Role of hepatocyte S6K1 in palmitic acid-induced endoplasmic reticulum stress, lipotoxicity, insulin resistance and in oleic acid-induced protection

Role of hepatocyte S6K1 in palmitic acid-induced endoplasmic reticulum stress, lipotoxicity, insulin resistance and in oleic acid-induced protection Virginia Pardo a,b, Águeda González-Rodríguez a,b,*,Jordi Muntané c,d, Sara C. Kozma e,f, Ángela M. Valverde a,b,** aInstituto de Investigaciones Biomédicas Alberto Sols (CSIC/UAM), 28029 Madrid, Spain bCentro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), ISCIII, Spain cOncology Surgery, Cell Therapy and Transplant Organs, Institute of Biomedicine of Seville (IBiS)/Virgen del Rocio Universitary Hospital/CSIC/University of Seville, Seville, Spain dCentro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Spain eDepartment of Cancer and Cell Biology, University of Cincinnati, Cincinnati, OH 45215, USA fBellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet, Barcelona, Spain ARTICLE INFO Article history: Received 3 November 2014 Accepted 29 March 2015 Available online 3 April 2015 Keywords: Lipotoxicity Insulin signaling Hepatocytes Free fatty acids ER stress Apoptosis ABSTRACT The excess of saturated free fatty acids, such as palmitic acid, that induces lipotoxicity in hepatocytes, has been implicated in the development of non-alcoholic fatty liver disease also associated with insulin resistance. By contrast, oleic acid, a monounsaturated fatty acid, attenuates the effects of palmitic acid. We evaluated whether palmitic acid is directly associated with both insulin resistance and lipoapoptosis in mouse and human hepatocytes and the impact of oleic acid in the molecular mechanisms that mediate both processes. In human and mouse hepatocytes palmitic acid at a lipotoxic concentration triggered early activation of endoplasmic reticulum (ER) stress-related kinases, induced the apoptotic transcription factor CHOP, activated caspase 3 and increased the percentage of apoptotic cells. These effects concurred with decreased IR/IRS1/Akt insulin pathway. Oleic acid suppressed the toxic effects of palmitic acid on ER stress activation, lipoapoptosis and insulin resistance. Besides, oleic acid suppressed palmitic acidinduced activation of S6K1. This protection was mimicked by pharmacological or genetic inhibition of S6K1 in hepatocytes. In conclusion, this is the first study highlighting the activation of S6K1 by palmitic acid as a common and novel mechanism by which its inhibition by oleic acid prevents ER stress, lipoapoptosis and insulin resistance in hepatocytes. © 2015 Elsevier Ltd. 1. Introduction Non-alcoholic fatty liver disease (NAFLD) is an emergent public health problem since it is the most common cause of chronic liver disease in Western countries. NAFLD is strongly associated with overweight/obesity, insulin resistance, type 2 diabetes (T2DM) and cardiovascular complications; therefore, it is considered the hepatic manifestation of the metabolic syndrome (Anstee et al., 2011; Malhi and Gores, 2008; Targher and Byrne, 2013). NAFLD is characterized by the accumulation of ectopic fat in the liver, initially triggering a benign steatosis. However, 10% of NAFLD patients develop more severe liver injuries directly associated with lipotoxicity such as nonalcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma (Ratziu and Poynard, 2006). In fact, elevated levels of circulating free fatty acids (FFAs) are linked to the progression of NAFLD and the pathogenesis of insulin resistance (Samuel et al., 2004; Savage et al., 2007). The excess of circulating FFAs is particularly toxic for the liver since saturated FFAs induce apoptosis in hepatocytes, a phenomenon known as lipoapoptosis (Zambo et al., 2013). Indeed, lipoapoptosis is increased in correlation with the severity of NAFLD (Feldstein