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O-1602 Promotes Hepatic Steatosis through GPR55 and PI3 Kinase/Akt/SREBP-1c Signaling in Mice

Non-alcoholic fatty liver disease is recognized as the leading cause of chronic liver disease. Overnutrition and obesity are associated with hepatic steatosis. G protein-coupled receptor 55 (GPR55) has not been extensively studied in hepatic steatosis, although its endogenous ligands have been implicated in liver disease progression. Therefore, the functions of GPR55 were investigated in Hep3B human hepatoma cells and mice fed high-fat diets. O-1602, the most potent agonist of GPR55, induced lipid accumulation in hepatocytes, which was reversed by treatment with CID16020046, an antagonist of GPR55. O-1602 also induced intracellular calcium rise in Hep3B cells in a GPR55-independent manner. O-1602-induced lipid accumulation was dependent on the PI3 kinase/Akt/SREBP-1c signaling cascade. Furthermore, we found increased levels of lysophosphatidylinositol species of 16:0, 18:0, 18:1, 18:2, 20:1, and 20:2 in the livers of mice fed a high-fat diet for 4 weeks. One-week treatment with CID16020046 suppressed high-fat diet-induced lipid accumulation and O-1602-induced increase of serum triglyceride levels in vivo. Therefore, the present data suggest the pro-steatotic function of GPR55 signaling in hepatocytes and provide a potential therapeutic target for non-alcoholic fatty liver disease.


Introduction
Non-alcoholic fatty liver disease is recognized as the leading cause of chronic liver disease. Overnutrition and obesity are associated with non-alcoholic hepatic steatosis, a condition characterized by extensive lipid accumulation in hepatocytes [1]. Lysolipids have been implicated in human obesity and liver disease progression. For example, postprandial lysophospholipid was shown to suppress hepatic fatty acid oxidation in group 1B phospholipase A2-deficient mice [2]. In addition, loss of the membrane bound O-acyltransferase domain-containing 7 gene was associated with the accumulation of its substrate lysophosphatidylinositols, and direct administration of lysophosphatidylinositols promoted hepatic inflammatory and fibrotic transcriptional changes [3]. Levels of lysophosphatidylinositol species such as 16:0, 18:0, and 20:4 have been found to increase in obesity and correlate with weight and fat percentage [4]. G protein-coupled receptor 55 (GPR55) is now recognized as a receptor for lysophosphatidylinositols [5,6] and its expression has been described in the liver of humans and mice [4,7]. However, the role of GPR55 in liver diseases such as hepatic steatosis has not been much studied. Therefore, the functions of GPR55 were investigated in human Hep3B hepatoma cells and in mice fed a high-fat diet using O-1602, the most potent agonist of GPR55, and CID16020046, an antagonist of GPR55 [6,8]. We also Int. J. Mol. Sci. 2021, 22, 3091 2 of 14 measured levels of lysophosphatidylinositol species in the serum and liver from mice fed a high-fat diet for 4 weeks. We found that GPR55 signaling in hepatocytes may promote lipid accumulation in the liver.

O-1602 Induces Lipid Accumulation through GPR55 in Hep3B Cells
First, we assessed the expression of GPR55 in human hepatoma cell lines Hep3B and HepG2. GPR55 was detected in both Hep3B and HepG2 cells ( Figure 1A). Thus, O-1602, the most potent synthetic GPR55 agonist, was used to test the role of GPR55 in lipid accumulation [6]. No significant cytotoxicity was observed in Hep3B cells treated with O-1602 up to 30 µM ( Figure 1B). O-1602 treatment strongly increased the size and number of lipid droplets in Hep3B cells ( Figure 1C). O-1602 treatment induced lipid accumulation in a concentration-dependent manner ( Figure 1D). steatosis has not been much studied. Therefore, the functions of GPR55 were investigated in human Hep3B hepatoma cells and in mice fed a high-fat diet using O-1602, the most potent agonist of GPR55, and CID16020046, an antagonist of GPR55 [6,8]. We also measured levels of lysophosphatidylinositol species in the serum and liver from mice fed a high-fat diet for 4 weeks. We found that GPR55 signaling in hepatocytes may promote lipid accumulation in the liver.

