Theaflavins attenuate hepatic lipid accumulation through activating AMPK in human HepG2 cells

Black tea is one of the world's most popular beverages, and its health-promoting effects have been intensively investigated. The antiobesity and hypolipidemic effects of black tea have attracted increasing interest, but the mechanisms underlying these phenomena remain unclear. In the present study, the black tea major component theaflavins were assessed for their hepatic lipid-lowering potential when administered in fatty acid overload conditions both in cell culture and in an animal experimental model. We found that theaflavins significantly reduced lipid accumulation, suppressed fatty acid synthesis, and stimulated fatty acid oxidation. Furthermore, theaflavins also inhibited acetyl-coenzyme A carboxylase activities by stimulating AMP-activated protein kinase (AMPK) through the LKB1 and reactive oxygen species pathways. These observations support the idea that AMPK is a critical component of decreased hepatic lipid accumulation by theaflavin treatments. Our results show that theaflavins are bioavailable both in vitro and in vivo and may be active in the prevention of fatty liver and obesity. Black tea is one of the world's most popular beverages, and its health-promoting effects have been intensively investigated. The antiobesity and hypolipidemic effects of black tea have attracted increasing interest, but the mechanisms underlying these phenomena remain unclear. In the present study, the black tea major component theaflavins were assessed for their hepatic lipid-lowering potential when administered in fatty acid overload conditions both in cell culture and in an animal experimental model. We found that theaflavins significantly reduced lipid accumulation, suppressed fatty acid synthesis, and stimulated fatty acid oxidation. Furthermore, theaflavins also inhibited acetyl-coenzyme A carboxylase activities by stimulating AMP-activated protein kinase (AMPK) through the LKB1 and reactive oxygen species pathways. These observations support the idea that AMPK is a critical component of decreased hepatic lipid accumulation by theaflavin treatments. Our results show that theaflavins are bioavailable both in vitro and in vivo and may be active in the prevention of fatty liver and obesity. acetyl-coenzyme A carboxylase AMP-activated protein kinase double-stranded RNA high-fat diet reactive oxygen species small interfering RNA Tea (Camellia sinensis), one of the world's most popular beverages, is consumed worldwide, and its health-promoting effects have been intensively investigated. In recent years, the antiobesity and hypolipidemic effects of tea have attracted increasing interest (1.Lin J.K. Lin-Shiau S.Y. Mechanisms of hypolipidemic and anti-obesity effects of tea and tea polyphenols.Mol. Nutr. Food Res. 2006; 50: 211-217Crossref PubMed Scopus (265) Google Scholar). Tea can be categorized into several types, depending on the level of fermentation during manufacturing. Black tea is consumed heavily throughout the world, and its polyphenols are thought to exert a possible inhibitory effect against tumorigenesis and tumor growth (2.Beltz L.A. Bayer D.K. Moss A.L. Simet I.M. Mechanisms of cancer prevention by green and black tea polyphenols.Anticancer Agents Med. Chem. 2006; 6: 389-406Crossref PubMed Scopus (193) Google Scholar). In particular, reports of significant hypolipidemic and antiobesity effects accompanying the use of black tea as a dietary supplement have increased interest in whether black tea components may be potent inhibitors of obesity. Among the black tea polyphenols, theaflavins are generally considered to be the more effective components. There are three major black tea theaflavins: theaflavin (TF-1), theaflavin-3-gallate (TF-2), and theaflavin-3,3-digallate (TF-3) (3.Weng M.S. Ho C.T. Ho Y.S. Lin J.K. Theanaphthoquinone inhibits fatty acid synthase expression in EGF-stimulated human breast cancer cells via the regulation of EGFR/ErbB-2 signaling.Toxicol. Appl. Pharmacol. 