© 2013, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(13)60099-5

 

 

Contributors: XGQ proposed the study. QY, LS and CHT performed research and wrote the first draft. QY collected and analyzed the data. YCH provided the scientific guide. TXD and YHT participated part of the research. All authors contributed to interpretation of the study and to further drafts. XGQ is the guarantor.

Funding: This study was supported by a grant from the National Natural Science Foundation of China (491010-N11026).

Ethical approval: The animals were subjected to experimental protocols adhered to ethical standards and under the care of animal and licensing guidelines.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

 

 

 

 

 

(Hepatobiliary Pancreat Dis Int 2013;12:630-636)

 

KEY WORDS: non-alcoholic fatty liver disease; caveolin-1; scavenger receptor class B type 1; pathogenesis; high fat and cholesterol diet

Non-alcoholic fatty liver disease (NAFLD) as a clinical syndrome without excess alcohol intake is characterized pathologically by such symptoms as liver steatosis. The prevalence of this disease increasingly exceedings that of hepatitis B, hepatitis C and alcoholic liver disease. It has been the most common liver disease. However, the mechanisms involving in the pathogenesis of NAFLD have not been thoroughly investigated.

 

Caveolin-1 (cav1) is the main structural protein of caveolae, which has now emerged as a plasma membrane protector, organizer and sensor that can respond to plasma membrane stresses and remodel the extracellular environment.^[1] Recent studies^[2-4] also identified the role of cav1 in cholesterol transport, lipogenesis and lipolysis. However, how cav1 implicates in the pathogenesis of NAFLD has not been clarified. Reports^[5,6] have shown that upregulation of cav1 enhances the oxidized low density lipoprotein (LDL) absorption of HepG2 cell markedly, and that downregulation of cav1 inhibits HepG2 uptake of long chain fatty [³H]-oleic acid. Although the involvement of cav1 in lipogenesis has been investigated, the role of cav1 in mice with NAFLD has rarely been found.

 

Recent evidences have shown that scavenger receptor class B type 1 (SR-B1) ameliorates hepatic lipid metabolism disorder.^[7] Moreover, SR-B1 is also implicated in plasma lipid metabolism. Apart from the surface receptor of high density lipoprotein (HDL) in the liver, SR-B1 binds to the receptors of non-HDL, i.e. very low density lipoprotein (VLDL) and LDL. Hoekstra et al^[8] found that SR-B1 regulated plasma VLDL and LDL levels in mice. But the role of SR-B1 in the formation of lipid droplets in NAFLD is still controversial.^{[9, 10]} Matveev and coworkers^[11] showed that cav1 is a negative regulator of SR-B1-dependent selective cholesteryl ester uptake.

 

Our study aimed to determine the mRNA and protein expressions of cav1 and SR-B1 in the liver of NAFLD mouse induced by HFC diet. We also detected the distribution associations of cav1 and SR-B1 by immunohistochemistry.

 

 

Eight-week C57BL/6 male mice were obtained from Shanghai Laboratory Animal Center (Shanghai, China). The mice were kept under room temperature (22 ��) and constant light-dark cycles with free access to water and normal diet (<0.02% cholesterol). Twelve mice were fed on a high fat and cholesterol (HFC) diet consisting of 15% fat and 1.25% cholesterol after receiving two-week normal diet, while another six mice were still fed with normal diet (control). Both normal diet and HFC chaw were purchased from the Zhejiang Academy of Medical Science (Hangzhou, China). All mice were subjected to experimental protocols adhered to ethical standards and under the care of animal and licensing guidelines. Samples were collected between 9 and 10 AM at the end of the dark phase of the diurnal cycle.

 

Standard molecular biological techniques were applied. TRIzol reagent was purchased from Invitrogen™(Carlsbad, CA, USA). ReverTra Ace qPCR RT Kit and Thunderbird SYBR qPCR Mix were obtained from TOYOBO (Osaka, Japan). Primers were designed by AlleleID 7.0 and synthesized by Sangon Biotech Co., Ltd., (Shanghai, China). Total protein extraction kit was also purchased from Sangon Biotech Co., Ltd. Rabbit monoclonal antibody to glyceraldehyde phosphate dehydrogenase (GAPDH) and rabbit antibody to cav1 were purchased from Cell Signaling Technology (Beverly, MA, USA). Rabbit antibody to SR-B1 and goat anti-rabbit IgG-HRP (horseradish peroxidase) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PageRuler prestained protein ladder was purchased from Fermentas Life Science (Shanghai, China). Diaminobenzidine was provided by Dr. HT Yao from the First Affiliated Hospital, Zhejiang University School of Medicine, China. Unless previous indicated, materials were obtained from Amresco (Ohio, USA) or BBI (Nova Scotis, Canada). Micro BCA™ protein assay kit was purchased from Thermo Pierce Scientific (Shanghai, China).

