Insulin-Stimulated Muscle Glucose Uptake and Insulin Signaling in Lean and Obese Humans

Abstract

Purpose: Skeletal muscle is the primary site for insulin-stimulated glucose disposal, and muscle insulin resistance is central to abnormal glucose metabolism in obesity. Whether muscle insulin signaling to the level of Akt/AS160 is intact in insulin-resistant obese humans is controversial.

Methods: We defined a linear range of insulin-stimulated systemic and leg glucose uptake in 14 obese and 14 nonobese volunteers using a 2-step insulin clamp (Protocol 1) and then examined the obesity-related defects in muscle insulin action in 16 nonobese and 25 obese male and female volunteers matched for fitness using a 1-step, hyperinsulinemic, euglycemic clamp coupled with muscle biopsies (Protocol 2).

Results: Insulin-stimulated glucose disposal (Si) was reduced by > 60% (P < 0.0001) in the obese group in Protocol 2; however, the phosphorylation of Akt and its downstream effector AS160 were not different between nonobese and obese groups. The increase in phosphorylation of Akt2 in response to insulin was positively correlated with Si for both the nonobese (r = 0.53, P = 0.03) and the obese (r = 0.55, P = 0.01) groups. Total muscle GLUT4 protein was 17% less (P < 0.05) in obese subjects.

Conclusions: We suggest that reduced muscle glucose uptake in obesity is not due to defects in the insulin signaling pathway at the level of Akt/AS160, which suggests there remain significant gaps in our knowledge of muscle insulin resistance in obesity. Our data imply that models of acute lipotoxicity do not replicate the pathophysiology of obesity.

Keywords: Akt signaling; glucose uptake; insulin clamp; insulin resistance; obesity; skeletal muscle.

Figure 1.

Relationships between plasma insulin concentrations and whole body and leg glucose disposal rates. Correlation between insulin concentrations and glucose disposal rates of nonobese and obese volunteers measured as mg • kg FFM⁻¹ • min⁻¹. A, Protocol 2 Whole body glucose disposal rates and leg glucose uptakes linearity. B, correlation between whole-body and leg glucose disposal (r = 0.80). C, Protocol 1 and Protocol 2 whole-body glucose disposal rates and fitness of linearity within range of insulin concentration. D, Protocol 2 glucose infusion rates from nonobese and obese during 5-hour hyperinsulinemic-euglycemic clamp.

Figure 2.

Early onset activation of the insulin signaling pathway. Correlation between the 30-minute and 5-hour fold increase from baseline in phosphorylation of Akt (Ser⁴⁷³), Akt2 (Ser⁴⁷⁴), AS160 (Thr⁶⁴²), and GSK3β(Ser⁹) in nonobese and obese volunteers. Muscle biopsies were collected at just prior to insulin clamp and 30 minutes and 5 hours after starting the clamp. A, The fold increase values for Akt (Ser⁴⁷³) phosphorylation from the 2 biopsies were correlated (r = 0.76, P ≤ 0.001) B, The fold increase values for Akt2 (Ser⁴⁷⁴) phosphorylation from the 2 biopsies were correlated (r = 0.75, P ≤ 0.001) C, The fold increase in AS160 (Thr⁶⁴²) phosphorylation from the 2 biopsies correlated (r = 0.70, P ≤ 0.001). D, The fold increase in GSK3β (Ser⁹) phosphorylation from the 2 biopsies were positively correlated (r = 0.55, P ≤ 0.001)

Figure 3.

Insulin signaling response in nonobese and obese subjects during a hyperinsulinemic-euglycemic clamp in relation to muscle insulin sensitivity. Akt (Ser⁴⁷³) (panel A), Akt2(Ser⁴⁷⁴) (panel B), AS160 (Thr⁶⁴²) (panel C), and GSK3β (Ser⁹) (panel D) phosphorylation was measured by means of capillary Western blot from vastus lateralis biopsies at baseline and the end of a 5-hour insulin clamp. Content of pan Akt and pAkt, pan AS160 and pAS160, as well as pan GSK3β and p GSK3β were assessed relative to tissue weight. The fold increase in the phosphorylation was calculated as a ratio of pSer^473/474 Akt/panAkt, pSer⁴⁷⁴Akt/Akt2, pAS160/panAS160, and pGSK3β/panGSK3β at 5 hours over the ratio at baseline. Insulin sensitivity (Si) is expressed as mg•kg FFM⁻¹•min⁻¹•µIU/mL⁻¹. There was not a statistically significant difference between groups in the relationships of Akt, AS160, or GSK3β fold increase and Si.

Figure 4.

Total GLUT4 protein content of nonobese and obese subjects. Total GLUT4 protein content was measured in muscle biopsy samples. The average of the 3 biopsies was taken per subject. The 17% reduction in GLUT4 total protein in obese compared with nonobese groups is significant (P = 0.047).

Figure 5.

VO₂ peak association with Akt2 response in nonobese subjects during a hyperinsulinemic-euglycemic clamp. Association between VO₂ peak and Akt2 specific phosphorylation for the nonobese group at 30 minutes (r = 0.59, P = 0.016) and at 5 hours (r = 0.41, P = 0.11). There was no association between either Akt phosphorylation and VO₂ peak for the obese group or in the responses of AS160 and GS3Kβ and VO₂ peak.