2007; 218: 107-118Crossref PubMed Scopus (19) Google Scholar). Theaflavins have been shown to be potent inhibitors of tumorigenesis in animal model systems. Furthermore, some previous reports suggested that theaflavin-enriched tea extract treatments could reduce the lipids and lipoproteins of subjects with mild to moderate hypercholesterolemia (4.Maron D.J. Lu G.P. Cai N.S. Wu Z.G. Li Y.H. Chen H. Zhu J.Q. Jin X.J. Wouters B.C. Zhao J. Cholesterol-lowering effect of a theaflavin-enriched green tea extract: a randomized controlled trial.Arch. Intern. Med. 2003; 163: 1448-1453Crossref PubMed Scopus (198) Google Scholar). Although these observations reveal that black tea components such as theaflavins can reduce lipid accumulation and exert some antiobese benefits, the mechanism underlying this phenomenon remains unclear. Approximately one-fifth of the population of the United States is afflicted with fatty liver (5.Angulo P. Nonalcoholic fatty liver disease.N. Engl. J. Med. 2002; 346: 1221-1231Crossref PubMed Scopus (4204) Google Scholar), which is a disease defined as hepatic fat accumulation of >5% of liver wet weight. The major causes of fatty liver are obesity, diabetes, hyperlipidemia, drugs, and metabolic disorders (6.Charlton M. Kasparova P. Weston S. Lindor K. Maor-Kendler Y. Wiesner R.H. Rosen C.B. Batts K.P. Frequency of nonalcoholic steatohepatitis as a cause of advanced liver disease.Liver Transpl. 2001; 7: 608-614Crossref PubMed Scopus (247) Google Scholar). Approximately 20–30% of adults are estimated to have excess liver fat accumulation in a normal population (7.Neuschwander-Tetri B.A. Caldwell S.H. Nonalcoholic steatohepatitis: summary of an AASLD Single Topic Conference.Hepatology. 2003; 37: 1202-1219Crossref PubMed Scopus (1808) Google Scholar). Currently, the mainstay of fatty liver treatment is weight loss, indicating that the prevalence of fatty liver is significantly coincident with obesity. It has been suggested that increased free FAs supplied to the liver play a major role in the early stage of this disease (8.Scheen A.J. Luyckx F.H. Obesity and liver disease.Best. Pract. Res. Clin. Endocrinol. Metab. 2002; 16: 703-716Crossref PubMed Scopus (103) Google Scholar), supporting the idea that high circulating FAs are the major risk factor of fatty liver (9.Sanyal A.J. Mechanisms of disease: pathogenesis of nonalcoholic fatty liver disease.Nat. Clin. Pract. Gastroenterol. Hepatol. 2005; 2: 46-53Crossref PubMed Scopus (184) Google Scholar). Although this common syndrome is usually considered benign and without crucial clinical significance, it may progress to fibrosis, cirrhosis, and even hepatocellular carcinoma. To date, some FAS inhibitors, such as cerulenin and C75, are being investigated to reduce hepatic fat content, but applications are limited by some side effects (10.Ronnett G.V. Kim E.K. Landree L.E. Tu Y. Fatty acid metabolism as a target for obesity treatment.Physiol. Behav. 2005; 85: 25-35Crossref PubMed Scopus (103) Google Scholar). Recent data collected in several laboratories indicate that AMP-activated protein kinase (AMPK) plays a key role in regulating carbohydrate and fat metabolism, serving as a metabolic master switch in response to alterations in cellular energy charge (11.Winder W.W. Hardie D.G. AMP-activated protein kinase, a metabolic master switch: possible roles in type 2 diabetes.Am. J. Physiol. 1999; 277: E1-E10PubMed Google Scholar). AMPK is known to play a major role in energy homeostasis by coordinating adaptive responses in low-energy metabolic states (12.Kemp B.E. Stapleton D. Campbell D.J. Chen Z.P. Murthy S. Walter M. Gupta A. Adams J.J. Katsis F. Denderen B.van et al.AMP-activated protein kinase, super metabolic regulator.Biochem. Soc. Trans. 2003; 31: 162-168Crossref PubMed Google Scholar). Based on this, AMPK cascades have emerged as novel targets for the treatment of obesity and fatty liver (13.Carling D. The AMP-activated protein kinase cascade—a unifying system for energy control.Trends Biochem. Sci. 2004; 29: 18-24Abstract Full Text Full Text PDF PubMed Scopus (963) Google Scholar). Indeed, AMPK has been proposed to play a role in the regulation of lipid metabolism. AMPK is also known to be activated with 5-amino-imidazole-4-carboxamide riboside (AICAR), which can be converted to a nucleotide that mimics the effect of AMP, and long-term treatment with AICAR has been proven to prevent obesity in animal models (14.Giri S. Rattan R. Haq E. Khan M. Yasmin R. Won J.S. Key L. Singh A.K. Singh I. AICAR inhibits adipocyte differentiation in 3T3L1 and restores metabolic alterations in diet-induced obesity mice model.Nutr. Metab. (Lond). 