Inhibition of GPR75 Alleviates Lipid Metabolism by Activating the AMPK-SIRT1 Signaling Pathway In Vitro and In Vivo

Abstract

The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is primarily driven by excessive lipid accumulation and metabolic dysregulation, necessitating a comprehensive investigation into the underlying mechanisms. This study employed an in vitro model, wherein Huh7 cells were induced with a palmitic acid/oleic acid mixture, and an in vivo model involving the provision of a high-fat diet to SD rats for six weeks. Employing techniques such as oil red O staining, immunofluorescence, and Western blotting, we examined lipid synthesis, metabolism, and the associated molecular pathways. The findings indicate that GPR75 overexpression markedly enhances lipid synthesis and impairs lipid metabolism. Conversely, GPR75 knockdown significantly diminished the fluorescence intensity of lipid synthesis factors FASN and SREBP1, concurrently elevating the expression of AMPK and SIRT1 proteins, which culminated in reduced lipid synthesis and improved lipid metabolism. Furthermore, inhibiting the AMPK-SIRT1 pathway following GPR75 knockdown led to a significant reversal of these lipid metabolic alterations. Overall, our study elucidates that GPR75 inhibition may diminish lipid accumulation and enhance lipid metabolism both in vitro and in vivo, primarily through the activation of the AMPK-SIRT1 signaling pathway.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12033-025-01451-3.

Introduction

NAFLD has become a notable public health issue globally due to its growing effects on health. This chronic condition of the liver is defined as a clinical syndrome mainly marked by hepatic steatosis [1–3], which occurs due to causes other than alcohol consumption. NAFLD is intricately linked with disturbances in glucose and lipid metabolism, alongside insulin resistance [4], with nearly 50% of patients with hypertension also presenting with NAFLD. Furthermore, there are significant connections between NAFLD and issues such as arterial stiffness, changes in myocardial structure, kidney ailments, and heart failure [5, 6]. While progress has been made in comprehending the mechanisms that drive NAFLD, numerous hurdles still exist concerning therapeutic targets and strategies, with no adequate pharmacological treatments available at this time. Consequently, it is vital to investigate new targets and molecular pathways to uncover potential avenues for drug development aimed at treating NAFLD.

The disturbances in lipid metabolism appear to be a key contributor to the onset of NAFLD [7]. Metabolic events in adipose tissue can drive the progression of NASH, making it a potential target for the treatment of NAFLD [8]. An exome sequencing investigation identified that variations in the G protein-coupled receptor 75 (GPR75) correlate with body mass index (BMI) and obesity prevalence. Silencing GPR75 significantly reduced weight gain and increased blood glucose levels in mice [9]. Schwartzman found [10] that mice lacking GPR75 were shielded from obesity linked to a high-fat diet. In a similar vein, studies by Murray and Heisler et al. indicated that GPR75 represents a promising target for both preventing NAFLD and mitigating body fat accumulation [11]. Together, these findings highlight the importance of GPR75 in the management and prevention of NAFLD. Nevertheless, the precise molecular pathways through which GPR75 influences the prevention and management of NAFLD are not yet fully elucidated.

In recent years, it has been shown that GPR75 can be activated by at least two ligands: chemokine ligand (CCL5) and 20-hydroxy-eicosatetraenoic acid (20-HETE). Upon its activation, GPR75 triggers the activation of numerous signaling molecules, similar to other members within the G protein-coupled receptor family. AMP-dependent protein kinase (AMPK), a serine/threonine kinase, plays a pivotal role in energy homeostasis as well as fatty acid metabolism [12, 13]. When activated, AMPK phosphorylates various downstream targets to regulate lipid metabolic balance. Silent information regulator (SIRT1), a NAD-dependent deacetylase, is crucial for lipid metabolism and energy management. The activation of both AMPK and SIRT1 is mutually dependent, creating a positive feedback mechanism that promotes lipid metabolism and energy balance. It has been noted that the activation of the AMPK pathway can reduce lipid accumulation both in vitro and in vivo [12, 14, 15]. However, during the onset and advancement of NAFLD, the activities of both AMPK and SIRT1 are suppressed. Nonetheless, the precise molecular mechanism through which GPR75 governs the AMPK-SIRT1 signaling pathway in the context of NAFLD has yet to be reported.

In this research, we developed models both in vivo and in vitro to confirm the impact of GPR75 on lipid buildup and to explore the molecular mechanisms involved. Our results indicate that the over-expression of GPR75 markedly boosts lipid synthesis and affects lipid metabolism induced by palmitic/oleic acid mixtures in vitro. In contrast, down-regulation of GPR75 expression may activate the AMPK-SIRT1 signaling pathway, which subsequently leads to a reduction in lipid accumulation in both in vivo and in vitro settings.

