GLUT4 and UBC9 Protein Expression Is Reduced in Muscle from Type 2 Diabetic Patients with Severe Insulin Resistance

Subgroups of patients with type 2 diabetes mellitus demand large insulin doses to maintain euglycemia. These patients are characterized by severe skeletal muscle insulin resistance and the underlying pathology remains unclear. The purpose of this study was to examine protein expression of the principal glucose transporter, GLUT4, and associated proteins in skeletal muscle from type 2 diabetic patients characterized by severe insulin resistance.Seven type 2 diabetic patients with severe insulin resistance (mean insulin dose 195 IU/day) were compared with seven age matched type 2 diabetic patients who did not require insulin treatment, and with an age matched healthy control group. Protein expression of GLUT4 and associated proteins was assessed in muscle and fat biopsies using standard western blotting techniques.GLUT4 protein expression was significantly reduced by ∼30 pct in skeletal muscle tissue from severely insulin resistant type 2 diabetic subjects, compared with both healthy controls and type 2 diabetic subjects that did not require insulin treatment. In fat tissue, GLUT4 protein expression was reduced in both diabetic groups. In skeletal muscle, the reduced GLUT4 expression in severe insulin resistance was associated with decreased ubiquitin-conjugating enzyme 9 (UBC9) expression while expression of GLUT1, TBC1D1 and AS160 was not significantly different among type 2 diabetic patients and matched controls.Type 2 diabetic patients with severe insulin resistance have reduced expression of GLUT4 in skeletal muscle compared to patients treated with oral antidiabetic drugs alone. GLUT4 protein levels may therefore play a role in the pathology behind type 2 diabetes mellitus among subgroups of patients, and this may explain the heterogeneous response to insulin treatment. This new finding contributes to the understanding of the underlying mechanisms for the development of extreme insulin resistance

GLUT4 protein content in fat biopsies.

<p>Protein content was assessed by western blotting analysis of GLUT4 and corrected for β-actin levels. Data are presented as mean + SE. *: P<0.05 versus control and T2DOAD.</p

Total protein content in muscle biopsies.

<p>Protein content was assessed by western blotting analysis of GLUT4 (A), UBC9 (B), GLUT1 (C), TBC1D1 (D), and AS160 (E). All protein expressions are corrected for β-actin levels. Data are presented as mean + SE. *: P<0.05 versus control and T2DOAD.</p

Comparison of clamp data (last 30 minutes of the hyperinsulinemic euglycemic clamp).

<p>All data are presented as means±SE or median (range) (n = 7). T2DSI  =  type 2 diabetes, severely insulin resistant, T2DOAD  =  type 2 diabetes, oral antidiabetics, diet.</p

Gene expression of GLUT4, SOCS3 and PTP1B.

<p>mRNA levels in skeletal muscle was measured by quantitative PCR using beta2-microglobulin as housekeeping gene. GLUT4 mRNA was reduced in skeletal muscle from both diabetic groups. GLUT4 gene expression is presented as mean + SE. SOCS3 and PTP1B expressions were not normally distributed and data are presented as median + 75% percentile *: P<0.05 versus control and T2DOAD.</p

Clinical and metabolic characteristics.

<p>All data are presented as means±SE or median (range), (n  = 7 unless indicated otherwise). T2DSI  =  type 2 diabetes, severely insulin resistant, T2DOAD  =  type 2 diabetes, oral antidiabetics, diet.</p><p>M-value, Insulin and C-peptide are mean values obtained during the last 30 minutes of the hyperinsulinemic euglycemic clamp.</p><p>P-value is result of ANOVA testing. Post-hoc test were performed when indicated:</p>a.<p>P<0.001 vs. T2DSI,</p>b<p>P<0.001 vs. controls,</p>c<p>P≤0.01 vs. controls,</p>d<p>P<0.05 vs. controls.</p