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doi:10.1111/jgh.12430
H E PAT O L O G Y
Apolipoprotein A-I and adenosine triphosphate-binding cassette transporter A1 expression alleviates lipid accumulation in hepatocytes Wei Liu,*,1 Ling Qin,†,1 Hao Yu,* Fangqiao Lv* and Yutong Wang* *Department of Cell Biology, Municipal Laboratory for Liver Protection and Regulation of Regeneration, School of Basic Medical Sciences and † Biomedical Information Center of Beijing Youan Hospital, Capital Medical University, Beijing, China
Key words apolipoprotein A-I, ATP-binding cassette transporter, non-alcoholic steatohepatitis. Accepted for publication 11 September 2013. Correspondence Dr Yutong Wang, Department of Cell Biology, Capital Medical University, 10 You An Men Wai Xi Tou Tiao, Beijing 100069, China. Email: [email protected] 1
These authors contributed equally to this work.
Abstract Background and Aim: Abnormal lipid metabolism may contribute to the pathogenesis of non-alcoholic steatohepatitis. ATP-binding cassette transporter A1 (ABCA1) mediates the transport of cholesterol and phospholipids from cells to high density lipoprotein apolipoproteins. The lipidation of apolipoprotein A-I (apoA-I) by ABCA1 is the ratelimiting step in reverse cholesterol transport and the generation of plasma high density lipoprotein. Here, we examined the effect of apoA-I or ABCA1 overexpression on hepatic lipid levels in BEL-7402 cells. Methods: Human ABCA1 or apoA-I was overexpressed in BEL-7402 hepatocytes by transfection and human apoA-I was overexpressed via adenoviral vector in C57BL/6J mice with MCD diet. Results: Overexpression of either apoA-I or ABCA1 resulted in an increase in cholesterol efflux and a decrease in cellular fatty acids and triglycerides. However, after repression of ABCA1 by its siRNA, overexpression of apoA-I failed to decrease both cellular fatty acids and triglycerides. ApoA-I or ABCA1 overexpression also resulted in a decrease in the expression of the endoplasmic reticulum stress-related proteins GRP78 and SREBP-1. Overexpression of apoA-I in mice also reduced hepatic lipid levels. Conclusions: Expression of apoA-I or ABCA1 can reduce steatosis by decreasing lipid storage in hepatocytes through lipid transport and may also reduce endoplasmic reticulum stress, further lessening hepatic steatosis.
Introduction Non-alcoholic fatty liver disease is a recently emerging obesityrelated disorder characterized by fatty infiltration of the liver in the absence of chronic alcohol consumption. This disorder is characterized by macrovesicular steatosis and has been increasingly recognized as the hepatic manifestation of insulin resistance and metabolic syndrome.1–3 Some patients with steatosis develop superimposed necroinflammatory activity with a non-specific inflammatory infiltrate, lobular inflammation, and hepatic cellular ballooning, called non-alcoholic steatohepatitis (NASH).4 The current working model for the pathogenesis of NASH is the “twohit” hypothesis,5 in which the first “hit” is steatosis and the second “hit” is oxidative stress and inflammation, ultimately resulting in NASH.6 More recent work has revealed that natural killer T-cell deficiency,7 regulatory T-cell dysregulation,8 or intestinal microbiota modification9 may also be involved in the second hit. Excessive accumulation of lipids within hepatocytes has been considered an important factor for hepatic steatosis and inflammation;10 therefore, efficient transportation or delivery of lipids 614
may be key to NASH prevention and treatment. Lipidation of apolipoprotein A-I (apoA-I) by ATP-binding cassette transporter A1 (ABCA1) is the rate-limiting step in reverse cholesterol transport and the generation of plasma high density lipoprotein (HDL).11,12 ABCA1 is an integral membrane protein that mediates the transport of cellular cholesterol and phospholipids to apoA-I to generate nascent HDL particles.13,14 The transcription of the ABCA1 gene is regulated by nuclear liver X receptors (LXRα and LXRβ) and retinoid X receptor15,16 Previous reports suggest posttranslational regulation plays a key role in ABCA1 expression.17 ApoA-I increases ABCA1 protein stability by decreasing the phosphorylation of the PEST sequence and reducing calpain-mediated proteolysis of ABCA1.18 The interaction of apoA-I with ABCA1 may also activate various signaling pathways, including protein kinase A,19 protein kinase C,20 and Janus kinase 2.21 ApoA-I is composed of 243 amino acids, including 10 amphipathic helices,22 and functions as the primary protein component of HDL, which accepts cellular cholesterol and phospholipids transported by ABCA1 as the initial step of reverse cholesterol transport. Liver-specific overexpression of apoA-I
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substantially reduces the progression of atherosclerosis in mice.22,23 Furthermore, adenoviral vector-mediated gene transfer of apoA-I to the liver reduces the progression24,25 and induces the regression26 of atherosclerosis. Through the use of apoA-I mimetics or apoA-I-based infusion therapies in animal models, apoA-I has been shown to mediate a number of important effects on reverse cholesterol transport. In addition to promoting cholesterol efflux, ABCA1, apoA-I, and apoA-I mimetics can enhance other aspects of reverse cholesterol transport, such as the activation of lecithin cholesterol acyltransferase27 and off-loading of cholesterol to the liver via the scavenger receptor B-1.28 Previous studies of apoA-I and ABCA1 have mainly focused on the relationship of these proteins with cardiovascular diseases; however, their role in the pathogenesis of NASH as a lipid transporter remains poorly understood. In this study, we examined the effect of apoA-I and ABCA1 on lipid content in the human hepatocyte cell line BEL-7402. Overexpression of apoA-I and ABCA1 significantly reduced cellular cholesterol, triglycerides, and fatty acids as well as markers of endoplasmic reticulum (ER) stress such as GRP78 and SREBP1. These results suggest that an increased supply of apoA-I and ABCA1 could promote clearance of excess cholesterol and phospholipids from hepatocytes and may be beneficial for the prevention and treatment of NASH.