Materials and Methods

Cell Culture

Huh7 cells were sourced from Wuhan Pricella, China, and the cells were exposed to a mixture of palmitic acid (PA) and oleic acid (OA) in a 1:2 ratio for a duration of 24–48 h to create an in vitro model of NAFLD. The cells were regularly maintained in Dulbecco’s modified Eagle medium (DMEM) complete medium (Gibco, USA) that included 10% fetal bovine serum (Gibco, USA). For the transfection of plasmids designed for overexpression or siRNA, Lipofectamine™ 3000 (Invitrogen, USA) was employed following the guidelines provided by the manufacturer. After twelve hours of transfection, the cells were treated with fatty acids for a period of 24 h. The cells were then harvested for additional biological assays.

Oil Red O Staining

Cells were cultivated until reaching an optimal condition, after which the culture medium was removed and the cells were rinsed once with PBS. Cells were then fixed using 4% paraformaldehyde for a duration of 10 min, followed by two rinses with PBS. Subsequently, oil red O staining solution was applied to the cells for a coverage period of 20 s. After removal of this solution, a suitable volume of working oil red O staining solution was introduced, staining the cells for 10 min. Once the oil red O working solution was aspirated, a staining wash solution was added to the cells and allowed to remain for 30 s. The cells were then washed with PBS for 20 s. Lastly, PBS was used to cover the cells. The cells were observed under a microscope and images were captured.

Animal

Healthy male SD rats of SPF grade, aged 8 weeks, were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. These rats were kept in a controlled environment at temperatures ranging from 20 to 23 °C and with a relative humidity of 40%–60%, following a 12-h light/dark cycle. After a 1-week period of acclimatization, the rats were randomly assigned to a control group (n = 6) and a model group (n = 18). The model group was fed a high-fat diet containing 60 kcal% from fat (Huayastron, Chongqing, China). Following 6 weeks of continuous feeding, rats that showed a body mass increase of 20% more than the control group were subsequently categorized into the high-fat diet (HFD) group (n = 6), the GPR75 shRNA group (n = 6), and the SIRT1 inhibitor group (n = 6). Starting from week 7, rats in the control group were injected intraperitoneally with saline. Rats in the HFD group received injections of control shRNA, while rats in the SIRT1 inhibitor group were injected with Nicotinamide. Additionally, lentiviral particles targeting GPR75 shRNA were administered to the rats in the GPR75 shRNA group. The HFD group continued with the high-fat diet while the control group maintained their standard diet. Approval was obtained from the Animal Experimentation Ethics Review Committee of Kunming Medical University before the study began (approval number: kmmu20241849). The current study's animal experiments adhered to the experimental protocol sanctioned by the Animal Ethics Committee of Kunming Medical University.

Metabolic Indicator Assay

All enzyme-linked immunosorbent assay (ELISA) and biochemical kits were procured from Beijing Solarbio Technology Co., Ltd. in China, and the experimental procedures were conducted in strict adherence to the manufacturers'instructions. Collected the supernatant from each group of cells and centrifuged at 4 °C for 20 min at 1000 × g. Collected the supernatant for subsequent testing. Added 100 µL of the samples to be tested into the appropriate wells of a 96-well ELISA plate, ensuring the setup of two duplicate wells for each sample. The plate was covered with a sealer and incubated for 90 min at 37 °C. After the incubation period, each well was blotted dry, and 100 µL of biotinylated detection antibody working solution was added. Incubated at 37 °C for 1 h. The plates were washed three times. Then, 100 µL of HRP enzyme conjugate working solution was added to each well. Incubated the plate at 37 °C for 30 min, then washed each well three times. added 90 µL of the substrate solution (TMB) to each well and covered the plate. Incubated at 37 °C for an additional 15 min. Added 50 µL of the termination solution to each well. Finally, measure the OD of each well at 450 nm using an enzyme marker immediately after adding the termination solution.

Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR)

RNA was extracted from Huh7 cells employing Trizol reagent (Invitrogen, USA). A reverse transcription kit (Tiangen, Beijing, China) was utilized for the synthesis of first strand cDNA. The GPR75 gene was amplified using SYBR Green Master Mix (Tiangen Biotech Co., Ltd., Beijing, China) under the following conditions: pre-denaturation at 95 °C for 8 min, followed by 40 cycles of amplification with 15 s at 95 °C and 30 s at 60 °C. Immediately after the amplification cycles, a melting curve analysis was conducted by heating from 60 °C to 95 °C to verify the specificity of the amplified products. Using GAPDH as the reference gene, the relative mRNA expression levels were determined using the 2-ΔΔCT method [16]. The sequences of the primers used: GPR75: forward: 5'-TGCTGGAAAAGGGAAAGCCA-3', reverse: 5'- TGTGGTCTGGAAGCATCGAC-3'; GAPDH: forward: 5'-CAGCCTCAAGATCATCAGCA-3', reverse: 5'- ATGATGTTCTGGAGAGCCCC-3'.

