GRAPHICAL ABSTRACT

Highlights

• Lactate promotes hepatic lipid accumulation through GPR81 activation.

• GPR81 knockdown reverses lactate-induced lipogenesis via AMPK restoration.

• NAFLD mouse models show increased hepatic lactate and GPR81 expression.

• Lactate exposure induces liver steatosis in zebrafish.

• GPR81 emerges as a potential therapeutic target for NAFLD.

INTRODUCTION

Nonalcoholic fatty liver disease (NAFLD) is the predominant chronic liver condition worldwide, affecting approximately 25% of the general population [1] and mainly involving individuals with obesity and type 2 diabetes mellitus (T2DM) [2]. NAFLD is characterized by the presence of excess triglycerides in the liver, particularly in the cytoplasm of hepatocytes in the absence of excessive alcohol consumption [3,4]. Simple steatosis is defined histologically as more than 5% hepatic fat in the absence of inflammation or hepatocellular damage [5] and can progress to nonalcoholic steatohepatitis with inflammation, leading to severe complications such as fibrosis, cirrhosis, or liver cancer [6]. Despite ongoing research, there are currently no approved medications for NAFLD treatment.

Lactate is traditionally viewed as a byproduct of anaerobic glycolysis and is associated with various harmful effects [7,8]. However, it is now recognized not only as a waste product of anaerobic metabolism but also as a signaling molecule and/or an essential energy source. Recent studies have suggested that lactate can function as a signaling molecule via its dedicated receptor, G-protein-coupled receptor 81 (GPR81) [9], or can be transported across cells by monocarboxylate transporters (MCTs) [10]. In the body, a healthy liver eliminates 70% of lactate, exhibiting the highest clearance rate among the organs [11]. In chronic liver disease, impaired hepatic function leads to reduced lactate clearance and subsequent accumulation. Recent studies have demonstrated that lactate accumulation in the liver, driven by the acetylation of lactate dehydrogenase B (LDHB), is associated with the progression from simple steatosis to non-alcoholic steatohepatitis (NASH) [12]. Notably, hepatic lactate accumulation correlates with lipid accumulation, and experimental interventions such as LDHB deacetylation or MCT1 haploinsufficiency reduce hepatic lipid content in high-fat diet (HFD) models [12,13], suggesting a mechanistic link between lactate and hepatic lipid metabolism. Patients with T2DM and obesity reportedly have increased plasma lactate levels [14-18]. In these chronic metabolic conditions, lactate may exert distinct effects from its acute role during exercise, contributing to altered cellular metabolism, insulin resistance, and disrupted glucose homeostasis [14,19]. Thus, delineating the physiological and pathological roles of chronically elevated L-lactate levels in obesity and T2DM is essential for understanding their metabolic sequelae.

In this study, we examined the effects of lactate and GPR81 on lipid metabolism in hepatocytes in vitro. In vivo, we evaluated liver lactate accumulation in diet-induced NAFLD mouse models. Lactate-induced lipid accumulation was also assessed in zebrafish. The zebrafish liver shares morphological and functional similarities with the human liver including analogous cell types [20]. Zebrafish have been shown to develop hepatic steatosis in response to high-fat high-cholesterol (HFHC) diets, resembling human NAFLD [21]. About 71.4% of zebrafish genes are shared with humans, including those related to liver function and lipid metabolism, supporting their use as a translational model [22].

METHODS

Reagents and antibodies

Sodium lactate, obtained from Sigma-Aldrich (#71718, St. Louis, MO, USA), was diluted in phosphate-buffered saline (PBS) to prepare a 1M solution, and AZD3965 (# HY-12750, MedChem Express, Monmouth Junction, NJ, USA) was diluted in dimethyl sulfoxide.

Antibodies for GPR81 (MBS821939, My BioSource, San Diego, CA, USA), MCT1 (sc-365501, Santa Cruz Biotechnology, Santa Cruz, CA, USA), MCT4 (sc-376140, Santa Cruz Biotechnology), β-actin (sc-47778, Santa Cruz Biotechnology), phosphorylation 5’ adenosine monophosphate-activated protein kinase (p-AMPK) (Thr172 #2535, Cell Signaling Technology, Danvers, MA, USA), AMPK (#2532, Cell Signaling Technology), peroxisome proliferator-activated receptor alpha (PPARα) (sc-398394 Santa Cruz Biotechnology), sterol regulatory element-binding protein 1 (SREBP1) (NB100-2215, Novus Biologicals, Centennial, CO, USA), stearoyl-CoA desaturase-1 (SCD1) (#2794, Cell Signaling Technology), acetyl-CoA carboxylase (ACC) (#3676, Cell Signaling Technology), and α-tubulin (#12351, Cell Signaling Technology) were used in this study.

Cell lines and cell culture

The alpha mouse liver 12 (AML12) mouse hepatocyte cell line was procured from the American Type Culture Collection (Manassas, VA, USA) and grown in Dulbecco’s Modified Eagle Medium (DMEM)/F-12 supplemented with 1X insulin-transferrin-selenium (ITS) supplement, 40 ng/mL dexamethasone, 100 U/mL penicillin, 100 mg/mL streptomycin, 0.25 g/L glutamine, and 10% fetal bovine serum at 37°C with 95% air and 5% CO2.