Methods Materials. BEL-7402 cells were obtained from the American Type Culture Collection. The total RNA extraction reagent TRIzol, a reverse transcription kit, Dulbecco’s Modified Eagle’s medium (DMEM), and fetal bovine serum were acquired from Invitrogen (Carlsbad, CA, USA). Fatty acid-free bovine serum albumin (BSA), 22(R)-hydroxycholesterol, 9-cis-retinoic acid, and protease inhibitor cocktail were purchased from Sigma (St. Louis, MO, USA). ApoA-I was obtained from Millipore (Billerica, MA, USA). Methanol, butanol, hexane, and isopropanol were acquired from Beijing Modern Eastern Finechemical (Beijing, China). Triton X-100 was purchased from Beijing Solarbio (Beijing, China). Cholesterol, fatty acid, and triglyceride quantification kits were obtained from BioVision (Mountain View, CA, USA). A Prism 7300 sequence detecting system for quantitative reverse transcription–polymerase chain reaction was purchased from Applied Biosystems (Foster City, CA, USA). FuGene HD was obtained from Roche (Basel, Switzerland). Antibodies against ABCA1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Novus Biologicals (Littleton, CO, USA) and Shanghai Kang Chen Biotech (Shanghai, China), respectively. Antibodies against apoA-I, GRP78, and SREBP-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cell culture and cholesterol efflux. BEL-7402 cells were maintained in DMEM containing 10% fetal bovine serum or were incubated in serum-free DMEM plus 1 mg/mL fatty acid-free BSA (DMEM/BSA). Washed cells were incubated for 16 h with medium containing 5 mg/mL BSA in the presence or absence of 250 μM fatty acids (molar ratios to BSA of 1.8 and 0). Fatty acids were added from a stock solution in which they were bound to
ApoA-I/ABCA1 expression reduces lipids
BSA at a 3.5 molar ratio and were adjusted to lower ratios by adding fatty acid-free BSA. To induce ABCA1 expression, 10 μM 22(R)-hydroxycholesterol and 10 μM 9-cis-retinoic acid were added in combination to this medium. To measure cholesterol efflux, cells were incubated with DMEM/BSA with or without 10 μg/mL apoA-I at 37°C for 4 h and chilled on ice. The medium was then collected and centrifuged to remove detached cells. Cholesterol was extracted from the medium and the cells with methanol/chloroform (1:2) and hexane/ isopropanol (1:1), respectively. Cholesterol content was assayed following the kit manufacturer’s guide. ApoA-I-mediated cholesterol efflux is expressed as the fraction of cholesterol released into the medium after subtraction of the values obtained in the absence of apoA-I. Western blot. Cells were washed and dislodged from the dish at 0°C in buffer containing protease inhibitors. Cellular proteins were solubilized in phosphate-buffered saline (PBS) containing 1% Triton X-100 plus protease inhibitors and resolved by sodium dodecylsulfate–polyacrylamide gel electrophoresis. The expression of apoA-I, ABCA1, and GAPDH was identified by Western blot analysis using antibodies against ABCA1 and GAPDH, respectively.29 Determination of fatty acids and triglycerides. Lipid mass analysis was conducted as previously described.30 BEL-7402 cells were cultured in 6-well plates at 80% confluency in DMEM. Total cellular lipids were extracted by hexane/ isopropanol (1:1). The chloroform/methanol extract was backextracted with water to remove contaminating proteins. The amounts of free fatty acids and triglycerides were measured by colorimetric enzyme assays using test reagents for serum lipids according to the manufacturer’s protocol and normalized with total cellular protein. Plasmid transfection. The ABCA1 shRNA plasmid and control plasmid were designed and constructed by Shanghai GenePharma Co. (Shanghai, China). CMV-ABCA1, pcDNA3.0/ apoA-I, shABCA1, and the control plasmid were transfected into BEL-7402 hepatocytes using FuGene HD as described in the manufacturer’s protocol. The target sequence of the ABCA1 shRNA was as follows: 5′-GGAACTGGACAATGCAGAACC-3′. Quantitative real-time polymerase chain reaction. Total RNA from BEL-7402 hepatocytes was extracted using TRIzol. Two micrograms of total RNA were reverse transcribed into cDNA using Superscript II Reverse Transcriptase. Gene expression was quantified using a SYBR-Green PCR kit with 18S rRNA as the control. Quantitative reverse transcription– polymerase chain reaction was performed with a Prism 7300 sequence detecting system. The primer sequence has been described previously.31,32 Oil Red O staining. BEL-7402 cells were cultivated on cover slips in 24-well plates at 10 000 cells/well with 5 mg/mL BSA in the presence or absence of 250 μM oleic acid (OA) for 24 h. Following cultivation, the medium was aspirated, the cells were rinsed twice with PBS, and staining with Oil Red O (Sigma) was performed. Cells were washed with PBS and fixed with 3.7%
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formaldehyde for 1 h. Then, 0.3% Oil Red O in isopropanol was added directly to the fixed cells and incubated for 1 h. The microscopy studies were performed with a microscope equipped with a digital camera (Leica, Wetzlar, Germany). Animal studies. C57BL/6J mice were purchased from Academy of Military Medical Sciences (Beijing, China). Male mice 4–6 weeks old were used in these studies. Before fed with methionine choline-deficient (MCD) diet, mice were injected with 1 × 108 pfu/mL adenoviral vector containing human apoA-I (ad-apoA-I) or vector alone (ad-Null) the femoral vein. The mice were divided into four groups and were fed with different diet for 1 week with or without the injection of adenoviral vector. Histological analysis of tissue samples. At the end of each experiment, mice were sacrificed and liver was collected and stored either at −80°C or fixed in 10% formalin or made frozen section. Four-micrometer thick sections were obtained from the formalin-fixed paraffin-embedded tissue for further histological analyses. Conventional hematoxylin and eosin histological staining was performed in order to evaluate the microscopic morphology of the liver tissue samples. Ten micrometer sections were obtained from frozen tissue for further oil red staining in order to evaluate the lipid levels in liver. Statistical analysis. The results of multiple observations are presented as the mean ± standard error of the mean. The data were analyzed with the statistics software SPSS 11.5 (SPSS Inc., Chicago, IL, USA) by a non-parametric analysis of variance test. Difference were considered significant if P < 0.05.
Results Cellular lipid accumulation by oleic acid treatment. OA has been reported to induce steatosis in HepG2 cells.33 To examine the effect of OA on BEL-7402 cells, we incubated BEL-7402 cells with 5 mg/mL BSA with or without 250 μM OA for 24 h, followed by Oil Red O staining to examine lipid accumulation. Compared with the control (Fig. 1a), OA treatment resulted in a significant increase in lipid accumulation, as revealed by Oil Red O staining (Fig. 1b). When the content of different lipids was analyzed, the results revealed that 24 h of OA treatment caused a marked increase in the cellular levels of triglycerides, free fatty acids, and cholesterol (Fig. 1c–e, respectively). Alteration of cellular free fatty acids and triglycerides by apoA-I and ABCA1 overexpression. ABCA1 can transport cellular cholesterol and phospholipids to lipid-poor apoA-I to generate nascent HDL.13,14 Thus, the expression levels of apoA-I or ABCA1 may have a significant impact on cellular lipid levels. To test this hypothesis, we altered apoA-I or ABCA1 expression levels in OA-treated BEL-7402 cells by transfecting the cells with ABCA1- or apoA-I-expressing plasmid. Forty-eight hours after transfection, ABCA1 mRNA and protein levels were measured. As expected, transfection of apoA-Iexpressing vector significantly increased cellular apoA-I protein levels (Fig. 1f). And transfection of the ABCA1- or apoA-Iexpressing vector significantly increased ABCA1 protein levels 616
(Fig. 1h) and cholesterol efflux (Fig. 1i) compared with control cells. Consistent with previous reports, apoA-I increased ABCA1 protein levels (Fig. 1h) without affecting its mRNA levels (Fig. 1g), presumably by altering the phosphorylation status of the PEST sequence in ABCA1 to prevent ABCA1 degradation. To evaluate the impact of ABCA1 expression on cellular lipids, the free fatty acid (Fig. 1j) and triglyceride (Fig. 1k) content were also measured. Consistent with the roles of ABCA1 as a cholesterol and phospholipid transporter and apoA-I as a lipid acceptor, when compared with control cells, apoA-I or ABCA1 overexpression caused a significant decrease in cellular-free fatty acids and triglycerides. These data indicate that alterations in apoA-I or ABCA1 expression levels lead to the modification of cellular lipid storage. To eliminate the possibility that apoA-I may alter the cellular lipid profile through mechanisms other than the ABCA1-mediated lipid transport pathway, we used shRNA to specifically inhibit ABCA1 expression. Seventy-two hours after transfection, ABCA1 mRNA and protein levels were quantified, and cholesterol efflux, cellular-free fatty acid levels, and cellular triglyceride levels were also measured. ABCA1 shRNA dramatically reduced ABCA1 mRNA and protein levels (Fig. 2a and b). Compared with the control vector, ABCA1 shRNA abolished the ability of apoA-I to increase cholesterol efflux (Fig. 2c) or decrease the level of cellular-free fatty acids (Fig. 2d) and triglycerides (Fig. 2e). These results suggest that apoA-I promotes cholesterol efflux and reduces cellular lipid levels through the ABCA1-mediated lipid transport pathway. Alteration of GRP78 and SREBP-1 by apoA-I and ABCA1 overexpression. Lipid toxicity may play a key role in ER stress.34 To evaluate the effect of ABCA1/apoA-I expression on ER stress, two proteins related to ER stress, GRP78 and SREBP-1, were measured by Western blot. OA treatment markedly increased the levels of GRP78 (Fig. 3a and b) and the precursor form of SREBP-1 (Fig. 3c and d). These results are consistent with previous reports that ER stress can lead to increased translation of RNAs that contain internal ribosome entry sites, such as GRP78 and SREBP-1.35,36 Upon the expression of apoA-I or ABCA1, the levels of GRP78 and the SREBP-1 precursor decreased significantly, indicating a reduction in ER stress. Moreover, transfection of the apoA-I or ABCA1 expression vector also resulted in a significant decrease in the mature form of SREBP-1 (Fig. 3e). Alteration hepatic lipid levels by apoA-I overexpression in vivo. To test the effect of apoA-I overexpression on hepatic levels in vivo, we injected mice with control or apoA-I expressing vector followed by 1 week of MCD diet. Consistent with the results from cultured cells, expressing of apoA-I in vivo significantly reduced hepatic lipid deposition induced by MCD (Fig. 4A and B) diet as well as hepatic cholesterol (Fig. 4D), triglyceride (Fig. 4E), and fatty acid (Fig. 4F) levels.
