Inhibition of metabotropic glutamate receptor-5 alleviates hepatic steatosis by enhancing autophagy via activation of the AMPK signaling pathway

Abstract

BACKGROUND

The global prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) has continued to increase annually. Recent studies have indicated that inhibition of metabotropic glutamate receptor 5 (mGluR5) may alleviate hepatic steatosis. However, the precise mechanism warrants further exploration.

AIM

To investigate the potential mechanism by which mGluR5 attenuates hepatocyte steatosis in vitro and in vivo.

METHODS

Free fatty acids (FFAs)-stimulated HepG2 cells were treated with the mGluR5 antagonist MPEP and the mGluR5 agonist CHPG. Oil Red O staining and a triglyceride assay kit were used to evaluate lipid content. Western blot analysis was conducted to detect the expression of the autophagy-associated proteins p62 and LC3-II, as well as the expression of the key signaling molecules AMPK and ULK1, in the treated cells. To further elucidate the contributions of autophagy and AMPK, we used chloroquine (CQ) to inhibit autophagy and compound C (CC) to inhibit AMPK activity. In parallel, wild-type mice and mGluR5 knockout (KO) mice fed a normal chow diet or a high-fat diet (HFD) were used to evaluate the effect of mGluR5 inhibition in vivo.

RESULTS

mGluR5 inhibition by MPEP attenuated hepatocellular steatosis and increased LC3-II and p62 protein expression. The autophagy inhibitor CQ reversed the effects of MPEP. In addition, MPEP promoted AMPK and ULK1 expression in HepG2 cells exposed to FFAs. MPEP treatment led to the nuclear translocation of transcription factor EB, which is known to promote p62 expression. This effect was negated by the AMPK inhibitor CC. mGluR5 KO mice presented reduced body weight, improved glucose tolerance and reduced hyperlipidemia when fed a HFD. Additionally, the livers of HFD-fed mGluR5 KO mice presented increases in LC3-II and p62.

CONCLUSION

Our results suggest that mGluR5 inhibition promoted autophagy and reduced hepatocyte steatosis through activation of the AMPK signaling pathway. These findings reveal a new functional mechanism of mGluR5 as a target in the treatment of MASLD.

Key Words: Metabolic dysfunction-associated steatotic liver disease; Hepatic steatosis; Metabotropic glutamate receptor 5; Autophagy; AMPK

Core Tip: The present work showed that inhibition of metabotropic glutamate receptor 5 can activate the AMPK signaling pathway, promoting the entry of transcription factor EB into the nucleus and enhancing autophagy, which will ameliorate hepatocyte steatosis.

INTRODUCTION

Metabolic dysfunction-associated fatty liver disease (MAFLD) is defined as hepatic steatosis accompanied by overweight/obesity, type 2 diabetes mellitus, or metabolic dysregulation, irrespective of alcohol consumption or other concurrent liver diseases[1]. However, the diagnosis of nonalcoholic fatty liver disease (NAFLD) is based on hepatic steatosis without excessive alcohol consumption or other known liver diseases[2]. The novel definition and diagnostic criteria for MAFLD highlight the importance of systemic metabolic dysregulation in disease progression. Approximately 20% of MAFLD patients develop hepatic cirrhosis or end-stage liver disease[3]. However, due to the increased metabolic risk, mortality among patients with this disease is predominantly due to cerebrovascular and cardiovascular disease. Obesity, type 2 diabetes mellitus, insulin resistance, hyperlipidemia, hypertension, and metabolic syndrome are closely related to MAFLD[4]. The global prevalence of MAFLD is approximately 40%, with the highest rates in Europe and Asia[5]. Recently, following the Delphi consensus, metabolic dysfunction-associated steatotic liver disease (MASLD) was proposed as an alternative term to replace NAFLD[6]. The novel nomenclature and diagnostic criteria are widely endorsed and non-stigmatizing, facilitating enhanced awareness and patient identification. MASLD indeed poses a serious threat to human health and imposes a significant economic burden. To date, no universal treatment or medication has been established specifically for MASLD[7]. Therefore, studying the pathogenesis of MASLD will help identify key therapeutic targets and provide a theoretical basis for the treatment of MASLD.