MTT assay

AML12 cells were seeded in a 24-well culture plate at a concentration of 5×10⁴ cells per well and allowed to adhere overnight. Subsequently, the cells were treated with sodium L-lactate at concentrations of 20 or 40 mM for a duration of 4 days. The medium was changed every 2 days in each group. After treatment, the AML12 cells were incubated with a fresh medium containing 250 ng/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for 2 hours. The MTT dye in its reduced form was dissolved in isopropanol and subsequently, 200 μL of the solution was transferred to each well of a 96-well plate to measure absorbance at 570 nm using the Spectra-Max 190 Microplate Reader (Molecular Devices LLC., San Jose, CA, USA).

Oil Red O staining

AML12 cells were cultured at a concentration of 2×10⁵ cells/mL (2 mL) in 6-well plates overnight, followed by treatment with either 20- or 40-mM sodium L-lactate for 4 days. The medium was changed every 2 days in each group. After exposure, the cells were rinsed twice with PBS and fixed in 4% paraformaldehyde solution diluted in PBS for 30 minutes. The cells were then exposed to a working solution of Oil Red O at 20°C to 25°C for 15 minutes. Non-stained Oil Red O was removed by washing the wells three times with distilled water. Afterward, cell images were captured using an OLYMPUS (Tokyo, Japan) optical microscope. To semi-quantitatively analyze the stained lipids, 600 μL of isopropanol was added to each well of the 6-well plate. Subsequently, the solution was transferred to a 96-well microplate and the absorbance was measured at 510 nm using a SpectraMax 190 Microplate Reader (Molecular Devices LLC.).

Triglyceride quantification

We treated AML12 cells using the same process as that used for Oil Red O staining. Lipid accumulation induced by L-lactate treatment was confirmed in AML12 cells by measuring triglyceride levels using a colorimetric adipogenesis absorbance assay (Biovision, Milpitas, CA, USA) and quantified according to the manufacturer’s instructions.

Transient transfection of small interfering RNA

The day before transfection, AML12 cells were plated in 6-well plates at a density of 2×10⁵ cells in each well. After overnight incubation, cells were transfected with scramble or GPR81 small interfering RNA (siRNA) using the Lipofectamine RNAiMAX transfection reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) following the manufacturer’s protocol. In brief, scramble or GPR81 siRNA and Lipofectamine RNAiMAX were each diluted in 50 μL DMEM/F12 free serum without antibiotics and incubated for 5 minutes at room temperature (RT). The diluted scramble or GPR81 siRNA and Lipofectamine RNAiMAX were combined and then incubated at RT for 20 minutes. After incubation, siRNA-Lipofectamine RNAiMAX complexes were added to each well. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2 for 6 hours. Subsequently, complexes were replaced with growth media with or without L-lactate (20 mM) and cells were cultured for 3 days for harvesting proteins or 4 days for staining with Oil Red O. GPR81 siRNA and negative control siRNA were purchased from Bioneer (AccuTarget predesigned siRNA; no. 243270, AccuTarget negative control siRNA; SN-1001, Daejeon, Korea). The concentration of siRNA was optimized at 50 nM per well.

Immunofluorescence

AML12 cells were initially seeded onto sterile glass coverslips and incubated overnight. Following a 3-day treatment with sodium L-lactate, fixation was performed using 4% paraformaldehyde for 10 minutes, and the coverslips were washed for 2 minutes with PBS. The cells were permeabilized with 0.2% Triton X-100 in PBS at RT for 5 minutes, followed by a 5-minute PBS wash. Blocking was performed using 10% normal goat serum for 1 hour at RT. Subsequently, the AML12 cells were incubated overnight at 4°C with the primary antibody for MCT1 (1:50; sc-365501, Santa Cruz Biotechnology). After three gentle 5-minute washes with PBS, the coverslips were incubated with the secondary antibody, goat anti-mouse Alexa Fluor 647, for 1 hour at RT. Following the completion of all the necessary washing steps, 4ʹ,6-diamidino-2-phenylindole was used for nuclear counterstaining. The samples were imaged using a fluorescence microscope.

Isolation of membrane-associated proteins

Membrane-associated proteins were isolated using the Minute Plasma Membrane Protein Isolation and Cell Fractionation Kit (Invent Biotechnologies Inc., Plymouth, MN, USA) following the manufacturer’s instructions. All procedures were conducted at 4°C. Briefly, cells from suspension cultures were harvested by centrifugation at 300 ×g for 5 minutes. The obtained cell pellets were lysed using buffer A and transferred to a filter cartridge. Following centrifugation at 14,000 rpm for 30 seconds, discard the filter and re-suspend the pellet by vigorously vortex for 10 seconds and centrifuged again at 3,000 rpm for 1 minute. The supernatant was collected and centrifuged at 14,000 rpm for 10 minutes. The resulting supernatant served as the cytosolic protein fraction, and the pellet represented the total membrane fraction. The membrane fraction was resuspended in buffer B and centrifuged at 10,000 rpm for 5 minutes, and the resulting pellet was collected as the organelle membrane protein. The supernatant was centrifuged at 14,000 rpm for 15 minutes and the resulting pellet was collected as the plasma membrane protein fraction for subsequent experiments. The isolated proteins were then examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detected using various antibodies by Western blotting.

