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Alpha-lipoic acid induces adipose triglyceride lipase expression and decreases intracellular lipid accumulation in HepG2 cells
European Journal of Pharmacology 692 (2012) 10–18
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
Alpha-lipoic acid induces adipose triglyceride lipase expression and decreases intracellular lipid accumulation in HepG2 cells Yung-Ting Kuo a,b,c, Ting-Han Lin c,d, Wei-Lu Chen c,d, Horng-Mo Lee c,d,e,n a
Department of Pediatrics, Shuang Ho Hospital, Taipei Medical University, Taiwan Department of Pediatrics, School of Medicine, College of Medicine, Taipei Medical University, Taiwan c Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taiwan d School of Medical Laboratory Sciences and Biotechnology, College of Medicine, Taipei Medical University, Taiwan e Institute of Pharmaceutical Science and Technology, Central Taiwan University of Science and Technology, Taichung, Taiwan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 July 2011 Received in revised form 5 July 2012 Accepted 7 July 2012 Available online 20 July 2012
Non-alcoholic fatty liver disease can be attributed to the imbalance between lipogenesis and lipolysis in the liver. Alpha-lipoic acid has been shown to activate the 50 -AMP-activated protein kinase (AMPK) signalling pathway and to effectively inhibit the lipogenesis pathway in liver. However, whether alphalipoic acid stimulates lipolysis remains unclear. Recently, adipose triglyceride lipase (ATGL) was shown to be responsible for triacylglycerol hydrolase activity in cells. In the present study, we established a fatty liver cell model by incubating HepG2 cells in a high glucose (30 mM glucose) and high fat (0.1 mM palmitate) medium. We found that the activation of the AMPK signalling pathway induced ATGL protein expression and enhanced lipid hydrolysis. Similarly, treatment of the fatty liver cell model with alpha-lipoic acid reduced intracellular lipid accumulation in HepG2 cells, increased AMPK phosphorylation, and induced ATGL expression. We showed that insulin phosphorylates the transcription factor forkhead box O1 (FOXO1), which regulates ATGL expression and inhibits FOXO1 translocation into the nucleus. In contrast, alpha-lipoic acid dephosphorylated FOXO1 and reversed the nuclear exclusion of FOXO1. These data suggest that alpha-lipoic acid can effectively ameliorate intracellular lipid accumulation and induce ATGL expression through the FOXO1/ATGL pathway in liver cells. Thus, alpha-lipoic acid may be a potential therapeutic agent for treating fatty liver disease. Crown Copyright & 2012 Published by Elsevier B.V. All rights reserved.
Keywords: Alpha-lipoic acid (ALA) 50 -AMP-activated protein kinase (AMPK) Adipose triglyceride lipase (ATGL) Forkhead box O1 (FOXO1)
1. Introduction Non-alcoholic fatty liver disease is an important feature of metabolic diseases such as chronic liver disease and is strongly associated with obesity and dysregulated insulin action in the liver (Feldstein et al., 2009; Gupta et al., 2011; Pacifico et al., 2011). Pathologic findings show that the parenchyma of the liver is characterised by increased intracellular triglyceride accumulation (Brunt, 2009; Carter-Kent et al., 2009; Patton et al., 2006). Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are the major triglyceride lipases in many tissues (Reid et al., 2008). ATGL and HSL expression are decreased in the obese, insulin-resistant state, suggesting that insulin resistance is associated with impaired lipolysis (Jocken et al., 2007; Zechner et al., 2009).
n Corresponding author at: Institute of Pharmaceutical Sciences and Technology, Central Taiwan University of Science and Technology, Taichung, Taiwan. Tel.: þ886 2 2736 1661; fax: þ 886 2 2732 4510. E-mail address: [email protected] (H.-M. Lee).
Current treatments for patients with fatty liver disease include change of lifestyle and improvement of insulin sensitivity to alleviate the associated metabolic syndrome (Angulo, 2007; Cortez-Pinto and Machado, 2008; Nobili et al., 2008). Alpha-lipoic acid is an endogenous cofactor in many multi-enzyme complexes that exerts beneficial effects on obesity, type 2 diabetes mellitus, and non-alcoholic fatty liver disease in patients with insulin resistance (Park et al., 2008; Reid et al., 2008). The 50 -AMP-activated protein kinase (AMPK) signalling pathway is thought to be a natural response to reduce dyslipidemia and to ameliorate insulin resistance (Ben Djoudi Ouadda et al., 2009). AMPK is a fuel-sensing enzyme that exerts effects on cellular energy metabolism and mitochondrial biogenesis (Jager et al., 2007; Winder and Holmes, 2000). Once activated, AMPK phosphorylates acetyl-CoA carboxylase, thereby blocking its activity. This in turn decreases the malonyl-CoA concentration, leading to enhanced mitochondrial fatty acid b-oxidation. Alphalipoic acid was shown to reduce body weight by suppressing hypothalamic AMPK (Kim et al., 2004). In diabetes-prone Otsuka Long Tokushima fatty rats, alpha-lipoic acid increased insulin sensitivity and skeletal muscle fatty acid oxidation via AMPK
0014-2999/$ - see front matter Crown Copyright & 2012 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2012.07.028
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activation (Evans and Goldfine, 2000; Lee et al., 2005). Treatment with alpha-lipoic acid ameliorated severe hypertriglyceridemia by inhibiting TG synthesis and very low density lipoproteintriglyceride (VLDL-TG) secretion in Zucker diabetic fatty rats (Butler et al., 2009). AMPK has been shown to phosphorylate ATGL (Narbonne and Roy, 2009). In adipose tissue, the activation of AMPK-dependent pathways favours energy dissipation rather than lipid storage (Gaidhu et al., 2009). The forkhead box gene group O (FOXO) subfamily of transcription factors regulates carbohydrate and lipid metabolism (Altomonte et al., 2004) and pancreatic cell function (Buteau and Accili, 2007). The protein FOXO1 can be phosphorylated via an insulin-dependent pathway; the phosphorylation of FOXO1
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leads to the nuclear exclusion and inhibition of FOXO1 activity (Chakrabarti and Kandror, 2009). In the insulin-resistant state, FOXO1 increases the hepatic gluconeogenic master genes, phosphoenolpyruvate carboxykinase and glucose 6-phosphatase, and increases ATGL expression (Chakrabarti and Kandror, 2009), thus protecting against excessive lipid accumulation in the liver during hyperglycaemia (Haeusler et al., 2010; Houde et al., 2010). Because of the aforementioned metabolic actions of AMPK and FOXO1, we investigated whether alpha-lipoic acid regulates lipid metabolism through the AMPK/FOXO1 signalling pathway. We demonstrated that alpha-lipoic acid enhances ATGL expression through the AMPK/FOXO1 pathway, which in turn reduces triacylglycerol accumulation in HepG2 cells exposed to high
Fig. 1. A fatty liver model—High glucose, high fat environment induces intracellular lipid formation in HepG2 cells and induces ATGL. A: Micrographs of HepG2 cells incubated for 24 h in high glucose medium with various concentration of palmitate to simulate a high fat environment. Oil red O staining at 1000 magnification. The concentration of intracellular lipid droplets increased with palmitate concentration. B: MTT colorimetric assay for cell viability in the high glucose, high fat environment after 24 h. No differences were observed between control and high fat (0.1 mM palmitate) conditions. C: Western blots of ATGL and GAPDH for HepG2 cells incubated with various concentrations (0.05, 0.1, 0.5, 1 mM) of palmitate at 37 1C for 24 h. NC, control cells incubated at normal glucose concentration (5.5 mM). HG, control cells incubated in high glucose medium (30 mM).
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glucose and palmitate. These data suggest that alpha-lipoic acid may exert beneficial effects in treating fatty liver disease.
and anti-phospho-Ser256 FOXO1 were purchased from Cell Signaling Technology (Beverly, MA). 2.2. Culture of human HepG2 cells and preparation of cell lysates
2. Materials and methods 2.1. Materials Dulbecco’s modified Eagle medium (DMEM), foetal calf serum, and glutamine were purchased from Life Technologies (Gaithersburg, MD). Rabbit polyclonal antibodies for AMPK-a1 and AMPKa2 were purchased from Bethyl Laboratories (Montgomery, TX). The anti-GAPDH antibody was from Gentex (Irvine, CA). The a-tubulin, horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). 5-Aminoimidazole-4carboxamide riboside (AICAR), compound C, metformin, and nicotinamide were purchased from Calbiochem-Novabiochem (San Diego, CA). The rabbit monoclonal anti-FOXO1 antibody
HepG2 cells were cultured in DMEM supplemented with 10% heat-inactivated foetal calf serum and sodium pyruvate (1 mM). After reaching 90% confluence, HepG2 cells were treated with various concentrations of the indicated agents and incubated for the indicated times in a 5% CO2-humidified incubator at 37 1C. Cells were removed using a rubber policeman in the presence of lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 100 ml/ml PMSF, 1% NP-40, and 4% protease inhibitor cocktail) to obtain cell lysates for western blotting. 2.3. Polyacrylamide gel electrophoresis (PAGE) and western blotting Cell lysates of equal protein concentration were separated by sodium dodecylsulphate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoridemembranes for
Fig. 2. AMPK activation by metformin increases ATGL expression and decreases intracellular lipid droplet accumulation. A, B, Western blots of phospho-AMPK (pAMPK) and ATGL expression. HepG2 cells were incubated with high glucose (HG, 30 mM) and high fat (HF, 1 mM palmitate) medium for 24 h. A: AMPK phosphorylation increased after AICAR (A) and metformin (M) treatment, whereas compound C (CC) decreased AMPK phosphorylation. B: ATGL was also induced by AICAR and metformin. C: HepG2 cells were treated with metformin for 1 h after incubation in high glucose, high fat (0.1 mM palmitate) medium for 24 h. Lipid droplets were stained with oil red O stain and observed under light microscopy at 1000 magnification. The concentration of intracellular lipid droplets decreased compared to the control cells.
