Acute and chronic leptin treatment mediate contrasting effects on signaling, glucose uptake, and GLUT4 translocation in L6‐GLUT4myc myotubes

Abstract

We have previously shown that in L6-GLUT4myc rat skeletal muscle cells, acute treatment with leptin reduced insulin-stimulated glucose uptake without altering insulin-stimulated GLUT4 translocation. In contrast, we show here that the ability of leptin to increase phosphorylation of its receptor and to reduce insulin-stimulated glucose uptake was lost in cells that were continuously exposed to leptin for 24 h. This desensitization correlated with an increase in expression of suppressor of cytokine signaling-3 (SOCS-3). Time course analysis demonstrated that the transition from acute to chronic effects of leptin occurs after 2 h. The desensitization of leptin action at 24 h was not reversed by 30 min washout period prior to re-exposing cells to leptin. However, despite insulin-stimulated glucose uptake being unaffected upon 24 h preincubation with leptin, a small but significant decrease (37%) in insulin-stimulated GLUT4 translocation and phosphorylation of Akt on T308 was detected. Insulin-stimulated phosphorylation of Akt on S473 or of p38 MAPK were unaffected. These results suggest that the chronic leptin treatment leads to desensitization of leptin signaling yet can simultaneously decrease the ability of insulin to phosphorylate Akt on T308 and translocate GLUT4. However, this does not manifest as a reduction in total glucose uptake into L6 myotubes.

Supplementation of pyruvate prevents palmitate-induced impairment of glucose uptake in C2 myotubes

“…However, the decreased ability of insulin to phosphorylate Akt on T308 and induce GLUT4 translocation did not result in reduced glucose uptake, which suspected the exclusive role of GLUT4 translocation in glucose uptake (Tajmir et al, 2003). In particular, a reduction of intrinsic GLUT4 activity has been suggested to be a contributor to insulin resistance in obese diabetic subjects (Alkhateeb et al, 2007).…”

Globular adiponectin increases GLUT4 translocation and glucose uptake but reduces glycogen synthesis in rat skeletal muscle cells

“…We employed cells transfected to stably overexpress GLUT4 harbouring a myc epitope on the first exofacial loop of the transporter (L6 GLUT4myc cells; a kind gift from A. Klip, The Hospital for Sick Children, Toronto, ON, Canada). This facilitated accurate quantitative analysis of GLUT4 translocation in intact cells [14,17,18].…”

“…Myotubes were subsequently washed twice, and glucose transport was assayed in HEPES-buffered saline solution (140 mmol/l NaCl, 20 mmol/l HEPES-Na, 2.5 mmol/l MgSO 4 , 1 mmol/l CaCl 2 , 5 mmol/l KCl, pH 7.4) containing 10 μmol/l 2-deoxyglucose (18,500 Bq/ml 2-deoxy-D-[ 3 H]glucose) as described previously [17]. For kinetic measurements, cells were grown and differentiated in 96-well plates and volumes were adjusted accordingly.…”

“…Levels of GLUT4myc at the cell surface were measured by an antibody-coupled colorimetric assay as described previously [17,21]. Briefly, L6 myotubes were cultivated in 24-well plates and serum-starved for 4 h before being incubated with or without gAd (60 μmol/l, equivalent to 1 μg/ml, 2 h) and/or insulin (100 nmol/l, 20 min) or AICAR (1 mmol/l, 30 min).…”

Regulation of insulin signalling, glucose uptake and metabolism in rat skeletal muscle cells upon prolonged exposure to resistin

“…Cell culture L6-GLUT4myc myoblasts (a kind gift from A. Klip, The Hospital for Sick Children, Toronto, ON, Canada) were cultured as described previously [26] in α-MEM supplemented with 10% (v/v) FBS and 1% antibiotic/antimycotic solution (100 units/ml penicillin, 100 μg/ml streptomycin, 250 ng/ml amphotericin B) in a humidified atmosphere of 95% air and 5% CO 2 at 37°C. Cells were passaged before reaching confluence.…”