Western blot analysis

The protein content of the cell lysates was determined using the BCA Protein Assay Kit (Thermo Scientific Pierce, Rockford, IL, USA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis separated equal amounts of total proteins, which were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Following the transfer, the membrane was incubated for 1 hour at RT in Tris-buffered saline containing 0.1% Tween-20 with either 5% nonfat dry milk or 5% bovine serum albumin. Afterward, the membranes underwent three washes with Tris-buffered saline containing 0.1% Tween-20 and were incubated with primary antibodies overnight at 4°C or for 1 to 2 hours at RT as per the manufacturer’s instructions. The membranes were then incubated with the appropriate horseradish peroxide-conjugated secondary antibodies for 1 to 2 hours at RT. Subsequently, the blots were exposed to WestGlow PICO PLUS chemiluminescent substrate (Biomax, Seoul, Korea) and images were captured using the ChemiDoc Imaging System (Bio-Rad Laboratories Inc., Hercules, CA, USA). The intensities of the bands were determined using Image Lab software version 6.1.0 (Bio-Rad Laboratories Inc.).

L-lactate colorimetric assay kit

L-lactate concentrations were determined using a colorimetric assay kit (ab65331, Abcam, Cambridge, UK). In this assay, lactate is oxidized by lactate dehydrogenase to produce a product that undergoes a color change upon interaction with the probe molecule. Samples were exposed to a reaction mixture consisting of lactate assay buffer, lactate enzyme mix, and lactate substrate mix for 30 minutes at RT. The absorbance was measured at 450 nm using a microplate reader (SpectraMax 190, Molecular Devices, Sunnyvale, CA, USA).

Glycolytic extracellular acidification rate analysis

AML-12 cells were plated in XF24 cell culture microplates (Seahorse Bioscience, North Billerica, MA, USA) and treated with sodium L-lactate (20 or 40 mM) for 72 hours. Extracellular acidification rates were measured utilizing Agilent Seahorse XF analyzer (Agilent, Santa Clara, CA, USA) following the manufacturer’s protocol. Glycolysis was assessed using the XF Glycolysis Stress Test kit according to the manufacturer’s instructions.

Mouse model

The Institutional Animal Care and Use Committee of the National Kangwon University approved the animal study (KW-220218-2), and all procedures were performed in compliance with the relevant guidelines. Four-week-old male C57BL/6N mice were purchased from Doo Yeol Biotech (Seoul, Korea). The mice were housed in the animal care facility at the National Kangwon University under standard conditions, including an ambient temperature of 22°C±1°C, and a lighting schedule of 12 hours of light followed by 12 hours of darkness was maintained. The mice had unrestricted access to food and water. A 1-week acclimation period was provided to the mice before commencement of the study.

As for a HFHC diet, the mice were randomly allocated into two groups: (1) the control group (n=8), which received a control diet (2018S, Envigo, Indianapolis, IN, USA), and (2) the nonalcoholic steatohepatitis model group (n=11), which received a HFHC diet (2018S 38.25%, cocoa butter 60%, cholesterol 1.25%, cholate 0.5%). The study lasted 12 weeks. As for the HFD diet mouse models, 8-week-old C57BL/6J male mice were housed in specific-pathogen-free mouse facilities under 12:12 hours light–dark cycles (6:00 AM–6:00 PM light, 6:00 PM–6:00 AM dark) at 23°C with 60%–70% humidity and free access to water. These mice were fed either a chow diet as control or an HFD (D12492, containing 60% fat [kcal%]; Research Diet, New Brunswick, NJ, USA) for 12 weeks.

Zebrafish model

Experiments using the zebrafish model were conducted with the approval of the Korea University Institutional Animal Care and Use Committee (KOREA-2021-0134). Zebrafish were maintained under standard conditions at 28°C, with a pH of 7.2–7.4 and a 14-hour light/10-hour dark cycle. All the zebrafish experiments were performed during the larval stage under fasting conditions. We used liver-specific cyan fluorescent protein (CFP) transgenic (Tg), Tg (fabp10a: CFP), zebrafish larvae were randomly divided into two groups. Lactate (10 mM, Sigma-Aldrich) or 0.1% dimethyl sulfoxide was added to the fish water at 6-day post-fertilization (dpf) for 24 hours. The zebrafish were fixed in 4% paraformaldehyde overnight at 4°C, followed by washing with 1× PBS with 0.1% Tween-20 (PBST) and staining with Nile red. Nile red (Sigma-Aldrich) was dissolved in acetone at 1.25 mg/mL as stock solution and stored at −20°C. The working concentration of Nile red was 0.5 μg/mL, placed in the dark at RT for 1 hour, and then washed with 1×PBS with 0.1% Tween-20. Images of the stained larvae were captured using a confocal microscope (Nikon A1Si, Nikon, Tokyo, Japan). Maximum intensity projection images were obtained by combining six Z-stack images at 4-μm intervals. All quantifications (lipid droplet counts and areas) were performed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Statistical analysis

GraphPad Prism 9.0 (GraphPad Software, Boston, MA, USA) software was used for the statistical analysis. The result columns were presented as mean±standard error of the mean or mean±standard deviation. Tukey’s t-test was used to compare data between two groups, and one-way analysis of variance was used to compare data between three or more groups. Statistical significance was stated as P<0.05, P<0.01, P<0.001, and P<0.0001.

