Advertisement

Epigallocatechin Gallate (EGCG) Stimulates Autophagy in Vascular Endothelial Cells

A POTENTIAL ROLE FOR REDUCING LIPID ACCUMULATION*
  • Hae-Suk Kim
    Affiliations
    From the Department of Medicine, Division of Endocrinology, Diabetes, and Metabolism, and
    Search for articles by this author
  • Vedrana Montana
    Affiliations
    Departments of Neurobiology, Center for Glial Biology in Medicine, Atomic Force Microscopy and Nanotechnology Laboratories, Civitan International Research Center, Evelyn F. McKnight Brain Institute, and
    Search for articles by this author
  • Hyun-Ju Jang
    Affiliations
    From the Department of Medicine, Division of Endocrinology, Diabetes, and Metabolism, and
    Search for articles by this author
  • Vladimir Parpura
    Affiliations
    Departments of Neurobiology, Center for Glial Biology in Medicine, Atomic Force Microscopy and Nanotechnology Laboratories, Civitan International Research Center, Evelyn F. McKnight Brain Institute, and

    the Department of Biotechnology, University of Rijeka, 51000 Rijeka, Croatia
    Search for articles by this author
  • Jeong-a Kim
    Correspondence
    To whom correspondence should be addressed: Dept. of Medicine, Division of Endocrinology, Diabetes, and Metabolism, University of Alabama at Birmingham, Birmingham, AL 35294. Tel.: 205-934-4128; Fax; 205-975-9372;
    Affiliations
    From the Department of Medicine, Division of Endocrinology, Diabetes, and Metabolism, and

    Departments of Molecular Cellular Pathology and

    Comprehensive Diabetes Center, University of Alabama at Birmingham, Birmingham, Alabama 35294 and
    Search for articles by this author
  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant P60 DK-079626 (to University of Alabama at Birmingham Diabetes Research Training Center-sponsored Pilot and Feasibility Program). This work was also supported by American Diabetes Association Grants 1-09-JF-33 and 1-12-BS-99 (to J. K.), American Heart Association Grant 13GRNT17220057 (to J. K.), and National Science Foundation Grant CBET 0943343 (to V. P.).
    This article contains supplemental Movies 1 and 2.
    ♦ This article was selected as a Paper of the Week.
Open AccessPublished:June 10, 2013DOI:https://doi.org/10.1074/jbc.M113.477505
      Epigallocatechin gallate (EGCG) is a major polyphenol in green tea that has beneficial effects in the prevention of cardiovascular disease. Autophagy is a cellular process that protects cells from stressful conditions. To determine whether the beneficial effect of EGCG is mediated by a mechanism involving autophagy, the roles of the EGCG-stimulated autophagy in the context of ectopic lipid accumulation were investigated. Treatment with EGCG increased formation of LC3-II and autophagosomes in primary bovine aortic endothelial cells (BAEC). Activation of calmodulin-dependent protein kinase kinase β was required for EGCG-induced LC3-II formation, as evidenced by the fact that EGCG-induced LC3-II formation was significantly impaired by knockdown of calmodulin-dependent protein kinase kinase β. This effect is most likely due to cytosolic Ca2+ load. To determine whether EGCG affects palmitate-induced lipid accumulation, the effects of EGCG on autophagic flux and co-localization of lipid droplets and autophagolysosomes were examined. EGCG normalized the palmitate-induced impairment of autophagic flux. Accumulation of lipid droplets by palmitate was markedly reduced by EGCG. Blocking autophagosomal degradation opposed the effect of EGCG in ectopic lipid accumulation, suggesting the action of EGCG is through autophagosomal degradation. The mechanism for this could be due to the increased co-localization of lipid droplets and autophagolysosomes. Co-localization of lipid droplets with LC3 and lysosome was dramatically increased when the cells were treated with EGCG and palmitate compared with the cells treated with palmitate alone. Collectively, these findings suggest that EGCG regulates ectopic lipid accumulation through a facilitated autophagic flux and further imply that EGCG may be a potential therapeutic reagent to prevent cardiovascular complications.
      Background: Green tea polyphenol (EGCG) has beneficial effects on cardiovascular dysfunction.
      Results: EGCG stimulates autophagy through a CaMKKβ-mediated mechanism, which contributes to degradation of lipid droplets.
      Conclusion: Regulation of autophagic flux by EGCG plays a role in intracellular lipid accumulation.
      Significance: Findings show a novel mechanism for beneficial effects of EGCG in cardiovascular complications.

