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Originally published In Press as doi:10.1074/jbc.M408149200 on September 14, 2004

J. Biol. Chem., Vol. 279, Issue 46, 47898-47905, November 12, 2004
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AMP-activated Protein Kinase Is Required for the Lipid-lowering Effect of Metformin in Insulin-resistant Human HepG2 Cells*

Mengwei Zang{ddagger}§, Adriana Zuccollo{ddagger}§, Xiuyun Hou{ddagger}§, Daisuke Nagata§, Kenneth Walsh§, Haya Herscovitz¶, Peter Brecher{ddagger}§, Neil B. Ruderman||, and Richard A. Cohen{ddagger}§**

From the {ddagger}Vascular Biology Unit, §Whitaker Cardiovascular Institute, ||Diabetes and Metabolism Unit, Department of Medicine, and the Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts 02118

Received for publication, July 19, 2004 , and in revised form, September 7, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The antidiabetic drug metformin stimulates AMP-activated protein kinase (AMPK) activity in the liver and in skeletal muscle. To better understand the role of AMPK in the regulation of hepatic lipids, we studied the effect of metformin on AMPK and its downstream effector, acetyl-CoA carboxylase (ACC), as well as on lipid content in cultured human hepatoma HepG2 cells. Metformin increased Thr-172 phosphorylation of the {alpha} subunit of AMPK in a dose- and time-dependent manner. In parallel, phosphorylation of ACC at Ser-79 was increased, which was consistent with decreasing ACC activity. Intracellular triacylglycerol and cholesterol contents were also decreased. These effects of metformin were mimicked or completely abrogated by adenoviral-mediated expression of a constitutively active AMPK{alpha} or a kinase-inactive AMPK{alpha}, respectively. An insulin-resistant state was induced by exposing cells to 30 mM glucose as indicated by decreased phosphorylation of Akt and its downstream effector, glycogen synthase kinase 3{alpha}/{beta}. Under these conditions, the phosphorylation of AMPK and ACC was also decreased, and the level of hepatocellular triacylglycerols increased. The inhibition of AMPK and the accumulation of lipids caused by high glucose concentrations were prevented either by metformin or by expressing the constitutively active AMPK{alpha}. The kinase-inactive AMPK{alpha} increased lipid content and blocked the ability of metformin to decrease lipid accumulation caused by high glucose concentrations. Taken together, these results indicate that AMPK{alpha} negatively regulates ACC activity and hepatic lipid content. Inhibition of AMPK may contribute to lipid accumulation induced by high concentrations of glucose associated with insulin resistance. Metformin lowers hepatic lipid content by activating AMPK, thereby mediating beneficial effects in hyperglycemia and insulin resistance.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
AMP-activated protein kinase (AMPK)1 is a phylogenetically conserved intracellular energy sensor that has been implicated in the regulation of glucose and lipid homeostasis (1–4). AMPK is activated by physiological stimuli, such as exercise, muscle contraction, and hormones including adiponectin and leptin, as well as by pathological stresses, glucose deprivation, hypoxia, oxidative stress, and osmotic shock (2, 5). AMPK serine/threonine protein kinase is a heterotrimeric complex consisting of a catalytic subunit ({alpha}) and two regulatory subunits ({beta} and {gamma}) (5). Regulation of AMPK activity is complex; it involves allosteric activation by AMP, which increases during states of stress where ATP is depleted, and phosphorylation via the presumptive upstream activator AMPK kinase (6–9), which may also be allosterically activated by AMP (5). Moreover, phosphorylation of Thr-172 within the activation loop of the catalytic domain of the {alpha} subunit is necessary for AMPK activity because site-directed mutagenesis of Thr-172 to Ala completely abolishes AMPK activity (10, 11). Once activated, AMPK phosphorylates its downstream substrates to reduce ATP-consuming anabolic pathways, including cholesterol, fatty acid, and triacylglycerol synthesis, and increases ATP-generating catabolic pathways, including fatty acid oxidation and lipolysis. Phosphorylation by AMPK of two key substrates, 3-hydroxy-3-methylglutaryl-coenzyme A reductase and acetyl-CoA carboxylase (ACC) (12), which are the rate-limiting enzymes in cholesterol and fatty acid biosynthesis, respectively, results in their inactivation and thus reduces cellular ATP consumption during metabolic stress.

Type II diabetes is associated with hyperinsulinemia and insulin resistance leading to elevated hepatic glucose production, hyperglycemia, and hyperlipidemia (13, 14). Administration of the AMPK activator, 5-amino-4-imidazolecarboxamide riboside (AICAR), improves glucose tolerance and lipid profiles in the insulin-resistant Zucker rat (15), suggesting that AMPK activity regulates insulin sensitivity and the associated dyslipidemia. Metformin is an oral biguanide antidiabetic drug that improves insulin sensitivity and reduces plasma glucose and lipids in patients with type II diabetes (16–18). In fructose-fed rats with hyperinsulinemia and hyperglycemia, metformin decreases liver triacylglycerol and free fatty acid levels and increases lipoprotein lipase activity (19). Metformin may increase insulin sensitivity through up-regulation of insulin signaling as reflected by increased tyrosine phosphorylation of the insulin receptor and insulin receptor substrate 1 (20). However, the precise mechanism by which metformin lowers lipids is unknown. Recently, activation of AMPK by metformin has been shown to decrease glucose production and increase fatty acid oxidation in the liver (21, 22).

