Cannabinoid receptor 1 and 2 agonists increase lipid accumulation in hepatocytes
Andrea De Gottardi, Laurent Spahr, Florence Ravier-Dall’Antonia and Antoine Hadengue
ABSTRACT
Background: Cannabinoid receptors CB1 and CB2 are expressed in the liver, but their regulation in fatty hepatocytes is poorly documented. The aim of this study was to investigate the effects of selective CB1 or CB2 agonists on the expression of key regulators of lipid metabolism. Methods: We used an in vitro model of fatty liver by treating immortalized human hepatocytes and HepG2 cells with oleic acid and the selective agonists arachidonyl-2-chloroethylamide (ACEA) (CB1, 12 nM) and (2-iodo-5-nitrophenyl)-[1-(1-methylpiperidin-2ylmethyl)-1H-indol-3-yl]-methanone (AM1241) (CB2, 16 nM). The quantity of intracellular lipids was assessed using Oil-Red-O and a biochemical triglyceride assay. The expression of several proteins regulating endocannabinoid signalling and lipid metabolism was quantified by real-time polymerase chain reaction and by Western blot. Results: Both CB1 and CB2 agonists dosedependently increased the degree of steatosis of oleic acid-treated fatty hepatocytes. Cannabinoid receptors were downregulated in the presence of steatosis, and treatment with a CB2 agonist increased the expression of CB1. Carnitine palmitoyltransferase 1 was significantly overexpressed and sterol response element-binding protein (SREBP)-1c, fatty acid synthase and lecithin–cholesterol acetyltransferase (LCAT) were downregulated in fatty immortalized human hepatocytes. Treatment with the CB agonists ACEA and AM1241 partially reversed these changes, except for SREBP-1c. CB2, but not CB1, agonism decreased the expression of apolipoprotein B. In HepG2 cells, only LCAT resulted increased after treatment with CB agonists. Conclusions: Not only CB1 but also CB2 participated in the regulation of lipid metabolism in human-derived immortalized hepatocytes by regulating the expression of key enzymes of lipid synthesis and transport.
Keywords cannabinoid receptor – cannabinoids – hepatocyte cell lines – steatosis
Introduction
Obesity, as part of the metabolic syndrome, constitutes one of the major risk factors for the development of fatty liver (1, 2). Increasing evidence suggests that a fatty liver is more vulnerable to factors that lead to inflammation and fibrosis (3). The presence of steatosis not only is an essential component of non-alcoholic steato-hepatitis, but also may negatively influence other liver diseases, such as viral hepatitis. Hence, a better understanding of the mechanisms leading to steatosis may help in improving liver injury and decreasing the progression of fibrosis (4).
Rupture of the fine balance between lipogenesis, mitochondrial b-oxidation and the export of lipid components has been proposed to explain the accumulation of lipid droplets in hepatocytes. For instance, both alcoholic and non-alcoholic fatty liver are associated with enhanced hepatic lipogenesis mediated by an upregulation of fatty acid synthase (FAS) and sterol response element-binding protein (SREBP)-1c and/or decreased fatty acid oxidation through a downregulation of AMPK and CPT-1 in animal models.
Recent findings indicate that these processes are regulated, in part, by endogenous cannabinoids (EC) (5, 6). EC are lipid mediators that produce effects similar to those of marijuana by acting on membrane-bound receptors and regulate appetite behaviour (7). Cannabinoid receptors are localized mainly in the brain, but they are also present in minor amounts in the liver and some other peripheral tissues (CB1) and in immune and haematopoietic cells (CB2) (8). Endocannabinoids may also regulate peripheral energy metabolism, as demonstrated by their CB1-mediated effect on lipoprotein lipase activity in adipocytes (9) and their ability to stimulate lipogenesis in hepatocytes (5). In addition, a novel EC receptor, the orphan receptor GPR55, has been recently identified (10), that could mediate the activity of some EC, for example cannabidiol, which exhibit no CB1 or CB2 activity.
In obese mice, liver CB1 receptors are upregulated, while the lipogenic transcription factor sterol response element-binding protein 1c (SREBP1c) is reduced in CB1 receptor knockout mice. In normal mice, CB1 agonists increased the expression of SREBP1c and its target enzymes acetyl CoA carboxylase-1 and FAS (11).