 

All mice fasted overnight were anesthetized with 4% chloral hydrate (8 mL/kg body weight, intraperitoneally) after 14-week feeding with HFC diet or normal diet (control). Blood was collected from the inferior vena cava, centrifuged at 3000 rpm for 8 minutes at room temperature and finally the supernatant was collected. The plasma samples were analyzed immediately. The harvested liver was weighed, cut into pieces, washed extensively in cold phosphate-buffer saline (PBS) and aliquoted. Part of the aliquots was snap-frozen in liquid nitrogen and stored at -80 �� until use, and part was fixed in formaldehyde solution.

 

Once the plasma of all mice was collected, it was aliquoted into special test tube with a fixed volume, and mounted to an automated analysis equipment (Hitachi 7600, Japan) which has been set parameters according to lipid test kit protocols, such as triglyceride test reagent kit, cholesterol test kit, and so on.

 

Total RNA was extracted from control and HFC liver according to TRIzol reagent protocol. The concentration of total RNA was measured using a Nanodrop Spectrophotometer 2000 (Thermo Scientific, USA). About 0.5 µg RNA was subjected to reverse transcription using ReverTra Ace qPCR Kit. Thunderbird SYBR qPCR Mix was used in a 7500 instrument (ABI Real-time system, USA) according to the manufacturer's instructions. The primers used are listed in Table 1. The expression of each gene of interest was normalized with the endogenous control, β-actin (2^-??Ct method).

 

Total protein was isolated from the liver according to the protocol of Sangon Biotech Co., Ltd. Protein concentrations were measured using BCA assay. Equal quantities of protein (20 µg) were subjected to 12% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were electrophoretically transferred onto 0.45 µm polyvinylidene difluoride membranes. The membranes were blocked overnight at 4 �� in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST) and 5% skim milk. After five-time wash in TBST, antigen detection was carried out using a 1:500 dilution of a rabbit polyclonal antibody against SR-B1 or monoclonal antibody against cav1 for 4 hours. After five-time wash, the membrane was incubated with HRP-labeled secondary antibodies for one hour; after another five-time wash, the immune-reactive proteins were detected with Immobilon™ Western Chemiluminescent HRP Substrate (Millipore, USA) and VersaDoc Imaging System IM5900 (Bio-Rad, Germany) and quantified by scanning densitometry. To show equal protein loading, the blots were probed with a 1:2000 dilution of rabbit monoclonal antibody to GAPDH as a control.

 

Mouse liver samples fixed in 10% buffered formalin solution were embedded in paraffin. Three-µm sections were cut and stained with hematoxylin and eosin (HE), and histological changes including steatosis were analyzed. As reported,^[12] steatosis was graded as follows: mild (less than 33% of hepatocytes), moderate (33%-66% of hepatocytes), and severe (more than 66% of hepatocytes). The rest sections were used for immunohisto­chemical analysis. They were rehydrated and treated with 0.3% H₂O₂ in PBS to block endogenous peroxidase activity, followed by blocking in 5% skim milk. After several time wash in PBS, the sections were incubated with mouse monoclonal antibody against mouse cav1 (BD Transduction Technology, USA) or rabbit polyclonal antibody against SR-B1 diluted 1:100 in blocking buffer at 4 ��. After several rinses in PBS, peroxidase-labeled secondary antibody at 1:20 dilution was used for 1 hour at room temperature. The activity of peroxidase was observed with diaminobenzidine, yielding a yellow-brown deposit. Negative controls included sections with the primary antibodies omitted. Three random sections of each liver were examined.

 

Statistical analysis was performed using Student's t test or nonparametric test with the SPSS 17.0 software. The results of this study were reported as mean±SD. Statistical significance was considered as a two-tailed alpha level of P<0.05.

 

 

There was no mortality during the whole experimental process. On HFC diet, livers of mice appeared to be enlarged, pale and fatty. Compared with the normal diet, HFC chaw increased the liver/body weight ratio significantly (Fig. 1).

HE staining showed liver steatosis in mice fed with HFC diet. The mice fed with HFC diet for 14 weeks developed NAFLD, i.e. moderate NAFLD (steatosis) in 3 mice and severe steatosis in 9 (Fig. 2). No abnormal histological changes were observed in the livers of the control mice.

 

Plasma lipid concentration was measured by an automatic biochemical analyser. No differences of plasma triglyceride were observed between the two groups (0.719±0.383 vs 0.710±0.487 mmol/L, P>0.05) (Table 2). However, total cholesterol level of NAFLD mice was 3.771±0.804 mmol/L and significantly higher than that of the control mice (1.940±0.300 mmol/L). The levels of plasma HDL-cholesterol (HDL-C) and LDL-cholesterol (LDL-C) of NAFLD mice increased compared with those of the control mice (HDL-C: 2.224±0.428 vs 1.120±0.066 mmol/L, P<0.01; LDL-C: 1.241±0.663 vs 0.510±0.191 mmol/L, P<0.01).

