- Email: [email protected]
Saturated free fatty acid, palmitic acid, induces apoptosis in fetal hepatocytes in culture
ARTICLE IN PRESS
Experimental and Toxicologic Pathology 56 (2005) 369–376
EXPERIMENTAL ANDTOXICOLOGIC PATHOLOGY www.elsevier.de/etp
Saturated free fatty acid, palmitic acid, induces apoptosis in fetal hepatocytes in culture Jun Jia, Li Zhanga,, Ping Wangb, Yi-Ming Mub, Xiao-Yu Zhua, Yuan-Yuan Wua, Huan Yua, Bin Zhanga, Shu-Min Chen, Xi-Zuo Suna a
Department of Central Laboratory, Dalian Municipal Central Hospital, Xuegong Street 42, Shahekou-qu, Dalian 116033, China Department of Endocrinology, Chinese PLA General Hospital, Beijing 100853, China
b
Received 11 November 2004; accepted 15 February 2005
Abstract To investigate the effects of saturated free fatty acid, palmitic acid (PA), on hepatocytes, we administered PA to rat hepatocytes. We demonstrated that PA inhibited the cell growth as a dose- and time-dependent manner in rat hepatocytes. PA-induced morphological changes including swelling, membrane dissolution and formation of debris, and apoptosis with appearance of sub-G1 fraction determined by cell cycle analysis after treatment for 4 days. The level of Bcl-2 was slightly decreased, in contrast, the level of Bax elevated markedly, which resulted in a significant decrease of Bcl-2/Bax ratio after PA treatment on HepG2 cells. These findings demonstrated that PA induces cell death on hepatocytes, perhaps via mitochondria-mediated apoptosis. Furthermore, the present study indicates that PA’s cell toxicity may play important roles in the transition from steatosis to steatohepatitis in human especially with obesity. r 2005 Elsevier GmbH. All rights reserved. Keywords: Palmatic acid; Hepatocytes; Cell viability; Cell toxicity; Apoptosis
Introduction Long chain free fatty acids (FFAs) play an important role in the cellular biological functions. They serve as a source of metabolic energy, as the substrates for cell membrane biogenesis (glyco- and phospholipid), and as precursors of many intracellular signaling molecules (Bergstrom, 1967). On the other hand, FFAs are often elevated in the patients with obesity and type 2 diabetes mellitus, and the accumulation of FFAs is reported as a common reason causing fatty liver. Fatty liver is the earliest, and the most common, form of both alcoholic fatty liver disease (AFLD) and Corresponding author. Tel./fax: +86 411 84441341.
E-mail address: [email protected] (L. Zhang). 0940-2993/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2005.02.003
nonalcoholic fatty liver disease (NAFLD). Inflammation and hepatocyte death (i.e., hepatitis) are inconspicuous in fatty livers and, hence, the prognosis of hepatic steatosis is generally benign. However, fatty livers are unusually vulnerable to injury from various causes, and when hepatitis develops, the probability of eventual, liver-related morbidity and mortality increase dramatically (Matteoni et al., 1999). Thus, the transition from steatosis to steatohepatitis is an important, rate-limiting step in the progression of both AFLD and NAFLD. To clarify the mechanisms involved in this process, some unsaturated FFAs, such as arachidonic acid (AA), linoleic acid (LA) and linolenic acid, have been reported to play a key role in injures of fatty liver through directly cytotoxicity. Furthermore, liver injury requires the proinflammatory cytokine tumor necrosis factor-alpha
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J. Ji et al. / Experimental and Toxicologic Pathology 56 (2005) 369–376
(TNF-a) (Mayeux, 1997). TNF-a kills cells by activating caspases that cause apoptosis (Heller and Kronke, 1994). However, healthy hepatocytes normally are not killed by TNF-a, because when they are exposed to TNF-a, they activate antiapoptotic transcription factors, such as nuclear factor-kappa-B (NF-kB) (Antwerp et al., 1996). NF-kB, in turn, up regulates the synthesis of antiapoptotic members of the Bcl-2 family that prevent the release of cytochrome c from mitochondria. Although the mechanism of the transition from steatosis of liver to steatohepatitis is involved in a lot of events in human, that unsaturated FFAs induce the injury of hepatocytes suggests FFAs may play an important role in the transition from steatosis of liver to steatohepatitis. However, the effect of saturated FFAs on human hepatocytes is largely unknown. The aim of the present investigation was to determine the effect of palmitic acid (PA), a common kind of saturated FFAs, on cell growth inhibition and cell toxicity in rat hepatocytes. In addition, the cell apoptosis in HepG2 cells induced by PA was also examined clarify its mechanism in fetal hepatocytes in culture.
Materials and methods
governing the use of animals for research. For a cell survival and toxicity assay, the cells were plated onto a 24-well plate at about 1 104 cells/well 24 h before treatment. The cells were treated with various FFAs, whereas control cells were incubated in medium with carrier (0.8 mN NaOH). After washing with PBS buffer, the cells were trypsinized and cell number of each well was counted on a hemocytometer under a microscope. All experiments were performed on triplicate wells and repeated at least three times.
Morphological observation by fluorescence microscopy Extent of cell killing via apoptosis was assessed by fluorescence microscopy, using the nuclear fluorophore Hoechst 33258 (Ho258) and a Leica inverted microscope (DM IRB, Leica, German). Rat hepatocytes were cultured in a 24-well plate for 24 h and then were examined after different periods of treatment with PA. Thirty minutes before viewing, the cells were treated with 5 mM Ho258. Images were acquired using 365–395 nm excitation, an emission range of 435–485 nm and a Spot-2 cooled CCD digital camera (DC300F, Leica).
Chemicals Cell cycle analysis by flow cytometry PA, stearic acid (SA), LA and AA, and Annexin VEGFP/PI Apoptosis Detection Kit were purchased from Sigma (St. Louis, MO). PA was dissolved in 0.1 N NaOH solution, and the final concentration of NaOH in the cell culture medium was 0.08 mN. Antibodies including antihuman Bcl-2 and Bax used for flowcytometric analysis were purchased from Beckman Dickinson (Coulter Corp., Miami, FL, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY).