2006; 3: 31Crossref PubMed Scopus (158) Google Scholar). It is well known that AMPK is physiologically activated by the phosphorylation of threonine 172 within the α subunit catalyzed by the kinase LKB1, the upstream kinase of AMPK (15.Taylor E.B. Ellingson W.J. Lamb J.D. Chesser D.G. Winder W.W. Long-chain acyl-CoA esters inhibit phosphorylation of AMP-activated protein kinase at threonine-172 by LKB1/STRAD/MO25.Am. J. Physiol. Endocrinol. Metab. 2005; 288: E1055-E1061Crossref PubMed Scopus (51) Google Scholar). In the current study, we examined whether theaflavins have inhibitory effects on the liver fat accumulation of HepG2 human hepatoblastoma cells. It had been reported that increased FAs might cause the accumulation of fat deposits in liver and disturb hepatic metabolic functions (16.Nehra V. Angulo P. Buchman A.L. Lindor K.D. Nutritional and metabolic considerations in the etiology of nonalcoholic steatohepatitis.Dig. Dis. Sci. 2001; 46: 2347-2352Crossref PubMed Scopus (140) Google Scholar). Our principal hypothesis was that the likely protective role of theaflavins would be through the marked reduction or alteration of fat present in the hepatocytes under a high number of FAs. We found that theaflavins significantly reduced hepatic lipid content and suppressed fatty acid synthesis both in vitro and in vivo. Furthermore, theaflavins also inhibited acetyl-coenzyme A carboxylase (ACC) activities by stimulating AMPK through the LKB1 and reactive oxygen species (ROS) pathways. Our results confirmed that theaflavins inhibit hepatic lipid accumulation and induce AMPK-induced fatty acid oxidation, findings that should contribute to a clearer understanding of obesity and fatty liver prevention by theaflavins. The tea-derived flavonoids (−)-epigallocatechin-3-gallate (EGCG), (−)-epigallocatechin (EGC), (−)-epicatechin-3-O-gallate (ECG), (−)-epigallocatechin (EC), and catechin (C) were purchased from Sigma (München, Germany). TF-1, TF-2, and TF-3 were provided by Dr. C. T. Ho. of Rutgers University. Theaflavins were separated by chromatography on an LH-20 column to a purity of >99%, as described previously (17.Menet M.C. Sang S. Yang C.S. Ho C.T. Rosen R.T. Analysis of theaflavins and thearubigins from black tea extract by MALDI-TOF mass spectrometry.J. Agric. Food Chem. 2004; 52: 2455-2461Crossref PubMed Scopus (132) Google Scholar). The structures of these tea polyphenols are shown in supplementary Fig. I. All theaflavins were dissolved in DMSO. Antibodies to β-actin, LKB1, and FAS were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-pAMPK and anti-AMPK antibodies were from Upstate Biotechnology (Lake Placid, NY). The anti-pACC and anti-ACC antibodies were purchased from Transduction Laboratory (Lexington, KY). The AMPK activator AICAR was obtained from Cell Signaling Technology, Inc. (Beverly, MA). HepG2 cells obtained from the American Type Culture Collection were maintained in DMEM supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM l-glutamine and kept at 37°C in a humidified atmosphere of 5% CO2. Cells were grown to 70% confluence and then incubated in serum-free medium for 24 h before treatments. To induce FA overloading, HepG2 cells at 70% confluence were exposed to a long-chain mixture of FAs (containing oleic acid, palmitic acid, linolic acid, linoleic acid, and arachidonic acid in proportions of 25:40:15:15:5). FA/BSA complex was prepared as reported previously (18.Cousin S.P. Hugl S.R. Wrede C.E. Kajio H. Myers Jr., M.G. Rhodes C.J. Free fatty acid-induced inhibition of glucose and insulin-like growth factor I-induced deoxyribonucleic acid synthesis in the pancreatic beta-cell line INS-1.Endocrinology. 