Discussion Modulation of ABCA1 activity could have a profound impact on cholesterol and phospholipid transport. Our previous studies
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Figure 1 Apolipoprotein A-I (ApoA-I) or ATP-binding cassette transporter A1 (ABCA1) overexpression alters cellular lipid levels in BEL-7402 cells. BEL-7402 cells were incubated for 24 h in DMEM with 5 mg/mL bovine serum albumin (BSA) in the presence or absence of 250 μM oleic acid (OA). (a) Lipids were stained with Oil Red O, as described in the “Methods”. (b) Twenty-four hours after adding 250 μM OA to the Dulbecco’s Modified Eagle Medium (DMEM) medium, cellular triglycerides (TG) (c), free fatty acids (FFA) (d), and cholesterols (e) were measured as described in the “Methods”. (f–k) BEL-7402 cells were then transfected with control vector with a CMV promoter or CMV-ABCA1 or CMV-apoA-I plasmid. Thirty-six hours after transfection, the cells were incubated in DMEM/BSA media with 10 μM 22(R)-hydroxycholesterol and 10 μM 9-cis-retinoic acid. Forty-eight hours after transfection, (g) ABCA1 mRNA levels were measured by quantitative real-time polymerase chain reaction as described in the “Methods”. Total cellular proteins were isolated, and immunoblot analysis of apoA-I (f) or ABCA1 (h) was conducted as described in the “Methods”. The immunoblot represents the result of a single experiment that is representative of three independent experiments. The immunoblot was quantified for from three independent experiments. Cholesterol efflux (i), cellular free fatty acids (j), and triglycerides (k) were measured as described in the “Methods”. The results are representative of three independent experiments and are presented as the mean ± standard error of the mean. *P < 0.05 versus control, **P < 0.01 versus control.
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Figure 2 ATP-binding cassette transporter A1 (ABCA1) repression alters cellular levels of triglycerides (TG) and free fatty acids (FFA). BEL-7402 cells were incubated for 24 h in Dulbecco’s Modified Eagle Medium (DMEM) with 5 mg/mL bovine serum albumin (BSA) and 250 μM oleic acid (OA). The cells were then co-transfected with a negative control containing a scrambled siRNA sequence, the plasmid pcDNA3.0, and/or the apolipoprotein A-I (apoA-I) expression plasmid. Fifty-six hours after transfection, cells were incubated in DMEM/BSA with 10 μM 22(R)-hydroxycholesterol and 10 μM 9-cis-retinoic acid. (a) Seventy-two hours after transfection, total cellular RNA was isolated, and relative ABCA1 mRNA levels were measured as described in the “Methods”. (b) Cellular proteins were isolated, and immunoblot analysis of ABCA1 was performed as described in the “Methods”. The immunoblot represents the result of a single experiment that is representative of three independent experiments. The immunoblot of ABCA1 was quantified for from three independent experiments. Seventy-two hours after transfection, cellular free fatty acids (c), triglycerides (d), and cholesterol efflux (e) were measured as described in the “Methods”. The results are representative of three independent experiments and are presented as the mean ± standard error of the mean. *P < 0.05 versus control.
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Figure 3 apolipoprotein A-I (ApoA-I) or ATP-binding cassette transporter A1 (ABCA1) expression alters levels of GRP78 and SREBP-1. BEL-7402 cells were incubated for 24 h in Dulbecco’s Modified Eagle Medium (DMEM) with 5 mg/mL bovine serum albumin (BSA) in the presence of absence of 250 μM oleic acid (OA). The cells were then transfected with the control plasmid pcDNA3.0, the ABCA1 expression plasmid, or the apoA-I expression plasmid. Thirty-six hours after transfection, the cells were incubated in DMEM/BSA with 10 μM 22(R)-hydroxycholesterol and 10 μM 9-cis-retinoic acid. Forty-eight hours after transfection, total cellular proteins were isolated, and immunoblot analysis of GRP78 (a) or SREBP-1 (c) was performed as described in the “Methods”. The immunoblot represents the result of a single experiment that is representative of three independent experiments. The immunoblots for GRP78 (b), the precursor form of SREBP-1 (d) or the mature form of SREBP-1 (e) were quantified from three independent experiments. The results are presented as the mean ± standard error of the mean. *P < 0.05 versus control.
revealed that unsaturated fatty acids suppress ABCA1 protein levels in HepG2 cells and that ABCA1 also regulates cellular fatty acid content.37 Here, we provide evidence that this increase in either apoA-I or ABCA1 expression can also effectively regulate cellular fatty acid and triglyceride content and alleviate ER stress. Incubation with OA induced hepatic steatosis in BEL-7402 cells. Under these conditions, hepatic fatty acid and triglyceride content was significantly decreased in cells upon apoA-I or ABCA1 overexpression and markedly increased after inhibition of ABCA1 by RNAi. Protein levels of GRP78 and SREBP-1, two proteins related to ER stress, also increased significantly upon OA treatment and then decreased when apoA-I or ABCA1 was overexpressed. These observations reveal an association between cellular fatty acid/triglyceride levels and hepatic ABCA1/apoA-I, strongly suggesting that upregulation of the ABCA1 pathway by expression of either apoA-I or ABCA1 can effectively reduce hepatic steatosis as well as decrease ER stress. ABCA1 is a large integral membrane protein with a molecular weight greater than 250 kD. Numerous studies have been conducted, almost exclusively in the field of atherosclerosis, to
attempt to promote reverse cholesterol transport by increasing ABCA1 expression. However, overexpression of ABCA1 by transfection for a prolonged period of time (more than 72 h) may cause cell viability problems (unpublished data), presumably because a large number of ABCA1 molecules may cause damage to the plasma membrane due to the large size of ABCA1. Thus, overexpression of apoA-I may be an excellent alternative to promote the ABCA1-mediated reverse cholesterol transport pathway. ApoA-I can not only function as a key lipoprotein to accept cholesterol and phospholipids from ABCA1 but can also stabilize the ABCA1 protein and increase membrane ABCA1 content through different pathways, including altering the phosphorylation status of the PEST sequence in ABCA1 and activating protein kinase A, protein kinase C, and Janus kinase 2.18–21 Although the most important site for the ABCA1/apoA-I interaction is located at the plasma membrane, apoA-I conjugated to nascent lipids has been isolated from endosomes in hepatocytes.38 In HepG2 cells, ∼20% of newly synthesized apoA-I is lipidated intracellularly,39 suggesting that the intracellular interaction between apoA-I and
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Figure 4 apolipoprotein A-I (ApoA-I) expression reduces mice hepatic lipid levels. Hematoxylin and eosin (a) and Oil Red O (b) staining for mice fed with normal diet (panel a), mice fed with MCD (panel b), mice injected with vector (panel c), and mice injected with apoA-I expressing vector (panel d). (c) Total hepatic cellular proteins were isolated, and immunoblot analysis of apoA-I was conducted as described in the “Materials and methods”. Hepatic cholesterol (d), triglyceride (TG) (e), and fatty acid (FFA) (f) levels were measured as described in the “Methods”. The results are representative of three independent experiments and are presented as the mean ± standard error of the mean. *P < 0.05 versus control.
