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. 2011 Jan 7;286(1):1-11.
doi: 10.1074/jbc.M110.121806. Epub 2010 Nov 8.

Metformin Activates AMP Kinase Through Inhibition of AMP Deaminase

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Free PMC article

Metformin Activates AMP Kinase Through Inhibition of AMP Deaminase

Jiangyong Ouyang et al. J Biol Chem. .
Free PMC article

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.

Figures

FIGURE 1.
FIGURE 1.
AMPK in muscle. The proposed mechanisms for the action of metformin in stimulating AMPK can be mapped to this diagram. The enzyme needs to be phosphorylated (1), which is catalyzed principally by LBK1 in liver and muscle. Dephosphorylation (2) inactivates the enzyme; however, binding to AMP leads to activation (3) by making AMPK a poorer substrate for the phosphatase. Normally, energy demand (4) can increase ADP and consequently AMP concentration by means of increased flow through adenylate kinase (5). Alternatively, AMP may arise by an increased ADP due to interruption of mitochondrial energy supply, which would also increase AMP concentration involving flow-through adenylate kinase (5). The present work supports a new site of action for metformin, the AMP deaminase (6). Also shown are several metabolic processes known to be affected by AMPK activation via metformin: glucose uptake, fatty acid oxidation, glycogen synthesis, and lactate formation.
FIGURE 2.
FIGURE 2.
Metabolic effects of metformin in L6 myotubes. A, effect of metformin on AMPKα phosphorylation in L6 myotubes. L6 cells were incubated with or without 10 mm metformin for 2 h, and then AMPKα phosphorylation at Thr172 was measured by Western blot; AMPKα was used as a control. P-AMPKα, phosphorylated AMPKα. AMPKα and P-AMPKα were probed with separate selective antibodies. Con, control; Met, metformin. B, effect of metformin on glucose uptake in L6 myotubes. L6 cells were incubated with low (0.5 mm) or high (5 mm) glucose with or without 10 mm metformin for 2 h, and then glucose uptake was measured as described under “Experimental Procedures.” *, p < 0.05 versus 5 mm glucose (5 mm Glu); *, p < 0.05 versus 0.5 mm glucose (0.5 mm Glu); Met, metformin. C, effect of metformin on palmitate oxidation in L6 myotubes. L6 cells were incubated with 60 or 120 μm palmitic acid with or without 10 mm metformin for 3 h, and then palmitate oxidation was measured as described under “Experimental Procedures.” *, p < 0.05 versus 60 μm palmitic acid (60 μm PA) and 120 μm palmitic acid (120 μm PA), respectively; Met, metformin. D, effect of metformin on glycogen synthesis in L6 myotubes. L6 cells were incubated with 0.5 or 5 mm glucose with or without 10 mm metformin for 2 h, and then glycogen synthesis was measured as described under “Experimental Procedures.” *, p < 0.05 versus 0.5 mm glucose (0.5 mm Glu) and 5 mm glucose (5 mm Glu), respectively; Met, metformin. E, effect of metformin on glycogen content in L6 myotubes. L6 cells were incubated with 0.5 or 5 mm glucose with or without 10 mm metformin for 4 h, and then total glycogen content was measured as described under “Experimental Procedures.” Glu, glucose; Met, metformin. F, effect of metformin on lactate formation in L6 myotubes. L6 cells were incubated with 0.5 or 5 mm glucose with or without 10 mm metformin for 4 h, and then lactate formation was measured as described under “Experimental Procedures.” *, p < 0.05 versus 0.5 mm glucose (0.5 mm Glu) and 5 mm glucose (5 mm Glu), respectively. Data are expressed as per mg of cellular protein.
FIGURE 3.
FIGURE 3.
Effect of rotenone on fatty acid oxidation and glucose uptake. A, effect of different concentrations of rotenone on metformin-induced palmitate oxidation in L6 myotubes. L6 cells were incubated with 120 μm palmitic acid with or without different concentrations of rotenone and 10 mm metformin for 3 h, then palmitate oxidation was measured as described under “Experimental Procedures.” Control (dark bar) or with metformin (light bar); *, p < 0.05. B, effect of rotenone on glucose uptake in L6 cells. L6 cells were incubated with 5 mm glucose with or without 250 μm rotenone. Data are expressed as per mg of cellular protein.
FIGURE 4.
FIGURE 4.
