Full-length adiponectin protects hepatocytes from palmitate-induced apoptosis via inhibition of c-Jun NH 2 terminal kinase Tae W. Jung 1 , Yong J. Lee 2 , Myung W. Lee 3,4 , Seon M. Kim 3 and Tae W. Jung 1 1 Samsung Biomedical Institute, Seoul, Korea 2 Division of Clinical Research, Seoul Medical Center Research Institute, Korea 3 Department of Family Medicine, Brain Korea 21 Project Medical Science, College of Medicine, Korea University, Seoul, Korea 4 Department of Anatomy, College of Medicine, Korea University, Seoul, Korea Non-alcoholic fatty liver disease (NAFLD) is a chronic disease that is initially characterized by steatosis, with progression in some individuals to non-alcoholic ste- atohepatitis (NASH) and last-stage hepatic disease [1,2]. NAFLD is a common cause of chronic liver enzyme elevation and cryptogenic cirrhosis. It has been proposed that the trigger for a progression into the more processed stages of NAFLD involves damage to Keywords adiponectin; AMPK; apoptosis; JNK; palmitate Correspondence T. W. Jung, Samsung Biomedical Institute, Seoul, Korea, Annex B235, 50 Ilwon-Dong, Kangnam-Ku, PO Box 135-710, Seoul, Korea Fax: +82 2 873 8071 Tel: +82 2 873 8071 E-mail: ohayo2030@hanmail.net (Received 24 December 2008, revised 31 January 2009, accepted 10 February 2009) doi:10.1111/j.1742-4658.2009.06955.x Hepatic apoptosis is elevated in patients with non-alcoholic steatohepatitis and is correlated with the severity of the disease. Long-chain saturated fatty acids, such as palmitate, induce apoptosis in liver cells. The present study examined adiponectin-mediated protection against saturated fatty acid-induced apoptosis in the human hepatoma cell line, HepG2. Cells were cultured in a control media (i.e. without fatty acids) or the same media containing 250 lmolÆL )1 of albumin-bound oleate or palmitate for 24 h. The adiponectin concentrations used were: 0, 1, 10 or 100 lgÆmL )1 (n = 4–6 per treatment). Palmitate and thapsigargin, but not oleate, acti- vated caspase-3 and decreased cell viability in the absence of adiponectin. Adiponectin reduced palmitate- and thapsigargin-induced activation of cas- pase-3 and cell death in a dose-dependent manner. Phosphatidylinositol 3-kinase and AMP-activated protein kinase inhibitors abolished the effects of adiponectin. Adiponectin-induced inhibition of palmitate- and thapsigar- gin-induced apoptosis was not the result of an augmentation in the unfolded protein response or the increased expression of genes encoding the inhibitor of apoptosis proteins, inhibitor of apoptosis protein-2 and X-linked mammalian inhibitor of apoptosis protein. Palmitate and thapsi- gargin, but not oleate, increased c-Jun NH 2 terminal kinase phosphoryla- tion in the absence of adiponectin. Adiponectin blocked palmitate- and thapsigargin-induced activation of c-Jun NH 2 terminal kinase and reduced apoptosis. These data suggest that adiponectin is an important determinant of saturated fatty acid-induced apoptosis in liver cells and may have impli- cations for fatty acid-mediated liver cell injury in adiponectin-deficient individuals. Abbreviations AMPK, AMP-activated protein kinase; CHOP, CCAAT ⁄ enhancer-binding protein homologous protein; ER, endoplasmic reticulum; IAP, inhibitor of apoptosis protein; JNK, c-Jun NH 2 terminal kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; PI3 kinase, phosphatidylinositol 3-kinase; UPR, unfolded protein response; XIAP, X-linked mammalian inhibitor of apoptosis protein. 