The present studies were performed to determine 1) the extent to which the effect of metformin on hepatocellular lipids is mediated by AMPK and 2) whether AMPK regulates lipid accumulation in insulin-resistant states. We utilized the cultured human hepatoma HepG2 cell line as a model system and presented biochemical evidence that AMPK serves as a critical regulator of hepatocellular lipid content. Metformin increased ACC phosphorylation in an AMPK-dependent manner, which in turn decreased intracellular triacylglycerol and cholesterol contents. Importantly, the ability of metformin to reduce lipids was mimicked by adenoviral-mediated expression of a constitutively active AMPK{alpha} and was blocked by a kinase-inactive AMPK{alpha}, suggesting that AMPK is required for the hypolipidemic actions of metformin. In addition, exposing cells to high glucose concentrations induced a model of insulin resistance in which AMPK was inhibited and lipids accumulated. Expression of the kinase-inactive AMPK{alpha} also led to triacylglycerol accumulation, suggesting a pivotal role for AMPK in lipid accumulation associated with insulin resistance. This study also provides a model for developing potential therapeutic agents to target AMPK in insulin resistance and dyslipidemia in type II diabetes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Metformin (1,1-dimethylbiguanide), Nonidet P-40, aprotinin, leupeptin, phenylmethylsulfonyl fluoride, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were purchased from Sigma. InfinityTM triacylglycerol and cholesterol reagents were from Thermo DMA (Louisville, CO). The cytotoxicity detection kit used to measure lactate dehydrogenase release was from Roche Applied Science. Phospho-AMPK{alpha} (Thr-172), phospho-Akt (Ser-473), and phospho-GSK3{alpha}/{beta} (Ser-21/9) antibodies were purchased from Cell Signaling Technology (Beverly, MA). AMPK antibodies against the {alpha}1 or {alpha}2 isoform were from Bethyl Laboratories, Inc. (Montgomery, TX). Phospho-ACC (Ser-79) antibody was purchased from Upstate Biotechnology (Lake Placid, NY). Anti-Myc (9E10) antibody and GSK3{beta} antibody were from BD Biosciences. {beta}-Actin antibody was from Abcam Inc. (Cambridge, MA). Anti-mouse and anti-rabbit antibodies conjugated to horseradish peroxidase were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum were purchased from Invitrogen. All other reagents were of analytical grade.

Cell Culture—Human hepatoma HepG2 cells were obtained from the American Type Culture Collection (Manassas, VA). HepG2 cells were cultured in DMEM containing 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 5.5 mM D-glucose. The cells were incubated in a humidified atmosphere of 5% CO2 at 37 °C and passaged every 3 days by trypsinization. For experiments, HepG2 cells were incubated in complete medium with 10% fetal bovine serum in 100-mm-diameter dishes, grown to 70% confluence, and maintained in serum-free DMEM overnight as described elsewhere (23–25). Cells were treated with metformin as indicated in the legends of Figs. 2, 3, 4, and 6. To ensure cell viability during prolonged incubation of metformin in serum-free medium, cell viability was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay performed according to the manufacturer's suggestions (Sigma). In addition, cytotoxicity was assessed by measuring lactate dehydrogenase release into the culture medium using the cytotoxicity detection kit (Roche Applied Science) as described previously (26). Under the conditions of our studies, treatment of HepG2 cells with metformin had no detectable cell toxicity.



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  		FIG. 2.
Metformin stimulates AMPK{alpha} and ACC phosphorylation and decreases intracellular lipid content in a dose- and time-dependent manner in cultured human hepatoma HepG2 cells. HepG2 cells were quiesced in serum-free medium overnight and treated with increasing doses of metformin (0.5–2 mM) for the indicated times. A, representative immunoblots of AMPK and ACC phosphorylation by metformin. Cell lysates were prepared, and 20–50 µg of protein were resolved by 8% SDS-PAGE as described under "Materials and Methods." The phosphorylation of AMPK and ACC was immunoblotted with anti-phospho-Thr-172 AMPK{alpha} ({alpha}-pAMPK) and anti-phospho-Ser-79 ACC ({alpha}-pACC) antibodies, respectively, and the expression of total AMPK protein was reprobed with anti-AMPK{alpha}2 antibody ({alpha}-AMPK{alpha}2). B, dose-response effect of metformin on AMPK and ACC phosphorylation. The phosphorylation levels of AMPK and ACC in cells treated with metformin for 24 h were quantified using model GS-700 Imaging Densitometer (Bio-Rad) and normalized to total AMPK protein. Data were expressed as mean ± S.E. relative to the basal phosphorylation level from at least three independent experiments. *, p < 0.05, compared with control. C, time course of the effect of metformin on AMPK{alpha} phosphorylation at Thr-172. D, time course of the effect of metformin on ACC phosphorylation at Ser-79. E, dose-response effect of metformin on hepatocellular lipid content. Levels of intracellular triacylglycerol and cholesterol in cells treated with metformin for 24 h were measured using spectrophotometric assays and expressed as µg of lipid/mg of protein as described under "Materials and Methods." The data are represented as the mean ± S.E. (n = 4). *, p < 0.05, compared with the untreated control.

