Autophagy protects against palmitate-induced apoptosis in hepatocytes
© Cai et al.; licensee BioMed Central Ltd. 2014
Received: 27 September 2013
Accepted: 22 March 2014
Published: 21 May 2014
Non-alcoholic fatty liver disease, one of the most common liver diseases, has obtained increasing attention. Palmitate (PA)-induced liver injury is considered a risk factor for the development of non-alcoholic fatty liver disease. Autophagy, a cellular degradative pathway, is an important self-defense mechanism in response to various stresses. In this study, we investigated whether autophagy plays a protective role in the progression of PA-induced hepatocytes injury.
Annexin V-FITC/PI staining by FCM analysis, TUNEL assay and the detection of PARP and cleaved caspase3 expression levels demonstrated that PA treatment prominently induced the apoptosis of hepatocytes. Meanwhile, treatment of PA strongly induced the formation of GFP-LC3 dots, the conversion from LC3I to LC3II, the decrease of p62 protein levels and the increase of autophagosomes. These results indicated that PA also induced autophagy activation. Autophagy inhibition through chloroquine pretreatment or Atg5shRNA infection led to the increase of cell apoptosis after PA treatment. Moreover, induction of autophagy by pretreatment with rapamycin resulted in distinct decrease of PA-induced apoptosis. Therefore, autophagy can prevent hepatocytes from PA-induced apoptosis. In the further study, we explored pathway of autophagy activation in PA-treated hepatocytes. We found that PA activated PKCα in hepatocytes, and had no influence on mammalian target of rapamycin and endoplasmic reticulum stress pathways.
These results demonstrated that autophagy plays a protective role in PA-induced hepatocytes apoptosis. And PA might induce autophagy through activating PKCα pathway in hepatocytes.
KeywordsAutophagy Palmitate Hepatocytes Apoptosis Protector
Non-alcoholic fatty liver disease (NAFLD) is usually considered the accumulation of extra fat in hepatocytes that is not caused by alcohol . In recent years, its incidence is rapidly rising and affects not only adults, but also children [2, 3]. NAFLD refers to a spectrum of disease ranging from steatosis to inflammation in nonalcoholic steatohepatitis (NASH) with different degrees of fibrosis that can progress to cirrhosis [4–6]. Accumulating evidence suggests that it is implicated with the levels of plasma free fatty acids (FFAs), the primary source for triacylglycerols (TAGs) in hepatocytes [3, 7–9]. Some studies demonstrated the condition that hepatocytes were exposed to elevated FFAs could promote steatosis and hepatic apoptosis via activation of Bim and PUMA [10, 11]. Hepatocytes apoptosis as a critical feature of NAFLD is correlated with disease severity [12, 13]. Moreover, diets with a high intake of fat, especially saturated fatty acids, promotes the development of NASH [14, 15]. Palmitate (PA) as a saturated fatty acid could induce intracellular steatosis and cellular damage , which would be a risk factor for NAFLD. However, NAFLD presents different developmental stages and degrees of severity. The different degrees of injury in NAFLD indicate that there might be some protective factors against the injury.
Nearly a decade, research in autophagy has become overwhelming. Autophagy is discovered as an evolutionarily conserved to have vast array of homeostatic, developmental, and other physiological functions [16, 17]. Autophagy, a cellular self-catabolic process, maintains cellular homeostasis by trafficking accumulation of damaged proteins and organelles to lysosomes for proteolytic degradation . The interesting role of “self-eating” means it can break down harmful components from itself, thus showing a survival benefit. Moreover, it is regarded as a self-protective mechanism, coping with the cellular stress. Increasing evidence suggests that autophagy is involved in a broad spectrum of diseases. The study of Dutta D shows that autophagy induction can resist oxidative stress-mediated damage in cardiomyocytes . Another research reported that human mesenchymal stem cells protected against apoptosis by enhancing autophagy in lung carcinoma cells . Besides, autophagy activation can reduce renal tubular injury induced by urinary proteins . According to the results from above studies, autophagy is taken as a benefit role in most situations. However, some researches also show that autophagy can promote cell death and the creation of apoptosis body . Therefore, it is important to make it clear to the effect of autophagy in various situations. In the present research, we attempted to investigate the effect of PA treatment in hepatocytes and the role of autophagy in this process.