2001; 142: 229-240Crossref PubMed Scopus (154) Google Scholar). Stock solutions of 50 mM FAs prepared in culture medium containing 1% BSA were conveniently diluted in culture medium to obtain the desired final concentrations. The FA/BSA complexed solution was sterile-filtered through a 0.45 μm pore membrane filter and stored at −20°C. To measure cellular neutral lipid droplet accumulation, HepG2 cells were stained by the Oil Red O method (19.Hwang J.T. Park I.J. Shin J.I. Lee Y.K. Lee S.K. Baik H.W. Ha J. Park O.J. Genistein, EGCG, and capsaicin inhibit adipocyte differentiation process via activating AMP-activated protein kinase.Biochem. Biophys. Res. Commun. 2005; 338: 694-699Crossref PubMed Scopus (414) Google Scholar). After treatments, cells were washed three times with iced PBS and fixed with 10% formalin for 60 min. After fixation, cells were washed and stained with Oil Red O solution (stock solution, 3 mg/ml in isopropanol; working solution, 60% Oil Red O stock solution and 40% distilled water) for 60 min at room temperature. After staining, cells were washed with water to remove unbound dye. To quantitate Oil Red O content levels, isopropanol was added to each sample shaken at room temperature for 5 min, and samples were read spectrophotometrically at 510 nm. HepG2 cells were harvested and homogenized in a lysis buffer (50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol) for 30 min at 4°C. Equal amounts of total cellular proteins (50 μg) were resolved by SDS-PAGE transferred onto polyvinylidene difluoride membranes (Amersham Biosciences) and then probed with primary antibody followed by secondary antibody conjugated with horseradish peroxidase. The immunocomplexes were visualized with enhanced chemiluminescence kits (Amersham Biosciences). For lipidic determinations, homogenates from cells or rat liver were extracted according to a modified Bligh and Dyer procedure (20.Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43132) Google Scholar). In brief, sample was homogenized with chloroform-methanol solution (chloroform-methanol-water, 8:4:3). The resulting mixture was shaken at 37°C for 1 h and then centrifuged at 1,100 g for 10 min. The bottom layer supernatant was collected and resuspended for analysis of hepatic lipid. Triacylglycerol, total cholesterol, NEFA, and total lipid contents were measured using enzymatic method kits from Randox Laboratories (Antrim, UK) in accordance with the manufacturer's instructions. Rates of de novo fatty acid synthesis were determined on the basis of the incorporation rates of [1-14C]acetate into fatty acids during a 2 h period, as described previously (21.Alberts A.W. Ferguson K. Hennessy S. Vagelos P.R. Regulation of lipid synthesis in cultured animal cells.J. Biol. Chem. 1974; 249: 5241-5249Abstract Full Text PDF PubMed Google Scholar). Cells were incubated for 24 h with the indicated compounds after treatment with [1-14C]acetate (0.1 μCi/ml). After treatment, cells were harvested and digested in a potassium hydroxide solution (30%) at 95°C for 30 min, followed by saponification in 30% KOH/50% alcohol at 95°C for 3 h. After removal of the nonsaponifiable lipids with petroleum ether, the sample solution containing the saponified fatty acids was acidified with sulfuric acid, and fatty acids were extracted with petroleum ether. Fatty acid oxidation was measured in cells incubated for 24 h with the indicated compounds, and then [1-14C]palmitate (0.2 μCi/ml) was added to the fatty acid-free medium for 1 h. Radiolabeled CO2 was collected from the center wells by filter paper soaked with methylbenzethonium hydroxide in methanol. The incubations were stopped with 5% perchloric acid, and all radioactivities were determined by scintillation counting (22.Tavridou A. Kaklamanis L. Megaritis G. Kourounakis A.P. Papalois A. Roukounas D. Rekka E.A. Kourounakis P.N. Charalambous A. Manolopoulos V.G. Pharmacological characterization in vitro of EP2306 and EP2302, potent inhibitors of squalene synthase and lipid biosynthesis.Eur. J. Pharmacol. 