A previously unrecognized role for CB2 receptors has recently been suggested by Deveaux et al., who showed that knocking out the CB2 receptor (Cnr2 / ) prevented steatosis and inflammation in ob/ob mice (11).
The regulation and the effects of CB agonists in human hepatocytes are poorly documented, and the aim of this study was to investigate the effects of activation of the CB system in relationship to the expression of key regulators of lipid metabolism in human cell lines. We chose to use both the hepatoma cell line HepG2, as a classical tool in cellular biology, and immortalized human hepatocytes (IHH), which maintain in vitro the primary features of human hepatocytes (12).
Material and methods
Cell cultures
Primary human hepatocytes, isolated and immortalized (IHH) by lentiviral transduction with the SV40T antigen and hTERT (13), were a kind gift from D. Trono and T. Nguyen. IHH were maintained at 37 1C and 5% CO2 in Dulbecco’s modified Eagle’s medium F12 (Gibco-BRL, Invitrogen Life Technologies, Basel, Switzerland) containing 10–6 M dexamethasone, 10–12 M human insulin (Humalog; Lilly Suisse, Vernier, Switzerland) and antibiotics (100 IU/ml penicillin, 100 mg/ml streptomycin) with 10% fetal bovine serum (Invitrogen Life Technologies). The human hepatoma cell line HepG2 (European Collection of Cell Cultures (ECACC)] was cultured in DMEM under the same conditions containing equal concentrations of antibiotics and fetal bovine serum, without dexamethasone and insulin.
Induction of steatosis was achieved using a 100 mM oleic acid (Sigma, St Louis, MO, USA) stock solution diluted in ethanol, which was added to the normal condition culture medium in order to obtain a final concentration of 50 mM. The duration of the treatment to induce steatosis was 7 days. Oleic acid was chosen to induce steatosis, because it was more effective in inducing fat droplets accumulation in cultured hepatocytes compared with palmitic or arachydonic acid (14).
Microscopy and intracellular lipid quantification
Semiconfluent IHH or HepG2 grown on a coverslip were fixed in buffered para-formaldehyde 4% for 20 min. at room temperature. Then, staining of intracellular neutral lipids was performed using Oil-Red-O (Sigma) according to the manufacturer’s instructions. The positively stained area was quantified in pixels using METAMORPH PROFESSIONAL IMAGE ANALYSIS software (Universal Imaging, Molecular Devices Corporation, Sunnyvale, CA, USA) and the values were then divided by the number of cellular nuclei counted in the selected area.
Triglyceride assay
After lysis of hepatocytes with digitonine 0.01% overnight at 4 1C, triglycerides were quantified with a GPO assay kit (Synchron LXs; Beckman Coulter, Brea, CA, USA) using a spectrophotometer with absorbance at 520 nm.
Cannabinoid receptor ligands
Selective CB agonists were used to treat IHH and HepG2 over 7 days in the presence or absence of oleic acid. Arachidonyl-2-chloroethylamide (ACEA), a high-affinity CB1 agonist (15), was added to cell cultures in con centrations ranging from 3 to 12 nM. AM1241 ((2-iodo5-nitrophenyl)-[1-(1-methylpiperidin-2-ylmethyl)-1Hindol-3-yl]-methanone) was used as a selective CB2 agonist (16) at concentrations from 4 to 16 nM (both compounds from Cayman chemical, Ann Arbor, MI, USA). If not otherwise specified, ACEA and AM1241 were used at the concentrations of 12 nM and 16 nM respectively.
Quantitative polymerase chain reaction
Total RNA was extracted from cell cultures using the Total RNA Isolation Nucleospins RNA II (MachereyNagel, Duren, Germany) according to the manufacturer’s¨ instructions. cDNA was synthesized from total RNA with the Omniscript reverse transcriptase (Qiagen, Hombrechtikon, Switzerland), an RNAse inhibitor and random hexamers (Promega, Charbonnie`res-les-Bains, France). Specific forward and reverse primers were designed using PRIMER EXPRESS 2.0 software (Applied Biosystems, Life Technologies, Basel, Switzerland) and are provided in supporting information Table S1.
Primer pairs were tested and efficiencies were measured using standard curves from serial dilutions of cDNA. cDNA was amplified by polymerase chain reaction (PCR) using a 7900HT SDS System (Applied Biosystems). Specificity of Sybr Green reactions was determined by the examination of product melting curves.