ABCA1 may also play an important role. Although the mechanisms involved are complex, the ability of apoA-I overexpression to reduce atherosclerosis has been observed in mice.40 In our studies, expression of apoA-I also significantly reduced cellular fatty acid and triglyceride content. Hepatic steatosis is the initial stage of NASH development. NASH is associated with suboptimal protein secretion by hepatocytes and induction of the unfolded protein response, an indicator of ER stress.41 Two hypotheses have been proposed to explain this finding: (i) lipid accumulation in hepatocytes causes unfolded protein response activation and ER dysfunction due to lipotoxicity;42 and (ii) ER dysfunction and unfolded protein accumulation precedes steatosis, causing the disease.41 However, both hypotheses may explain the pathogenesis of NASH, that is lipotoxicity promotes ER dysfunction, and ER dysfunction promote steatosis, thus forming a vicious cycle. ApoA-I/ABCA1 expression can promote lipid transport and effectively reduce cellular lipid content (Fig. 1). As a result, apoA-I/ABCA1 expression may reduce ER stress and thus reduce the levels of ER stressrelated proteins such as GRP78 and SREBP-1 (Fig. 3). Because ER stress can promote hepatic steatosis through the activation of SREBP-1,43 the reduction of cellular lipid levels may also affect 620
SREBP-1 activation. As expected, the mature form of SREBP-1 increased significantly after 24 h of OA treatment and then decreased to the level prior to OA treatment when apoA-I or ABCA1 was overexpressed. This evidence indicates that expression of apoA-I or ABCA1 can decrease cellular lipid levels and exhibit beneficial effects by reducing ER stress. Eventually, the reduction of ER stress may further reduce steatosis, creating a positive cycle. Since SREBP-1 can regulate the transcription of lipogenic genes such as fatty acid synthase and acetyl-CoA carboxylase,44 reduction in SREBP-1 may cause a decrease in the expression of these genes. As a result, the reduced level of fatty acid synthesis may then affect the levels of triglycerides, which may also contribute to overall reduction of lipid accumulation caused by apoA-I. This study has important therapeutic implications for treating NASH. By mediating the expression of apoA-I and ABCA1, levels of ER stress as well as cellular lipids including cholesterol, triglycerides, and fatty acids may be significantly reduced. As a result, the ABCA1/apoA-I pathway lipid-removal pathway may be a feasible means of NASH prevention and treatment. Understanding the mechanisms by which apoA-I reduces cellular lipid levels through the ABCA1-mediated pathway will be useful for
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designing therapeutic interventions that enhance the activity of this lipid-removal pathway and prevent the development of NASH.
Acknowledgments This work was supported by the National Natural Science Foundation of China (30740094, 30871223) and the Beijing Nova Program (2007B064).
References 1 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. 2 Okamoto M, Takeda Y, Yoda Y, Kobayashi K, Fujino MA, Yamagata Z. The association of fatty liver and diabetes risk. J. Epidemiol. 2003; 13: 15–21. 3 Hamaguchi M, Kojima T, Takeda N et al. The metabolic syndrome as a predictor of nonalcoholic fatty liver disease. Ann. Intern. Med. 2005; 143: 722–8. 4 Ludwig J, Viggiano TR, McGill DB, Oh BJ. Nonalcoholic steatohepatitis: Mayo Clinic experiences with a hitherto unnamed disease. Mayo Clin. Proc. 1980; 55: 434–8. 5 Day CP, James OF. Steatohepatitis: a tale of two “hits”. Gastroenterology 1998; 114: 842–5. 6 Youssef W, McCullough AJ. Diabetes mellitus, obesity, and hepatic steatosis. Semin. Gastrointest. Dis. 2002; 1: 17e30. 7 Kremer M, Thomas E, Milton RJ et al. Kupffer cell and interleukin-12-dependent loss of natural killer T cells in hepatosteatosis. Hepatology 2010; 51: 130–41. 8 Ma X, Hua J, Mohamood AR, Hamad AR, Ravi R, Li Z. A high-fat diet and regulatory T cells influence susceptibility to endotoxin-induced liver injury. Hepatology 2007; 46: 1519–29. 9 Musso G, Gambino R, Cassader M. Gut microbiota as a regulator of energy homeostasis and ectopic fat deposition: mechanisms and implications for metabolic disorders. Curr. Opin. Lipidol. 2010; 21: 76–83. 10 Sheth SG, Gordon FD, Chopra S. Nonalcoholic steatohepatitis. Ann. Intern. Med. 1997; 126: 137–45. 11 Joyce C, Freeman L, Brewer HB, Jr, Santamarina-Fojo S. Study of ABCA1 function in transgenic mice. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 965–71. 12 Aiello RJ, Brees D, Francone OL. ABCA1-deficient mice: insights into the role of monocyte lipid efflux in HDL formation and inflammation. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 972–80. 13 Oram JF, Heinecke JW. ATP-binding cassette transporter A1: a cell cholesterol exporter that protects against cardiovascular disease. Physiol. Rev. 2005; 85: 1343–72. 14 Wang N, Tall AR. Regulation and mechanisms of ATP-binding cassette transporter A1-mediated cellular cholesterol efflux. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1178–84. 15 Langmann T, Klucken J, Reil M et al. Molecular cloning of the human ATP-binding cassette transporter 1 (hABC1): evidence for sterol-dependent regulation in macrophages. Biochem. Biophys. Res. Commun. 1999; 257: 29–33. 16 Costet P, Luo Y, Wang N, Tall AR. Sterol-dependent transactivation of the ABC1 promoter by the liver X receptor/retinoid X receptor. J. Biol. Chem. 2000; 275: 28240–5. 17 Wellington CL, Walker EK, Suarez A et al. ABCA1 mRNA and protein distribution patterns predict multiple different roles and levels of regulation. Lab. Invest. 2002; 82: 273–83.
