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Epigallocatechin-3-gallate (EGCG), A Green Tea Polyphenol, Suppresses Hepatic Gluconeogenesis through 5′-AMP-activated Protein Kinase

安普克 糖异生 蛋白激酶A AMP活化蛋白激酶 化学 生物化学 激活剂(遗传学) 激酶 生物 新陈代谢 受体
作者
Qu Fan Collins,Hui-Yu Liu,Jingbo Pi,Zhenqi Liu,Michael J. Quon,Wenhong Cao
出处
期刊:Journal of Biological Chemistry [Elsevier]
卷期号:282 (41): 30143-30149 被引量:319
标识
DOI:10.1074/jbc.m702390200
摘要

Epigallocatechin-3-gallate (EGCG), a main catechin of green tea, has been suggested to inhibit hepatic gluconeogenesis. However, the exact role and related mechanism have not been established. In this study, we examined the role of EGCG in hepatic gluconeogenesis at concentrations that are reachable by ingestion of pure EGCG or green tea, and are not toxic to hepatocytes. Our results show in isolated hepatocytes that EGCG at relatively low concentrations (≤1 μm) inhibited glucose production via gluconeogenesis and expression of key gluconeogenic genes. EGCG was not toxic at these concentrations while demonstrating significant cytotoxicity at 10 μm and higher concentrations. EGCG at 1 μm or lower concentrations effective in suppressing hepatic gluconeogenesis did not activate the insulin signaling pathway, but activated 5′-AMP-activated protein kinase (AMPK). The EGCG suppression of hepatic gluconeogenesis was prevented by blockade of AMPK activity. In defining the mechanism by which EGCG activates AMPK, we found that the EGCG activation of AMPK was mediated by the Ca2+/calmodulin-dependent protein kinase kinase (CaMKK). Furthermore, our results show that the EGCG activation of AMPK and EGCG suppression of hepatic gluconeogenesis were both dependent on production of reactive oxygen species (ROS), which was a known activator of CaMKK. Together, our results demonstrate an inhibitory role for EGCG in hepatic gluconeogenesis and shed new light on the mechanism by which EGCG suppresses gluconeogenesis. Epigallocatechin-3-gallate (EGCG), a main catechin of green tea, has been suggested to inhibit hepatic gluconeogenesis. However, the exact role and related mechanism have not been established. In this study, we examined the role of EGCG in hepatic gluconeogenesis at concentrations that are reachable by ingestion of pure EGCG or green tea, and are not toxic to hepatocytes. Our results show in isolated hepatocytes that EGCG at relatively low concentrations (≤1 μm) inhibited glucose production via gluconeogenesis and expression of key gluconeogenic genes. EGCG was not toxic at these concentrations while demonstrating significant cytotoxicity at 10 μm and higher concentrations. EGCG at 1 μm or lower concentrations effective in suppressing hepatic gluconeogenesis did not activate the insulin signaling pathway, but activated 5′-AMP-activated protein kinase (AMPK). The EGCG suppression of hepatic gluconeogenesis was prevented by blockade of AMPK activity. In defining the mechanism by which EGCG activates AMPK, we found that the EGCG activation of AMPK was mediated by the Ca2+/calmodulin-dependent protein kinase kinase (CaMKK). Furthermore, our results show that the EGCG activation of AMPK and EGCG suppression of hepatic gluconeogenesis were both dependent on production of reactive oxygen species (ROS), which was a known activator of CaMKK. Together, our results demonstrate an inhibitory role for EGCG in hepatic gluconeogenesis and shed new light on the mechanism by which EGCG suppresses gluconeogenesis. Epigallocatechin-3-gallate (EGCG) 2The abbreviations used are: EGCG, epigallocatechin-3-gallate; ROS, reactive oxygen species; DMEM, Dulbecco's modified Eagle's medium; AMPK, 5′-AMP-activated protein kinase; ECG, epicatechin-3-gallate; CaMKK, calmodulin-dependent protein kinase kinase; GAPDH, glyceraldehyde-3-phosphate; PEG, polyethyleneglycol; ACC, acetyl-CoA carboxylase. is the most abundant catechin contained in green tea (1Lambert J.D. Yang C.S. J. Nutr. 2003; 133: 3262S-3267SCrossref PubMed Google Scholar). Catechins including epicatechin (EC), epicatechin-3-gallate (ECG), and EGCG account for 30-40% of the dry weight of green tea (reviewed in Refs. 1Lambert J.D. Yang C.S. J. Nutr. 2003; 133: 3262S-3267SCrossref PubMed Google Scholar, 2Yang C.S. Sang S. Lambert J.D. Hou Z. Ju J. Lu G. Mol. Nutr. Food Res. 2006; 50: 170-175Crossref PubMed Scopus (90) Google Scholar). EGCG has been shown to be involved in regulation of a variety of metabolic processes and has been used as an anti-obesity reagent in animal models and in humans (3Nelson-Dooley C. Della-Fera M.A. Hamrick M. Baile C.A. Curr. Med. Chem. 2005; 12: 2215-2225Crossref PubMed Scopus (57) Google Scholar, 4Wolfram S. Raederstorff D. Wang Y. Teixeira S.R. Elste V. Weber P. Ann. Nutr. Metab. 