression in muscle as compared to wild-type littermates. Rad tg mice grow normally and have normal glucose tolerance and insulin sensitivity, but have reduced plasma triglyceride levels. On a high-fat diet, Rad tg mice develop more severe glucose intolerance than the wild-type mice; this is due to increased insulin resistance in muscle, as exemplified by a rightward shift in the dose-response curve for insulin stimulated 2-deoxyglucose uptake. There is also a unexpected further reduction of the plasma triglyceride levels that is associated with increased levels of lipoprotein lipase in the Rad tg mice. These results demonstrate a potential synergistic interaction between increased expression of Rad and high-fat diet in creation of insulin resistance and altered lipid metabolism present in type 2 diabetes.

Fig. 1.

Construction of the Rad transgene and Rad expression in the Rad-Tg mice. (a) Schematic structure of the Rad transgene. (b) Southern blot analysis of the two transgenic lines with the highest expression of Rad. Equal amounts of tail DNA digested with EcoRI were blotted with a 1.1-kb MCK probe. (c) Western blotting with anti-Rad serum of quadriceps muscle lysates from the same two lines. Line 51 was used for all studies. (d) Western blot analysis comparing Rad protein level in different muscles from two pairs of WT and Rad-Tg mice. (e) Comparison of Rad protein level in the heart, lung, and skeletal muscle of a WT and a Rad-Tg mouse by Western blotting.

Fig. 2.

Effects of Rad overexpression of body weight and glucose and insulin levels. (a) Comparison between WT (black bars) and Rad-Tg (gray bars) mice body weight at 4–5 months of age on normal and high-fat diets. High-fat diet was started at 6 weeks of age. (b) Fasting blood glucose levels at 5–6 months of age. (c) Fasting insulin levels. After overnight fasting, mice were anesthetized, and blood samples were taken by retro-orbital bleeding. Each bar represent the mean ± SE of 26–39 mice.

Fig. 3.

Glucose tolerance tests. (a) Glucose tolerance test of WT (circles) and Rad-Tg (triangles) mice on normal chow (filled symbols) and high-fat (open symbols) diet. The GTTs were performed at the age of 5–6 months after overnight fasting. (b) Area under the curves for GTTs. Each bar represents the mean ± SE of 18–28 mice. *, P < 0.05 for Rad-Tg on high-fat diet vs. the other groups.

Fig. 4.

Insulin tolerance tests. Insulin tolerance tests for WT (circles) and Rad-Tg (triangles) mice on either normal chow (filled symbols) or high fat (open symbols) diets at 5 months of age. Results are expressed as mean percent of basal blood glucose concentration ± SEM from at least seven mice of each group.

Fig. 5.

Glucose transport in isolated muscles. (a) Glucose transport in isolated muscles from WT (open circle) and Rad-Tg (filled circle) fed a normal diet. Results are expressed as mean ± SEM of muscles from at least five mice. (b) Glucose transport on high-fat diet. Results are from at least five mice.

Fig. 6.

Amino acid transport into isolated muscles. (a) Amino acid transport in isolated muscles from WT (black bars) and Rad-Tg (hatched bars) fed a normal diet. Results are expressed as mean ± SEM of muscle from at least five male mice. (b) Amino acid transport on high-fat diet. Results are from at least five replicate studies.

Fig. 7.

Triglyceride and free fatty acid levels. (a) Fasting triglyceride levels of WT (black bars) and Rad-Tg (gray bars) mice on normal chow and high-fat diets at 5–6 months of age. Each bar represents the mean from 8–40 mice ± SE. *, P < 0.05 for the combined effect of diet and Rad overexpression. (b) FFA levels after overnight fasting at the same age. Each bar represents the mean of 9–18 mice ± SE.

Fig. 8.

LPL level in muscle. (a) Western blot analysis of LPL level in muscle. Thigh muscles were taken from mice after overnight fasting, homogenized, and blotted with anti-LPL antibody. (b) Quantitative analysis of LPL level in muscle by using imagequant. The data are expressed as mean ± SEM of Western blots from at least three mice.