RESULTS

Lactate-induced lipid accumulation in hepatocytes in vitro

Treatment of AML12 cells with 20- and 40-mM sodium L-lactate for 4 days did not significantly affect cell viability (Fig. 1A). However, Oil Red O staining revealed a marked increase in lipid droplet accumulation in the treated cells (Fig. 1B). This was further confirmed by quantitative analysis, which showed a statistically significant increase in intracellular triglyceride content in the cells treated with sodium L-lactate compared to the control groups (Fig. 1C).

GPR81 may enhance MCT1 expression in hepatocytes in vitro

Treatment of AML12 cells with sodium L-lactate led to a significant, time- and dose-dependent increase in MCT1 and GPR81 protein levels (Fig. 2A). Notably, MCT4 expression remained unchanged under the same conditions. Furthermore, knockdown of GPR81 resulted in a decrease in MCT1 expression (Fig. 2B).

GPR81 played a major role in increasing lipid accumulation in lactate-treated AML12 cells

Treatment of AML12 cells with 20-mM sodium L-lactate and MCT1 inhibitor AZD3965 (100 nM) did not significantly alter lipid accumulation compared with lactate treatment alone (Fig. 3A). Interestingly, MCT1 expression decreased in the plasma membrane but increased within the cytosol of lactate-treated cells compared to the control (Fig. 3B). Immunofluorescence staining confirmed the significant cytosolic presence of MCT1 in the lactate-treated AML12 cells (Fig. 3C). siGPR81 treatment alleviated the lipid accumulation induced by sodium L-lactate (Fig. 3D).

Lactate-induced GPR81 activation promoted hepatocyte lipogenesis and fatty acid storage in vitro

To investigate the relationship between GPR81 activation and lipid accumulation, we examined the expression levels of specific protein markers involved in lipogenesis, fatty acid uptake, and lipid metabolism. The results showed an increased expression of the mature form of SREBP1c in lactate-treated AML12 cells (Fig. 4A), whereas the expression decreased when treated with siGPR81 (Fig. 4B). Furthermore, the expression of proteins involved in fatty acid synthesis, such as ACC and SCD1, and those associated with fatty acid uptake, such as CD36 and fatty acid binding protein 4 (FABP4), increased in response to lactate treatment (Fig. 4A). Meanwhile, siGPR81 treatment reduced the expression of these proteins (Fig. 4B). Interestingly, PPARα and carnitine palmitoyltransferase I (CPT1), proteins associated with fatty acid oxidation, showed reduced expression only when treated with 40-mM lactate under the same conditions (Fig. 4A). However, siGPR81 treatment did not significantly impact PPARα and CPT1 levels (Fig. 4B).

Lactate inhibits the AMPK phosphorylation via GPR81 activation and induces lipogenesis and glycolysis in AML12 cells

Our data revealed that lactate treatment led to dose-dependent downregulation of AMPK phosphorylation (Fig. 5A). Intriguingly, SiGPR81 resulted in a significantly enhanced AMPK phosphorylation level in AML12 cells (Fig. 5B).

To further investigate the role of AMPK activation in the mitigation of lactate-induced lipid accumulation, we utilized 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an AMPK activator. The results demonstrated that AICAR at 100 μM effectively ameliorated lactate-induced lipid accumulation, as evidenced by reduced Oil Red O staining (Fig. 5C). Additionally, the expression levels of SREBP1, CD36, and FABP4 proteins were reduced in cells co-treated with AICAR and lactate compared to those treated with lactate alone (Fig. 5D). Furthermore, treatment of AML12 cells with 20- and 40-mM sodium L-lactate for 3 days induce significant glycolysis in Seahorse analysis (Fig. 6A-E).

Lactate-induced hepatic lipid accumulation in zebrafish model

Furthermore, 10 mM lactate was added to fish water 6 dpf for 24 hours in zebrafish and a notable increase in lipid accumulation in the liver was noticed compared to the control group, as indicated by Nile red staining (Fig. 6G). To better observe the liver and its surrounding structures, further experiments were conducted using an endothelial-specific enhanced green fluorescent protein (EGFP) Tg zebrafish, Tg (fli1a:EGFP), model expressing green, fluorescent color in blood vessels. As shown in the figure (Fig. 6F), when 10 mM of lactate was treated in the zebrafish model at 6 dpf for 24 hours, lipid accumulation in the liver was increased compared to that in the control group. However, there was no significant increase in lipid accumulation in other tissues (e.g., muscle tissue around the vertebral artery or mesenteric adipose tissue around the supra-intestinal artery).

Upregulation of GPR81 protein expression in the liver of NAFLD mouse model

In this study, we examined liver samples from mice fed an HFHC diet for 12 weeks and a HFD for 12 weeks. The results revealed that diet-induced NAFLD was significantly associated with higher lactate accumulation in the liver than the control group (Supplementary Fig. 1A). Moreover, there was a noticeable increase in GPR81 expression in liver samples from the HFHC group (Supplementary Fig. 1B) and from the HFD groups both at 12 weeks (Supplementary Fig. 1C) compared to the control group. However, no significant differences were observed in the expression of MCT1. We checked the p-AMPK in the liver tissue of HFHC diet and HFD fed mice. As expected, p-AMPK decreased in the liver tissue of HFD fed mice, but the expression of p-AMPK was surprisingly increased in the liver of HFHC diet (Supplementary Fig. 1D).