2278 FEBS Journal 276 (2009) 2278–2284 ª 2009 The Authors Journal compilation ª 2009 FEBS liver by oxidative stress, or a second effect, in addition to hepatic steatosis and abnormal apoptosis [3]. Hepatic apoptosis is present in patients with high calorie-induced hepatic steatosis and correlates with the severity of the disease [4,5]. Excess circulating and non-adipose tissue lipids, in particular long-chain satu- rated fatty acids, induce apoptosis in a number of cell types, including hepatocytes [6–11]. Obesity and insulin resistance, which are both conditions associated with and determined by excess lipids, play important roles in the development and progression of NAFLD [12,13]. Notably, insulin and several growth factors inhibit apoptosis and promote cell survival via phos- phatidylinositol 3-kinase (PI3 kinase)- and Akt-depen- dent mechanisms [14–17]. Adiponectin is a known adipokine in which plasma levels are decreased in hyperlipidemic conditions such as obesity and type 2 diabetes [18]. It has been reported that intravenous injection of adiponectin normalizes decreased insulin signaling and sensitivity [19]. The effect of adiponectin, an antidiabetic adipokine, has also been suggested to involve the PI3 kinase ⁄ Akt signaling pathway, and the ability of PI3 kinase ⁄ Akt to suppress the c-Jun NH 2 terminal kinase (JNK) pathway has been studied in a variety of cell types [20]. Therefore, excess lipid deliv- ery together with the role of reduced adiponectin may comprise an environment that promotes apoptosis and the development and ⁄ or severity of NASH. The pres- ent study aimed to determine whether adiponectin restricts lipid-mediated apoptosis in hepatocytes and, if so, whether this involved: (a) augmentation of the unfolded protein response (UPR); (b) up-regulation of members of the inhibitor of apoptosis protein (IAP) family; and ⁄ or (c) inhibition of JNK activity [8,11,17]. Results and Discussion Adiponectin reduces endoplasmic reticulum (ER) stress-mediated apoptosis Hyperlipidemia has been reported to induce ER stress, which may phosphorylate JNK and contribute to the development of insulin resistance and cell death [21]. Therefore, we treated HepG2 cells with thapsigargin and palmitate to confirm the inhibitory effect of adipo- nectin in chemically induced- or palmitate-induced ER stress. In the absence of adiponectin, elevated caspase- 3 activity (Fig. 1A) and decreased cell viability in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay (Fig. 1B) were observed in HepG2 cells incubated with thapsigargin or palmitate. Adiponectin inhibits thapsigargin- and palmitate- induced caspase-3 activity (Fig. 1A) and recovered cell viability in the MTT assay (Fig. 1B) in a dose-depen- dent manner. AMP-activated protein kinase (AMPK) inhibitor and PI3 kinase inhibitor inhibit adiponectin-mediated inhibition of apoptosis Adiponectin has been reported to be an AMPK activa- tor [18] and there is a known connection between AMPK and the PI3 kinase ⁄ Akt signaling pathway [20]. Therefore, we verified the signaling pathway of AMPK-ER stress-induced cell death using compound c, as an AMPK inhibitor, and wortmannin, as a PI3 kinase inhibitor. In the absence of adiponectin, thapsi- gargin and palmitate elevated caspase-3 activity in HepG2 cells (Fig. 2). Wortmannin (Fig. 2A) or com- pound c (Fig. 2B) interrupted the protective effects of 7 6 * * * ! ! ! ! ! ! ! ! ! * 0 µg·mL –1 10 µg·mL –1 100 µg·mL –1 Adiponectin A B 0 µg·mL –1 10 µg·mL –1 1 µg·mL –1 100 µg·mL –1 Adiponectin 5 4 3 Caspase 3 activity (folds of control) Cell viability (%) 2 1 0 120 100 80 60 40 20 0 Con TG O300 P300 Con TG O300 P300 Fig. 1. Adiponectin inhibits thapsigargin- and palmitate-induced apoptosis in a dose-dependent manner. (A) Caspase-3 activity is presented as the mean ± SD (n = 5). (B) Cell death was measured by the MTT assay from a total of three independent experiments. Treatments were carried out for 24 h. Con, not treated; TG, 250 n M thapsigargin; O300, 300 lM oleate; P300, 300 lM palmitate. *Signif- icantly different from Con and O250. Significantly different from the same treatment in the absence of adiponectin. T. W. Jung et al. Effects of adiponectin on hepatic apoptosis FEBS Journal 276 (2009) 2278–2284 ª 2009 The Authors Journal compilation ª 2009 FEBS 2279 adiponectin on thapsigargin- and palmitate-mediated apoptosis. Adiponectin is unable to augment the UPR The UPR is a signaling pathway that serves to reduce the protein load degradative ability of the ER in response to the accumulation of mis- and unfolded proteins [22]. An inappropriate