A E Clark

Chronic insulin effects on insulin signalling and GLUT4 endocytosis are reversed by metformin.

Decreases in insulin-responsive glucose transport and associated levels of cell surface GLUT4 occur in rat adipocytes maintained in culture for 20 h under hyperinsulinaemic and hyperglycaemic conditions. We have investigated whether this defect is due to reduced signalling from the insulin receptor, GLUT4 expression or impaired GLUT4 trafficking. The effects of chronic insulin treatment on glucose transport and GLUT4 trafficking were ameliorated by inclusion of metformin in the culture medium. In comparison with the ic insulin treatment attenuated changes in signalling processes leading to glucose transport. These included insulin receptor tyrosine phosphorylation, phosphoinositide 3-kinase activity and Akt activity, which were all reduced by 60-70%. Inclusion of metformin in the culture medium prevented the effects of the chronic insulin treatment on these signalling processes. In comparison with cells maintained in culture without insulin, the total expression of GLUT4 protein was not significantly altered by chronic insulin treatment, although the level of GLUT1 expression was increased. Trafficking rate constants for wortmannin-induced cell-surface loss of GLUT4 and GLUT1 were assessed by 2-N-4-(1-azi-2, 2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannose-4-yloxy)-2-propyla min e (ATB-BMPA) photolabelling. In comparison with cells acutely treated with insulin, chronic insulin treatment resulted in a doubling of the rate constants for GLUT4 endocytosis. These results suggest that the GLUT4 endocytosis process is very sensitive to the perturbations in signalling that occur under hyperinsulinaemic and hyperglycaemic conditions, and that the resulting elevation of endocytosis accounts for the reduced levels of net GLUT4 translocation observed.

The effects of insulin on the level and activity of the GLUT4 present in human adipose cells

SummaryHuman adipose cells are much less responsive to insulin stimulation of glucose transport activity than are rat adipocytes. To assess and characterize this difference, we have determined the rates of 3-O-methyl-D-glucose transport in human adipose cells and have compared these with the levels of glucose transporter 4 (GLUT4) assessed by using the bis-mannose photolabel, 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos-4-yloxy)-2-propylamine, ATB-BMFA. The rates of 3-O-methyl-D-glucose transport and the cell-surface level of GLUT4 are very similar in the human and rat adipocyte in the basal state. The Vmax for 3-O-methyl-D-glucose transport in fully insulin-stimulated human adipose cells is 15-fold lower than in rat adipose cells. Photolabelling of GLUT4 suggests that this low transport activity is associated with a low GLUT4 abundance (39·104 sites/cell; 19.9·104 sites at the cell surface). The turnover number for human adipose cell GLUT4 (5.8·104 min−1) is similar to that observed for GLUT4 in rat adipose cells and the mouse cell line, 3T3L1. Since 50% of the GLUT4 is at the cell surface of both human and rat adipose cells in the fully insulin-stimulated state, an inefficient GLUT4 exocytosis process cannot account for the low transport activity. The intracellular retention process appears to have adapted to release, in the basal state, a greater proportion of the total-cellular pool of GLUT4 to the cell surface of the larger human adipocytes. These cell-surface transporters are presumably necessary to provide the basal metabolic needs of the adipocyte. As a consequence of this adaptation to cell size and surface area, the residual intracellular-reserve pool of GLUT4 that is available to respond to insulin is lower in the human than in the rat adipocyte.

Effect of Insulin on GLUT4 Cell Surface Content and Turnover Rate in Human Skeletal Muscle as Measured by the Exofacial Bis-Mannose Photolabeling Technique

Insulin-stimulated glucose transport across the skeletal muscle cell membrane is a major regulatory step in postprandial glucose disposal. To estimate the total molar concentration of GLUT4 as well as the turnover rate of GLUT4 in human vastus lateralis muscles at the cell surface in the basal state and after insulin exposure, we have applied the sensitive exofacial bis-mannose photolabeling technique on in vitro incubated human skeletal muscle strips from healthy subjects. In addition, we have measured 3-O-methylglucose transport in other muscle strips prepared from the same surgically removed human skeletal muscle biopsies to compare glucose transport with cell surface level of GLUT4. Maximal in vitro insulin stimulation (2,400 pmol/l) resulted in a twofold increase compared with basal in both surface GLUT4 content (0.38 ± 0.05 vs. 0.19 ± 0.03 pmol/g wet muscle wt, P < 0.005) and 3-O-methylglucose transport (1.24 ± 0.13 vs. 0.63 ± 0.08 umol · ml−1 · h−1 P < 0.005). The insulin-induced increment in 3-O-methylglucose transport was strongly correlated with the insulin-induced increase in cell surface GLUT4 content (r2 = 0.91; P < 0.005). The calculated turnover rate of human skeletal muscle GLUT4 amounted to ∼8 × 104 min−1 at 35δC and was unaffected by insulin. In conclusion, maximal in vitro insulin stimulation of vastus lateralis muscle strips from healthy subjects resulted in a twofold rise in glucose transport as well as in cell surface content, whereas the turnover rate of GLUT4 was unaffected by insulin under the chosen experimental conditions.