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immunoblotting. Membranes were blocked with blocking solution containing 3% bovine serum albumin in phosphate-buffered saline (PBS) containing 0.1% Tween 20 for 1 h at room temperature. This was followed by sequential incubation with solutions containing primary and secondary antibodies with PBS washes in between. Immunodetection was performed using a LumiGLO chemiluminescence kit (GE Healthcare Life Sciences). Levels of protein expression were quantified by scanning densitometry using a model GS-700 imaging densitometer (Bio-Rad) and normalised to a-tubulin levels as a control. 2.4. Oil red O staining HepG2 cells were grown in 6-well plates for 24 h. At the end of drug treatment, the cells were washed with PBS and fixed with 4% PBS-buffered formaldehyde for 15 min. Subsequently, the cells were rinsed with water, dipped for a few seconds in 60% isopropanol, stained in oil red O for 15 min, and rinsed again in 60% isopropanol to remove excess stain. Cell nuclei were stained
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for a few seconds in haematoxylin solution, washed with water, and mounted with commercially available Mounting Medium (Dako, Glostrup, Denmark). The samples were photographed by microscope (Olympus B 60) using the Olympus MicroImage software. 2.5. Immunofluorescence HepG2 cells were seeded in 6-well plates containing sterilised coverslips and incubated at 37 1C for defined periods with drug stimulation. After treatment, the cells were washed twice with PBS (137 mM NaCl, 1.4 mM KH2PO4, 4.3 mM Na2HPO4, 2.7 mM KCl, pH 7.2) and fixed with 4% PBS-buffered formaldehyde for 10 min at room temperature. After fixation, the cells were washed three times with PBS for 5 min each time. Thereafter, Triton-X 100 was added for 10 min, and the cells were washed three times with PBS. The coverslips were then incubated with skimmed milk containing PBS blocking buffer at room temperature for 1 h and subsequently with specific antibodies in PBS blocking buffer
Fig. 3. Alpha-lipoic acid induces AMPK phosphorylation and enhances ATGL expression. A: HepG2 cells were incubated for various times with 0.25 mM a-lipoic acid in high glucose (30 mM), high fat (0.1 mM palmitate) medium and then lysed. Phosphorylation of AMPK (pAMPK-Thr172) was analysed by Western blots. The ratios of pAMPK to tAMPK were quantified from three independent experiments per condition. The data are expressed as the mean 7S.E.M.; n¼ 3; *P o0.05, relative to controls (0 mM a-lipoic acid). B: Western blots of phospho-AMPK (pAMPK) induced by alpha-lipoic acid in high glucose, high fat medium. The data are expressed as the mean 7S.E.M.; n¼ 3; *Po 0.05, relative to controls (0 mM alpha-lipoic acid). C: Western blots of ATGL induced by alpha-lipoic acid (ALA) in high glucose, high fat medium. The data are expressed as the mean 7 S.E.M.; n¼3; *Po 0.05, relative to controls (0 mM alpha-lipoic acid). D: Micrographs of HepG2 cells in high glucose, high fat medium treated with alpha-lipoic acid. 1000 magnification. Oil red O staining shows that the concentration of neutral lipid droplets in the intracellular cytoplasm decreased inversely with an increase in alpha-lipoic acid concentration.
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overnight at 4 1C. The following day, the cells were washed three times with PBS, incubated with fluorescein-conjugated goat antirabbit IgG antibody for 1 h at 37 1C, and washed three times. The nuclei were stained with DAPI.
3. Results
2.6. MTT assay
Lipid droplet-associated triglycerides were found in many non-adipose tissues when they were supplied with excessive nutrients (Son et al., 2010). To determine whether liver cells esterify and deposit fatty acid as lipid droplets, HepG2 cells were incubated in high glucose (30 mM) media with various concentrations of palmitate (0.05–1 mM) for 24 h. After incubation, intracellular lipid accumulation was determined by the oil red O staining. Oil red O staining revealed that intracellular lipid droplets had accumulated in HepG2 cells when incubated in medium with high concentrations of palmitate (0.1 mM) (Fig. 1A). Incubation of HepG2 cells with high concentrations of palmitate did not affect cell viability as demonstrated by the MTT assay (Fig. 1B). ATGL is responsible for the catabolism of cellular lipid stores (Zechner et al., 2009). ATGL expression levels were examined by western blotting of HepG2 cells. HepG2 cells expressed substantial levels of ATGL at a normal glucose concentration (NC, 5.5 mM). However, the expression level of ATGL was lower when cells were incubated at a high glucose concentration (HG, 30 mM). Treatment of cells with a high palmitate concentration (0.5 mM) for 24 h increased expression levels of ATGL under high glucose conditions. Palmitate at 0.1 mM did not increase ATGL protein levels in HepG2 cells (Fig. 1C). Thus, we established the parameters for a fatty liver cell model by incubating HepG2 cells in high glucose (30 mM), high fat (0.1 mM palmitate) medium.
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is a test of metabolic competence and assesses mitochondrial performance. The colourimetric assay relies on the conversion of yellow tetrazolium bromide to the purple formazan derivative by mitochondrial succinate dehydrogenase in viable cells. HepG2 cells were seeded onto 24-well plates at a density of 2 104 cells/well in serum-free medium. The medium was removed after the cells were cultured for 24 h in the reagent medium (100 ml). The cell viability was then measured by the MTT assay. After reagent treatment for 24 h, the cells were washed once with PBS. Subsequently, fresh growth medium was added to each well, and the cells were cultured for 24 h. MTT was then added at a final concentration 0.5 mg/ml, and the cells were further incubated for 2 h at 37 1C. The medium was removed, and the formazan crystals were dissolved in DMSO (200 ml). The optical density (OD) was measured at 570 nm (reference filter 690 nm) using an ELISA microplate reader. Survival (viability) was determined by comparing the OD of the wells containing treated cells to those containing vehicle-treated (0.1% ethanol) cells.
3.1. A fatty liver cell model—High glucose, high fat environment increases intracellular lipid formation in HepG2 cells and induces ATGL expression
2.7. Statistical analysis All data are expressed as the mean 7S.E.M., with the number of experiments denoted by n. Statistical significance (P o0.05) between experimental groups was determined by single-factor analysis of variance (ANOVA) for multiple groups or an unpaired t-test for two groups.
3.2. AMPK activation increased ATGL expression and decreased intracellular lipid accumulation under high fat conditions To determine whether activation of AMPK leads to ATGL protein expression, AMPK activators, AICAR and metformin, were used to treat HepG2 cells. Both AICAR and metformin increased
Fig. 4. Inhibition of sirtuins does not affect alpha-lipoic acid-mediated induction of AMPK phosphorylation or ATGL expression. HepG2 cells in high glucose, high fat medium were pretreated with compound C (20 mM) or nicotinamide (NA; 1 mM) for 30 min prior to the addition of alpha-lipoic acid (0.25 mM) for 24 h. A: Pretreatment with nicotinamide, a sirtuin inhibitor, prior to addition of alpha-lipoic acid (ALA), induced AMPK phosphorylation (pAMPK); in contrast, pretreatment with compound C (CC) inhibited AMPK phosphorylation. B: ATGL expression was induced by alpha-lipoic acid alone or with nicotinamide pretreatment. However, compound C (CC) lowered the alpha-lipoic acid-mediated induction of ATGL. These data are expressed as the mean 7S.E.M.; n¼3; *P o 0.05, relative to control; #P o0.05, relative to ALA treated alone.
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AMPK phosphorylation in HepG2 cells, and these effects were inhibited by pretreatment with compound C, a selective AMPK inhibitor (Fig. 2A). Activation of AMPK is associated with increased phosphorylation of acetyl-CoA carboxylase at Ser79 in HepG2 cells (data not shown). AMPK activators induced ATGL protein expression (Fig. 2B) and decreased intracellular lipid droplet accumulation in HepG2 cells (Fig. 2C). 3.3. Alpha-lipoic acid stimulates AMPK phosphorylation and ATGL expression Alpha-lipoic acid has been shown to activate AMPK in skeletal muscle cells (Lee et al., 2005). Thus, the effects of alpha-lipoic acid on AMPK phosphorylation in HepG2 cells needed to be determined. As expected, treatment of HepG2 cells with alpha-lipoic acid increased AMPK phosphorylation in a time-dependent and concentration-dependent manner (Figs. 3A and B). To determine whether alpha-lipoic acid induced ATGL expression, cells were incubated in high glucose (30 mM), high fat (0.1 mM palmitate) medium in the presence of various concentrations of alpha-lipoic acid (0.25–1.0 mM). As shown in Fig. 3C, treatment with alphalipoic acid increased ATGL protein levels in HepG2 cells. In agreement with these findings, treatment with alpha-lipoic acid reduced intracellular lipid accumulation as shown by oil red O and haematoxylin staining (Fig. 3D). 3.4. SIRT pathway does not affect alpha-lipoic acid-induced AMPK phosphorylation or ATGL expression The NAD(þ)-dependent deacetylase and gene repressor SIRT1 has been shown to play a role in regulating AMPK and the functional consequences of its activation. To determine whether SIRT1 is involved in alpha-lipoic acid-induced ATGL expression, a pharmacological SIRT1 inhibitor was used to block SIRT1 activity. HepG2 cells were pretreated with nicotinamide, an inhibitor of sirtuins, or compound C, an inhibitor of AMPK, for 30 min before alpha-lipoic acid was added. As shown in Fig. 4A, treatment with alpha-lipoic acid increased AMPK phosphorylation, which was blocked by pretreatment with compound C, but not nicotinamide. Similar effects were seen in alpha-lipoic acid-induced ATGL expression (Fig. 4B). These data suggested that SIRT1 is not involved in alpha-lipoic acid-induced ATGL expression. 3.5. Alpha-lipoic acid decreases FOXO1 phosphorylation and reverses insulin-induced FOXO1 nuclear exclusion in HepG2 cells FOXO1 plays an important role in transcriptional control of ATGL. To determine whether alpha-lipoic acid regulates ATGL expression through FOXO1, the effects of alpha-lipoic acid on FOXO1 phosphorylation were examined. Fig. 5A shows that alpha-lipoic acid suppressed endogenous FOXO1 phosphorylation in HepG2 cells. Insulin is one of the most important anabolic hormones involved in regulating hepatic lipogenesis. It increases lipogenesis through the phosphorylation and nuclear exclusion of FOXO1. When cells were pretreated with insulin for 16 h under high glucose, high fat conditions, FOXO1 was primarily located in the cytosol (Fig. 5B, middle panel). Incubation of cells with alphalipoic acid (0.25 mM) for an additional 16 h retained FOXO1 in the nucleus, reversing the insulin-stimulated nuclear exclusion in HepG2 cells (Fig. 5B, lower panel). 3.6. Alpha-lipoic acid decreases FOXO1 phosphorylation through AMPK-dependent pathways To determine whether FOXO1 phosphorylation and nuclear localisation is secondary to AMPK activation, compound C, an
Fig. 5. Alpha-lipoic acid decreases FOXO1 phosphorylation and concentrates FOXO1 in the nucleus. A: Western blot analysis of FOXO1 phosphorylation with alpha-lipoic acid treatment of HepG2 cells in a high glucose, high fat environment. FOXO1 phosphorylation was inhibited by alpha-lipoic acid. The data are expressed as the mean 7 S.E.M.; n¼3; *Po 0.05, relative to controls (0 mM alpha-lipoic acid). B: FOXO1 expression was localised by immunohistochemical staining with antiFOXO1 and DAPI (staining the nucleus). When the endogenous PI3K/Akt pathway was inhibited by serum starvation, FOXO1 was localised almost entirely within the nucleus. After treatment with insulin for 16 h, FOXO1 was localised almost entirely within the nucleus (upper panels). After treatment with insulin for 16 h, FOXO1 translocated into the cytoplasm (middle panels). When co-treated with insulin and alpha-lipoic acid (0.25 mM), FOXO1 was found to be in the nucleus (lower panels).