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18 Martinez LO, Agerholm-Larsen B, Wang N, Chen W, Tall AR. Phosphorylation of a pest sequence in ABCA1 promotes calpain degradation and is reversed by ApoA-I. J. Biol. Chem. 2003; 278: 37368–74. 19 Haidar B, Denis M, Marcil M, Krimbou L, Genest JJ. Apolipoprotein A-I activates cellular cAMP signaling through the ABCA1 transporter. J. Biol. Chem. 2004; 279: 9963–9. 20 Yamauchi Y, Hayashi M, Abe-Dohmae S, Yokoyama S. Apolipoprotein A-I activates protein kinase C alpha signaling to phosphorylate and stabilize ATP binding cassette transporter A1 for the high density lipoprotein assembly. J. Biol. Chem. 2003; 278: 47890–7. 21 Tang C, Vaughan AM, Oram JF. Janus kinase 2 modulates the apolipoprotein interactions with ABCA1 required for removing cellular cholesterol. J. Biol. Chem. 2004; 279: 7622–8. 22 Law SW, Gray G, Brewer HB Jr. cDNA cloning of human apoA-I: amino acid sequence of preproapoA-I. Biochem. Biophys. Res. Commun. 1983; 112: 257–64. 23 Rubin EM, Krauss RM, Spangler EA, Verstuyft JG, Clift SM. Inhibition of early atherogenesis in transgenic mice by human apolipoprotein AI. Nature 1991; 353: 265–7. 24 Plump AS, Scott CJ, Breslow JL. Human apolipoprotein A-I gene expression increases high density lipoprotein and suppresses atherosclerosis in the apolipoprotein E-deficient mouse. Proc. Natl Acad. Sci. U. S. A. 1994; 91: 9607–11. 25 Benoit P, Emmanuel F, Caillaud JM et al. Somatic gene transfer of human ApoA-I inhibits atherosclerosis progression in mouse models. Circulation 1999; 99: 105–10. 26 Belalcazar LM, Merched A, Carr B et al. Long-term stable expression of human apolipoprotein A-I mediated by helper-dependent adenovirus gene transfer inhibits atherosclerosis progression and remodels atherosclerotic plaques in a mouse model of familial hypercholesterolemia. Circulation 2003; 107: 2726–32. 27 Ohnsorg PM, Mary JL, Rohrer L, Pech M, Fingerle J, von Eckardstein A. Trimerized apolipoprotein A-I (TripA) forms lipoproteins, activates lecithin: cholesterol acyltransferase, elicits lipid efflux, and is transported through aortic endothelial cells. Biochim. Biophys. Acta 2011; 1811: 1115–23. 28 Song X, Fischer P, Chen X, Burton C, Wang J. An apoA-I mimetic peptide facilitates off-loading cholesterol from HDL to liver cells through scavenger receptor BI. Int. J. Biol. Sci. 2009; 5: 637–46. 29 Oram JF, Lawn RM, Garvin MR, Wade DP. ABCA1 is the cAMP-inducible apolipoprotein receptor that mediates cholesterol secretion from macrophages. J. Biol. Chem. 2000; 275: 34508–11. 30 Abe-Dohmae S, Suzuki S, Wada Y, Aburatani H, Vance DE, Yokoyama S. Characterization of apolipoprotein-mediated HDL generation induced by cAMP in a murine macrophage cell line. Biochemist 2000; 39: 11092–9. 31 Cignarella A, Engel T, von Eckardstein A et al. Pharmacological regulation of cholesterol efflux in human monocyte-derived macrophages in the absence of exogenous cholesterol acceptors. Atherosclerosis 2005; 179: 229–36. 32 Li C, Kong Y, Wang H et al. Homing of bone marrow mesenchymal stem cells mediated by sphingosine 1-phosphate contributes to liver fibrosis. J. Hepatol. 2009; 50: 1174–83. 33 Cui W, Chen SL, Hu KQ. Quantification and mechanisms of oleic acid-induced steatosis in HepG2 cells. Am. J. Transl. Res. 2010; 2: 95–104. 34 Fu S, Watkins SM, Hotamisligil GS. The role of endoplasmic reticulum in hepatic lipid homeostasis and stress signaling. Cell Metab. 2012; 15: 623–34. 35 Yang Q, Sarnow P. Location of the internal ribosome entry site in the 5′ non-coding region of the immunoglobulin heavy-chain binding
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protein (BiP)mRNA: evidence for specific RNA-protein interactions. Nucleic Acids Res. 1997; 25: 2800–7. Damiano F, Alemanno S, Gnoni GV, Siculella L. Translational control of the sterol-regulatory transcription factor SREBP-1 mRNA in response to serum starvation or ER stress is mediated by an internal ribosome entry site. Biochem. J. 2010; 429: 603–12. Yang Y, Jiang Y, Wang Y, An W. Suppression of ABCA1 by unsaturated fatty acids leads to lipid accumulation in HepG2 cells. Biochimie 2010; 92: 958–63. Hamilton RL, Moorehouse A, Havel RJ. Isolation and properties of nascent lipoproteins from highly purified rat hepatocytic Golgi fractions. J. Lipid Res. 1991; 32: 529–43. Chisholm JW, Burleson ER, Shelness GS, Parks JS. ApoA-I secretion from HepG2 cells: evidence for the secretion of both lipid-poor apoA-I and intracellularly assembled nascent HDL. J. Lipid Res. 2002; 43: 36–44.
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40 Lebherz C, Sanmiguel J, Wilson JM, Rader DJ. Gene transfer of wild-type apoA-I and apoA-I Milano reduce atherosclerosis to a similar extent. Cardiovasc. Diabetol. 2007; 6: 15. 41 Malhi H, Gores GJ. Molecular mechanisms of lipotoxicity in nonalcoholic fatty liver disease. Semin. Liver Dis. 2008; 28: 360–9. 42 Malhi H, Kaufman RJ. Endoplasmic reticulum stress in liver disease. J. Hepatol. 2011; 54: 795–809. 43 Werstuck GH, Lentz SR, Dayal S et al. Homocysteine-induced endoplasmic reticulum stress causes dysregulation of the cholesterol and triglyceride biosynthetic pathways. J. Clin. Invest. 2001; 107: 1263–73. 44 Shimano H, Yahagi N, Amemiya-Kudo M et al. Sterol regulatory element-binding protein-1 as a key transcription factor for nutritional induction of lipogenic enzyme genes. J. Biol. Chem. 1999; 274: 35832–9.
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doi:10.1111/jgh.12430
H E PAT O L O G Y
Apolipoprotein A-I and adenosine triphosphate-binding cassette transporter A1 expression alleviates lipid accumulation in hepatocytes Wei Liu,*,1 Ling Qin,†,1 Hao Yu,* Fangqiao Lv* and Yutong Wang* *Department of Cell Biology, Municipal Laboratory for Liver Protection and Regulation of Regeneration, School of Basic Medical Sciences and † Biomedical Information Center of Beijing Youan Hospital, Capital Medical University, Beijing, China
Key words apolipoprotein A-I, ATP-binding cassette transporter, non-alcoholic steatohepatitis. Accepted for publication 11 September 2013. Correspondence Dr Yutong Wang, Department of Cell Biology, Capital Medical University, 10 You An Men Wai Xi Tou Tiao, Beijing 100069, China. Email: [email protected] 1
These authors contributed equally to this work.
Abstract Background and Aim: Abnormal lipid metabolism may contribute to the pathogenesis of non-alcoholic steatohepatitis. ATP-binding cassette transporter A1 (ABCA1) mediates the transport of cholesterol and phospholipids from cells to high density lipoprotein apolipoproteins. The lipidation of apolipoprotein A-I (apoA-I) by ABCA1 is the ratelimiting step in reverse cholesterol transport and the generation of plasma high density lipoprotein. Here, we examined the effect of apoA-I or ABCA1 overexpression on hepatic lipid levels in BEL-7402 cells. Methods: Human ABCA1 or apoA-I was overexpressed in BEL-7402 hepatocytes by transfection and human apoA-I was overexpressed via adenoviral vector in C57BL/6J mice with MCD diet. Results: Overexpression of either apoA-I or ABCA1 resulted in an increase in cholesterol efflux and a decrease in cellular fatty acids and triglycerides. However, after repression of ABCA1 by its siRNA, overexpression of apoA-I failed to decrease both cellular fatty acids and triglycerides. ApoA-I or ABCA1 overexpression also resulted in a decrease in the expression of the endoplasmic reticulum stress-related proteins GRP78 and SREBP-1. Overexpression of apoA-I in mice also reduced hepatic lipid levels. Conclusions: Expression of apoA-I or ABCA1 can reduce steatosis by decreasing lipid storage in hepatocytes through lipid transport and may also reduce endoplasmic reticulum stress, further lessening hepatic steatosis.