 

qPCR revealed that cav1 and SR-B1 mRNA transcriptions were significantly increased in the liver of NAFLD mice, with a 1.57-fold increase in cav1 (1.536±0.226 vs 0.980±0.272, P<0.05) and a 1.44-fold increase in SR-B1 (1.377±0.125 vs 0.956±0.151, P<0.05) (Fig. 3).

 

Western blotting analysis demonstrated that the hepatic expression of cav1 protein in NAFLD mice increased by 5.43 times (0.643±0.240 vs 0.100±0.130, P<0.01) (Fig. 4A). In agreement with upregulation of SR-B1 mRNA expression as described above, SR-B1 protein expression also increased significantly (2.156±0.507 vs 0.211±0.211, P<0.01) (Fig. 4B).

 

The distributions of cav1 and SR-B1 in the liver were investigated by immunohistochemistry. The expression of cav1 and SR-B1 in NAFLD mice was significantly higher than that in the control mice (Fig. 5). In the mice fed with normal diet, however, cav1 positivity was only found in the baso-lateral membrane of hepatocytes but not in the cytoplasm of hepatocytes (Fig. 5A). The distribution of SR-B1 was the same as that of cav1 (Fig. 5E). In the fatty liver, the immunoreactivity of cav1 (Fig. 5B, D) and SR-B1 (Fig. 5F, G) was shown not only in the plasma membrane of hepatocytes, but also in cytoplasm and membrane of lipid droplets. Furthermore, the density of cav1 and SR-B1 in NAFLD mice was in conformity with the severity of steatosis and the immunoreactivity of cav1 and SR-B1 in zone 3 where fatty liver is more serious in the hepatic acini than that in the other two zones (Fig. 5C, D and 5C, G, respectively).^[13]

 

 

NAFLD is characterized by abnormal accumulation of intrahepatic lipid without excessive alcohol intake and has a potential risk of evolution to non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), hepatic fibrosis, cirrhosis and even hepatocellular carcinoma.^[14] With an increasing morbidity, NAFLD has been a worldwide health problem. Clinically, NAFLD is always accompanied by other abnormalities such as insulin resistance, visceral obesity, type 2 diabetes mellitus and dyslipidaemia.^[15] Meanwhile, animal model has demonstrated a pivotal role of the liver in the development of metabolic syndrome.^[16] Therefore, NAFLD is considered as a hepatic manifestation of the metabolic syndrome.

 

This study has successfully established a mouse model of NAFLD through persistent feeding of the C57BL/6 mice with HFC diet for 14 weeks. The HFC diet-induced mouse model of NAFLD mimics human NAFLD in etiology, biochemistry and pathology. The 14-week feeding is based on our pilot data and the literature.^[17]

 

After 14-week HFC diet was given to the C57BL/6 mice, plasma concentrations of triglyceride, cholesterol, HDL-C, and LDL-C were determined. Among these variables, cholesterol, HDL-C and LDL-C were increased significantly. These data indicate that cholesterol, HDL-C and LDL-C may be related to the pathogenesis of NAFLD. Our results are consistent with other studies.^{[18, 19]} It is known that lipid metabolism starts with the intestinal absorption of dietary lipids. Lipids are emulsified and hydrolyzed within the lumen. Hydrolyzed lipids are then absorbed by enterocytes, secreted into the lymphatic system, where they bypass the liver and enter the systemic circulation. Serum cholesterol is mainly carried by LDL, which can be recognized by apoB-100 receptor in the hepatocyte surface and processed by lysosome and hydrolyzed into cholesterol and fatty acid. Hydrolyzed cholesterol has an important regulatory role in the intracellular cholesterol metabolism, including the formation of cellular membrane. One of its regulatory roles is to decrease cellular uptake of LDL by inhibiting LDL receptor gene transcription to down-regulate its protein expression. Using rat as an animal model, Wang et al^[20] and Xin et al^[9] demonstrated that LDL receptor was decreased in NAFLD rats, indicating the impaired re-uptake of LDL in circulation. Furthermore, SR-B1 could influence plasma LDL-C through VLDL remnant metabolism when LDL receptor is low.^[18] Matveev et al^[11] found that cav1 inhibited hepatic uptake of cholesterol from HDL. Truong et al^[21] revealed that high serum HDL-C stimulated the expression of cav1 and SR-B1, which have a synergistic effect.

 

Blouin et al^[22] proved that cav1 concentration on adipocytes lipid droplets is positively related to the size of lipid droplets in obese mice models and human adipocytes. In addition, recent studies^{[23, 24]} have established the role of cav1 in lipid droplets formation in the liver, indicating that cav1 is indispensable to the formation of lipid droplet and indirectly inferring that cav1 should be up-regulated in NAFLD. Our results confirmed the significant increase of cav1 both in mRNA and protein levels in the NAFLD mice.