AMPK inhibitor, was used to block the AMPK signalling pathway. As shown in Fig. 6A, alpha-lipoic acid decreased FOXO1 phosphorylation in HepG2 cells, and this effect was reversed after pretreatment with compound C, but not nicotinamide. Consistent with these results, alpha-lipoic acid stimulated nuclear localisation of FOXO1, and this effect was inhibited by pre-treatment with an AMPK inhibitor (Fig. 6B). Taken together, the data suggest that alpha-lipoic acid induces ATGL expression through the AMPK/FOXO1 signalling pathway (Fig. 6C).
4. Discussion Non-alcoholic fatty liver disease may result in progressive liver disease with risks for cirrhosis and hepatocellular carcinoma (Brunt, 2005; 2010; Richardson et al., 2007). Several lines of evidence suggest that insulin sensitisers may be used to control non-alcoholic fatty liver disease—associated hepatocellular damage (Marchesini et al., 2001; Nair et al., 2004; Uygun et al.,
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Fig. 6. Alpha-lipoic acid decreases FOXO1 phosphorylation through AMPK-dependent pathways. A: HepG2 cells in high glucose, high fat medium were treated by cumulative addition of nicotinamide (1 mM) for 24 h followed by compound C for 30 min and subsequently alpha-lipoic acid (ALA, 0.25 mM) for 1 h; the cells were then lysed. Western blots revealed that FOXO1 phosphorylation decreased when cells were treated with Compound C. These data are expressed as the mean 7 S.E.M.; n ¼3; *Po 0.05, relative to control; #P o0.05, relative to ALA treated alone. B: Immunohistochemical analysis of FOXO1 expression. FOXO1 expression was found in the nucleus in the high glucose, high fat medium with alpha-lipoic acid (ALA) treatment but translocated into cytoplasm when treated with compound C. C: Proposed schematic—Alpha-lipoic acid decreases intracellular lipid levels through an increase in ATGL expression. Alpha-lipoic acid (ALA) activates AMPK by phosphorylation, which suppresses FOXO1 phosphorylation to maintain FOXO1 in the nucleus; FOXO1 is a transcription factor that mediates an increase in ATGL expression. AGTL along with HSL co-ordinately catabolise stored triglycerides into free fatty acids. In contrast, compound C (CC) inhibition of AMPK inactivates FOXO1 by phosphorylation and translocates FOXO1 to the cytoplasm.
2004). However, the mechanisms by which insulin sensitisers exert their lipid lowering effect are not clear, and a cell model is required to investigate the underlying mechanisms. In the present study, we demonstrated that under high glucose and high fat acid conditions, human HepG2 cells can be used as a cell model for non-alcoholic fatty liver disease. We showed that alpha-lipoic acid activates the AMPK signalling pathway, which enhances ATGL expression and decreases intracellular lipid accumulation in HepG2 cells. We also provide evidence that alpha-lipoic acidinduced ATGL expression is regulated via the FOXO1. The regulation of ATGL expression is impaired with age and could contribute to the increased difficulty to metabolise lipids (Caimari et al., 2008). ATGL is the rate-limiting enzyme in triacylglycerol hydrolysis, which produces free fatty acids (Reid et al., 2008). An increase of free fatty acid availability is linked to decreased glucose utilisation in skeletal muscle and increased
triglyceride storage in the liver (Krebs and Roden, 2005; Matsumoto et al., 2006; Watt et al., 2004). Importantly, alphalipoic acid-activation of AMPK may increase acetyl-CoA carboxylase phosphorylation, which subsequently enhances fatty acid transport across the mitochondrial membrane and facilitates fatty acid oxidation. Thus, free fatty acids released from ATGL-catalysed triglyceride hydrolysis are not likely to be accumulated. This hypothesis agrees with the fact that caloric restriction and exercise can be used to treat non-alcoholic fatty liver disease because both activate AMPK. These data are in agreement with those reported by Park et al. (2008), where alpha-lipoic acid decreases hepatic lipogenesis through AMPK-dependent pathways (Park et al., 2008) and the down-regulation of liver triglyceride secretion (Butler et al., 2009). In addition to the direct lipid-lowering effect, (Butler et al., 2009) alpha-lipoic acid has also been shown to exert anti-obesity effects by suppression of
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hypothalamic AMP-activated protein kinase (Kim et al., 2004; Park et al., 2008). SIRT1 is a NAD-dependent deacetylase that participates in cellular control of gene expression, metabolism, genomic stability, and anti-ageing. Alpha-lipoic acid can be reduced to dihydrolipoate either by mitochondrial lipoamide dehydrogenase or by the thiredoxin/thioredoxin reductase system to increase the NAD þ / NADH ratio. Increase of the NAD þ /NADH ratio may increase SIRT1 activity. Because alpha-lipoic acid activates AMPK, it is possible that AMPK activation is secondary to SIRT1 activation. However, alpha-lipoic acid-stimulated AMPK phosphorylation was not inhibited by the SIRT1 inhibitor nicotinamide in HepG2 cells, suggesting that SIRT1 does not act as an upstream activator for AMPK. In fact, inhibition of SIRT1 by nicotinamide increased the phosphorylation of AMPK (Fig. 4A) and the expression level of ATGL (Fig. 4B). These data suggest an inverse relationship between AMPK and SIRT1 pathways. However, AMPK and SIRT1 are both metabolic sensors that can increase each other’s activity and are both down-regulated in the liver under conditions of nutrient excess (Suchankova et al., 2009). Why would nicotinamide enhance AMPK phosphorylation and ATGL expression remains to be determined. This study also provides evidence that alpha-lipoic acidinduced ATGL expression is regulated via the forkhead transcription factor, FOXO1. FOXO1 is thought to play a pivotal role in the metabolic switch from the utilisation of carbohydrate to the oxidation of lipids (Gross et al., 2008). The promoter region of ATGL has two FOXO1 binding sites (Chakrabarti and Kandror, 2009). We showed that incubation of HepG2 cells with insulin resulted in the phosphorylation and nuclear exclusion of FOXO1. This result was associated with lower ATGL protein levels. Incubation with alpha-lipoic acid reversed the insulin action on FOXO1 phosphorylation and translocation and countered the effect of insulin on intracellular lipid accumulation in HepG2 cells. Our results suggest that alpha-lipoic acid may attenuate fat deposition by increasing lipolysis via the AMPK/FOXO1 pathway. Thus, alpha-lipoic acid may have application in the treatment of non-alcoholic fatty liver disease.
References Altomonte, J., Cong, L., Harbaran, S., Richter, A., Xu, J., Meseck, M., Dong, H.H., 2004. Foxo1 mediates insulin action on apoC-III and triglyceride metabolism. J. Clin. Invest. 114, 1493–1503. Angulo, P., 2007. Obesity and nonalcoholic fatty liver disease. Nutr. Rev. 65, S57–63. Ben Djoudi Ouadda, A., Levy, E., Ziv, E., Lalonde, G., Sane, A.T., Delvin, E., Elchebly, M., 2009. Increased hepatic lipogenesis in insulin resistance and Type 2 diabetes is associated with AMPK signalling pathway up-regulation in Psammomys obesus. Biosci. Rep. 29, 283–292. Brunt, E.M., 2005. Pathology of nonalcoholic steatohepatitis. Hepatol. Res. 33, 68–71. Brunt, E.M., 2009. Histopathology of non-alcoholic fatty liver disease. Clin. Liver Dis. 13, 533–544. Brunt, E.M., 2010. Pathology of nonalcoholic fatty liver disease. Nat. Rev. Gastroenterol. Hepatol. 7, 195–203. Buteau, J., Accili, D., 2007. Regulation of pancreatic beta-cell function by the forkhead protein FoxO1. Diabetes Obes. Metab. 9 (Suppl. 2), 140–146. Butler, J.A., Hagen, T.M., Moreau, R., 2009. Lipoic acid improves hypertriglyceridemia by stimulating triacylglycerol clearance and downregulating liver triacylglycerol secretion. Arch. Biochem. Biophys. 485, 63–71. Caimari, A., Oliver, P., Palou, A., 2008. Impairment of nutritional regulation of adipose triglyceride lipase expression with age. Int. J. Obes. (London) 32, 1193–1200. Carter-Kent, C., Yerian, L.M., Brunt, E.M., Angulo, P., Kohli, R., Ling, S.C., Xanthakos, S.A., Whitington, P.F., Charatcharoenwitthaya, P., Yap, J., Lopez, R., McCullough, A.J., Feldstein, A.E., 2009. Nonalcoholic steatohepatitis in children: a multicenter clinicopathological study. Hepatology 50, 1113–1120. Chakrabarti, P., Kandror, K.V., 2009. FoxO1 controls insulin-dependent adipose triglyceride lipase (ATGL) expression and lipolysis in adipocytes. J. Biol. Chem. 284, 13296–13300.