      Introduction

      Ectopic accumulation of lipids, including neutral lipid and cholesterol esters, contributes to inflammatory status and endoplasmic reticulum (ER)
      The abbreviations used are: ER, endoplasmic reticulum; EGCG, epigallocatechin-gallate; CaMKKβ, calmodulin-dependent protein kinase kinase β; BAEC, bovine aortic endothelial cell; AMPK, AMP-activated protein kinase; ULK, uncoordinated-51-like kinase; LAMP-1, lysosomal associated membrane protein1; CPA, cyclopiazonic acid; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester); NH4Cl/Leu, ammonium chloride/leupeptin; mTOR, mammalian target of rapamycin.
      stress in vascular endothelium that is associated with endothelial dysfunction and atherosclerosis (
      • Kolattukudy P.E.
      • Niu J.
      Inflammation, endoplasmic reticulum stress, autophagy, and the monocyte chemoattractant protein-1/CCR2 pathway.
      ). Degradation of lipid droplets by stimulation of autophagy (lipophagy) reduces ER stress and inflammation (
      • Younce C.
      • Kolattukudy P.
      MCP-1-induced protein promotes adipogenesis via oxidative stress, endoplasmic reticulum stress, and autophagy.
      ). Autophagy is a catabolic process that plays pivotal roles in metabolism, cell death, and differentiation (
      • Chimal-Monroy J.
      • Abarca-Buis R.F.
      • Cuervo R.
      • Diaz-Hernandez M.
      • Bustamante M.
      • Rios-Flores J.A.
      • Romero-Suarez S.
      • Farrera-Hernandez A.
      Molecular control of cell differentiation and programmed cell death during digit development.
      ,
      • Sridhar S.
      • Botbol Y.
      • Macian F.
      • Cuervo A.M.
      Autophagy and disease: always two sides to a problem.
      ). An excess amount of lipids, aggregated proteins, and organelles is degraded through the autophagic process, which is one of the protective mechanisms used to remove unused cellular materials (
      • Cuervo A.M.
      Autophagy: in sickness and in health.
      ,
      • Singh R.
      Autophagy and regulation of lipid metabolism.
      ). Macroautophagy (hereafter autophagy) occurs through a series of events forming membrane-like structure, compartmentalization, and fusion of vesicles that generate autophagolysosome (
      • Cuervo A.M.
      Autophagy: in sickness and in health.
      ). Impairment of the lysosomal degradation process causes reduced autophagic flux leading to serious disorders in cardiovascular and metabolic tissues (
      • Singh R.
      • Cuervo A.M.
      Autophagy in the cellular energetic balance.
      ,
      • Gustafsson A.B.
      • Gottlieb R.A.
      Autophagy in ischemic heart disease.
      ,
      • De Meyer G.R.
      • Martinet W.
      Autophagy in the cardiovascular system.
      ).
      An 11-year follow-up study shows that green tea consumption is associated with reduced mortality due to cardiovascular diseases but not with mortality due to cancer (
      • Kuriyama S.
      • Shimazu T.
      • Ohmori K.
      • Kikuchi N.
      • Nakaya N.
      • Nishino Y.
      • Tsubono Y.
      • Tsuji I.
      Green tea consumption and mortality due to cardiovascular disease, cancer, and all causes in Japan: the Ohsaki study.
      ). We and others have shown that the most abundant green tea polyphenol, epigallocatechin-3-gallate (EGCG), has beneficial health effects in various pathophysiological conditions, including insulin resistance, endothelial dysfunction, and ischemia-reperfusion injuries (
      • Bose M.
      • Lambert J.D.
      • Ju J.
      • Reuhl K.R.
      • Shapses S.A.
      • Yang C.S.
      The major green tea polyphenol, (−)-epigallocatechin-3-gallate, inhibits obesity, metabolic syndrome, and fatty liver disease in high-fat-fed mice.
      ,
      • Potenza M.A.
      • Marasciulo F.L.
      • Tarquinio M.
      • Tiravanti E.
      • Colantuono G.
      • Federici A.
      • Kim J.A.
      • Quon M.J.
      • Montagnani M.
      EGCG, a green tea polyphenol, improves endothelial function and insulin sensitivity, reduces blood pressure, and protects against myocardial I/R injury in SHR.
      ,
      • Kim J.A.
      • Formosa G.
      • Li Y.
      • Potenza M.A.
      • Marasciulo F.L.
      • Montagnani M.
      • Quon M.J.
      Epigallocatechin gallate, a green tea polyphenol, mediates NO-dependent vasodilation using signaling pathways in vascular endothelium requiring reactive oxygen species and Fyn.
      ). One of the molecular mechanisms for EGCG-mediated protective effects is through activation of adenosine monophosphate-activated protein kinase (AMPK) (
      • 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.
      ,
      • Moon H.S.
      • Chung C.S.
      • Lee H.G.
      • Kim T.G.
      • Choi Y.J.
      • Cho C.S.
      Inhibitory effect of (−)-epigallocatechin-3-gallate on lipid accumulation of 3T3-L1 cells.
      ). However, the molecular mechanisms for linking EGCG-stimulated AMPK and autophagy with regard to lipid metabolism are not known. Furthermore, although polyphenols, including EGCG and resveratrol, have effects on lipolysis, it is not known whether the lipolysis is associated with lipophagy (
      • Ahn J.
      • Cho I.
      • Kim S.
      • Kwon D.
      • Ha T.
      Dietary resveratrol alters lipid metabolism-related gene expression of mice on an atherogenic diet.
      ,
      • Sohle J.
      • Knott A.
      • Holtzmann U.
      • Siegner R.
      • Gronniger E.
      • Schepky A.
      • Gallinat S.
      • Wenck H.
      • Stab F.
      • Winnefeld M.
      White tea extract induces lipolytic activity and inhibits adipogenesis in human subcutaneous (pre)-adipocytes.
      ).
      In this study, we investigated the mechanism of EGCG-induced autophagy and its role in accumulation of intracellular lipid accumulation. Here, we show that EGCG-stimulated autophagy is (at least in part) through a CaMKKβ/AMPK-mediated mechanism and has a role in degradation of lipid droplets in vascular endothelial cells. Our data demonstrate a novel mechanism for polyphenols to regulate lipid metabolism in vascular endothelial cells.

      DISCUSSION

      This study demonstrates that EGCG stimulates autophagy through a CaMKKβ/AMPK-dependent mechanism and facilitates autophagic flux (Fig. 9). Furthermore, EGCG-stimulated lysosomal degradation leads to reduced accumulation of intracellular lipid droplets in vascular endothelial cells. These EGCG effects in vascular endothelium may contribute to protection from lipid-mediated endothelial dysfunction and cardiovascular complications.
      Figure thumbnail gr9
      FIGURE 9Schematic diagram of proposed EGCG-stimulated signaling pathway to activate lipophagy. EGCG-induced oscillation of cytosolic Ca2+ levels by stimulating Ca2+ store in ER that activates CaMKKβ/AMPK pathways and facilitation of autophagic flux. Saturated fatty acid (palmitate) causes impairment of autophagic flux, which leads to accumulation of ectopic lipid accumulation. The facilitated autophagic flux by EGCG affects degradation of accumulated lipid droplets in vascular endothelial cells, which may contribute to improvement of cardiovascular function and prevention of cardiovascular disease. This EGCG-stimulated lipophagy may explain the beneficial health effect of green tea consumption.

