- Open Access
Effect of α-linolenic acid on endoplasmic reticulum stress-mediated apoptosis of palmitic acid lipotoxicity in primary rat hepatocytes
© Zhang et al; licensee BioMed Central Ltd. 2011
Received: 8 June 2011
Accepted: 25 July 2011
Published: 25 July 2011
Hepatic inflammation and degeneration induced by lipid depositions may be the major cause of nonalcoholic fatty liver disease (NAFLD). In this study, we investigated the effects of saturated and unsaturated fatty acids (FA) on apoptosis in primary rat hepatocytes.
The primary rat hepatocytes were treated with palmitic acid and/or α-linolenic acid in vitro. The expression of proteins associated with endoplasmic reticulum (ER) stress, apoptosis, caspase-3 levels were detected after the treatment.
The treatment with palmitic acid produced a significant increase in cell death. The unfolded protein response (UPR)-associated genes CHOP, GRP78, and GRP94 were induced to higher expression levels by palmitic acid. Co-treatment with α-linolenic acid reversed the apoptotic effect and levels of all three indicators of ER stress exerted by palmitic acid. Tunicamycin, which induces ER stress produced similar effects to those obtained using palmitic acid; its effects were also reversed by α-linolenic acid.
α-Linolenic acid may provide a useful strategy to avoid the lipotoxicity of dietary palmitic acid and nutrient overload accompanied with obesity and NAFLD.
Nonalcoholic fatty liver disease (NAFLD) is a multifactorial disease [1, 2] that illustrates a variety of symptoms, ranging from mild steatosis, nonalcoholic steatohepatitis to cirrhosis in the liver. Although it affects millions of people all over the world, the etiology of NAFLD is still unknown. However, hepatic inflammation and degeneration induced by the deposition of lipid droplets in the organ is considered as one of the major reasons of the disease [1–3]. In particular, certain saturated fatty acids (FA) such as palmitic acid can induce endoplasmic reticulum (ER) stress and apoptosis in rat and human liver cell lines [4–8], leading to inflammation and/or degeneration in the liver. This hypothesis is further demonstrated by the fact that ER stress and apoptosis could be induced by palmitic acid in both primary cells and cell lines derived from mice and rats [9, 10].
Different fatty acids have different effects on ER stress. Palmitic acid is an effective inducer of ER stress . Palmitic acid stimulates the synthesis of ceramides and increases reactive oxygen species , either of which may induce ER stress [12–14]. Palmitate modulates intracellular signaling, induces endoplasmic reticulum stress, and leads to apoptosis in mouse 3T3-L1 and rat primary preadipocytes .
We hypothesized that the cytoprotection provided by α-linolenic acid was a common function of rat primary hepatocytes and would be effective with clinically-relevant palmitic acid lipotoxicity. We have previously proved that α-linolenic acid protects against endoplasmic reticulum stress-mediated apoptosis of stearic acid lipotoxicity . In this paper, we report that: (1) The characteristics of palmitic acid-mediated ER stress and apoptosis in primary hepatocytes; (2) α-linolenic acid could provide protection against the cell death induced by palmitic acid; (3) Take the role of GRP78, GRP94 expression and induction of CHOP into consideration, the beneficial effects were mediated via modification of the ER stress process with specific attention.
2. Materials and methods
2.1 Materials and cells
Rat hepatocytes were isolated from newborn (1-day-old) Sprague-Dawley rats using the modified two-step collagenase perfusion technique as previously described . Freshly prepared hepatocytes were seeded at a density of 2 × 105 cells/well on 24-well multidishes precoated with rat tail collagen type I and grown in Williams Medium E containing 5% of fetal calf serum, 100 nM insulin, 2.5 μg/ml amphotericin B, 0.1 mg/ml gentamicin, 30 nM Na2SeO3, and 0.1 μM dexamethasone (Sigma-Aldrich, St. Louis, MO). Calf serum and amphotericin B were present for the first 24 h then omitted.