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Cortez-Pinto, H., Machado, M., 2008. Impact of body weight, diet and lifestyle on nonalcoholic fatty liver disease. Expert Rev. Gastroenterol. Hepatol. 2, 217–231. Evans, J.L., Goldfine, I.D., 2000. Alpha-lipoic acid: a multifunctional antioxidant that improves insulin sensitivity in patients with type 2 diabetes. Diabetes Technol. Ther. 2, 401–413. Feldstein, A.E., Charatcharoenwitthaya, P., Treeprasertsuk, S., Benson, J.T., Enders, F.B., Angulo, P., 2009. The natural history of non-alcoholic fatty liver disease in children: a follow-up study for up to 20 years. Gut 58, 1538–1544. Gaidhu, M.P., Fediuc, S., Anthony, N.M., So, M., Mirpourian, M., Perry, R.L., Ceddia, R.B., 2009. Prolonged AICAR-induced AMP-kinase activation promotes energy dissipation in white adipocytes: novel mechanisms integrating HSL and ATGL. J. Lipid Res. 50, 704–715. Gross, D.N., van den Heuvel, A.P., Birnbaum, M.J., 2008. The role of FoxO in the regulation of metabolism. Oncogene 27, 2320–2336. Gupta, R., Bhangoo, A., Matthews, N.A., Anhalt, H., Matta, Y., Lamichhane, B., Malik, S., Narwal, S., Wetzler, G., Ten, S., 2011. The prevalence of non-alcoholic fatty liver disease and metabolic syndrome in obese children. J. Pediatr. Endocrinol. Metab. 24, 907–911. Haeusler, R.A., Han, S., Accili, D., 2010. Hepatic FoxO1 ablation exacerbates lipid abnormalities during hyperglycemia. J. Biol. Chem. 285, 26861–26868. Houde, V.P., Brule, S., Festuccia, W.T., Blanchard, P.G., Bellmann, K., Deshaies, Y., Marette, A., 2010. Chronic rapamycin treatment causes glucose intolerance and hyperlipidemia by upregulating hepatic gluconeogenesis and impairing lipid deposition in adipose tissue. Diabetes 59, 1338–1348. Jager, S., Handschin, C., St-Pierre, J., Spiegelman, B.M., 2007. AMP-activated protein kinase (AMPK) action in skeletal muscle via direct phosphorylation of PGC1alpha. Proc. Natl. Acad. Sci. U. S. A. 104, 12017–12022. Jocken, J.W., Langin, D., Smit, E., Saris, W.H., Valle, C., Hul, G.B., Holm, C., Arner, P., Blaak, E.E., 2007. Adipose triglyceride lipase and hormone-sensitive lipase protein expression is decreased in the obese insulin-resistant state. J. Clin. Endocrinol. Metab. 92, 2292–2299. Kim, M.S., Park, J.Y., Namkoong, C., Jang, P.G., Ryu, J.W., Song, H.S., Yun, J.Y., Namgoong, I.S., Ha, J., Park, I.S., Lee, I.K., Viollet, B., Youn, J.H., Lee, H.K., Lee, K.U., 2004. Anti-obesity effects of alpha-lipoic acid mediated by suppression of hypothalamic AMP-activated protein kinase. Nat. Med. 10, 727–733. Krebs, M., Roden, M., 2005. Molecular mechanisms of lipid-induced insulin resistance in muscle, liver and vasculature. Diabetes Obes. Metab. 7, 621–632. Lee, D.H., Park, D.B., Lee, Y.K., An, C.S., Oh, Y.S., Kang, J.S., Kang, S.H., Chung, M.Y., 2005. The effects of thiazolidinedione treatment on the regulations of aquaglyceroporins and glycerol kinase in OLETF rats. Metabolism 54, 1282–1289. Marchesini, G., Brizi, M., Bianchi, G., Tomassetti, S., Bugianesi, E., Lenzi, M., McCullough, A.J., Natale, S., Forlani, G., Melchionda, N., 2001. Nonalcoholic fatty liver disease: a feature of the metabolic syndrome. Diabetes 50, 1844–1850. Matsumoto, M., Han, S., Kitamura, T., Accili, D., 2006. Dual role of transcription factor FoxO1 in controlling hepatic insulin sensitivity and lipid metabolism. J. Clin. Invest. 116, 2464–2472. Nair, S., Diehl, A.M., Wiseman, M., Farr Jr., G.H., Perrillo, R.P., 2004. Metformin in the treatment of non-alcoholic steatohepatitis: a pilot open label trial. Aliment Pharmacol. Ther. 20, 23–28. Narbonne, P., Roy, R., 2009. Caenorhabditis elegans dauers need LKB1/AMPK to ration lipid reserves and ensure long-term survival. Nature 457, 210–214. Nobili, V., Manco, M., Devito, R., Di Ciommo, V., Comparcola, D., Sartorelli, M.R., Piemonte, F., Marcellini, M., Angulo, P., 2008. Lifestyle intervention and antioxidant therapy in children with nonalcoholic fatty liver disease: a randomized, controlled trial. Hepatology 48, 119–128. Pacifico, L., Nobili, V., Anania, C., Verdecchia, P., Chiesa, C., 2011. Pediatric nonalcoholic fatty liver disease, metabolic syndrome and cardiovascular risk. World J. Gastroenterol. 17, 3082–3091. Park, K.G., Min, A.K., Koh, E.H., Kim, H.S., Kim, M.O., Park, H.S., Kim, Y.D., Yoon, T.S., Jang, B.K., Hwang, J.S., Kim, J.B., Choi, H.S., Park, J.Y., Lee, I.K., Lee, K.U., 2008. Alpha-lipoic acid decreases hepatic lipogenesis through adenosine monophosphate-activated protein kinase (AMPK)-dependent and AMPK-independent pathways. Hepatology 48, 1477–1486. Patton, H.M., Sirlin, C., Behling, C., Middleton, M., Schwimmer, J.B., Lavine, J.E., 2006. Pediatric nonalcoholic fatty liver disease: a critical appraisal of current data and implications for future research. J. Pediatr. Gastroenterol. Nutr. 43, 413–427. Reid, B.N., Ables, G.P., Otlivanchik, O.A., Schoiswohl, G., Zechner, R., Blaner, W.S., Goldberg, I.J., Schwabe, R.F., Chua Jr., S.C., Huang, L.S., 2008. Hepatic overexpression of hormone-sensitive lipase and adipose triglyceride lipase promotes fatty acid oxidation, stimulates direct release of free fatty acids, and ameliorates steatosis. J. Biol. Chem. 283, 13087–13099. Richardson, M.M., Jonsson, J.R., Powell, E.E., Brunt, E.M., Neuschwander-Tetri, B.A., Bhathal, P.S., Dixon, J.B., Weltman, M.D., Tilg, H., Moschen, A.R., Purdie, D.M., Demetris, A.J., Clouston, A.D., 2007. Progressive fibrosis in nonalcoholic steatohepatitis: association with altered regeneration and a ductular reaction. Gastroenterology 133, 80–90. Son, N.H., Yu, S., Tuinei, J., Arai, K., Hamai, H., Homma, S., Shulman, G.I., Abel, E.D., Goldberg, I.J., 2010. PPARgamma-induced cardiolipotoxicity in mice is ameliorated by PPARalpha deficiency despite increases in fatty acid oxidation. J. Clin. Invest. 120, 3443–3454.
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Suchankova, G., Nelson, L.E., Gerhart-Hines, Z., Kelly, M., Gauthier, M.S., Saha, A.K., Ido, Y., Puigserver, P., Ruderman, N.B., 2009. Concurrent regulation of AMP-activated protein kinase and SIRT1 in mammalian cells. Biochem. Biophys. Res. Commun. 378, 836–841. Uygun, A., Kadayifci, A., Isik, A.T., Ozgurtas, T., Deveci, S., Tuzun, A., Yesilova, Z., Gulsen, M., Dagalp, K., 2004. Metformin in the treatment of patients with nonalcoholic steatohepatitis. Aliment Pharmacol. Ther. 19, 537–544. Watt, M.J., Southgate, R.J., Holmes, A.G., Febbraio, M.A., 2004. Suppression of plasma free fatty acids upregulates peroxisome proliferator-activated
receptor (PPAR) alpha and delta and PPAR coactivator 1alpha in human skeletal muscle, but not lipid regulatory genes. J. Mol. Endocrinol. 33, 533–544. Winder, W.W., Holmes, B.F., 2000. Insulin stimulation of glucose uptake fails to decrease palmitate oxidation in muscle if AMPK is activated. J. Appl. Physiol. 89, 2430–2437. Zechner, R., Kienesberger, P.C., Haemmerle, G., Zimmermann, R., Lass, A., 2009. Adipose triglyceride lipase and the lipolytic catabolism of cellular fat stores. J. Lipid Res. 50, 3–21.