2005; 49: 54-63Crossref PubMed Scopus (196) Google Scholar, 5Wolfram S. Wang Y. Thielecke F. Mol. Nutr. Food Res. 2006; 50: 176-187Crossref PubMed Scopus (368) Google Scholar, 6Murase T. Haramizu S. Shimotoyodome A. Tokimitsu I. Int. J. Obes. (Lond). 2006; 30: 561-568Crossref PubMed Scopus (53) Google Scholar). Although its effectiveness in the treatment of human diabetes has not been established, EGCG has been shown in rodents to be effective in preventing the development of Type I diabetes and treatment of Type II diabetes (7Song E.K. Hur H. Han M.K. Arch. Pharm. Res. 2003; 26: 559-563Crossref PubMed Scopus (107) Google Scholar, 8Wolfram S. Raederstorff D. Preller M. Wang Y. Teixeira S.R. Riegger C. Weber P. J. Nutr. 2006; 136: 2512-2518Crossref PubMed Scopus (290) Google Scholar). The mechanism of EGCG regulation of metabolism remains to be established although a variety of different roles for EGCG have been suggested. EGCG has been shown to reduce food intake, plasma levels of glucose, and body weight (9Kao Y.H. Hiipakka R.A. Liao S. Endocrinology. 2000; 141: 980-987Crossref PubMed Scopus (370) Google Scholar, 10Lin J.K. Lin-Shiau S.Y. Mol. Nutr. Food Res. 2006; 50: 211-217Crossref PubMed Scopus (267) Google Scholar). EGCG inhibits proliferation and adipose differentiation of the 3T3-L1 preadipocyte cell line, and induces apoptosis in 3T3-L1 adipocytes (11Furuyashiki T. Nagayasu H. Aoki Y. Bessho H. Hashimoto T. Kanazawa K. Ashida H. Biosci. Biotechnol. Biochem. 2004; 68: 2353-2359Crossref PubMed Scopus (235) Google Scholar, 12Hung P.F. Wu B.T. Chen H.C. Chen Y.H. Chen C.L. Wu M.H. Liu H.C. Lee M.J. Kao Y.H. Am. J. Physiol. Cell Physiol. 2005; 288: C1094-C1108Crossref PubMed Scopus (128) Google Scholar, 13Lin J. Della-Fera M.A. Baile C.A. Obes. Res. 2005; 13: 982-990Crossref PubMed Scopus (194) Google Scholar). Pure EGCG and green tea extracts have both been shown in humans to stimulate brown fat thermogenesis (14Dulloo A.G. Duret C. Rohrer D. Girardier L. Mensi N. Fathi M. Chantre P. Vandermander J. Am. J. Clin. Nutr. 1999; 70: 1040-1045Crossref PubMed Scopus (762) Google Scholar, 15Dulloo A.G. Seydoux J. Girardier L. Chantre P. Vandermander J. Int. J. Obes. Relat. Metab. Disord. 2000; 24: 252-258Crossref PubMed Scopus (348) Google Scholar). EGCG can modulate insulin secretion and insulin sensitivity (16Li C. Allen A. Kwagh J. Doliba N.M. Qin W. Najafi H. Collins H.W. Matschinsky F.M. Stanley C.A. Smith T.J. J. Biol. Chem. 2006; 281: 10214-10221Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 17Anderson R.A. Polansky M.M. J. Agric. Food Chem. 2002; 50: 7182-7186Crossref PubMed Scopus (316) Google Scholar). EGCG has also been shown in skeletal muscle to promote fatty acid oxidation (18Murase T. Haramizu S. Shimotoyodome A. Nagasawa A. Tokimitsu I. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2005; 288: R708-R715Crossref PubMed Scopus (180) Google Scholar). Furthermore, EGCG has been suggested to reduce blood pressure through enhancing vascular endothelial function and insulin sensitivity (19Potenza M.A. Marasciulo F.L. Tarquinio M. Tiravanti E. Colantuono G. Federici A. Kim J.A. Quon M.J. Montagnani M. Am. J. Physiol. Endocrinol. Metab. 2007; 292: E1378-E1387Crossref PubMed Scopus (319) Google Scholar). Of our interest, EGCG has previously been shown to inhibit hepatic gluconeogenesis by mimicking insulin function (20Waltner-Law M.E. Wang X.L. Law B.K. Hall R.K. Nawano M. Granner D.K. J. Biol. Chem. 2002; 277: 34933-34940Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). However, concentrations used in previous studies were at high levels (>10 μm) that were capable of killing various kinds of tumor cells (1Lambert J.D. Yang C.S. J. Nutr. 2003; 133: 3262S-3267SCrossref PubMed Google Scholar, 2Yang C.S. Sang S. Lambert J.D. Hou Z. Ju J. Lu G. Mol. Nutr. Food Res. 2006; 50: 170-175Crossref PubMed Scopus (90) Google Scholar, 20Waltner-Law M.E. Wang X.L. Law B.K. Hall R.K. Nawano M. Granner D.K. J. Biol. Chem. 2002; 277: 34933-34940Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). Furthermore, catechins such as EGCG, once ingested by