Glucose uptake in rat extraocular muscles: effect of insulin and contractile activity

Abstract

Purpose: Extraocular muscles show specific adaptations to fulfill the metabolic demands imposed by their constant activity. One aspect that has not been explored is the availability of substrate for energy pathways in extraocular muscles. In limb muscles, glucose enters by way of GLUT1 and GLUT4 transporters in a process regulated by insulin and contractile activity to match metabolic supply to demand. This mechanism may not apply to extraocular muscles because their constant activity may require high basal (insulin- and activity-independent) glucose uptake. The authors tested the hypothesis that glucose uptake by extraocular muscles is not regulated by insulin or contractile activity.

Methods: Extraocular muscles from adult male Sprague-Dawley rats were incubated with 100 nM insulin or were electrically stimulated to contract (activity); glucose uptake was measured with 2-deoxy-d[1,2-(3)H]glucose. The contents of GLUT1, GLUT4, total and phosphorylated protein kinase B (Akt), phosphorylated AMP-activated protein kinase (AMPK), and glycogen synthase kinase 3 (GSK3) underwent Western blot analysis.

Results: Insulin and activity increased glucose uptake over the basal rate to 108% and 78%, respectively. GLUT1 and GLUT4 were detectable in extraocular muscles. Phosphorylated AKT/total AKT increased by twofold after insulin stimulation, but there was no change with activity. AMPK phosphorylation increased 35% with activity. Phosphorylated-GSK3/total GSK3 did not change with insulin or activity.

Conclusions: Glucose uptake in extraocular muscles is regulated by insulin and contractile activity. There is evidence of differences in the insulin signaling pathway that may explain the low glycogen content in these muscles.

Figure 1.

Glucose uptake in rat extraocular muscles. (A) Basal glucose uptake was 1.2 ± 0.3 μmol/g/h, comparable to values in other muscles. Insulin increased glucose uptake to 2.5 ± 0.3 μmol/g/h (*P = 0.001, Insulin greater than Basal). (B) Contractile activity increased glucose uptake in a similar manner: Basal, 1.3 ± 0.2 versus Activity, 2.4 ± 1.4 μmol/g/h; *P = 0.009. Values are mean ± SEM; n = 6 rats per group.

Figure 2.

GLUT1 and GLUT4, the glucose transporters typically found in skeletal muscle, were also present in the extraocular muscles. Insets: representative Western blots for both transporters. Each inset shows transporter content from two independent samples.

Figure 3.

AKT signaling in extraocular muscles stimulated with insulin and contractile activity. (A) Phosphorylated and total AKT in extraocular muscles under basal conditions and after insulin stimulation were measured by Western blot. The ratio of phosphorylated AKT to total AKT increased twofold in insulin-stimulated muscles (*P = 0.008, Insulin greater than Basal; n = 4 rats). Inset: representative Western blots (Basal, left lane; Insulin, right lane) for phosphorylated AKT (P-AKT, top) and total AKT (middle). The Ponceau S–stained membrane (bottom) demonstrates equal protein loading. (B) Phosphorylated and total AKT in extraocular muscles under basal conditions and after activity. Contractile activity did not change the ratio of phosphorylated AKT to total AKT (n = 4 rats). Inset: representative Western blots (Basal, left lane; Activity, right lane) for phosphorylated AKT (P-AKT, top) and total AKT (middle). The Ponceau S–stained membrane (bottom) demonstrates equal protein loading.

Figure 4.

GSK3 signaling in extraocular muscles after insulin and contractile activity. (A) Phosphorylated and total GSK3 under basal conditions and after insulin stimulation. Insulin did not change the phosphorylated to total GSK3 ratio (n = 4 rats). Inset: representative Western blots (Basal, left lane; Insulin, right lane) for phosphorylated GSK3 (P-GSK3, top) and total GSK3 (middle). The Ponceau S–stained membrane (bottom) demonstrates equal protein loading. (B) Phosphorylated and total GSK3 under basal conditions and after contractile activity. The phosphorylated to total GSK3 ratio was not changed by activity (n = 4 rats). Inset: representative Western blots (Basal, left lane; Activity, right lane) for phosphorylated GSK3 (P-GSK3, top) and total GSK3 (middle). The Ponceau S–stained membrane demonstrates equal protein loading (bottom).

Figure 5.

AMPK signaling in extraocular muscles after contractile activity. Phosphorylated AMPK under basal and after contractile activity. Phosphorylated AMPK increased 35% in muscles stimulated with contractile activity. (*P = 0.019, Activity greater than Basal; n = 5 rats). Inset: representative Western blots (Basal, left lane; activity, right lane) for phosphorylated AMPK. The Ponceau S–stained membrane (bottom) demonstrates equal protein loading.