Culture of fetal hepatocytes and cell toxicity assay Hepatocytes from 20-day-old fetal Wistar rats were isolated by collagenase disruption following the method described by Roncero et al. (Leevy, 1962), and plated in DMEM on culture dishes, supplemented with 10% FBS, streptomycin (100 mg/ml) and penicillin (100 mg/ml) under an atmosphere of 90% air and 10% CO2 at 37 1C. Cells were incubated in 5% CO2 at 37 1C for 24 h to allow attachment. The medium was changed at that time and replaced by the same DMEM with 10% FBS. All procedures were approved by the Animal Care Committee of the Dalian Medical Collage and conformed to European Union Animal Care Regulations
Cells cultured with or without PA were analyzed by flow cytometry at 0, 24, 48 and 72 h, respectively, after the beginning of the treatment. Floating and trypsinized adherent cells were collected and adjusted to 2 106 cells. The cells were washed with PBS for three times and fixed in 70% ethanol for 4 h at room temperature. After fixation, the cells were washed with PBS again, and incubated in phosphate-citrate buffer for 30 min, and then treated with RNase A for 30 min at room temperature. Finally, the cells were stained with PI (50 mg/ml). After 1 h of incubation, cells were analyzed on a Beckman EPICS XL (Coulter Corp., Miami, FL).
Detection of apoptosis by flow cytometry Cells were plated and treated as described above and then were stained with biotin-conjugated Annexin V, FITC-conjugated streptavidin and PI using the Annexin V-Biotin Apoptotic Detection Kit according to the manufacturer’s protocol. Apoptotic cells were subsequently counted by flow cytometry, and the data were analyzed with the Winlist Software (Variety Software House, Top Sham, ME).
ARTICLE IN PRESS J. Ji et al. / Experimental and Toxicologic Pathology 56 (2005) 369–376
Determination of the levels of Bcl-2 and Bax by flow cytometry
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Results PA induce a dose-dependent reduction in hepatocytes cell survival
To quantify the amounts of Bcl-2 and Bax, flow cytometry analysis was performed in HepG2 cells. Cells were treated similarly by the method as described above. The samples contained 50 ml cell suspension (1 106 cells) were incubated for 15 min at 18–25 1C in the dark with 100 ml of intraPrep reagent 1 (Fixation, IntraPrep Permeabization Regent, Immunotech, Bechman Coulter). After washed with PBS, 100 ml of intraPrep reagent 2 (Permeabization, IntraPrep Permeabization Regent, Immunotech, Bechman Coulter) was added to the samples. After incubating for 5 min at room temperature and gently agitating for 1–2 s, 20 ml of intracellular unconjugated specific antibody was added to the assay tubes. After incubation for 15 min at room temperature, the samples were mixed with recommended volume of conjugated secondary antibody and incubated for 15 min at room temperature in the dark. Then the samples were washed with PBS again, and resuspended in 500 ml of PBS containing 0.5% formaldehyde and proceed to flow cytometry analysis (Tsujimoto et al., 1985).
To determine the effects of various FFAs on rat hepatocytes cell survival, we investigated the cell viability of rat hepatocytes analyzed by Trypan Blue exclusion, using a hemocytometer. The cells were treated with or without FFAs for 2 days, and then the cell viability was determined. As shown in Fig. 1, saturated FFA including PA (C16:0) and SA (C18:0) induced a dose-dependent reduction in cell survival at 200–800 mM. PA and SA seemed to be similar in the potency to reduce the cell survival. The cell survival rates were 12% and 17% after exposure to 800 mM of PA and SA, respectively, for 2 days. Unsaturated FFA LA (C18:2) had little effect on the cell survival at 50–400 mM, while AA (C20:4) instead showed a stimulatory effect on cell proliferation at concentrations of 5 and 10 mM, respectively. The cell survival rate was 124% of the control after treatment with 10 mM AA for 2 days. On the other hand, the control cells treated with 0.08 mN NaOH had no effect on cell viability compared with the cells incubated in medium without NaOH.
Data analysis
Time course of the effects of PA on rat hepatocytes cell survival
For the cell survival assay, experiments were performed in triplicate, and the results were expressed as the mean7SD from three or four independent experiments. Statistical significance was analyzed with the Mann–Whitney t-test by Statview software. po0:05 was considered to be statistically significant.
Time course of the effects of FFAs on the cell proliferation was assessed by Trypan Blue exclusion after the treatment with various FFAs for 4 days.
140
** *
120
Cell survival (%)
100 80
**
**
60
**
**
40
**
20
**
0
Control
200 400 600 800
200 400 600 800
PA µM
SA µM
50
100 200 400
LA µM
1
2
5
10
AA µM
Fig. 1. Effects of FFAs on hepatocytes cell survival. The cultured rat hepatocytes were exposed to various concentrations of FFAs as indicated for 2 days and then the cell viability was determined by Trypan Blue staining as described in Materials and methods. The data represent the mean7SD of three independent experiments with triplicate wells. *po0.05 vs. control cells; **po0.01 vs. control cells. PA: palmitic acid; SA: stearic acid; LA: linoleic acid; AA: arachidonic acid.
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160
Control LA 200 µmol/L
PA 400 µmol/L
SA 400 µmol/L
AA 10 µmol/L
Cell survival rate (%)
140 120 100 80 60 40 20 0
24
48
72
96
Time (h)
Fig. 2. Time-dependent effects of FFAs on cell survival. Rat hepatocytes were exposed to various FFAs as indicated for 96 h and then the cell viability was determined by Trypan Blue staining as described in Materials and methods. The data represent the mean7SD of three independent experiments with triplicate wells. po0.05 vs. control cells; po0.01 vs. control cells. PA: palmitic acid; SA: stearic acid; LA: linoleic acid; AA: arachidonic acid.
Fig. 3. Morphological change of cells treated with PA. After incubating the hepatocytes with various concentrations of PA (A, control; B, PA 100 mM; C, 200 mM; D, 400 mM) for 4 days, as indicated, the cells were stained by Hoechst as described in Materials and methods. The cells were then observed by a fluorescence microscope. Number of apoptotic cells with nuclear fragmentation appeared increasingly as arising of concentration of PA (100–400 mM) and indicated by the arrows. Magnification, 400.
