Genetic downregulation of AMPK- isoforms uncovers the mechanism by which metformin decreases FA uptake and oxidation in skeletal muscle cells

Bogachus LD, Turcotte LP. Genetic downregulation of AMPKisoforms uncovers the mechanism by which metformin decreases FA uptake and oxidation in skeletal muscle cells. Am J Physiol Cell Physiol 299: C1549–C1561, 2010. First published September 15, 2010; doi:10.1152/ajpcell.00279.2010.—Metformin is known to improve insulin sensitivity in part via a rise in AMP-activated protein kinase (AMPK) activity and alterations in muscle metabolism. However, a full understanding of how metformin alters AMPK1 vs. AMPK2 activation remains unknown. To study this question, L6 skeletal muscle cells were treated with or without RNAi oligonucleotide sequences to downregulate AMPK1 or AMPK2 protein expression and incubated with or without 5-aminoimidazole-4-carboxamide-1-D-ribofuranoside (AICAR) or metformin and/or insulin. In contrast to AICAR, which preferentially activated AMPK2, metformin preferentially activated AMPK1 in a doseand timedependent manner. Metformin increased (P 0.05) glucose uptake and plasma membrane (PM) Glut4 in a doseand time-dependent manner. Metformin significantly reduced palmitate uptake (P 0.05) and oxidation (P 0.05), and this was accompanied by a similar decrease (P 0.05) in PM CD36 content but with no change in acetyl-CoA carboxylase (ACC) phosphorylation (P 0.05). AICAR and metformin similarly increased (P 0.05) nuclear silent matingtype information regulator 2 homolog 1 (SIRT1) activity. Downregulation of AMPK1 completely prevented the metformin-induced reduction in palmitate uptake and oxidation but only partially reduced the metformin-induced increase in glucose uptake. Downregulation of AMPK2 had no effect on metformin-induced glucose uptake, palmitate uptake, and oxidation. The increase in SIRT1 activity induced by metformin was not affected by downregulation of either AMPK1 or AMPK2. Our data indicate that, in muscle cells, the inhibitory effects of metformin on fatty acid metabolism occur via preferential phosphorylation of AMPK1, and the data indicate that cross talk between AMPK and SIRT1 does not favor either AMPK isozyme.