PA induces hepatocytes apoptosis
PA induces autophagy activation in hepatocytes
Autophagy inhibition augments apoptosis of PA-induced in hepatocytes
Autophagy activation reduces apoptosis of PA-induced in hepatocytes
PA induces PKCα activation, but has no influenced on mTOR and ER stress pathways in hepatocytes
In the present research, we found that PA could not only induce cell apoptosis but also activate autophagy in hepatocytes. Moreover, we also found that autophagy inhibition resulted in the elevated cell apoptosis of PA treatment, and in contrast activating autophagy brought about the decrease of PA-induced apoptosis in hepatocytes. In addition, it was also discovered that PA activated PKCα, and had no influence on mTOR and ER stress signaling pathways in hepatocytes. Together with these findings, we conclude that autophagy has an important role in protecting PA-induced hepatocytes apoptosis, and PA might activate autophagy through PKCα pathway in hepatocytes.
Laura L. Listenberger et al. reported that PA-induced apoptosis occurred in Chinese hamster ovary cells via the generation of reactive oxygen species . Taheripak G and his colleagues found that PA could induce mitochondrial dysfunction and apoptosis in skeletal muscle cells . In addition, some research reports that PA induces hepatocytes lipoapoptosis [27–29]. These reports are identified with the damage effect of PA. We also found that PA led to apoptosis in hepatocytes, and autophagy could be activated with PA treatment. Moreover, through the effect of regulating autophagy, we have proved that autophagy had a protective effect in PA-treated hepatocytes. Autophagy was reported that it has a pro-survival function under stressful “life-threatening” conditions in most liver disease . Song MY et al. discovered that dimethyl sulfoxide reduced hepatocellular lipid accumulation by autophagy induction . Consequently, autophagy played a protective role in PA-induced hepatocytes apoptosis.
The reason why PA was able to activate autophagy in hepatocytes was speculative. Blocking mTOR signaling is the best pathway for activating autophagy . P70S6K and 4E-BP1 are two crucial downstream substrates of mTOR signaling. When sufficient nutrients are available, mTOR is phosphorylated and transmits a positive signal to p70S6K and the inactivation effect of 4E-BP1 . We found that PA treatment caused no significant difference in phosphorylation levels of mTOR, p70S6K and 4E-BP1 in hepatocytes, in comparison to control treatment. Therefore, PA-induced autophagy activation in hepatocytes was independent of mTOR signaling pathway. Accumulating data indicated that ER stress was a potent trigger of autophagy [34–37], and FFAs have been reported to have a function of generating ER stress in hepatocytes . Nevertheless, our result was not consistent with these findings, since PA had no influence on ER stress markers in hepatocytes, suggesting that autophagy activation was independent of ER stress pathway. Then PKCα, as a member of the classical PKC family, was found played a critical mediator in PA-induced autophagy in MEF cells . We investigated the role of PKCα in hepatocytes with PA treatment. It was found that PA treatment activated p-PKCα in hepatocytes. Taken together, PA might activate PKCα pathway for activating autophagy in hepatocytes.
In conclusion, PA can induce hepatocytes apoptosis and during the process autophagic system is activated, and the activated autophagy plays a protective role against PA-induced apoptosis. Besides, PA might induce autophagy through activating PKC α pathway in hepatocytes. However, the detailed mechanism involved in the protective effect of autophagy in PA-treated hepatocytes has yet to be further research.
Materials and methods
PA, Albumin from bovine serum (BSA, fatty acid free) and CQ were purchased from Sigma-Aldrich (St.Louise, MO). Rapamycin was purchased from Gene Operation Datasheet. Cell counting kit-8 (CCK-8) assay kit was purchased from DOJINDO (Japan). AnnexinV/PI analysis kit was purchased from KeyGen Biotechnology (China). DAPI staining solution was purchased from Beyotime Institute of Biotechnology (China). GAPDH was purchased from HuaAn Biotechnology (China). RIPA buffer and other all antibodies were purchased from Cell Signaling Technology (Beverly, MA). Pierce BCA Protein Assay Kit was purchased from Thermo Fisher Scientific. Fugene HD transfection reagent was purchased from Roche (04709705001). Odyssey Blocking Buffer was purchased from LI-COR Biosciences. DNA fragmentation detection kit was purchased from Calbiochem (America).