et al., 2003; Garcia-Monzon et al., 2011). Different mechanisms contribute to hepatocyte dysfunction such as endoplasmic reticulum (ER) stress, oxidative stress and metabolic alterations (Cheung and Sanyal, 2010; Ozcan et al., 2004). Among them, ER stress is activated when the unfolded protein response (UPR) cannot re-establish the homeostasis in the ER. ER stress signaling involves three main proteins, X-box binding protein 1 * Corresponding author. Instituto de Investigaciones Biomédicas Alberto Sols (CSIC/UAM), C/Arturo Duperier 4, 28029 Madrid, Spain. Tel.: +34 91 5854497; fax: +34 91 5854401. E-mail address: [email protected] (Á. González-Rodríguez). ** Corresponding author. Instituto de Investigaciones Biomédicas Alberto Sols (CSIC/UAM), C/Arturo Duperier 4, 28029 Madrid, Spain. Tel.: +34 91 5854497; fax: +34 91 5854401. E-mail address: aval[email protected] (Á.M. Valverde). http://dx.doi.org/10.1016/j.fct.2015.03.029 0278-6915/© 2015 Elsevier Ltd. Food and Chemical Toxicology 80 (2015) 298–309 Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox This is an open access article under the CC BY license. This is an open access article under the CC BY license. (X-BP1), PRKR-like endoplasmic reticulum kinase (PERK) and activating transcription factor-6 (ATF6), which cooperate to alleviate ER stress. These proteins remain inactive while sensors are bound to the intraluminal chaperone glucose-regulated protein 78 (GRP78). If the cell fails to adapt, oxidative and inflammatory signaling pathways are triggered leading to apoptosis through the expression of PERK-mediated C/EBP homologous protein (CHOP), IRE1α-mediated recruitment of tumor necrosis factor receptor associated factor 2 (TRAF2) and apoptosis signal regulated kinase 1 (ASK1)/C-jun N terminal kinase (JNK) (Hotamisligil, 2010). In fact, the activation of JNK has been revealed as a central mediator of FFA-induced hepatic lipoapoptosis (Cazanave et al., 2009; Schattenberg et al., 2006). Activation of ER stress signaling induced by FFAs plays also an important role in the hepatic insulin resistance associated to the progression of NAFLD (Ozcan et al., 2004). It has been demonstrated that chemical chaperones (tauroursodeoxycholic acid (TUDCA) and 4-phenylbutryate), which mitigate ER stress through protein stabilization, improve systemic glucose homeostasis (Ozcan et al., 2006). Moreover, obese subjects treated with TUDCA showed increasedinsulin sensitivity in liver and skeletal muscle by approximately 30% compared to those receiving placebo therapies (Kars et al., 2010). Atamolecular level, FFAs can impair insulin action through the down-regulation of early mediators of the insulin signaling cascade such as the insulin receptor (IR) or insulin receptor substrate 1 (IRS1). In this regard, it has been demonstrated that ER stress induces IR down-regulation and dysfunction, and contributes to obesityinduced insulin resistance in peripheral tissues (Zhou et al., 2009). Particularly, palmitic acid (PA), a representative saturated FFA, inhibits insulin signaling acting on IR expression level in two welldifferentiated hepatocyte cell lines (Ruddock et al., 2008). Moreover, after saturated FFA challenge in hepatic cells, the activation of stress kinases such as JNK, ribosomal protein S6 kinase 1 (S6K1), IκB kinase (IKK) and also protein kinase C (PKC) increases serine/threonine phosphorylation of IRS1, reducing its tyrosine (stimulatory) phosphorylation and targets it for proteosomal degradation (Liu et al., 2011; Mordier and Iynedjian, 2007; Ravichandran et al., 2001; Solinas et al., 2006). The most abundant FFAs present in the diet and in serum are PA, a saturated FA, and oleic acid (OA), a monounsaturated one (Baylin et al., 2002). Some studies have demonstrated that monounsaturated FFAs are less toxic and attenuate PA-induced toxicity