 

The upregulation of cav1 in the liver was also displayed by immunohistochemistry. Cav1 was widely spread in the plasma membrane, cytoplasm of hepatocytes and membrane of lipid droplets. Whether upregulation of cav1 in liver cytoplasm is related to the elevated cav1 expression in the organelles like mitochondria and endoplasmic reticulum (ER) is still unknown. However, Mastrodonato et al^[25] reported that intracytoplasmic immunoreactivity for cav1 was present mostly in the baso-lateral membrane of hepatocytes, inner mitochondrial membrane, micro/macrovesicles, and lipid droplets. To our knowledge, lipid droplets consist of a core of apolar lipids encapsulated in a single monolayer of phospholipids and cholesterol^[26] associated with a limited group of proteins including cav1.^[22] Cav1 is required to maintain the cholesterol content of lipid droplets, and increased cholesterol level stimulated the translocation of cav1 to lipid droplets, which is in agreement with other studies showing that cav1 is indispensable to lipid droplet size^[22] and that cav1 can be relocated between plasma membrane and lipid droplets.^[27] Blouin et al^[22] explained the trafficking of cav1 in aberrant situation in detail, that is, when stimulated by cholesterol, cav1 synthesized in the ER and stored in Golgi could be transferred to plasma membrane. Only when the cells are loaded with lipid especial for fatty acid, cav1 in the plasma membrane can be translocated to lipid droplet. Our results showed that HFC diet increased blood cholesterol, resulting in cav1 transfer from Golgi complex to plasma membrane. Wang and colleagues^[20] demonstrated the increase of fatty acid in NAFLD, which led to the relocation of cav1 to lipid droplet. Pol et al^[27] found that cav1 was up-regulated and redistributed from the cell surface to the newly formed lipid bodies in a partial hepatectomy model. These data support the cav1 translocation of liver cytoplasma, plasma membrane, and the membrane of lipid droplets.

 

Cav1 immunoactivity in the present study also showed zonal differences: the more serious the fatty liver, the stronger the cav1 immunoactivity. These differences strongly indicate that cav1 positive expression is associated with the severity of hepatic steatosis.

 

SR-B1 is also involved in cholesterol transportation. It is a cell surface HDL receptor that mediates selective uptake of the lipid from HDL, which is an important process in reverse cholesterol transport in hepatocytes.^[7] Thus, SR-B1 is thought to be an anti-NAFLD receptor. Xin^[9] and Jourdan^[28] revealed that the upregulation of SR-B1 may ameliorate NAFLD. But we did not find this phenomenon. We speculated that inconsistent results may be due to the following reasons. First, it might be related to unsaturated fatty acid exposure. A study showed that administration of polyunsaturated fatty acids increases hepatic SR-B1 levels in hamster.^[29] And the HFC diet increases the serum polyunsaturated fatty acids.^[29] Second, hepatic steatosis is an intricate pathological process. But Inoue et al^[30] demonstrated that high fat diet increases the expression of PPAR-γ markedly in the fatty liver and that the activity of PPAR-γ could up-regulate SR-B1.^{[31, 32]} Third, the upregulation of SR-B1 mRNA and protein might be a confrontation response to NAFLD.

 

Surprisingly, our immunohistochemistry of SR-B1 showed the same distribution as cav1, that is, increased SR-B1 protein located in the plasma membrane of hepatocytes, cytoplasm and the membrane of lipid droplets. Our results are consistent with the recent study showing that SR-B1 localized in caveolae, and interestingly SR-B1 colocalized with cav1.^[33] Meanwhile, SR-B1 positive expression is also associated with the severity of hepatic steatosis. In the present study we found the similar gene, protein and distribution changes of cav1 and SR-B1 in NAFLD, showing that they are possibly associated with the formation of NAFLD. Frank et al^[34] demonstrated that SR-B1 stabilizes the cav1 protein, while cav1 has little effect on SR-B1 stability, implying that SR-B1 plays a role in the regulation of cav1 function.

 

In conclusion, the results of this study showed that HFC diet could induce a NAFLD model in mice and increase the plasma levels of cholesterol, HDL-C and LDL-C significantly. The levels of cav1 and SR-B1 mRNA and protein are increased significantly in fatty liver induced by HFC diet. Cav1 and SR-B1 are mainly located in the plasma membrane of hepatocytes, cytoplasm and the membrane of lipid droplets, and their expressions are associated with the severity of hepatic steatosis. All of these indicate that cav1 and SR-B1 may play pivotal roles in the formation of NAFLD. Therefore, cav1 and SR-B1 might be potential therapeutic targets in the treatment of NAFLD.

 

 

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