2006; 535: 34-42Crossref PubMed Scopus (37) Google Scholar). ROS production was monitored by flow cytometry using dichlorofluorescin diacetate (DCFH-DA). Briefly, cells were incubated with DCFH-DA at a final concentration of 10 μM in medium at 37°C followed by our experimental treatments. Cells were washed twice with ice-cold buffer solution 2 h later and resuspended in PBS for analysis with FACScan using excitation and emission wavelengths of 475 and 525 nm, respectively. For each analysis, there were 10,000 events to be recorded. The LKB1 small interfering RNA (siRNA) gene silencer double-stranded RNA was obtained from Santa Cruz Biotechnology (sc-25816). HepG2 cells were transfected with double-stranded RNAs using siRNA Transfection Reagent (Santa Cruz Biotechnology) and incubated for 6 h. They were then analyzed by immunoblot for LKB1 expression after 24 h. Nontargeting scramble-siRNA directed against 5′-UAGCGACUAAACACAUCAAUU-3′ was the negative control. No mammalian mRNAs contained this sequence in the National Center for Biotechnology Information database. Male Wistar rats (5 weeks old) were purchased from the National Laboratory Animal Breeding Research Center (Taipei, Taiwan). Rats were housed in stainless steel wire-bottomed cages and acclimated under laboratory conditions for at least 1 week before each study. Rat weights at the beginning of the study ranged from 150 to 200 g. All rats were fed with appropriate diet and free access to water and were weighed every week. After 1 week of acclimation, the rats were fed different diets: in the control group (n = 8), rats were fed a standard diet; a calorie-rich-fat diet was used in the high-fat diet (HFD) group (n = 8). The HFD was manufactured commercially (Fwusow Industry, Taiwan) and containing 1% cholesterol, 18% lipid (lard), 40% sucrose, 1% AIN-93G vitamins, and 19% casein, with equal quantities of fiber and minerals as in the rat maintenance diet. Rats in the theaflavin-rich group (n = 8) were fed the HFD and 4% (w/w) dry black tea leaves (∼50 mg total theaflavins/kg/day) (23.Yeh C.W. Chen W.J. Chiang C.T. Lin-Shiau S.Y. Lin J.K. Suppression of fatty acid synthase in MCF-7 breast cancer cells by tea and tea polyphenols: a possible mechanism for their hypolipidemic effects.Pharmacogenomics J. 2003; 3: 267-276Crossref PubMed Scopus (116) Google Scholar). Rats were housed, and the experiment was terminated after 12 weeks. At experiment end, serum samples were collected for the estimation of cholesterol, triglyceride, and liver lipid content, and tissues were homogenized for Western blotting. Animals were housed and maintained at the College of Medicine Animal Facility of National Taiwan University, and all experiments were in compliance with protocols and policies approved by the National Taiwan University Institutional Animal Care and Use Committee. Data are presented as means ± SEM. Statistical significance was set at P < 0.05. Statistically significant differences were determined by the ANOVA in SPSS statistical software (SPSS, Inc., Chicago, IL). Previous studies have suggested that tea components might have antiobesity potential (24.Yang M. Wang C. Chen H. Green, oolong and black tea extracts modulate lipid metabolism in hyperlipidemia rats fed high-sucrose diet.J. Nutr. Biochem. 2001; 12: 14-20Crossref PubMed Scopus (150) Google Scholar). To evaluate the antiobesity effects, we used five major polyphenols present in green tea, known as EGCG, EGC, ECG, EC, and C, and three major polyphenols in black tea, known as TF-1, TF-2, and TF-3, and cotreated them with mixed FAs (1 mM) in the human HepG2 cell line. Cultured HepG2 cells were exposed to FAs cotreated with different tea components, and fat decrease levels were detected by Oil Red O staining after 24 h. As shown in Fig. 1, the intracellular lipid content could be reduced significantly by treatment with gallate forms of tea polyphenols, such as EGCG, EGC, and ECG. In contrast, nongallate forms of tea polyphenols, such as C and EC, displayed fewer effects against cellular lipid accumulation. Among these polyphenols, theaflavins (TF-1, TF-2, and TF-3) were the most effective tea ingredients in our assay. These results were further confirmed by the quantification of intracellular triglyceride and cholesterol contents. In Table 1, theaflavins also showed a significant inhibitory effect on triglyceride accumulation in HepG2 cells. TF-2 and TF-3 in particular exhibited more significant effects on the amounts of triglycerides, with ∼80% reduced levels. However, the cholesterol in FA-overloaded cultured HepG2 cells showed slightly reduced levels