in hepatocytes (Malhi et al., 2006; Ricchi et al., 2009). Moreover, it has been demonstrated that OA improves insulin sensitivity in human diet treatments (Vessby et al., 2001). However, the molecular mechanism implicated in the protective role elicited in the liver by OA remains unclear and, therefore, the identification of further mechanisms of PA-induced lipotoxicity and whether OA can counteract these effects is needed. Taken together, in this study we have investigated the effects of PA and OA on lipotoxicity and insulin signaling in hepatocytes to elucidate new mechanisms involved. 2. Materials and methods 2.1. Reagents Fetal bovine serum (FBS) (#10270), culture media DMEM (#41966-029) and 4, 6-diamidino-2-phenylindole (DAPI) (D1306) were obtained from Invitrogen (Grand Island, NY). Palmitic acid (P0500), oleic acid (O1008), bovine serum albumin (BSA) (A6003), rapamycin (37094), tunicamycin (T7765), puromycin (8833), polybrene (S2667) and insulin (I0516) were from Sigma-Aldrich (St. Louis, MO). Bradford reagent, acrylamide, immunoblotting PVDF membrane and Immobilon Western Chemiluminescent HRP Substrate were purchased from Bio-Rad (Hercules, CA). 2.2. Cell culture Human hepatocytes were isolated by the two-step collagenase procedure from non-tumor areas of liver biopsies from patients submitted to a surgical resection for liver tumors after obtaining patients’ written consent (Pichard et al., 2006). The generation and characterization of immortalized mouse hepatocyte cell line has been previously described (Gonzalez-Rodriguez et al., 2009). Briefly, pooled (4–6) livers from wild-type and S6K1-deficient neonates (3.5–4 days old) in the C57BL/6 genetic background were digested and hepatocytes were isolated. All animal experimentation was conducted in accord with accepted standards of animal care. Viral Bosc- 23 packaging cells were transfected at 70% confluence with 3 μg/6 cm-dish of the puromycin-resistance retroviral vector pBabe encoding SV40 Large T antigen (provided by J. de Caprio, Dana Farber Cancer Institute, Boston, MA). Neonatal hepatocytes (60% confluence) were infected with polybrene (4 μg/ml)-supplemented virus and maintained in culture medium for 72 h before selection with puromycin (1 μg/ml) for 10–15 days. Pools of infected cells rather than individual clones were selected to avoid potential clone-to-clone variations. These pools of immortalized neonatal hepatocytes were further cultured with arginine-free medium supplemented with 10% FBS for at least 2 weeks to avoid growth of non-parenchymal cells. Cells were grown in DMEM plus 10% heat inactivated FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM glutamine. 2.3. Cell treatment Palmitic acid (PA), dissolved in isopropyl alcohol at a stock concentration of 80 mM, and oleic acid (OA), dissolved in chloroform at a stock concentration of 3540 mM, were added to DMEM containing 5% FBS and 1% bovine serum albumin to assure a physiological ratio between bound and unbound FFA in the medium (Richieri and Kleinfeld, 1995). The concentration of PA used in the experiments corresponds to the fasting PA plasma concentrations observed in human non-alcoholic steatohepatitis (Belfort et al., 2006; Sanyal et al., 2001) and is used to obtain hepatic lipoapoptosis (Cazanave et al., 2010). Confluent cells were treated with PA (200–800 μM) and/or OA (400 μM) for the indicated time periods. In all experiments, control cells were treated with the corresponding vehicle of each FFA used (chloroform 0.01% (v/v) for OA or isopropanol 0.75% (v/v) for PA). Vehicles used did not induce hepatotoxic effects and did not affect any of the parameters analyzed. 