In this study, we investigated the possible mechanisms underlying the protective effect of the GLP-1R agonist Ex-4 on hepatic steatosis in an in vitro cell model. We used the HepG2 cell line treated with oleic acid as a steatosis model and confirmed that Ex-4 significantly reduces OA-induced lipid accumulation. GLP-1R agonists have a wide range of complex physiological effects due to the widespread expression of the GLP-1 receptors throughout the body [14]. Because of this pleiotropic effect, distinguishing between direct, i.e., via agonist-receptor interaction, and indirect effects of these agonists in vivo is challenging. Therefore, it remains unclear whether the reduction of steatosis observed in animal and human trials in response to treatment with GLP-1R agonists results from direct activation of hepatic GLP-1R or the indirect impact such as weight loss, increased insulin sensitivity, brain-liver signals such as brain leptin [52], or other hormonal signals that these agonists might trigger [14]. To overcome this challenge, we opted for the in vitro model to ascertain that Ex-4's effect on steatosis results from direct activation of the GLP-1R.
The most important finding of our study is the significantly lower expression of FABP1 (also known as liver-type fatty acid-binding protein or L-FABP) in Ex-4-treated cells compared to steatotic cells. Fatty acid-binding proteins (FABPs) are small cytoplasmic proteins involved in intracellular lipid metabolisms such as fatty acid uptake, transport to mitochondria or peroxisome for oxidation, lipid synthesis, storage in lipid droplets, and regulation of nuclear receptors [53]. FABP1 is highly expressed in hepatocytes and is required for FFA uptake and shuttling [54]. Previously, Wolfrum and coworkers [55] elegantly showed that increasing the FABP1 expression by treating HepG2 cells with the potent peroxisome proliferators bezafibrate and Pirinixic acid leads to increased uptake of radio-labeled oleic acid by 38% and 78%, respectively. Conversely, decreasing FABP1 expression by antisense FABP1 mRNA to one-sixth of its regular expression reduces the ratio-labeled oleic acid uptake rate by 66%. Similar results were obtained in FABP1−/− mice following intravenous bolus administration of OA [56]. These findings indicate a direct correlation between FABP1 expression and fatty acid uptake in the liver.
The Ex-4-induced FABP1 downregulation we see correlates with the significant reduction in TGs content observed under the same treatment. Interestingly, the silencing of β-catenin with siRNA abrogates the effect of Ex-4 on FABP1 expression, indicating its dependency on β-catenin signaling. To our knowledge, this is the first time a reduced FABP1 expression in response to direct activation of the GLP-1R is shown in hepatocytes. Previously, Panjwani and colleagues reported significantly reduced levels of TGs and FABP1 in liver cells from high-fat diet-fed male ApoE(−/−) mice treated with taspoglutide, a long-lasting GLP-1R agonist [Panjwani, 2013 #146]. However, the authors suggested the effect of taspoglutide was indirect as they could detect neither the protein nor the mRNA of GLP-1R in liver cells. However, it is worth noting that several studies have reported GLP-1R expression in both human and rodent hepatocytes [57, 58]. We have also detected GLP-1R expression in HepG2 cells by western blotting and quantitative PCR (data not shown). Additionally, a recent study investigating the effect of the GLP-1R agonist liraglutide on obesity-induced chronic kidney injury in obese rats showed that the agonist significantly reduced the lipid content and, concomitantly, the expression level of FABP1 protein in the obese kidney, relative to untreated rats [59].
In principle, four separate mechanisms may lead to hepatic lipid accumulation: (a) enhanced uptake of circulating free fatty acids, (b) increased hepatic de novo lipogenesis, (c) diminished hepatic β-oxidation, and (d) decreased hepatic lipid export via VLDL [40, 41]. Therefore, one explanation for the Ex-4-induced improvement in steatosis observed in our model could be a decreased fatty acid uptake by FABP1. This explanation is consistent with the fact that FABP1 silencing in mice reduces liver weight and hepatic TG content [60, 61], whereas FABP1 overexpression increases hepatic fatty acid uptake [Wang, 2019 #152}. Moreover, the expression of FABP1 is significantly higher in the liver in obese patients with simple steatosis than in the obese healthy group [62].