humans, are rapidly metabolized through glucuronidation, sulfation, methylation, and ring fission (1Lambert J.D. Yang C.S. J. Nutr. 2003; 133: 3262S-3267SCrossref PubMed Google Scholar), and the peak plasma concentration of EGCG after ingestion of a large amount of green tea or pure EGCG can only reach ∼1 μm (21Lee M.J. Maliakal P. Chen L. Meng X. Bondoc F.Y. Prabhu S. Lambert G. Mohr S. Yang C.S. Cancer Epidemiol. Biomarkers. Prev. 2002; 11: 1025-1032PubMed Google Scholar). Therefore, in this study we set to examine whether EGCG is effective in suppressing hepatic gluconeogenesis at or lower than 1 μm. Our results show that EGCG indeed inhibited hepatic gluconeogenesis at 1 μm or lower concentrations without cytotoxicity, but was toxic to hepatocytes at 10μm or higher concentrations. EGCG at 1 μm, and lower concentrations did not activate insulin signaling, instead activated AMPK through CaMKK and ROS. Chemicals and Antibodies—EGCG (Cat. E4143), 8-(4-chlorophenyl-thio) adenosine 3′,5′-cyclic monophosphate sodium salt (cAMP, cat. C3912), dexamethasone (cat. D4902), catalase (cat. C1345), PEG-catalase (PEG-CAT, cat. C4963), STO-609 acetic acid (cat. C1318), NADPH, H2O2, and β-actin antisera (cat. A-5441) were from Sigma. LY2294002 (cat. 440202) and Compound C (cat. 171260) were from Calbiochem. Antibodies against AMPK (cat. 2532), phospho-AMPK (cat. 2531), LKB1 (cat. 3054), ACC (cat. 3661), and AKT/total (cat. 9297 and 9191) were from Cell Signaling Technology. Phospho-IRS-1pY612 was from Abcam (cat. ab4868-50). The CaMKK antibody (cat. 610544), anti-phosphoserine/threonine antibody (cat. 612548) were from BD Transduction Laboratories. The siRNA duplexes against AMPKα were from Santa Cruz Biotechnology (cat. sc-45313). The cytotoxicity detection kit (cat. 1644793) was from Roche Applied Sciences. Isolation of Primary Hepatocytes—Primary hepatocytes were isolated from C57BL/6 mice as previously described (22Collins Q.F. Xiong Y. Lupo J. Liu H.Y. Cao W. J. Biol. Chem. 2006; 281: 24336-24344Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). All mice used for isolation of hepatocytes were fed with a normal chow diet at a regular schedule unless otherwise noted. Briefly, under anesthesia with pentobarbital (intraperitoneal, 30 mg/kg body weight), livers were perfused with a Ca2+-free Hanks' balanced solution (Invitrogen) at 5 ml/min for 8 min, followed by a continuous perfusion with the serum-free Williams' E medium containing collagenase (Worthington, type II, 50 units/ml), HEPES (10 mm), and NaOH (0.004 n) at 5 ml/min for 12 min. Hepatocytes were harvested and purified with Percoll and centrifugation. The viability of hepatocytes was examined with Trypan blue exclusion. Only cells isolated with a viability >95% were used. Hepatocytes were inoculated into collagen-coated 6-well plates (5 × 105/well) or 24-well plates in the Williams' E medium with 10% fetal bovine serum, and were incubated for 24 h before any experimentation. Measurement of Glucose Production in Primary Hepatocytes—The glucose production from primary hepatocytes was measured as previously described (22Collins Q.F. Xiong Y. Lupo J. Liu H.Y. Cao W. J. Biol. Chem. 2006; 281: 24336-24344Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 23Liu H.Y. Collins Q.F. Xiong Y. Moukdar F. Lupo J. Liu Z. Cao W. J. Biol. Chem. 2007; 282: 14205-14212Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Briefly, cells were treated with EGCG for 3 h at concentrations as noted in the serum-free Williams' E medium. Cells were then washed with a pre-warmed glucose-free DMEM medium three times, and stimulated by cAMP (100 μm)/dexamethasone (500 nm) in the presence of EGCG at concentrations as noted for another 3 h in the glucose-free DMEM. Gluconeogenic substrates including 20 mm sodium lactate and 2mm sodium pyruvate were added to some cells. Glucose in the media was quantified by using a glucose assay kit from Roche Applied Sciences (cat. 0716251) and normalized to cellular protein concentrations. The total glucose production was derived from both glycogenolysis and gluconeogenesis. The glucose production from glycogenolysis was measured in the absence of gluconeogenic substrates. The glucose production via gluconeogenesis is defined as the difference between the total glucose production and the glucose from glycogenolysis. Immunoblotting—Immunoblotting was performed as previously described (22Collins Q.F. Xiong Y. Lupo