The isoquinoline alkaloid berberine has been shown to decrease serum cholesterol and triglycerides levels in hypercholesterolemic patients and animals. Whether BBR could decrease fatty acid induced TG accumulation in hepatocyte is uncertain. The aim of this study is to investigate whether BBR can alleviate fat accumulation in human hepatocytes.
Methods: HepG2 cells were treated with BBR with/without oleic acid (400 Î¼M) for 24h. The intracellular TG level was measured with fluorescent BODIPY staining, and with western blotting to determine expression level of adipocyte differentiation-related protein (ADRP)/adipophilin.
Results: Oleic acid significantly enhanced the accumulation of intracellular lipid droplets (141%, P < 0.05) and elevated the expression level of ADRP (207%, P < 0.05) in 24h comparing to vehicle control. However, BBR did not exert an inhibitory effect on oleic acid-induced lipid droplet content (101%, P = 0.920), nor significantly decreased the expression level of ADRP (84.09%, P = 0.265) in HepG2 cells.
Conclusion: BBR has no significant effects in suppressing fatty acid induced hepatic TG accumulation, which is of practical implication in choosing the appropriate stimuli for the in vitro study of berberine.
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Berberine, fatty acid, Triglyceride accumulation, ADRP, lipid droplet
Non-alcoholic fatty liver disease (NAFLD) is characteristic of fatty liver (steatosis), steatonecrosis, and non-alcoholic steatohepatitis (NASH).1-3) Fatty liver is the accumulation of excessive lipid (predominantly triglycerides (TG)) in hepatocytes.4, 5) . Diet is an important contributor to the pathogenesis of NAFLD, and excessive intake of saturated fat is associated with NAFLD.2, 6) There are multiple molecular mechanisms leading to the development of hepatic steatosis in the pathogenesis of NAFLD, which include enhanced nonesterified fatty acid release from adipose tissue, increased de novo fatty acid and TG synthesis, and decreased Î²-oxidation.2, 7) The exposure of hepatocytes to increased free fatty acids (FFA) results in enhanced lipid accumulation and a wide responses including inflammation, increase of oxidative stress, apoptosis and the production of fibrogenic cytokines which are similar to those observed in NAFLD and NASH patients.8-11)
Berberine (BBR) is a natural isoquinoline alkaloid extracted from the root of a Chinese herb Rhizoma Coptidis (Huanglian, Coptis Chinensis). Emerging evidences demonstrated that Berberine exerts a pleiotropic regulatory effect on lipid metabolism through multipathway mechanism, which includes 1) upregulating LDLR expression mediated by an extracellular signal regulated kinase (ERK) mechanism12) or transcriptionally suppressing pro-protein convertase subtilisin/kexin type 9 (PCSK9);13, 14) 2) inhibiting lipid synthesis and increasing fatty acid oxidation by activating AMP-activated protein kinase (AMPK) and inactivating acetyl-coenzyme A carboxylase (ACC);15-17) 3) stimulating mitochondrial biogenesis via a SIRT1-mediated mechanism and preventing the diet-induced insulin resistance.18) Berberine decreases fasting blood glucose, total cholesterol, and TG levels through mechanisms which include AMP-activated protein kinase- (AMPK-) p38 mitogen-activated protein kinase- (MAPK-) glucose transporter 4 (GLUT4), c-jun N-terminal kinase (JNK) pathway, and peroxisome proliferator-activated receptor Î± (PPARÎ±) pathway.21)
It has been well described the in vivo/in vitro discrepancy in various biological processes, which involving drug metabolism,19) gene expression,20) T-cell proliferation,21) liposome-complement interaction,22) etc. In vitro model is widely used in the studies of lipid metabolism. It is, therefore, of great significance to modify and assess in vitro model so that they more closely mimic the in vivo situation, and the results can be interpreted properly in vivo.
BBR has been shown to have antidiabetic properties and defined as "a novel cholesterol-lowering drug",12) and ameliorate high-fat diet-induced fatty liver in animal23). For example, BBR reduces plasma TG levels16, 24, 25), hepatic TG contents15, 23, 26) in high-fat-fed rats, and the TG-lowering effect is found in skeletal muscle of rats17) as well, whereas in in vitro models, the evidences are limited. Although previous studies have demonstrated that berberine decreases triglyceride accumulation during differentiation from 3T3-L1 fibroblast to adipocyte,16, 27-29) few studies have addressed the effect of berberine in suppressing fatty acid, especially unsaturated fatty acid, induced TG accumulation in vitro. Intriguingly, Brusq et al. reported that the inhibitory effect of berberine on TG synthesis induced by oleate in HepG2 cells is negligible,15) which contradicted that of in vivo studies on high-fat diet-induced fatty livers. The aim of the study is to study whether berberine has effect on reducing TG level in hepatocytes from phenotype to protein changes, in the presence or absence of oleic acid (OA). Attempts are also made to measure the intracellular TG level by fluorescent image processing to quantitate the content of BODIPY stained lipid droplets, and western blotting to determine the expression level of adipocyte differentiation-related protein (ADRP).