      EGCG Induces Autophagy through AMPK and CaMKKβ

      In this study, we demonstrate that EGCG induces autophagosome and autophagolysosome formation (Fig. 2) through a CaMKKβ/AMPK signaling pathway leading to facilitation of autophagic flux (FIGURE 3, FIGURE 5).
      Previously, it has been shown that a high concentration (50–100 μm) of EGCG stimulates autophagy leading to cell death in cancer cells (
      • Satoh M.
      • Takemura Y.
      • Hamada H.
      • Sekido Y.
      • Kubota S.
      EGCG induces human mesothelioma cell death by inducing reactive oxygen species and autophagy.
      ,
      • Zhang Y.
      • Yang N.D.
      • Zhou F.
      • Shen T.
      • Duan T.
      • Zhou J.
      • Shi Y.
      • Zhu X.Q.
      • Shen H.M.
      (−)-Epigallocatechin-3-gallate induces non-apoptotic cell death in human cancer cells via ROS-mediated lysosomal membrane permeabilization.
      ). Another study reported that EGCG stimulates autophagy, which leads to inhibition of endotoxin-induced septic shock through EGCG-induced degradation of HMGB1, a late lethal inflammatory factor (
      • Li W.
      • Zhu S.
      • Li J.
      • Assa A.
      • Jundoria A.
      • Xu J.
      • Fan S.
      • Eissa N.T.
      • Tracey K.J.
      • Sama A.E.
      • Wang H.
      EGCG stimulates autophagy and reduces cytoplasmic HMGB1 levels in endotoxin-stimulated macrophages.
      ). However, a molecular mechanism for the EGCG-stimulated autophagy with regard to Ca2+/CaMKKβ and lipid droplet is unknown. In this study, we demonstrate a novel mechanism for EGCG-stimulated autophagy and its functional consequence in degradation of lipid droplets.
      AMPK is a key mediator for the initial process of autophagy by stimulating the phosphorylation of ULK and formation of its protein complex with multiple autophagic proteins (
      • Zhao M.
      • Klionsky D.J.
      AMPK-dependent phosphorylation of ULK1 induces autophagy.
      ). Thus, activation of AMPK is crucial for initiation of autophagy. Because AMPK is an energy-sensing enzyme recognizing the AMP/ATP ratio, starvation conditions activate autophagy through an AMPK-dependent mechanism (
      • Birkenfeld A.L.
      • Lee H.Y.
      • Guebre-Egziabher F.
      • Alves T.C.
      • Jurczak M.J.
      • Jornayvaz F.R.
      • Zhang D.
      • Hsiao J.J.
      • Martin-Montalvo A.
      • Fischer-Rosinsky A.
      • Spranger J.
      • Pfeiffer A.F.
      • Jordan J.
      • Fromm M.F.
      • König J.
      • Lieske S.
      • Carmean C.M.
      • Frederick D.W.
      • Weismann D.
      • Knauf F.
      • Irusta P.M.
      • De Cabo R.
      • Helfand S.L.
      • Samuel V.T.
      • Shulman G.I.
      Deletion of the mammalian INDY homolog mimics aspects of dietary restriction and protects against adiposity and insulin resistance in mice.
      ). Thus, EGCG may be mimicking starvation or caloric restriction conditions, which are consistent with the beneficial health effects of polyphenols, including EGCG and resveratrol (
      • de Boer V.C.
      • de Goffau M.C.
      • Arts I.C.
      • Hollman P.C.
      • Keijer J.
      SIRT1 stimulation by polyphenols is affected by their stability and metabolism.
      ,
      • Huang C.H.
      • Tsai S.J.
      • Wang Y.J.
      • Pan M.H.
      • Kao J.Y.
      • Way T.D.
      EGCG inhibits protein synthesis, lipogenesis, and cell cycle progression through activation of AMPK in p53 positive and negative human hepatoma cells.
      ,
      • Baur J.A.
      • Pearson K.J.
      • Price N.L.
      • Jamieson H.A.
      • Lerin C.
      • Kalra A.
      • Prabhu V.V.
      • Allard J.S.
      • Lopez-Lluch G.
      • Lewis K.
      • Pistell P.J.
      • Poosala S.
      • Becker K.G.
      • Boss O.
      • Gwinn D.
      • Wang M.
      • Ramaswamy S.
      • Fishbein K.W.
      • Spencer R.G.
      • Lakatta E.G.
      • Le Couteur D.
      • Shaw R.J.
      • Navas P.
      • Puigserver P.
      • Ingram D.K.
      • de Cabo R.
      • Sinclair D.A.
      Resveratrol improves health and survival of mice on a high-calorie diet.
      ,
      • Park S.J.
      • Ahmad F.
      • Philp A.
      • Baar K.
      • Williams T.
      • Luo H.
      • Ke H.
      • Rehmann H.
      • Taussig R.
      • Brown A.L.
      • Kim M.K.
      • Beaven M.A.
      • Burgin A.B.
      • Manganiello V.
      • Chung J.H.
      Resveratrol ameliorates aging-related metabolic phenotypes by inhibiting cAMP phosphodiesterases.
      ). EGCG also has an anti-diabetic effect that is similar to metformin, an anti-diabetic drug that activates AMPK (
      • Chen D.
      • Pamu S.
      • Cui Q.
      • Chan T.H.
      • Dou Q.P.
      Novel epigallocatechin gallate (EGCG) analogs activate AMP-activated protein kinase pathway and target cancer stem cells.
      ). This suggests that EGCG and metformin may have a common mechanism to ameliorate metabolic and cardiovascular disorders. Knockdown of ATG5 or CaMKKβ was significantly but not completely able to block the LC3-II formation (Figs. 1E and 5D). This may be due to the incomplete removal of ATG5 or CaMKKβ, because siRNAs were able to knock down only 40 and 48% of ATG5 and CaMKKβ, respectively. However, it is possible that the remaining LC3-II after knockdown of ATG5 or CaMKKβ could be due to the CaMKKβ- or ATG5-independent mechanism. Using primary cells from knock-out mice may help to understand the precise mechanism. In this study, our results suggest that EGCG stimulates LC3-II formation at least in part through an ATG5- and CaMKKβ-mediated mechanism.
      We observed that EGCG activates AMPK, whereas EGCG did not stimulate the phosphorylation of mTOR (Fig. 5C). Activation of AMPK inhibits mTOR in starvation-induced autophagy (
      • Alers S.
      • Löffler A.S.
      • Wesselborg S.
      • Stork B.
      Role of AMPK-mTOR-Ulk1/2 in the regulation of autophagy: cross-talk, shortcuts, and feedbacks.
      ,
      • Kim J.
      • Kundu M.
      • Viollet B.
      • Guan K.L.
      AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1.
      ). However, we were not able to observe that mTOR is inhibited by EGCG, which may be a cell type-specific or a stimulus-specific response. Interestingly, we observed that EGCG was not able to induce LC3-II formation in mouse embryonic fibroblasts (data not shown). In addition, other studies have shown that palmitate induces autophagy through an mTOR-independent mechanism (
      • Tan S.H.
      • Shui G.
      • Zhou J.
      • Li J.J.
      • Bay B.H.
      • Wenk M.R.
      • Shen H.M.
      Induction of autophagy by palmitic acid via protein kinase C-mediated signaling pathway independent of mTOR (mammalian target of rapamycin).
      ). This unexpected result suggests that EGCG-stimulated accumulation of LC3-II is independent of the mTOR pathway.

