Diabetes is accompanied by changes in the levels of proteins involved in endosomal GLUT4 trafficking in obese human skeletal muscle

Abstract

Introduction: The regulated delivery of the glucose transporter GLUT4 from intracellular stores to the plasma membrane underpins insulin-stimulated glucose transport. Insulin-stimulated glucose transport is impaired in skeletal muscle of patients with type-2 diabetes, and this may arise because of impaired intracellular trafficking of GLUT4. However, molecular details of any such impairment have not been described. We hypothesized that GLUT4 and/or levels of proteins involved in intracellular GLUT4 trafficking may be impaired in skeletal muscle in type-2 diabetes and tested this in obese individuals without and without type-2 diabetes.

Methods: We recruited 12 participants with type-2 diabetes and 12 control participants. All were overweight or obese with BMI of 25-45 kg/m² . Insulin sensitivity was measured using an insulin suppression test (IST), and vastus lateralis biopsies were taken in the fasted state. Cell extracts were immunoblotted to quantify levels of a range of proteins known to be involved in intracellular GLUT4 trafficking.

Results: Obese participants with type-2 diabetes exhibited elevated fasting blood glucose and increased steady state glucose infusion rates in the IST compared with controls. Consistent with this, skeletal muscle from those with type-2 diabetes expressed lower levels of GLUT4 (30%, p = .014). Levels of Syntaxin4, a key protein involved in GLUT4 vesicle fusion with the plasma membrane, were similar between groups. By contrast, we observed reductions in levels of Syntaxin16 (33.7%, p = 0.05), Sortilin (44%, p = .006) and Sorting Nexin-1 (21.5%, p = .039) and -27 (60%, p = .001), key proteins involved in the intracellular sorting of GLUT4, in participants with type-2 diabetes.

Conclusions: We report significant reductions of proteins involved in the endosomal trafficking of GLUT4 in skeletal muscle in obese people with type 2 diabetes compared with age- and weight-matched controls. These abnormalities of intracellular GLUT4 trafficking may contribute to reduced whole body insulin sensitivity.

Keywords: clinical medicine; diabetes; metabolic disease.

FIGURE 1

IST insulin sensitivity. Plasma glucose and plasma insulin were obtained in the fasted state prior to commencing the IST and compared between obese participants with and without T2D. Fasting results are shown in panels A and B. Plasma insulin and glucose samples were obtained every 10 min from 150 to 180 min of the IST and the mean calculated to determine steady state levels (SSPG and SSPI) which are shown in C and D respectively. Individual values are presented along with the mean and standard deviation (S.D). Panels A and B represent 12 people with T2D (Ob‐T2D) and 12 people without T2D (Ob); Panels C and D are from 11 participants without T2D as one patient withdrew after muscle biopsy but before the IST (see Methods.) Statistical analysis was performed using a two‐tailed unpaired t‐test, with significant differences indicated by *p < .0001.

FIGURE 2

GLUT4 levels in skeletal muscle from people with and without T2D. Vastus lateralis skeletal muscle biopsy samples were obtained from study participants in the fasted state. Samples were subjected to immunoblotting. (A) shows representative immunoblots from six participants without T2D (Ob) and six with T2D (Ob‐T2D) (labelled 1–6) for GLUT4, respectively, loaded as two separate amounts, 10 and 20 μg, along with similar amounts of 3 T3‐L1 adipocyte lysate for comparison. (B) is a box and whisker plot comparing the ratio of skeletal muscle GLUT4 to 3 T3‐L1 GLUT4 levels from all subjects with (Ob‐T2D) and without (Ob) T2D (n = 12) repeated three times on separate gels. Each point is the mean of triplicate determinations and thus represents an individual subject. Statistical analysis was performed using an unpaired t‐test. *p = .014

FIGURE 3

Syntaxin levels in skeletal muscle from people with and without T2D. Skeletal muscle samples (20 μg) from 6 participants (numbered 1–6) in each group were immunoblotted for Sx4 (panel A) and Sx16 (panel B) as indicated. (C and D) are box and whisker plots of the ratio of skeletal muscle Sx4 and Sx16, respectively, to 3 T3‐L1 levels in 12 people with and 12 people without T2D. Each point is the mean of triplicate determinations and thus represents an individual subject. Statistical analysis was performed using an unpaired t‐test (*p = .05; n.s., not significantly different, p = .37).

FIGURE 4

Levels of Sortilin in skeletal muscle from people with and without T2D. Skeletal muscle samples (20 μg) from 6 participants (numbered 1–6) in each group were probed for Sortilin as described in Figure 2. A typical immunoblot is shown in A and quantification of the entire dataset is presented in panel B as a box and whisker plot. Each point is the mean of triplicate determinations from each subject. Statistical analysis was performed using an unpaired t‐test, significant differences are shown by *p = .006).

FIGURE 5

Sorting nexins in skeletal muscle from people with and without T2D. Skeletal muscle samples (20 μg) from 6 participants (numbered 1–6) in each group were probed using anti‐SNX1 (panels A), anti‐SNX27 (panels B) or anti‐Vps35 antibodies (panel C). Typical blots (A–C) are shown, together with box and whisker plots of the entire data set (panels D–F). Each point is the mean of triplicate determinations from each study subject. Statistical significance between the groups is indicated by *p = .039, **p = .0001; n.s., not significantly different.