 



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  		FIG. 3.
Metformin reverses the suppression of AMPK{alpha} and ACC phosphorylation and the accumulation of triacylglycerol by high concentrations of glucose. HepG2 cells were quiesced in serum-free medium overnight and incubated in DMEM containing either normal (5.5 mM) or high (30 mM) glucose concentrations in the absence or presence of 2 mM metformin for an additional 24 h. A, metformin restores the suppression of AMPK signaling by high glucose concentrations. Total cell extracts were subjected to immunoblot analysis with phospho-Thr-172 AMPK ({alpha}-pAMPK), total AMPK{alpha}2 ({alpha}-AMPK{alpha}2), and phospho-Ser-79 ACC ({alpha}-pACC) antibodies, respectively. All Western blots shown represent three independent experiments. B and C, metformin lowers the lipid accumulation caused by high glucose concentrations. Levels of intracellular triacylglycerols and cholesterol were measured as in Fig. 1. Each bar represents the mean ± S.E. (n = 4). *, p < 0.05, compared between two groups as indicated.

 



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  		FIG. 4.
Expression of a constitutively active form of AMPK{alpha} mimics the effect of metformin on ACC phosphorylation and lipids. HepG2 cells were transfected with adenoviral vectors encoding GFP (Ad-GFP) or a Myc-tagged constitutively active form of AMPK{alpha}1 (Ad-CA-AMPK) in serum-free medium for 24 h, followed by incubation with or without 2 mM metformin for an additional 24 h. A, expression of the CA-AMPK is sufficient to increase baseline ACC phosphorylation as well as enhance metformin-induced ACC phosphorylation. Expression of the CA-AMPK recombinant protein (~31 kDa) was detected by immunoblots using anti-Myc antibody ({alpha}-Myc). The phosphorylation of AMPK and ACC and expression of total AMPK protein were analyzed by immunoblots with the phospho-Thr-172 AMPK{alpha} antibody ({alpha}-pAMPK), phospho-Ser-79 ACC antibody ({alpha}-pACC), and anti-AMPK{alpha}2 antibody ({alpha}-AMPK{alpha}2), respectively. Immunoblots shown are representative of at least three independent experiments. B and C, inhibition of intracellular triacylglycerol and cholesterol contents by metformin was mirrored by expression of the CA-AMPK. Each bar represents the mean ± S.E. (n = 4). *, p < 0.05, compared between two groups as indicated.

 



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  		FIG. 6.
Metformin prevents lipid accumulation induced by high glucose concentrations in an AMPK-dependent manner. HepG2 cells were transfected with the adenovirus vector encoding either Ad-GFP or a Myc-tagged dominant-negative AMPK{alpha}2 (Ad-DN-AMPK) in serum-free medium for 24 h, followed by incubation with or without 2 mM metformin in medium containing either normal (5.5 mM) or high (30 mM) glucose concentrations for an additional 24 h. A, the ability of metformin to phosphorylate AMPK and ACC is diminished by the DN-AMPK in cells exposed to normal or high glucose concentrations. Expression of the DN-AMPK recombinant protein (~64 kDa) was detected by immunoblots using anti-Myc antibody ({alpha}-Myc) or anti-AMPK{alpha}2 antibody ({alpha}-AMPK{alpha}2), respectively. The phosphorylation of AMPK and ACC was immunoblotted with the phospho-Thr-172 AMPK{alpha} ({alpha}-pAMPK) or phospho-Ser-79 ACC ({alpha}-pACC) antibodies. All Western blots shown represent three independent experiments. B and C, inhibition of lipid levels by metformin was abrogated by the DN-AMPK. Each bar represents the mean ± S.E. (n = 4). *, p < 0.05, compared between two groups as indicated.