mGluRs are G protein-coupled receptors that can modulate downstream signaling pathways. mGluRs are divided into three different subgroups: Group I (mGluR1 and mGluR5), group II (mGluR2 and mGluR3) and group III (mGluR4, mGluR6, mGluR7 and mGluR8). In addition to the central nervous system, mGluRs are also widely expressed in peripheral tissues; for example, liver tissue primarily expresses mGluR5 and mGluR3[8]. mGluR5 is considered a key pharmacological target in neurodegenerative diseases, such as Alzheimer's disease (AD)[9], Huntington's disease (HD)[10] and Parkinson's disease[11]. Furthermore, the role of mGluR5 in the regulation of metabolism suggests that it may be a potential therapeutic target in MASLD. Previous studies have shown that mGluR5 regulates body weight and glucose metabolism. According to one study, knockdown or pharmacological inhibition of mGluR5 reduced food intake and promoted weight loss in rats, whereas the stimulation of mGluR5 promoted weight gain[12,13]. Negative modulation of mGluR5 improved obesity and binge-like eating behavior[14]. In one study, artificial mutation of mGluR5 in ventromedial hypothalamic SF1 neurons severely impaired insulin sensitivity, glycemic control, fat metabolism and sympathetic output in female mice[15]. Our previous study revealed that mGluR5 regulated hepatic steatosis in vitro. We also found that the inhibition of mGluR5 by mGluR5 knockdown or by using the mGluR5 antagonist MPEP reduced lipid accumulation and inflammation in hepatocytes[16]. Consistent with our results, a recent in vivo study revealed that MPEP reduced both fat accumulation and oxidative stress in a 7-week murine model of steatosis[17]. However, the mechanisms underlying the effects of mGluR5 on hepatic steatosis require further exploration.

Autophagy is an important lysosome-mediated process by which intracellular components, including damaged organelles and misfolded proteins, are degraded to maintain the intracellular energy balance[18]. Lipophagy is a selective autophagic process that involves the catabolism of lipid droplets in lysosomes[19]. Our previous study indicated that promoting lipophagy alleviates intracellular lipid accumulation in liver cells[20]. Studies on neurodegenerative diseases such as AD and HD have shown that the inhibition or knockout (KO) of mGluR5 can promote autophagy, thereby clearing abnormally accumulated protein aggregates and slowing disease progression[21-23]. Given the critical role of autophagy in hepatic steatosis and the capacity of mGluR5 to regulate autophagy, the present study aimed to investigate whether mGluR5 can affect hepatic steatosis by modulating autophagy.

MATERIALS AND METHODS

Animal model

Male mGluR5 KO mice were purchased from Chengdu Yaokang Biological Technology Co., Ltd. As shown in Supplementary Figure 1A, after a week of adaptive feeding, the 9-week-old mice were randomly divided into four groups: Wild-type (WT)/normal chow diet (NCD) (n = 5), KO/NCD (n = 5), WT/high-fat diet (HFD) (n = 7), and KO/HFD (n = 5). The animals were fed a NCD (D12450B, research diets) or a HFD (D12492, research diets) for 13 consecutive weeks. The mice were housed in a temperature-controlled facility under a 12-hour light/dark cycle and were provided unrestricted access to food and water. The intraperitoneal glucose tolerance test (IPGTT) was conducted via glucose injection in the eleventh week. Mice were euthanized with 10 g/L pentobarbital sodium at the end of the thirteenth week. Retroorbital blood was collected in a 1.5 mL EP tube and then centrifuged at 5000 rpm at 4 °C after standing for a period of time. The supernatant was collected, stored separately and maintained at -80 °C until subsequent serological detection. Liver tissue was collected and placed in a 1.5 mL cryopreservation tube and stored in a liquid nitrogen dewar until use. All animal experiments were approved by the Animal Ethics Committee of Chongqing Medical University (Approval number: IACUC-CQMU-2024-0085).

Intraperitoneal glucose tolerance test in mice

After fasting for 8 hours, the blood glucose level at 0 minutes was detected with a blood glucose meter. Subsequently, an intraperitoneal injection of 1 g/kg glucose was administered, after which the blood glucose levels were measured at 15, 30, 60, and 120 minutes.