DISCUSSION

Lactate functions not only as a glycolytic byproduct but also as a metabolic fuel and signaling molecule [23,24]. Our study identifies lactate as a key regulator of hepatic lipid accumulation through the GPR81–AMPK signaling axis. In AML12 hepatocytes, lactate promotes lipogenesis and fatty acid uptake while suppressing AMPK activity. These effects were dependent on GPR81, as its knockdown restored AMPK phosphorylation and abolished lactate-induced lipid accumulation. In vivo, we observed increased hepatic lactate and GPR81 expression in both HFD- and HFHC-fed mice, accompanied by lipid accumulation. Zebrafish exposed to lactate similarly demonstrated hepatic steatosis, suggesting a conserved mechanism across species.

In AML12 cells, MCT1 was strongly expressed and upregulated by lactate in a time- and dose-dependent manner. However, MCT1 inhibition did not reduce lipid accumulation, and no consistent changes in MCT1 expression were observed in vivo. This discrepancy may reflect compensatory mechanisms or differences in cellular context between in vitro and in vivo systems. Additionally, lactate induced relocalization of MCT1 from the plasma membrane to the cytosol, suggesting a potential adaptive response with unclear functional relevance. These findings support the idea that lactate primarily acts via extracellular signaling through GPR81 rather than intracellular transport via MCT1.

Interestingly, lactate treatment also increased GPR81 expression, and GPR81 silencing not only reduced MCT1 expression but also abolished lactate-induced lipid accumulation, suggesting a regulatory role for GPR81 in both MCT1 expression and lipid metabolism. Previous studies identified GPR81 as an anti-lipolytic receptor in adipose tissue and a modulator of hepatic lipid homeostasis [25-27], with expression increased during fasting and decreased in obesity [28]. While Wu et al. [28] emphasized GPR81’s role in fasting-induced lipid mobilization, our study highlights its involvement in lactate-driven lipogenesis, underscoring the context-dependent functions of GPR81 in hepatic metabolism. GPR81 knockdown reduced expression of lipogenic genes (SREBP1c, ACC, SCD1) and lipid uptake proteins (FABP4, CD36), while β-oxidation genes (PPARα, CPT1) were only affected at supraphysiologic lactate levels (40 mM) and remained unchanged by GPR81 silencing. This suggests that GPR81 selectively promotes lipogenesis and lipid uptake without significantly altering fatty acid oxidation.

Although our data shows a strong link between lactate, GPR81, and steatosis, the in vivo causality remains to be confirmed. Liver-specific GPR81 knockout models would help clarify its tissue-specific role. Moreover, the downstream signaling of GPR81 (e.g., cyclic adenosine monophosphate, β-arrestin, protein kinase A) warrants further investigation. We focused on MCT1 due to its hepatic expression and included MCT4 based on reports of its inducibility under pathological conditions such as hypoxia or hepatocellular carcinoma [29,30]. MCT2 and MCT3 were excluded due to minimal expression in liver tissue. Further exploration of other transporters and compartment-specific signaling will help define whether lactate primarily functions as a receptor ligand or a metabolic substrate in hepatocytes. Finally, differences observed with 2-day versus 4-day lactate treatment suggest dynamic and time-dependent regulations. Future time-course and dose-response studies will be essential to better understand these temporal effects.

Mechanistically, lactate suppressed AMPK phosphorylation in hepatocytes, and this inhibition was reversed by GPR81 knockdown or AICAR treatment. AMPK activation attenuated lactate-induced lipid accumulation and gene expression, confirming the role of GPR81–AMPK signaling in promoting steatosis. These data position the lactate–GPR81–AMPK axis as a key driver of hepatic lipogenesis and a potential therapeutic target in NAFLD.

Although AMPK typically inhibits lipogenesis and enhances fatty acid oxidation, lactate-induced lipid accumulation in AML12 cells occurred with minimal changes in β-oxidation, suggesting compartment-specific AMPK regulation. In vivo, AMPK phosphorylation decreased in HFD fed mice but increased in HFHC-fed mice, potentially due to differences in inflammation, bile acid signaling, or energy balance. These results highlight the dietary context-dependence of AMPK signaling in NAFLD and indicate the need for further studies on upstream regulators (liver kinase B1, calcium/calmodulin-dependent protein kinase kinase 2, transforming growth factor beta–activated kinase 1) and in vivo validation using liver-specific GPR81 knockout mice to confirm the role of GPR81-AMPK signaling.

In both HFD- and HFHC-fed mice, lactate exposure increased hepatic lipid accumulation and GPR81 expression. These findings are consistent with previous reports linking lactate accumulation (e.g., due to LDHB mutation) to hepatic steatosis [12]. Using zebrafish as a complementary in vivo model, we confirmed that lactate administration led to significant hepatic lipid accumulation, supporting a conserved pathogenic role for lactate in NAFLD across species.

In summary, our findings highlight the lactate–GPR81–AMPK axis as a central driver of hepatic lipid accumulation. GPR81 promotes lipogenesis and lipid uptake while suppressing AMPK, with minimal impact on fatty acid oxidation. These results offer mechanistic insight into lactate’s role in NAFLD and support GPR81 as a potential therapeutic target. Further mechanistic and genetic studies are needed to validate its translational relevance.

SUPPLEMENTARY MATERIALS

NOTES

CONFLICTS OF INTEREST

Eun-Hee Cho has been an associate editor of the Diabetes & Metabolism Journal since 2022. Ki Woo Kim has been an associate editor of the Diabetes & Metabolism Journal since 2024. They were not involved in the review process of this article. Otherwise, there was no conflict of interest.