response to these stres- sors results in apoptotic cell death [22]. We hypothe- sized that adiponectin might inhibit thapsigargin- and palmitate-mediated apoptosis via augmentation of the UPR. However, in the absence of adiponectin, thasi- gargin and palmitate elevated the expression of several genes involved in the UPR in HepG2 cells (Fig. 3). In the presence of thapsigargin or palmitate, adiponectin was unable to decrease the expression of these genes (Fig. 3). Thapasigargin, palmitate and adiponectin are unable to influence the expression of Bcl-2, cIAP 2 and the IAP family Bcl-2 proteins play important roles in caspase-depen- dent apoptosis, and the IAP family, cIAP 2 and X-linked mammalian inhibitor of apoptosis protein (XIAP) all play a protective role in ER stress-induced apoptosis in human breast cancer cells [23]. Therefore, we evaluated the expression levels of Bcl-2, cIAP 2 and XIAP in HepG2 cells in the presence and absence of adiponectin. Thapsigargin and palmitate, as well as adiponectin, were unable to affect the expression of Bcl-2, cIAP 2 and XIAP (Fig. 4). Adiponectin inhibits thapsigargin- and palmitate-induced JNK phosphorylation Palmitate has been reported to induce JNK dependent apoptosis in liver cells [8]. Thus, we evaluated the 1.2 A B Control Wortmannin Adiponectin Adipo + Wort Control Compound c Adiponectin Adipo + Com c 1.0 0.8 0.6 0.4 0.2 0.0 Con TG O300 P300 Con TG O300 P300 Caspase 3 activity (U·mg –1 total protein) 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Caspase 3 activity (U·mg –1 total protein) ! * * ! ! ! ! * * * * * * ! ! ! * * * * Fig. 2. Adiponectin inhibits thapsigargin- and palmitate-induced cas- pase-3 activity via PI3 kinase and AMPK in HepG2 cells. (A) The effects of 10 lgÆmL )1 of adiponectin and 1 lM of wortmannin, or both, on caspase-3 activity. (B) The effects of 10 ugÆmL )1 of adipo- nectin and 10 l M of compound c, or both, on caspase-3 activity. Treatments were carried out for 24 h. These data were obtained from a total of three independent experiments or represent the mean ± SD (n = 3). Con, not treated; TG, 250 n M thapsigargin; O300, 300 l M oleate; P300, 300 lM palmitate. *Significantly differ- ent from Con and O250. Significantly different from same treat- ment in the absence of adiponectin. A B Con GRP78 CHOP GRP78 CHOP * * 8 6 4 2 0 Beta actin TG TG + A P AP + A Con TG TG + A P AP + A Relative expressions (folds of control) Fig. 3. Adiponectin is unable to inhibit thapsigargin- and palmitate- induced ER stress markers (GRP78 and CHOP) in HepG2 cells. The effects of adiponectin on the expression of GRP78 and CHOP mRNA were measured by semiquantitative RT-PCR. Treatments were car- ried out for 24 h. These data were obtained from a total of three independent experiments or represent the mean ± SD (n = 3). Con, not treated; TG, 250 n M thapsigargin; P, 300 l M palmitate; A, 10 lgÆmL )1 adiponectin. *Significantly different from Con and A. Effects of adiponectin on hepatic apoptosis T. W. Jung et al. 2280 FEBS Journal 276 (2009) 2278–2284 ª 2009 The Authors Journal compilation ª 2009 FEBS effect of adiponectin on thapsigargin- and palmitate- induced phosphorylation of JNK in HepG2 cells. Thapsigargin and palmitate elevated JNK phosphory- lation and activity. As expected, adiponectin inhibited these inductions (Fig. 5). An elevation of plasma free fatty acids and fat accu- mulation in the liver are the cause of hepatic insulin resistance and liver disease [24–26]. Adiponectin induces fatty acid oxidation and insulin sensitivity [27]. Therefore, an adequate adiponectin signaling pathway in the liver may prove to be important in provoking apoptosis, which is a cause of hepatic inflammation and fibrosis. In the present study, we evaluated the ability of adiponectin to prevent palmitate-induced apoptosis in HepG2 cells. The results obtained demon- strate that adiponectin partially inhibits both palmi- tate- and thapsigargin-induced apoptosis via JNK phosphorylation. In the present study, adiponectin inhibits caspase-3 and cell