O-1602 Induces Lipid Accumulation through GPR55 in Hep3B Cells
First, we assessed the expression of GPR55 in human hepatoma cell lines Hep3B and HepG2. GPR55 was detected in both Hep3B and HepG2 cells ( Figure 1A). Thus, O-1602, the most potent synthetic GPR55 agonist, was used to test the role of GPR55 in lipid accumulation [6]. No significant cytotoxicity was observed in Hep3B cells treated with O-1602 up to 30 M ( Figure 1B). O-1602 treatment strongly increased the size and number of lipid droplets in Hep3B cells ( Figure 1C). O-1602 treatment induced lipid accumulation in a concentration-dependent manner ( Figure 1D). Data are from three individual experiments and expressed as mean ± SD. ** p < 0.01, *** p < 0.001, compared with the nontreated group. CID16020046, a selective antagonist of GPR55, was used to verify whether the pro-lipogenesis effect of O-1602 is mediated by GPR55 [8]. As shown in Figure 2A,B, CID16020046 significantly inhibited O-1602-induced lipid accumulation in a concentrationdependent manner.
CID16020046, a selective antagonist of GPR55, was used to verify whether the prolipogenesis effect of O-1602 is mediated by GPR55 [8]. As shown in Figure 2A,B, CID16020046 significantly inhibited O-1602-induced lipid accumulation in a concentration-dependent manner.

O-1602 Induces Intracellular Ca 2+ Increase in a GPR55-Independent Manner in Hep3B Cells
Second, we evaluated the effects of O-1602 on intracellular Ca 2+ levels in Hep3B ( Figure 3A). O-1602 treatment induced an increase in intracellular Ca 2+ levels in a concentration-dependent manner from 15 M ( Figure 3B). However, CID16020046 pretreatment did not inhibit the O-1602-induced Ca 2+ increase ( Figure 3C,D), suggesting GPR55-independent signaling of O-1602 for the Ca 2+ response in Hep3B cells.

PI3K/Akt Signaling in the O-1602-Induced Lipid Accumulation in Hep3B Cells
To investigate the signaling pathway of the O-1602-GPR55 response, phosphorylation of serine 374 of Akt was measured by western blot analysis as an index of Akt activation. As shown in Figure 4A

PI3K/Akt Signaling in the O-1602-Induced Lipid Accumulation in Hep3B Cells
To investigate the signaling pathway of the O-1602-GPR55 response, phosphorylation of serine 374 of Akt was measured by western blot analysis as an index of Akt activation. As shown in Figure 4A

O-1602 Induces SREBP-1c through GPR55 and PI3K in Hep3B Cells
SREBP-1c is the main transcription factor for hepatic lipogenic genes in hepatic steatosis [9,10]. Thus, the effects of O-1602 on SREBP-1c expression was evaluated. O-1602 treatment induced the expression of preform and mature form SREBP-1c proteins in a concentration-dependent manner ( Figure 5A,B). In addition, the O-1602-mediated induction of SREBP-1c was markedly inhibited by treatment of CID16020046 or LY294002 ( Figure 5C,D). Therefore, these data suggest that activation of GPR55 by O-1602 induces the expression of SREBP-1c through PI3K/Akt in hepatocytes, leading to lipid accumulation.

O-1602 Induces SREBP-1c through GPR55 and PI3K in Hep3B Cells
SREBP-1c is the main transcription factor for hepatic lipogenic genes in hepatic steatosis [9,10]. Thus, the effects of O-1602 on SREBP-1c expression was evaluated. O-1602 treatment induced the expression of preform and mature form SREBP-1c proteins in a concentration-dependent manner ( Figure 5A,B). In addition, the O-1602-mediated induction of SREBP-1c was markedly inhibited by treatment of CID16020046 or LY294002 ( Figure 5C,D). Therefore, these data suggest that activation of GPR55 by O-1602 induces the expression of SREBP-1c through PI3K/Akt in hepatocytes, leading to lipid accumulation.

Increase of Lysophosphatidylinosiltol Levels in Livers from High-Fat Diet-Fed Mice In Vivo
To determine the in vivo significance of the O-1602-GPR55 response in hepatocytes, we measured the levels of lysophosphatidylinositols in the livers of mice fed a normal chow diet or a high-fat diet for 4 weeks. The levels of lysophosphatidylinositol species of 18:0, 18:1, 18:2, 18:3, 20:1, and 20:2 in the livers of mice fed high-fat diets were significantly higher than those in mice fed normal chow diets ( Figure 6, Table 1). Although levels of 20:4 lysophosphatidylinositol were lower in the high-fat diet group, total levels of lysophosphatidylinositols were significantly higher in the high-fat diet group than iin the normal chow diet group ( Figure 6). We also measured the levels of lysophosphatidylinositol species in the serum, but there was no significant difference between the two groups (data not shown).