AUTHOR CONTRIBUTIONS

Conception or design: D.H.C., E.H.C.

Acquisition, analysis, or interpretation of data: G.N., J.H.Y., J.H.L.

Drafting the work or revising: P.T.M.P., T.L.L., S.Y.P., K.W.K., S.S.I., J.H., Y.L., E.H.C.

Final approval of the manuscript: all authors.

FUNDING

None

ACKNOWLEDGMENTS

This study was supported by the Korean Government (NRF-2020R1I1A3074388 and 2020R1F1A1074265). We would like to extend our thanks to Professor Seung-Soon Im at Kyemyung University for their generosity in providing liver samples from mice fed an HFD for 12 weeks.

Fig. 1.

Lactate-induced lipid accumulation in hepatocytes in vitro. Alpha mouse liver 12 (AML12) cells were treated with different doses of sodium L-lactate for 4 days. (A) Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (B) Intracellular lipid is stained with Oil Red O. The upper images representative gross morphology of Oil Red O staining of AML12 cells. The below representative pictures of cells were taken by a microscope at 200× original magnification. Scale bar: 100 μm. (C) Quantification of intracellular triglyceride (TG) content. Statistical significance compared with control is indicated by ^aP<0.05, ^bP<0.01, ^cP<0.0001.

Fig. 2.

G-protein-coupled receptor 81 (GPR81) may regulate monocarboxylate transporter 1 (MCT1) expression in hepatocytes in vitro. (A) Alpha mouse liver 12 (AML12) cells were treated with sodium L-lactate 20 and 40 mM for different time course. Western blot analysis was performed to assess the expression levels of GPR81, MCT1, and MCT4 in AML12 cells. (B) AML12 cells were treated with small interfering RNA (siRNA) 50 nM with or without lactate 20 mM for 3 days. Western blot analysis was conducted to evaluate the expression levels of GPR81 and MCT1 in AML12 cells. The intensities of the bands in the Western blot images were quantified using Image Lab software and are displayed in the corresponding plot alongside the representative blot images. The protein levels were normalized to β-actin expression. Statistical significance compared with control is indicated by ^aP<0.05, ^bP<0.01, ^cP<0.001, lactate 20 mM treated group vs. control; ^dP<0.05, ^eP<0.01, ^fP<0.001, lactate 40 mM treated group vs. control; statistical significance compared with si-scramble is indicated by ^gP<0.05, ^hP<0.01, ⁱP<0.001, with siGPR81 indicated ^jP<0.01.

Fig. 3.

G-protein-coupled receptor 81 (GPR81) played a major role in regulating lipid accumulation in lactate-treated alpha mouse liver 12 (AML12) cells. (A) AML12 cells were treated with L-lactate at 20 mM with or without AZD3965 at 100 nM for 4 days. Lipid accumulation was evaluated using Oil Red O staining. (B) AML12 cells were treated with sodium L-lactate 20 and 40 mM for 3 days. The isolation of plasma membrane (PM) and cytosol fractions was performed, and the expression of GPR81 and monocarboxylate transporter 1 (MCT1) in AML12 cells was assessed. Na/K ATPase served as a housekeeping marker for the PM, while tubulin served as a housekeeping marker for the cytosol. (C) Immunofluorescence of MCT1 (red) and nuclei (4ʹ,6-diamidino2-phenylindole [DAPI] blue). Scale bar: 20 μm. (D) AML12 cells were treated with small interfering RNA (siRNA) 50 nM with or without lactate 20 mM for 4 days. The accumulation of lipids in AML12 cells was visualized using Oil Red O staining. Statistical significance compared with control is indicated by ^aP<0.05, ^bP<0.01. Statistical significance compared with si-scramble is indicated by ^cP<0.05, ^dP<0.001, with siGPR81 indicated ^eP<0.001.

Fig. 4.

Lactate-induced G-protein-coupled receptor 81 (GPR81) activation promotes hepatocyte lipogenesis and fatty acid storage in vitro. (A) Alpha mouse liver 12 (AML12) cells were treated with sodium L-lactate 20 and 40 mM for 3 days. (B) AML12 cells were treated with small interfering RNA (siRNA) 50 nM with or without lactate 20 mM for 3 days. Cell lysates were then analyzed via Western blot to determine protein levels. Representative images of immunoblots of lipogenesis markers. β-Actin is a loading control. SREBP1c, sterol regulatory element-binding protein 1c; ACC, acetyl-CoA carboxylase; SCD1, stearoyl-CoA desaturase-1; FABP4, fatty acid binding protein 4; PPARα, peroxisome proliferator-activated receptor alpha; CPT1, carnitine palmitoyltransferase I; NS, not significant. Statistical significance compared with control is indicated by ^aP<0.05, ^bP<0.01, ^cP<0.0001. Statistical significance compared with si-scramble is indicated by ^dP<0.05, ^eP<0.01, with siGPR81 indicated ^fP<0.05, ^gP<0.01.

Fig. 5.