death induced by thapsigargin and palmitate (Fig. 1). Interestingly, the addition of either wortman- nin or compound c in the presence of adiponectin pre- vented the effects of adiponectin (Fig. 2). These results suggest that adiponectin inhibits apoptotic cell death via AMPK and PI3 kinase activation. Adiponectin reduced palmitate- and thapsigargin- induced apoptosis, although it did not inhibit elevated ER stress markers, suggesting that adiponectin-medi- ated protection involves a pathway independent of ER stress markers (Fig. 3). A B Con Bcl-2 clAP 2 XIAP Bcl-2 clAP 2 XIAP Beta actin 2.0 1.5 1.0 0.0 0.5 TG TG + A P AP + A Con TG TG + A P AP + A Relative expressions (folds of control) Fig. 4. Thapsigargin, palmitate and adiponectin are unable to affect the expression of Bcl-2 and inhibitor of apoptosis family members in HepG2 cells. The effects of adiponectin on the expression of Bcl-2, cIAP 2 and XIAP mRNA were measured by semiquantitative RT-PCR. Treatments were carried out for 24 h. These data were obtained from a total of three independent experiments or repre- sent the mean ± SD (n = 3). Con, not treated; TG, 250 n M thapsi- gargin; P, 300 l M palmitate; A, 10 lgÆmL )1 adiponectin. A B Con P-JNK 54 kDa 46 kDa 54 kDa 46 kDa 42 kDa T-JNK Beta actin 6 5 4 3 JNK phosphorylation (folds of control) 2 1 0 5 4 3 JNK activity (folds of control) 2 1 0 TG TG + A P P + A Con TG TG + A P * * # # * * ! ! P + A Con TG TG + A P P + A Fig. 5. Adiponectin inhibits thapsigargin- and palmitate-induced JNK phosphorylation in HepG2 cells. (A) The effects of adiponectin on JNK phosphorylation were measured by western blot analysis. (B) The effects of adiponectin on enzymatic JNK activity were mea- sured using the JNK activity assay kit. Treatments were carried out for 24 h. These data were obtained from a total of three indepen- dent experiments or represent the mean ± SD (n = 4). There was no effect of sole adiponectin on JNK phosphorylation. Con, not treated; TG, 250 n M thapsigargin; P, 300 lM palmitate; A, 10 lgÆmL )1 adiponectin. *Significantly different from Con. Signifi- cantly different from TG. #Significantly different from P. T. W. Jung et al. Effects of adiponectin on hepatic apoptosis FEBS Journal 276 (2009) 2278–2284 ª 2009 The Authors Journal compilation ª 2009 FEBS 2281 Bcl-2, XIAP and the IAP family are related to caspase- dependent cell death [17]. However, thapsigargin and palmitate did not induce Bcl-2, XIAP and the IAP fam- ily. Moreover, adiponectin was also unable to influence their expression (Fig. 4). These results suggest that the adiponectin-mediated protective effects of thapsigargin- and palmitate-induced apoptosis occur independently of the expression of Bcl-2, XIAP and the IAP family. The mitogen-activated protein kinase family responds to a variety of stressors [28]. Especially, JNK is a criti- cal metabolic regulator and plays a role in lipoapoptosis in a variety of cell types, including hepatocytes [29]. In the present study, palmitate and thapsigargin induced JNK phosphorylation. Adiponectin inhibited palmitate- and thapsigargin-mediated JNK phosphorylation. These results coincide with the findings of a study per- formed in mouse hepatocyte and HepG2 cells in which free fatty acid-induced apoptosis was reported to be partially dependent on JNK [30]. The present data sug- gest that adiponectin-mediated protection from apopto- sis may involve a JNK-dependent pathway. In conclusion, the results obtained in the present study demonstrate that both the AMPK and PI3 kinase signaling pathways are critical factors for the protective effects of adiponectin with respect to palmi- tate- and thapsigargin-induced apoptosis via JNK phosphorylation in HepG2 cells. These data may be valuable for identifying adiponectin as a candidate for the treatment of NASH, which is characterized by abnormal hepatic apoptosis. Experimental procedures Culture media and reagents HepG2 cells were plated at a density of 2 · 10 5 cellsÆmL )1 and grown in DMEM medium supplemented with heat inactivated 