Cell culture materials and routine chemicals were obtained from Sigma (Oakville, ON, Canada) or Fischer Scientific (Nepean, ON, Canada). Primary antibodies were obtained from Stressgen (Victoria, ON, Canada) unless otherwise stated.
The experimental protocols were approved by the Animal Care and Protection Committee of Xi'an Jiaotong University.
2.2. Incubation of primary hepatocytes
Cultured hepatocytes at 80-90% confluence, were incubated with palmitic acid (250 μmol/l) for up to 16 h. Hepatocytes were also incubated with tunicamycin (5 μg/ml). Further incubations were also performed in which hepatocytes were incubated with palmitic acid (250 μmol/l) in the absence or presence of α-linolenic acid (150 or 250 μmol/l) for up to 16 h.
2.3. Measurement of cell viability and death
Cell viability and death were assessed as described previously by measurement of the enzymatic conversion of the yellow tetrazolium salt 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) into purple formazan and the release of lactate dehydrogenase (LDH) from lysed cells, respectively . Primary rat hepatocytes were stained with Hoechst 33342-propidium iodide (HPI) to assess cell death by apoptosis and necrosis, respectively . Specifically, apoptotic cells were distinguished as those with characteristic nuclear fragmentation and intense staining of condensed chromatin. Propidium iodide does not enter cells with intact plasma membranes, however, after entering damaged apoptotic or non-apoptotic cells it stains nuclear DNA pink. One thousand, randomly distributed nuclei were counted per sample and were scored as morphologically normal, apoptotic and necrotic using an inverted fluorescence microscope (Axiovert 25, Zeiss) set at excitation and emission wavelengths of 365 and 397 nm, respectively.
2.4. Measurement of caspase-3 activation
Caspase-3 activity was evaluated using a DEVD-NucViewTM 488 Caspase-3 substrate kit (Biotium Inc., Cambridge, UK). In the presence of active caspase-3 enzyme, the substrate dissociates from its bound fluorogenic DNA-binding dye and the latter binds to DNA and emits fluorescence. Caspase-3 was detected by microscopic examination and also by adapting the kit for microplate fluorescence reading. For this, cells were incubated with 50 μL of 5 μmol/L DEVD-NucView™ 488 Caspase-3 substrate for 30 min. Fluorescence was measured in a microplate reader (Cary Eclipse, Varian Inc.) set at wavelengths of 490 nm excitation and 520 nm emission.
2.5. Western blot analysis
Western blot analysis was performed as described in detail previously . Membranes were incubated with antibodies against glucose-regulated protein 78 (GRP78; Stressgen, Victoria BC, Canada), glucose-regulated protein 94 (GRP94; Stressgen, Victoria BC, Canada), CCAAT/enhancer-binding protein homologous protein (CHOP; Santa Cruz Biotechnology, Santa Cruz, CA), and actin (Sigma). Proteins were detected with horseradish peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biotech, Piscataway, NJ) and an enhanced chemiluminescence reagent (Pierce, Rockford, IL). Density was quantified using a UVP Bioimaging system (Upland, CA).
2.6. Statistical analysis
Results are expressed as mean ± standard error of the mean (S.E.M.) for n independent observations as indicated. Statistical differences between mean values of groups have been determined using one way analysis of variance (ANOVA) followed by a Dunnett's post-significance test for comparison of multiple means using the SPSS version 11.5. The level of significance was set at P < 0.05.
3.1. Palmitic acid causes obvious cellular death of hepatocytes - protection by α-linolenic acid
Palmitic-mediated apoptosis of primary rat hepatocytes coincided with a significant increase in caspase-3 activation (Figure 1C). Tunicamycin as well as palmitic acid increased fluorescence in the caspase-3 assay confirming activation of apoptosis pathways (Figure 1C).