Cell culture and activity experiments

Human HepG2 obtained from ATCC were cultured at 37 °C in a CO₂ incubator (Thermo Fisher Scientific, United States) with Dulbecco's Modified Eagle's Medium (DMEM, C0891, Gibco) supplemented with 10% fetal bovine serum (C0235, Gibco) and 1% penicillin-streptomycin (C0222, Beyotime, Shanghai, China). When the cell confluence reached approximately 80%-90%, the cells were seeded in a 96-well plate at a density of 5000 cells per well and incubated for 24 hours. After they adhered to the plate, the cells were exposed to 100 μL of 0.5 mmol/L free fatty acids (FFAs) [oleate (OA): palmitate (PA) = 2: 1, 143-19-1, 57-10-3, Sigma-Aldrich, St. Louis, MO, United States] for 24 hours. Subsequently, cells were treated with FFAs supplemented with the mGluR5 antagonist MPEP (M5435, Sigma-Aldrich) (0, 2.5, 5, 10, 20, 50 μM) or the mGluR5 agonist CHPG (HY-101364, MedChemExpress) (0, 50, 100, 200, 500, 1000 μM) and incubated for an additional 24 hours. Finally, the cells were incubated with CCK-8 solution in complete medium (medium: CCK8 = 9: 1) for 30 minutes (CCK-8 cell proliferation and cytotoxicity assay kit, CA1210, Solarbio), after which the absorbance of each well was detected at 450 nm with a plate reader.

Cell treatment

Upon reaching 80%-90% confluence, the cells were seeded in a 6-well plate. After they were allowed to adhere for 24 hours, the cells were exposed to 0.5 mmol/L FFAs (OA:PA = 2:1) for an additional 24 hours to induce steatosis in vitro. As shown in Supplementary Figure 1B, an antagonist (MPEP) and an agonist (CHPG) were subsequently added in the presence of FFAs for 24 hours to explore the role of mGluR5 in steatosis. As shown in Supplementary Figure 1C, to investigate the involvement of autophagy and AMPK in the inhibition of mGluR5, chloroquine (CQ) (HY-17589A, MedChemExpress) and compound C (CC) (HY-13418A, MedChemExpress) were separately added to the cells in the presence of FFAs for 24 hours after successful induction of steatosis.

Oil Red O staining

HepG2 cells cultured in 6-well plates were subjected to staining with an Oil Red O Stain Kit (G1262, Solarbio) according to the manufacturer’s instructions. Images of the cells were captured with a light microscope (Olympus, Tokyo, Japan).

Measurements of triglyceride, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol contents

A triglyceride (TG) assay kit (A110-1-1), total cholesterol (TC) assay kit (A111-1-1), high-density lipoprotein cholesterol (HDL-C) assay kit (A112-1-1), and low-density lipoprotein cholesterol (LDL-C) assay kit (A113-1-1) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China) and were used to quantify TG, TC, HDL-C, and LDL-C, respectively, according to the manufacturer’s instructions. Finally, the data were obtained via a spectrophotometer (Thermo Fisher Scientific, United States).