Western Blot

A kit for total protein extraction was employed to employed to obtain total cellular protein, after which a BCA kit was applied to measure the protein concentration, loading 50 μg of the sample into each well. Proteins were resolved on a 4–20% SDS-PAGE gel before being transferred to a PVDF membrane. To prevent nonspecific binding, the membrane was incubated with PBS buffer containing 5% skimmed milk powder for one hour. Primary antibodies specific to each protein of interest, along with a β-actin antibody as a loading control, were introduced and allowed to incubate overnight at 4 °C. The membrane was washed three times with PBST buffer, with each wash lasting 5 min. Subsequently, the relevant secondary antibody was added and allowed to incubate at 37 °C for 2 h. After three additional washes with PBST buffer, chemiluminescent development was performed using ECL, and the grayscale values of the target proteins were quantified using Image J along with a protein gel imaging system. Antibodies targeting PGC1α、SIRT1 and Phosphorylated AMPK were acquired Abcam (Cambridge, UK). The internal control antibody β-actin and HRP secondary antibody were obtained from Proteintech (Wuhan, China).

Immunofluorescence Assay

Tissue sections embedded in paraffin were baked for 30 min, then deparaffinized using xylene, and subsequently hydrated in graded ethanol for 5 min each. Following this, they were rinsed with distilled water for an additional 5 min before being placed in a microwave for antigen retrieval, allowing them to cool to room temperature afterward. The tissues were then dried with a blotting technique, and a drop of normal sheep serum was applied, which was followed by a 2-h incubation period in a wet chamber at room temperature. Next, the sections were rinsed three times with PBS and treated with the appropriate fluorescent secondary antibody for 1 h at room temperature, ensuring protection from light exposure. For nuclear staining, DAPI was added and the sections were incubated for 15 min at room temperature, followed by three washes with PBS. Finally, the sections were scanned using a laser confocal microscope (Leica TCS-SP5), where green fluorescence was excited by laser light at a wavelength of 488 nm, and the resulting data were captured and digitally processed by a computer.

Hematoxylin and Eosin (HE) Staining

The tissue sections were first baked for 30 min, then immersed in xylene two times for 5 min each. Next, the sections were subjected to 5-min treatments in 100%, 90%, 80%, and 70% ethanol, followed by a 5-min exposure to distilled water. After excess water was removed, Hematoxylin Stain was applied for 5 min. The sections were then rinsed under running water to eliminate surplus stain, and the staining intensity was examined using a microscope. Differentiation of the sections occurred with 1% hydrochloric acid in alcohol for a duration of 1 to 3 s, followed by washing in water and submersion in tap water for 5 to 10 min to regain the blue hue. Afterwards, the sections were treated with 0.5% eosin stain for a period of 10 to 15 s, rinsed with distilled water, and inspected under a microscope. For dehydration, the sections underwent a progressive immersion in 80%, 90%, 95%, and finally 100% ethanol. They were then placed in xylene two times for 5 min each and left to dry at room temperature. Ultimately, the sections were covered with glycerol and photographed under a microscope after 24 h.

Statistical Analysis

The experimental data were analyzed statistically and illustrated using GraphPad Prism 9.0. Results are presented as mean ± standard deviation. One-way ANOVA with post-hoc Tukey’s test was utilized for data analysis, with p < 0.05 regarded as statistically significant.

Results

Overexpression of GPR75 Promotes PA-OA-Induced Lipid Synthesis in Huh7 Cells

To examine the impact of GPR75 on lipid synthesis, Huh7 cells were treated with a blend of PA and OA for 24 h, after which the cells were transfected using plasmids designed for GPR75 overexpression. The results from RT-qPCR validated the successful establishment of the GPR75-overexpressing Huh7 cell line (Fig. 1A). As a result, one of the overexpressing cell lines was chosen for subsequent experiments. The ELISA revealed marked increases in FFA, triglycerides (TG), total cholesterol (TC), and malondialdehyde (MDA)—a marker of lipid peroxidation—in cells overexpressing GPR75 (Fig. 1B–D, G). Moreover, Oil Red O staining demonstrated a considerable rise in intracellular lipids in Huh7 cells treated with PA-OA compared to the control group, with lipid accumulation being notably greater following GPR75 overexpression (Fig. 1E, F). Additionally, immunofluorescence analysis of critical adipogenic factors showed a significant rise in the fluorescence intensity of fatty acid synthase (FASN) and sterol regulatory element-binding protein 1 (SREBP1) in GPR75-overexpressing Huh7 cells, relative to their counterparts induced by PA-OA (Fig. 1H–K).