 
Induction of Insulin Resistance Caused by High Glucose Concentrations—To develop a model of insulin resistance, HepG2 cells were incubated in serum-free DMEM containing either normal concentrations of glucose (5.5 mM D-glucose) or high concentrations of glucose (30 mM D-glucose) for 24 h, and the response to insulin (100 nM for 10 min) was tested as described (25). The phosphorylation of Akt and its down-stream target, GSK3{alpha}/{beta}, was analyzed with anti-phospho-Ser-473 Akt and anti-phospho-Ser-21/9 GSK3{alpha}/{beta} antibodies. Insulin stimulated 6- and 3-fold increases in the phosphorylation of Akt and GSK3{alpha}/{beta}, respectively, over the basal level in cells maintained in normal glucose concentrations (Fig. 1, lanes 1 and 2). In contrast, high glucose concentrations diminished the insulin-induced phosphorylation of Akt as well as both basal and insulin-stimulated GSK3{alpha}/{beta} phosphorylation. There was no change in the expression of total Akt and GSK3{beta} protein or {beta}-actin (Fig. 1, lanes 2 and 4).



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  		FIG. 1.
Induction of insulin resistance caused by high glucose concentrations as indicated by the decrease in insulin-induced Akt and its downstream GSK3{alpha}/{beta} phosphorylation. HepG2 cells were quiesced in serum-free medium overnight and incubated in serum-free medium containing either normal (5.5 mM) or high (30 mM) concentrations of D-glucose (high glucose) for an additional 24 h. Before harvesting, cells were stimulated with 100 nM insulin for 10 min. Total cell extract was analyzed for the phosphorylation of Akt and GSK3{alpha}/{beta} by immunoblots with phospho-Ser-479 Akt ({alpha}-pAkt), total Akt ({alpha}-Akt), phospho-Ser-21/9GSK3{alpha}/{beta} ({alpha}-pGSK3{alpha}/{beta}), total GSK3{beta} ({alpha}-GSK3{beta}), or loading control {beta}-actin ({alpha}-{beta}-Actin) antibodies, respectively. A representative blot from three independent experiments is shown.

 
Adenoviral-mediated Gene Transfer—A replication-defective adenoviral vector expressing green fluorescence protein (Ad-GFP) was used as a control (27). The adenoviral vector expressing a dominant-negative mutant of AMPK{alpha}2 (Ad-DN-AMPK{alpha}2) was constructed from AMPK{alpha}2 bearing a mutation of lysine 45 to arginine (K45R) as described previously (27–29). To generate the adenoviral vector expressing a constitutively active mutant of AMPK{alpha}1 (Ad-CA-AMPK{alpha}1), a rat cDNA encoding residues 1–312 of AMPK{alpha}1 and bearing a mutation of threonine 172 into aspartic acid (T172D) was subcloned into a shuttle vector (p-shuttle cytomegalovirus). The c-Myc epitope tag was fused in frame to the 5' terminus of the coding sequence. The resulting plasmid was linearized by digestion with PmeI and co-transformed into Escherichia coli BJ5183 with the adenoviral backbone plasmid, pAdEasy-1. Homologous recombinants were selected with kanamycin. The linearized recombinant plasmid was transfected into transformed human embryonic kidney 293 cells. Recombinant adenoviruses were amplified on human embryonic kidney 293 cells and purified by two ultracentrifugation steps on cesium chloride gradients. The number of viral particles was assessed by measuring the optical density at 260 nm. HepG2 cells were transfected overnight with 30–100 plaque-forming units/cell in serum-free medium. After fresh serum-free medium was added, cells were treated with metformin (0.5–2 mM) for the indicated times. Preliminary studies revealed that within 48 h of transfection with control Ad-GFP, 80–90% of HepG2 cells expressed green fluorescent protein.

Western Blot Analysis—Western blot analysis was carried out as described previously (30, 31). In brief, HepG2 cells were lysed in buffer (20 mM Tris-HCl, pH 8.0, 1% Nonidet P-40, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml leupeptin, and 1 µg/ml pepstatin). Cell debris was removed by centrifugation at 14,000 x g for 15 min at 4 °C, and the resulting supernatant (cell lysate) was used for Western blotting and analysis of lipid content. Protein concentrations in cell lysates were measured using a Bio-Rad protein assay kit. For Western blotting, 20–50 µg of protein were separated by 8% SDS-polyacrylamide gel electrophoreses (SDS-PAGE) and electrophoretically transferred to polyvinylidene difluoride membranes in a transfer buffer consisting of 20 mM Tris-HCl, 154 mM glycine, and 20% methanol. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20 and incubated with specific antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were visualized by the enhanced chemiluminescence detection system. The intensity of bands was quantified using a model GS-700 Imaging Densitometer (Bio-Rad).

Determination of Triacylglycerol and Cholesterol Contents—Triacylglycerol and total cholesterol contents were determined in cell lysates using a colorimetric assay and expressed as µg of lipid/mg of cellular protein as described (32). In brief, HepG2 cells were maintained in serum-free medium overnight and incubated with either normal or high glucose concentrations in the absence or presence of metformin. Cell lysates were prepared as described above. Triacylglycerols and total cholesterol levels in cell lysates were measured according to the manufacturer's instructions for InfinityTM reagents.