Introduction Non-alcoholic fatty liver disease is a recently emerging obesityrelated disorder characterized by fatty infiltration of the liver in the absence of chronic alcohol consumption. This disorder is characterized by macrovesicular steatosis and has been increasingly recognized as the hepatic manifestation of insulin resistance and metabolic syndrome.1–3 Some patients with steatosis develop superimposed necroinflammatory activity with a non-specific inflammatory infiltrate, lobular inflammation, and hepatic cellular ballooning, called non-alcoholic steatohepatitis (NASH).4 The current working model for the pathogenesis of NASH is the “twohit” hypothesis,5 in which the first “hit” is steatosis and the second “hit” is oxidative stress and inflammation, ultimately resulting in NASH.6 More recent work has revealed that natural killer T-cell deficiency,7 regulatory T-cell dysregulation,8 or intestinal microbiota modification9 may also be involved in the second hit. Excessive accumulation of lipids within hepatocytes has been considered an important factor for hepatic steatosis and inflammation;10 therefore, efficient transportation or delivery of lipids 614
may be key to NASH prevention and treatment. Lipidation of apolipoprotein A-I (apoA-I) by ATP-binding cassette transporter A1 (ABCA1) is the rate-limiting step in reverse cholesterol transport and the generation of plasma high density lipoprotein (HDL).11,12 ABCA1 is an integral membrane protein that mediates the transport of cellular cholesterol and phospholipids to apoA-I to generate nascent HDL particles.13,14 The transcription of the ABCA1 gene is regulated by nuclear liver X receptors (LXRα and LXRβ) and retinoid X receptor15,16 Previous reports suggest posttranslational regulation plays a key role in ABCA1 expression.17 ApoA-I increases ABCA1 protein stability by decreasing the phosphorylation of the PEST sequence and reducing calpain-mediated proteolysis of ABCA1.18 The interaction of apoA-I with ABCA1 may also activate various signaling pathways, including protein kinase A,19 protein kinase C,20 and Janus kinase 2.21 ApoA-I is composed of 243 amino acids, including 10 amphipathic helices,22 and functions as the primary protein component of HDL, which accepts cellular cholesterol and phospholipids transported by ABCA1 as the initial step of reverse cholesterol transport. Liver-specific overexpression of apoA-I
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substantially reduces the progression of atherosclerosis in mice.22,23 Furthermore, adenoviral vector-mediated gene transfer of apoA-I to the liver reduces the progression24,25 and induces the regression26 of atherosclerosis. Through the use of apoA-I mimetics or apoA-I-based infusion therapies in animal models, apoA-I has been shown to mediate a number of important effects on reverse cholesterol transport. In addition to promoting cholesterol efflux, ABCA1, apoA-I, and apoA-I mimetics can enhance other aspects of reverse cholesterol transport, such as the activation of lecithin cholesterol acyltransferase27 and off-loading of cholesterol to the liver via the scavenger receptor B-1.28 Previous studies of apoA-I and ABCA1 have mainly focused on the relationship of these proteins with cardiovascular diseases; however, their role in the pathogenesis of NASH as a lipid transporter remains poorly understood. In this study, we examined the effect of apoA-I and ABCA1 on lipid content in the human hepatocyte cell line BEL-7402. Overexpression of apoA-I and ABCA1 significantly reduced cellular cholesterol, triglycerides, and fatty acids as well as markers of endoplasmic reticulum (ER) stress such as GRP78 and SREBP1. These results suggest that an increased supply of apoA-I and ABCA1 could promote clearance of excess cholesterol and phospholipids from hepatocytes and may be beneficial for the prevention and treatment of NASH.
Methods Materials. BEL-7402 cells were obtained from the American Type Culture Collection. The total RNA extraction reagent TRIzol, a reverse transcription kit, Dulbecco’s Modified Eagle’s medium (DMEM), and fetal bovine serum were acquired from Invitrogen (Carlsbad, CA, USA). Fatty acid-free bovine serum albumin (BSA), 22(R)-hydroxycholesterol, 9-cis-retinoic acid, and protease inhibitor cocktail were purchased from Sigma (St. Louis, MO, USA). ApoA-I was obtained from Millipore (Billerica, MA, USA). Methanol, butanol, hexane, and isopropanol were acquired from Beijing Modern Eastern Finechemical (Beijing, China). Triton X-100 was purchased from Beijing Solarbio (Beijing, China). Cholesterol, fatty acid, and triglyceride quantification kits were obtained from BioVision (Mountain View, CA, USA). A Prism 7300 sequence detecting system for quantitative reverse transcription–polymerase chain reaction was purchased from Applied Biosystems (Foster City, CA, USA). FuGene HD was obtained from Roche (Basel, Switzerland). Antibodies against ABCA1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Novus Biologicals (Littleton, CO, USA) and Shanghai Kang Chen Biotech (Shanghai, China), respectively. Antibodies against apoA-I, GRP78, and SREBP-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cell culture and cholesterol efflux. BEL-7402 cells were maintained in DMEM containing 10% fetal bovine serum or were incubated in serum-free DMEM plus 1 mg/mL fatty acid-free BSA (DMEM/BSA). Washed cells were incubated for 16 h with medium containing 5 mg/mL BSA in the presence or absence of 250 μM fatty acids (molar ratios to BSA of 1.8 and 0). Fatty acids were added from a stock solution in which they were bound to
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BSA at a 3.5 molar ratio and were adjusted to lower ratios by adding fatty acid-free BSA. To induce ABCA1 expression, 10 μM 22(R)-hydroxycholesterol and 10 μM 9-cis-retinoic acid were added in combination to this medium. To measure cholesterol efflux, cells were incubated with DMEM/BSA with or without 10 μg/mL apoA-I at 37°C for 4 h and chilled on ice. The medium was then collected and centrifuged to remove detached cells. Cholesterol was extracted from the medium and the cells with methanol/chloroform (1:2) and hexane/ isopropanol (1:1), respectively. Cholesterol content was assayed following the kit manufacturer’s guide. ApoA-I-mediated cholesterol efflux is expressed as the fraction of cholesterol released into the medium after subtraction of the values obtained in the absence of apoA-I. Western blot. Cells were washed and dislodged from the dish at 0°C in buffer containing protease inhibitors. Cellular proteins were solubilized in phosphate-buffered saline (PBS) containing 1% Triton X-100 plus protease inhibitors and resolved by sodium dodecylsulfate–polyacrylamide gel electrophoresis. The expression of apoA-I, ABCA1, and GAPDH was identified by Western blot analysis using antibodies against ABCA1 and GAPDH, respectively.29 Determination of fatty acids and triglycerides. Lipid mass analysis was conducted as previously described.30 BEL-7402 cells were cultured in 6-well plates at 80% confluency in DMEM. Total cellular lipids were extracted by hexane/ isopropanol (1:1). The chloroform/methanol extract was backextracted with water to remove contaminating proteins. The amounts of free fatty acids and triglycerides were measured by colorimetric enzyme assays using test reagents for serum lipids according to the manufacturer’s protocol and normalized with total cellular protein. Plasmid transfection. The ABCA1 shRNA plasmid and control plasmid were designed and constructed by Shanghai GenePharma Co. (Shanghai, China). CMV-ABCA1, pcDNA3.0/ apoA-I, shABCA1, and the control plasmid were transfected into BEL-7402 hepatocytes using FuGene HD as described in the manufacturer’s protocol. The target sequence of the ABCA1 shRNA was as follows: 5′-GGAACTGGACAATGCAGAACC-3′. Quantitative real-time polymerase chain reaction. Total RNA from BEL-7402 hepatocytes was extracted using TRIzol. Two micrograms of total RNA were reverse transcribed into cDNA using Superscript II Reverse Transcriptase. Gene expression was quantified using a SYBR-Green PCR kit with 18S rRNA as the control. Quantitative reverse transcription– polymerase chain reaction was performed with a Prism 7300 sequence detecting system. The primer sequence has been described previously.31,32 Oil Red O staining. BEL-7402 cells were cultivated on cover slips in 24-well plates at 10 000 cells/well with 5 mg/mL BSA in the presence or absence of 250 μM oleic acid (OA) for 24 h. Following cultivation, the medium was aspirated, the cells were rinsed twice with PBS, and staining with Oil Red O (Sigma) was performed. Cells were washed with PBS and fixed with 3.7%
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formaldehyde for 1 h. Then, 0.3% Oil Red O in isopropanol was added directly to the fixed cells and incubated for 1 h. The microscopy studies were performed with a microscope equipped with a digital camera (Leica, Wetzlar, Germany). Animal studies. C57BL/6J mice were purchased from Academy of Military Medical Sciences (Beijing, China). Male mice 4–6 weeks old were used in these studies. Before fed with methionine choline-deficient (MCD) diet, mice were injected with 1 × 108 pfu/mL adenoviral vector containing human apoA-I (ad-apoA-I) or vector alone (ad-Null) the femoral vein. The mice were divided into four groups and were fed with different diet for 1 week with or without the injection of adenoviral vector. Histological analysis of tissue samples. At the end of each experiment, mice were sacrificed and liver was collected and stored either at −80°C or fixed in 10% formalin or made frozen section. Four-micrometer thick sections were obtained from the formalin-fixed paraffin-embedded tissue for further histological analyses. Conventional hematoxylin and eosin histological staining was performed in order to evaluate the microscopic morphology of the liver tissue samples. Ten micrometer sections were obtained from frozen tissue for further oil red staining in order to evaluate the lipid levels in liver. Statistical analysis. The results of multiple observations are presented as the mean ± standard error of the mean. The data were analyzed with the statistics software SPSS 11.5 (SPSS Inc., Chicago, IL, USA) by a non-parametric analysis of variance test. Difference were considered significant if P < 0.05.