Contents lists available at SciVerse ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
Alpha-lipoic acid induces adipose triglyceride lipase expression and decreases intracellular lipid accumulation in HepG2 cells Yung-Ting Kuo a,b,c, Ting-Han Lin c,d, Wei-Lu Chen c,d, Horng-Mo Lee c,d,e,n a
Department of Pediatrics, Shuang Ho Hospital, Taipei Medical University, Taiwan Department of Pediatrics, School of Medicine, College of Medicine, Taipei Medical University, Taiwan c Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taiwan d School of Medical Laboratory Sciences and Biotechnology, College of Medicine, Taipei Medical University, Taiwan e Institute of Pharmaceutical Science and Technology, Central Taiwan University of Science and Technology, Taichung, Taiwan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 July 2011 Received in revised form 5 July 2012 Accepted 7 July 2012 Available online 20 July 2012
Non-alcoholic fatty liver disease can be attributed to the imbalance between lipogenesis and lipolysis in the liver. Alpha-lipoic acid has been shown to activate the 50 -AMP-activated protein kinase (AMPK) signalling pathway and to effectively inhibit the lipogenesis pathway in liver. However, whether alphalipoic acid stimulates lipolysis remains unclear. Recently, adipose triglyceride lipase (ATGL) was shown to be responsible for triacylglycerol hydrolase activity in cells. In the present study, we established a fatty liver cell model by incubating HepG2 cells in a high glucose (30 mM glucose) and high fat (0.1 mM palmitate) medium. We found that the activation of the AMPK signalling pathway induced ATGL protein expression and enhanced lipid hydrolysis. Similarly, treatment of the fatty liver cell model with alpha-lipoic acid reduced intracellular lipid accumulation in HepG2 cells, increased AMPK phosphorylation, and induced ATGL expression. We showed that insulin phosphorylates the transcription factor forkhead box O1 (FOXO1), which regulates ATGL expression and inhibits FOXO1 translocation into the nucleus. In contrast, alpha-lipoic acid dephosphorylated FOXO1 and reversed the nuclear exclusion of FOXO1. These data suggest that alpha-lipoic acid can effectively ameliorate intracellular lipid accumulation and induce ATGL expression through the FOXO1/ATGL pathway in liver cells. Thus, alpha-lipoic acid may be a potential therapeutic agent for treating fatty liver disease. Crown Copyright & 2012 Published by Elsevier B.V. All rights reserved.
Keywords: Alpha-lipoic acid (ALA) 50 -AMP-activated protein kinase (AMPK) Adipose triglyceride lipase (ATGL) Forkhead box O1 (FOXO1)
1. Introduction Non-alcoholic fatty liver disease is an important feature of metabolic diseases such as chronic liver disease and is strongly associated with obesity and dysregulated insulin action in the liver (Feldstein et al., 2009; Gupta et al., 2011; Pacifico et al., 2011). Pathologic findings show that the parenchyma of the liver is characterised by increased intracellular triglyceride accumulation (Brunt, 2009; Carter-Kent et al., 2009; Patton et al., 2006). Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are the major triglyceride lipases in many tissues (Reid et al., 2008). ATGL and HSL expression are decreased in the obese, insulin-resistant state, suggesting that insulin resistance is associated with impaired lipolysis (Jocken et al., 2007; Zechner et al., 2009).
n Corresponding author at: Institute of Pharmaceutical Sciences and Technology, Central Taiwan University of Science and Technology, Taichung, Taiwan. Tel.: þ886 2 2736 1661; fax: þ 886 2 2732 4510. E-mail address: [email protected] (H.-M. Lee).
Current treatments for patients with fatty liver disease include change of lifestyle and improvement of insulin sensitivity to alleviate the associated metabolic syndrome (Angulo, 2007; Cortez-Pinto and Machado, 2008; Nobili et al., 2008). Alpha-lipoic acid is an endogenous cofactor in many multi-enzyme complexes that exerts beneficial effects on obesity, type 2 diabetes mellitus, and non-alcoholic fatty liver disease in patients with insulin resistance (Park et al., 2008; Reid et al., 2008). The 50 -AMP-activated protein kinase (AMPK) signalling pathway is thought to be a natural response to reduce dyslipidemia and to ameliorate insulin resistance (Ben Djoudi Ouadda et al., 2009). AMPK is a fuel-sensing enzyme that exerts effects on cellular energy metabolism and mitochondrial biogenesis (Jager et al., 2007; Winder and Holmes, 2000). Once activated, AMPK phosphorylates acetyl-CoA carboxylase, thereby blocking its activity. This in turn decreases the malonyl-CoA concentration, leading to enhanced mitochondrial fatty acid b-oxidation. Alphalipoic acid was shown to reduce body weight by suppressing hypothalamic AMPK (Kim et al., 2004). In diabetes-prone Otsuka Long Tokushima fatty rats, alpha-lipoic acid increased insulin sensitivity and skeletal muscle fatty acid oxidation via AMPK
0014-2999/$ - see front matter Crown Copyright & 2012 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2012.07.028
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activation (Evans and Goldfine, 2000; Lee et al., 2005). Treatment with alpha-lipoic acid ameliorated severe hypertriglyceridemia by inhibiting TG synthesis and very low density lipoproteintriglyceride (VLDL-TG) secretion in Zucker diabetic fatty rats (Butler et al., 2009). AMPK has been shown to phosphorylate ATGL (Narbonne and Roy, 2009). In adipose tissue, the activation of AMPK-dependent pathways favours energy dissipation rather than lipid storage (Gaidhu et al., 2009). The forkhead box gene group O (FOXO) subfamily of transcription factors regulates carbohydrate and lipid metabolism (Altomonte et al., 2004) and pancreatic cell function (Buteau and Accili, 2007). The protein FOXO1 can be phosphorylated via an insulin-dependent pathway; the phosphorylation of FOXO1
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leads to the nuclear exclusion and inhibition of FOXO1 activity (Chakrabarti and Kandror, 2009). In the insulin-resistant state, FOXO1 increases the hepatic gluconeogenic master genes, phosphoenolpyruvate carboxykinase and glucose 6-phosphatase, and increases ATGL expression (Chakrabarti and Kandror, 2009), thus protecting against excessive lipid accumulation in the liver during hyperglycaemia (Haeusler et al., 2010; Houde et al., 2010). Because of the aforementioned metabolic actions of AMPK and FOXO1, we investigated whether alpha-lipoic acid regulates lipid metabolism through the AMPK/FOXO1 signalling pathway. We demonstrated that alpha-lipoic acid enhances ATGL expression through the AMPK/FOXO1 pathway, which in turn reduces triacylglycerol accumulation in HepG2 cells exposed to high
Fig. 1. A fatty liver model—High glucose, high fat environment induces intracellular lipid formation in HepG2 cells and induces ATGL. A: Micrographs of HepG2 cells incubated for 24 h in high glucose medium with various concentration of palmitate to simulate a high fat environment. Oil red O staining at 1000 magnification. The concentration of intracellular lipid droplets increased with palmitate concentration. B: MTT colorimetric assay for cell viability in the high glucose, high fat environment after 24 h. No differences were observed between control and high fat (0.1 mM palmitate) conditions. C: Western blots of ATGL and GAPDH for HepG2 cells incubated with various concentrations (0.05, 0.1, 0.5, 1 mM) of palmitate at 37 1C for 24 h. NC, control cells incubated at normal glucose concentration (5.5 mM). HG, control cells incubated in high glucose medium (30 mM).
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glucose and palmitate. These data suggest that alpha-lipoic acid may exert beneficial effects in treating fatty liver disease.
and anti-phospho-Ser256 FOXO1 were purchased from Cell Signaling Technology (Beverly, MA). 2.2. Culture of human HepG2 cells and preparation of cell lysates
2. Materials and methods 2.1. Materials Dulbecco’s modified Eagle medium (DMEM), foetal calf serum, and glutamine were purchased from Life Technologies (Gaithersburg, MD). Rabbit polyclonal antibodies for AMPK-a1 and AMPKa2 were purchased from Bethyl Laboratories (Montgomery, TX). The anti-GAPDH antibody was from Gentex (Irvine, CA). The a-tubulin, horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). 5-Aminoimidazole-4carboxamide riboside (AICAR), compound C, metformin, and nicotinamide were purchased from Calbiochem-Novabiochem (San Diego, CA). The rabbit monoclonal anti-FOXO1 antibody
HepG2 cells were cultured in DMEM supplemented with 10% heat-inactivated foetal calf serum and sodium pyruvate (1 mM). After reaching 90% confluence, HepG2 cells were treated with various concentrations of the indicated agents and incubated for the indicated times in a 5% CO2-humidified incubator at 37 1C. Cells were removed using a rubber policeman in the presence of lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 100 ml/ml PMSF, 1% NP-40, and 4% protease inhibitor cocktail) to obtain cell lysates for western blotting. 2.3. Polyacrylamide gel electrophoresis (PAGE) and western blotting Cell lysates of equal protein concentration were separated by sodium dodecylsulphate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoridemembranes for
Fig. 2. AMPK activation by metformin increases ATGL expression and decreases intracellular lipid droplet accumulation. A, B, Western blots of phospho-AMPK (pAMPK) and ATGL expression. HepG2 cells were incubated with high glucose (HG, 30 mM) and high fat (HF, 1 mM palmitate) medium for 24 h. A: AMPK phosphorylation increased after AICAR (A) and metformin (M) treatment, whereas compound C (CC) decreased AMPK phosphorylation. B: ATGL was also induced by AICAR and metformin. C: HepG2 cells were treated with metformin for 1 h after incubation in high glucose, high fat (0.1 mM palmitate) medium for 24 h. Lipid droplets were stained with oil red O stain and observed under light microscopy at 1000 magnification. The concentration of intracellular lipid droplets decreased compared to the control cells.