3.2. α-Linolenic acid reduces ER stress mediated by palmitic acid
In the presence of α-linolenic acid, ER stress mediated by palmitic acid was significantly reduced. Co-incubation of hepatocytes with 250 μmol/l palmitic acid and 250 μmol/l α-linolenic acid produced a significant reduction in levels of GRP78, GRP94 and CHOP after 16 h (Figure 2).
3.3. Effects of α-linolenic acid on primary rat hepatocytes death mediated by tunicamycin
3.4. Effects of α-linolenic acid on ER stress induced by tunicamycin
The present study was undertaken to elucidate the hypotheses that palmitic acid would induce ER stress and then cell death in liver cells, and that α-linolenic acid would inhibit these outcomes. Our study has three main findings. First, we have shown that the effects of a saturated and an unsaturated fatty acid, singly or in combination, upon induction of cell death, cell apoptosis, caspase-3 activity, ER stress in primary rat hepatocytes. Secondly, we have also illustrated the protein expression of three ER stress-associated genes in response to fatty acids treatment. The delivery and accumulation of lipids in non-adipose tissues leads to cellular dysfunction and death. This phenomenon, termed lipotoxicity, has been demonstrated in the pathogenesis of diabetes, cardiac failure and NAFLD [20–22]. Studies has found that damage of ER homeostasis and activation of the UPR in murine models of cardiac dysfunction, obesity and NAFLD [20, 23, 24].
Increased long chain saturated fatty acids induce ER stress, activate the UPR and promote cell death in a great amount of cell types, such as hepatocytes [26, 27]. Thus, disruption in ER function seems lead to the pathogenesis of some diseases and to cellular impairments coupled with lipotoxicity. This was supported by our observations. Previously, we found that palmitic acid causes obvious cell death in primary rat hepatocytes. To elucidate the underlying mechanism of these effects, we found that the palmitic acid causes an obvious degree of ER stress in primary rat hepatocytes. Our results demonstrated that the ER stress response contributes to palmitic acid lipotoxicity. In addition, the studies showed that α-linolenic acid protects primary rat hepatocytes against palmitic acid lipotoxicity via reducing ER stress and apoptosis. Comparing with our previous study about stearic acid, we prove that 150 μmol/l α-linolenic acid provides very little benefits, which was presumed as the result of palmitic acid's stronger ability to elicited ER stress . Furthermore, α-linolenic acid can reduce cellular dysfunction and apoptosis caused by tunicamycin. Tunicamycin, a well-known ER stress inducer, leads to apoptosis in a number of cells, including intestinal epithelial cells, renal cells and liver cells [28–30]. The mechanism on the basis of the cell necrosis induced by tunicamycin has not been clarified. These observations, together with our findings, suggest that α-linolenic acid protect primary rat hepatocytes through alleviation of tunicamycin-induced apoptosis.
The previous study sought to determine whether the ER stress and cell death in hepatocytes contributed to cell death in hepatocytes and to establish a link between these two . Chop is among the best characterized of the UPR-regulated proapoptotic proteins . Chop plays a role in disruption of ER homeostasis and apoptosis induced by long-chain saturated fatty acids. In several cell types, including liver, co-supplementation of oleate and palmitate reduces palmitate-mediated ER stress and apoptosis [33, 34]. For this purpose, the ability of long chain saturated fatty acids to activate the ER-associated caspase-3 was examined.
Our work sheds new light on the mechanisms behind that ER stress produced by palmitic acid in primary rat hepatocytes can be considerably reduced by α-linolenic acid, an unsaturated fatty acid. We also manifest that an unsaturated fatty acid involves a reduction of ER stress which result in protection. Moreover, we examined the effects of a reduction in the raised levels of caspase-3 and CHOP coupled with palmitic acid. However, the results of our investigation seems to exclude a protective mechanism regulated by GRP78 as α-linolenic acid did not significantly affect levels of this chaperone molecule whereas levels of CHOP were substantialy reduced. In short, we concluded that α-linolenic acid may provide a useful strategy to avoid the lipotoxicity of dietary palmitic acid and nutrient overload accompanied with obesity and NAFLD.