2.4. Preparation of protein extracts and Western blot Cell lysates were obtained by scraping off cultured dishes with ice cold lysis buffer (25 mM HEPES, 2.5 nM EDTA, 0.1% Triton X-100, 1 mM PMSF and 5 μg/ml leupeptin) followed by 10 min incubation on ice. After protein content determination with Bradford reagent, total protein were boiled in Laemmli sample buffer and submitted to 8–15% SDS-PAGE. Proteins were transferred to Immunoblot PVDF membrane and, after blocking with 3% BSA or 5% non-fat dry milk, membranes were incubated overnight with several antibodies as indicated. Immunoreactive bands were visualized using the ECL Western blotting protocol. Densitometric analysis of the bands was performed using Image J software. The anti-phospho-PERK (Thr 980) (#3179), antiphospho-eIF2α (Ser 51) (#9721), anti-phospho-JNK (#9251), anti-phospho-STAT3 (#9131), anti-phospho-ERK1/2 (#9101), anti-S6K1 (#9202), anti-active caspase 3 (#9661) and anti-ERK1/2 (#4695) antibodies were from Cell Signaling Technology (Danvers, MA). The anti-JNK (sc-571), anti-phospho-S6K1 (Thr 389) (sc-11759), anti- IRβ (sc-711), anti-PERK (sc-13073), anti-eIF2α (sc-11386), anti-GRP78 (sc-376768), anti-CHOP (sc-7351), anti-phospho-p38 (sc-17852), anti-p38 (sc-9212), anti-IRS1 (sc-559), anti-IRS2 (sc-8299), anti-phospho-IR (sc-25103), anti-phospho-Akt (Thr 308) (sc-16646), anti-phospho-Akt (Ser 473) (sc-7985) and anti-Akt (sc-8312) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-α-tubulin (T5168) antibody (A5441) was from Sigma-Aldrich. 2.5. Quantification of apoptosis Cells were grown on glass coverslips and treated as described above. Then, cells were washed twice with PBS, fixed in p-formaldehyde (4%) for 10 min and characteristic morphological changes of apoptosis were assessed by fluorescence microscopy after staining nuclei with DAPI. 2.6. Analysis of caspase 3 activity Cells were scraped off, collected by centrifugation at 2500 ×gfor 5 minutes and lysed at 4 °C in 5 mM Tris/HCl pH 8, 20 mM EDTA, 0.5% Triton X-100. Lysates were clarified by centrifugation at 13,000 ×gfor 10 min. Reaction mixture contained 25 μl cellular lysates, 325 μl assay buffer (20 mM HEPES pH 7.5, 10% glycerol, 2 mM dithiothreitol) and 20 μM caspase 3 substrate (Ac-DEVD-AMC). After 2 h incubation in the dark, enzymatic activity was measured in a luminescence spectrophotometer (Perkin Elmer LS-50, Norwalk, CT) (λexcitation, 380 nm; λemission, 440 nm). 2.7. Cell electron microscopy For electron microscopy (EM), mouse hepatocytes were grown in chamber slides and treated as described above. Then, cells were washed twice with PBS, fixed in a glutaraldehyde (2%) plus p-formaldehyde (4%) for 60 min. Samples were postfixed in 1% osmium tetroxide for 60 min at 25 °C, stained with uranyl acetate (5 mg/ ml) for 1 h at 25 °C, dehydrated in acetone and embedded in Epon 812 (EMbed 812; Electron Microscopy Science, Hatfield, PA). Ultra-thin sections, unstained or poststained with uranyl acetate and lead hydroxide, were examined under a Morgagni 299V. Pardo et al./Food and Chemical Toxicology 80 (2015) 298–309 268D transmission electron microscope (FEI, Hillsboro, OR) equipped with a Mega View II charge-coupled device camera (SIS, Soft Imaging System GmbH, Munster, Germany) and analyzed with AnalySIS software (SIS). 2.8. Statistical analysis Data are presented as mean ±SEM, and were compared by using the Bonferroni ANOVA test. All statistical analyses were performed using the IBM SPSS Statistics 21.0 (SPSS Inc. IBM, Armonk, NY) software with 2-sided tests. Differences were considered statistically significant at p <0.05. 