AMPK/AS160 mediates tiliroside derivatives-stimulated GLUT4 translocation in muscle cells

Abstract

Introduction: The Chinese herb Potentilla chinensis can reduce blood glucose level of diabetic mice. Tiliroside is the main effective component, but the detailed mechanism is not clear. Skeletal muscles play an important role in whole body glucose homeostasis. Insulin and exercise/contraction stimulate glucose uptake by muscle cells via redistribution of glucose transporter GLUT4 to the cell surface.

Materials and methods: We explored the effects of tiliroside derivatives on cell surface GLUT4 level (GLUT4 translocation) and the underlying mechanism in L6-GLUT4myc muscle cells.

Results: We showed that tiliroside derivatives D1-22 stimulated GLUT4myc translocation in L6-GLUT4myc skeletal muscle cells. Derivatives D1, D8 and D18 regulated GLUT4myc translocation in a time- and dose-dependent manner. Their effects on GLUT4 were additive with that of acute insulin stimulation. Moreover, they increased phosphorylated adenosine monophosphate-activated protein kinase (AMPK), but not protein kinase B (PKB, also called Akt). Their effects on GLUT4 were inhibited by Compound C. In addition, derivative D8 significantly stimulated AMPK and Akt substrate of 160 kDa (AS160) phosphorylation and GLUT4myc translocation in L6-GLUT4myc cells, but not in L6-AS160 4A-GLUT4myc cells.

Conclusion: Tiliroside derivatives D1, D8 and D18 stimulated GLUT4myc translocation by a mechanism different to that of insulin in skeletal muscle cells. The effect of derivative D8 on GLUT4myc translocation is mediated by AMPK/AS160 signaling pathway.

Keywords: AMPK; insulin resistance; skeletal muscle cells; type 2 diabetes.

Figure 1

Chemical structures of tiliroside (A) and 22 tiliroside derivatives (B).

Figure 2

Effects of derivatives D1, D8, and D18 on GLUT4myc translocation in L6-GLUT4myc myotubes. Dose-dependent (A) and time-dependent (B) measurements of GLUT4myc translocation following tiliroside derivative treatment. Myotubes were serum starved for 4 h followed by incubation with derivative D1, D8, or D18 for indicated dose and time. Cell surface GLUT4myc level was measured as detailed in the “Materials and methods” section. Results are the mean±SE of 4 independent experiments in triplicate. Note: *p<0.05, **p<0.01, ***p<0.001 vs basal (untreated). Abbreviation: SE, standard error.

Figure 3

Additivity of the derivatives with insulin and the role of AMPK in the effects of the derivatives on GLUT4 in L6-GLUT4myc myotubes. Myotubes were serum starved for 4 h followed by the indicated treatment with D1, D8 or D18 for 24 h or with insulin (Ins), metformin (Met) or Compound C, as detailed in the “Material and methods” section. The role of insulin in the derivatives-regulated cell surface GLUT4myc levels and immunoblotted for Akt and pAkt (A). Cell lysates were immunoblotted for AMPK, pAMPK, pACC and GLUT4 (B). The role of Compound C, which is the inhibitor of AMPK, in the derivatives-regulated cell surface GLUT4myc levels (C). The role of insulin in the derivatives D8 regulated cell surface GLUT4myc levels. Insulin resistance was induced by 25 mM glucose and 100 nM insulin for 24 h (D). Then the cells were treated with insulin or/and derivative D8 as A. Results are the mean±SE of 4 independent experiments. *p<0.05, **p<0.01, ***p<0.001 vs basal (untreated) or as indicated. Abbreviations: DMSO, dimethyl sulfoxide; SE, standard error; Bas, untreated basal.

Figure 4

Effects of derivatives on AS160 phosphorylation and GLUT4myc translocation in L6-GLUT4myc myotubes or L6-GLUT4myc myotubes stably expressing AS160-4A. Myotubes were treated with 1 µg/mL D8 for 24 h, or with 100 nM insulin for 10 min, respectively. (A) Immunoblots of pAMPK, pAS160, pAkt and actinin-1 (as loading control) in cell lysates. (B) Cell surface GLUT4myc levels. Results are the mean±SE of 4 independent experiments. Note: *p<0.05, **p<0.01, ***p<0.001 vs each basal (untreated) or as indicated. Abbreviations: Ins, insulin; SE, standard error; Bas, untreated basal.