      EGCG Induces Cytosolic Ca2+ Dynamics Implicated in Autophagy

      EGCG induces intracellular Ca2+ dynamics, and chelating cytosolic calcium by BAPTA-AM or reducing availability of extracellular Ca2+ by EGTA suppressed autophagy (Fig. 6F). This suggests that cytosolic Ca2+ load is necessary for EGCG-induced autophagy. Consistent with our data, previous reports show that the heightened free cytosolic calcium induces autophagy through activation of CaMKKβ and AMPK (
      • Abbott M.J.
      • Edelman A.M.
      • Turcotte L.P.
      CaMKK is an upstream signal of AMP-activated protein kinase in regulation of substrate metabolism in contracting skeletal muscle.
      ,
      • Witczak C.A.
      • Fujii N.
      • Hirshman M.F.
      • Goodyear L.J.
      Ca2+/calmodulin-dependent protein kinase kinase-alpha regulates skeletal muscle glucose uptake independent of AMP-activated protein kinase and Akt activation.
      ,
      • Hurley R.L.
      • Anderson K.A.
      • Franzone J.M.
      • Kemp B.E.
      • Means A.R.
      • Witters L.A.
      The Ca2+/calmodulin-dependent protein kinase kinases are AMP-activated protein kinase kinases.
      ). EGCG stimulates Ca2+ release from the ER store, because treatment with CPA, a blocker of ER Ca2+-ATPase, reduced cytosolic Ca2+ load. It has been shown that Ca2+ is required for CaMKKβ to activate AMPK, although CaMKKβ can autonomously activate other substrates, including CaMKI and CaMKIV, without Ca2+/CaM binding (
      • Racioppi L.
      • Means A.R.
      Calcium/calmodulin-dependent protein kinase kinase 2: roles in signaling and pathophysiology.
      ). Our data suggest that the elevated cytosolic Ca2+ dynamics are necessary for EGCG-stimulated autophagy. Resveratrol, a major polyphenol in red wine, activates CaMKKβ/AMPK through inhibition of cAMP-degrading phosphodiesterase and PKCϵ that leads to the activation of ryanodine receptor to increase intracellular calcium levels (
      • Park S.J.
      • Ahmad F.
      • Philp A.
      • Baar K.
      • Williams T.
      • Luo H.
      • Ke H.
      • Rehmann H.
      • Taussig R.
      • Brown A.L.
      • Kim M.K.
      • Beaven M.A.
      • Burgin A.B.
      • Manganiello V.
      • Chung J.H.
      Resveratrol ameliorates aging-related metabolic phenotypes by inhibiting cAMP phosphodiesterases.
      ). However, the link between autophagy and polyphenol-induced intracellular calcium is not known. Here, we show for the first time that EGCG elicits an increase in cytosolic Ca2+, which contributes to autophagy. In addition to the signaling molecules to stimulate the CaMKKβ/AMPK axis, increased cytosolic Ca2+ may contribute to autophagosomal membrane formation from the ER membrane. Previously, it has been shown that ER membranes co-localize with calcium phosphate precipitate-induced LC3-positive autophagosome (
      • Chen X.
      • Li M.
      • Chen D.
      • Gao W.
      • Guan J.L.
      • Komatsu M.
      • Yin X.M.
      Autophagy induced by calcium phosphate precipitates involves endoplasmic reticulum membranes in autophagosome biogenesis.
      ). This suggests that calcium-induced autophagosome formation may be attributable to interaction with the ER membrane. Further studies are necessary to reveal the role of cytosolic Ca2+ and CaMKKβ in autophagy.