compared with that measured in other lipids. At this concentration, HepG2 cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and annexin V staining was not compromised by theaflavin treatments after 24 h of exposure (see supplementary Fig. II).TABLE 1.Comparison of intracellular total lipid, triglyceride, and cholesterol levels in in vitro FA-overloaded HepG2 cells by various polyphenolsTreatmentTotal LipidsTriglycerideCholesterolμg/mg proteinVehicle (no FA)258.2 ± 53.577.5 ± 12.1102.7 ± 9.6FA only575.2 ± 64.1220.6 ± 11.7149.8 ± 16.0(−)-Epigallocatechin-3-gallate + FA364.6 ± 21.2aP < 0.05, with respect to FA-only-treated control cells.114.0 ± 8.8aP < 0.05, with respect to FA-only-treated control cells.130.7 ± 11.1(−)-Epigallocatechin + FA429.8 ± 49.6157.6 ± 14.1aP < 0.05, with respect to FA-only-treated control cells.145.9 ± 8.0(−)-Epicatechin-3-O-gallate + FA406.3 ± 64.5129.8 ± 10.3aP < 0.05, with respect to FA-only-treated control cells.152.1 ± 21.9(−)-Epigallocatechin + FA547.0 ± 14.1200.6 ± 18.5136.5 ± 15.3Catechin + FA570.7 ± 32.3215.9 ± 22.2139.9 ± 11.8Theaflavin + FA292.1 ± 20.8bP < 0.01, with respect to FA-only-treated control cells.121.4 ± 7.9bP < 0.01, with respect to FA-only-treated control cells.114.3 ± 6.8Theaflavin-3-gallate + FA119.8 ± 10.0bP < 0.01, with respect to FA-only-treated control cells.48.5 ± 5.1bP < 0.01, with respect to FA-only-treated control cells.97.4 ± 9.9aP < 0.05, with respect to FA-only-treated control cells.Theaflavin-3,3-digallate + FA106.2 ± 5.9bP < 0.01, with respect to FA-only-treated control cells.58.4 ± 3.5bP < 0.01, with respect to FA-only-treated control cells.87.8 ± 9.0aP < 0.05, with respect to FA-only-treated control cells.Values are expressed as means ± SD of three independent samples. HepG2 cells were treated for 24 h with 1 mM FA or coincubated with 50μM tea polyphenols as indicated.a P < 0.05, with respect to FA-only-treated control cells.b P < 0.01, with respect to FA-only-treated control cells. Open table in a new tab Values are expressed as means ± SD of three independent samples. HepG2 cells were treated for 24 h with 1 mM FA or coincubated with 50μM tea polyphenols as indicated. AMPK activation is thought to be a key proximal event in the cellular energy balance response, and AMPK phosphorylation levels in threonine 172 are currently accepted as a marker of AMPK activity. Therefore, we first determined the phosphorylation of AMPK by cotreated FAs and theaflavins in HepG2 cells. Compared with the initial level, HepG2 cells already had reduced levels of phosphorylated AMPK (∼30%; P < 0.05) after 24 h at high FA conditions (Fig. 2A). However, these levels increased under coincubation with 50 μM theaflavins, particularly TF-2 and TF-3. In contrast to theaflavin cotreatments, green tea EGCG had a relatively small effect on AMPK phosphorylation. These increased levels of phosphorylated AMPK could be attributable to the reported anti-hepatic-fat accumulation effects of black teas that contain high levels of polyphenols (24.Yang M. Wang C. Chen H. Green, oolong and black tea extracts modulate lipid metabolism in hyperlipidemia rats fed high-sucrose diet.J. Nutr. Biochem. 2001; 12: 14-20Crossref PubMed Scopus (150) Google Scholar). The enzyme ACC involved in fatty acid synthesis has been identified as the primary target of AMPK and is inactivated by downstream phosphorylated serine 79 upon AMPK activation, leading to lipid synthesis inhibition. Because AMPK phosphorylation was upregulated in HepG2 cells incubated with theaflavins, the effects on ACC phosphorylation by theaflavin treatment were also studied.Fig. 2.Theaflavin treatments increase AMP-activated protein kinase (AMPK) and acetyl-coenzyme A carboxylase (ACC) phosphorylation. Cells were coexposed to FAs (1 mM) and various polyphenols (50 μM) for 24 h. A: AMPK phosphorylation (pThr172-AMPK) and its substrate ACC (pSer79-ACC) phosphorylation are detected by Western blot analysis. The FAS protein levels were also examined under the same conditions. The numbers below the panels represent quantification of the immunoblot by densitometry. B: Representative immunoblot and densitometric quantification of AMPK threonine 172 phosphorylation shows a