As shown in Fig. 2, 400 mM of PA and SA induced a time-dependent decline of cell proliferation. The cell survival rates were 30% and 24% after exposure to 400 mM of PA and SA, respectively, for 4 days. LA had little effect at 200 mM after treatment for 4 days. The stimulatory effect of AA seamed to be also timedependent, because the cell survival increased dramatically as the time of treatment increased from 1 to 4 days. The cell viabilities were 130% and 134% in the cells exposed to 10 mM for 3 and 4 days, respectively.
Morphological change of cells treated with PA After incubating the rat hepatocytes with various concentrations of PA, the cells were stained by Hoechst as described in Materials and methods, and were then observed by a microscope. As shown in Fig. 3, at 4 days after treatment with PA, the cells showed a damage including swelling, membrane dissolution and formation of debris, some cells showed nuclear fragmentation, which are the
ARTICLE IN PRESS J. Ji et al. / Experimental and Toxicologic Pathology 56 (2005) 369–376
manifestation of apoptosis. These damage effects were aggravated as arising of concentration of PA from 100 to 400 mM.
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Observation of appearance of sub-G1 cells after treatment with PA Cell cycle progression of rat hepatocytes exposure to 200 mM PA was shown in Fig. 4. Fragmented DNA was clearly revealed as sub-G1 fraction at 48 h (7.6%) after the treatment of 200 mM PA, and increased to 13.5% at 72 h. The increase in sub-G1 fraction by time course was compatible to the results of increasing dead cells determined by Trypan Blue and morphological evidence.
Observation of early apoptosis induced by PA
Fig. 4. Cell cycle analysis of hepatocytes treated with PA. Hepatocytes cultured in 9 in dishes were exposed to 400 mM of PA for 0, 24, 48 and 72 h, respectively, and then were harvested and analyzed by flow cytometry as described in Materials and methods.
In an attempt to characterize PA-induced cell death, all cells including adherent cells and floating cells in the medium of each well were collected, and then were labeled with Annexin V and PI for the flow cytometric analysis (Fig. 5). Phosphatidylserine externalization is a characteristic of cells undergoing apoptosis. Annexin V has a strong affinity for phosphatidylserine. Staining cells simultaneously with Annexin V and PI allows the resolution of intact cells (double-negative), early apoptotic cells (Annexin V-positive and PI-negative), and late apoptotic and necrotic cells (double-positive), which can be located in the lower left, upper left and upper right quadrants of the cytograms, respectively. Because only cells with Annexin V-positive and PI-negative are truly representative of apoptotic cells, the percentage of this
Fig. 5. Quantitation of apoptotic cells by flow cytometric analysis. Hepatocytes were exposed to 400 mM for 24, 48, 72, 96, 120 and 144 h, respectively, and then were harvested and labeled with Annexin V and PI. Intact cells, early apoptotic cells and late apoptotic or necrotic cells are located in the lower left, upper left and upper right quadrants of the cytograms, respectively.
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Table 1.
J. Ji et al. / Experimental and Toxicologic Pathology 56 (2005) 369–376
Percentage of cells stained with Annexin V and PI treated with PA 400 mM
%
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Annexin V-positive and PI-negative cells Double-positive cells Total dead cells
8.70 1.49 10.19
8.82 3.85 12.67
11.03 9.01 20.04
12.20 23.54 35.74
13.91 52.53 66.44
22.37 57.82 80.19
cell population was quantitated for time course, as shown in Table 1. It is clear that PA was able to induce progressive apoptosis in a time-dependent manner, at the concentration of 400 mM.
PA induced the decrease in Bcl-2:Bax ratio To determine whether PA-induced apoptosis in hepatocytes is related to the suppression of an apoptotic suppressor, Bcl-2, or the induction of an apoptotic effector, Bax, we measured the expression level of Bcl-2 and Bax in HepG2 cells, either treated or not treated with PA, by a flow cytometric analysis. As shown in Table 2, PA treatment resulted in a down-regulation of Bcl-2 (po0:05) and a dramatic up-regulation of Bax (po0:01). The ratio of Bcl-2 and Bax is critical for regulating the release of cytochrome c from mitochondria. Therefore, we calculated the Bcl-2:Bax ratio and found it was significantly decreased after the treatment of PA at 200 and 400 mM concentrations (po0:001) for 4 days, suggesting the apoptosis induced by PA might have occurred through mitochondriamediated pathway.
Discussion The presence of fatty liver in patients with obesity and type 2 diabetes mellitus has long been reported (Stone and Thiel, 1985). It is usually considered an incidental pathologic finding, with scarce or no clinical significance. Only recently did Ludwig et al. (1980) identify a syndrome characterized by the association of fatty liver and lobular hepatitis and chronically elevated alanine aminotransferase plasma levels in patients with negligible alcohol intake. The syndrome is mainly associated with obesity, diabetes and dyslipidemia, but a few patients are lean, and have normal fasting glucose and glucose tolerance, and show no evidence of increased plasma lipids. Patients with fatty liver and hepatitis are identified as having nonalcoholic steatohepatitis (NASH). They are part of the broad spectrum of NAFLDs, which also include pure steatosis. The limits between NAFLD and NASH are set only by liver histology and cannot be predicted on clinical or laboratory grounds (Matteoni et al., 1999).
Table 2. Expression (%) of Bcl-2 and Bax on HepG2 cells treated with PAa %
Control
PA 200 mM
PA 200 mM
Bcl-2 Bax Bcl-2/Bax
0.5570.002 0.2970.001 1.9070.067
0.4970.011b 0.5670.008c 0.8870.089d
0.4870.009b 0.6370.012c 0.7670.072d
a
The expression of Bcl-2 and Bax was determined 4 days after treatment with PA. b po0.05 vs. control. c po0.01 vs. control. d po0.001 vs. control.