3. Results 3.1. Palmitic acid induces lipotoxicity and insulin resistance in mouse and human hepatocytes In order to elucidate the molecular mechanisms involved in the toxic effects induced by FFAs in the liver, we assessed whether both primary human hepatocytes and immortalized mouse hepatocytes, generated in our laboratory from 3- to 5-day-old wild-type mice (Gonzalez- Rodriguez et al., 2009), are sensitive to the lipotoxic effects induced by palmitic acid (PA), a saturated FFA that is elevated in obesity (Bertola et al., 2010). We first treated primary human hepatocytes for 8 h with 600 μM PA, dose at which lipoapoptosis was previously reported (Cazanave et al., 2009). To test whether stress and inflammatory signaling pathways were triggered, we analyzed the phosphorylation of PERK, eIF2α and JNK, and found them enhanced in PA-treated- compared to vehicle-treated human primary hepatocytes (Fig. 1A). Following incubation for 24 h, PA treatment increased the expression of CHOP, an ER stress marker, and induced the activation of caspase 3, detected by the presence of the active fragment (Fig. 1B). Moreover, the analysis of nuclear morphology by fluorescence microscopy following DAPI staining showed an increase in the incidence of condensed and/or fragmented nuclei (Fig. 1C). Altogether, these results demonstrate that human primary hepatocytes are sensitive to lipotoxic doses of PA. Next, we assessed these responses in immortalized mouse hepatocytes treated with PA at increasing concentrations (200–800 μM). In these cells, maximal phosphorylation of JNK, induction of CHOP and activation of caspase 3 was detected at the lipotoxic PA concentration of 600 μM (Fig. 2A). We then characterized the PA-mediated lipoapoptosis in immortalized mouse hepatocytes at the molecular level since this cell line was not previously used for in vitro studies of FFA-mediated toxicity. These cells were highly sensitive to PA since we already detected the phosphorylation of kinases related to ER stress (PERK and eIF2α) and inflammation-linked stress kinases (STAT3, JNK, p38 MAPK and ERK) in hepatocytes treated with PA for only 6 h (Fig. 2B). Notably, activation of these kinases was not detected at earlier timeperiods (up to 4 h of treatment, results not shown). However, after 16 h PA triggered an elevation of CHOP expression and cleavage of caspase 3, which correlated with increased caspase 3 enzymatic activity (Fig. 2C and D). In this case, phase-contrast microscopy revealed the lipotoxic effects of PA since many hepatocytes showed morphological characteristics of apoptotic cells compared with control cells that did not receive PA (Fig. 2E). Activation of ER stress has been shown to interfere with the early steps of insulin signaling by multiple feed-back mechanisms (Ozcan et al., 2004). On that basis, we explored if the impact of PA on the insulin signaling cascade was similar in primary human hepatocytes and in our immortalized mouse cell line. For this goal, primary human and immortalized mouse hepatocytes were pretreated with 600 μM PA for 24 and 16 h, respectively, and then, stimulated with insulin for a further 10 min. As reported in Fig. 3, insulin-induced IR tyrosine phosphorylation was reduced in both kinds of hepatocytes pretreated with PA compared to hepatocytes treated with vehicle. Likewise, PA induced IR and IRS1 degradation and decreased Akt phosphorylation at both catalytic (Thr308) and regulatory (Ser473) phosphorylation sites. Notably, IRS2 levels were not affected after PA treatment. Altogether, these results indicate that immortalized neonatal hepatocytes are a suitable cellular model to study the responses to PA overload since they recapitulate all of the qualitative features of lipotoxicity and insulin resistance observed in primary human hepatocytes. Fig. 1. Palmitic acid (PA) induces ER stress and lipotoxicity in primary human hepatocytes. Primary human hepatocytes were treated with 600 μM PA for several time periods (A, 8 h; B and C, 24 h). (A and B) Total protein was analyzed by Western blot using the indicated antibodies. Representative blots are shown (n =3). (C) Representative nuclear morphology images after staining of DNA with DAPI. After quantification of apoptotic nuclei from all images, results