Statistics—Results are expressed as the means ± the standard error of the mean (S.E.). Significance was analyzed using a two-tailed unpaired Student's t test. p < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Metformin Stimulates AMPK and ACC Phosphorylation, Decreases Lipid Content, and Attenuates the Hyperlipidemic Effects of High Glucose Concentrations in Insulin-resistant HepG2 Cells—We first determined the phosphorylation state of the {alpha} subunit of AMPK (AMPK{alpha}) by using a specific antiphospho-Thr-172 AMPK antibody. In HepG2 cells exposed to increasing concentrations of metformin for various times (Fig. 2A), phosphorylation of AMPK{alpha} at Thr-172 was stimulated by as much as 3.6-fold by metformin (2 mM) at 24 h (Fig. 2B). No change in the expression of endogenous AMPK{alpha} protein was noted by immunoblotting with AMPK{alpha}2 antibody (Fig. 2A). At the lower concentrations used (0.5 mM and 1 m M), no change in phosphorylation was evident at 6 h, but there was a significant increase at 24 h (Fig. 2C). Metformin (2 mM) caused a more rapid and potent increase in AMPK{alpha} phosphorylation, with a significant increase noted at 6 h, reaching levels comparable with those caused by 1 mM at 24 h (Fig. 2C).

To assess AMPK activity, we next determined the phosphorylation of its downstream target, ACC. Western blot analysis showed that metformin markedly induced phosphorylation of ACC at Ser-79 in a dose- and time-dependent fashion, which paralleled the stimulation of Thr-172 phosphorylation of AMPK{alpha} at 6 and 24 h (Fig. 2, A and D). When cells were treated with metformin for 24 h, phosphorylation of ACC was significantly increased by 3-fold at 1 mM and up to 5.5-fold at 2 mM (Fig. 2B). Metformin at 0.5 mM had little effect, whereas the effect of metformin at 1 mM caused a 3.2-fold rise at 6 h and was sustained for 24 h. Metformin at 2 mM caused a robust and sustained increase in ACC phosphorylation over 24 h (Fig. 2D).

Intracellular levels of triacylglycerol and cholesterol in HepG2 cells exposed to metformin for 1, 6, and 24 h were also measured. Increasing concentrations of metformin (0.5–2 mM) decreased intracellular triacylglycerol and cholesterol content at 24 h in a dose-dependent manner (Fig. 2E). Treatment for 1 h had little effect, but significant reductions in both triacylglycerol and cholesterol content were observed at 6 h, although they were less pronounced than those shown at 24 h (data not shown). At the highest metformin concentration utilized (2 mM), there was approximately a 30 and 40% decrease in triacylglycerol content and a 21 and 33% decrease in cholesterol content at 6 and 24 h, respectively (Fig. 2E and data not shown).

Using a model of insulin resistance induced by high concentrations of glucose (Fig. 1), we determined the effects of these high glucose concentrations on the phosphorylation of AMPK{alpha} at Thr-172, on the phosphorylation of ACC at Ser-79, and on lipid levels. Exposure of HepG2 cells to glucose (30 mM, 24 h) decreased phosphorylation of AMPK and ACC without a change in total AMPK protein (Fig. 3A, lanes 1 and 3). In concert with these changes in phosphorylation of AMPK and ACC, triacylglycerol content dramatically increased by almost 3-fold in insulin-resistant hepatocytes without a significant change in cholesterol content (Fig. 3, B and C).

To determine whether metformin reverses lipid accumulation induced by high glucose concentrations, HepG2 cells were incubated with either normal or high glucose concentrations in the absence or presence of metformin (2 mM) for 24 h. Consistent with our earlier observations, the phosphorylation of AMPK{alpha} and ACC was markedly up-regulated by metformin in normal concentrations of glucose (Fig. 3A, lanes 1 and 2), and the intracellular contents of triacylglycerol and cholesterol were lowered ~40% by metformin (Fig. 3, B and C). Moreover, the inhibition of AMPK and ACC phosphorylation in cells exposed to high glucose concentrations was restored by metformin (Fig. 3A, lanes 3 and 4). Consistent with this, the intracellular contents of triacylglycerol that were increased by high glucose concentrations were reduced 40% by metformin (Fig. 3, B and C).