dose-dependent effect by theaflavin treatment in the presence of 1 mM FAs. C: De novo lipogenesis is decreased by theaflavin treatment. HepG2 cells were incubated with [14C]acetate for 2 h cotreated with theaflavins or 5-amino-imidazole-4-carboxamide riboside (AICAR) in the presence of 1 mM FAs, and the radioactivity in the saponifiable fatty acid fractions was measured. This result demonstrated that the reduced acetate incorporation rates may be attributable to the increased ACC phosphorylation by theaflavin treatments. D: Fatty acid oxidation rate was determined by cotreatment with theaflavins and AICAR in the presence of 1 mM FAs. Data depict means ± SEM of at least three experiments. Asterisks represent statistically significant differences from the FA-treated control group (* P < 0.05, ** P < 0.01).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To evaluate ACC activities, ACC serine 79 phosphorylation was detected by Western blot analysis. As observed in Fig. 2A, parallel to AMPK phosphorylation, a 24 h incubation with theaflavins resulted in increased ACC serine 79 phosphorylation. It is known that some enzymes may also contribute to the regulation of fat accumulation and fuel metabolism. For instance, FAS is a key enzyme in the lipid synthesis pathway, and our previous work suggested that FAS protein levels are reduced by tea theaflavin compounds (3.Weng M.S. Ho C.T. Ho Y.S. Lin J.K. Theanaphthoquinone inhibits fatty acid synthase expression in EGF-stimulated human breast cancer cells via the regulation of EGFR/ErbB-2 signaling.Toxicol. Appl. Pharmacol. 2007; 218: 107-118Crossref PubMed Scopus (19) Google Scholar). Consistent with previous reports, FAS total protein content in HepG2 cells decreased significantly under theaflavin treatment. Similarly, a decrease in the condensations of acetyl-CoA corresponded to theaflavin-induced hepatic FAS inhibition (data not shown). Based upon our observations above, we hypothesized that the inhibition of ACC by AMPK could affect hepatic lipid synthesis and accumulation. To further confirm the association of ACC with AMPK, we performed Western blot analysis to verify these observations. As expected, a dose-dependent increase in AMPK and ACC phosphorylation under theaflavin treatment was observed (Fig. 2B). Finally, to measure fatty acid synthesis by ACC, we tested the rates of incorporation of [14C]acetate into hepatic total fatty acids. We found treatments with TF-1, TF-2, and TF-3 led to decreased values of 14.9, 41.0, and 48.9%, respectively, suggesting that the rate of fatty acid synthesis decreased significantly (Fig. 2C). Moreover, we showed that theaflavins also increased fatty acid oxidation, particularly for TF-2 and TF-3 (Fig. 2D). As seen in previous reports, activated AMPK can block anabolic pathways and promote the catabolic pathway in mammalian cells (25.Fediuc S. Gaidhu M.P. Ceddia R.B. Regulation of AMP-activated protein kinase and acetyl-CoA carboxylase phosphorylation by palmitate in skeletal muscle cells.J. Lipid Res. 2006; 47: 412-420Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). This implies that under theaflavin treatments, inhibition of lipid synthesis and activation of fatty acid oxidation may be involved in AMPK activation. It was known that the AMPK signaling cascade might also involve LKB1 activation, stimulating cellular energy expenditure (26.Shaw R.J. Lamia K.A. Vasquez D. Koo S.H. Bardeesy N. Depinho R.A. Montminy M. Cantley L.C. The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin.Science. 2005; 310: 1642-1646Crossref PubMed Scopus (1570) Google Scholar). To further explore these effects with theaflavins, we investigated whether LKB1 was essential to theaflavin-induced AMPK phosphorylation. We used the suppression RNA interference (RNAi) method to inhibit LKB1 expression in HepG2 cells. As shown in Fig. 3A, transfected HepG2 cells with LKB1-RNAi interfered with LKB1 protein expression, in contrast to mock or scramble control groups. In agreement with the previous findings, increasing levels of phosphorylated AMPK and ACC were observed by theaflavins without RNAi transfection. Under RNAi transfection, although we added theaflavins to RNAi-treated cel