From a clinical point of view, a few patients have ongoing liver injury: 50% of NASH patients develop liver fibrosis, 15% develop cirrhosis and 3% may progress to terminal liver failure, requiring liver transplantation. In 15–50% of cases, liver fibrosis or cirrhosis may also be diagnosed at presentation (Falchuk et al., 1980). The main reason of hepatitis and liver fibrosis or cirrhosis on obesity was reported to be directly injury to liver cells by a lot of content of unsaturated FFAs, such as AA. The effects in human liver cells of saturated FFAs, acting as a main content of FFAs on obesity from either diet or endogenous, have not been reported. Because saturated FFAs, such as PAand SA-induced apoptosis in human granulosa cells (Mu et al., 2001) and rat testicular Leydig cells (Lu et al., 2003); thus, the primary goal of the present study was to investigate the toxicity of PA in human liver cells. The nonfasting serum levels of PA, SA, LA and AA in patients with hyperlipidemia were reported to be 140.9777.7, 109770.0, 45.9744.0 and 0.871.3 mM, respectively (Mitropoulos et al., 1997). In our study, PA and SA induced a cell growth inhibition in hepatocytes, and promoted a significant cell death by a dose- and time-dependent manner at the doses of PA and SA ranging from 200 to 800 mM. Especially, as a mimic hyperlipidemia condition of 200–400 mM, PA induced more than 50% cell death within 5 days after the treatment, and a typical morphological change of apoptosis with fragmented nuclear was observed at 4 days. The results of present study indicate that PA at a 3–4-fold of normal plasma level (Mitropoulos et al., 1997) with a long period can significantly inhibits the cell survival and induces cell death of hepatocytes
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possibly via apoptosis. Unsaturated FFA LA had little effect on the cell survival at a 1–8-fold of normal plasma level within 2 days, while AA presented a dosedependent stimulatory effect on the cell proliferation at 1–10-fold of normal plasma level. These results may clinically suggested that not only the circulating levels of FFAs, but also the composition of FFAs, may play an important role in the damage of hepatocytes induced by hyperlipidemia. On the other hand, ethanol and AA were shown to be toxic and cause apoptosis in rat hepatocytes and HepG2 cells which express CYP2E1 through increasing release of cytochrome c into the cytosol fraction, and activation of p38MAPK and caspase 3 but not in control rat hepatocytes and HepG2 cell lines. This discrepancy between our study and the previous reports could be related to different doses of AA, which more than 30 mM were used in their experiments, and whether overexpressing CYP2E1. FFA-induced apoptosis has been reported in the hepatocytes (Wu and Cederbaum, 2003). However, most of the research was about the unsaturated FFAs, and there have been very few investigations about that saturated FFAs induce injury of hepatocytes. Here, we investigated the effect of PA on hepatocytes, and confirmed that PA-induced apoptosis in hepatocytes. It has been reported that FFAs-induced apoptosis is not only a direct effect of FFAs but also an indirect effect through enhancement to lipid peroxidation of mitochondria (Diehl, 1999). Mitochondrial function has been implicated as a critical early regulator of apoptosis in many cells including hepatocytes. Bcl-2, a mitochondrial protein, inhibits the apoptotic process and promotes cell survival (Hockenbery et al., 1990). While Bax, also a mitochondrial protein and a member of Bcl2 family, contributes to mitochondria-mediated apoptotic pathway. Although it appears that both Bcl-2 and Bax can regulate apoptosis independently, there also seems to be a competition that exists between the two. Bcl-2 is a 25 kDa protein that has extensive amino acid homology with Bax. It forms homodimers or heterodimers with Bax and the apoptotic activity depends on the balance of both molecules. And the ratio of the two, Bcl-2/Bax, regulates the release of cytochrome c which proceeds the mitochondrial apoptotic pathway (Micheldes, 1999). However, in human hepatocytes, the mechanism of PA-induced apoptosis has not been understood. But, it is increasingly recognized a variety of key events in apoptosis converge on mitochondria, and that mitochondrial release of cytochrome c plays a central role in triggering apoptosis (Takehara et al., 2001). Antiapoptotic members of the Bcl-2 family, such as Bcl-2 and Bcl-xl, are mainly located on the outer membrane of mitochondria and inhibit a common pathway of apoptosis, at least in part, by preventing the release of cytochrome c into cytosol (Takehara et al., 2001). And it was reported that PA decreased Bcl-2
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expression and induced release of cytochrome c from the mitochondria into the cytosol in Human Pancreatic bCells (Maedler et al., 2003). Here, we found a slight decrease in mitochondrial Bcl-2 and a markedly increase in mitochondrial level of Bax in the HepG2 cells treated with PA from 200 to 400 mM concentrations. This resulted in a significant decrease in Bcl-2/Bax ratio (po0:001), and suggested that PA would advance apoptosis through mitochondria-mediated apoptotic pathway. In summary, this study investigated, for the first time, that saturated FFA, such as PA, induced apoptosis in hepatocytes, perhaps not only by the toxicity of PA but also through mitochondria-mediated apoptosis pathway by regulating Bcl-2/Bax ratio. These effects of PA on hepatocytes proliferation and cell killing may therefore be a possible mechanism for both AFLD and NAFLD, and especially may play an important role in the transition from steatosis to steatohepatitis in human.
Acknowledgments The authors would like to thank Dr. Li P and Dr. Zhang QH for their technical assistance.
References Antwerp DJV, Martin SJ, Kafri TD, Green R, Verma IM. Suppression of TNF-alpha-induced apoptosis by NFkappa-B. Science 1996;274:787–9. Bergstrom S. Prostaglandins: members of a new hormonal system. These physiologically very potent compounds of ubiquitous occurrence are formed from essential fatty acid. Science 1967;157:382–91. Diehl AM. Nonalcholic steatohepatitis. Semin Liver Dis 1999; 19:221–9. Falchuk KR, Fiske SC, Haggitt RC, Federman R. Trey C: pericentral hepatic fibrosis and intracellular hyalin in diabetes mellitus. Gastroenterology 1980;78:535–41. Heller RA, Kronke M. Tumor necrosis factor receptormediated signaling pathways. J Cell Biol 1994;126:5–9. Hockenbery D, Nunez G, Milliman C, Schreiber RD, Korsmeyer SJ. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 1990;348:334–6. Leevy CM. Fatty liver: a study of 270 patients with biopsy proven fatty liver and review of the literature. Medicine 1962;41:249–76. Lu ZH, Mu YM, Li BA, Wang XL, Lu JM, Li JY. Saturated free fatty acids, palmitic acid and stearic acid, induce apoptosis by stimulation of ceramide generatin in rat testicular Leydig cell. Biochem Biophys Res Commun 2003; 303:1002–7. Ludwig J, Viaggiano TR, McGill DB, Oh BJ. Nonalcoholic steatohepatitis: Mayo clinic experience with a hitherto unnamed disease. Mayo Clin Proc 1980;55:434–8.