J. Liu H.Y. Cao W. J. Biol. Chem. 2006; 281: 24336-24344Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 24Cao W. Luttrell L.M. Medvedev A.V. Pierce K.L. Daniel K.W. Dixon T.M. Lefkowitz R.J. Collins S. J. Biol. Chem. 2000; 275: 38131-38134Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 25Cao W. Medvedev A.V. Daniel K.W. Collins S. J. Biol. Chem. 2001; 276: 27077-27082Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 26Cao W. Daniel K.W. Robidoux J. Puigserver P. Medvedev A.V. Bai X. Floering L.M. Spiegelman B.M. Collins S. Mol. Cell Biol. 2004; 24: 3057-3067Crossref PubMed Scopus (459) Google Scholar, 27Cao W.H. Collins Q.F. Becker T.C. Robidoux J. Lupo J.E.G. Xiong Y. Daniel K. Floering L. Collins S. J. Biol. Chem. 2005; 280: 42731-42737Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 28Xiong Y. Collins Q.F. Lupo J. Liu H.Y. Jie A. Liu D. Cao W. J. Biol. Chem. 2006; 282: 4975-4982Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Briefly, cell lysates were prepared by the homogenization and sonication, followed by adding 2× Laemmli sample buffer. Aliquots (5 μg of proteins/well) were resolved with mini Tris-glycine gradient gels (4-20%, Invitrogen) and transferred to nitrocellulose membranes. Levels of phospho-/total AKT, AMPK and phospho-IRS-1pY615, -ACC, -LKB1 were detected with a 1:1,000 dilution of each specific antiserum, followed by incubation with a 1:10,000 dilution of second immunoglobulin G conjugated with the alkaline phosphatase (RPN5783, Amersham Biosciences). Fluorescent bands were visualized with a Typhoon PhosphorImager (Molecular Dynamics). Immunoprecipitation—Immunoprecipitations were performed as previously described (24Cao W. Luttrell L.M. Medvedev A.V. Pierce K.L. Daniel K.W. Dixon T.M. Lefkowitz R.J. Collins S. J. Biol. Chem. 2000; 275: 38131-38134Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 29Robidoux J. Cao W.H. Quan Q. Daniel K.W. Moukdar F. Bai B. Floering L.M. Collins S. Mol. Cell Biol. 2005; 25: 5466-5479Crossref PubMed Scopus (96) Google Scholar, 30Liu H.Y. MacDonald J.I. Hryciw T. Li C. Meakin S.O. J. Biol. Chem. 2005; 280: 19461-19471Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 31Liu H.Y. Meakin S.O. J. Biol. Chem. 2002; 277: 26046-26056Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Target cells were dissolved with ice-cold Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mm NaCl, 10% glycerol, 2 mm EDTA, 20 mm Tris (pH 8.0), 1 mm dithiothreitol, 1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 2 g/ml leupeptin, and 10 g/ml aprotinin) at 4 °C for 10 min. CaMKK proteins were precipitated with specific antibodies and protein G-agarose at 4 °C for overnight. Phosphorylation of CaMKK was detected by immunoblotting with antibodies against phosphorylated serine/threonine. RNA Isolation and TaqMan Real-time PCR—Total RNAs from hepatocytes were prepared by using a RNA purification kit from Qiagen. mRNAs were reversely transcribed into cDNAs, and quantified with Taqman real-time PCR and normalized to the endogenous GADPH. Probes, primers, and other related reagents were from Applied Biosystems. Reactions were performed according to manuals from the manufacturer. Catalogue numbers are: Mm00440636-m1 for PEPCK, Mm00839363-m1 for G6Pase, and Mm99999915-g1 for GAPDH. Measurement of H2O2 Production in Vitro—Levels of H2O2 were measured as described (32Bai Y. Onuma H. Bai X. Medvedev A.V. Misukonis M. Weinberg J.B. Cao W. Robidoux J. Floering L.M. Daniel K.W. Collins S. J. Biol. Chem. 2005; 280: 19062-19069Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Briefly, increasing amounts of EGCG were incubated in PBS in the presence or absence of 100 μm NADPH or NADPH plus catalase (20 units/ml) at 37 °C for 30 min. Levels of H2O2 in the media were measured using an Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Molecular Probes) with H2O2 solution as a standard. The absorbance at 535 nm was measured by VICTOR 3 1420 Mutilabel Counter (Perkin Elmer). Introduction of siRNA Duplexes into Primary Hepatocytes—The siRNA duplexes were introduced into primary hepatocytes via transient transfection as previously described (22Collins Q.F. Xiong Y. Lupo J. Liu H.Y. Cao W. J. Biol. Chem. 2006; 281: 24336-24344Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Briefly, siRNA duplexes as indicated in each experiment were mixed with 4 μl of Lipofactamine RNAiMAX Reagents (Invitrogen, Cat. 