The Effect of Metformin on Hepatic Lipids Is Mimicked by a Constitutively Active Form of AMPK—To confirm the role of the catalytic {alpha} subunit of AMPK in regulating lipid metabolism, we determined the effect of adenoviral-mediated expression of a Myc-tagged constitutively active mutant of AMPK{alpha}1 (Ad-CA-AMPK{alpha}1) on the changes in ACC phosphorylation and lipid levels. As expected, after adenoviral transfection, the recombinant AMPK{alpha}1 protein that contained a T172D mutation and was truncated at residue 312 (~31 kDa) was expressed as detected by immunoblotting for the Myc tag at its N terminus (Figs. 4A and 5A, lanes 3 and 4). In contrast, endogenous {alpha}1or {alpha}2 AMPK protein level was not obviously affected by the transfection (Figs. 4A and 5A). As predicted, ACC phosphorylation was higher in the basal state following transfection with the CA-AMPK (Figs. 4A and 5A, lane 3). It was evident that the cells remained sensitive to metformin because expression of the CA-AMPK potentiated the effect of metformin on ACC phosphorylation over the effect observed in Ad-GFP transfected cells (Fig. 4A, lanes 2 and 4). Similar to the effect of metformin in GFP-transfected cells, the CA-AMPK transfection also reduced triacylglycerol and cholesterol levels by ~35%, and the levels were further significantly reduced by metformin (Fig. 4, B and C), suggesting that there is a synergistic effect of metformin and the CA-AMPK on the phosphorylation of ACC and reduction of lipids.



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  		FIG. 5.
Modulation of AMPK activity is sufficient to alter hepatocellular lipid contents. Ad-GFP or Ad-CA-AMPK were transfected into HepG2 cells for 24 h. Cells were subsequently incubated in DMEM containing either normal (5.5 mM) or high (30 mM) glucose concentrations for another 24 h. A, suppression of AMPK signaling by high glucose concentrations is prevented by expression of the CA-AMPK. Total cell extracts were subjected to immunoblot with phospho-Thr-172 AMPK{alpha} ({alpha}-pAMPK), total AMPK{alpha}1 ({alpha}-AMPK{alpha}1), total AMPK{alpha}2 ({alpha}-AMPK{alpha}2), phospho-Ser-79 ACC ({alpha}-pACC), and anti-Myc antibodies, respectively. A representative blot from three independent experiments is shown. B and C, expression of the CA-AMPK is sufficient to decrease intracellular triacylglycerol and cholesterol content. Each bar represents the mean ± S.E. (n = 4). *, p < 0.05, compared between two groups as indicated.

 
The effect of the CA-AMPK transfection was also tested in cells exposed to high concentrations of glucose. Although the CA-AMPK had no appreciable effect on total or phosphorylated forms of endogenous AMPK, the phosphorylation of ACC was increased, and the level in cells exposed to high glucose concentrations was comparable with that in cells exposed to normal glucose concentrations and transfected with GFP (Fig. 5A, lanes 1 and 4). Exposing CA-AMPK-transfected cells to high glucose concentrations elevated triacylglycerols level significantly, but the level was significantly less than that in cells transfected with GFP and exposed to high glucose concentrations (Fig. 5B). The CA-AMPK also reduced the cholesterol content in cells exposed to high glucose concentrations, although cholesterol levels were not statistically affected by high glucose concentrations (Fig. 5C). These results indicate that the expression of the CA-AMPK promotes the phosphorylation of ACC and lowers lipid accumulation caused by high glucose concentrations in insulin-resistant HepG2 cells, thus mimicking the effect of metformin.

Metformin Reduces Lipid Accumulation Caused by High Glucose Concentrations in an AMPK-dependent Manner—The functional relationship between AMPK activation, ACC phosphorylation, and lipid content was further examined by overexpression of a dominant-negative form of AMPK{alpha}2. AMPK activity was inhibited by adenoviral-mediated expression of a catalytically inactive AMPK{alpha}2 bearing a K45R mutation, which has a dominant-negative effect on both {alpha}1 and {alpha}2 AMPK (29). As shown in Fig. 6A, transfection with the DN-AMPK increased expression of the ~64-kDa {alpha}2 subunit mutant, as estimated by immunoreactivity with anti-AMPK{alpha}2 or anti-Myc antibodies. Expression of the DN-AMPK suppressed levels of phosphorylated AMPK and ACC in cells exposed to normal glucose concentrations and further suppressed these levels in cells exposed to high glucose concentrations (Fig. 6A). Furthermore, similar to the effect of high concentrations of glucose, cells expressing the DN-AMPK displayed an ~50% increase in the triacylglycerol levels and a smaller but significant increase in cholesterol content (Fig. 6, B and C). These results suggest not only that exposure to high concentrations of glucose inhibits AMPK and ACC phosphorylation, but also that modulation of AMPK by high concentrations of glucose contributes to the increase in lipid content that accompanies insulin resistance caused by high concentrations of glucose.