We thank laboratory of Tissue Engineering, the Fourth Military Medical University, for guidance of primary cell culture.
- Solis Herruzo JA, Garcia Ruiz I, Perez Carreras M, Munoz Yague MT: Non-alcoholic fatty liver disease. From insulin resistance to mitochondrial dysfunction. Rev Esp Enferm Dig. 2006, 98 (11): 844-874.View ArticlePubMedGoogle Scholar
- Byrne CD: Fatty liver: role of inflammation and fatty acid nutrition. Prostaglandins Leukot Essent Fatty Acids. 2010, 82 (4-6): 265-271. 10.1016/j.plefa.2010.02.012View ArticlePubMedGoogle Scholar
- Straub BK, Schirmacher P: Pathology and biopsy assessment of non-alcoholic fatty liver disease. Dig Dis. 2010, 28 (1): 197-202. 10.1159/000282086View ArticlePubMedGoogle Scholar
- Wei Y, Wang D, Topczewski F, Pagliassotti MJ: Saturated fatty acids induce endoplasmic reticulum stress and apoptosis independently of ceramide in liver cells. Am J Physiol Endocrinol Metab. 2006, 291 (2): E275-281. 10.1152/ajpendo.00644.2005View ArticlePubMedGoogle Scholar
- Wei Y, Wang D, Pagliassotti MJ: Saturated fatty acid-mediated endoplasmic reticulum stress and apoptosis are augmented by trans-10, cis-12-conjugated linoleic acid in liver cells. Mol Cell Biochem. 2007, 303 (1-2): 105-113. 10.1007/s11010-007-9461-2View ArticlePubMedGoogle Scholar
- Wei Y, Wang D, Gentile CL, Pagliassotti MJ: Reduced endoplasmic reticulum luminal calcium links saturated fatty acid-mediated endoplasmic reticulum stress and cell death in liver cells. Mol Cell Biochem. 2009, 331 (1-2): 31-40. 10.1007/s11010-009-0142-1PubMed CentralView ArticlePubMedGoogle Scholar
- Kim DS, Jeong SK, Kim HR, Chae SW, Chae HJ: Metformin regulates palmitate-induced apoptosis and ER stress response in HepG2 liver cells. Immunopharmacol Immunotoxicol. 2010, 32 (2): 251-257. 10.3109/08923970903252220View ArticlePubMedGoogle Scholar
- Pfaffenbach KT, Gentile CL, Nivala AM, Wang D, Wei Y, Pagliassotti MJ: Linking endoplasmic reticulum stress to cell death in hepatocytes: roles of C/EBP homologous protein and chemical chaperones in palmitate-mediated cell death. Am J Physiol Endocrinol Metab. 2010, 298 (5): E1027-1035. 10.1152/ajpendo.00642.2009PubMed CentralView ArticlePubMedGoogle Scholar
- Guo W, Wong S, Xie W, Lei T, Luo Z: Palmitate modulates intracellular signaling, induces endoplasmic reticulum stress, and causes apoptosis in mouse 3T3-L1 and rat primary preadipocytes. Am J Physiol Endocrinol Metab. 2007, 293 (2): E576-586. 10.1152/ajpendo.00523.2006View ArticlePubMedGoogle Scholar
- Ricchi M, Odoardi MR, Carulli L, Anzivino C, Ballestri S, Pinetti A, Fantoni LI, Marra F, Bertolotti M, Banni S: Differential effect of oleic and palmitic acid on lipid accumulation and apoptosis in cultured hepatocytes. J Gastroenterol Hepatol. 2009, 24 (5): 830-840. 10.1111/j.1440-1746.2008.05733.xView ArticlePubMedGoogle Scholar
- Ota T, Gayet C, Ginsberg HN: Inhibition of apolipoprotein B100 secretion by lipid-induced hepatic endoplasmic reticulum stress in rodents. J Clin Invest. 2008, 118 (1): 316-332. 10.1172/JCI32752PubMed CentralView ArticlePubMedGoogle Scholar