We have also observed that the presence of Ex-4 decreases the expression of ACC and DGAT1, which are critical rate-limiting enzymes for fatty acid biosynthesis and TG formation, respectively [63, 64]. Previous research on DGAT1−/− mice demonstrated that DGAT1 was required for hepatic steatosis caused by a high-fat diet or fasting, both of which promote hepatic uptake of exogenous FAs, but not for hepatic steatosis caused by upregulation of endogenous de novo FA synthesis [65]. As a result, the low DGAT1 expression observed in the presence of Ex-4 is most likely a response to reduced FAs uptake rather than reduced de novo lipogenesis, ruling out a role for reduced de novo lipogenesis in the Ex-4-induced steatosis improvement.
A decrease in ACC expression stimulates lipid β-oxidation by reducing the production of the β-oxidation inhibitor malonyl-CoA [66]. Thus, an increased β-oxidation might explain the improved steatosis we observe in the presence of Ex-4. Nevertheless, this possibility is ruled out by the fact that Ex-4 decreases the expression of CPT1, the rate-limiting enzyme for mitochondrial β-oxidation [67].
OA treatment significantly increases the expression of ApoB, an essential protein for the assembly and secretion of TG-rich ApoB-containing lipoproteins, such as VLDL [68]. This increase in ApoB expression likely reflects a compensatory mechanism to enhance the secretion of VLDL and hence reduce the content of TGs. Nonetheless, Ex-4 significantly reduces the OA-induced upregulation of ApoB. This finding is in line with a previous study, which reported that continuous administration of fat diet-fed APOE*3-Leiden transgenic mice with Ex-4 or CNTO3649, a GLP-1 peptide analog, results in reduced hepatic TGs, cholesterol, and phospholipids in addition to down-regulation of ApoB expression [69]. Thereby, this observation excludes the significant contribution of enhanced lipid export to the Ex-4-induced steatosis reduction. Interestingly, the Ex-4-induced reduction of ApoB expression was blunted by the silencing of β-catenin, indicating its dependency on β-catenin signaling.
The transcription factor FOXA1 is among the most effective activators of human FABP1 [70]. We show that the presence of Ex-4 significantly reduces the FOXA1 expression relative to OA alone, which may, in turn, decrease FABP1 expression. Interestingly, FOXA1 is downregulated in liver samples from humans and rats with simple steatosis [71], probably as a feedback mechanism to reduce FAs uptake by FABP1. Furthermore, FOXA1 promotes fatty acid breakdown by inducing peroxisomal fatty acid b-oxidation.[71]. Nonetheless, given the reduced FOXA1 expression induced by Ex-4 in our study, it is unlikely that the observed Ex-4-induced TG content reduction is due to the stimulation of peroxisomal fatty acid -oxidation. Ex-4 induces a significant downregulation of FOXA1 (Fig. 2C) compared to steatotic cells. However, this downregulation is abrogated upon silencing of β-catenin, suggesting a role of the Wnt/β-catenin pathway in this process.
The involvement of the β-catenin signaling in the Ex-4-induced improvement in hepatic steatosis was suggested previously by Seo and coworkers [36], who showed that the β-catenin inhibitor IWR-1 abrogates the protective effect of Ex-4 against palmitate-induced steatosis. Our results also indicate the potential involvement of the β-catenin signaling pathway by showing the impact of Ex-4 on the expression of nuclear transcription factors SREBP-1, a key regulator of lipid metabolism in the liver [72], and TCF4, a central transcription factor in the β-catenin pathway, when β-catenin is silenced. Hence, after β-catenin knockdown, OA treatment significantly upregulates both SREBP-1 and TCF4. However, the presence of Ex-4 drastically reduces this upregulation. Interestingly, in the context of Wnt/β-catenin signaling-dependent liver tumorigenesis, it was suggested that TCF4 might act in concert with the FOXA factors to regulate hepatocellular carcinoma-specific Wnt target gene expression [73]. Therefore, GLP-1R stimulation may activate the β-catenin pathway, which may result in a concerted action by TCF4 and FOXA1 to regulate the expression of FABP1 and hence prevent the lipid accumulation induced by OA. It is worth noting that FABP1was suggested as a critical driver gene in hepatitis B X-protein-induced hepatic lipid accumulation [74]. However, further investigations are warranted to decipher the complete mechanism underlying the protective effect of GLP1R agonists against hepatic steatosis.
In conclusion, the present study proposes that the direct activation of GLP-1R by Ex-4 reduces OA-induced steatosis in HepG2 cells by stimulating the Wnt/β-catenin signaling pathway, which reduces FOXA1 expression. FOXA1 downregulation, in turn, reduces FABP1 expression, which ultimately leads to a decrease in FFAs uptake. Targeting FABP1 expression in the liver could be beneficial as a medical treatment for fatty liver disease.