      EGCG Facilitates Lipophagy

      In this study, we demonstrate that EGCG facilitates autophagic flux, which contributes to oppose palmitate-induced accumulation of lipid droplets in endothelial cells (FIGURE 7, FIGURE 8). Reduction of lipid droplets in endothelial cells through autophagic flux suggests a novel mechanism for EGCG-mediated beneficial health effects. Autophagy (or lipophagy) plays a role in lipid metabolism both in adipose tissue and in non-adipose tissue (
      • Singh R.
      • Kaushik S.
      • Wang Y.
      • Xiang Y.
      • Novak I.
      • Komatsu M.
      • Tanaka K.
      • Cuervo A.M.
      • Czaja M.J.
      Autophagy regulates lipid metabolism.
      ,
      • Weidberg H.
      • Shvets E.
      • Elazar Z.
      Lipophagy: selective catabolism designed for lipids.
      ,
      • Singh R.
      Hypothalamic lipophagy and energetic balance.
      ). Reduction of lipid accumulation in adipose tissue leads to weight loss and improvement of whole body metabolism. In contrast, reduced lipid accumulation in vascular endothelium may contribute to cardiovascular function, because triglyceride and cholesterols are the major components of lipid droplets that are associated with atherosclerosis and coronary heart disease. Chronic high fat diet and acute high cholesterol diet lead to impaired lysosomal degradation (
      • Rodriguez-Navarro J.A.
      • Kaushik S.
      • Koga H.
      • Dall'Armi C.
      • Shui G.
      • Wenk M.R.
      • Di Paolo G.
      • Cuervo A.M.
      Inhibitory effect of dietary lipids on chaperone-mediated autophagy.
      ), and fatty acid inhibits autophagic flux due to the failure of lysosomal degradation in beta cells (
      • Las G.
      • Serada S.B.
      • Wikstrom J.D.
      • Twig G.
      • Shirihai O.S.
      Fatty acids suppress autophagic turnover in β-cells.
      ). Consistent with these reports, our data show that palmitate impairs autophagic flux in primary aortic endothelial cells, and EGCG enhances degradation of lipid droplets through facilitation of autophagic flux.
      Despite the reduced number of lipid droplets in the EGCG-treated cells, a larger number of lipid droplets co-localize with LC3 and LAMP-1 in the presence of NH4Cl/Leu (Fig. 8). The reason for the smaller number of lipid droplets and the fewer co-localizations of lipid droplets and LC3 or LAMP-1 in the presence of EGCG without NH4Cl/Leu seems to be due to the rapid degradation of lipid droplets.
      The accumulation of lipid droplets is much more prominent in the cells treated with palmitate alone than the cells treated with palmitate along with EGCG. One possibility is that triglyceride synthesis could be slower in EGCG-treated cells. However, we observed that reduction of the lipid droplet was almost identical whether the cells were co-treated or post-treated with EGCG with palmitate (Fig. 8, H and I). Moreover, co-localization of lipid droplets with autophagosomes was markedly increased in the EGCG-treated cells when lysosomal degradation was blocked (Fig. 8). These suggest that treatment with palmitate seems to inhibit the fusion process of autophagosome and lysosome, and the fusion is facilitated by EGCG.
      Autophagic flux can be divided into three steps as follows: (i) formation of autophagosome; (ii) formation of autolysosome by fusion of lysosome and autophagosome; and (iii) lysosomal degradation. Formation of autophagosome was dramatically increased by EGCG as shown the samples with lysosomal inhibitors (Fig. 3, A and B, 2nd and 4th lanes), and lysosomal degradation was increased (Fig. 3, C–E). Nonetheless, our results suggest that EGCG stimulates lipophagy through facilitation of autophagosome formation, lysosomal fusion, and degradation. This does not exclude the possibility that other lipases, including hormone-sensitive lipase and endothelial lipase, may contribute to reduction of lipid accumulation. In fact, EGCG stimulates hormone-sensitive lipase in adipocytes and pancreatic lipases in the serum that are associated with weight loss in adipose tissue (
      • Grove K.A.
      • Sae-tan S.
      • Kennett M.J.
      • Lambert J.D.
      (−)-Epigallocatechin-3-gallate inhibits pancreatic lipase and reduces body weight gain in high fat-fed obese mice.
      ,
      • Lee M.S.
      • Kim C.T.
      • Kim I.H.
      • Kim Y.
      Inhibitory effects of green tea catechin on the lipid accumulation in 3T3-L1 adipocytes.
      ). In contrast, our data present for the first time that EGCG reduces endothelial ectopic lipid accumulation. We previously reported that EGCG intake protects from insulin resistance, hypertension, and ischemia-reperfusion injury in the heart in spontaneously hypertensive rats (
      • Potenza M.A.
      • Marasciulo F.L.
      • Tarquinio M.
      • Tiravanti E.
      • Colantuono G.
      • Federici A.
      • Kim J.A.
      • Quon M.J.
      • Montagnani M.
      EGCG, a green tea polyphenol, improves endothelial function and insulin sensitivity, reduces blood pressure, and protects against myocardial I/R injury in SHR.
      ). These beneficial health effects of EGCG in cardiovascular tissues may be associated with the EGCG-induced facilitation of autophagy. Further studies are required to understand the more detailed regulatory mechanisms for EGCG-stimulated autophagic flux.
      In summary, EGCG induces autophagy through a Ca2+/CaMKKβ/AMPK-mediated mechanism, which contributes to reduction in the palmitate-induced accumulation of lipid droplets in endothelial cells. These findings suggest the following: 1) heightened intracellular calcium dynamics activating CaMKKβ/AMPK may play an important role in the beneficial health effect of green tea; 2) EGCG stimulates autophagic flux, a key step for autophagic degradation, which may help reduce the accumulation of lipid; 3) supplementation of green tea may have the a beneficial effect in endothelial function through facilitation of lipophagy. These effects of green tea polyphenol may help prevent metabolic and cardiovascular disorders.