13778-150) in the OPTI medium (Invitrogen) and then added to hepatocytes. Six hours later, the media were replaced with fresh Williams' E Media containing 10% fetal bovine serum; and the cells were cultured for another 20 h. Statistical Analysis—Results were analyzed using GraphPad Prism (version 4.0), and were presented as mean ± S.D. of at least three independent experiments. Statistical significance was determined by Student's t tests or one way analysis of variance analysis. EGCG Inhibits Gluconeogenesis in Primary Hepatocytes—To examine the effect of EGCG on hepatic gluconeogenesis, isolated hepatocytes were stimulated by cAMP/dexamethasone in the presence or absence of increasing amounts of EGCG, followed by measuring the glucose production via gluconeogenesis and expression of key gluconeogenic genes. As shown in Fig. 1A, the glucose production via gluconeogenesis was stimulated by cAMP/dexamethasone as expected, but the stimulation was attenuated by EGCG in a concentration-dependent manner. Similarly, the glucose production via gluconeogenesis was blocked by insulin as anticipated. Consistently, expression of PEPCK and G6Pase genes was also blocked by either EGCG or insulin (Fig. 1B). These results show that EGCG inhibits hepatic gluconeogenesis at or lower than 1 μm, and support the results from a previous study with higher concentrations that EGCG inhibits hepatic gluconeogenesis (20Waltner-Law M.E. Wang X.L. Law B.K. Hall R.K. Nawano M. Granner D.K. J. Biol. Chem. 2002; 277: 34933-34940Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). To control the possibility that EGCG suppresses hepatic gluconeogenesis through a toxic effect, LDH released from hepatocytes was measured. As shown in Fig. 1C, EGCG did not cause significant release of LDH at 1 μm, which was effective in suppressing hepatic gluconeogenesis. However, EGCG at concentrations higher than 10 μm caused significant release of LDH suggesting that EGCG is toxic to cells at concentration higher than 10 μm. It is noteworthy that EGCG caused an even stronger toxicity in H4IIE hepatoma cells (data not shown). These results are consistent with previous reports that EGCG is capable of killing cancer cells at concentrations higher than 10 μm (1Lambert J.D. Yang C.S. J. Nutr. 2003; 133: 3262S-3267SCrossref PubMed Google Scholar, 2Yang C.S. Sang S. Lambert J.D. Hou Z. Ju J. Lu G. Mol. Nutr. Food Res. 2006; 50: 170-175Crossref PubMed Scopus (90) Google Scholar). EGCG Inhibition of Hepatic Gluconeogenesis Is Independent of Insulin Signaling—Because a previous study has shown that EGCG at concentrations higher than 10 μm represses hepatic gluconeogenesis by mimicking the insulin function (20Waltner-Law M.E. Wang X.L. Law B.K. Hall R.K. Nawano M. Granner D.K. J. Biol. Chem. 2002; 277: 34933-34940Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar), we examined the effect of EGCG at lower concentrations on the insulin signaling pathway. As shown in Fig. 2A, EGCG did not stimulate the tyrosine phosphorylation of IRS1 or Akt phosphorylation at 1 μm, which was effective in suppressing hepatic gluconeogenesis (Fig. 1, A and B). In contrast, insulin robustly activated the tyrosine phosphorylation of IRS1 and Akt phosphorylation as expected. Furthermore, EGCG suppression of the glucose production via gluconeogenesis and expression of PEPCK and G6Pase genes were not influenced by a PI3K inhibitor, LY294002 (Fig. 2, B and C). However, LY294002 completely blocked the insulin-induced suppression of the glucose production via gluconeogenesis and the expression of PEPCK and G6Pase genes. Together, these results indicate that EGCG inhibits hepatic gluconeogenesis independent of the insulin signaling pathway. EGCG Activates AMPK Signaling Pathway in Primary Hepatocytes—AMPK is another known suppressor of hepatic gluconeogenesis in addition to the insulin signaling, and poly-phenolic compounds including resveratrol, apigenin, S17834 (a synthetic polyphenol), and EGCG have previously been shown to activate AMPK in HepG2 hepatoma cells, HT-29 colon cancer cells, or 3T3-L1 cells (33Hwang J.T. Park I.J. Shin J.I. Lee Y.K. Lee S.K. Baik H.W. Ha J. Park O.J. Biochem. Biophys. Res. Commun. 2005; 338: 694-699Crossref PubMed Scopus (420) Google Scholar, 34Zang M. Xu S. Maitland-Toolan K.A. Zuccollo A. Hou X. Jiang B. Wierzbicki M. Verbeuren T.J. Cohen R.A. Diabetes. 