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immunoblotting. Membranes were blocked with blocking solution containing 3% bovine serum albumin in phosphate-buffered saline (PBS) containing 0.1% Tween 20 for 1 h at room temperature. This was followed by sequential incubation with solutions containing primary and secondary antibodies with PBS washes in between. Immunodetection was performed using a LumiGLO chemiluminescence kit (GE Healthcare Life Sciences). Levels of protein expression were quantified by scanning densitometry using a model GS-700 imaging densitometer (Bio-Rad) and normalised to a-tubulin levels as a control. 2.4. Oil red O staining HepG2 cells were grown in 6-well plates for 24 h. At the end of drug treatment, the cells were washed with PBS and fixed with 4% PBS-buffered formaldehyde for 15 min. Subsequently, the cells were rinsed with water, dipped for a few seconds in 60% isopropanol, stained in oil red O for 15 min, and rinsed again in 60% isopropanol to remove excess stain. Cell nuclei were stained
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for a few seconds in haematoxylin solution, washed with water, and mounted with commercially available Mounting Medium (Dako, Glostrup, Denmark). The samples were photographed by microscope (Olympus B 60) using the Olympus MicroImage software. 2.5. Immunofluorescence HepG2 cells were seeded in 6-well plates containing sterilised coverslips and incubated at 37 1C for defined periods with drug stimulation. After treatment, the cells were washed twice with PBS (137 mM NaCl, 1.4 mM KH2PO4, 4.3 mM Na2HPO4, 2.7 mM KCl, pH 7.2) and fixed with 4% PBS-buffered formaldehyde for 10 min at room temperature. After fixation, the cells were washed three times with PBS for 5 min each time. Thereafter, Triton-X 100 was added for 10 min, and the cells were washed three times with PBS. The coverslips were then incubated with skimmed milk containing PBS blocking buffer at room temperature for 1 h and subsequently with specific antibodies in PBS blocking buffer
Fig. 3. Alpha-lipoic acid induces AMPK phosphorylation and enhances ATGL expression. A: HepG2 cells were incubated for various times with 0.25 mM a-lipoic acid in high glucose (30 mM), high fat (0.1 mM palmitate) medium and then lysed. Phosphorylation of AMPK (pAMPK-Thr172) was analysed by Western blots. The ratios of pAMPK to tAMPK were quantified from three independent experiments per condition. The data are expressed as the mean 7S.E.M.; n¼ 3; *P o0.05, relative to controls (0 mM a-lipoic acid). B: Western blots of phospho-AMPK (pAMPK) induced by alpha-lipoic acid in high glucose, high fat medium. The data are expressed as the mean 7S.E.M.; n¼ 3; *Po 0.05, relative to controls (0 mM alpha-lipoic acid). C: Western blots of ATGL induced by alpha-lipoic acid (ALA) in high glucose, high fat medium. The data are expressed as the mean 7 S.E.M.; n¼3; *Po 0.05, relative to controls (0 mM alpha-lipoic acid). D: Micrographs of HepG2 cells in high glucose, high fat medium treated with alpha-lipoic acid. 1000 magnification. Oil red O staining shows that the concentration of neutral lipid droplets in the intracellular cytoplasm decreased inversely with an increase in alpha-lipoic acid concentration.
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overnight at 4 1C. The following day, the cells were washed three times with PBS, incubated with fluorescein-conjugated goat antirabbit IgG antibody for 1 h at 37 1C, and washed three times. The nuclei were stained with DAPI.
3. Results
2.6. MTT assay
Lipid droplet-associated triglycerides were found in many non-adipose tissues when they were supplied with excessive nutrients (Son et al., 2010). To determine whether liver cells esterify and deposit fatty acid as lipid droplets, HepG2 cells were incubated in high glucose (30 mM) media with various concentrations of palmitate (0.05–1 mM) for 24 h. After incubation, intracellular lipid accumulation was determined by the oil red O staining. Oil red O staining revealed that intracellular lipid droplets had accumulated in HepG2 cells when incubated in medium with high concentrations of palmitate (0.1 mM) (Fig. 1A). Incubation of HepG2 cells with high concentrations of palmitate did not affect cell viability as demonstrated by the MTT assay (Fig. 1B). ATGL is responsible for the catabolism of cellular lipid stores (Zechner et al., 2009). ATGL expression levels were examined by western blotting of HepG2 cells. HepG2 cells expressed substantial levels of ATGL at a normal glucose concentration (NC, 5.5 mM). However, the expression level of ATGL was lower when cells were incubated at a high glucose concentration (HG, 30 mM). Treatment of cells with a high palmitate concentration (0.5 mM) for 24 h increased expression levels of ATGL under high glucose conditions. Palmitate at 0.1 mM did not increase ATGL protein levels in HepG2 cells (Fig. 1C). Thus, we established the parameters for a fatty liver cell model by incubating HepG2 cells in high glucose (30 mM), high fat (0.1 mM palmitate) medium.
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is a test of metabolic competence and assesses mitochondrial performance. The colourimetric assay relies on the conversion of yellow tetrazolium bromide to the purple formazan derivative by mitochondrial succinate dehydrogenase in viable cells. HepG2 cells were seeded onto 24-well plates at a density of 2 104 cells/well in serum-free medium. The medium was removed after the cells were cultured for 24 h in the reagent medium (100 ml). The cell viability was then measured by the MTT assay. After reagent treatment for 24 h, the cells were washed once with PBS. Subsequently, fresh growth medium was added to each well, and the cells were cultured for 24 h. MTT was then added at a final concentration 0.5 mg/ml, and the cells were further incubated for 2 h at 37 1C. The medium was removed, and the formazan crystals were dissolved in DMSO (200 ml). The optical density (OD) was measured at 570 nm (reference filter 690 nm) using an ELISA microplate reader. Survival (viability) was determined by comparing the OD of the wells containing treated cells to those containing vehicle-treated (0.1% ethanol) cells.
3.1. A fatty liver cell model—High glucose, high fat environment increases intracellular lipid formation in HepG2 cells and induces ATGL expression
2.7. Statistical analysis All data are expressed as the mean 7S.E.M., with the number of experiments denoted by n. Statistical significance (P o0.05) between experimental groups was determined by single-factor analysis of variance (ANOVA) for multiple groups or an unpaired t-test for two groups.
3.2. AMPK activation increased ATGL expression and decreased intracellular lipid accumulation under high fat conditions To determine whether activation of AMPK leads to ATGL protein expression, AMPK activators, AICAR and metformin, were used to treat HepG2 cells. Both AICAR and metformin increased
Fig. 4. Inhibition of sirtuins does not affect alpha-lipoic acid-mediated induction of AMPK phosphorylation or ATGL expression. HepG2 cells in high glucose, high fat medium were pretreated with compound C (20 mM) or nicotinamide (NA; 1 mM) for 30 min prior to the addition of alpha-lipoic acid (0.25 mM) for 24 h. A: Pretreatment with nicotinamide, a sirtuin inhibitor, prior to addition of alpha-lipoic acid (ALA), induced AMPK phosphorylation (pAMPK); in contrast, pretreatment with compound C (CC) inhibited AMPK phosphorylation. B: ATGL expression was induced by alpha-lipoic acid alone or with nicotinamide pretreatment. However, compound C (CC) lowered the alpha-lipoic acid-mediated induction of ATGL. These data are expressed as the mean 7S.E.M.; n¼3; *P o 0.05, relative to control; #P o0.05, relative to ALA treated alone.
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AMPK phosphorylation in HepG2 cells, and these effects were inhibited by pretreatment with compound C, a selective AMPK inhibitor (Fig. 2A). Activation of AMPK is associated with increased phosphorylation of acetyl-CoA carboxylase at Ser79 in HepG2 cells (data not shown). AMPK activators induced ATGL protein expression (Fig. 2B) and decreased intracellular lipid droplet accumulation in HepG2 cells (Fig. 2C). 3.3. Alpha-lipoic acid stimulates AMPK phosphorylation and ATGL expression Alpha-lipoic acid has been shown to activate AMPK in skeletal muscle cells (Lee et al., 2005). Thus, the effects of alpha-lipoic acid on AMPK phosphorylation in HepG2 cells needed to be determined. As expected, treatment of HepG2 cells with alpha-lipoic acid increased AMPK phosphorylation in a time-dependent and concentration-dependent manner (Figs. 3A and B). To determine whether alpha-lipoic acid induced ATGL expression, cells were incubated in high glucose (30 mM), high fat (0.1 mM palmitate) medium in the presence of various concentrations of alpha-lipoic acid (0.25–1.0 mM). As shown in Fig. 3C, treatment with alphalipoic acid increased ATGL protein levels in HepG2 cells. In agreement with these findings, treatment with alpha-lipoic acid reduced intracellular lipid accumulation as shown by oil red O and haematoxylin staining (Fig. 3D). 3.4. SIRT pathway does not affect alpha-lipoic acid-induced AMPK phosphorylation or ATGL expression The NAD(þ)-dependent deacetylase and gene repressor SIRT1 has been shown to play a role in regulating AMPK and the functional consequences of its activation. To determine whether SIRT1 is involved in alpha-lipoic acid-induced ATGL expression, a pharmacological SIRT1 inhibitor was used to block SIRT1 activity. HepG2 cells were pretreated with nicotinamide, an inhibitor of sirtuins, or compound C, an inhibitor of AMPK, for 30 min before alpha-lipoic acid was added. As shown in Fig. 4A, treatment with alpha-lipoic acid increased AMPK phosphorylation, which was blocked by pretreatment with compound C, but not nicotinamide. Similar effects were seen in alpha-lipoic acid-induced ATGL expression (Fig. 4B). These data suggested that SIRT1 is not involved in alpha-lipoic acid-induced ATGL expression. 3.5. Alpha-lipoic acid decreases FOXO1 phosphorylation and reverses insulin-induced FOXO1 nuclear exclusion in HepG2 cells FOXO1 plays an important role in transcriptional control of ATGL. To determine whether alpha-lipoic acid regulates ATGL expression through FOXO1, the effects of alpha-lipoic acid on FOXO1 phosphorylation were examined. Fig. 5A shows that alpha-lipoic acid suppressed endogenous FOXO1 phosphorylation in HepG2 cells. Insulin is one of the most important anabolic hormones involved in regulating hepatic lipogenesis. It increases lipogenesis through the phosphorylation and nuclear exclusion of FOXO1. When cells were pretreated with insulin for 16 h under high glucose, high fat conditions, FOXO1 was primarily located in the cytosol (Fig. 5B, middle panel). Incubation of cells with alphalipoic acid (0.25 mM) for an additional 16 h retained FOXO1 in the nucleus, reversing the insulin-stimulated nuclear exclusion in HepG2 cells (Fig. 5B, lower panel). 3.6. Alpha-lipoic acid decreases FOXO1 phosphorylation through AMPK-dependent pathways To determine whether FOXO1 phosphorylation and nuclear localisation is secondary to AMPK activation, compound C, an
Fig. 5. Alpha-lipoic acid decreases FOXO1 phosphorylation and concentrates FOXO1 in the nucleus. A: Western blot analysis of FOXO1 phosphorylation with alpha-lipoic acid treatment of HepG2 cells in a high glucose, high fat environment. FOXO1 phosphorylation was inhibited by alpha-lipoic acid. The data are expressed as the mean 7 S.E.M.; n¼3; *Po 0.05, relative to controls (0 mM alpha-lipoic acid). B: FOXO1 expression was localised by immunohistochemical staining with antiFOXO1 and DAPI (staining the nucleus). When the endogenous PI3K/Akt pathway was inhibited by serum starvation, FOXO1 was localised almost entirely within the nucleus. After treatment with insulin for 16 h, FOXO1 was localised almost entirely within the nucleus (upper panels). After treatment with insulin for 16 h, FOXO1 translocated into the cytoplasm (middle panels). When co-treated with insulin and alpha-lipoic acid (0.25 mM), FOXO1 was found to be in the nucleus (lower panels).