- Listenberger LL, Ory DS, Schaffer JE: Palmitate-induced apoptosis can occur through a ceramide-independent pathway. J Biol Chem. 2001, 276 (18): 14890-14895. 10.1074/jbc.M010286200View ArticlePubMedGoogle Scholar
- Brookheart RT, Michel CI, Listenberger LL, Ory DS, Schaffer JE: The non-coding RNA gadd7 is a regulator of lipid-induced oxidative and endoplasmic reticulum stress. J Biol Chem. 2009, 284 (12): 7446-7454. 10.1074/jbc.M806209200PubMed CentralView ArticlePubMedGoogle Scholar
- Boslem E, MacIntosh G, Preston AM, Bartley C, Busch AK, Fuller M, Laybutt DR, Meikle PJ, Biden TJ: A lipidomic screen of palmitate-treated MIN6 beta-cells links sphingolipid metabolites with endoplasmic reticulum (ER) stress and impaired protein trafficking. Biochem J. 2011, 435 (1): 267-276. 10.1042/BJ20101867View ArticlePubMedGoogle Scholar
- Zhang Y, Dong L, Yang X, Shi H, Zhang L: alpha-Linolenic acid prevents endoplasmic reticulum stress-mediated apoptosis of stearic acid lipotoxicity on primary rat hepatocytes. Lipids Health Dis. 2011, 10 (1): 81- 10.1186/1476-511X-10-81PubMed CentralView ArticlePubMedGoogle Scholar
- Seglen PO: Preparation of isolated rat liver cells. Methods Cell Biol. 1976, 13: 29-83.View ArticlePubMedGoogle Scholar
- Samai M, Sharpe MA, Gard PR, Chatterjee PK: Comparison of the effects of the superoxide dismutase mimetics EUK-134 and tempol on paraquat-induced nephrotoxicity. Free Radic Biol Med. 2007, 43 (4): 528-534. 10.1016/j.freeradbiomed.2007.05.014View ArticlePubMedGoogle Scholar
- Beeharry N, Lowe JE, Hernandez AR, Chambers JA, Fucassi F, Cragg PJ, Green MH, Green IC: Linoleic acid and antioxidants protect against DNA damage and apoptosis induced by palmitic acid. Mutat Res. 2003, 530 (1-2): 27-33.View ArticlePubMedGoogle Scholar
- Pagliassotti MJ, Kang J, Thresher JS, Sung CK, Bizeau ME: Elevated basal PI 3-kinase activity and reduced insulin signaling in sucrose-induced hepatic insulin resistance. Am J Physiol Endocrinol Metab. 2002, 282 (1): E170-176.PubMedGoogle Scholar
- Ozcan U, Cao Q, Yilmaz E, Lee AH, Iwakoshi NN, Ozdelen E, Tuncman G, Gorgun C, Glimcher LH, Hotamisligil GS: Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes. Science. 2004, 306 (5695): 457-461. 10.1126/science.1103160View ArticlePubMedGoogle Scholar
- Borradaile NM, Han X, Harp JD, Gale SE, Ory DS, Schaffer JE: Disruption of endoplasmic reticulum structure and integrity in lipotoxic cell death. J Lipid Res. 2006, 47 (12): 2726-2737. 10.1194/jlr.M600299-JLR200View ArticlePubMedGoogle Scholar
- Clark JM, Diehl AM: Nonalcoholic fatty liver disease: an underrecognized cause of cryptogenic cirrhosis. JAMA. 2003, 289 (22): 3000-3004. 10.1001/jama.289.22.3000View ArticlePubMedGoogle Scholar
- Wang D, Wei Y, Pagliassotti MJ: Saturated fatty acids promote endoplasmic reticulum stress and liver injury in rats with hepatic steatosis. Endocrinology. 2006, 147 (2): 943-951.View ArticlePubMedGoogle Scholar