2006; 55: 2180-2191Crossref PubMed Scopus (591) Google Scholar, 35Hwang J.T. Ha J. Park I.J. Lee S.K. Baik H.W. Kim Y.M. Park O.J. Cancer Lett. 2007; 247: 115-121Crossref PubMed Scopus (246) Google Scholar). It is currently unknown whether or not EGCG activates AMPK in primary hepatocytes. To address this question and the possible role of AMPK in the EGCG suppression of hepatic gluconeogenesis, we treated primary hepatocytes with EGCG and measured levels of phosphorylated AMPK and an AMPK substrate, acetyl-CoA carboxylase (ACC). As shown in Fig. 3, A and B, EGCG elevated the phosphorylation of AMPK and ACC in a time- and concentration-dependent manner. These data show that EGCG activates AMPK in hepatocytes suggesting that EGCG suppresses hepatic gluconeogenesis through AMPK. AMPK Mediates the EGCG Inhibition of Hepatic Gluconeogenesis—To examine the role of AMPK in EGCG inhibition of hepatic gluconeogenesis, the activity of AMPK in primary hepatocytes was blocked by either a selective chemical inhibitor, Compound C (36McCullough L.D. Zeng Z. Li H. Landree L.E. McFadden J. Ronnett G.V. J. Biol. Chem. 2005; 280: 20493-20502Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 37Fediuc S. Gaidhu M.P. Ceddia R.B. J. Lipid Res. 2006; 47: 412-420Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), or siRNA duplexes against the AMPKα gene prior to the treatment with EGCG, followed by the measurement of the glucose production via gluconeogenesis and expression of PEPCK and G6Pase. As showed in Fig. 4, EGCG activation of AMPK was prevented by the Compound C, and EGCG suppression of the glucose production via hepatic gluconeogenesis and expression of PEPCK and G6Pase genes were prevented by either Compound C or the specific siRNA. The scrambled siRNA had not effect. Together, these results demonstrate that AMPK is a mediator of the EGCG inhibition of hepatic gluconeogenesis. The EGCG Activation of AMPK and Suppression of Hepatic Gluconeogenesis is CaMKK-dependent—There are currently two known activators of AMPK, LKB1, and CaMKK (see Ref. 38Witters L.A. Kemp B.E. Means A.R. Trends Biochem. Sci. 2006; 31: 13-16Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar for review). Therefore, we examined whether or not these kinases were involved in the EGCG activation of AMPK and suppression of gluconeogenesis. Our results from primary hepatocytes indicate that EGCG did not initiate detectable phosphorylation of LKB1 (data not shown). However, EGCG stimulated phosphorylation of both CaMKK and AMPK (Fig. 5, A and B). EGCG-induced phosphorylation of both CaMKK and AMPK was blocked by a CaMKK inhibitor, STO-609 (Fig. 5, A and B). Furthermore, EGCG suppression of the glucose production via gluconeogenesis and expression of PEPCK and G6Pase genes were all attenuated by STO-609 (Fig. 5, C and D). Together, these data indicated that hepatic CaMKK is the upstream activator of AMPK induced by EGCG. EGCG Activation of AMPK and Suppression of Hepatic Gluconeogenesis Is ROS-dependent—ROS is know to be able to activate AMPK through CaMKK (33Hwang J.T. Park I.J. Shin J.I. Lee Y.K. Lee S.K. Baik H.W. Ha J. Park O.J. Biochem. Biophys. Res. Commun. 2005; 338: 694-699Crossref PubMed Scopus (420) Google Scholar, 35Hwang J.T. Ha J. Park I.J. Lee S.K. Baik H.W. Kim Y.M. Park O.J. Cancer Lett. 2007; 247: 115-121Crossref PubMed Scopus (246) Google Scholar) In particular, ROS has been shown to be involved in activation of AMPK by EGCG in HT-29 cancer cells (35Hwang J.T. Ha J. Park I.J. Lee S.K. Baik H.W. Kim Y.M. Park O.J. Cancer Lett. 2007; 247: 115-121Crossref PubMed Scopus (246) Google Scholar). Therefore, we postulated that ROS was involved in EGCG activation of AMPK and suppression of hepatic gluconeogenesis. To test the hypothesis, we examined whether or not EGCG can induce ROS production. Our results show that in the absence of EGCG there was no detectable H2O2 in the PBS buffer (data not shown). However, EGCG alone induced production of H2O2 in a concentration-dependent manner; and the EGCG-induced H2O2 production was enhanced in the presence of NADPH as a proton donor (Fig. 6A). Furthermore, the H2O2 induced by EGCG and NADPH was neutralized by a catalase. To examine the role of ROS induced by EGCG in gluconeogenesis, primary hepatocytes as noted were treated with EGCG in the presence of a cell membrane permeable catalase (PEG-CAT), which was presumably able to eliminate H2O2. As shown in Fig. 5B, EGCG induction of AMPK phosphorylation was completely