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Maedler K, Oberholzer J, Bucher P, Giatgen Spinas A, Donath YM. Monounsaturated fatty acids prevent the deleterious effects of palmitate and high glucose on human pancreatic b-cell turnover and function. Diabetes 2003;52: 726–33. Matteoni C, Younossi ZM, McCullough A. Nonalcoholic fatty liver disease: a spectrum of clinical pathological severity. Gastroenterology 1999;116:1413–9. Matteoni CA, Younossi ZM, Gramlich T, Boparai N, Liu YC, McCullough AJ. Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity. Gastroenterology 1999;116:1413–9. Mayeux PR. Pathobiology of lipopolysaccharide. J Toxicol Environ Health 1997;51:415–35. Micheldes RJ. Cell cycle regulators: mechanisms and their role in antiology, prognosis and treatment of cancer. J Clin Pathol 1999;52:555–68. Mitropoulos KA, Armitage JM, Collins R, Meade TW, Reeves BE, Wallendszus KR, et al. Randomized placebo
controlled study of the effects of simvastatin on haemostatic variables, lipoproteins and free fatty acids. The Oxford Cholesterol Study Group. Eur Heart J 1997;18: 235–41. Mu YM, Yanase TH, Nishi YH, Tanaka A, Saito M, Jin CH. Saturated FFAs, palmitic acid and steaeic acid, induce apoptosis in human granulosa cells. Endocrinology 2001; 142:3590–7. Stone BG, Thiel DHV. Diabetes mellitus and the liver. Semin Liver Dis 1985;5:8–28. Takehara T, Liu X, Fujimoto J, Friedman SL, Takahashi H. Expression and role of Bcl-xL in human hepatocellular carcinomas. Hepatology 2001;34:55–61. Tsujimoto Y, Cossman J, Jaffe E, Croce CM. Involvement of the Bcl-2 gene in human follicular lymphoma. Science 1985;228:1440–3. Wu D, Cederbaum AI. Role of p38 MAPK in CYP2E1dependent arachidonic acid toxicity. J Biol Chem 2003; 278:1115–24.
Experimental and Toxicologic Pathology 56 (2005) 369–376
EXPERIMENTAL ANDTOXICOLOGIC PATHOLOGY www.elsevier.de/etp
Saturated free fatty acid, palmitic acid, induces apoptosis in fetal hepatocytes in culture Jun Jia, Li Zhanga,, Ping Wangb, Yi-Ming Mub, Xiao-Yu Zhua, Yuan-Yuan Wua, Huan Yua, Bin Zhanga, Shu-Min Chen, Xi-Zuo Suna a
Department of Central Laboratory, Dalian Municipal Central Hospital, Xuegong Street 42, Shahekou-qu, Dalian 116033, China Department of Endocrinology, Chinese PLA General Hospital, Beijing 100853, China
b
Received 11 November 2004; accepted 15 February 2005
Abstract To investigate the effects of saturated free fatty acid, palmitic acid (PA), on hepatocytes, we administered PA to rat hepatocytes. We demonstrated that PA inhibited the cell growth as a dose- and time-dependent manner in rat hepatocytes. PA-induced morphological changes including swelling, membrane dissolution and formation of debris, and apoptosis with appearance of sub-G1 fraction determined by cell cycle analysis after treatment for 4 days. The level of Bcl-2 was slightly decreased, in contrast, the level of Bax elevated markedly, which resulted in a significant decrease of Bcl-2/Bax ratio after PA treatment on HepG2 cells. These findings demonstrated that PA induces cell death on hepatocytes, perhaps via mitochondria-mediated apoptosis. Furthermore, the present study indicates that PA’s cell toxicity may play important roles in the transition from steatosis to steatohepatitis in human especially with obesity. r 2005 Elsevier GmbH. All rights reserved. Keywords: Palmatic acid; Hepatocytes; Cell viability; Cell toxicity; Apoptosis
Introduction Long chain free fatty acids (FFAs) play an important role in the cellular biological functions. They serve as a source of metabolic energy, as the substrates for cell membrane biogenesis (glyco- and phospholipid), and as precursors of many intracellular signaling molecules (Bergstrom, 1967). On the other hand, FFAs are often elevated in the patients with obesity and type 2 diabetes mellitus, and the accumulation of FFAs is reported as a common reason causing fatty liver. Fatty liver is the earliest, and the most common, form of both alcoholic fatty liver disease (AFLD) and Corresponding author. Tel./fax: +86 411 84441341.
E-mail address: [email protected] (L. Zhang). 0940-2993/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2005.02.003
nonalcoholic fatty liver disease (NAFLD). Inflammation and hepatocyte death (i.e., hepatitis) are inconspicuous in fatty livers and, hence, the prognosis of hepatic steatosis is generally benign. However, fatty livers are unusually vulnerable to injury from various causes, and when hepatitis develops, the probability of eventual, liver-related morbidity and mortality increase dramatically (Matteoni et al., 1999). Thus, the transition from steatosis to steatohepatitis is an important, rate-limiting step in the progression of both AFLD and NAFLD. To clarify the mechanisms involved in this process, some unsaturated FFAs, such as arachidonic acid (AA), linoleic acid (LA) and linolenic acid, have been reported to play a key role in injures of fatty liver through directly cytotoxicity. Furthermore, liver injury requires the proinflammatory cytokine tumor necrosis factor-alpha
ARTICLE IN PRESS 370
J. Ji et al. / Experimental and Toxicologic Pathology 56 (2005) 369–376
(TNF-a) (Mayeux, 1997). TNF-a kills cells by activating caspases that cause apoptosis (Heller and Kronke, 1994). However, healthy hepatocytes normally are not killed by TNF-a, because when they are exposed to TNF-a, they activate antiapoptotic transcription factors, such as nuclear factor-kappa-B (NF-kB) (Antwerp et al., 1996). NF-kB, in turn, up regulates the synthesis of antiapoptotic members of the Bcl-2 family that prevent the release of cytochrome c from mitochondria. Although the mechanism of the transition from steatosis of liver to steatohepatitis is involved in a lot of events in human, that unsaturated FFAs induce the injury of hepatocytes suggests FFAs may play an important role in the transition from steatosis of liver to steatohepatitis. However, the effect of saturated FFAs on human hepatocytes is largely unknown. The aim of the present investigation was to determine the effect of palmitic acid (PA), a common kind of saturated FFAs, on cell growth inhibition and cell toxicity in rat hepatocytes. In addition, the cell apoptosis in HepG2 cells induced by PA was also examined clarify its mechanism in fetal hepatocytes in culture.