are expressed as % of number of apoptotic nuclei and are mean ±SEM (n =3), ***p <0.001, PA vs C. 300 V. Pardo et al./Food and Chemical Toxicology 80 (2015) 298–309 3.2. Oleic acid prevents ER stress and lipotoxicity induced by palmitic acid in immortalized mouse hepatocytes It is well known that the two most abundant dietary fatty acids, oleic acid (OA) and PA, play a different role in hepatic metabolism (Mei et al., 2011; Noguchi et al., 2011; Ricchi et al., 2009). This prompted us to investigate whether OA was able to prevent PA- induced ER stress and the subsequent lipotoxicity. First, we evaluated the differential effects of OA and PA on ER stress. Of note, OA itself did not induce the activation of ER stress-mediated signaling in hepatocytes (Fig. 4A). Interestingly, co-treatment with 400 μM OA was sufficient to abolish PA-induced PERK and eIF2α phosphorylation and also significantly blocked JNK and p38 MAPK phosphorylation (Fig. 4A). Moreover, co-treatment of PA and OA prevented the increase of CHOP expression and the accumulation of the active fragment of caspase 3 (Fig. 4B). Next, we investigated the role of OA in PA-induced lipoapoptosis in hepatocytes. Phase-contrast microscopy revealed that OA did not elicit any effect on cell morphology. Moreover, after 16 h of co-treatment with PA and OA, most of the immortalized hepatocytes remained intact whereas as stated above, hepatocytes treated with PA alone displayed an apoptotic phenotype due to lipotoxicity (Fig. 4C). These results strongly suggest that OA elicits a protective effect against lipoapoptosis induced by PA. As ER undergoes dramatic changes after treatment with ER stress initiators, we analyzed ER morphology using transmission electron microscopy (EM). As expected, we observed an ER expansion in immortalized hepatocytes incubated with PA for 16 h. OA treatment did not affect ER morphology and, importantly, blocked PA- induced ER expansion (Fig. 4D). 3.3. Insulin resistance induced by palmitic acid is prevented by oleic acid in immortalized mouse hepatocytes Regarding insulin sensitivity, we investigated whether OA protects against the inhibitory effects of PA. As observed in Fig. 5A, OA alone did not affect insulin-induced IR and Akt phosphorylation in immortalized mouse hepatocytes compared to cells treated with vehicle. Next, Fig. 2. Palmitic acid (PA) triggers ER stress and lipotoxicity in immortalized mouse hepatocytes. (A) Immortalized mouse hepatocytes were treated with 200–800 μM PA for several time periods (left panel, 6 h; right panel, 16 h). Total protein was analyzed by Western blot using the indicated antibodies. Representative blots are shown (n =3). (B and C) Immortalized mouse hepatocytes were treated with 600 μM PA for several time periods (B, 6 h; C, 16 h). Total protein was analyzed by Western blot using the indicated antibodies. Representative blots are shown (n ≥4). (D) Analysis of caspase-3 enzymatic activity. Results are expressed as fold increase relative to C condition (1) and are mean ±SEM (n =3), ***p <0.001. (E) Representative nuclear morphology images after staining of DNA with DAPI. After quantification of apoptotic nuclei from all images, results are expressed as % of number of apoptotic nuclei and are mean ±SEM (n =3), ***p <0.001, PA vs C. 301V. Pardo et al./Food and Chemical Toxicology 80 (2015) 298–309 hepatocytes were incubated with PA in the presence or absence of OA before insulin stimulation. As depicted in Fig. 5B, both IR and Akt phosphorylation were recovered in hepatocytes incubated with PA plus OA as compared to cells treated exclusively with PA. In fact, co-treatment with PA and OA induced similar phosphorylation levels to those detected in control hepatocytes treated with vehicle. Moreover, after co-treatment with PA and OA, the expression of IR and IRS1 was maintained