To ascertain whether AMPK activity is required for metformin to phosphorylate ACC and lower lipids, HepG2 cells were transfected with Ad-DN-AMPK or Ad-GFP, followed by incubation with either normal or high glucose concentrations in the absence or presence of metformin (2 mM) for 24 h. In GFP-transfected cells, the effects of metformin on AMPK and ACC phosphorylation, as well as on triacylglycerol and cholesterol content, were similar to the effects observed in non-transfected cells as shown in Fig. 3. However, the ability of metformin to phosphorylate AMPK and ACC was diminished by the DN-AMPK (Fig. 6A), indicating that metformin up-regulates ACC phosphorylation in an AMPK-dependent manner. Moreover, although metformin significantly decreased the triacylglycerol content by ~40% in cells exposed to normal or high glucose concentrations, the expression of the DN-AMPK abolished the inhibitory effect of metformin on triacylglycerols (Fig. 6B). The DN-AMPK also blocked the smaller inhibitory effect of metformin on cholesterol content in cells exposed to normal or high glucose concentrations (Fig. 6C). Together, these data support our hypothesis that AMPK mediates the effect of metformin on hepatic lipid content.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The primary goal of this study was to assess the role of AMPK activation by metformin in the regulation of hepatocellular lipids. We showed not only that metformin increased phosphorylation of AMPK and its downstream lipid regulator, ACC, but also that its actions were mimicked by overexpression of a constitutively active AMPK (CA-AMPK). In addition, an AMPK dominant-negative AMPK (DN-AMPK) blocked both the increase in ACC phosphorylation and the decrease in lipid content of HepG2 cells caused by the antidiabetic agent. Furthermore, in cells in which an insulin-resistant, hyperlipidemic state was induced by high glucose concentrations, the lipid-lowering effects of metformin were also mimicked by overexpressing the CA-AMPK or prevented by the DN-AMPK. These studies strongly suggest that the salutary effects of metformin on hepatocellular lipids are mediated predominantly by AMPK.

Stimulation of AMPK and Lipid-lowering Effects of Metformin—Considerable evidence has been accumulated to show that the phosphorylation of Thr-172, the major stimulatory phosphorylation site of the {alpha} subunit, is essential for AMPK activity (10, 11). Several groups have generated antibodies that specifically recognize the phosphorylated form of Thr-172 and have demonstrated that the phosphorylation status of Thr-172 mirrors AMPK activity under all conditions tested (6, 7, 33, 34). In the present study, it was observed that the time- and concentration-dependent effects of metformin increase the phosphorylation of AMPK at Thr-172, which confirms previous observations that metformin increases the catalytic activity of AMPK in rat primary hepatocytes, the H4IIE hepatocyte cell line, skeletal muscle, and rat pancreatic {beta} cells (21, 22, 35, 36).

In the present study, the phosphorylation state of ACC at Ser-79 was also used to assess AMPK activity. ACC, which plays a pivotal role in hepatic lipid metabolism, is controlled by allosteric regulation by citrate and glutamate and by covalent modification by phosphorylation (37). AMPK inhibits ACC by phosphorylation of Ser-79 (38–40). Although other protein kinases can phosphorylate ACC, the increase in ACC phosphorylation at Ser-79 caused by metformin in this study was inhibited by the overexpression of the DN-AMPK, which is consistent with AMPK being the mediator. This finding is in agreement with the observation that metformin negatively controls ACC activity via AMPK in skeletal muscle (36). ACC catalyzes the biosynthesis of malonyl-CoA, which serves as the initial substrate for fatty acid biosynthesis as well as a potent inhibitor of carnitine palmitoyltransferase I, the rate-limiting step for mitochondrial fatty acid oxidation. The important role of ACC in lipid metabolism has been demonstrated by increased fatty acid oxidation and leanness in mice deficient in ACC2 (41). Previous studies have shown that AMPK activation by either AICAR or metformin stimulates fatty acid oxidation, and AICAR reduces [14C]oleate and [3H]glycerol incorporation into triacylglycerol in rat hepatocytes (21, 42). This suggests that the reduction in triglyceride levels caused by metformin observed in HepG2 cells, which was coincident with stimulation of ACC phosphorylation by metformin, can be explained by increased fatty acid oxidation and/or decreased fatty acid synthesis. In addition to regulating ACC, it is also possible that metformin regulates other factors that affect fatty acid synthesis or oxidation, such as sterol regulatory element-binding proteins, which could affect the lipid levels measured in the present study. In metformin-treated rats and hepatocytes, gene and protein expression of sterol regulatory element-binding protein 1 and other lipogenic enzymes is suppressed (21). Further studies will be needed to delineate critical mediators of the lipid-lowering effect of metformin via AMPK.

Because the increase in ACC phosphorylation, as well as the reduction in triacylglycerols caused by metformin, were inhibited in the present study by overexpression of the DN-AMPK, it is likely that ACC is the main determinant of the decrease in lipids. This conclusion is also strengthened by the fact that overexpression of the CA-AMPK mimicked both the increase in ACC phosphorylation and the decrease in triacylglycerols caused by metformin. In addition, the AMPK activator, AICAR, decreases synthesis of triacylglycerols and their precursor, diacylglycerol, in hepatocytes (42). Metformin also decreased HepG2 cell content of cholesterol. The effect of metformin on cholesterol content may be explained by the fact that 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-limiting enzyme in cholesterol biosynthesis, is also phosphorylated and inhibited by AMPK (43). Like the decrease in triacylglycerols, the effect of metformin on cholesterol was mimicked by overexpression of the CA-AMPK and blocked by the DN-AMPK, suggesting a direct action of metformin on hepatocellular cholesterol metabolism via AMPK. Thus, the effects of metformin on both ACC and 3-hydroxy-3-methylglutaryl-coenzyme A reductase via AMPK can explain its inhibitory effects on lipid content of HepG2 cells.