      REFERENCES

        • Kolattukudy P.E.
        • Niu J.
        Inflammation, endoplasmic reticulum stress, autophagy, and the monocyte chemoattractant protein-1/CCR2 pathway.
        Circ. Res. 2012; 110: 174-189
        • Younce C.
        • Kolattukudy P.
        MCP-1-induced protein promotes adipogenesis via oxidative stress, endoplasmic reticulum stress, and autophagy.
        Cell. Physiol. Biochem. 2012; 30: 307-320
        • Chimal-Monroy J.
        • Abarca-Buis R.F.
        • Cuervo R.
        • Diaz-Hernandez M.
        • Bustamante M.
        • Rios-Flores J.A.
        • Romero-Suarez S.
        • Farrera-Hernandez A.
        Molecular control of cell differentiation and programmed cell death during digit development.
        IUBMB Life. 2011; 63: 899-906
        • Sridhar S.
        • Botbol Y.
        • Macian F.
        • Cuervo A.M.
        Autophagy and disease: always two sides to a problem.
        J. Pathol. 2012; 226: 255-273
        • Cuervo A.M.
        Autophagy: in sickness and in health.
        Trends Cell Biol. 2004; 14: 70-77
        • Singh R.
        Autophagy and regulation of lipid metabolism.
        Results Probl. Cell Differ. 2010; 52: 35-46
        • Singh R.
        • Cuervo A.M.
        Autophagy in the cellular energetic balance.
        Cell Metab. 2011; 13: 495-504
        • Gustafsson A.B.
        • Gottlieb R.A.
        Autophagy in ischemic heart disease.
        Circ. Res. 2009; 104: 150-158
        • De Meyer G.R.
        • Martinet W.
        Autophagy in the cardiovascular system.
        Biochim. Biophys. Acta. 2009; 1793: 1485-1495
        • Kuriyama S.
        • Shimazu T.
        • Ohmori K.
        • Kikuchi N.
        • Nakaya N.
        • Nishino Y.
        • Tsubono Y.
        • Tsuji I.
        Green tea consumption and mortality due to cardiovascular disease, cancer, and all causes in Japan: the Ohsaki study.
        JAMA. 2006; 296: 1255-1265
        • Bose M.
        • Lambert J.D.
        • Ju J.
        • Reuhl K.R.
        • Shapses S.A.
        • Yang C.S.
        The major green tea polyphenol, (−)-epigallocatechin-3-gallate, inhibits obesity, metabolic syndrome, and fatty liver disease in high-fat-fed mice.
        J. Nutr. 2008; 138: 1677-1683
        • Potenza M.A.
        • Marasciulo F.L.
        • Tarquinio M.
        • Tiravanti E.
        • Colantuono G.
        • Federici A.
        • Kim J.A.
        • Quon M.J.
        • Montagnani M.
        EGCG, a green tea polyphenol, improves endothelial function and insulin sensitivity, reduces blood pressure, and protects against myocardial I/R injury in SHR.
        Am. J. Physiol. Endocrinol. Metab. 2007; 292: E1378-E1387
        • Kim J.A.
        • Formosa G.
        • Li Y.
        • Potenza M.A.
        • Marasciulo F.L.
        • Montagnani M.
        • Quon M.J.
        Epigallocatechin gallate, a green tea polyphenol, mediates NO-dependent vasodilation using signaling pathways in vascular endothelium requiring reactive oxygen species and Fyn.
        J. Biol. Chem. 2007; 282: 13736-13745
        • 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: 694-699
        • Moon H.S.
        • Chung C.S.
        • Lee H.G.
        • Kim T.G.
        • Choi Y.J.
        • Cho C.S.
        Inhibitory effect of (−)-epigallocatechin-3-gallate on lipid accumulation of 3T3-L1 cells.
        Obesity. 2007; 15: 2571-2582
        • Ahn J.
        • Cho I.
        • Kim S.
        • Kwon D.
        • Ha T.
        Dietary resveratrol alters lipid metabolism-related gene expression of mice on an atherogenic diet.
        J. Hepatol. 2008; 49: 1019-1028
        • Sohle J.
        • Knott A.
        • Holtzmann U.
        • Siegner R.
        • Gronniger E.
        • Schepky A.
        • Gallinat S.
        • Wenck H.
        • Stab F.
        • Winnefeld M.
        White tea extract induces lipolytic activity and inhibits adipogenesis in human subcutaneous (pre)-adipocytes.
        Nutr. Metab. 2009; 6: 20
        • Montana V.
        • Ni Y.
        • Sunjara V.
        • Hua X.
        • Parpura V.
        Vesicular glutamate transporter-dependent glutamate release from astrocytes.
        J. Neurosci. 2004; 24: 2633-2642
        • Lee W.
        • Malarkey E.B.
        • Reyes R.C.
        • Parpura V.
        Micropit: A new cell culturing approach for characterization of solitary astrocytes and small networks of these glial cells.
        Front. Neuroeng. 2008; 1: 2
        • Stout Jr., R.F.
        • Parpura V.
        Voltage-gated calcium channel types in cultured C. elegans CEPsh glial cells.
        Cell Calcium. 2011; 50: 98-108
        • Malarkey E.B.
        • Ni Y.
        • Parpura V.
        Ca2+ entry through TRPC1 channels contributes to intracellular Ca2+ dynamics and consequent glutamate release from rat astrocytes.
        Glia. 2008; 56: 821-835
        • Fujita N.
        • Itoh T.
        • Omori H.
        • Fukuda M.
        • Noda T.
        • Yoshimori T.
        The Atg16L complex specifies the site of LC3 lipidation for membrane biogenesis in autophagy.
        Mol. Biol. Cell. 2008; 19: 2092-2100
        • Tanida I.
        • Sou Y.S.
        • Ezaki J.
        • Minematsu-Ikeguchi N.
        • Ueno T.
        • Kominami E.
        HsAtg4B/HsApg4B/autophagin-1 cleaves the carboxyl termini of three human Atg8 homologues and delipidates microtubule-associated protein light chain 3- and GABAA receptor-associated protein-phospholipid conjugates.
        J. Biol. Chem. 2004; 279: 36268-36276
        • Tanida I.
        • Ueno T.
        • Kominami E.
        LC3 conjugation system in mammalian autophagy.
        Int. J. Biochem. Cell Biol. 2004; 36: 2503-2518
        • Mizushima N.
        • Yoshimori T.
        How to interpret LC3 immunoblotting.
        Autophagy. 2007; 3: 542-545
        • Hubbard V.M.
        • Valdor R.
        • Patel B.