Lactate-mediated G-protein-coupled receptor 81 (GPR81) activation regulates 5’ adenosine monophosphate-activated protein kinase (AMPK) in alpha mouse liver 12 (AML12) cells. (A) AML12 cells were treated with sodium L-lactate 20 and 40 mM for 3 days. (B) AML12 cells were treated with small interfering RNA (siRNA) 50 nM with or without lactate 20 mM for 4 days. Cell lysates were analyzed by Western blot to measure the protein levels of phosphorylation AMPK (p-AMPK) and AMPK. (C) AML12 cells were treated with lactate for 2 days and then co-treated with or without 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) 100 µM for next 2 days. Lipid accumulation was evaluated using Oil Red O staining. (D) AML12 cells were treated with lactate for 2 days and then co-treated with or without AICAR 100 µM for next 1 day. Representative images of immunoblots of mature form of sterol regulatory element-binding protein 1c (SREBP1c), CD36, and fatty acid binding protein 4 (FABP4). β-Actin or tubulin is a loading control. NS, not significant. Statistical significance compared with control is indicated by ^aP<0.05, ^bP<0.01, ^cP<0.0001. Statistical significance compared with si-scramble is indicated by ^dP<0.01, with siGPR81 indicated ^eP<0.01. Statistical significance compared with lactate 20 mM is indicated by ^fP<0.05, ^gP<0.01, ^hP<0.001.

Fig. 6.

Lactate- induced glycolysis in alpha mouse liver 12 (AML12) cells and hepatic lipid accumulation zebrafish. (A) Seahorse analysis of extracellular acidification rate (ECAR), (B) non-glycolytic acidification, (C) glycolysis, (D) glycolytic capacity, and (E) glycolytic reserve were assessed in AML12 cells treated with sodium L-lactate 20 and 40 mM for 3 days. (F) To detect the hepatic response to lactate, we utilized selective fluorescent staining (Nile red) for intracellular lipid droplets in transgenic (Tg) (fabp10a: cyan fluorescent protein [CFP]) zebrafish larvae treated with or without lactate 10 mM. Figures are magnified as ×200. Quantitative analysis of the area of lipid droplet in liver based on Nile Red staining. (G) Lactate-induced lipid accumulation mostly in the liver not in muscle or adipose tissue in zebrafish model Nile red staining for intracellular lipid droplets in Tg (fabp10a: CFP) zebrafish larvae treated with or without lactate 10 mM. Scale bar indicated 100 µm. OD, optical density; 2-DG, 2-deoxy-d-glucose; DMSO, dimethyl sulfoxide; DA, dorsal aorta; L, liver; SB, swim bladder; SIA, supra-intestinal artery; VTA, vertebral artery. Statistical significance compared with control is indicated by ^aP<0.05, ^bP<0.01, ^cP<0.001, ^dP<0.0001.