Fig. 1.

GPR75 overexpression promotes PA-OA-induced lipid synthesis in Huh7 cells A RT-qPCR validation of GPR75 overexpression vector. B–D ELISA for free fatty acids, cholesterol and triglycerides in cell supernatants, respectively. E, F Oil red O staining to detect lipid droplet area in Huh7 cells. G MDA in cells was detected by ELISA. H, I Cellular immunofluorescence was used to detect the expression of FASN and its fluorescence intensity was counted. J, K The fluorescence intensity of SREBP1 was detected by cell immunofluorescence and counted. NC group: normal Huh7 cells. OE-NC group: Huh7 cells that were transfected with a negative control overexpression plasmid. FL group: a mixture of PA and OA at a 1:2 ratio. GPR75-OE group: Huh7 cells transfected with GPR75 overexpression plasmid. #P < 0.05, ##P < 0.01, ###P < 0.001, vs. NC. *P < 0.05, **P < 0.01, ***P < 0.001, vs. FL. Scale bars = 10 μm

Knockdown of GPR75 Activates the AMPK-SIRT1 Pathway to Attenuate Lipid Metabolism In Vitro

To investigate the molecular mechanism by which GPR75 affects lipid synthesis, the Huh7 cell line was transfected with three different siRNAs targeting GPR75. Then, the knockdown efficiency was evaluated using RT-qPCR. The results showed that GPR75 siRNA1 had the lowest knockdown efficiency compared to the other two siRNAs; therefore, GPR75 siRNA1 was used for subsequent experiments (Fig. 2A). Western blot analyses revealed that levels of SIRT1, p-AMPK, and peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) markedly rose after GPR75 knockdown. The suppression of AMPK or SIRT1 caused a varying degree of reduction in the expression levels of SIRT1 or AMPK. When we compared the GPR75 knockdown group to those undergoing dual treatment with both GPR75 knockdown and AMPK or SIRT1 inhibition, a significant decrease in PGC1α expression was noted (Fig. 2B–E). The results from Oil Red O staining indicated a marked reduction in lipid levels within the GPR75 knockdown cohort in comparison to the cells induced by PA-OA. Importantly, this decrease in lipid content was reversed with the inhibition of either AMPK or SIRT1 expression (Fig. 2F, G). Furthermore, our cellular immunofluorescence assays showed that knockdown of GPR75 resulted in diminished fluorescence intensity for FASN and SREBP1. Conversely, the inhibition of AMPK or SIRT1 led to an increase in the activities of both FASN and SREBP1 (Fig. 2H–J). These results imply that, in vitro, the AMPK-SIRT1 signaling pathway is activated following GPR75 knockdown, consequently enhancing the expression of the downstream factor PGC1α and facilitating lipid metabolism.

Fig. 2.

Knockdown of GPR75 promotes AMPK-SIRT1 pathway activation and attenuates lipid metabolism A Three different sequences of GPR75 siRNA were transfected into Huh7 cells, and RT-qPCR was used to detect the efficiency of GPR75 knockdown. B Western blot for changes in expression of PGC1α, SIRT1 and p-AMPK. C–E Grey scale analysis of PGC1α, p-AMPK and SIRT1 protein bands respectively (compared to the internal reference β-actin). F, G Oil red O detection of lipid droplets in cells and counting their area. H–J Cellular immunofluorescence was used to detect the expression of FASN and SREBP1, and the fluorescence intensities were counted by using image J software. NC group: Huh7 cells transfected with only negative control siRNA. PA-OA group: Huh7 cells treated with a 1:2 mixture of PA and OA. GPR75 siRNA group: treating them with PA and OA before transfecting Huh7 cells with GPR75 siRNA. AMPK inhibitor group was treated with an AMPK inhibitor in conjunction with PA and OA. Similarly, the SIRT1 inhibitor group received a SIRT1 inhibitor along with their PA and OA treatment. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars = 10 μm

Inhibition of SIRT1 Reverses the Attenuation of Lipid Accumulation in NAFLD Rats Following GPR75 Reduction