Materials and methods
governing the use of animals for research. For a cell survival and toxicity assay, the cells were plated onto a 24-well plate at about 1 104 cells/well 24 h before treatment. The cells were treated with various FFAs, whereas control cells were incubated in medium with carrier (0.8 mN NaOH). After washing with PBS buffer, the cells were trypsinized and cell number of each well was counted on a hemocytometer under a microscope. All experiments were performed on triplicate wells and repeated at least three times.
Morphological observation by fluorescence microscopy Extent of cell killing via apoptosis was assessed by fluorescence microscopy, using the nuclear fluorophore Hoechst 33258 (Ho258) and a Leica inverted microscope (DM IRB, Leica, German). Rat hepatocytes were cultured in a 24-well plate for 24 h and then were examined after different periods of treatment with PA. Thirty minutes before viewing, the cells were treated with 5 mM Ho258. Images were acquired using 365–395 nm excitation, an emission range of 435–485 nm and a Spot-2 cooled CCD digital camera (DC300F, Leica).
Chemicals Cell cycle analysis by flow cytometry PA, stearic acid (SA), LA and AA, and Annexin VEGFP/PI Apoptosis Detection Kit were purchased from Sigma (St. Louis, MO). PA was dissolved in 0.1 N NaOH solution, and the final concentration of NaOH in the cell culture medium was 0.08 mN. Antibodies including antihuman Bcl-2 and Bax used for flowcytometric analysis were purchased from Beckman Dickinson (Coulter Corp., Miami, FL, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY).
Culture of fetal hepatocytes and cell toxicity assay Hepatocytes from 20-day-old fetal Wistar rats were isolated by collagenase disruption following the method described by Roncero et al. (Leevy, 1962), and plated in DMEM on culture dishes, supplemented with 10% FBS, streptomycin (100 mg/ml) and penicillin (100 mg/ml) under an atmosphere of 90% air and 10% CO2 at 37 1C. Cells were incubated in 5% CO2 at 37 1C for 24 h to allow attachment. The medium was changed at that time and replaced by the same DMEM with 10% FBS. All procedures were approved by the Animal Care Committee of the Dalian Medical Collage and conformed to European Union Animal Care Regulations
Cells cultured with or without PA were analyzed by flow cytometry at 0, 24, 48 and 72 h, respectively, after the beginning of the treatment. Floating and trypsinized adherent cells were collected and adjusted to 2 106 cells. The cells were washed with PBS for three times and fixed in 70% ethanol for 4 h at room temperature. After fixation, the cells were washed with PBS again, and incubated in phosphate-citrate buffer for 30 min, and then treated with RNase A for 30 min at room temperature. Finally, the cells were stained with PI (50 mg/ml). After 1 h of incubation, cells were analyzed on a Beckman EPICS XL (Coulter Corp., Miami, FL).
Detection of apoptosis by flow cytometry Cells were plated and treated as described above and then were stained with biotin-conjugated Annexin V, FITC-conjugated streptavidin and PI using the Annexin V-Biotin Apoptotic Detection Kit according to the manufacturer’s protocol. Apoptotic cells were subsequently counted by flow cytometry, and the data were analyzed with the Winlist Software (Variety Software House, Top Sham, ME).
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Determination of the levels of Bcl-2 and Bax by flow cytometry
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Results PA induce a dose-dependent reduction in hepatocytes cell survival
To quantify the amounts of Bcl-2 and Bax, flow cytometry analysis was performed in HepG2 cells. Cells were treated similarly by the method as described above. The samples contained 50 ml cell suspension (1 106 cells) were incubated for 15 min at 18–25 1C in the dark with 100 ml of intraPrep reagent 1 (Fixation, IntraPrep Permeabization Regent, Immunotech, Bechman Coulter). After washed with PBS, 100 ml of intraPrep reagent 2 (Permeabization, IntraPrep Permeabization Regent, Immunotech, Bechman Coulter) was added to the samples. After incubating for 5 min at room temperature and gently agitating for 1–2 s, 20 ml of intracellular unconjugated specific antibody was added to the assay tubes. After incubation for 15 min at room temperature, the samples were mixed with recommended volume of conjugated secondary antibody and incubated for 15 min at room temperature in the dark. Then the samples were washed with PBS again, and resuspended in 500 ml of PBS containing 0.5% formaldehyde and proceed to flow cytometry analysis (Tsujimoto et al., 1985).
To determine the effects of various FFAs on rat hepatocytes cell survival, we investigated the cell viability of rat hepatocytes analyzed by Trypan Blue exclusion, using a hemocytometer. The cells were treated with or without FFAs for 2 days, and then the cell viability was determined. As shown in Fig. 1, saturated FFA including PA (C16:0) and SA (C18:0) induced a dose-dependent reduction in cell survival at 200–800 mM. PA and SA seemed to be similar in the potency to reduce the cell survival. The cell survival rates were 12% and 17% after exposure to 800 mM of PA and SA, respectively, for 2 days. Unsaturated FFA LA (C18:2) had little effect on the cell survival at 50–400 mM, while AA (C20:4) instead showed a stimulatory effect on cell proliferation at concentrations of 5 and 10 mM, respectively. The cell survival rate was 124% of the control after treatment with 10 mM AA for 2 days. On the other hand, the control cells treated with 0.08 mN NaOH had no effect on cell viability compared with the cells incubated in medium without NaOH.
Data analysis
Time course of the effects of PA on rat hepatocytes cell survival
For the cell survival assay, experiments were performed in triplicate, and the results were expressed as the mean7SD from three or four independent experiments. Statistical significance was analyzed with the Mann–Whitney t-test by Statview software. po0:05 was considered to be statistically significant.
Time course of the effects of FFAs on the cell proliferation was assessed by Trypan Blue exclusion after the treatment with various FFAs for 4 days.