REFERENCES

• 1. Cotter TG, Rinella M. Nonalcoholic Fatty liver disease 2020: the state of the disease. Gastroenterology 2020;158:1851-64.ArticlePubMedPMC
• 2. Roden M. Mechanisms of disease: hepatic steatosis in type 2 diabetes--pathogenesis and clinical relevance. Nat Clin Pract Endocrinol Metab 2006;2:335-48.ArticlePubMedPDF
• 3. Angulo P. Nonalcoholic fatty liver disease. N Engl J Med 2002;346:1221-31.ArticlePubMed
• 4. Kawano Y, Cohen DE. Mechanisms of hepatic triglyceride accumulation in non-alcoholic fatty liver disease. J Gastroenterol 2013;48:434-41.ArticlePubMedPMCPDF
• 5. Neuschwander-Tetri BA, Caldwell SH. Nonalcoholic steatohepatitis: summary of an AASLD Single Topic Conference. Hepatology 2003;37:1202-19.ArticlePubMed
• 6. Belfort R, Harrison SA, Brown K, Darland C, Finch J, Hardies J, et al. A placebo-controlled trial of pioglitazone in subjects with nonalcoholic steatohepatitis. N Engl J Med 2006;355:2297-307.ArticlePubMedPMC
• 7. Fletcher WM. Lactic acid in amphibian muscle. J Physiol 1907;35:247-309.PubMedPMC
• 8. Wasserman K. The anaerobic threshold measurement to evaluate exercise performance. Am Rev Respir Dis 1984;129(2 Pt 2):S35-40.ArticlePubMed
• 9. Brown TP, Ganapathy V. Lactate/GPR81 signaling and proton motive force in cancer: role in angiogenesis, immune escape, nutrition, and Warburg phenomenon. Pharmacol Ther 2020;206:107451.ArticlePubMed
• 10. Felmlee MA, Jones RS, Rodriguez-Cruz V, Follman KE, Morris ME. Monocarboxylate transporters (SLC16): function, regulation, and role in health and disease. Pharmacol Rev 2020;72:466-85.ArticlePubMedPMC
• 11. Phypers B, Pierce JMT. Lactate physiology in health and disease. Contin Educ Anaesth Crit Care Pain 2006;6:128-32.Article
• 12. Wang T, Chen K, Yao W, Zheng R, He Q, Xia J, et al. Acetylation of lactate dehydrogenase B drives NAFLD progression by impairing lactate clearance. J Hepatol 2021;74:1038-52.PubMed
• 13. Carneiro L, Asrih M, Repond C, Sempoux C, Stehle JC, Leloup C, et al. AMPK activation caused by reduced liver lactate metabolism protects against hepatic steatosis in MCT1 haploinsufficient mice. Mol Metab 2017;6:1625-33.ArticlePubMedPMC
• 14. Lovejoy J, Newby FD, Gebhart SS, DiGirolamo M. Insulin resistance in obesity is associated with elevated basal lactate levels and diminished lactate appearance following intravenous glucose and insulin. Metabolism 1992;41:22-7.ArticlePubMed
• 15. Crawford SO, Hoogeveen RC, Brancati FL, Astor BC, Ballantyne CM, Schmidt MI, et al. Association of blood lactate with type 2 diabetes: the Atherosclerosis Risk in Communities Carotid MRI Study. Int J Epidemiol 2010;39:1647-55.ArticlePubMedPMC
• 16. Juraschek SP, Selvin E, Miller ER, Brancati FL, Young JH. Plasma lactate and diabetes risk in 8045 participants of the atherosclerosis risk in communities study. Ann Epidemiol 2013;23:791-6.e4.ArticlePubMedPMC
• 17. Juraschek SP, Shantha GP, Chu AY, Miller ER, Guallar E, Hoogeveen RC, et al. Lactate and risk of incident diabetes in a case-cohort of the atherosclerosis risk in communities (ARIC) study. PLoS One 2013;8:e55113.ArticlePubMedPMC
• 18. DiGirolamo M, Newby FD, Lovejoy J. Lactate production in adipose tissue: a regulated function with extra-adipose implications. FASEB J 1992;6:2405-12.ArticlePubMedPDF
• 19. Crandall DL, Fried SK, Francendese AA, Nickel M, DiGirolamo M. Lactate release from isolated rat adipocytes: influence of cell size, glucose concentration, insulin and epinephrine. Horm Metab Res 1983;15:326-9.ArticlePubMed
• 20. Goessling W, Sadler KC. Zebrafish: an important tool for liver disease research. Gastroenterology 2015;149:1361-77.ArticlePubMedPMC
• 21. Chen B, Zheng YM, Zhang JP. Comparative study of different diets-induced NAFLD models of zebrafish. Front Endocrinol (Lausanne) 2018;9:366.ArticlePubMedPMC
• 22. Shimizu N, Shiraishi H, Hanada T. Zebrafish as a useful model system for human liver disease. Cells 2023;12:2246.ArticlePubMedPMC
• 23. Mozaffarian D, Hao T, Rimm EB, Willett WC, Hu FB. Changes in diet and lifestyle and long-term weight gain in women and men. N Engl J Med 2011;364:2392-404.ArticlePubMedPMC
• 24. Rabinowitz JD, Enerback S. Lactate: the ugly duckling of energy metabolism. Nat Metab 2020;2:566-71.ArticlePubMedPMCPDF
• 25. Liu C, Wu J, Zhu J, Kuei C, Yu J, Shelton J, et al. Lactate inhibits lipolysis in fat cells through activation of an orphan G-protein-coupled receptor, GPR81. J Biol Chem 2009;284:2811-22.ArticlePubMed
• 26. Jeninga EH, Bugge A, Nielsen R, Kersten S, Hamers N, Dani C, et al. Peroxisome proliferator-activated receptor gamma regulates expression of the anti-lipolytic G-protein-coupled receptor 81 (GPR81/Gpr81). J Biol Chem 2009;284:26385-93.PubMedPMC
• 27. Wanders D, Graff EC, Judd RL. Effects of high fat diet on GPR109A and GPR81 gene expression. Biochem Biophys Res Commun 2012;425:278-83.ArticlePubMed
• 28. Wu G, Dai Y, Yan Y, Zheng X, Zhang H, Li H, et al. The lactate receptor GPR81 mediates hepatic lipid metabolism and the therapeutic effect of metformin on experimental NAFLDs. Eur J Pharmacol 2022;924:174959.ArticlePubMed
• 29. Shen J, Wu Z, Zhou Y, Yang D, Wang X, Yu B, et al. Knockdown of SLC16A3 decreases extracellular lactate concentration in hepatocellular carcinoma, alleviates hypoxia and induces ferroptosis. Biochem Biophys Res Commun 2024;733:150709.ArticlePubMed
• 30. Mukai Y, Yamaguchi A, Sakuma T, Nadai T, Furugen A, Narumi K, et al. Involvement of SLC16A1/MCT1 and SLC16A3/MCT4 in l-lactate transport in the hepatocellular carcinoma cell line. Biopharm Drug Dispos 2022;43:183-91.PubMed

Figure & Data

References

Citations

Citations to this article as recorded by  

• LncRNA LELE enhances lactate efflux and reduces lipid deposition via upregulating MCT1
  Kang Xiao, Yingying Zhou, Qiyong Qiu, Chenguang Zhu, Xiaoxue Shen, Le Chang, Wei Qiang, Hengtong Liu, Guangzhen Jiang, Xiangfei Li, Wenbin Liu, Dingdong Zhang
  Aquaculture.2026; 622: 744043.     CrossRef

About this article

Nguyen G, Yu JH, Pham PTM, Lai TL, Park SY, Kim KW, Im SS, Hong J, Lee Yh, Lee JH, Kang SM, Choi DH, Cho EH. Lactate-Induced Lipid Accumulation in Hepatocytes through GPR81 Activation. Diabetes Metab J. 2026;50(2):307-319.

Received: Sep 03, 2024; Accepted: Jul 22, 2025

DOI: https://doi.org/10.4093/dmj.2024.0531.

Download citation ↓