Western blot analysis

The cell lysate was prepared by lysing the sample in RIPA buffer (P0013B, Beyotime) supplemented with a 50:1 ratio of protease and phosphatase inhibitor cocktails for general use (P1045, Beyotime). An ultrasonic apparatus (Dahon Biotech Lab Instruments Co., Ltd., Beijing, China) was used to ensure complete cell disruption, followed by centrifugation of the samples in a centrifuge (Thermo Fisher Scientific, United States) at 12000 rpm for 15 minutes at 4 °C. The protein concentration was determined using an Enhanced BCA Protein Assay Kit (P0010, Beyotime), which confirmed the presence of 20 μg of immobilized protein. A Western blot electrophoresis apparatus (Bio-Rad, United States) was used to transfer proteins onto a PVDF membrane (IPSV00010, Millipore, America), which was subsequently blocked with skim milk (P0216-300 g, Beyotime) for 2 hours. The membrane was incubated with primary antibodies at 4 °C for more than 12 hours. Following three wash steps with TBST (T1081, Solarbio) for 10 minutes each time, the membrane was incubated with secondary antibodies on a shaker for 1 hour. The membrane was again washed three times with TBST, and the reagent from the New-SUPER ECL Detection Kit (KGC4602-100, Kaiki Biology Co., Ltd., Jiangsu, China) was added. Finally, the membrane was developed via a chemiluminescence imaging and analysis system (Bio-Rad, United States). The antibodies used for western blotting were as follows: Anti-mGluR5 (ab76316, Abcam), anti-p62 (#5114T, CST), LC3I/II (#12741S, CST), AMPK (#5832T, CST), P-AMPK^Thr172 (#2535T, CST), ULK1 (#8054T, CST), p-ULK1^Ser555 (#5869T, CST), anti-rabbit IgG (#7074, CST), tubulin (#5568S, CST), actin (bs-10966R, Bioss), GAPDH (#5174T, CST), histone H3 (#4499T, CST), and transcription factor EB (TFEB) (#A303--673A, Bethyl Laboratories Inc., United States).

Cytoplasmic and nuclear protein extraction

Cytoplasmic and nuclear proteins were isolated from HepG2 cells using the Nuclear and Cytoplasmic Protein Extraction Kit (P0028, Beyotime) according to the manufacturer’s instructions.

Statistical analysis

The data obtained from all experiments were analyzed via GraphPad Prism version 8.3. IBM SPSS Statistics 23 software was also employed for statistical analysis. The data are reported as the mean ± SE. Student’s t test was used to compare two groups, and one-way analysis of variance (ANOVA) followed by the least significant difference test was used to compare multiple groups. A P value less than 0.05 indicated statistical significance.

RESULTS

Inhibition of mGluR5 attenuated hepatocyte steatosis in vitro

First, we detected the expression of mGluR5 in an in vitro steatosis model. HepG2 cells were treated with different concentrations of FFAs for 24 hours or with 0.5 mM FFAs for various durations, after which the expression of mGluR5 was determined. The protein expression of mGluR5 tended to increase with increasing concentrations of FFAs and prolonged exposure time (Figure 1A and B). Next, we measured the effect of mGluR5 on lipid accumulation in HepG2 cells. The CCK8 assay was used to evaluate the cytotoxic effects of agonists or antagonists on hepatocytes. When the concentrations of MPEP and CHPG reached 20 and 1000 μM, respectively, the cell viability significantly decreased (Figure 1C and D). Therefore, for subsequent experiments, we selected concentrations of 10 μM for MPEP and 500 μM for CHPG. Oil Red O staining was used to evaluate the effect of mGluR5 on steatosis in HepG2 cells. The cells were initially treated with 0.5 mM FFAs for 24 hours, followed by MPEP or CHPG for an additional 24 hours. Exposure to FFAs increased fat accumulation in HepG2 cells, and MPEP treatment significantly decreased FFAs-induced fat accumulation and intracellular TG content, whereas CHPG did not affect fat accumulation or TG levels (Figure 1E and F).

Figure 1 Inhibition of metabotropic glutamate receptor type 5 attenuates hepatocyte steatosis in vitro. A: Protein expression of metabotropic glutamate receptor type 5 (mGluR5) in HepG2 cells treated with different concentrations of free fatty acids (FFAs) for 24 hours; B: Protein expression of mGluR5 in HepG2 cells treated with 0.5 mmol/L FFAs for different Durations; C: CCK-8 assays revealed the effects of MPEP (2.5, 5, 10, 20, and 50 μM) on HepG2 cell viability; D: CCK-8 assays revealed the effects of CHPG (50, 100, 200, 500, and 1000 μM) on HepG2 cell viability; E and F: HepG2 cells were stimulated with 0.5 mmol/L FFAs for 24 hours and then treated with MPEP (10 μM) or CHPG (500 μM) for another 24 hours. The cells were stained with Oil Red O, and the intracellular triglyceride concentration was quantitatively determined. ^aP < 0.05 vs free fatty acids, ^bP < 0.01 vs free fatty acids, ^cP < 0.001 vs free fatty acids; ^dP < 0.001 vs bovine serum albumin. The data are presented as the mean ± SE. All cell experiments were repeated three times. mGluR5: Metabotropic glutamate receptor type 5; FFAs: Free fatty acids; BSA: Bovine serum albumin.

