Muscle-specific vascular endothelial growth factor deletion induces muscle capillary rarefaction creating muscle insulin resistance

Abstract

Muscle insulin resistance is associated with a reduction in vascular endothelial growth factor (VEGF) action and muscle capillary density. We tested the hypothesis that muscle capillary rarefaction critically contributes to the etiology of muscle insulin resistance in chow-fed mice with skeletal and cardiac muscle VEGF deletion (mVEGF(-/-)) and wild-type littermates (mVEGF(+/+)) on a C57BL/6 background. The mVEGF(-/-) mice had an ~60% and ~50% decrease in capillaries in skeletal and cardiac muscle, respectively. The mVEGF(-/-) mice had augmented fasting glucose turnover. Insulin-stimulated whole-body glucose disappearance was blunted in mVEGF(-/-) mice. The reduced peripheral glucose utilization during insulin stimulation was due to diminished in vivo cardiac and skeletal muscle insulin action and signaling. The decreased insulin-stimulated muscle glucose uptake was independent of defects in insulin action at the myocyte, suggesting that the impairment in insulin-stimulated muscle glucose uptake was due to poor muscle perfusion. The deletion of VEGF in cardiac muscle did not affect cardiac output. These studies emphasize the importance for novel therapeutic approaches that target the vasculature in the treatment of insulin-resistant muscle.

FIG. 1.

VEGF-A protein levels in tissue homogenates from cardiac and skeletal muscle (A) and VEGF-A plasma concentration (B). Skeletal muscle VEGF levels were quantified in the gastrocnemius. Values are expressed as means ± SE (n = 5). *P ≤ 0.05 vs. mVEGF^+/+.

FIG. 2.

Arterial glucose (A) and glucose infusion rate (B) during the hyperinsulinemic-euglycemic clamp. Mice were fasted 5 h before the onset of the clamp. Blood glucose was maintained at ∼150 mg/dL during steady-state (80–120 min), and the time course is displayed to demonstrate quality of the clamp. Glucose (50%) was infused to maintain euglycemia. EndoR_a (C), whole-body R_d, (D), and insulin-stimulated glucose disposal (E) were determined during the hyperinsulinemic-euglycemic clamp. Basal values were determined from samples at −15 and −5 min before the onset of the clamp, and insulin clamp levels were calculated from steady-state values (80–120 min). Insulin-stimulated R_d was calculated by subtracting the basal R_d from the clamp R_d, which measures the increase in insulin-stimulated glucose disposal. Data are expressed as mean ± SE (n = 7–9). *P ≤ 0.05 vs. mVEGF^+/+.

FIG. 3.

Glucose tolerance was determined (A), and corresponding plasma insulin levels were quantified (B) on 5-h fasted mice. C: The area under the curve was determined from 0 to 30 min and normalized to fasting arterial glucose concentration. D: RT-PCR was performed on hepatic RNA extracts from mice fasted for 5 h for the relative expression of the gluconeogenic genes PEPCK and G6Pase. Values are expressed as mean ± SE (n = 5–6). *P ≤ 0.05 vs. mVEGF^+/+.

FIG. 4.

Insulin-stimulated R_g in skeletal (A) and cardiac (B) muscle. To quantify insulin-stimulated R_g, we performed a time control in a cohort of mVEGF^+/+ and mVEGF^−/− mice in which saline was infused in lieu of insulin to quantify basal R_g. The R_g values from the saline infusion were averaged and subtracted from the R_g values during the insulin clamp. Data are expressed as mean ± SE (n = 7–9). *P ≤ 0.05 vs. mVEGF^+/+.

FIG. 5.

Skeletal muscle and hepatic insulin signaling after the hyperinsulinemic-euglycemic clamp. Western blot analysis was performed on extracts of the gastrocnemius and liver for the phosphorylation (p) of Akt at Ser473 and total Akt. Infrared imaging was performed on 4–12% SDS-PAGE gel in skeletal muscle (A and C) and liver (E). A: Immunoprecipitation (IP) from gastrocnemius extracts were performed with total IRS-1; then, immunoblots (IB) were completed for p-IRS-1 at Tyr612 and the p85 subunit of PI3K. B: Insulin-stimulated p85 association with p-IRS-1 was quantified as the ratio of p85–to–p-IRS-1. Insulin activation of Akt (C and E) was quantified as the ratio of p-Akt to total Akt in skeletal muscle (D) and liver (F). GAPDH was used as a loading control. Data are expressed as mean ± SE (n = 5–6). *P ≤ 0.05 vs. mVEGF^+/+.

FIG. 6.

Skeletal muscle insulin action was assessed in the isolated soleus (A) and extensor digitorum longus (B). Mice were fasted for 5 h before the excision of the muscles. Values are expressed as mean ± SE (n = 6).