HL-7702 cell was maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 g/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. HepG2 cell was maintained in DMEM medium supplemented with the same conditions. Above reagents were purchased from Gibco Life Technologies.
Preparation of PA
Briefly, 0.103 g Palmitic acid was prepared in 0.1 M 200 ml NaOH at 70°C and filtered. Five percent FFAs-free BSA solution was prepared in double-distilled H2O and filtered. The solution of PA was conjugated to 5% BSA in a 70°C water bath. The above solution was then cooled to room temperature and diluted in RPMI 1640/DMEM to final concentrations . Cells were treated at the concentration of 500 μM PA in the present research normally. Cells were cultured in RPMI 1640/DMEM with 3% FBS as control.
Cell viability assay
Cells (5 × 103 cells/well) were seeded in 96-wells plate, and cultured overnight. After treatments as indicated, cells were incubated with the mixed liquor (10 μL CCK-8 reagent + 90 μL RPMI 1640/DMEM medium) at 37°C for 1 hour. Then the value was measured at 450 nm of light absorption.
Cells were seeded in microscope slides, and then were placed in 24-wells plate. After treated as indicated, cells were fixed using 4% paraformaldehyde, and the manufacturer’s protocol was followed. TUNEL positive cells were observed under confocal microscopy.
Western blot analysis
Cellular protein was extracted with 1× cell RIPA buffer. Density of proteins was determined by Pierce BCA Protein Assay Kit. According to the routine, equivalent amounts of protein (30 μg) were loaded onto poly-acrylamide gels, electrophoresed, and then transferred onto nitrocellulose NC membranes (Whatman). After blocking these membranes with odyssey blocking buffer for 1 hour, target antigens were reacted with primary antibodies and subsequently secondary antibodies. At last, the membranes were scanned by the Odyssey infrared imaging system.
Transfection of GFP-LC3 plasmids
Cells were seeded in 96-well plates, then GFP-LC3 expression plasmids were transfected into the cells using Fugene HD transfection reagent. After 24 hour, cells were treated with PA (500 μM) or non-PA for 24 hours. Autofluorescence GFP-LC3 was observed under fluorescence microscope.
Gene silencing with lentivirus-delivered shRNA
shRNA candidate target sequence to Atg5 is 5′-CCTTTCATTCAGAAGCTGTTT-3′. Scrambled shRNA sequence, as a negative control, is 5′-TTCTCCGAACGTGTCACGT-3′. The oligonucleotides encoding the Atg5-shRNA or Scrambled shRNA sequence were inserted into the GFP express vector pGCL-GFP (Shanghai GeneChem, shanghai, china). The recombinant virus was packaged using Lentivector Expression Systems (Shanghai GeneChem). HL-7702 and HepG2 cells were infected, and observed under fluorescence microscope after 72h.
Annexin V-FITC and PI Staining Analysis
In order to assess apoptosis, 1× 106 cells were plated onto 6-well culture plates and treated with ligands previously. Following staining according to manufacturer’s protocol, the apoptosis analysis of cell was performed by flow cytometry (FCM).
All the data were expressed as mean ± SEM deviation of at least three independent experiments. Statistical differences between the various groups were compared by using Student’s t test and one-way ANONA. P values less than 0.05 were considered statistically significant.
Non-alcoholic fatty liver disease
Free fatty acids
Microtubule-associated protein 1 light chain 3
mammalian target of rapamycin
p70 S6 kinase
4E- binding protein 1
Protein kinase C
Cell counting kit-8.
This project was supported by the Key Basic Research Project of China (Grant NO. 2011CB966203); Major State Scientific Research Program of China (Grant NO. 2012CBA01303); Key Project of National Natural Science Foundation of China (Grant NO. 81030041); National Natural Science Foundation of China (Grant NO. 31171321, 81101622, 81372312, 81301715); Special Funds for National key Sci-Tech Sepcial Project of China (Grant NO.2012ZX10002-016, 2012ZX10002011-011); Shanghai Science and Technology Committee (Grant NO. 12431900802); Innovation Program of Shanghai Municipal Education Commission (14YZ041) and Science Fund for Creative Research Groups, NSFC, China (Grant NO. 81221061).
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