Inhibition of mGluR5 improved lipid accumulation by increasing autophagy in FFAs-stimulated HepG2 cells

To explore how mGluR5 regulates lipid accumulation, we measured changes in autophagy in FFAs-stimulated HepG2 cells. Autophagy-related proteins were detected by protein blot analysis. The results revealed that the inhibition of mGluR5 promoted the protein expression of LC3-II and p62 in HepG2 cells, whereas CHPG suppressed p62 (Figure 2A and B). These data indicate that mGluR5 regulates autophagy in HepG2 cells. We then used CQ to block the fusion of autophagosomes with lysosomes. CQ cotreatment abolished the effect of MPEP on the reduction of fat accumulation and the decrease in TG levels (Figure 2C and D), which suggests that autophagy is involved in the ameliorative effect of MPEP on hepatic fat deposition.

Figure 2 Inhibition of metabotropic glutamate receptor type 5 improves lipid accumulation by increasing autophagy in free fatty acids-stimulated HepG2 cells. A and B: HepG2 cells were stimulated with 0.5 mmol/L free fatty acids (FFAs) for 24 hours and then treated with MPEP (10 μM) or CHPG (500 μM) for another 24 hours. Western blotting was used to detect the protein expression of LC3 and p62 in the cells; C and D: HepG2 cells were stimulated with 0.5 mmol/L FFAs for 24 hours and then treated with MPEP (10 μM) or MPEP (10 μM) +chloroquine (20 μM) for another 24 hours. The cells were stained with Oil Red O, and the intracellular triglyceride concentration was quantitatively determined. ^aP < 0.05 vs free fatty acids; ^bP < 0.01 vs free fatty acids; ^cP < 0.001 vs bovine serum albumin; ^dP < 0.001 vs MPEP; ^eP < 0.05 vs bovine serum albumin. The data are presented as the mean ± SE. All cell experiments were repeated three times. FFAs: Free fatty acids; BSA: Bovine serum albumin; CQ: Chloroquine.

Inhibition of mGluR5 induced autophagy by activating the AMPK signaling pathway

To test whether autophagy was activated via the AMPK signaling pathway, the levels of AMPK and the downstream protein ULK1 were assayed in vitro. The inhibition of mGluR5 by MPEP significantly increased the P-AMPK/T-AMPK ratio and tended to increase the P-ULK1/T-ULK1 ratio (Figure 3A and B). In contrast, CHPG reduced the P-ULK1/T-ULK1 ratio (Figure 3B). To investigate the relationship between AMPK activation and MPEP-induced autophagy, the AMPK inhibitor CC was used to block AMPK phosphorylation in hepatocytes. Treatment with CC reversed the attenuation of hepatocellular steatosis caused by MPEP (Figure 3C and D). Generally, an increase in p62 suggests the inhibition of autophagy since p62 is an autophagic substrate. However, the TFEB-induced increase in p62 has been reported to be accompanied by the activation of autophagy. Therefore, we examined the protein expression of TFEB in the nucleus (Figure 3E). Compared with the FFAs group, the MPEP group presented an increase in the TFEB protein in the nucleus (P < 0.05). Furthermore, compared with that in the MPEP group, the nuclear TFEB protein content in the MPEP and CC cotreatment group was decreased (P < 0.01), which indicates that the AMPK signaling pathway is involved in TFEB and p62 expression and regulates MPEP-induced autophagy.