10% (v ⁄ v) fetal bovine serum, 100 UÆmL )1 pen- icillin and 100 lgÆmL )1 streptomycin. Palmitate was pur- chased from Sigma (St Louis, MO, USA). Palmitate was conjugated to BSA at a 2 : 1 molar ratio [11]. Thapsigargin, which was used to chemically induce the misfolded or unfolded protein response and apoptosis, and wortmannin, a PI3 kinase inhibitor, were purchased from Sigma. AMPK inhibitor compound c was purchased from Calbiochem (San Diego, CA, USA). Human full-length adiponectin was purchased from BioVision (Mountain View, CA, USA). RNA extraction and analysis Total RNA was isolated using TRIzol according to the manufacture’s instructions (Invitrogen, Carlsbad, CA, USA). Primer sequences and their respective PCR fragment lengths were: CCAAT ⁄ enhancer-binding protein homolo- gous protein (CHOP), forward: 5¢-ATGAGGACCTGC AAGAGGTCC-3¢, reverse: 5¢-TCCTCCTCAGTCAGCCA AGC-3¢ (137 bp); glucose regulated protein 78, forward: 5¢-GTTCTTGCCGTTCAAGGTGG-3¢, reverse: 5¢-TGGTA CAGTAACAACTGCATG-3¢ (182 bp); b-actin, forward: 5¢-GAGACCTTCAACACCCCAGCC-3¢, reverse: 5¢-GGA TCTTCATGAGGTAGTCAG-3¢ (206 bp); Bcl-2, forward: 5¢-TTTTAGGAGACCGAAGTCCG-3¢, reverse: 5¢-AGCC AACGTGCCATGTGCTA-3¢ (392 bp); cIAP 2 , forward: 5¢-TTTATCCTAATTTGGTTTCC-3¢, reverse: 5¢-AATTCT TAAAGGTTAACTC-3¢ (253 bp); and XIAP, forward: 5¢-GAAGACCCTTGGGAACAGCA-3¢, reverse: 5¢-CGCC TTAGCTGCTCTTCAGT-3¢ (383 bp). Immunoblot analysis Cells were washed with NaCl ⁄ P i and harvested using lysis buffer contatining 20 mm Hepes (pH 7.4), 1% Triton X-100, 15% glycerol, 2 mm EGTA, 1 mm sodium vanadate, 2 mm dithiothreitol, 10 lm leupeptin and 5 lm pepstatin. Equiva- lent amounts of total extracts (20–30 lg) were loaded onto SDS ⁄ PAGE, transferred to Hybond-P membranes (Amer- sham Pharmacia Biotech, Piscataway, NJ, USA) and the membranes were incubated with antibodies against phosphor- ylated JNK (Cell Signaling Technology, Beverly, MA, USA), total JNK (Cell Signaling Technology) and b-actin (Sigma). Proteins were detected using horseradish peroxidase conju- gated secondary antibodies and reacted with ECL solution (Amersham Pharmacia Biotech). Signals were detected using horseradish peroxidase conjugated secondary antibodies and a chemoluminescence reagent (Pierce, Rockford, IL, USA). Determination of JNK activity Cell lysates were assayed for JNK phosphorylation using the Phospho-JNK DuoSet IC ELISA kit (R&D Systems, Minneapolis, MN, USA). Determination of caspase-3 activity and cell death Activity of the caspase-3 class of cysteine protease was deter- mined with the colorimetric activity assay (R&D Systems). Caspase-3 activity was normalized to the total extracted pro- tein concentration. After treatment, culture medium was removed and cells were incubated in NaCl ⁄ P i containing 2mgÆmL )1 of MTT. After 4 h of incubation at 37 °C, HepG2 cells were solubilized with dimethyl sulfoxyde. Statistical analysis Statistical comparisons were calculated using analysis of variance. P < 0.05 was considered statistically significant. All data are reported as the mean ± SD. Effects of adiponectin on hepatic apoptosis T. W. 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J Clin Invest 115, 1111–1119. 30 Malhi H, Bronk SF, Werneburg NW & Gores GJ (2006) Free fatty acids induce JNK-dependent hepato- cyte lipoapoptosis. J Bio Chem 17, 12093–12101. Effects of adiponectin on hepatic apoptosis T. W. Jung et al. 2284 FEBS Journal 276 (2009) 2278–2284 ª 2009 The Authors Journal compilation ª 2009 FEBS . Full-length adiponectin protects hepatocytes from palmitate-induced apoptosis via inhibition of c-Jun NH 2 terminal kinase Tae W. Jung 1 , Yong J. Lee 2 ,. increased c-Jun NH 2 terminal kinase phosphoryla- tion in the absence of adiponectin. Adiponectin blocked palmitate- and thapsigargin-induced activation of c-Jun NH 2 terminal kinase and reduced apoptosis. . AMP-activated protein kinase inhibitors abolished the effects of adiponectin. Adiponectin- induced inhibition of palmitate- and thapsigar- gin-induced apoptosis was not the result of an augmentation