The relative quantification of gene expression was normalized to the expression of the housekeeping genes d-aminolevulinate synthase 1, b-2-microglobulin and eukaryotic translation elongation factor 1.
Western blot
Hepatocytes were lysed in buffer (40 mM Tris-HCl pH 6.8, 0.1 M DTT, 2% SDS, 40% glycerol, bromophenol blue) with 1 mM PMSF, 1 mM ortho-vanadate and the cocktail of protease inhibitors Complete (Roche). Then, samples were sonicated at 4 1C and heated for 5min at 95 1C. Equal amounts of proteins for each sample were resolved on acrylamide gels at different concentrations and blotted onto PVDF membranes (Immobilon-P, Millipore, Bedford, MA, USA) for Western blots. Membranes were incubated with primary antibodies according to the parameters presented in supporting information Table S2. After blocking with a 5% defatted milk or 5% bovine serum albumine TBS-Triton X100 0.1% solution, primary antibodies were detected using appropriated HRP-conjugated secondary antibodies (Bio-Rad, Reinach, Switzerland) and the enhanced chemiluminescence detection system (ECL; GE Healthcare, Glattbrugg, Switzerland). Quantifications were performed using the OPTIQUANT software (Packard Instruments, Meriden, CT, USA).
Statistics
Experiments were always conducted at least in triplicate. For gene expression quantification, data were obtained from quintuplicate experiments. Comparisons between groups were performed using the Student test or the Welch test, when appropriate. A value of P o 0.05 was considered to be significant.
Results
Effects of cannabinoid receptor 1 and cannabinoid receptor 2 agonists on lipid content in fatty hepatocytes
Treatment with ACEA, a CB1 agonist with a Kd of 1.4 nM for CB1 and 2000nM for CB2, during 7 days induced a significant dose-dependent increase (3–12 nM) in the lipid content of both immortalized human hepatocytes IHH and HepG2 cells in the presence of oleic acid (50 mM, 7 days). Similarly, we observed that steatosis further increased after treatment with AM1241 (Kd 7 nM for CB2 and 580 nM for CB1) at concentrations from 4 to 16 nM. However, it must be stressed that the lipid accumulation reached statistical significance only at the highest dose of both CB1 and CB2 agonists. These findings confirmed that agonism induced lipid accumulation and suggested a similar role for CB2 agonists (Fig. 1). For all further experiments, ACEA and AM1241 were used at the concentrations of 12 and 16 nM respectively. The treatment with ACEA or AM1241 did not induce any lipid accumulation in the absence of oleic acid.
Cannabinoid receptors in cultured hepatocytes
The cannabinoid receptor CB1 was detectable by polymerase chain reaction in IHH and its expression was lowest in oleic acid-treated cells. Treatment with AM1241 increased the expression of CB1 in oleic acid-treated IHH (Fig. 2). In HepG2 cells, CB1 expression was very low and could not be quantified, because the Ct value was beyond 35 PCR amplification cycles.
CB2 was clearly detectable in untreated IHH, but its expression was under the detection level of quantitative PCR in fatty IHH or in HepG2, indicating that in hepatocytes, the presence of a lipid overload is associated with a strong downregulation of CB2.
The expression of GPR55, another cannabinoid receptor, was detectable neither in IHH nor in HepG2, suggesting that, at least in the hepatocyte cell lines studied here, this receptor is not involved in the cannabinoid signalling pathway.
Further, we investigated the expression of effector genes regulating cannabinoid signalling. Endocannabinoids are generated from membrane phospholipids through the action of diacylglycerol lipase and phospholipase D. The quantitative real-time PCR assessment of the expression of these enzymes did not show any significant change induced by CB agonists. However, the expression of fatty acid amide hydrolase, an enzyme degrading the endocannabinoid 2-arachidonyl glycerol, was decreased after treatment with ACEA in HepG2 cells (0.87-fold, P= 0.02).
Taken together, these results demonstrated that the effects of the treatment with CB1 or CB2 agonists on the intracellular lipid accumulation could not be ascribed to gene expression changes in this pathway, except for a possible reduced degradation of 2-arachidonyl-glycerol.