MATERIALS AND METHODS:
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Dulbecco's Modified Eagle's Medium (low glucose, DMEM) and fetal bovine serum (FBS) were purchased from Hyclone (Logan, UT). Penicillin/streptomycin solution was from Gibco (Grand Island, NY). Fatty-acid free bovine serum albumin (BSA), 4â€², 6-diamidino-2-phenylindole, dihydrochloride (DAPI), berberine chloride, oleic acid, Rabbit anti-human ADRP antibody, protease inhibitor cocktail, and phosphatase inhibitor cocktail were from Sigma (St. Louis, MO). Oleic acid was prepared in a form of fatty acid-BSA complex with 10% fatty-acid free BSA as previously described.30-32) Vehicle control was prepared with equal amount of BSA and phosphate-buffered saline in parallel. Berberine was dissolved in DMEM at a concentration of 1mM as stock solution. 4,4-diï¬‚uoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503) and 0.4% Trypan Blue solution were purchased from Invitrogen (Eugene, OR). Beta-Actin Mouse Monoclonal antibody, IRDye 800CW Goat anti-Rabbit IgG and IRDye 680RD Donkey anti-Mouse IgG (1.0 mg/mL) were the products of LI-COR (St. Lincoln, NE).
Cell Culture and treatments:
Human hepatoma cell line HepG2 was purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China) and maintained in 25 cm2 flasks with 5 mL low glucose DMEM supplemented with 10% (v/v) FBS, penicillin (100 units/mL) and streptomycin (100 Î¼g/mL) at 37 oC in equilibration with 5% CO2. The medium was replaced with fresh medium of identical composition every three days. After 6-7 days, dense monolayers were formed in the flask and preceded to subculture for subsequent experiments. To induce intracellular lipid accumulation and assess the effect of berberine, cells were seeded on a 12-well plate at a density of 1 Ã- 106 cells/well and treated with 400 Î¼M oleic acid and varying concentrations of berberine for 24h after reaching complete confluence. Every treatment has been performed in duplicate wells and repeated for at least 3 individual experiments.
Cells were washed twice with phosphate-buffered saline and fixed with 10% formalin for 10 min. Intracellular lipid droplets and nuclei were stained with 2 Î¼g/mL of BODIPY 493/503 for 20 min and 1.2 Î¼M DAPI for 30 min, respectively. The cells were viewed with a Nikon Eclipse Ti-S microscope using a 20 x objective lens with a 0.45 numerical aperture. Acquisition and processing parameters (e.g. exposure time and analog gain) were adjusted empirically and maintained equivalent through each experiment. DAPI was imaged in the DAPI channel (EX/DM/BA: 340-380/400/435-485), while BODIPY493/503 was imaged in the FITC channel (EX/DM/BA: 465-495/505/515-555). Images (1280 Ã- 1024 pixel2) of 13 random fields in each well were captured for both DAPI and FITC channels. The BODIPY 493/503 fluorescence was captured after the DAPI image acquisition.
MatlabÂ® software was employed for the measurement of mean gray value (MGV) of each image. An algorithm was created in MatlabÂ®, and consisted of three steps for MGV quantification: 1) read the original BODIPY stained image using command "imread"; 2) convert the true color image to the grayscale intensity image using "rgb2gray"; 3) using "mean2" command to compute the mean of the values in image from step2.
Cell Counting Assay:
To determine the effect of oleic acid and berberine treatments on HepG2 cell numbers, cells were seeded on a 24-well plate, and pretreated with 400 Î¼M oleic acid and berberine (0 Î¼M, 10 Î¼M, 20 Î¼M, 80 Î¼M, and 100 Î¼M) for 24h. After detaching and staining with 0.4% Trypan Blue, cell number was counted using Countessâ„¢ Automated Cell Counter (Invitrogen, Carlsbad, CA). Total, live and dead cell numbers along with cell viabilities were obtained, among which live cell number of each treatment was taken into calculation. Each treatment was performed twice in duplicates.
Cells were washed twice with ice-cooled PBS and lyzed with lysis buffer (200 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 1 mM DTT). The protein concentration was determined by Bradford protein assay.33) Approximate 40 Î¼g of protein was diluted with sample buffer (0.625 M Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate, 0.5% Î²-mercaptoethanol, 10% glycerol, 0.1% bromophenol bule) and seperated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Following this step, protein was transferred electrophoretically onto a PVDF membrane (pore size: 0.45 Î¼m, Millipore, Billerica, MA) with a Semi-dry Transfer Cell (Bio-Rad). PVDF membranes were blocked using blocking buffer (50 mM Tris, 100 mM NaCl, 0.02% Tween-20, 3% BSA) overnight and immunoblotted with primary antibodies (Mouse anti-human Î²-actin monoclonal antibody and rabbit anti-human ADFP antibody) and secondary antibodies (goat anti-rabbit antibody and donkey anti-mouse antibody). The bound antibodies were detected using OdysseyÂ® Infrared Image System (LI-COR).
