Abstract
The mechanism for how metformin activates AMPK (AMP-activated kinase) was investigated in isolated skeletal muscle L6 cells. A widely held notion is that inhibition of the mitochondrial respiratory chain is central to the mechanism. We also considered other proposals for metformin action. As metabolic pathway markers, we focused on glucose transport and fatty acid oxidation. We also confirmed metformin actions on other metabolic processes in L6 cells. Metformin stimulated both glucose transport and fatty acid oxidation. The mitochondrial Complex I inhibitor rotenone also stimulated glucose transport but it inhibited fatty acid oxidation, independently of metformin. The peroxynitrite generator 3-morpholinosydnonimine stimulated glucose transport, but inhibited fatty acid oxidation. Addition of the nitric oxide precursor arginine to cells did not affect glucose transport. These studies differentiate metformin from inhibition of mitochondrial respiration and from active nitrogen species. Knockdown of adenylate kinase also failed to affect metformin stimulation of glucose transport. Hence, any means of increase in ADP appears not to be involved in the metformin mechanism. Knockdown of LKB1, an upstream kinase and AMPK activator, did not affect metformin action. Having ruled out existing proposals, we suggest a new one: metformin might increase AMP through inhibition of AMP deaminase (AMPD). We found that metformin inhibited purified AMP deaminase activity. Furthermore, a known inhibitor of AMPD stimulated glucose uptake and fatty acid oxidation. Both metformin and the AMPD inhibitor suppressed ammonia accumulation by the cells. Knockdown of AMPD obviated metformin stimulation of glucose transport. We conclude that AMPD inhibition is the mechanism of metformin action.
Highlights
Transfection of siRNA—L6 cells were transfected with 100 nM AK1/LKB1/AMP deaminase (AMPD) siRNA for 3 days by the following procedure: 104 cells/well were seeded in 12-well plates that were incubated with Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) for 2 h before transfection with 2 M siRNA solution in 1ϫ siRNA buffer or control
Metabolic Characterization of Metformin in L6 Skeletal Muscle Cell Cultures—To demonstrate characteristic metformin action in our model, we measured a number of parameters linked to metformin
For the key processes of glucose uptake and fatty acid oxidation, we incubated cells with substrate concentrations representing submaximal and maximal concentrations to assess this variable in metformin action
Summary
Materials—Metformin, and most chemicals, including BSA (bovine serum albumin, essentially fatty acid free), were obtained from Sigma. The cells were first incubated for 15 min with KHB, glucose, and other agents as indicated. At this point, the labeled deoxyglucose (0.6 Ci) was added to each well and the incubation continued for 45 min. The KHB for these incubations was supplemented with fatty acid-poor albumin dialyzed against the same buffer (three changes). Cells were washed with ice-cold KHB and lysed in 0.5 ml of KOH (30%). Aliquots of 0.2 ml were spotted onto Whatman 3T paper, and glycogen was precipitated by immersing the papers in ice-cold 66% (v/v) ethanol overnight. Transfection of siRNA—L6 cells were transfected with 100 nM AK1/LKB1/AMPD siRNA for 3 days by the following procedure: 104 cells/well were seeded in 12-well plates that were incubated with DMEM with 10% FBS for 2 h before transfection with 2 M siRNA solution in 1ϫ siRNA buffer or control
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