Expression of genes regulating lipid metabolism
The effects of CB1 or CB2 agonists on gene expression in steatotic hepatocytes are presented in Table 1A for IHH and Table 1B for HepG2 cells. The quantitative assessment of genes involved in lipid metabolism showed that the increase of steatosis induced by the CB agonists was associated with a significant upregulation of the expression of FAS in IHH. In HepG2 cells, FAS was also increased, however, without reaching statistical significance.
Together with the observation that the treatment with oleic acid alone induced a significant decrease in the expression of FAS, these findings suggest that CB agonists partially counteract the physiological downregulation of FAS in the presence of excess intracellular lipids.
CB agonist treatment did not significantly affect the expression of SREBP-1c, a transcription factor controlling lipogenic gene expression. However, similarly to FAS and independently from either treatment with CB agonists, SREBP-1c was considerably downregulated in hepatocytes with an excess of lipids induced by oleic acid. In HepG2 cells, these changes were not observed, possibly because these cells already present an intracellular lipid overload in basal conditions.
The expression of ApoB decreased in IHH after treatment with the CB2 agonist at approximately 75%. Because ApoB is involved in the cellular export of triglycerides, a decreased transport outside the hepatocyte induced by AM1241 may contribute to the accumulation of intracellular lipid droplets that we observed.
However, this mechanism was limited to IHH (in Importantly, we did not detect any significant up- or HepG2, ApoB resulted unchanged) and appeared to be downregulation of two enzymes regulating fatty acid selectively mediated by CB2. oxidation. Indeed, AMPK and CPT1 resulted unchanged after treatment with CB1 or CB2 receptor agonist in both hepatocyte cell lines.
These findings suggest that the enhanced steatosis induced by the stimulation of CB receptors was not related to a decreased oxidation of fatty acids. Similarly, the expressions of monoacylglycerol lipase, hepatic lipase and microsomal triglyceride transfer protein remained unchanged in both cell lines after treatment with CB agonists (data not shown).
Finally, the expression of the reverse cholesterol transport enzyme LCAT was upregulated significantly by CB agonists in both cell lines, possibly resulting in an increased selective cholesterol uptake by hepatocytes.
Regulation of proteins involved in lipid homeostasis by cannabinoid receptor agonists
Because we observed in IHH an increased expression of FAS and LCATand a downregulation of ApoB induced by CB agonists, we aimed at examining these results at the protein level using Western blots.
Our results showed that FAS was unchanged in HepG2 cells, confirming the findings from quantitative PCR experiments. However, in contrast with the findings from PCR measurements, the protein levels of FAS resulted moderately decreased in IHH treated with CB agonists (Figs 3a and 3b), suggesting the presence of a posttranscriptional downregulation of this protein. Nevertheless, we confirmed the important decrease of FAS (at mRNA and at protein level) induced by oleic acid in IHH, but not in HepG2, possibly because these cells contain lipid droplets also in basal culture conditions without oleic acid.
The changes in the expression of LCAT in Western blots (Figs 3c and 3d) showed differences at the protein level, which were less evident than those at the mRNA level.
In both IHH and HepG2 cells, Western blots of SREBP-1c protein showed that the treatment with CB agonists was not associated with any significant changes compared with cells treated with oleic acid only, confirming the data obtained using PCR.
ApoB resulted not significantly affected by CB agonists. However, the quantification of this protein in IHH was not possible because it was below the detection level (data not shown). Based on these findings, we propose a scheme illustrating the differential regulation of mediators of lipid metabolism in fatty human immortalized hepatocytes treated with ACEA (CB1 agonist) or with AM1241 (CB2 agonist, Fig. 4). This model is compatible with the results of this study, but deserves further confirmatory investigations with molecular tools (e.g. siRNAs) modifying the expression of LCAT, FAS or ApoB.
Discussion
The main findings of this study underlined the role of both CB1 and CB2 cannabinoid receptors as mediators of lipid metabolism and in particular of steatosis in human hepatocyte cell cultures. Our results on the role of CB1 in the pathogenesis of steatosis were in keeping with previous findings obtained in animal models of fatty liver induced by a high-fat diet (5) or by ethanol feeding (6) and add novel findings related to steatosis in human hepatocytes. We showed that human hepatocytes in culture can reproduce the steatosis seen in animals and can be used to delineate its mechanisms. In addition, we identified some common and some selective effects on regulators of lipid metabolism according to the specific receptor subtype CB1 or CB2.