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Data are presented as mean Â± S.E. Multiple group comparisons were performed using one-way ANOVA followed by Student's Dunnett t test as well as Student Newman-Keuls test when appropriate. P < 0.05 was considered statistically significant.
Effects of oleic acid and berberine on HepG2 cell number
Oleic acid (400 Î¼M) had no significant effects on cell numbers upon incubation for 24 hrs, compared to cells treated with the vehicle control group (9.6Ã-106 vs. 8.8Ã-106, P = 0.438, n = 8; Fig. 1A). Berberine (0-100 Î¼M) also had no significant effects on cell numbers after 24h, compared to the negative control group (0 Î¼M) (P = 0.779, 0.453, 0.12, 0.317, 0.94 for berberine at concentrations of 10 Î¼M, 20 Î¼M, 80 Î¼M, 100 Î¼M respectively, Fig. 1B). These data indicated there was no adverse effect using oleic acid and BBR under current experimental conditions.
Oleic acid enhanced TG levels in HepG2 cells
Oleic acid (at 400 Î¼M) increased TG levels by approx 141.28% (P < 0.01, n = 5), quantified with BODIPY fluorescence microscopy (Fig. 2A). Consistently, Oleic acid increased protein levels of ADRP by approx 222.74% (P < 0.001, n = 8, Fig. 2B) in 24h, compared to the controls.
Berberine had no significant effects on TG levels in HepG2 cells
In the presence of 400 Î¼M oleic acid, berberine (5-20 Î¼M) had no significant effects on intracellular TG levels in HepG2 cells (P = 0.984, 1.000, n = 4, for berberine treated groups at concentrations of 5 Î¼M, 10 Î¼M, compared to the vehicle control group, respectively; Fig. 3A). In the absence of oleic acid, there was a mild dose-dependent but not significant reduction in lipid droplet accumulation over 24h (P = 0.444, 0.253, n = 4, for berberine treated groups at concentrations of 5 Î¼M, 10 Î¼M compared to the normal incubation group, negative control; Fig. 3B). Interestingly, 20 Î¼M of berberine significantly reduced lipid droplet accumulation in the absence of oleic acid (88.18%, P = 0.002, n = 4; Fig. 3B), which was in contrast with the circumstance in oleic acid presence (96.70%, P= 0.991, n = 4; Fig. 3A).
In cells treated with oleic acid showing increased intra-cellular TG levels, BBR (at 10 Î¼M) had no significant effects on intracellular TG level (P = 0.920,n = 4; Fig. 4A). In the absence of oleic acid pretreatment, BBR (at 10 Î¼M) showed no effects on TG levels (P = 0.779, n = 4, Fig. 4A). Consistently, BBR had no significant effects on ADRP levels in cells either pre-treated with oleic acids or without oleic acids (P = 0.265, n=4 and P = 0.775, n = 4, respectively; Fig. 4B).
We have presented data showing oleic acids increase intracellular TG levels and ADRP levels; and to our surprise BBR had no significant effects on both intracellular TG and ADRP levels in cells with or without pre-incubation with oleic acid.
Previous studies have demonstrated that berberine reduces TG levels in vivo. For example, in hypercholesterolemic patients, berberine treatment results in a marked decrease in serum TG levels12). In high-fat-fed rats, berberine reduces plasma and hepatic TG levels, due to that BBR increases hepatic TG export and inhibits TG synthesis 15, 16, 23-26). Similarly, BBR decreases TG levels during the adipocyte differentiation, but to date, few in vitro fatty liver models establish to elucidate the beneficial effect of berberine on ameliorating TG level. Brusq et al.15) described that berberine does not prevent [14C]oleate incorporation into TG, but inhibits intracellular [14C]Triglycerides synthesis labeled with [14C]acetate and [14C]glycerol, suggesting that TG assembly is not affected during the berberine treatment. For de novo lipogenesis, glucose or cellular glycogen and acetate are proposed to act as the major substrates in HepG2 cells and rat colonic epithelial cells respectively.11, 34) It is well established that HepG2 cells have a far higher capacity for TG synthesis from extracellular oleic acid than from endogenous synthesized fatty acid, and the de novo fatty acid synthesis is inhibited in the presence of extracellular oleic acid.11) Through the phosphatidate pathway, exogenous fatty acids are incorporated into diacylglycerols (DAG), which are then partitioned between either the synthesis of cytosolic TAG or, after permeation through the endoplasmic reticulum (ER) membrane, into TAG destined for secretion.35) The secretion level of the above newly synthesized TG as lipoprotein is very low with a defect in lipid mobilization (oxidation, lipolysis, secretion) in HepG2 cells which results in a high level of intracellular TG accumulation.11, 36-38) In our study, we find out the first time that berberine did not exert an inhibitory effect on suppressing TG accumulation elevated by oleic acid in hepatocytes, which is partially in agreement with the study of Brusq et al..15) Taken together, it is conceivable to speculate that berberine could inhibit the de novo TG synthesis from substrates as acetate, glycerol, or endogenous fatty acid but may not exert beneficial effects on the TG synthesis from exogenous free fatty acid through the phosphatidate pathway.