Actions of Metformin in a Hepatocellular Model of Insulin Resistance—To investigate the importance of AMPK in the actions of metformin in insulin-resistant states, HepG2 cells were exposed to high glucose concentrations for 24 h, and resistance to insulin signaling was demonstrated by decreased insulin-induced Akt phosphorylation. This observation is in agreement with the finding that high concentrations of glucose attenuate insulin-induced phosphorylation of tyrosine residues of the insulin receptor substrate 1 as well as association of insulin receptor substrate 1 and phosphatidylinositol 3-kinase (25). Exposure of HepG2 cells to high glucose concentrations also decreased AMPK and ACC phosphorylation, which was similar to findings in pancreatic {beta} cell lines MIN-6 and INS-1 as well as in multiple hypothalamic regions (44, 45). In addition, high glucose concentrations dramatically increased the triacylglycerol content of HepG2 cells, supporting the role of ACC in hepatocellular triacylglycerol accumulation. It is notable that overexpression of the DN-AMPK also increased triacylglycerol content of HepG2 cells. This supports the role of decreased ACC phosphorylation, which indicates increased activity, in those cells as well as those exposed to high glucose concentrations as an important cause for the increased triacylglycerol content. No change in AMPK expression occurred in this short term model of insulin resistance, suggesting that changes in phosphorylation of AMPK were the primary mediators of the effects of high glucose concentrations on triacylglycerol accumulation. In contrast to triacylglycerols, cholesterol content did not increase in HepG2 cells exposed to high concentrations of glucose, suggesting that the changes in AMPK and ACC phosphorylation were not sufficient to increase the levels; this was perhaps also reflected in the smaller changes in cholesterol content that occurred in response to overexpression of the DN-AMPK.

Metformin overcame the decrease in phosphorylation of AMPK and ACC caused by high glucose concentrations, and lowered the elevated triacylglycerol levels in cells exposed to high glucose concentrations to levels that were observed in untreated cells exposed to normal glucose concentrations. The effects of metformin could be attributed to changes in AMPK, as they were completely abrogated by overexpression of the DN-AMPK and mimicked by the CA-AMPK. It is possible that the effects of metformin are also mediated by the altered expression of other enzymes that regulate lipid biosynthesis, such as fatty acid synthase and sterol regulatory element-binding proteins (21, 46, 47). Nevertheless, the fact that DN-AMPK prevented the effects of metformin is strong evidence that its actions are primarily mediated by AMPK. This is also consistent with accumulating evidence that AMPK regulates the key enzymes that control lipid biosynthesis, such as glycerol-3-phosphate acyltransferase and malonyl-CoA decarboxylase (42, 48, 49). The fact that the actions of metformin in HepG2 cells were completely abrogated by the DN-AMPK makes this hepatocellular model of insulin resistance ideal for identifying other agents that affect lipid metabolism via AMPK.

In summary, this study provides strong biochemical evidence that the effects of metformin on the lipid content of HepG2 cells depend on activation of AMPK. Despite the fact that the mechanism by which metformin stimulates AMPK remains controversial, this study demonstrates that AMPK is the principal mediator of the effects of metformin on lipid biosynthesis. Our results also suggest that the salutary effects of metformin on the elevated lipids associated with insulin-resistant states also depend on AMPK. Therefore, this study demonstrates an excellent model both to explore the mechanisms by which AMPK regulates lipids as well as to identify more potent agents than metformin that are capable of stimulating AMPK and controlling lipid biosynthesis.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants P01 HL68758 (to M. Z., N. B. R., and R. A. C.) and AR40197 (to D. N. and K. W.) and by a Strategic Alliance with Institut de Recherches Servier. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** To whom correspondence should be addressed: Vascular Biology Unit, X708, Boston University School of Medicine, 650 Albany St., Boston, MA 02118-2393. Tel.: 617-638-7115; Fax: 617-638-7113; E-mail: racohen{at}bu.edu.

1 The abbreviations used are: AMPK, AMP-activated protein kinase; Ad, adenoviral vector; CA-AMPK, constitutively active AMPK; DN-AMPK, dominant-negative AMPK; ACC, acetyl-CoA carboxylase; AICAR, 5-amino-4-imidazolecarboxamide riboside; GSK, glycogen synthase kinase; DMEM, Dulbecco's modified Eagle's medium; GFP, green fluorescent protein. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Bingbing Jiang and Tyler Heibeck for helpful suggestions.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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