Results Cellular lipid accumulation by oleic acid treatment. OA has been reported to induce steatosis in HepG2 cells.33 To examine the effect of OA on BEL-7402 cells, we incubated BEL-7402 cells with 5 mg/mL BSA with or without 250 μM OA for 24 h, followed by Oil Red O staining to examine lipid accumulation. Compared with the control (Fig. 1a), OA treatment resulted in a significant increase in lipid accumulation, as revealed by Oil Red O staining (Fig. 1b). When the content of different lipids was analyzed, the results revealed that 24 h of OA treatment caused a marked increase in the cellular levels of triglycerides, free fatty acids, and cholesterol (Fig. 1c–e, respectively). Alteration of cellular free fatty acids and triglycerides by apoA-I and ABCA1 overexpression. ABCA1 can transport cellular cholesterol and phospholipids to lipid-poor apoA-I to generate nascent HDL.13,14 Thus, the expression levels of apoA-I or ABCA1 may have a significant impact on cellular lipid levels. To test this hypothesis, we altered apoA-I or ABCA1 expression levels in OA-treated BEL-7402 cells by transfecting the cells with ABCA1- or apoA-I-expressing plasmid. Forty-eight hours after transfection, ABCA1 mRNA and protein levels were measured. As expected, transfection of apoA-Iexpressing vector significantly increased cellular apoA-I protein levels (Fig. 1f). And transfection of the ABCA1- or apoA-Iexpressing vector significantly increased ABCA1 protein levels 616
(Fig. 1h) and cholesterol efflux (Fig. 1i) compared with control cells. Consistent with previous reports, apoA-I increased ABCA1 protein levels (Fig. 1h) without affecting its mRNA levels (Fig. 1g), presumably by altering the phosphorylation status of the PEST sequence in ABCA1 to prevent ABCA1 degradation. To evaluate the impact of ABCA1 expression on cellular lipids, the free fatty acid (Fig. 1j) and triglyceride (Fig. 1k) content were also measured. Consistent with the roles of ABCA1 as a cholesterol and phospholipid transporter and apoA-I as a lipid acceptor, when compared with control cells, apoA-I or ABCA1 overexpression caused a significant decrease in cellular-free fatty acids and triglycerides. These data indicate that alterations in apoA-I or ABCA1 expression levels lead to the modification of cellular lipid storage. To eliminate the possibility that apoA-I may alter the cellular lipid profile through mechanisms other than the ABCA1-mediated lipid transport pathway, we used shRNA to specifically inhibit ABCA1 expression. Seventy-two hours after transfection, ABCA1 mRNA and protein levels were quantified, and cholesterol efflux, cellular-free fatty acid levels, and cellular triglyceride levels were also measured. ABCA1 shRNA dramatically reduced ABCA1 mRNA and protein levels (Fig. 2a and b). Compared with the control vector, ABCA1 shRNA abolished the ability of apoA-I to increase cholesterol efflux (Fig. 2c) or decrease the level of cellular-free fatty acids (Fig. 2d) and triglycerides (Fig. 2e). These results suggest that apoA-I promotes cholesterol efflux and reduces cellular lipid levels through the ABCA1-mediated lipid transport pathway. Alteration of GRP78 and SREBP-1 by apoA-I and ABCA1 overexpression. Lipid toxicity may play a key role in ER stress.34 To evaluate the effect of ABCA1/apoA-I expression on ER stress, two proteins related to ER stress, GRP78 and SREBP-1, were measured by Western blot. OA treatment markedly increased the levels of GRP78 (Fig. 3a and b) and the precursor form of SREBP-1 (Fig. 3c and d). These results are consistent with previous reports that ER stress can lead to increased translation of RNAs that contain internal ribosome entry sites, such as GRP78 and SREBP-1.35,36 Upon the expression of apoA-I or ABCA1, the levels of GRP78 and the SREBP-1 precursor decreased significantly, indicating a reduction in ER stress. Moreover, transfection of the apoA-I or ABCA1 expression vector also resulted in a significant decrease in the mature form of SREBP-1 (Fig. 3e). Alteration hepatic lipid levels by apoA-I overexpression in vivo. To test the effect of apoA-I overexpression on hepatic levels in vivo, we injected mice with control or apoA-I expressing vector followed by 1 week of MCD diet. Consistent with the results from cultured cells, expressing of apoA-I in vivo significantly reduced hepatic lipid deposition induced by MCD (Fig. 4A and B) diet as well as hepatic cholesterol (Fig. 4D), triglyceride (Fig. 4E), and fatty acid (Fig. 4F) levels.
Discussion Modulation of ABCA1 activity could have a profound impact on cholesterol and phospholipid transport. Our previous studies
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Figure 1 Apolipoprotein A-I (ApoA-I) or ATP-binding cassette transporter A1 (ABCA1) overexpression alters cellular lipid levels in BEL-7402 cells. BEL-7402 cells were incubated for 24 h in DMEM with 5 mg/mL bovine serum albumin (BSA) in the presence or absence of 250 μM oleic acid (OA). (a) Lipids were stained with Oil Red O, as described in the “Methods”. (b) Twenty-four hours after adding 250 μM OA to the Dulbecco’s Modified Eagle Medium (DMEM) medium, cellular triglycerides (TG) (c), free fatty acids (FFA) (d), and cholesterols (e) were measured as described in the “Methods”. (f–k) BEL-7402 cells were then transfected with control vector with a CMV promoter or CMV-ABCA1 or CMV-apoA-I plasmid. Thirty-six hours after transfection, the cells were incubated in DMEM/BSA media with 10 μM 22(R)-hydroxycholesterol and 10 μM 9-cis-retinoic acid. Forty-eight hours after transfection, (g) ABCA1 mRNA levels were measured by quantitative real-time polymerase chain reaction as described in the “Methods”. Total cellular proteins were isolated, and immunoblot analysis of apoA-I (f) or ABCA1 (h) was conducted as described in the “Methods”. The immunoblot represents the result of a single experiment that is representative of three independent experiments. The immunoblot was quantified for from three independent experiments. Cholesterol efflux (i), cellular free fatty acids (j), and triglycerides (k) were measured as described in the “Methods”. The results are representative of three independent experiments and are presented as the mean ± standard error of the mean. *P < 0.05 versus control, **P < 0.01 versus control.