Increase of Lysophosphatidylinosiltol Levels in Livers from High-Fat Diet-Fed Mice In Vivo
To determine the in vivo significance of the O-1602-GPR55 response in hepatocytes, we measured the levels of lysophosphatidylinositols in the livers of mice fed a normal chow diet or a high-fat diet for 4 weeks. The levels of lysophosphatidylinositol species of 18:0, 18:1, 18:2, 18:3, 20:1, and 20:2 in the livers of mice fed high-fat diets were significantly higher than those in mice fed normal chow diets ( Figure 6, Table 1). Although levels of 20:4 lysophosphatidylinositol were lower in the high-fat diet group, total levels of lysophosphatidylinositols were significantly higher in the high-fat diet group than iin the normal chow diet group ( Figure 6). We also measured the levels of lysophosphatidylinositol species in the serum, but there was no significant difference between the two groups (data not shown).   To determine the therapeutic potential of GPR55, CID16020046 was administrated in 4-week high-fat diet-fed mice or in 4-week (3 times for a week) O-1602 treated mice for 5 days in the last week ( Figure 7A).  Hepatic steatosis was induced by both high-fat diet feeding and O-1602 treatment ( Figure 7B,C). CID16020046 administration for the last week reduced the lipid accumulation in livers in high-fat diet fed mice but not in O-1602-treated mice, which were judged by oil red O staining ( Figure 7B,C). Furthermore, CID16020046 administration also reduced the ratio of liver/body weight significantly in high-fat diet fed mice ( Figure 7D). On the other hand, the increased serum triglycerides levels by O-1602 administration was reduced by CID16020046 administration but was not blunted in high-fat diet-fed mice ( Figure 7E).

Discussion
The present study reports GPR55-dependent fat accumulation in hepatocytes and steatosis in the liver. Four key findings are reported. First, O-1602 induced lipid accumulation in Hep3B cells via GPR55. Second, O-1602-induced lipid accumulation was mediated by the expression of lipid-synthesizing SREBP-1c via the PI3K and Akt signaling pathways. Third, high-fat diet feeding increased the levels of lysophosphatidylinositols, the endogenous ligands of GPR55 in the liver. Fourth, the high-fat diet feeding in vivo induced lipid accumulation in the liver, which could be reversed by administration of CID16020046 ( Figure 8). Liver/body weight ratio. (E) Triglycerids contents in serum. Green color means NCD, red color for HFD, red hatching or arrows for O-1602 administration, blue hatching or arrows for CID16020046 administration. Results are presented as the means ± SDs of 5 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the NCD group; # p < 0.05, ## p < 0.01, vs. O-1602 group.
Hepatic steatosis was induced by both high-fat diet feeding and O-1602 treatment ( Figure 7B,C). CID16020046 administration for the last week reduced the lipid accumulation in livers in high-fat diet fed mice but not in O-1602-treated mice, which were judged by oil red O staining ( Figure 7B,C). Furthermore, CID16020046 administration also reduced the ratio of liver/body weight significantly in high-fat diet fed mice ( Figure 7D). On the other hand, the increased serum triglycerides levels by O-1602 administration was reduced by CID16020046 administration but was not blunted in high-fat diet-fed mice ( Figure 7E).