AMPK inhibitor, was used to block the AMPK signalling pathway. As shown in Fig. 6A, alpha-lipoic acid decreased FOXO1 phosphorylation in HepG2 cells, and this effect was reversed after pretreatment with compound C, but not nicotinamide. Consistent with these results, alpha-lipoic acid stimulated nuclear localisation of FOXO1, and this effect was inhibited by pre-treatment with an AMPK inhibitor (Fig. 6B). Taken together, the data suggest that alpha-lipoic acid induces ATGL expression through the AMPK/FOXO1 signalling pathway (Fig. 6C).
4. Discussion Non-alcoholic fatty liver disease may result in progressive liver disease with risks for cirrhosis and hepatocellular carcinoma (Brunt, 2005; 2010; Richardson et al., 2007). Several lines of evidence suggest that insulin sensitisers may be used to control non-alcoholic fatty liver disease—associated hepatocellular damage (Marchesini et al., 2001; Nair et al., 2004; Uygun et al.,
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Fig. 6. Alpha-lipoic acid decreases FOXO1 phosphorylation through AMPK-dependent pathways. A: HepG2 cells in high glucose, high fat medium were treated by cumulative addition of nicotinamide (1 mM) for 24 h followed by compound C for 30 min and subsequently alpha-lipoic acid (ALA, 0.25 mM) for 1 h; the cells were then lysed. Western blots revealed that FOXO1 phosphorylation decreased when cells were treated with Compound C. These data are expressed as the mean 7 S.E.M.; n ¼3; *Po 0.05, relative to control; #P o0.05, relative to ALA treated alone. B: Immunohistochemical analysis of FOXO1 expression. FOXO1 expression was found in the nucleus in the high glucose, high fat medium with alpha-lipoic acid (ALA) treatment but translocated into cytoplasm when treated with compound C. C: Proposed schematic—Alpha-lipoic acid decreases intracellular lipid levels through an increase in ATGL expression. Alpha-lipoic acid (ALA) activates AMPK by phosphorylation, which suppresses FOXO1 phosphorylation to maintain FOXO1 in the nucleus; FOXO1 is a transcription factor that mediates an increase in ATGL expression. AGTL along with HSL co-ordinately catabolise stored triglycerides into free fatty acids. In contrast, compound C (CC) inhibition of AMPK inactivates FOXO1 by phosphorylation and translocates FOXO1 to the cytoplasm.
2004). However, the mechanisms by which insulin sensitisers exert their lipid lowering effect are not clear, and a cell model is required to investigate the underlying mechanisms. In the present study, we demonstrated that under high glucose and high fat acid conditions, human HepG2 cells can be used as a cell model for non-alcoholic fatty liver disease. We showed that alpha-lipoic acid activates the AMPK signalling pathway, which enhances ATGL expression and decreases intracellular lipid accumulation in HepG2 cells. We also provide evidence that alpha-lipoic acidinduced ATGL expression is regulated via the FOXO1. The regulation of ATGL expression is impaired with age and could contribute to the increased difficulty to metabolise lipids (Caimari et al., 2008). ATGL is the rate-limiting enzyme in triacylglycerol hydrolysis, which produces free fatty acids (Reid et al., 2008). An increase of free fatty acid availability is linked to decreased glucose utilisation in skeletal muscle and increased
triglyceride storage in the liver (Krebs and Roden, 2005; Matsumoto et al., 2006; Watt et al., 2004). Importantly, alphalipoic acid-activation of AMPK may increase acetyl-CoA carboxylase phosphorylation, which subsequently enhances fatty acid transport across the mitochondrial membrane and facilitates fatty acid oxidation. Thus, free fatty acids released from ATGL-catalysed triglyceride hydrolysis are not likely to be accumulated. This hypothesis agrees with the fact that caloric restriction and exercise can be used to treat non-alcoholic fatty liver disease because both activate AMPK. These data are in agreement with those reported by Park et al. (2008), where alpha-lipoic acid decreases hepatic lipogenesis through AMPK-dependent pathways (Park et al., 2008) and the down-regulation of liver triglyceride secretion (Butler et al., 2009). In addition to the direct lipid-lowering effect, (Butler et al., 2009) alpha-lipoic acid has also been shown to exert anti-obesity effects by suppression of
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hypothalamic AMP-activated protein kinase (Kim et al., 2004; Park et al., 2008). SIRT1 is a NAD-dependent deacetylase that participates in cellular control of gene expression, metabolism, genomic stability, and anti-ageing. Alpha-lipoic acid can be reduced to dihydrolipoate either by mitochondrial lipoamide dehydrogenase or by the thiredoxin/thioredoxin reductase system to increase the NAD þ / NADH ratio. Increase of the NAD þ /NADH ratio may increase SIRT1 activity. Because alpha-lipoic acid activates AMPK, it is possible that AMPK activation is secondary to SIRT1 activation. However, alpha-lipoic acid-stimulated AMPK phosphorylation was not inhibited by the SIRT1 inhibitor nicotinamide in HepG2 cells, suggesting that SIRT1 does not act as an upstream activator for AMPK. In fact, inhibition of SIRT1 by nicotinamide increased the phosphorylation of AMPK (Fig. 4A) and the expression level of ATGL (Fig. 4B). These data suggest an inverse relationship between AMPK and SIRT1 pathways. However, AMPK and SIRT1 are both metabolic sensors that can increase each other’s activity and are both down-regulated in the liver under conditions of nutrient excess (Suchankova et al., 2009). Why would nicotinamide enhance AMPK phosphorylation and ATGL expression remains to be determined. This study also provides evidence that alpha-lipoic acidinduced ATGL expression is regulated via the forkhead transcription factor, FOXO1. FOXO1 is thought to play a pivotal role in the metabolic switch from the utilisation of carbohydrate to the oxidation of lipids (Gross et al., 2008). The promoter region of ATGL has two FOXO1 binding sites (Chakrabarti and Kandror, 2009). We showed that incubation of HepG2 cells with insulin resulted in the phosphorylation and nuclear exclusion of FOXO1. This result was associated with lower ATGL protein levels. Incubation with alpha-lipoic acid reversed the insulin action on FOXO1 phosphorylation and translocation and countered the effect of insulin on intracellular lipid accumulation in HepG2 cells. Our results suggest that alpha-lipoic acid may attenuate fat deposition by increasing lipolysis via the AMPK/FOXO1 pathway. Thus, alpha-lipoic acid may have application in the treatment of non-alcoholic fatty liver disease.
References Altomonte, J., Cong, L., Harbaran, S., Richter, A., Xu, J., Meseck, M., Dong, H.H., 2004. Foxo1 mediates insulin action on apoC-III and triglyceride metabolism. J. Clin. Invest. 114, 1493–1503. Angulo, P., 2007. Obesity and nonalcoholic fatty liver disease. Nutr. Rev. 65, S57–63. Ben Djoudi Ouadda, A., Levy, E., Ziv, E., Lalonde, G., Sane, A.T., Delvin, E., Elchebly, M., 2009. Increased hepatic lipogenesis in insulin resistance and Type 2 diabetes is associated with AMPK signalling pathway up-regulation in Psammomys obesus. Biosci. Rep. 29, 283–292. Brunt, E.M., 2005. Pathology of nonalcoholic steatohepatitis. Hepatol. Res. 33, 68–71. Brunt, E.M., 2009. Histopathology of non-alcoholic fatty liver disease. Clin. Liver Dis. 13, 533–544. Brunt, E.M., 2010. Pathology of nonalcoholic fatty liver disease. Nat. Rev. Gastroenterol. Hepatol. 7, 195–203. Buteau, J., Accili, D., 2007. Regulation of pancreatic beta-cell function by the forkhead protein FoxO1. Diabetes Obes. Metab. 9 (Suppl. 2), 140–146. Butler, J.A., Hagen, T.M., Moreau, R., 2009. Lipoic acid improves hypertriglyceridemia by stimulating triacylglycerol clearance and downregulating liver triacylglycerol secretion. Arch. Biochem. Biophys. 485, 63–71. Caimari, A., Oliver, P., Palou, A., 2008. Impairment of nutritional regulation of adipose triglyceride lipase expression with age. Int. J. Obes. (London) 32, 1193–1200. Carter-Kent, C., Yerian, L.M., Brunt, E.M., Angulo, P., Kohli, R., Ling, S.C., Xanthakos, S.A., Whitington, P.F., Charatcharoenwitthaya, P., Yap, J., Lopez, R., McCullough, A.J., Feldstein, A.E., 2009. Nonalcoholic steatohepatitis in children: a multicenter clinicopathological study. Hepatology 50, 1113–1120. Chakrabarti, P., Kandror, K.V., 2009. FoxO1 controls insulin-dependent adipose triglyceride lipase (ATGL) expression and lipolysis in adipocytes. J. Biol. Chem. 284, 13296–13300.