140
** *
120
Cell survival (%)
100 80
**
**
60
**
**
40
**
20
**
0
Control
200 400 600 800
200 400 600 800
PA µM
SA µM
50
100 200 400
LA µM
1
2
5
10
AA µM
Fig. 1. Effects of FFAs on hepatocytes cell survival. The cultured rat hepatocytes were exposed to various concentrations of FFAs as indicated for 2 days and then the cell viability was determined by Trypan Blue staining as described in Materials and methods. The data represent the mean7SD of three independent experiments with triplicate wells. *po0.05 vs. control cells; **po0.01 vs. control cells. PA: palmitic acid; SA: stearic acid; LA: linoleic acid; AA: arachidonic acid.
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160
Control LA 200 µmol/L
PA 400 µmol/L
SA 400 µmol/L
AA 10 µmol/L
Cell survival rate (%)
140 120 100 80 60 40 20 0
24
48
72
96
Time (h)
Fig. 2. Time-dependent effects of FFAs on cell survival. Rat hepatocytes were exposed to various FFAs as indicated for 96 h and then the cell viability was determined by Trypan Blue staining as described in Materials and methods. The data represent the mean7SD of three independent experiments with triplicate wells. po0.05 vs. control cells; po0.01 vs. control cells. PA: palmitic acid; SA: stearic acid; LA: linoleic acid; AA: arachidonic acid.
Fig. 3. Morphological change of cells treated with PA. After incubating the hepatocytes with various concentrations of PA (A, control; B, PA 100 mM; C, 200 mM; D, 400 mM) for 4 days, as indicated, the cells were stained by Hoechst as described in Materials and methods. The cells were then observed by a fluorescence microscope. Number of apoptotic cells with nuclear fragmentation appeared increasingly as arising of concentration of PA (100–400 mM) and indicated by the arrows. Magnification, 400.
As shown in Fig. 2, 400 mM of PA and SA induced a time-dependent decline of cell proliferation. The cell survival rates were 30% and 24% after exposure to 400 mM of PA and SA, respectively, for 4 days. LA had little effect at 200 mM after treatment for 4 days. The stimulatory effect of AA seamed to be also timedependent, because the cell survival increased dramatically as the time of treatment increased from 1 to 4 days. The cell viabilities were 130% and 134% in the cells exposed to 10 mM for 3 and 4 days, respectively.
Morphological change of cells treated with PA After incubating the rat hepatocytes with various concentrations of PA, the cells were stained by Hoechst as described in Materials and methods, and were then observed by a microscope. As shown in Fig. 3, at 4 days after treatment with PA, the cells showed a damage including swelling, membrane dissolution and formation of debris, some cells showed nuclear fragmentation, which are the
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manifestation of apoptosis. These damage effects were aggravated as arising of concentration of PA from 100 to 400 mM.
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Observation of appearance of sub-G1 cells after treatment with PA Cell cycle progression of rat hepatocytes exposure to 200 mM PA was shown in Fig. 4. Fragmented DNA was clearly revealed as sub-G1 fraction at 48 h (7.6%) after the treatment of 200 mM PA, and increased to 13.5% at 72 h. The increase in sub-G1 fraction by time course was compatible to the results of increasing dead cells determined by Trypan Blue and morphological evidence.
Observation of early apoptosis induced by PA
Fig. 4. Cell cycle analysis of hepatocytes treated with PA. Hepatocytes cultured in 9 in dishes were exposed to 400 mM of PA for 0, 24, 48 and 72 h, respectively, and then were harvested and analyzed by flow cytometry as described in Materials and methods.
In an attempt to characterize PA-induced cell death, all cells including adherent cells and floating cells in the medium of each well were collected, and then were labeled with Annexin V and PI for the flow cytometric analysis (Fig. 5). Phosphatidylserine externalization is a characteristic of cells undergoing apoptosis. Annexin V has a strong affinity for phosphatidylserine. Staining cells simultaneously with Annexin V and PI allows the resolution of intact cells (double-negative), early apoptotic cells (Annexin V-positive and PI-negative), and late apoptotic and necrotic cells (double-positive), which can be located in the lower left, upper left and upper right quadrants of the cytograms, respectively. Because only cells with Annexin V-positive and PI-negative are truly representative of apoptotic cells, the percentage of this
Fig. 5. Quantitation of apoptotic cells by flow cytometric analysis. Hepatocytes were exposed to 400 mM for 24, 48, 72, 96, 120 and 144 h, respectively, and then were harvested and labeled with Annexin V and PI. Intact cells, early apoptotic cells and late apoptotic or necrotic cells are located in the lower left, upper left and upper right quadrants of the cytograms, respectively.
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Table 1.
J. Ji et al. / Experimental and Toxicologic Pathology 56 (2005) 369–376
Percentage of cells stained with Annexin V and PI treated with PA 400 mM
%
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Annexin V-positive and PI-negative cells Double-positive cells Total dead cells
8.70 1.49 10.19
8.82 3.85 12.67
11.03 9.01 20.04
12.20 23.54 35.74
13.91 52.53 66.44
22.37 57.82 80.19
cell population was quantitated for time course, as shown in Table 1. It is clear that PA was able to induce progressive apoptosis in a time-dependent manner, at the concentration of 400 mM.
PA induced the decrease in Bcl-2:Bax ratio To determine whether PA-induced apoptosis in hepatocytes is related to the suppression of an apoptotic suppressor, Bcl-2, or the induction of an apoptotic effector, Bax, we measured the expression level of Bcl-2 and Bax in HepG2 cells, either treated or not treated with PA, by a flow cytometric analysis. As shown in Table 2, PA treatment resulted in a down-regulation of Bcl-2 (po0:05) and a dramatic up-regulation of Bax (po0:01). The ratio of Bcl-2 and Bax is critical for regulating the release of cytochrome c from mitochondria. Therefore, we calculated the Bcl-2:Bax ratio and found it was significantly decreased after the treatment of PA at 200 and 400 mM concentrations (po0:001) for 4 days, suggesting the apoptosis induced by PA might have occurred through mitochondriamediated pathway.
Discussion The presence of fatty liver in patients with obesity and type 2 diabetes mellitus has long been reported (Stone and Thiel, 1985). It is usually considered an incidental pathologic finding, with scarce or no clinical significance. Only recently did Ludwig et al. (1980) identify a syndrome characterized by the association of fatty liver and lobular hepatitis and chronically elevated alanine aminotransferase plasma levels in patients with negligible alcohol intake. The syndrome is mainly associated with obesity, diabetes and dyslipidemia, but a few patients are lean, and have normal fasting glucose and glucose tolerance, and show no evidence of increased plasma lipids. Patients with fatty liver and hepatitis are identified as having nonalcoholic steatohepatitis (NASH). They are part of the broad spectrum of NAFLDs, which also include pure steatosis. The limits between NAFLD and NASH are set only by liver histology and cannot be predicted on clinical or laboratory grounds (Matteoni et al., 1999).
