Normal insulin-dependent activation of Akt/protein kinase B, with diminished activation of phosphoinositide 3-kinase, in muscle in type 2 diabetes

Abstract

To determine whether the serine/threonine kinase Akt (also known as protein kinase B) is activated in vivo by insulin administration in humans, and whether impaired activation of Akt could play a role in insulin resistance, we measured the activity and phosphorylation of Akt isoforms in skeletal muscle from 3 groups of subjects: lean, obese nondiabetic, and obese type 2 diabetic. Vastus lateralis biopsies were taken in the basal (overnight fast) and insulin-stimulated (euglycemic clamp) states. Insulin-stimulated glucose disposal was reduced 31% in obese subjects and 63% in diabetic subjects, compared with lean subjects. Glycogen synthase (GS) activity in the basal state was reduced 28% in obese subjects and 49% in diabetic subjects, compared with lean subjects. Insulin-stimulated GS activity was reduced 30% in diabetic subjects. Insulin treatment activated the insulin receptor substrate-1-associated (IRS-1-associated) phosphoinositide 3-kinase (PI 3-kinase) 6.1-fold in lean, 3.7-fold in obese, and 2.4-fold in diabetic subjects. Insulin also stimulated IRS-2-associated PI 3-kinase activity 2.2-fold in lean subjects, but only 1.4-fold in diabetic subjects. Basal activity of Akt1/Akt2 (Akt1/2) and Akt3 was similar in all groups. Insulin increased Akt1/2 activity 1.7- to 2. 0-fold, and tended to activate Akt3, in all groups. Insulin-stimulated phosphorylation of Akt1/2 was normal in obese and diabetic subjects. In lean subjects only, insulin-stimulated Akt1/2 activity correlated with glucose disposal rate. Thus, insulin activation of Akt isoforms is normal in muscle of obese nondiabetic and obese diabetic subjects, despite decreases of approximately 50% and 39% in IRS-1- and IRS-2-associated PI 3-kinase activity, respectively, in obese diabetic subjects. It is therefore unlikely that Akt plays a major role in the resistance to insulin action on glucose disposal or GS activation that is observed in muscle of obese type 2 diabetic subjects.

Figure 1

GDR during hyperinsulinemic, euglycemic clamp in lean, obese nondiabetic, and obese diabetic subjects. Data are mean ± SEM for 8–12 subjects per group. **P < 0.001 vs. lean. ‡P < 0.001 vs. obese nondiabetic.

Figure 2

Akt1/2 kinase activity (a), Akt1/2 phosphorylation (b), Akt1/2 protein levels (c), and Akt3 kinase activity (d) in skeletal muscle of lean, obese nondiabetic, and obese diabetic subjects. Twenty-six subjects had biopsies of vastus lateralis muscle performed before and at the end of a 3-hour hyperinsulinemic, euglycemic clamp. One additional lean and 1 additional obese subject have data only in the insulin-stimulated state, because biopsies before the clamp gave very low yields. (a and d) Akt kinase activity was measured in muscle lysates (500 μg) that were subjected to immunoprecipitation with either an antibody that recognizes both Akt1 and Akt2, or an Akt3 antibody. The immunoprecipitated pellets were assayed for kinase activity using Crosstide as substrate. In a, each ellipse shows values from a single subject; rectangles show mean ± SEM for 7–12 subjects per group. In d, bars represent mean ± SEM for 5 lean, 4 obese, and 8 diabetic subjects. (b and c) Proteins in muscle lysates (100 μg) were separated by SDS-PAGE on 8% gels and transferred to nitrocellulose membranes. Akt1/2 was visualized using immunoblotting. (b) Akt1/2 gel mobility shift in response to insulin in 3 representative subjects. (c) Representative autoradiogram of Akt1/2 levels in muscle of 6 subjects; bars show densitometric quantitation of Akt protein levels in 8–12 subjects per group. The autoradiograms in b and c are each representative of 3 different gels.

Figure 3

IRS-1–associated (a and b) and IRS-2–associated (c and d) PI 3-kinase activity and p85 protein levels (e) in skeletal muscle of lean, obese nondiabetic, and obese diabetic subjects. All subjects underwent a 3-hour hyperinsulinemic, euglycemic clamp, and biopsies of vastus lateralis muscle were performed before and at the end of the clamp. (a and c) PI 3-kinase activity was measured in muscle lysates (500 μg) that were subjected to immunoprecipitation with an IRS-1 (a) or IRS-2 (c) antibody. The origin and position of phosphatidylinositol 3-phosphate (PI3P) are indicated on the left. Each lane contains a sample from a single subject; the basal (–) and insulin-stimulated (+) samples from each subject were loaded in adjacent lanes. The autoradiogram is representative of 3 independent experiments. (b and d) Quantitation of IRS-1–associated (b) or IRS-2–associated (d) PI 3-kinase activity in muscle using a PhosphorImager. Data are mean ± SEM for 6–12 subjects per group. *P < 0.05 vs. insulin-stimulated obese nondiabetic subjects. §P < 0.01 vs. insulin-stimulated lean subjects. #P < 0.05 vs. insulin-stimulated lean subjects. (e) Proteins in muscle lysates (100 μg) were separated by SDS-PAGE on 8% gels and transferred to nitrocellulose membranes. The p85 regulatory subunit of PI 3-kinase was visualized by immunoblotting with a p85α antibody, and levels were densitometrically quantitated. The autoradiogram is representative of 3 independent experiments. Each lane contains muscle from 1 subject; bars show mean ± SEM for 6–12 subjects per group.

Figure 4

GS activity in skeletal muscle of lean, obese nondiabetic, and obese diabetic subjects. All subjects underwent a 3-hour hyperinsulinemic, euglycemic clamp; biopsies of vastus lateralis muscle were performed before and at the end of the clamp. GS activity was determined at a subsaturating concentration of substrate (0.3 mM UDP-glucose) and is expressed as fractional velocity (FV; GS activity at 0.1 mM glucose-6-phosphate divided by the activity at 10 mM glucose-6-phosphate). Data are mean ± SEM for 6–12 subjects per group. †Basal values in diabetic subjects are different from those in lean subjects (P < 0.05). §Insulin-stimulated values in diabetic subjects are different from those in lean subjects (P < 0.01). *Insulin-stimulated values in diabetic subjects are different from those in obese nondiabetic subjects (P < 0.05).

Figure 5

Relationship between insulin-stimulated Akt1/2 kinase activity in skeletal muscle and GDR in vivo in lean, obese nondiabetic, and obese diabetic subjects. Each square represents data from 1 subject.