Figure 3 Inhibition of metabotropic glutamate receptor type 5-induced autophagy via activation of the AMPK signaling pathway. A and B: HepG2 cells were stimulated with 0.5 mmol/L free fatty acids (FFAs) for 24 hours and then treated with MPEP (10 μM) or CHPG (500 μM) for another 24 hours. Western blotting was used to detect the protein expression of P-AMPK, AMPK, P-ULK1 and ULK1 in the cells; C-E: HepG2 cells were stimulated with 0.5 mmol/L FFAs for 24 hours and then treated with MPEP (10 μM), CHPG or MPEP (10 μM) + Compound C (10 μM) for another 24 hours. The cells were stained with Oil Red O (C), and the level of intracellular triglyceride was quantitatively determined (D). Western blot was used to determine transcription factor EB expression in the cytoplasmic and nuclear fractions of HepG2 cells (E). Histone H3 was used as a reference control for nuclear expression. ^aP < 0.05 vs free fatty acids; ^bP < 0.01 vs free fatty acids; ^cP < 0.001 vs bovine serum albumin; ^dP < 0.01 vs bovine serum albumin; ^eP < 0.01 vs MPEP. The data are presented as the mean ± SE. All cell experiments were repeated three times. FFAs: Free fatty acids; BSA: Bovine serum albumin; TFEB: Transcription factor EB.

KO of the mGluR5 gene improved glucose and lipid metabolism and induced hepatic autophagy in HFD-fed mice

Compared with that of WT/NCD mice, the body weight of WT/HFD mice was significantly increased beginning on the second week (Figure 4A). Compared with that of the WT/HFD group, the body weight of the KO/HFD group was significantly lower (Figure 4A). To examine the influence of mGluR5 on glucose tolerance, an IPGTT was conducted on the mice after an 8-hour fasting period (Figure 4B). As shown in Figure 4C, the area under the curve (AUC) revealed that glucose tolerance was impaired in the WT/HFD group compared with the WT/NCD group. In both the NCD and HFD groups, compared with WT mice, mGluR5 KO mice had decreased AUC values (Figure 4C). No significant difference was observed in the serum insulin levels among the different groups of mice (Figure 4D).

Figure 4 Knockout of the metabotropic glutamate receptor type 5 gene improved glucose and lipid metabolism and induced hepatic autophagy in high-fat diet-fed mice. A: Weekly weights of male mice; B and C: Blood glucose curve and area under the curve of the Intraperitoneal glucose tolerance test in male mice; D: Serum insulin levels in male mice; E and F: Serum triglyceride and total cholesterol levels in male mice; G and H: Serum low-density lipoprotein and high-density lipoprotein levels in male mice; I: Western blot analysis was used to evaluate the protein expression levels of LC3 and p62 in mice livers. The data are presented as the mean ± SE. ^aP < 0.05 vs WT/NCD, ^bP < 0.01 vs WT/NCD, ^cP < 0.001 vs WT/NCD; ^dP < 0.05 vs WT/HFD, ^eP < 0.01 vs WT/HFD, ^fP < 0.001 vs WT/HFD. WT: Wild-type; KO: Knockout; HFD: High-fat diet; NCD: Normal chow diet; AUC: Area under the curve; TG: Triglyceride; TC: Total cholesterol; HDL-C: High-density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol.

To assess the impact of mGluR5 on lipid metabolism, we analyzed serum lipid profiles, which included measurements of TG, TC, LDL-C, and HDL-C. Compared with those of the WT/NCD mice, the serum TG, TC, LDL-C and HDL-C levels of the WT/HFD-fed mice were significantly higher (Figure 4E-H). Compared with WT/HFD-fed mice, mGluR5 KO mice presented significantly lower serum TC, LDL-C and HDL-C levels, although the TG levels were not significantly different (Figure 4E-H). These results indicate that the inhibition of mGluR5 promotes weight loss and improves glucose tolerance and lipid metabolism in these mice.

We further examined the changes in autophagy in the livers of the mice. The results revealed that the level of LC3-II was lower in WT/HFD mice than in WT/NCD mice. KO of the mGluR5 gene increased the protein expression of LC3-II in KO/HFD mice, and while the protein expression of p62 also tended to increase, the difference was not statistically significant (Figure 4I). These data suggest that KO of mGluR5 promotes hepatic autophagy in HFD-fed mice.

