The GLP-1 receptor agonists exenatide and liraglutide activate Glucose transport by an AMPK-dependent mechanism

Abstract

Aims/hypothesis: Potentiation of glucose-induced insulin secretion is the main mechanism of exenatide (EXE) antidiabetic action, however, increased glucose utilization by peripheral tissues has been also reported. We here studied the effect of EXE on glucose uptake by skeletal muscle cells.

Methods: 2-deoxy-glucose (2DG) uptake and intracellular signal pathways were measured in rat L6 skeletal muscle myotubes exposed to 100 nmol/l EXE for up to 48 h. Mechanisms of EXE action were explored by inhibiting AMPK activity with compound C (CC, 40 μmol/l) or siRNAs (2 μmol/l).

Results: Time course experiments show that EXE increases glucose uptake up to 48 h achieving its maximal effect, similar to that induced by insulin, after 20 min (2- vs 2.5-fold-increase, respectively). Differently from insulin, EXE does not stimulate: (i) IR β-subunit- and IRS1 tyrosine phosphorylation and binding to p85 regulatory subunit of PI-3kinase; (ii) AKT activation; and (iii) ERK1/2 and JNK1/2 phosphorylation. Conversely, EXE increases phosphorylation of α-subunit of AMPK at Thr172 by 2.5-fold (p < 0.01). Co-incubation of EXE and insulin does not induce additive effects on 2DG-uptake. Inhibition of AMPK with CC, and reduction of AMPK protein expression by siRNA, completely abolish EXE-induced 2DG-uptake. Liraglutide, another GLP-1 receptor agonist, also stimulates AMPK phosphorylation and 2DG-uptake. Moreover, EXE stimulates 2DG-uptake also by L6 myotubes rendered insulin-resistant with methylglyoxal. Finally, EXE also induces glucose transporter Glut-4 translocation to the plasma membrane.

Conclusions/interpretation: In L6 myotubes, EXE and liraglutide increase glucose uptake in an insulin-independent manner by activating AMPK.

Keywords: AMPK; Exenatide; Glucose uptake; Insulin signaling; Liraglutide; Skeletal muscle cells.

Fig. 1

GLP-1R expression in different species and tissues: a Murine endocrine islet cells (αTC1 and βTC3), rat L6 myoblast (Bl) and myotube (Tb), human skeletal muscle (M) and baboon adipose (A) muscle (M) liver (L) and brain (B). Time course of EXE-induced 2DG uptake in L6 myotubes b Myotubes were stimulated with 100 nmol/l for 20 min, and 2, 4, 24, and 48 h and with 100 nmol/l insulin (INS) for 30 min as control. Effects of co-incubation with EXE and insulin on 2DG-glucose uptake c Myotubes were stimulated with either 100 nmol/l EXE, 100 nmo/l INS and both. Data are shown as mean ± SD of three independent experiments. (*p < 0.001, **p < 0.01 vs control)

Fig. 2

IRβ/IRS1/p85 signaling pathway in L6 myotubes. Cells were stimulated with either 100 nmol/l EXE, 100 nmol/l INS and both. The cells were then lysed and equal amounts of total proteins were immunoprecipitated with anti-IRS-1 antibody and immunoblotted with antiphosphotyrosine antibody (a, b) and anti-p85 subunit of PI 3-kinase (c). To normalize for protein levels the blots were stripped and re-probed with anti-IRS-1 antibody (A and C, top panel). Data are shown as mean ± SD of three independent experiments. (*p < 0.01 vs control)

Fig. 3

AMPKα activation in L6 myotubes. Cells were stimulated with either 100 nmol/l EXE, 100 nmol/l INS and both. After stimulation, whole lysates were immunoblotted with the phospho-specific antibodies: p-Akt S473 (a), p-GSK3β S21/9 (b), p-ERK1/2 T202/Y204 (c) and p-AMPK T172 (d). In order to normalize for protein levels, blots were stripped and re-probed with anti-AKT, anti ERK1/2, anti-GSK3β and anti-AMPK antibodies. Data are shown as mean ± SD of three independent experiments. (*p < 0.001 vs basal)

Fig. 4

Time-course and dose–response experiments of EXE effects on AMPK and ACC phosphorylation in L6 cells. Phosphorylated and total (a, b, c, upper panels) ACC S78/80 (a) and AMPKa T172 (b) protein levels were measured in cells exposed to EXE for increasing time periods. For the dose response curve (c) cells were incubated for 20 min with 0, 1, 10 and 100 nmol/l EXE. Thr172 phosphorylation and total levels of AMPK were shown. Each bar represents the mean ± SD of three independent experiments. (*P < 0.01 and **P < 0.02 vs. control)

Fig. 5

Effects of EXE (20 min or 48 h) and exendin 9–39 on AS-160, TBCD1D and AMPK in differentiated L6 cells. L6 myotubes were stimulated for 20 min with 100 nmol/l EXE, 100 nmol/l exendin 9–39 or the combination of both. Where indicated cells were stimulated with 100 nmol/l INS for 30 min and 100 nmol/l EXE for 20 min or 48 h. The blots were probed with phospho-specific AS160 T642 (a), TBCD1 (b) and AMPKα T172 (c). To normalize for protein levels, the blots were stripped and re-probed with β-actin antibodies. Each bar represents the mean ± SD of three independent experiments. (*p < 0.001 and **p < 0.01 vs. control; ^#p < 0.02 vs. EXE)

Fig. 6

EXE induces 2DG-uptake via AMPKα activation in L6 myotubes. L6 myotubes were transfected with specific AMPK siRNAs. After transfection, the cells were stimulated with 100 nmol/l EXE or 100 nmol/l INS. Proteins were then extracted and probed with AMPKα (a) and phospho-specific AMPKα T172 (b) antibodies. To normalize the blots for protein levels, the blots were stripped and re-probed with anti-tubulin (a, lower panel) and AMPKα antibodies (b, lower panel). 2DG-uptake was measured in cells after stimulation with INS or EXE (c). Data are represented as fold increase over control ± SD of three independent experiments (*p < 0.001, **p < 0.01 vs basal). 2DG-uptake was also measured in cells transfected with the AMPK siRNA 2 or control SiNR, before and after stimulation with EXE. Data are shown as fold increase over control ± SD of three independent experiments (d) (**p < 0.01 vs basal)

Fig. 7

Effects of EXE and Lira on AMPK and AKT phosphorylation in differentiated L6 cells. After 20 min- and 48 h incubation with EXE and Lira, both at 100 nmol/l, levels of Thr172 (a) and Ser 473 (b) phosphorylation were measured. 2DG-uptake (c) was measured in L6 myotubes treated for 30 min with 100 nmol/l INS and 100 nmol/l EXE and Lira for 20 min and 48 h. Each bar represents the mean ± SD of three independent experiments as shown. (*p < 0.01 and **p < 0.02 vs. control)

Fig. 8

Effects of EXE on MGO-induced insulin resistance in L6 myotubes. 2DG-uptake in cultured L6 myotubes pre-incubated with 2.5 mmol/l MGO and stimulated with INS and EXE both at 100 nmol/l. Data are shown as mean ± SD of three independent experiments (*p < 0.01 vs basal and MGO)