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Figure 2 ATP-binding cassette transporter A1 (ABCA1) repression alters cellular levels of triglycerides (TG) and free fatty acids (FFA). BEL-7402 cells were incubated for 24 h in Dulbecco’s Modified Eagle Medium (DMEM) with 5 mg/mL bovine serum albumin (BSA) and 250 μM oleic acid (OA). The cells were then co-transfected with a negative control containing a scrambled siRNA sequence, the plasmid pcDNA3.0, and/or the apolipoprotein A-I (apoA-I) expression plasmid. Fifty-six hours after transfection, cells were incubated in DMEM/BSA with 10 μM 22(R)-hydroxycholesterol and 10 μM 9-cis-retinoic acid. (a) Seventy-two hours after transfection, total cellular RNA was isolated, and relative ABCA1 mRNA levels were measured as described in the “Methods”. (b) Cellular proteins were isolated, and immunoblot analysis of ABCA1 was performed as described in the “Methods”. The immunoblot represents the result of a single experiment that is representative of three independent experiments. The immunoblot of ABCA1 was quantified for from three independent experiments. Seventy-two hours after transfection, cellular free fatty acids (c), triglycerides (d), and cholesterol efflux (e) were measured as described in the “Methods”. The results are representative of three independent experiments and are presented as the mean ± standard error of the mean. *P < 0.05 versus control.
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Figure 3 apolipoprotein A-I (ApoA-I) or ATP-binding cassette transporter A1 (ABCA1) expression alters levels of GRP78 and SREBP-1. BEL-7402 cells were incubated for 24 h in Dulbecco’s Modified Eagle Medium (DMEM) with 5 mg/mL bovine serum albumin (BSA) in the presence of absence of 250 μM oleic acid (OA). The cells were then transfected with the control plasmid pcDNA3.0, the ABCA1 expression plasmid, or the apoA-I expression plasmid. Thirty-six hours after transfection, the cells were incubated in DMEM/BSA with 10 μM 22(R)-hydroxycholesterol and 10 μM 9-cis-retinoic acid. Forty-eight hours after transfection, total cellular proteins were isolated, and immunoblot analysis of GRP78 (a) or SREBP-1 (c) was performed as described in the “Methods”. The immunoblot represents the result of a single experiment that is representative of three independent experiments. The immunoblots for GRP78 (b), the precursor form of SREBP-1 (d) or the mature form of SREBP-1 (e) were quantified from three independent experiments. The results are presented as the mean ± standard error of the mean. *P < 0.05 versus control.
revealed that unsaturated fatty acids suppress ABCA1 protein levels in HepG2 cells and that ABCA1 also regulates cellular fatty acid content.37 Here, we provide evidence that this increase in either apoA-I or ABCA1 expression can also effectively regulate cellular fatty acid and triglyceride content and alleviate ER stress. Incubation with OA induced hepatic steatosis in BEL-7402 cells. Under these conditions, hepatic fatty acid and triglyceride content was significantly decreased in cells upon apoA-I or ABCA1 overexpression and markedly increased after inhibition of ABCA1 by RNAi. Protein levels of GRP78 and SREBP-1, two proteins related to ER stress, also increased significantly upon OA treatment and then decreased when apoA-I or ABCA1 was overexpressed. These observations reveal an association between cellular fatty acid/triglyceride levels and hepatic ABCA1/apoA-I, strongly suggesting that upregulation of the ABCA1 pathway by expression of either apoA-I or ABCA1 can effectively reduce hepatic steatosis as well as decrease ER stress. ABCA1 is a large integral membrane protein with a molecular weight greater than 250 kD. Numerous studies have been conducted, almost exclusively in the field of atherosclerosis, to
attempt to promote reverse cholesterol transport by increasing ABCA1 expression. However, overexpression of ABCA1 by transfection for a prolonged period of time (more than 72 h) may cause cell viability problems (unpublished data), presumably because a large number of ABCA1 molecules may cause damage to the plasma membrane due to the large size of ABCA1. Thus, overexpression of apoA-I may be an excellent alternative to promote the ABCA1-mediated reverse cholesterol transport pathway. ApoA-I can not only function as a key lipoprotein to accept cholesterol and phospholipids from ABCA1 but can also stabilize the ABCA1 protein and increase membrane ABCA1 content through different pathways, including altering the phosphorylation status of the PEST sequence in ABCA1 and activating protein kinase A, protein kinase C, and Janus kinase 2.18–21 Although the most important site for the ABCA1/apoA-I interaction is located at the plasma membrane, apoA-I conjugated to nascent lipids has been isolated from endosomes in hepatocytes.38 In HepG2 cells, ∼20% of newly synthesized apoA-I is lipidated intracellularly,39 suggesting that the intracellular interaction between apoA-I and
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Figure 4 apolipoprotein A-I (ApoA-I) expression reduces mice hepatic lipid levels. Hematoxylin and eosin (a) and Oil Red O (b) staining for mice fed with normal diet (panel a), mice fed with MCD (panel b), mice injected with vector (panel c), and mice injected with apoA-I expressing vector (panel d). (c) Total hepatic cellular proteins were isolated, and immunoblot analysis of apoA-I was conducted as described in the “Materials and methods”. Hepatic cholesterol (d), triglyceride (TG) (e), and fatty acid (FFA) (f) levels were measured as described in the “Methods”. The results are representative of three independent experiments and are presented as the mean ± standard error of the mean. *P < 0.05 versus control.
ABCA1 may also play an important role. Although the mechanisms involved are complex, the ability of apoA-I overexpression to reduce atherosclerosis has been observed in mice.40 In our studies, expression of apoA-I also significantly reduced cellular fatty acid and triglyceride content. Hepatic steatosis is the initial stage of NASH development. NASH is associated with suboptimal protein secretion by hepatocytes and induction of the unfolded protein response, an indicator of ER stress.41 Two hypotheses have been proposed to explain this finding: (i) lipid accumulation in hepatocytes causes unfolded protein response activation and ER dysfunction due to lipotoxicity;42 and (ii) ER dysfunction and unfolded protein accumulation precedes steatosis, causing the disease.41 However, both hypotheses may explain the pathogenesis of NASH, that is lipotoxicity promotes ER dysfunction, and ER dysfunction promote steatosis, thus forming a vicious cycle. ApoA-I/ABCA1 expression can promote lipid transport and effectively reduce cellular lipid content (Fig. 1). As a result, apoA-I/ABCA1 expression may reduce ER stress and thus reduce the levels of ER stressrelated proteins such as GRP78 and SREBP-1 (Fig. 3). Because ER stress can promote hepatic steatosis through the activation of SREBP-1,43 the reduction of cellular lipid levels may also affect 620
SREBP-1 activation. As expected, the mature form of SREBP-1 increased significantly after 24 h of OA treatment and then decreased to the level prior to OA treatment when apoA-I or ABCA1 was overexpressed. This evidence indicates that expression of apoA-I or ABCA1 can decrease cellular lipid levels and exhibit beneficial effects by reducing ER stress. Eventually, the reduction of ER stress may further reduce steatosis, creating a positive cycle. Since SREBP-1 can regulate the transcription of lipogenic genes such as fatty acid synthase and acetyl-CoA carboxylase,44 reduction in SREBP-1 may cause a decrease in the expression of these genes. As a result, the reduced level of fatty acid synthesis may then affect the levels of triglycerides, which may also contribute to overall reduction of lipid accumulation caused by apoA-I. This study has important therapeutic implications for treating NASH. By mediating the expression of apoA-I and ABCA1, levels of ER stress as well as cellular lipids including cholesterol, triglycerides, and fatty acids may be significantly reduced. As a result, the ABCA1/apoA-I pathway lipid-removal pathway may be a feasible means of NASH prevention and treatment. Understanding the mechanisms by which apoA-I reduces cellular lipid levels through the ABCA1-mediated pathway will be useful for
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designing therapeutic interventions that enhance the activity of this lipid-removal pathway and prevent the development of NASH.
Acknowledgments This work was supported by the National Natural Science Foundation of China (30740094, 30871223) and the Beijing Nova Program (2007B064).
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