DISCUSSION

Previous studies have demonstrated that mGluR5 regulates hepatic lipid metabolism and may play a role in the development of MASLD[16,17,24]. mGluR5 is a potential novel drug target for the treatment of MASLD, but its mechanism of action remains unclear. In the present study, we treated HepG2 cells with FFAs to establish a cell model of steatosis. We detected the protein expression of mGluR5 in FFAs-stimulated HepG2 cells. The results revealed that the expression of the mGluR5 protein in the stimulated cells did not significantly differ from that in the control cells; however, mGluR5 expression appeared to increase upon treatment with higher FFAs concentrations and with longer exposure times. In our previous in vitro cell model of hepatic steatosis, mGluR5 expression was increased in HepG2 cells treated with 0.3 mM PA for 24 hours[16], but variations in the proportion of the high-fat mixture used may have impacted mGluR5 expression. Moreover, as a receptor, the inhibition or activation of mGluR5 could have physiological and pathological effects even if its expression is unchanged. In this study, we selected a different cellular model of hepatic steatosis from that used in previous studies to further elucidate the role of mGluR5. We treated FFAs-stimulated HepG2 cells with the mGluR5 antagonist MPEP or the agonist CHPG to investigate the effect of mGluR5 on lipid metabolism. The cytotoxic effects of an mGluR5 agonist and an antagonist on hepatocytes were evaluated by CCK8 cell viability assays. Oil Red O staining revealed that MPEP could reduce FFAs-induced lipid droplet formation and the intracellular TG content, which was consistent with our previous findings that the inhibition of mGluR5 reduced lipid accumulation in hepatocytes. Different studies have reported that mGluR5 regulates lipid synthesis genes and/or lipid oxidation genes in hepatocytes[16,24,25]. In this part of the study, the cells were treated with FFAs for 24 hours, after which the cells were treated with drugs for another 24 hours. We successfully established a cell model in which pharmacological blockade of mGluR5 alleviated hepatic steatosis. Next, we further explored the potential mechanism involved.

Previous studies have shown that autophagy plays a role in lipid metabolism in the liver. Lipid droplets are wrapped in autophagosomes, which fuse with lysosomes, and here, the TGs decompose into FFAs[26]. Growing evidence indicates that defects in autophagy exacerbate liver steatosis[27,28]. Promoting autophagy in steatotic hepatocytes can reduce fat accumulation[29]. In neurodegenerative diseases such as HD and AD, mGluR5 inhibition can slow disease progression by promoting autophagy[21-23]. However, researchers have not clearly determined whether mGluR5 affects fat accumulation in hepatocytes by regulating autophagy. Therefore, we detected the levels of the autophagy-associated proteins p62 and LC3-II in FFAs-stimulated HepG2 cells treated with MPEP or CHPG. We found that both p62 and LC3-II were increased in MPEP-treated cells. LC3-II is involved in autophagosome formation and is commonly used as an indicator of autophagy onset. LC3-II was increased in FFAs-treated cells, which was likely due to increased autophagy as a stress response during the early stage of steatosis. mGluR5 inhibition further increased LC3-II expression, which promoted autophagy. p62 is considered a marker of autophagy flux because it is degraded during autophagy[30]. While increased p62 is typically assumed to indicate inhibition of autophagy, it also suggests the activation of autophagy under certain conditions[31]. In addition, some studies have shown that p62 itself can protect the liver from lipotoxicity and fibrosis[32,33]. Our results are consistent with those of a recent study in which anthelmintic nitazoxanide increased the protein expression of LC3-II and p62, which promoted autophagy and reduced hepatocyte steatosis[34]. To further demonstrate the effect of autophagy, MPEP-treated cells were also exposed to the autophagy inhibitor CQ. The alleviating effect of MPEP on steatosis was blocked by the addition of CQ, which indicates that autophagy is indeed involved in MPEP-regulated fat accumulation.