        • Singh R.
        • Cuervo A.M.
        • Macian F.
        Macroautophagy regulates energy metabolism during effector T cell activation.
        J. Immunol. 2010; 185: 7349-7357
        • Bjørkøy G.
        • Lamark T.
        • Brech A.
        • Outzen H.
        • Perander M.
        • Overvatn A.
        • Stenmark H.
        • Johansen T.
        p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death.
        J. Cell Biol. 2005; 171: 603-614
        • Bjørkøy G.
        • Lamark T.
        • Johansen T.
        p62/SQSTM1: a missing link between protein aggregates and the autophagy machinery.
        Autophagy. 2006; 2: 138-139
        • Ichimura Y.
        • Kominami E.
        • Tanaka K.
        • Komatsu M.
        Selective turnover of p62/A170/SQSTM1 by autophagy.
        Autophagy. 2008; 4: 1063-1066
        • Reiter C.E.
        • Kim J.A.
        • Quon M.J.
        Green tea polyphenol epigallocatechin gallate reduces endothelin-1 expression and secretion in vascular endothelial cells: roles for AMP-activated protein kinase, Akt, and FOXO1.
        Endocrinology. 2010; 151: 103-114
        • Egan D.F.
        • Shackelford D.B.
        • Mihaylova M.M.
        • Gelino S.
        • Kohnz R.A.
        • Mair W.
        • Vasquez D.S.
        • Joshi A.
        • Gwinn D.M.
        • Taylor R.
        • Asara J.M.
        • Fitzpatrick J.
        • Dillin A.
        • Viollet B.
        • Kundu M.
        • Hansen M.
        • Shaw R.J.
        Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy.
        Science. 2011; 331: 456-461
        • Lee J.W.
        • Park S.
        • Takahashi Y.
        • Wang H.G.
        The association of AMPK with ULK1 regulates autophagy.
        PLoS One. 2010; 5: e15394
        • Jin B.Y.
        • Sartoretto J.L.
        • Gladyshev V.N.
        • Michel T.
        Endothelial nitric oxide synthase negatively regulates hydrogen peroxide-stimulated AMP-activated protein kinase in endothelial cells.
        Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 17343-17348
        • Shang L.
        • Wang X.
        AMPK and mTOR coordinate the regulation of Ulk1 and mammalian autophagy initiation.
        Autophagy. 2011; 7: 924-926
        • Ravikumar B.
        • Vacher C.
        • Berger Z.
        • Davies J.E.
        • Luo S.
        • Oroz L.G.
        • Scaravilli F.
        • Easton D.F.
        • Duden R.
        • O'Kane C.J.
        • Rubinsztein D.C.
        Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease.
        Nat. Genet. 2004; 36: 585-595
        • Hawley S.A.
        • Pan D.A.
        • Mustard K.J.
        • Ross L.
        • Bain J.
        • Edelman A.M.
        • Frenguelli B.G.
        • Hardie D.G.
        Calmodulin-dependent protein kinase kinase-β is an alternative upstream kinase for AMP-activated protein kinase.
        Cell Metab. 2005; 2: 9-19
        • Reyes R.C.
        • Parpura V.
        The trinity of Ca2+ sources for the exocytotic glutamate release from astrocytes.
        Neurochem. Int. 2009; 55: 2-8
        • Las G.
        • Serada S.B.
        • Wikstrom J.D.
        • Twig G.
        • Shirihai O.S.
        Fatty acids suppress autophagic turnover in β-cells.
        J. Biol. Chem. 2011; 286: 42534-42544
        • Lara-Castro C.
        • Garvey W.T.
        Intracellular lipid accumulation in liver and muscle and the insulin resistance syndrome.
        Endocrinol. Metab. Clin. North Am. 2008; 37: 841-856
        • Suganami T.
        • Tanaka M.
        • Ogawa Y.
        Adipose tissue inflammation and ectopic lipid accumulation.
        Endocr. J. 2012; 59: 849-857
        • Gustafson B.
        Adipose tissue, inflammation, and atherosclerosis.
        J. Atheroscler. Thromb. 2010; 17: 332-341
        • Singh R.
        • Kaushik S.
        • Wang Y.
        • Xiang Y.
        • Novak I.
        • Komatsu M.
        • Tanaka K.
        • Cuervo A.M.
        • Czaja M.J.
        Autophagy regulates lipid metabolism.
        Nature. 2009; 458: 1131-1135
        • Weidberg H.
        • Shvets E.
        • Elazar Z.
        Lipophagy: selective catabolism designed for lipids.
        Dev. Cell. 2009; 16: 628-630
        • Satoh M.
        • Takemura Y.
        • Hamada H.
        • Sekido Y.
        • Kubota S.
        EGCG induces human mesothelioma cell death by inducing reactive oxygen species and autophagy.
        Cancer Cell Int. 2013; 13: 19
        • Zhang Y.
        • Yang N.D.
        • Zhou F.
        • Shen T.
        • Duan T.
        • Zhou J.
        • Shi Y.
        • Zhu X.Q.
        • Shen H.M.
        (−)-Epigallocatechin-3-gallate induces non-apoptotic cell death in human cancer cells via ROS-mediated lysosomal membrane permeabilization.
        PLoS One. 2012; 7: e46749
        • Li W.
        • Zhu S.
        • Li J.
        • Assa A.
        • Jundoria A.
        • Xu J.
        • Fan S.
        • Eissa N.T.
        • Tracey K.J.
        • Sama A.E.
        • Wang H.
        EGCG stimulates autophagy and reduces cytoplasmic HMGB1 levels in endotoxin-stimulated macrophages.
        Biochem. Pharmacol. 2011; 81: 1152-1163
        • Zhao M.
        • Klionsky D.J.
        AMPK-dependent phosphorylation of ULK1 induces autophagy.
        Cell Metab. 2011; 13: 119-120
        • Birkenfeld A.L.
        • Lee H.Y.
        • Guebre-Egziabher F.
        • Alves T.C.
        • Jurczak M.J.
        • Jornayvaz F.R.
        • Zhang D.
        • Hsiao J.J.
        • Martin-Montalvo A.
        • Fischer-Rosinsky A.
        • Spranger J.
        • Pfeiffer A.F.
        • Jordan J.
        • Fromm M.F.
        • König J.
        • Lieske S.
        • Carmean C.M.
        • Frederick D.W.
        • Weismann D.
        • Knauf F.
        • Irusta P.M.
        • De Cabo R.
        • Helfand S.L.
        • Samuel V.T.
        • Shulman G.I.