Next, we explored how GPR75 influences lipid metabolism in a living organism. In this study, NAFLD rats were injected with shRNA designed to target GPR75, aiming to down-regulate its expression. RT-qPCR analysis revealed a significant reduction in GPR75 expression in both abdominal and inguinal adipose tissues following the injection of GPR75 shRNA into NAFLD rats (Fig. 3A, B). Following the 12 weeks on the high-fat diet, HE staining demonstrated that the sizes of inguinal and abdominal adipocytes were considerably larger in NAFLD rats than in normal rats; however, this enlargement was significantly diminished after inhibition of GPR75 expression and was only observed again following the inhibition of SIRT1 (Fig. 3C). In addition, body weight, epididymal fat mass, perirenal fat mass, and abdominal fat mass were significantly increased in the NAFLD rats (Fig. 3D–G). Specifically, the inhibition of GPR75 activity alone significantly reduced the increase in both body weight and adipose tissue mass among the NAFLD rats. The suppression of SIRT1 led to a reversal of adipose tissue and body mass when compared to the results seen with GPR75 inhibition only. The synthesis of adipose tissue is affected not only by the availability of nutrients but is also closely controlled by metabolic hormones [17]. Consequently, we measured fasting blood glucose (FBG) and insulin concentrations in the rats to assess islet functionality and resistance to insulin. The findings revealed that the NAFLD rats displayed notable insulin resistance and reduced pancreatic islet functionality, both of which were improved by inhibiting GPR75. Importantly, the blocking of SIRT1 diminished the impact on islet functionality that followed GPR75 inhibited (Fig. 3H–I). We also analyzed lipid parameters, including TG, TC, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in the NAFLD rats. The findings suggest that inhibiting GPR75 led to a notable decrease in serum TG, total TC, and low-density lipoprotein cholesterol (LDL-C) levels, alongside a significant rise in high-density lipoprotein cholesterol (HDL-C) when compared to the group with the NAFLD induced by a HFD. After SIRT1 was inhibited, the lipid levels showed a reversal (Fig. 3J–M). In summary, these findings collectively indicate that inhibition of GPR75 leads to substantial enhancements in lipid metabolism, a reduction in lipid synthesis, and mitigates fat accumulation in HFD-induced NAFLD rats, whereas the inhibition of SIRT1 counteracts these positive effects.

Fig. 3.

Inhibition of SIRT1 reverses the attenuation of lipid accumulation in NAFLD rats following GPR75 reduction A, B RT-qPCR was used to detect GPR75 mRNA expression levels in inguinal and abdominal adipose tissue of NAFLD rats (Control shRNA represents the HFD group; GPR75 shRNA represents the GPR75 shRNA group). C Pathological analysis of inguinal and abdominal white adipose tissue by HE staining. D Rat body weight statistics. E Adipose tissue mass statistics of epididymis. F Perirenal adipose tissue mass statistics. G Abdominal adipose tissue mass statistics. H ELISA of ELISA of blood glucose in fasting rats in fasting rats. I Serum insulin levels in rats by ELISA. J, K TG and TC in rat serum were detected by ELISA. L, M LDL-C and HDL-C in rat serum were measured by ELISA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars = 10 μm

Inhibition of GPR75 Activates AMPK-SIRT1 Pathway In Vivo to Improve Lipid Metabolism in Rats

To further investigate the molecular mechanisms by which GPR75 influences lipid metabolism in HFD-induced NAFLD rats, we evaluated the expression levels of SIRT1 and AMPK—key regulators in this process—in both inguinal and abdominal adipose tissues. Our Western blot analysis revealed that the levels of SIRT1 and p-AMPK, along with PGC1α, were significantly reduced in the adipose tissue of NAFLD rats compared to controls. Following in vivo inhibition of GPR75, the expression of SIRT1, p-AMPK, and PGC-1α was restored. Subsequently, with further inhibition of SIRT1 activity, the expression of p-AMPK and PGC-1α decreased again compared to the group that solely inhibited GPR75 (Fig. 4A–D). Tissue immunofluorescence results additionally indicated that GPR75 inhibition enhanced AMPK activity in the adipose tissue of NAFLD rats. However, after the dual inhibition of GPR75 and SIRT1 activities, AMPK activity was relatively diminished (Fig. 4E–H). These findings suggest that GPR75 inhibition activates the AMPK-SIRT1 signaling pathway, thereby enhancing PGC-1α activity and regulating lipid metabolism in vivo.

Fig. 4.