- Podolin DA, Sutherland E, Iwahashi M, Simon FR, Pagliassotti MJ: A high-sucrose diet alters the lipid composition and fluidity of liver sinusoidal membranes. Horm Metab Res. 1998, 30 (4): 195-199. 10.1055/s-2007-978865View ArticlePubMedGoogle Scholar
- Kharroubi I, Ladriere L, Cardozo AK, Dogusan Z, Cnop M, Eizirik DL: Free fatty acids and cytokines induce pancreatic beta-cell apoptosis by different mechanisms: role of nuclear factor-kappaB and endoplasmic reticulum stress. Endocrinology. 2004, 145 (11): 5087-5096. 10.1210/en.2004-0478View ArticlePubMedGoogle Scholar
- Moffitt JH, Fielding BA, Evershed R, Berstan R, Currie JM, Clark A: Adverse physicochemical properties of tripalmitin in beta cells lead to morphological changes and lipotoxicity in vitro. Diabetologia. 2005, 48 (9): 1819-1829. 10.1007/s00125-005-1861-9View ArticlePubMedGoogle Scholar
- Lai E, Bikopoulos G, Wheeler MB, Rozakis-Adcock M, Volchuk A: Differential activation of ER stress and apoptosis in response to chronically elevated free fatty acids in pancreatic beta-cells. Am J Physiol Endocrinol Metab. 2008, 294 (3): E540-550. 10.1152/ajpendo.00478.2007View ArticlePubMedGoogle Scholar
- Berger E, Haller D: Structure-function analysis of the tertiary bile acid TUDCA for the resolution of endoplasmic reticulum stress in intestinal epithelial cells. Biochem Biophys Res Commun. 2011, 409 (4): 610-615. 10.1016/j.bbrc.2011.05.043View ArticlePubMedGoogle Scholar
- Katsoulieris E, Mabley JG, Samai M, Green IC, Chatterjee PK: alpha-Linolenic acid protects renal cells against palmitic acid lipotoxicity via inhibition of endoplasmic reticulum stress. Eur J Pharmacol. 2009, 623 (1-3): 107-112. 10.1016/j.ejphar.2009.09.015View ArticlePubMedGoogle Scholar
- Chang YS, Tsai CT, Huangfu CA, Huang WY, Lei HY, Lin CF, Su IJ, Chang WT, Wu PH, Chen YT: ACSL3 and GSK-3beta are essential for lipid upregulation induced by endoplasmic reticulum stress in liver cells. J Cell Biochem. 2011, 112 (3): 881-893. 10.1002/jcb.22996View ArticlePubMedGoogle Scholar
- Malhi H, Bronk SF, Werneburg NW, Gores GJ: Free fatty acids induce JNK-dependent hepatocyte lipoapoptosis. J Biol Chem. 2006, 281 (17): 12093-12101. 10.1074/jbc.M510660200View ArticlePubMedGoogle Scholar
- Oyadomari S, Mori M: Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ. 2004, 11 (4): 381-389. 10.1038/sj.cdd.4401373View ArticlePubMedGoogle Scholar
- Listenberger LL, Han X, Lewis SE, Cases S, Farese RV, Ory DS, Schaffer JE: Triglyceride accumulation protects against fatty acid-induced lipotoxicity. Proc Natl Acad Sci USA. 2003, 100 (6): 3077-3082. 10.1073/pnas.0630588100PubMed CentralView ArticlePubMedGoogle Scholar
- Busch AK, Gurisik E, Cordery DV, Sudlow M, Denyer GS, Laybutt DR, Hughes WE, Biden TJ: Increased fatty acid desaturation and enhanced expression of stearoyl coenzyme A desaturase protects pancreatic beta-cells from lipoapoptosis. Diabetes. 2005, 54 (10): 2917-2924. 10.2337/diabetes.54.10.2917View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.