AMPK is a serine/threonine protein kinase that regulates various cellular processes, including metabolic homeostasis, cell proliferation and cell death. Activated AMPK can promote autophagy either directly or indirectly[35]. AMPK serves as an important energy sensor for the regulation of hepatic lipid metabolism[36]. In the liver, AMPK activation promotes catabolism (such as autophagy)[37], stimulates fatty acid oxidation[38], and suppresses anabolic pathways, such as lipid synthesis[39]. ULK1, which serves as the key initiator of autophagy, triggers the activation of beclin1 during the formation of autophagic vacuoles[40]. AMPK can directly phosphorylate ULK1 and indirectly activate ULK1 by inhibiting the mammalian target of rapamycin[41]. Our results revealed that mGluR5 inhibition increased the levels of phosphorylated AMPK (Thr172) and ULK1 (Ser555). TFEB has been identified as a key regulator of lysosomal biogenesis and autophagy[42]. The nuclear translocation of TFEB promotes the expression of the p62 gene so that the p62 Level increases along with autophagy[31]. Our results showed that mGluR5 inhibition by MPEP promoted TFEB nuclear translocation, which potentially explains the increase in the p62 protein level observed in this study. In addition, the AMPK inhibitor CC reversed the MPEP-induced reduction in lipid droplet deposition and TG levels and decreased the nuclear translocation of TFEB induced by MPEP. These results suggest that the inhibition of mGluR5 reduces hepatocyte steatosis and promotes the nuclear translocation of the TFEB protein via activation of AMPK (Figure 5).

Figure 5 Schematic diagram summarizing the underlying mechanism by which metabotropic glutamate receptor type 5 alleviates hepatic steatosis. Metabotropic glutamate receptor type 5 inhibition promotes AMPK phosphorylation, which leads to increased LC3II expression and nuclear translocation of transcription factor EB and the induction of p62 transcription, thereby activating autophagy and reducing lipid accumulation in hepatocytes. mGluR5: Metabotropic glutamate receptor type 5; TFEB: Transcription factor EB.

We further verified the effects of mGluR5 inhibition in vivo using mGluR5 KO mice. Our data suggested that KO of the mGluR5 gene reduced body weight and improved impaired glucose tolerance and hyperlipidemia in HFD-fed C57BL/6J mice. However, we did not observe significant changes in liver weight or liver lipid levels (data not shown), which might be due to the inadequate modeling of MASLD. Ferrigno et al[17] reported that MPEP attenuates body weight, liver weight, liver fat and serum TG content in obese mice. In another study, KO of the mGluR5 gene in obese mice with HD (BACHD mice) resulted in a significant reduction in body weight[43]. mGluR5 was also reported to be a mediator of appetite and energy balance[12,13], and thus the inhibition of mGluR5 might improve obesity and metabolism by suppressing appetite. However, considering the in vitro results provided by our team and others, we propose that mGluR5 directly regulates hepatic lipid metabolism. We also found that LC3-II was inhibited in HFD-fed WT mice and was increased in mGluR5-KO mice, whereas p62 expression tended to increase in KO/HFD mice, which suggests that the inhibition of mGluR5 promotes autophagy-related protein expression in these mice.

Our study has several limitations that should be noted. First, our experiment was conducted using only male mice. To achieve a more comprehensive understanding, it is essential to replicate the experiment with female mice to determine whether any sex-dependent effects occur in the mGluR5-mediated regulation of autophagy in alleviating hepatic steatosis. Second, due to the high variability among the mice and our limited resources, which prevented us from conducting multiple repetitions of the animal study, the data from the animal experiments were not as comprehensive or satisfactory as expected. It would be beneficial to refine the MASLD mouse model to gather more substantial evidence and to test the mechanisms of action we identified in HepG2 cells within an animal model. Furthermore, since mGluR5 inhibition improved liver steatosis, additional studies are necessary to investigate its role at different stages of MASLD, including metabolic dysfunction-associated steatohepatitis (MASH), both in mice and in cellular models. This information would help determine the timing of mGluR5 involvement in MASLD. Finally, whether improvements in fatty liver are accompanied by systemic metabolic benefits, such as increased hepatic insulin sensitivity and reduced atherosclerosis, remain to be determined.

CONCLUSION

In conclusion, the present work revealed that mGluR5 inhibition ameliorated hepatocyte steatosis by activating the AMPK signaling pathway, which promoted TFEB entry into the nucleus and enhanced autophagy. Since the spectrum of MASLD ranges from simple steatosis to MASH and potentially advances to cirrhosis and liver carcinoma, the regulation of autophagy by mGluR5 in other disease stages of MASLD requires further investigation.