Inhibition of GPR75 activates AMPK-SIRT1 pathway in vivo to improve lipid metabolism in rats A, B Expression of SIRT1 and p-AMPK as well as PGC-1α in inguinal white adipose tissue was detected by Western blot and grey scale analysis statistics were done. C, D Expression of SIRT1 and p-AMPK as well as PGC-1α in abdominal white adipose tissue was detected by Western blot and grey scale analysis statistics were done and plotted in tables. E–H Fluorescence intensity of phosphorylated AMPK in inguinal and abdominal white adipose tissue was detected by tissue immunofluorescence, respectively. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars = 10 μm

Discussion

Metabolic dysfunction-associated steatohepatopathy (MASLD), previously known as NAFLD, is identified by conditions such as obesity, visceral fat accumulation, insulin resistance, and dyslipidemia, which adversely affect various organs [18]. Lipids serve as an essential energy reservoir for the body, and the onset of NAFLD is intricately associated with lipid metabolism, especially the handling of triglycerides and cholesterol involved in fatty acid synthesis [19]. GPCRs serve as upstream regulators of adipogenesis and lipolysis balance, being crucial for adipocyte and overall metabolic homeostasis. Studies have shown that GPCRs such as β3-AR, GPR120, and A2 A receptor are abundantly expressed in specific types of adipose tissue, suggesting that drugs targeting these GPCRs can act more precisely on specific adipose depots, potentially reducing side effects on other tissues. Notably, GPCR pharmacology has been extensively studied and recognized, with approximately one-third of approved drugs targeting GPCRs. GPCRs like A2 A receptor and GPR120 have been confirmed to improve insulin resistance and lipid metabolism [20], holding potential therapeutic value for metabolic diseases. Among them, GPR75, a unique GPCR, has garnered significant attention in recent years for its potential roles in obesity and metabolic disorders. Unlike other GPCRs, GPR75 is specifically expressed in the brain and central nervous system. In the hypothalamus, GPR75 significantly influences the activity of neurons involved in appetite control, thereby regulating food intake and energy balance [11]. This central regulatory mechanism represents a unique advantage of GPR75 in the treatment of obesity and metabolic disorders. Our study found that overexpression of GPR75 significantly enhanced the expression of lipid metabolism-related molecules SREBP1, FASN, and AMPK, while inhibition of GPR75 expression reversed these molecular effects. While the significance of GPR75 in relation to obesity and metabolic syndrome is becoming more apparent [9], its molecular pathways concerning lipid accumulation are not yet fully understood. This research explored the impact and underlying molecular mechanisms of GPR75 on lipid metabolism through both in vitro and in vivo experiments.

To create an in vitro model of NAFLD, researchers used PA-OA to induce lipoatrophy in Huh7 cells. Following this treatment, biological assays revealed a notable increase in the intracellular concentrations of FFA, TG, total TC, and MDA, in addition to heightened lipid droplet formation. Importantly, when comparing PA-OA-treated Huh7 cells, a marked rise in these cellular indicators was noted after the overexpression of GPR75. This observation indicates that GPR75 may promote cellular steatosis and lipid peroxidation by PA-OA. Overexpression of GPR75 significantly enhanced the expression of lipid metabolism-related molecules SREBP1 and FASN, while inhibition of GPR75 expression reversed these molecular effects.

Disordered lipid metabolism is a central pathological mechanism in NAFLD progression, involving complex interactions among lipid synthesis, mitochondrial dysfunction, and oxidative stress. Insulin resistance and imbalanced lipid synthesis are key drivers of this metabolic dysregulation. In this study, a marked increase in the fluorescence intensity of FASN and SREBP1 in cells treated with PA-OA, with an even more pronounced fluorescence noted upon GPR75 overexpression. In contrast, reducing GPR75 levels led to a decrease in the expression of both FASN and SREBP1. FASN acts as an indicator of lipid production and holds a crucial role in regulating NAFLD [21]. The expression of FASN is induced by SREBP1, which in turn facilitates the production of fatty acids and triglycerides. Numerous studies have identified an increase in SREBP1 levels among patients with NAFLD, and experiments on mice show that enhanced SREBP1 expression correlates with elevated triglyceride concentrations in the liver [22, 23]. Studies have shown that under insulin-resistant conditions, hypomethylation of the SREBP-1c promoter elevates its expression, promoting de novo lipogenesis (DNL), lipid metabolic imbalance, and hepatic steatosis, thereby driving NAFLD development [24, 25]. This is consistent with our research findings.

