Insulin down-regulates the expression of ubiquitin E3 ligases partially by inhibiting the activity and expression of AMP-activated protein kinase in L6 myotubes

Abstract

While insulin is an anabolic hormone, AMP-activated protein kinase (AMPK) is not only a key energy regulator, but it can also control substrate metabolism directly by inducing skeletal muscle protein degradation. The hypothesis of the present study was that insulin inhibits AMPK and thus down-regulates the expression of the ubiquitin E3 ligases, muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1) in skeletal muscle cells. Differentiated L6 myotubes were treated with 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) and/or compound C to stimulate and/or block AMPK respectively. These treatments were also conducted in the presence or absence of insulin and the cells were analysed by western blot and quantitative real-time PCR. In addition, nucleotide levels were determined using HPLC. The activation of AMPK with AICAR enhanced the mRNA levels of MAFbx and MuRF1. Insulin reduced the phosphorylation and activity AMPK, which was accompanied by reduced MAFbx and MuRF1 mRNA levels. Using a protein kinase B (PKB/Akt) inhibitor, we found that insulin regulates AMPK through the activation of Akt. Furthermore, insulin down-regulated AMPK α2 mRNA. We conclude that insulin inhibits AMPK through Akt phosphorylation in L6 myotubes, which may serve as a possible signalling pathway for the down-regulation of protein degradation. In addition, decreased expression of AMPK α2 may partially participate in inhibiting the activity of AMPK.

Keywords: AMP-activated protein kinase (AMPK); insulin; muscle RING finger 1 (MuRF1); muscle atrophy F-box (MAFbx); myotubes.

Figure 1. Effects of AICAR and compound C on p-AMPK, p-ACC, MAFbx and MuRF1

Effects of AICAR and compound C (CC) on p-AMPK, p-ACC (A) and mRNA expression of MAFbx and MuRF1 (B) in L6 myotubes. L6 myotubes were pre-treated with 20 μM CC or without CC for 1 h, followed by treatment with AICAR (1 mM) for 30 min (A) or 24 h (B). (A) After stimulation with AICAR for 30 min, p-AMPK at Thr¹⁷² and p-ACC at Ser⁷⁹ were measured by western blotting, with GAPDH as the internal standard. Shown are representative blots from three independent experiments. (B) At 24 h after AICAR treatment, extracted RNA from L6 myotubes were assayed with qRT-PCR. Data were normalized to GAPDH and the values for the control group set at 1.0. Data are expressed as means ± S.D. (n=3). *P<0.05 compared with control. #P<0.05 compared with AICAR treatment group.

Figure 2. Effects of insulin on p-AMPK, p-ACC, MAFbx and MuRF1

Effects of insulin on p-AMPK, p-ACC (A) and mRNA expression of MAFbx and MuRF1 (B) in L6 myotubes. L6 myotubes were pre-treated with AICAR (1 mM), compound C (CC; 20 μM) or not with AICAR and CC for 1 h, followed by addition of insulin (100 nM) for 30 min (A) or 24 h (B). (A) At 30 min, p-AMPK and p-ACC were measured by western blotting, with GAPDH as the internal standard. Shown are representative blots from three independent experiments. (B) At 24 h, extracted RNA from L6 myotubes were assayed with qRT-PCR. Data were normalized to GAPDH and the values for the control group set at 1.0. Data are expressed as means ± S.D. (n=3). *P<0.05 compared with control. #P<0.05 compared with insulin only treatment group.

Figure 3. Effect of Akt inhibitor on inhibition of p-AMPK and p-ACC by insulin

The effect of Akt inhibitor (Akt inhibitor IV) on inhibition of p-AMPK and p-ACC by insulin in L6 myotubes. L6 myotubes were pre-treated with Akt inhibitor IV (1 μM) for 1 h, followed by addition of insulin (100 nM) or without insulin for 30 min. p-AMPK, p-ACC and phosphor-Akt (p-Akt) were measured by western blotting, with GAPDH as the internal standard. Shown are representative blots from three independent experiments.

Figure 4. Effect of insulin on AMPK α1 and α2

Effects of insulin on mRNA expression of AMPK α1 and α2. L6 myotubes were either untreated (control) or pre-treated with 1 mM AICAR or 20 μM CC or not with AICAR and CC for 1 h, followed by stimulation with 100 nM insulin for 24 h. Extracted RNA from L6 myotubes were assayed with qRT-PCR. Data were normalized to GAPDH and the values for the control group set at 1.0. Values are expressed as means ± S.D. (n=3). *P<0.05 compared with control. #P<0.05 compared with insulin only group.

Figure 5. Insulin has no influece on AMP–ATP ratio, ADP–ATP ratio and EC

Effects of insulin exerted no influence on AMP–ATP ratio, ADP–ATP ratio and EC 1 h after the treatment. The differentiated L6 myotubes were treated with insulin (100 nM) for 1 h. The nts were extracted from the treated L6 myotubes and determined with HPLC. (A) The order of eluted nts was ATP (2.5 min), ADP (4 min) and AMP (5.3 min). (B) AMP–ATP ratio, ADP–ATP ratio and EC are expressed as means ± S.D. (n=2).