17
Cortez-Pinto, H., Machado, M., 2008. Impact of body weight, diet and lifestyle on nonalcoholic fatty liver disease. Expert Rev. Gastroenterol. Hepatol. 2, 217–231. Evans, J.L., Goldfine, I.D., 2000. Alpha-lipoic acid: a multifunctional antioxidant that improves insulin sensitivity in patients with type 2 diabetes. Diabetes Technol. Ther. 2, 401–413. Feldstein, A.E., Charatcharoenwitthaya, P., Treeprasertsuk, S., Benson, J.T., Enders, F.B., Angulo, P., 2009. The natural history of non-alcoholic fatty liver disease in children: a follow-up study for up to 20 years. Gut 58, 1538–1544. Gaidhu, M.P., Fediuc, S., Anthony, N.M., So, M., Mirpourian, M., Perry, R.L., Ceddia, R.B., 2009. Prolonged AICAR-induced AMP-kinase activation promotes energy dissipation in white adipocytes: novel mechanisms integrating HSL and ATGL. J. Lipid Res. 50, 704–715. Gross, D.N., van den Heuvel, A.P., Birnbaum, M.J., 2008. The role of FoxO in the regulation of metabolism. Oncogene 27, 2320–2336. Gupta, R., Bhangoo, A., Matthews, N.A., Anhalt, H., Matta, Y., Lamichhane, B., Malik, S., Narwal, S., Wetzler, G., Ten, S., 2011. The prevalence of non-alcoholic fatty liver disease and metabolic syndrome in obese children. J. Pediatr. Endocrinol. Metab. 24, 907–911. Haeusler, R.A., Han, S., Accili, D., 2010. Hepatic FoxO1 ablation exacerbates lipid abnormalities during hyperglycemia. J. Biol. Chem. 285, 26861–26868. Houde, V.P., Brule, S., Festuccia, W.T., Blanchard, P.G., Bellmann, K., Deshaies, Y., Marette, A., 2010. Chronic rapamycin treatment causes glucose intolerance and hyperlipidemia by upregulating hepatic gluconeogenesis and impairing lipid deposition in adipose tissue. Diabetes 59, 1338–1348. Jager, S., Handschin, C., St-Pierre, J., Spiegelman, B.M., 2007. AMP-activated protein kinase (AMPK) action in skeletal muscle via direct phosphorylation of PGC1alpha. Proc. Natl. Acad. Sci. U. S. A. 104, 12017–12022. Jocken, J.W., Langin, D., Smit, E., Saris, W.H., Valle, C., Hul, G.B., Holm, C., Arner, P., Blaak, E.E., 2007. Adipose triglyceride lipase and hormone-sensitive lipase protein expression is decreased in the obese insulin-resistant state. J. Clin. Endocrinol. Metab. 92, 2292–2299. Kim, M.S., Park, J.Y., Namkoong, C., Jang, P.G., Ryu, J.W., Song, H.S., Yun, J.Y., Namgoong, I.S., Ha, J., Park, I.S., Lee, I.K., Viollet, B., Youn, J.H., Lee, H.K., Lee, K.U., 2004. Anti-obesity effects of alpha-lipoic acid mediated by suppression of hypothalamic AMP-activated protein kinase. Nat. Med. 10, 727–733. Krebs, M., Roden, M., 2005. Molecular mechanisms of lipid-induced insulin resistance in muscle, liver and vasculature. Diabetes Obes. Metab. 7, 621–632. Lee, D.H., Park, D.B., Lee, Y.K., An, C.S., Oh, Y.S., Kang, J.S., Kang, S.H., Chung, M.Y., 2005. The effects of thiazolidinedione treatment on the regulations of aquaglyceroporins and glycerol kinase in OLETF rats. Metabolism 54, 1282–1289. Marchesini, G., Brizi, M., Bianchi, G., Tomassetti, S., Bugianesi, E., Lenzi, M., McCullough, A.J., Natale, S., Forlani, G., Melchionda, N., 2001. Nonalcoholic fatty liver disease: a feature of the metabolic syndrome. Diabetes 50, 1844–1850. Matsumoto, M., Han, S., Kitamura, T., Accili, D., 2006. Dual role of transcription factor FoxO1 in controlling hepatic insulin sensitivity and lipid metabolism. J. Clin. Invest. 116, 2464–2472. Nair, S., Diehl, A.M., Wiseman, M., Farr Jr., G.H., Perrillo, R.P., 2004. Metformin in the treatment of non-alcoholic steatohepatitis: a pilot open label trial. Aliment Pharmacol. Ther. 20, 23–28. Narbonne, P., Roy, R., 2009. Caenorhabditis elegans dauers need LKB1/AMPK to ration lipid reserves and ensure long-term survival. Nature 457, 210–214. Nobili, V., Manco, M., Devito, R., Di Ciommo, V., Comparcola, D., Sartorelli, M.R., Piemonte, F., Marcellini, M., Angulo, P., 2008. Lifestyle intervention and antioxidant therapy in children with nonalcoholic fatty liver disease: a randomized, controlled trial. Hepatology 48, 119–128. Pacifico, L., Nobili, V., Anania, C., Verdecchia, P., Chiesa, C., 2011. Pediatric nonalcoholic fatty liver disease, metabolic syndrome and cardiovascular risk. World J. Gastroenterol. 17, 3082–3091. Park, K.G., Min, A.K., Koh, E.H., Kim, H.S., Kim, M.O., Park, H.S., Kim, Y.D., Yoon, T.S., Jang, B.K., Hwang, J.S., Kim, J.B., Choi, H.S., Park, J.Y., Lee, I.K., Lee, K.U., 2008. Alpha-lipoic acid decreases hepatic lipogenesis through adenosine monophosphate-activated protein kinase (AMPK)-dependent and AMPK-independent pathways. Hepatology 48, 1477–1486. Patton, H.M., Sirlin, C., Behling, C., Middleton, M., Schwimmer, J.B., Lavine, J.E., 2006. Pediatric nonalcoholic fatty liver disease: a critical appraisal of current data and implications for future research. J. Pediatr. Gastroenterol. Nutr. 43, 413–427. Reid, B.N., Ables, G.P., Otlivanchik, O.A., Schoiswohl, G., Zechner, R., Blaner, W.S., Goldberg, I.J., Schwabe, R.F., Chua Jr., S.C., Huang, L.S., 2008. Hepatic overexpression of hormone-sensitive lipase and adipose triglyceride lipase promotes fatty acid oxidation, stimulates direct release of free fatty acids, and ameliorates steatosis. J. Biol. Chem. 283, 13087–13099. Richardson, M.M., Jonsson, J.R., Powell, E.E., Brunt, E.M., Neuschwander-Tetri, B.A., Bhathal, P.S., Dixon, J.B., Weltman, M.D., Tilg, H., Moschen, A.R., Purdie, D.M., Demetris, A.J., Clouston, A.D., 2007. Progressive fibrosis in nonalcoholic steatohepatitis: association with altered regeneration and a ductular reaction. Gastroenterology 133, 80–90. Son, N.H., Yu, S., Tuinei, J., Arai, K., Hamai, H., Homma, S., Shulman, G.I., Abel, E.D., Goldberg, I.J., 2010. PPARgamma-induced cardiolipotoxicity in mice is ameliorated by PPARalpha deficiency despite increases in fatty acid oxidation. J. Clin. Invest. 120, 3443–3454.
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Y.-T. Kuo et al. / European Journal of Pharmacology 692 (2012) 10–18
Suchankova, G., Nelson, L.E., Gerhart-Hines, Z., Kelly, M., Gauthier, M.S., Saha, A.K., Ido, Y., Puigserver, P., Ruderman, N.B., 2009. Concurrent regulation of AMP-activated protein kinase and SIRT1 in mammalian cells. Biochem. Biophys. Res. Commun. 378, 836–841. Uygun, A., Kadayifci, A., Isik, A.T., Ozgurtas, T., Deveci, S., Tuzun, A., Yesilova, Z., Gulsen, M., Dagalp, K., 2004. Metformin in the treatment of patients with nonalcoholic steatohepatitis. Aliment Pharmacol. Ther. 19, 537–544. Watt, M.J., Southgate, R.J., Holmes, A.G., Febbraio, M.A., 2004. Suppression of plasma free fatty acids upregulates peroxisome proliferator-activated
receptor (PPAR) alpha and delta and PPAR coactivator 1alpha in human skeletal muscle, but not lipid regulatory genes. J. Mol. Endocrinol. 33, 533–544. Winder, W.W., Holmes, B.F., 2000. Insulin stimulation of glucose uptake fails to decrease palmitate oxidation in muscle if AMPK is activated. J. Appl. Physiol. 89, 2430–2437. Zechner, R., Kienesberger, P.C., Haemmerle, G., Zimmermann, R., Lass, A., 2009. Adipose triglyceride lipase and the lipolytic catabolism of cellular fat stores. J. Lipid Res. 50, 3–21.