Metformin promotes glucose metabolism in the absence of adenosine monophosphate-activated protein kinase activation

Abstract

Objective To examine whether metformin promotes glucose metabolism in the absence of adenosine monophosphate-activated protein kinase (AMPK) activation. Methods Glucose consumption and lactate production were used to evaluate the glucose-lowering and anaerobic glycolysis effects of metformin on HepG2 hepatocytes and C2C12 myotubes after treated with different concentration of metformin(2 mmol/L, 5 mmol/L) for 24 h. C2C12 cells were divided into 6 groups as blow: group 1(control group), group 2(2 mmol/L metformin treated for 24 h), group 3 (5 mmol/L metformin treated for 24 h), group 4 (10 μmol/L Compound C treated for 24 h), group 5 (10 μmol/L Compound C treated for 30 min then 2 mmol/L metformin treated for 24 h) and group 6(10 μmol/L Compound C treated for 30 min then 5 mmol/L metformin treated for 24 h). HepG2 cells were also divided into 6 groups as blow: group 1 (Ad-GFP transfected ), group 2(Ad-GFP transfected + 2 mmol/L metformin treated fro 24 h), group 3(Ad-GFP transfected + 5 mmol/L metformin treated fro 24 h), group 4(Ad-DN-AMPK transfected), group 5(Ad-DN-AMPK transfected + 2 mmol/L metformin treated for 24 h) and group 6 (Ad-DN-AMPK transfected + 5 mmol/L metformin treated for 24 h). AMPK phosphorylation and ACC phosphorylation were measured with Western blotting to assess the activity of AMPK pathway, and detected the glucose consumption and lactate production. Oxygen consumption rate of C2C12 myotubes was determined in Seahorse XF24 analyzer to estimate the production of ATP and function of respiratory chain complex I. Variance analysis was used to compare among multi-groups, and t test was used to compare two groups. Results (1) 2 mmol/L and 5 mmol/L metformin significantly increased glucose consumption(F=104.7, P<0.05) and lactate production (F=280.1, P<0.05) in HepG2 hepatocyte and also increased glucose consumption (F=38.53, P<0.05) and lactate production (F=172.90, P<0.05) in C2C12 myotubes in a dose-dependent manner. (2)AMPK and ACC phosphorylation were stimulated in group 2 and group 3 of HepG2 and C2C12 cells, but Compound C or Ad-DN-AMPK could inhibited the activity of AMPK. However, compare with the group 4, the group 5 and group 6 still had the ability to enhanced glucose consumption and lactate production by 39.5%, 62.6% and 39.0%, 61.0% in C2C12 cells(t=8.727, 21.38, 12.69, 27.31, all P<0.05) and 29.0%, 39.3% and 49.3%, 67.3% in HepG2 cells(t=9.96, 15.61, 23.32, 30.24, all P<0.05). (3)Metformin significantly inhibited the activity of mitochondrial respiratory chain complex I(t=36.08, P<0.05) and reduced ATP production(t=73.32, P<0.05). The inhibition of AMPK activity by Compound C failed to diminish the inhibition of activity of mitochondrial respiratory chain complex I(t=53.18, P<0.05)and the reduction of ATP production(t=246.10, P<0.05) which was stimulated by metformin. Conclusion Metformin promotes glucose metabolism by stimulation of glycolysis, which is probably resulted from inhibition of mitochondrial respiratory chain complex I, independent of AMPK activation. Key words: Metformin; Adenosine monophosphate-activated protein kinase; Glycolysis; Lactate