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.
Highlights
Insulin is a main anabolic hormone involved in the regulation of glucose, protein and lipid metabolisms [1,2,3]
AICAR induces the phosphorylation of AMP-activated protein kinase (AMPK) and increases the mRNA levels of muscle atrophy F-box (MAFbx) and MuRF-1 Previously, we found that at 1 mM or higher AICAR significantly stimulated the phosphorylation of AMPKα at Thr172 and the maximum phosphorylation of AMPK was attained 30 min after the addition of 1 mM AICAR
L6 myotubes were pre-treated with 20 μM compound C (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-acetyl CoA carboxylase (ACC) at Ser79 were measured by western blotting, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal standard
Summary
Insulin is a main anabolic hormone involved in the regulation of glucose, protein and lipid metabolisms [1,2,3]. Most of the effects of insulin depend on its ability to activate the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, subsequently phosphorylate glycogen synthase kinase 3-β (GSK3-β), tuberous sclerosis protein-2 (TSC2), bcl-2 antagonist of cell death (BAD), forkhead box O (Foxo) etc. Elevated AMP–ATP ratios makes AMPK vulnerable to phosphorylation at Thr172 and activation by several upstream kinases, such as serine/threonine kinase 11 (LKB1) or calcium/calmodulin-dependent protein kinase kinase (CaMKK)
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