at similar levels of those detected in the control condition. Altogether, these results indicate that OA improved insulin signaling by relieving PA-induced ER stress. Fig. 3. Palmitic acid (PA) induces insulin resistance in both primary human and immortalized mouse hepatocytes. A. Primary human hepatocytes were treated with 600 μM PA for 24 h, followed by serum starvation (2 h) and insulin stimulation (10 min, 10 nM). Total protein was analyzed by Western blot using the indicated antibodies. Representative blots are shown (n =3). (B) Immortalized mouse hepatocytes were treated with 600 μM PA for 16 h, followed by serum starvation (2 h) and insulin stimulation (10 min, 10 nM). After quantification of all blots, results are expressed as % of insulin stimulation or % of protein expression relative to C condition (100%) and are mean ±SEM (n ≥6), **p <0.01, ***p <0.001, PA vs C. 302 V. Pardo et al./Food and Chemical Toxicology 80 (2015) 298–309 3.4. S6K1 inhibition reduces ER stress and lipotoxicity induced by palmitic acid in immortalized mouse hepatocytes In addition to stress kinases such as JNK and p38 MAPK, S6K1, an mTORC1 target, also modulates insulin signaling after saturated FFA challenge in hepatic cells by increasing serine phosphorylation of IRS1 and targeting it for proteosomal degradation (Mordier and Iynedjian, 2007). Interestingly, phosphorylation of S6K1 was detected in hepatocytes treated with PA for 6 h and this event was prevented in the presence of OA (Fig. 6A), suggesting that S6K1 might be involved in PA-induced lipotoxicity and insulin resistance in mouse hepatocytes. To delineate the role of S6K1 in hepatic lipoapoptosis, we used two different cellular models of S6K1 inhibition and checked levels Fig. 4. Oleic acid (OA) prevents ER stress and lipotoxicity induced by palmitic acid (PA) in immortalized mouse hepatocytes. Immortalized mouse hepatocytes were treated with 600 μM PA, 400 μM OA and 600 μM PA plus 400 μM OA for 6 h (A) and 16 h (B–D). (A and B) Total protein was analyzed by Western blot using the indicated antibodies. Representative blots are shown (n ≥3). (C) (upper panel) Representative phase-contrast microscopy images. (lower panel) Representative nuclear morphology images after staining of DNA with DAPI. After quantification of apoptotic nuclei from all DAPI staining images, results are expressed as % of number of apoptotic nuclei and are mean ±SEM (n ≥4), ***p <0.001, PA vs C; ###p<0.001, PA+OA vs PA. (D) Representative electron microscopy (EM) images are shown. Black arrows indicate expanded ER. Scale bar represents 1 μm. 303V. Pardo et al./Food and Chemical Toxicology 80 (2015) 298–309 of CHOP and active caspase 3 as markers of ER stress and apoptosis, respectively. Firstly, we treated immortalized mouse hepatocytes with PA combined with rapamycin, a well-known inhibitor of mTORC1/S6K1 signaling pathway. As it was expected, rapamycin inhibited S6K1 phosphorylation (Fig. 6B). Regarding lipotoxicity, PA treatment increased CHOP expression and the accumulation of the active fragment of caspase 3 in hepatocytes compared to the untreated controls. Co-treatment with PA and rapamycin (R, 25 ng/ml) significantly reduced these effects induced by PA. Moreover, the number of cells which displayed an apoptotic phenotype after PA treatment was significantly reduced and normal ER structure was observed in the presence of rapamycin (Fig. 6C and D). Next, we confirmed these data using an immortalized neonatal hepatocyte cell line with genetic ablation of S6K1 generated from livers of S6K1-deficient mice. We have previously investigated in this cellular model whether S6K1 deficiency alters the susceptibility of hepatocytes to undergo apoptosis induced by death receptor activation or trophic factors withdrawal (Gonzalez-Rodriguez et al., 2009). After 16 h of PA