Table 2. Expression (%) of Bcl-2 and Bax on HepG2 cells treated with PAa %
Control
PA 200 mM
PA 200 mM
Bcl-2 Bax Bcl-2/Bax
0.5570.002 0.2970.001 1.9070.067
0.4970.011b 0.5670.008c 0.8870.089d
0.4870.009b 0.6370.012c 0.7670.072d
a
The expression of Bcl-2 and Bax was determined 4 days after treatment with PA. b po0.05 vs. control. c po0.01 vs. control. d po0.001 vs. control.
From a clinical point of view, a few patients have ongoing liver injury: 50% of NASH patients develop liver fibrosis, 15% develop cirrhosis and 3% may progress to terminal liver failure, requiring liver transplantation. In 15–50% of cases, liver fibrosis or cirrhosis may also be diagnosed at presentation (Falchuk et al., 1980). The main reason of hepatitis and liver fibrosis or cirrhosis on obesity was reported to be directly injury to liver cells by a lot of content of unsaturated FFAs, such as AA. The effects in human liver cells of saturated FFAs, acting as a main content of FFAs on obesity from either diet or endogenous, have not been reported. Because saturated FFAs, such as PAand SA-induced apoptosis in human granulosa cells (Mu et al., 2001) and rat testicular Leydig cells (Lu et al., 2003); thus, the primary goal of the present study was to investigate the toxicity of PA in human liver cells. The nonfasting serum levels of PA, SA, LA and AA in patients with hyperlipidemia were reported to be 140.9777.7, 109770.0, 45.9744.0 and 0.871.3 mM, respectively (Mitropoulos et al., 1997). In our study, PA and SA induced a cell growth inhibition in hepatocytes, and promoted a significant cell death by a dose- and time-dependent manner at the doses of PA and SA ranging from 200 to 800 mM. Especially, as a mimic hyperlipidemia condition of 200–400 mM, PA induced more than 50% cell death within 5 days after the treatment, and a typical morphological change of apoptosis with fragmented nuclear was observed at 4 days. The results of present study indicate that PA at a 3–4-fold of normal plasma level (Mitropoulos et al., 1997) with a long period can significantly inhibits the cell survival and induces cell death of hepatocytes
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possibly via apoptosis. Unsaturated FFA LA had little effect on the cell survival at a 1–8-fold of normal plasma level within 2 days, while AA presented a dosedependent stimulatory effect on the cell proliferation at 1–10-fold of normal plasma level. These results may clinically suggested that not only the circulating levels of FFAs, but also the composition of FFAs, may play an important role in the damage of hepatocytes induced by hyperlipidemia. On the other hand, ethanol and AA were shown to be toxic and cause apoptosis in rat hepatocytes and HepG2 cells which express CYP2E1 through increasing release of cytochrome c into the cytosol fraction, and activation of p38MAPK and caspase 3 but not in control rat hepatocytes and HepG2 cell lines. This discrepancy between our study and the previous reports could be related to different doses of AA, which more than 30 mM were used in their experiments, and whether overexpressing CYP2E1. FFA-induced apoptosis has been reported in the hepatocytes (Wu and Cederbaum, 2003). However, most of the research was about the unsaturated FFAs, and there have been very few investigations about that saturated FFAs induce injury of hepatocytes. Here, we investigated the effect of PA on hepatocytes, and confirmed that PA-induced apoptosis in hepatocytes. It has been reported that FFAs-induced apoptosis is not only a direct effect of FFAs but also an indirect effect through enhancement to lipid peroxidation of mitochondria (Diehl, 1999). Mitochondrial function has been implicated as a critical early regulator of apoptosis in many cells including hepatocytes. Bcl-2, a mitochondrial protein, inhibits the apoptotic process and promotes cell survival (Hockenbery et al., 1990). While Bax, also a mitochondrial protein and a member of Bcl2 family, contributes to mitochondria-mediated apoptotic pathway. Although it appears that both Bcl-2 and Bax can regulate apoptosis independently, there also seems to be a competition that exists between the two. Bcl-2 is a 25 kDa protein that has extensive amino acid homology with Bax. It forms homodimers or heterodimers with Bax and the apoptotic activity depends on the balance of both molecules. And the ratio of the two, Bcl-2/Bax, regulates the release of cytochrome c which proceeds the mitochondrial apoptotic pathway (Micheldes, 1999). However, in human hepatocytes, the mechanism of PA-induced apoptosis has not been understood. But, it is increasingly recognized a variety of key events in apoptosis converge on mitochondria, and that mitochondrial release of cytochrome c plays a central role in triggering apoptosis (Takehara et al., 2001). Antiapoptotic members of the Bcl-2 family, such as Bcl-2 and Bcl-xl, are mainly located on the outer membrane of mitochondria and inhibit a common pathway of apoptosis, at least in part, by preventing the release of cytochrome c into cytosol (Takehara et al., 2001). And it was reported that PA decreased Bcl-2
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expression and induced release of cytochrome c from the mitochondria into the cytosol in Human Pancreatic bCells (Maedler et al., 2003). Here, we found a slight decrease in mitochondrial Bcl-2 and a markedly increase in mitochondrial level of Bax in the HepG2 cells treated with PA from 200 to 400 mM concentrations. This resulted in a significant decrease in Bcl-2/Bax ratio (po0:001), and suggested that PA would advance apoptosis through mitochondria-mediated apoptotic pathway. In summary, this study investigated, for the first time, that saturated FFA, such as PA, induced apoptosis in hepatocytes, perhaps not only by the toxicity of PA but also through mitochondria-mediated apoptosis pathway by regulating Bcl-2/Bax ratio. These effects of PA on hepatocytes proliferation and cell killing may therefore be a possible mechanism for both AFLD and NAFLD, and especially may play an important role in the transition from steatosis to steatohepatitis in human.
Acknowledgments The authors would like to thank Dr. Li P and Dr. Zhang QH for their technical assistance.
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