blocked by PEG-CAT. Accordingly, EGCG suppression of the glucose production via gluconeogenesis and expression of PEPCK and G6Pase genes were also prevented by PEG-CAT (Fig. 6, B and C). Together, these results indicate that EGCG activates AMPK and consequently suppresses hepatic gluconeogenesis through the production of ROS. EGCG has previously been shown to suppress hepatic gluconeogenesis both in cultured cells and in animal models; and the role of EGCG has been suggested to be mediated by Akt (20Waltner-Law M.E. Wang X.L. Law B.K. Hall R.K. Nawano M. Granner D.K. J. Biol. Chem. 2002; 277: 34933-34940Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar, 39Koyama Y. Abe K. Sano Y. Ishizaki Y. Njelekela M. Shoji Y. Hara Y. Isemura M. Planta. Med. 2004; 70: 1100-1102Crossref PubMed Scopus (73) Google Scholar, 40Anton S. Melville L. Rena G. Cell Signal. 2006; 19: 378-383Crossref PubMed Scopus (62) Google Scholar). Here we have confirmed that EGCG is indeed inhibitory to gluconeogenesis in isolated hepatocytes at non-toxic concentrations. However, our results show that EGCG at these concentrations dose not activate Akt and IRS1, which are required for insulin inhibition of hepatic gluconeogenesis, but instead activates AMPK. We further show that the blockade of AMPK activation prevents EGCG suppression of hepatic gluconeogenesis. The role of EGCG in activation of Akt can be either stimulatory or inhibitory. The inhibitory role of EGCG is associated with its capability of counteracting the growth of tumor/cancer cells (see Refs. 1Lambert J.D. Yang C.S. J. Nutr. 2003; 133: 3262S-3267SCrossref PubMed Google Scholar, 41Zaveri N.T. Life Sci. 2006; 78: 2073-2080Crossref PubMed Scopus (721) Google Scholar for review). EGCG cannot only directly down regulate Akt activation in various tumor/cancer cell, but also blocks the activation of Akt induced by either EGF or PDGF in non-tumor cells (42Sah J.F. Balasubramanian S. Eckert R.L. Rorke E.A. J. Biol. Chem. 2004; 279: 12755-12762Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 43Masamune A. Kikuta K. Satoh M. Suzuki N. Shimosegawa T. World J. Gastroenterol. 2005; 11: 3368-3374Crossref PubMed Scopus (41) Google Scholar). The inhibitory effect of EGCG on Akt activation is mostly initiated by high concentrations, which are usually higher than 10 μm, typically at 50-100 μm (1Lambert J.D. Yang C.S. J. Nutr. 2003; 133: 3262S-3267SCrossref PubMed Google Scholar). Studies have shown in pancreatic stellate cells and epidermal keratinocytes that EGCG is toxic at concentrations higher than 10 μm (43Masamune A. Kikuta K. Satoh M. Suzuki N. Shimosegawa T. World J. Gastroenterol. 2005; 11: 3368-3374Crossref PubMed Scopus (41) Google Scholar, 44Chung J.H. Han J.H. Hwang E.J. Seo J.Y. Cho K.H. Kim K.H. Youn J.I. Eun H.C. Faseb. J. 2003; 17: 1913-1915Crossref PubMed Google Scholar). Similarly, we have found that EGCG is toxic to hepatocytes at 10 μm or higher concentrations. On the other hand, EGCG has been shown in epidermal keratinocytes, vascular endothelial cells, and neuronal cells to activate Akt; and the activation of Akt is protective to these cells against various stresses such as oxidative stress and ultraviolet light irradiation (19Potenza M.A. Marasciulo F.L. Tarquinio M. Tiravanti E. Colantuono G. Federici A. Kim J.A. Quon M.J. Montagnani M. Am. J. Physiol. Endocrinol. Metab. 2007; 292: E1378-E1387Crossref PubMed Scopus (319) Google Scholar, 44Chung J.H. Han J.H. Hwang E.J. Seo J.Y. Cho K.H. Kim K.H. Youn J.I. Eun H.C. Faseb. J. 2003; 17: 1913-1915Crossref PubMed Google Scholar, 45Lorenz M. Wessler S. Follmann E. Michaelis W. Dusterhoft T. Baumann G. Stangl K. Stangl V. J. Biol. Chem. 2004; 279: 6190-6195Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 46Koh S.H. Kim S.H. Kwon H. Kim J.G. Kim J.H. Yang K.H. Kim J. Kim S.U. Yu H.J. Do B.R. Kim K.S. Jung H.K. Neurotoxicology. 2004; 25: 793-802Crossref PubMed Scopus (84) Google Scholar, 47Koh S.H. Kwon H. Kim K.S. Kim J. Kim M.H. Yu H.J. Kim M. Lee K.W. Do B.R. Jung H.K. Yang K.W. Appel S.H. Kim S.H. Toxicology. 