Effect of arginine and reactive nitrogen species on metformin action. A, effect of different concentrations of arginine on glucose uptake in L6 myotubes. L6 cells were incubated with 5 mm glucose with or without different concentrations of arginine for 2 h. B, effect of arginase inhibitors on metformin-induced glucose uptake in L6 myotubes. L6 cells were incubated with 5 mm glucose with or without 10 mm metformin, and combined with 10 μm NOHA or 1 μm BEC for 2 h, and then glucose uptake was measured. Met, metformin; NOHA, Nω-hydroxy-nor-l-arginine; BEC, (S)-(2-boronoethyl)-l-cysteine, HCl. *, p < 0.05 versus control. C, effect of SIN-1 on glucose uptake in L6 myotubes. L6 cells were incubated with 5 mm glucose with or without 1 mm SIN-1 for 2 h, and then glucose uptake was measured as described under “Experimental Procedures.” *, p < 0.05 versus control. D, effect of SIN-1 on palmitate oxidation in L6 myotubes. L6 cells were incubated with 120 μm palmitic acid with or without 1 mm SIN-1 for 3 h, and then palmitate oxidation was measured as described under “Experimental Procedures.” *, p < 0.05 versus control. Data are expressed as per mg of cellular protein.
FIGURE 5.
FIGURE 5.
Effect of LKB1 on metformin action. A, LKB1 protein expression treated with LKB1 siRNA. B, effect of LKB1 siRNA on metformin-induced glucose uptake in L6 myotubes. After LKB1 siRNA transfection, L6 cells were incubated with 5 mm glucose with or without 10 mm metformin for 2 h, and then glucose uptake was measured as described under “Experimental Procedures.” Data are expressed as per mg of cellular protein. *, p < 0.05 versus no metformin.
FIGURE 6.
FIGURE 6.
Effect of adenylate kinase on metformin action. A, AK1 protein expression after ablation with AK1 siRNA. B, effect of adenylate kinase knockdown on potassium gluconate actions on glucose uptake. Potassium gluconate was present at 40 mm (40K), 80 mm (80K), and 120 mm (120K). C, control; A, AK1 siRNA; K, potassium gluconate. #, p < 0.05 compared with control. *, p < 0.05 compare with 80 and 120 K without AK1 siRNA. C, metformin-induced glucose uptake in L6 myotubes. After transfected by AK1 siRNA, L6 cells were incubated with 5 mm glucose with or without 10 mm metformin for 2 h, and then glucose uptake was measured as described under “Experimental Procedures.” Met, metformin. *, p < 0.05 compared with no metformin. Data are expressed as per mg of cellular protein.
FIGURE 7.
FIGURE 7.
Effect of metformin on AMP deaminase activity. A, the effect of 10 mm metformin on enzyme activity of isolated AMP deaminase at various substrate (AMP) concentrations. *, p < 0.05 versus control at different concentration of AMP, respectively. Data are expressed as per mg of soluble protein. B, effect of metformin on ammonia production in L6 myotubes. L6 cells were incubated with 5 mm glucose with or without 10 mm metformin for 2 h, and ammonia production was measured as described under “Experimental Procedures.” *, p < 0.05 versus control. Data are expressed as per mg of cellular protein.
FIGURE 8.
FIGURE 8.
Effect of EHNA, metformin, or both on glucose uptake in L6 myotubes. A, L6 cells were incubated with 5 mm glucose with or without 10 mm metformin, 100 μm EHNA, or both for 2 h, and then glucose uptake was measured as described under “Experimental Procedures.” *, p < 0.05 versus control. B, effect of EHNA, metformin, or both on palmitate oxidation in L6 myotubes. L6 cells were incubated with 120 μm palmitic acid with or without 10 mm metformin, 100 μm EHNA, or both for 3 h, and then palmitate oxidation was measured as described under “Experimental Procedures.” *, p < 0.05 versus control. C, effect of EHNA on AMPKα phosphorylation in L6 myotubes. L6 cells were incubated with or without 100 μm EHNA for 2 h, and then AMPKα phosphorylation at Thr172 was measured by Western blot; AMPKα was used as a control. P-AMPKα, phosphorylated AMPKα. AMPKα and P-AMPKα were probed with separate selective antibodies. D, effect of EHNA on ammonia production in L6 myotubes. L6 cells were incubated with 5 mm glucose with or without 100 μm EHNA for 2 h, and then ammonia production was measured as described under “Experimental Procedures.” *, p < 0.05 versus control. Data are expressed as per mg of cellular protein.
FIGURE 9.
FIGURE 9.
Effect of AMPD on metformin action. A, AMP deaminase protein expression treated with AMPD siRNA. B, effect of AMPD siRNA on metformin-induced glucose uptake in L6 myotubes. After AMPD siRNA transfection, L6 cells were incubated with 5 mm glucose with or without 10 mm metformin for 2 h, and then glucose uptake was measured as described under “Experimental Procedures.” Data are expressed as per mg of cellular protein. *, p < 0.05 versus no metformin.

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