treatment, CHOP expression was reduced in S6K1- deficient hepatocytes compared to control cells. Moreover, cleavage of caspase 3 was increased in immortalized mouse hepatocytes after PA challenge, but not in those lacking S6K1 (Fig. 7A). Accordingly, S6K1 deficiency also reduced both apoptotic cells and the incidence of condensed and/or fragmented nuclei and avoided ER expansion after PA treatment (Fig. 7B and C). These data suggest that PA-induced ER stress and lipoapoptosis are suppressed in S6K1-deficient hepatocytes. In fact, S6K1 deficiency protects also against ER stress in response to tunicamycin, a classical ER stress-inducing agent. Fig. 7D shows that both CHOP and active caspase 3 were almost undetectable in S6K1- deficient hepatocytes after 16 h of tunicamycin (10 μg/ml) treatment compared to control cells. Taken together, these results indicate that Fig. 5. Insulin resistance induced by palmitic acid (PA) is prevented by oleic acid (OA) in immortalized mouse hepatocytes. Immortalized mouse hepatocytes were treated with 400 μM OA (A), with 600 μM PA and 600 μM PA plus 400 μM OA (B) for 16 h, followed by serum starvation (2 h) and insulin stimulation (10 min, 10 nM). Total protein was analyzed by Western blot using the indicated antibodies. Representative blots are shown. After quantification of all blots, results are expressed as % of insulin stimulation or % of protein expression relative to C condition (100%) and are mean ±SEM (n ≥3), **p <0.01, ***p <0.001, PA vs C; ##p<0.01, ###p<0.001, PA+OA vs PA. 304 V. Pardo et al./Food and Chemical Toxicology 80 (2015) 298–309 Fig. 6. PA-induced ER stress and lipoapoptosis are reduced in rapamycin-treated immortalized mouse hepatocytes. Immortalized mouse hepatocytes were treated with 400 μM OA, 600 μM PA and 400 μM OA plus 600 μM PA for 6 h (A) or co-treated with 600 μM PA and rapamycin (R, 25 ng/ml) for 16 h (B, C and D). (A and B) Total protein was analyzed by Western blot using the indicated antibodies. Representative blots are shown. After quantification of all blots, results are expressed as % of protein expression relative to C condition (100%) and are mean ±SEM (n =3). (C) (upper panel) Representative phase-contrast microscopy images. (lower panel) Representative nuclear morphology images after staining of DNA with DAPI. After quantification of apoptotic nuclei from all DAPI staining images, results are expressed as % of number of apoptotic nuclei and are mean ±SEM (n ≥4). (D) Representative electron microscopy (EM) images are shown. Black arrows indicate expanded ER. Scale bar represents 1 μm. ***p <0.001, PA vs C; #p<0.05, ##p<0.01, ###p<0.001, PA+RvsPA. 305V. Pardo et al./Food and Chemical Toxicology 80 (2015) 298–309 Fig. 7. S6K1 deficiency prevented PA-mediated ER stress and lipoapoptosis in immortalized mouse hepatocytes. Wild-type (WT) and S6K1-deficient (S6K1KO) immortalized mouse hepatocytes were treated with 600 μM PA (A, B and C) and with tunicamycin (T, 10 μg/ml) (D) for 16 h. (A and D) Total protein was analyzed by Western blot using the indicated antibodies. Representative blots are shown. After quantification of all blots, results are expressed as % of protein expression relative to the C condition (100%) and are mean ±SEM (n =3). (B) (upper panel) Representative phase-contrast microscopy images. (lower panel) Representative nuclear morphology images after staining of DNA with DAPI. After quantification of apoptotic nuclei from all DAPI staining images, results are expressed as % of number of apoptotic nuclei and are mean ±SEM (n ≥4). (C) Representative electron microscopy (EM) images are shown. Black arrows indicate expanded ER. Scale bar represents 1 μm. *p <0.05, **p <0.01, ***p <0.001, PA vsCandTvsC;#p<0.05, ##p<0.01, ###p<0.001, S6K1KO vs WT. 306 V. Pardo et al./Food and Chemical Toxicology 80 (2015) 298–309