        Deletion of the mammalian INDY homolog mimics aspects of dietary restriction and protects against adiposity and insulin resistance in mice.
        Cell Metab. 2011; 14: 184-195
        • de Boer V.C.
        • de Goffau M.C.
        • Arts I.C.
        • Hollman P.C.
        • Keijer J.
        SIRT1 stimulation by polyphenols is affected by their stability and metabolism.
        Mech. Ageing Dev. 2006; 127: 618-627
        • Huang C.H.
        • Tsai S.J.
        • Wang Y.J.
        • Pan M.H.
        • Kao J.Y.
        • Way T.D.
        EGCG inhibits protein synthesis, lipogenesis, and cell cycle progression through activation of AMPK in p53 positive and negative human hepatoma cells.
        Mol. Nutr. Food Res. 2009; 53: 1156-1165
        • Baur J.A.
        • Pearson K.J.
        • Price N.L.
        • Jamieson H.A.
        • Lerin C.
        • Kalra A.
        • Prabhu V.V.
        • Allard J.S.
        • Lopez-Lluch G.
        • Lewis K.
        • Pistell P.J.
        • Poosala S.
        • Becker K.G.
        • Boss O.
        • Gwinn D.
        • Wang M.
        • Ramaswamy S.
        • Fishbein K.W.
        • Spencer R.G.
        • Lakatta E.G.
        • Le Couteur D.
        • Shaw R.J.
        • Navas P.
        • Puigserver P.
        • Ingram D.K.
        • de Cabo R.
        • Sinclair D.A.
        Resveratrol improves health and survival of mice on a high-calorie diet.
        Nature. 2006; 444: 337-342
        • Park S.J.
        • Ahmad F.
        • Philp A.
        • Baar K.
        • Williams T.
        • Luo H.
        • Ke H.
        • Rehmann H.
        • Taussig R.
        • Brown A.L.
        • Kim M.K.
        • Beaven M.A.
        • Burgin A.B.
        • Manganiello V.
        • Chung J.H.
        Resveratrol ameliorates aging-related metabolic phenotypes by inhibiting cAMP phosphodiesterases.
        Cell. 2012; 148: 421-433
        • Chen D.
        • Pamu S.
        • Cui Q.
        • Chan T.H.
        • Dou Q.P.
        Novel epigallocatechin gallate (EGCG) analogs activate AMP-activated protein kinase pathway and target cancer stem cells.
        Bioorg. Med. Chem. 2012; 20: 3031-3037
        • Alers S.
        • Löffler A.S.
        • Wesselborg S.
        • Stork B.
        Role of AMPK-mTOR-Ulk1/2 in the regulation of autophagy: cross-talk, shortcuts, and feedbacks.
        Mol. Cell. Biol. 2012; 32: 2-11
        • Kim J.
        • Kundu M.
        • Viollet B.
        • Guan K.L.
        AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1.
        Nat. Cell Biol. 2011; 13: 132-141
        • Tan S.H.
        • Shui G.
        • Zhou J.
        • Li J.J.
        • Bay B.H.
        • Wenk M.R.
        • Shen H.M.
        Induction of autophagy by palmitic acid via protein kinase C-mediated signaling pathway independent of mTOR (mammalian target of rapamycin).
        J. Biol. Chem. 2012; 287: 14364-14376
        • Abbott M.J.
        • Edelman A.M.
        • Turcotte L.P.
        CaMKK is an upstream signal of AMP-activated protein kinase in regulation of substrate metabolism in contracting skeletal muscle.
        Am. J. Physiol. Regul. Integr. Comp. Physiol. 2009; 297: R1724-R1732
        • Witczak C.A.
        • Fujii N.
        • Hirshman M.F.
        • Goodyear L.J.
        Ca2+/calmodulin-dependent protein kinase kinase-alpha regulates skeletal muscle glucose uptake independent of AMP-activated protein kinase and Akt activation.
        Diabetes. 2007; 56: 1403-1409
        • Hurley R.L.
        • Anderson K.A.
        • Franzone J.M.
        • Kemp B.E.
        • Means A.R.
        • Witters L.A.
        The Ca2+/calmodulin-dependent protein kinase kinases are AMP-activated protein kinase kinases.
        J. Biol. Chem. 2005; 280: 29060-29066
        • Racioppi L.
        • Means A.R.
        Calcium/calmodulin-dependent protein kinase kinase 2: roles in signaling and pathophysiology.
        J. Biol. Chem. 2012; 287: 31658-31665
        • Chen X.
        • Li M.
        • Chen D.
        • Gao W.
        • Guan J.L.
        • Komatsu M.
        • Yin X.M.
        Autophagy induced by calcium phosphate precipitates involves endoplasmic reticulum membranes in autophagosome biogenesis.
        PLoS One. 2012; 7: e52347
        • Singh R.
        Hypothalamic lipophagy and energetic balance.
        Aging. 2011; 3: 934-942
        • Rodriguez-Navarro J.A.
        • Kaushik S.
        • Koga H.
        • Dall'Armi C.
        • Shui G.
        • Wenk M.R.
        • Di Paolo G.
        • Cuervo A.M.
        Inhibitory effect of dietary lipids on chaperone-mediated autophagy.
        Proc. Natl. Acad. Sci. U.S.A. 2012; 109: E705-E714
        • Grove K.A.
        • Sae-tan S.
        • Kennett M.J.
        • Lambert J.D.
        (−)-Epigallocatechin-3-gallate inhibits pancreatic lipase and reduces body weight gain in high fat-fed obese mice.
        Obesity. 2012; 20: 2311-2313
        • Lee M.S.
        • Kim C.T.
        • Kim I.H.
        • Kim Y.
        Inhibitory effects of green tea catechin on the lipid accumulation in 3T3-L1 adipocytes.
        Phytother. Res. 2009; 23: 1088-1091

      Linked Article

      • Green Tea Compound Reduces Lipid Accumulation in Cells
        Journal of Biological ChemistryVol. 288Issue 31
        • Preview
          Green tea has an abundant polyphenol called epigallocatechin-3-gallate (EGCG), which has been shown to alleviate cardiovascular problems. In this Paper of the Week, a team led by Jeong-a Kim at the University of Alabama at Birmingham tested to see if EGCG is involved in autophagy, a protective mechanism that comes into play during cellular stress. By doing a series of analyses in vascular endothelial cells, the investigators demonstrated that EGCG stimulated autophagy by a mechanism that involved AMP-activated protein kinase and calcium/calmodulin-dependent protein kinase kinase.
        • Full-Text
        • PDF
        Open Access