Moreover, berberine treatment does not modulate ADRP expression level in hepatocyte, which to our knowledge for the first time has been investigated. Triacylglycerol-containing lipid droplets (LDs, also known as lipid bodies or adiposomes) are the lipid storage organelles in all organisms and serve as energy reservoirs, lipid source for membrane biosynthesis, or storage sites for toxic lipid species.39) The associated proteins embedded with the phospholipid monolayer of lipid droplets have been described as PAT family proteins, perilipin (P), adipocyte differentiation-related protein (ADRP)/adipophilin (A), and TIP47 (T) named after their constituents.40) ADRP is expressed in a variety of cells and predominantly contributes to the major lipid droplet binding protein.40-44) Based on its localization characteristics, ADRP can serve as a lipid droplet marker for TG levels. More importantly, ADRP plays a critical role in regulation of lipid metabolism. Studies demonstrate an increase in ADRP expression level in fatty liver of humans45) and rats46), where suppression of ADRP reduces expression of lipogenic genes, attenuates TG secretion and enhances insulin sensitivity.43, 44) Therefore, ADRP may act as a promising target for the treatment of NAFLD and associated lipid and glucose abnormalities.43) We show in this study that oleic acids increase intracellular TG levels, which is in accordance with previously studies.10) Consistently, our results reveal that free fatty acid markedly enhances ADRP expression level in hepatocytes, with concordant increase in lipid droplet content, and suggest ADRP serves as a reliable marker for lipid accumulation.
In conclusion, BBR had no significant effects on both intracellular TG and ADRP levels in cells with or without pre-incubation with oleic acid, suggesting that berberine may not exert beneficial effects on the de novo TG synthesis from exogenous free fatty acid and it is of practical implication to choose the appropriate stimuli for the in vitro study of berberine.
This study was supported by Department of Biological Science of Xi'an Jiaotong -Liverpool University.
Fig. 1. Oleic acid (400 Î¼M) and berberine (0-100 Î¼M) did not exert a cytotoxicity effect on HepG2 cell growth. Confluent HepG2 cells were incubated with (A) oleic acid (400 Î¼M) or (B) varying concentrations of berberine (0-100 Î¼M) for 24h. Cells were detached and cell numbers were counted automatically using Countessâ„¢, and expressed as means Â± SEM (n=8 for OA, n=2 for berberine, from three or two independent experiments, respectively; VC, vehicle control; OA, oleic acid treated group; NC, negative control, normal incubation).
Fig. 2. Oleic acid enhanced TG level in HepG2 cells. Cells were incubated with Oleic acid (400 Î¼M) for 24h. TG level was measured using (A) fluorescent image processing to quantitate the content of BODIPY stained lipid droplets, and (B) western blotting to determine the expression level of ADRP. Data were expressed as percentages of vehicle control and were the mean Â± SEM (n=5 for lipid content, and n=8 for ADRP expression level, from five and four independent experiments, respectively). ** P < 0.01, *** P < 0.001.
Fig. 3. Effects of berberine on lipid droplet content in HepG2 cells dose-dependently. Cells were treated with berberine (at 5-20 Î¼M) in the (A) presence and (B) absence of oleic acid for 24h. Data were expressed as percentages of vehicle control and negative control, respectively and were the mean Â± S.E. (n=4). ** P < 0.01.
Fig. 4. Berberine had no significant effects on oleic acid enhanced triglyceride level in HepG2 cells. Cells were incubated with BBR (at 10 Î¼M) and/or oleic acid (at 400 Î¼M) for 24h. TG level was measured using (A) fluorescent image processing to quantitate the content of BODIPY stained lipid droplets, and (B) western blotting to determine the expression level of ADRP, . Data were expressed as percentages of vehicle control and were the mean Â± S.E. (n=4 for all groups in ADRP experiments, n = 4 and 5 in lipid content experiments for OA+BBR, and OA group). * P < 0.05, ** P < 0.01, *** P < 0.001 (compared to the vehicle control), ns indicated no significance.