Discussion
The present study reports GPR55-dependent fat accumulation in hepatocytes and steatosis in the liver. Four key findings are reported. First, O-1602 induced lipid accumulation in Hep3B cells via GPR55. Second, O-1602-induced lipid accumulation was mediated by the expression of lipid-synthesizing SREBP-1c via the PI3K and Akt signaling pathways. Third, high-fat diet feeding increased the levels of lysophosphatidylinositols, the endogenous ligands of GPR55 in the liver. Fourth, the high-fat diet feeding in vivo induced lipid accumulation in the liver, which could be reversed by administration of CID16020046 ( Figure 8).  Recently, Fondevila et al. reported increased expression levels of liver GPR55 in human patients with non-alcoholic fatty liver diseases [11]. In several animal models of non-alcoholic hepatic steatosis and steatohepatitis, GPR55 was found to increase lipid content by inducing de novo fatty acid synthesis and decreasing fatty acid β oxidation, which is consistent with our results [11]. Serum levels of lysophosphatidylinositol species of 16:0, 18:1, and 18:1 isomer were found to be higher in patients with non-alcoholic steatohepatitis than in those with only steatosis [11]. The increased levels of lysophosphatidylinositols might contribute to the development of non-alcoholic hepatic steatohepatitis through the activation of GPR55 in hepatic stellate cells [11]. Our measurement of lysophosphatidylinositols also suggests that high-fat diets increase the levels of endogenous GPR55 agonists in the liver, which may activate GPR55 in hepatocytes and stellate cells, resulting in the development of hepatic steatosis and steatohepatitis. Indeed, in the present study, high-fat diet-fed mice showed the hepatic steatosis, which was reversed by CID16020046 administration, implying that increased lysophosphatidylinositols may be a cause of hepatic steatosis in over-nutrition conditions. O-1602 administration in vivo induced significant hepatic steatosis by itself but mild. The O-1602-induced hepatic steatosis was not reversed by administration of CID16020046. Considering the degree of hepatic steatosis induced by O-1602 was not severe as compared to high-fat diet feeding and CID16020046 administration duration and dose were fixed as one week and 1 mg/kg, further investigation is necessary.
Because insulin and insulin-like growth factors enhance not only glucose metabolism but also differentiation and survival in hepatocytes through the PI3K/Akt pathway [12,13], it is also important to consider the effects of GPR55 signaling through PI3K/Akt on insulin signaling on the metabolism, differentiation, and survival. Further investigation is necessary to clarify the cross-interaction betweeen insulin signaling and lysophosphatidylinositol signaling.
In this study, we found O-1602-induced Ca 2+ increase in Hep3B cells in a GPR55independent manner. GPR55-mediated Ca 2+ increase has been observed in other cell types like HEK293 cells and mouse pancreatic β cells [14,15]. O-1602-induced Ca 2+ increase was observed in isolated pancreatic β cells from wild-type mice but not from GPR55 gene-deficient mice [15], which proved O-1602-induced Ca 2+ increase is mediated through GPR55 activation in pancreatic β cells. However, O-1602-induced Ca 2+ increase in 3T3-L1 adipocytes was observed, but involvement of GPR55 was not assessed [16]. Therefore, it is necessary to verify carefully whether GPR55 is involved in Ca 2+ increase in other cell types, because O-1602 may cause Ca 2+ increase GPR55-independently as like Hep3B cells.
GPR55 in visceral and subcutaneous adipose tissue has been implicated in human obesity, and lysophosphatidylinositols increase the expression of lipogenic genes (fatty acid synthase and acetyl CoA carboxylase) and promote adipocyte differentiation by increasing PPARγ expression in visceral adipose tissues [4]. Conversely, these findings indicate that activation of GPR55 by increased lysophosphatidylinositols in hepatocytes and adipocytes results in the accumulation of fats and is associated with human obesity and metabolic disorders, suggesting the therapeutic potential of GPR55 in obesity-related diseases.

Cell Culture and Treatment
Human Hep3B hepatocytes were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Hep3B cells were maintained in Dulbecco's modified Eagle medium with high glucose (Welgene, Daegu, Korea) with 10% (v/v) fetal bovine serum, 100 units/mL penicillin, 50 µg/mL streptomycin at 37 • C in a humidified atmosphere containing 5% CO 2 . Cells were seeded onto 6-well culture plates and allowed to adhere overnight (18 h).

Oil Red O Staining
Oil red O staining was performed according to a previously described method [17]. Briefly, cells were fixed with 10% formalin for 15 min at room temperature and then rinsed with PBS. The slides were immersed in Oil red O working solution for 1 h. After rinsing in tap water, slides were counterstained with hematoxylin for 5 s, rinsed with tap water, and mounted with an aqueous mounting medium.