2004; 202: 213-225Crossref PubMed Scopus (73) Google Scholar). Although a study with epidermal keratinocytes show that EGCG activates Akt at concentrations lower than 1 μm (44Chung J.H. Han J.H. Hwang E.J. Seo J.Y. Cho K.H. Kim K.H. Youn J.I. Eun H.C. Faseb. J. 2003; 17: 1913-1915Crossref PubMed Google Scholar), activation of Akt by EGCG in most previous studies has been elicited by concentrations higher than 10 μm. In this study, we found that EGCG did not initiate detectable phosphorylation of Akt in hepatocytes at concentrations at or lower than 1 μm, but was effective in suppressing hepatic gluconeogenesis. EGCG did not elevate tyrosine phosphorylation of IRS1, either. Therefore, EGCG appears to inhibit hepatic gluconeogenesis through a signaling pathway distinct from the insulin signaling. AMPK is one of the two kinases that are known to be able to suppress hepatic gluconeogenesis (48Koo S.H. Flechner L. Qi L. Zhang X. Screaton R.A. Jeffries S. Hedrick S. Xu W. Boussouar F. Brindle P. Takemori H. Montminy M. Nature. 2005; 437: 1109-1111Crossref PubMed Scopus (837) Google Scholar, 49Shaw R.J. Lamia K.A. Vasquez D. Koo S.H. Bardeesy N. Depinho R.A. Montminy M. Cantley L.C. Science. 2005; 310: 1642-1646Crossref PubMed Scopus (1604) Google Scholar, 50Yamauchi J. Nagao M. Kaziro Y. Itoh H. J. Biol. Chem. 1997; 272: 27771-27777Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The other one is Akt described above and known to be activated by insulin (reviewed in Ref. 51Whiteman E.L. Cho H. Birnbaum M.J. Trends Endocrinol. Metab. 2002; 13: 444-451Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar). Interestingly, EGCG has previously been shown to activate AMPK, and the activation of AMPK is associated with the EGCG-induced apoptosis in tumor/cancer cells and the EGCG inhibition of adipose differentiation (33Hwang J.T. Park I.J. Shin J.I. Lee Y.K. Lee S.K. Baik H.W. Ha J. Park O.J. Biochem. Biophys. Res. Commun. 2005; 338: 694-699Crossref PubMed Scopus (420) Google Scholar, 35Hwang J.T. Ha J. Park I.J. Lee S.K. Baik H.W. Kim Y.M. Park O.J. Cancer Lett. 2007; 247: 115-121Crossref PubMed Scopus (246) Google Scholar). Other polyphenols including resveratrol (a major polyphenol in red wine), apigenin, and S17834 (a synthetic polyphenol) have been shown in HepG2 hepatoma cells and in mouse liver to activate AMPK and consequently prevent lipid accumulation (34Zang M. Xu S. Maitland-Toolan K.A. Zuccollo A. Hou X. Jiang B. Wierzbicki M. Verbeuren T.J. Cohen R.A. Diabetes. 2006; 55: 2180-2191Crossref PubMed Scopus (591) Google Scholar). In this study, we show that AMPK is activated in primary hepatocytes by EGCG, and is required for EGCG suppression of hepatic gluconeogenesis. AMPK can be activated by at least two known signaling pathways (reviewed in Ref. 38Witters L.A. Kemp B.E. Means A.R. Trends Biochem. Sci. 2006; 31: 13-16Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). First, LKB1 is an activator of AMPK (see Ref. 52Towler M.C. Hardie D.G. Circ. Res. 2007; 100: 328-341Crossref PubMed Scopus (1086) Google Scholar for review). We examined the possible role of LKB1 in EGCG activation of AMPK, and found that EGCG did not promote detectable phosphorylation of LKB1 (data not shown). Second, CaMKK is another activator of AMPK (52Towler M.C. Hardie D.G. Circ. Res. 2007; 100: 328-341Crossref PubMed Scopus (1086) Google Scholar). In this study, our results show that EGCG promotes phosphorylation of CaMKK, and blockade of CaMKK activity prevents EGCG activation of AMPK and mitigates the inhibitory role of EGCG in hepatic gluconeogenesis. EGCG activation of AMPK has previously been shown in colon cancer cells to be associated with ROS production (35Hwang J.T. Ha J. Park I.J. Lee S.K. Baik H.W. Kim Y.M. Park O.J. Cancer Lett. 2007; 247: 115-121Crossref PubMed Scopus (246) Google Scholar). Our results from this study show that EGCG can directly promote ROS production; and EGCG-induced ROS is required for the activation of AMPK and the consequent inhibition of hepatic gluconeogenesis by EGCG. In summary, results from this study demonstrate that EGCG suppresses hepatic gluconeogenesis at concentrations that are not toxic to hepatocytes and are reachable by ingestion of green tea or pure EGCG. We have also revealed a new mechanism by which EGCG suppresses hepatic gluconeogenesis through ROS, CaMKK, and AMPK. It is currently still unclear how EGCG exactly activates CaMKK through production of ROS. Future investigation on this matter and the in vivo role of CaMKK and AMPK in EGCG suppression of hepatic gluconeogenesis may provide new therapeutic approaches for the management of diabetes. We thank Drs. Sheila Collins and Jacques Robidoux for their instrumental suggestions.
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