SIRT1, a NAD⁺-dependent deacetylase and key metabolic regulator, plays a critical role in coordinating cellular energy metabolism networks and is involved in multiple metabolic processes, including glucose metabolism, lipid metabolism, biooxidation, and amino acid metabolism [26]. For example, Xu et al. demonstrated that SIRT1 levels are closely associated with bone turnover markers in type 2 diabetes mellitus (T2DM) patients, serving as an independent risk factor for diagnosing bone metabolic disorders in T2DM [27]. Xiang et al. showed that SIRT1 activation reduces mitochondrial oxidative stress and improves thermogenic capacity in brown adipose tissue of progeria mice [28]. Consistent with these studies, our research found that SIRT1 inhibition reversed the improved lipid metabolism effects induced by GPR75 knockdown. Notably, this effect may be related not only to lipid synthesis but also to SIRT1-mediated hepatic inflammation and glucose metabolism. Studies have revealed that SIRT1 activation reverses excessive acetylation of IRS-1 at serine 307, restoring insulin-mediated glucose uptake in a HFD-induced NAFLD mouse model. Additionally, downregulating SIRT1 expression leads to excessive acetylation of PGC-1α, decreased mitochondrial membrane potential, increased reactive oxygen species (ROS) production, and further exacerbation of lipid peroxidation and hepatocyte damage. Furthermore, SIRT1 alleviates interference of inflammatory factors (such as TNF-α) with insulin signaling by inhibiting nuclear factor κB (NF-κB) activity, thereby improving hepatic insulin resistance [29, 30]. These findings indicate that SIRT1 may serve as a key factor in the interaction between lipid metabolism and other pathological factors of NAFLD, such as hepatic inflammation and mitochondrial dysfunction.

Studies indicate that the mitophagy receptor BNIP3L can ameliorate lipid metabolism by clearing damaged mitochondria [31]. Furthermore, mitochondrial tissue heterogeneity may play a pivotal role in NAFLD development. In animal models, nuclear-encoded mitochondrial genes and proteins exhibit distinct expression patterns across metabolically active tissues such as the kidney, heart, and brain [32–34], with pronounced heterogeneity in mitochondrial activity and transcriptional profiles [35]. This mirrors pathological mitochondrial alterations observed in the livers of NAFLD patients. For instance, hepatic expression of the lipogenic gene SREBP-1c is twice as high in NAFLD patients compared to healthy individuals, correlating negatively with reduced mitochondrial DNA (mtDNA) copy numbers [36]. Additionally, adipose triglyceride lipase (ATGL) levels in NAFLD patients are 30% lower in adipose tissue, associated with elevated methylation levels in the promoter region of mitochondrial transcription factor A (TFAM) [37]. These findings suggest that mitochondrial heterogeneity impacts not only the liver but also other tissues in NAFLD. Future studies should evaluate the correlation between lipid metabolism markers and mitochondrial function across multiple tissues to fully elucidate the role of the GPR75-AMPK-SIRT1 axis in the multifactorial pathology of NAFLD.

It is important to note that the use of nicotinamide as a SIRT1 inhibitor in this study carries inherent limitations. While nicotinamide effectively inhibits SIRT1 activity, it may also competitively bind to other NAD⁺-dependent deacetylases, such as SIRT2, thereby interfering with mitochondrial function and energy metabolism [38]. Although the SIRT1 inhibitor was employed here to validate the causal relationship between the SIRT1-AMPK pathway and lipid metabolism, we cannot fully exclude potential confounding effects from other NAD⁺-dependent pathways. Future studies should utilize tissue-specific SIRT1 knockout animal models to more precisely delineate the tissue-specific roles of SIRT1 in GPR75-mediated lipid regulation. Furthermore, the potential adverse effects of long-term SIRT1 inhibition warrant rigorous evaluation in preclinical studies.

Several GPCRs have been demonstrated to mediate AMPK activity via the signaling intermediate cAMP to regulate whole-body energy homeostasis. Furthermore, AMPK can stimulate SIRT1 through the elevation of NAD⁺ concentrations [39–41]. Consistent with previous studies, pathological and biological analyses of WAT in abdominal and inguinal fat of rats revealed that inhibition of GPR75 was associated with a reduction in white adipocyte size and an increase in the expression of SIRT1, p-AMPK, and the downstream active protein PGC1α when compared to obese rats. Furthermore, in the group that solely inhibited GPR75, there was a rebound in white adipocyte size, while the expression of PGC1α and p-AMPK was suppressed in the GPR75 and SIRT1 inhibition group. These results indicate that knockdown of GPR75 leads to activation of the AMPK-SIRT1 signaling, thereby reducing lipid accumulation and improving lipid metabolism; inhibition of AMPK or SIRT1 reverses these effects. Although the present study does not clarify the direct interaction between GPR75 and AMPK/SIRT1, some research suggests a potential relationship between GPR75 and the AMPK pathway. Studies have shown that CCL5, one of the ligands for GPR75, may exert its promoting effects on bioenergetic metabolism through GPR75 [42]. CCL5 can regulate insulin signaling via the PI3 kinase and Akt pathway to increase glucose uptake and reduce AMPKα phosphorylation [43]. Additionally, AMPK activation can enhance SIRT1 activity through NAD⁺. Therefore, GPR75 knockdown might amplify the AMPK/SIRT1 signaling pathway through certain signal transduction mechanisms. Future experiments such as co-immunoprecipitation, transcriptomics, or proteomics analysis could be conducted to further determine whether interactions exist between GPR75 and AMPK-SIRT1, as well as the downstream signals of GPR75, in order to fully elucidate the molecular mechanisms by which GPR75 regulates the downstream signaling cascade.