MTT Cytotoxicity Assay
Hep3B cells (4 × 10 5 cells per well) were plated in 48-well flasks and starved for 24 h in DMEM containing 10% FBS. The cells were treated with O-1602 at the indicated concentrations for 24 h. Thirty microliters of 3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl-2Htetrazolium bromide (MTT, 5 mg/mL) was added to the cell cultures and cultured for an additional 4 h in a humidified atmosphere. The cell culture media containing cells were collected and centrifuged, the supernatants were carefully removed, and the pellets were resuspended in 0.5 mL of DMSO:EtOH (1:1) solution and shaken for 10 min. Absorbance was measured at 570 nm by a SpectraCount microplate reader (Packard Instrument Co., Meriden, IL, USA); the optical density (OD) of untreated cells was defined as 100%.

Reverse Transcription-PCR
Total RNA was isolated from cells using Trizol reagent (Invitrogen, Waltham, MA, USA). RNA concentrations were determined by a NanoDrop ND-1000 spectrophotometer. One microgram of RNA was used for transcription, which was performed with the Promega ImProm-II Reverse Transicription System (Madision, WI, USA), according to the manufacturer's protocol. Synthesized cDNA products and primers for each gene were used for PCR using Promega Go-Taq DNA polymerase (Madision, WI, USA). Specific primers for β-actin (sense 5 -CAC CAC ACC TTC TAC AAT GAG CTG-3 , antisense 5 -GAG GAG  CAA TGA TCT TGA TCT TCA TT-3 ), GPR55 (sense 5 -ATT ATG CTG CCA CCT CCA TC-3 antisense 5 -TGA AGC AGA TGG TGA AGA CG-3 ) were used to amplify gene fragments. PCR product aliquots (7 µL) were electrophoresed in 1.2% agarose gels and stained with nucleic acid gel stain (Real Biotech, Taiwan) [19].

Western Blot
Hep3B cells were harvested and resuspended in a lysis buffer. Protein content was determined using a BCA protein assay kit (Thermo scientific, Rockford, IL, USA) according to the manufacturer's protocol. Cell lysates (30 µg protein) were separated by 8% SDS-PAGE, electrophoretically transferred to nitrocellulose paper, blocked with 5% skim milk, and then incubated with specific primary antibodies recognizing SREBP-1c (Santa Cruz Biotechnology, CA, USA) or Akt (pan), p-Akt (S473), β-actin (Cell Signaling Technology, Danvers, MA, USA) at 4 • C overnight. Blots were incubated with HRP-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA, USA) and subsequently developed with ECL detection reagents [20]. Luminescence was detected using a ChemiDoc Touch Imaging System (BioRad, Hercules, CA, USA), followed by analysis with ImageLab software (BioRad).

Measurement of Triglycerides
Lipids were extracted with methanol/chloroform (1:2; v/v). The solvent was evaporated in 60 • C, and the lipids were resuspended in deionized water. Triglyceride levels were determined using a commercial kit from Asan Pharm (Chungcheong, South Korea).

High Fat Diet Feeding
Male C57BL/6 mice were obtained from Daehan Biolink (DBL, Seoul, Korea). The mice had ad libitum access to water and food in the laboratory animal facility at PNU. Eightweek-old mice were randomly divided into 2 groups for lysophosphatidylinositol analysis ( Figure 6). Control C57BL/6 mice (n = 5) were fed with a normal chow diet for 4 weeks while high-fat diet C57BL/6 mice (n = 5) were fed a synthetic diet supplemented with 60% (w/w) fat (HFD, Efeed, Korea) for 4 weeks (Figure 6). The other sets were randomly divided into 5 groups for fatty liver induction and CID16020046 treatment (Figure 7): Control C57BL/6 mice fed a normal chow diet for 4 weeks (n = 5), high-fat diet C57BL/6 mice fed a synthetic diet supplemented with 60% (w/w) fat (HFD, Efeed, Korea) for 4 weeks (n = 5), high-fat diet C57BL/6 mice treated with CID16020046 treatment for the last week (n = 5), C57BL/6 mice treated with O-1602 (1 mg/kg, i.p. injection three times per week) for 4 weeks (n = 5), and C57BL/6 mice treated with O-1602 for 4 weeks plus CID16020046 (1 mg/kg, i.p. injection, five consecutive days for the last week) (n = 5) (Figure 7). The animal protocol used in this study was reviewed and approved by the PNU Institutional Animal Care Committee with respect to the ethics of the procedures and animal care (PNU-20192335).

Statistical Analysis
All results were expressed as mean ± SD. Differences among groups were tested for statistical significance using analysis of variance (ANOVA) followed by Turkey's post hoc test. A p value < 0.05 was considered statistically significant.