Volume 127, Issue 6, Pages 1798-1808 (December 2004) The role of AMP-activated protein kinase in the action of ethanol in the liver  Min You, Michinaga Matsumoto, Christine M. Pacold, Won Kyoo Cho, David W. Crabb  Gastroenterology  Volume 127, Issue 6, Pages 1798-1808 (December 2004) DOI: 10.1053/j.gastro.2004.09.049 Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 1 Effect of AMPK activators on the activation of pSyn SRE reporter activity by ethanol in rat hepatoma McARH7777 cells. (A) McA RH7777 cells were plated in MEM supplement with 10% delipidated FBS and transfected with pSyn SRE reporter plasmid and internal control (pSV2CAT). Metformin or AICAR was added 24 hours later, and the cells were exposed to ethanol at the concentrations indicated, beginning at the same time. (B) McA RH7777 cells were transfected with a constitutively active form of AMPK mutant, AMPK α1312 (10 μg) or a catalytically inactive AMPK mutant, α1DNAMPK (10 μg), pSyn SRE reporter plasmid, and internal control (pSV2CAT); ethanol was added at the concentrations indicated 24 hours later. Forty-eight hours after transfection, the cells were harvested for assay of the reporter enzyme. Luciferase, and CAT activities were determined as described in the Materials and Methods section. The data are expressed as percentages of control (mean ± SE) from at least 3 experiments performed in duplicate. a, significant difference vs. control; b, significant difference compared with ethanol treatment. *P < .05, **P < .01 by 1-way ANOVA. Gastroenterology 2004 127, 1798-1808DOI: (10.1053/j.gastro.2004.09.049) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 2 Immunoblot analysis of SREBP-1 in cell nuclear extracts of rat hepatoma H4IIEC3 cells. H4IIEC3 cells were maintained in modified Eagle’s medium (MEM) supplemented with 10% fetal bovine serum. Cells were then washed with PBS, refed with MEM supplemented with 10% delipidated FBS and after incubation for 16 hours followed by treatment as follows at times indicated: E, ethanol (100 mmol/L); M, Metformin (1 mmol/L); and A, AICAR (0.5 mmol/L). After treatment, cells received 25 μg/mL ALLN and were incubated further for an additional 1 hour before being harvested. Aliquots of cell nuclear extracts (50 μg protein) were electrophoresed on 10% SDS gel. SREBP-1 protein was visualized by using anti-SREBP-1 antibody. Western blots were quantified by a PhosphorImager and ImageQuant software analysis. Mean ± SEM (n = 3 replicate assays) are shown. a, significant difference vs. control; b, significant difference compared with ethanol treatment. *P < .05, **P < .01 by 1-way ANOVA. Gastroenterology 2004 127, 1798-1808DOI: (10.1053/j.gastro.2004.09.049) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 3 Effect of ethanol on phosphorylation levels of AMPK and ACC in rat hepatoma H4IIEC3 cells and rat hepatocytes. H4IIEC3 cells were grown in modified Eagle’s medium (MEM) supplemented with 10% fetal bovine serum. Cells were then cultured in serum-free MEM for 16 hours, followed by treatment for times indicated. (A) C, control; E, ethanol (100 mmol/L); M, metformin (1 mmol/L); and E+M, ethanol (100 mmol/L) plus metformin (1 mmol/L). (B) Rat Hepatocytes were grown in DMEM containing 10% fetal bovine serum, 100 nmol/L insulin, 100 nmol/L dexamethasone, and 5 μg/mL transferrin for 4 hours. Cells were then cultured in serum-free DMEM for 16 hours followed by treatment. C, control; E, ethanol (100 mmol/L); M, metformin (1 mmol/L); and E+M, ethanol (100 mmol/L) plus metformin (1 mmol/L). (C) H4IIEC3 cells were treated as described in A. C, control; E, ethanol (100 mmol/L); M, metformin (1 mmol/L); and E+M, ethanol (100 mmol/L) plus metformin (1 mmol/L). (D) H4IIEC3 cells were cultured in serum-free MEM for 16 hours, followed by treatment as follows: C, control; Cy, cyanamide (0.1 mmol/L); 4Me, 4-methylpyrazole (0.1 mmol/L); E, ethanol (100 mmol/L); E+Cy, ethanol (100 mmol/L) + cyanamide (0.1 mmol/L); and E+4Me, ethanol (100 mmol/L) + 4-methylpyrazole (0.1 mmol/L). (E) H4IIEC3 cells were cultured in serum-free MEM for 16 hours, followed by treatment as follows: C, control; Cy+4Me, cyanamide (0.1 mmol/L) and 4-methylpyrazole (0.1 mmol/L); and Ach, acetaldehyde (250 μmol/L). Western blots were performed using anti-P-AMPKα, AMPKα, and anti-P-ACC antibodies. ALDH2 antibody was used to confirm equal loading. The data represent 3 or 4 replications. Gastroenterology 2004 127, 1798-1808DOI: (10.1053/j.gastro.2004.09.049) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 4 Effect of ethanol on rate of fatty acid β-oxidation in rat hepatocytes and rat hepatoma H4IIEC3 cells. (A) Rat hepatocytes were treated with ethanol at the concentrations indicated, metformin (1 mmol/L) or AICAR (0.5 mmol/L). (B) H4IIEC3 cells were transfected with a constitutively active form of AMPK mutant, AMPK α1312 (10 μg). The transfected cells were then treated with ethanol at concentrations indicated. Palmitic acid oxidation assays were performed as described in the Materials and Methods section. Values are expressed as nmol [14C]-palmitate oxidized/h/106 cells. a, significant difference vs. control; b, significant difference compared with ethanol treatment. *P < .05, **P < .01. Gastroenterology 2004 127, 1798-1808DOI: (10.1053/j.gastro.2004.09.049) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 5 Effect of ethanol feeding on hepatic AMPK, ACC activities, and liver malonyl-CoA content. (A) Western blots were performed on protein extracts from liver extracts of mice fed a low-fat diet without (LF) or with ethanol (LF+ ethanol) using anti-P-AMPKα or anti-AMPKα antibodies. Antiactin antibody was used to confirm equal loading. (B) AMPK activity was analyzed using livers of mice fed a low-fat diet without or with ethanol. The AMPK activity in the livers of control mice was 48.3 ± 3.2 pmol/min/mg protein (mean ± SD, n = 5 animals). (C) Western blots were performed using livers from mice fed a low-fat diet without (LF) or with ethanol (LF + ethanol) using anti-P-ACC antibody. (D) ACC activity was analyzed in the presence or absence of the indicated citrate concentrations using liver extracts from mice fed a low-fat diet without (LF) or with ethanol (LF + E). (E) Malonyl CoA concentrations were measured using livers from ethanol-fed mice and pair-fed control mice. *P < .05, **P < .01 by paired t test, compared with control. Gastroenterology 2004 127, 1798-1808DOI: (10.1053/j.gastro.2004.09.049) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 6 Potential role of AMPK in alcoholic fatty liver. Ethanol feeding may lead to inhibition of AMP-activated protein kinase activity, resulting in activation of sterol regulatory element-binding protein 1, which up-regulates hepatic lipid synthesis. Inhibition of hepatic AMP-activated kinase activity by ethanol feeding may lead to increased acetyl CoA carboxylase activity and enhance malonyl CoA levels, thereby inhibiting the rate of hepatic fatty acid oxidation via inhibition of carnitine palmitoytransferase I. Gastroenterology 2004 127, 1798-1808DOI: (10.1053/j.gastro.2004.09.049) Copyright © 2004 American Gastroenterological Association Terms and Conditions