GPR75 deficiency alleviates HFD-induced obesity in mice by affecting metabolic energy expenditure [10]. Recent studies report that genetic variants or deficiency of GPR75 are associated with significant reductions in human body fat and weight, and GPR75 deficiency largely suppresses the development of fatty liver. Importantly, while limiting adipose tissue expansion under overnutrition generally leads to worsening of metabolic health, GPR75 deficiency does not reduce fat mass by restricting the expansion of adipose tissue itself [11]. These findings highlight the therapeutic potential, safety, and importance of targeting GPR75 in metabolic diseases, presenting a potential new therapy for NAFLD. However, cautious consideration is necessary for translating GPR75-targeted therapy to human subjects. Future studies should continue to thoroughly evaluate and validate the multifaceted effects and consequences of targeting GPR75 in preclinical animal models to improve its feasibility and safety; developing selective GPR75 antagonists with minimal off-target effects is also critical. Additionally, combining GPR75 antagonists with SIRT1/AMPK agonists may represent a novel strategy to enhance the improvement of lipid metabolism.

Epidemiological research has shown a strong link between excess adipose tissue and the occurrence of metabolic illnesses [44, 45]. Mammalian species have three main types of fat: white, beige, and brown. WAT is the primary type of fat tissue found in the body [46]. To investigate the molecular pathways through which GPR75 affects fat accumulation in a living organism, we administered a high-fat diet to induce adipose tissue changes in rats, thereby creating an animal model of NAFLD, and obtained WAT samples from these subjects for further analysis. Studies suggest that an increase in visceral fat is associated with an elevated risk of metabolic disorders, such as insulin resistance [47], which is consistent with our experimental outcomes. In this investigation, we found that the inhibition of GPR75 reduced the increases in body weight caused by the high-fat diet, as well as decreases in both visceral fat (including epididymal and perirenal fat) and subcutaneous fat mass (specifically inguinal fat) in obese rats. Moreover, this inhibition also led to lower levels of serum markers linked to metabolic conditions. The concomitant inhibition of SIRT1, achieved through the inhibition of GPR75, significantly reversed the reduction in fat mass caused by the inhibition of GPR75, and reintroduced metabolic dysfunction.

Notably, this study presents for the first time evidence that inhibiting GPR75 may enhances lipid deposition in NAFLD by promoting the activation of the AMPK-SIRT1 pathway, thereby increasing the expression of key regulators of energy metabolism and modulating lipid metabolism and synthesis both in vitro and in vivo. However, although this study demonstrates that regulating the GPR75-SIRT1-AMPK signaling axis can affect lipid accumulation and metabolism, this conclusion is derived from short-term research. Given the widespread distribution of GPR75 in the brain, which may influence lipid accumulation through multiple brain-related pathways [11], the risks and benefits of long-term inhibition of GPR75 expression in animals and humans remain undetermined. Future studies may need to evaluate the multifaceted consequences of sustained GPR75 inhibition on the body and various organs in animal models that mimic the progression of human NAFLD.

Supplementary Information

Below is the link to the electronic supplementary material.

Authors Contribution

Conceptualization, B.W., K.L.L., J.Y.W.; Methodology, B.W., K.L.L., G.S.S. and J.Y.W.; Data curation, B.W. and K.L.L.; Writing – Original Draft Preparation, B.W. and K.L.L. Validation, G.S.S. and J.Y.W.; Formal analysis, S.W.L.; Software, S.W.L.; Visualization, J.Y.W. and X.H.; Investigation, R.Z .Z.; Project administration, B.W., K.L.L.; Writing – Review & Editing, B.W., K.L.L., G.S.S.; Funding acquisition, B.W.; Resources, B.W., K.L.L.; Supervision, B.W., K.L.L. All authors have reviewed and approved the final version of the manuscript.

Funding

This study was funded by the Yunnan Provincial Science and Technology Department Major Science and Technology Project (202402 AA310006), the Kunming Medical University Joint Yunnan Provincial Science and Technology Major Project (202301 AY070001-058), the Yunnan Provincial Geriatric Disease Clinical Research Centre and the Yunnan Provincial People's Hospital Open Project for Clinical Medicine Centre (2023YJZX-LN19).

Data Availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest in this article.