An enhanced expression of FAS, a key enzyme in lipid synthesis, was observed after treatment with both CB1 and CB2 agonists. These findings were in line with the reportedly increased FAS activity mediated by CB1 (5, 6) and expanded this result to CB2, identifying it as a novel mediator of fat accumulation in hepatocytes. However, this finding remained limited to IHH, because in HepG2 cells, FAS expression was unchanged after CB1 or CB2 agonist treatment. As expected, the FAS-regulator gene SREBP-1c resulted also downregulated in oleic acidtreated IHH. Nevertheless, its expression was not significantly different in hepatocytes treated with CB agonists. It may be hypothesized that other factors contribute to the CB-dependent regulation of FAS independently from SREBP-1c.
In contrast with animal models, where it resulted decreased, the expression of fatty acid oxidation regulators such as AMPK and CPT-1 was unchanged after treatment with CB agonists, suggesting that in cultured human hepatocytes, the accumulation of intracellular lipids was not (or only marginally) regulated through this mechanism.
Further, we identified another possible mechanism through which CB agonists may enhance steatosis with the finding that ApoB was downregulated by the CB2 agonist AM1241. ApoB is involved in the processing of LDL and participates in the extracellular export of triglycerides, which are the main components of lipid droplets in steatosis. Similar findings have been observed in the setting of viral hepatitis C, where the downregulation of ApoB in a cellular model of infection by HCV had been demonstrated and linked to an accumulation of lipids into hepatocytes (17). In IHH, an identical mechanism may contribute to the lipid accumulation induced by CB2 agonism, while in HepG2, the expression of ApoB did not change significantly. In a very recent report, Deveaux et al. described the steatogenic effects of the CB2 agonist JWH-133 in mice, clearly indicating that not only CB1 but also CB2 participates in the regulation of lipid homeostasis (18). We must, however, underline that although CB2 was present on normal IHH, steatosis induced by oleic acid was associated with a very marked downregulation of this gene, so that its precise function in fatty hepatocytes certainly warrants additional investigation. The presence of a significant steatosis even under basal culture conditions in Hep-G2 cells may explain, in part, why the results obtained in this cell line were minimally influenced by the stimulation of CB receptors.
We found that LCAT, an enzyme controlling the reverse cholesterol transport from plasma HDL to the liver, was significantly overexpressed in hepatocytes treated with the CB1 agonist ACEA. Elevated intracellular cholesterol levels may be associated with the increased steatosis that we observed, but whether or not the cannabinoid system may significantly contribute to this mechanism remains, at this point, speculative.
Taken together, these results highlight the fact that not only CB1 but also CB2 receptors appear to be involved in the control of lipid metabolism in the models we used. Although the levels of expression of the CB2 receptors were very low, the specific agonist AM1241 could enhance their activity and further increase steatosis.
The CB2 agonist AM1241 increased the expression of CB1 in hepatocytes compared with oleic acid treatment alone, suggesting the presence of a mechanism of crossregulation induced by CB2 on CB1, hence further enhancing the effects of CB1 agonism.
Interestingly, while CB1 and CB2 appear to partially share their role as regulators of lipid metabolism in hepatocytes by controlling the expression of FAS, they also exert selective effects, as is the case with the regulation of LCAT and ApoB expressions. The mechanisms of cross-regulation governing the interaction between CB1 and CB2 are only partially understood. They are not specific for hepatocytes, because non-redundant functions have been described also in other cell types including macrophages (19) and mast cells (20). Because not only the effects of CB agonists but also the presence of steatosis per se may affect the expression CB1 and CB2, the regulation of these receptors is presumably related to both direct and indirect effects. Further investigation is required to elucidate the mechanisms by which CB1 and CB2 interact to each other in normal physiology as well as in pathological conditions.
These findings suggest that, in addition to CB1, CB2 signalling may also contribute significantly to the mechanisms that regulate lipid accumulation in hepatocytes. Confirmatory studies are required because we were able to identify significant changes in IHH but not in HepG2 cells. Studies in animal models of steatosis and in CB2 knockout mice will help elucidate the relevance of the in vitro results of the present study.
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