Growth differentiation factor 15 mitigates lipotoxic steatosis by preserving mitochondrial morphodynamics and augmenting fatty acid oxidation in hepatocytes and liver organoids

Abstract

Aims

Growth differentiation factor 15 (GDF15) has emerged as a promising metabolic regulator with hepatoprotective properties in metabolic dysfunction‐associated steatotic liver disease (MASLD), yet its underlying mechanisms remain elusive. Given that mitochondria are the primary site of fatty acid oxidation (FAO) and that mitochondrial morphodynamics are critical for normal hepatic lipid metabolism, we investigated how GDF15 regulates hepatic lipid homeostasis through mitochondrial dynamics.

Materials and Methods

We established cellular steatosis models using primary rat hepatocytes exposed to lipotoxic palmitate (PA) or non‐lipotoxic free fatty acid mixture (FFA, oleate: palmitate = 2: 1). Following GDF15 administration, we quantified lipid droplet content, expression of lipid metabolism genes, mitochondrial fatty acid translocation, and mitochondrial morphodynamics and function. The mechanistic role of ERK1/2 signalling was assessed through pharmacological inhibition. These findings were subsequently validated in adult progenitor cell‐derived human liver organoids.

Results

GDF15 significantly mitigated both PA‐ and FFA‐induced lipid accumulation by upregulating key FAO genes and down regulating lipid synthesis genes. Importantly, GDF15 corrected PA‐induced mitochondrial fusion‐fission imbalance by increasing mitochondrial fusion proteins MFN1 and OPA1 while modulating the activation of fission regulator DRP1. GDF15 enhanced fatty acid translocation into mitochondria and improved FAO. Mechanistically, GDF15 exerted these effects partially through inhibition of the ERK1/2 signalling pathway. Human liver organoid models further corroborated this protective mechanism of GDF15 against hepatic steatosis.

Conclusions

Our study reveals that, specifically under lipotoxic conditions, GDF15 alleviates hepatocyte steatosis by preserving mitochondrial morphodynamics homeostasis and enhancing mitochondrial FAO capacity via ERK1/2 inhibition. These condition‐specific mechanisms provide critical insights into GDF15's hepatoprotective effects and support its further investigation as a potential therapeutic target for MASLD.

1. INTRODUCTION

Metabolic dysfunction‐associated steatotic liver disease (MASLD) represents a multifactorial condition characterised by hepatic steatosis, with a clinical spectrum ranging from simple fat accumulation to metabolic dysfunction‐associated steatohepatitis (MASH), which can progress to cirrhosis and hepatocellular carcinoma. The pathogenesis of MASLD is rooted in the dysregulation of hepatic lipid metabolism, where an imbalance between fatty acid acquisition and disposal leads to excessive triglyceride (TG) accumulation in hepatocytes. Consequently, strategies aimed at restoring lipid homeostasis, particularly by enhancing the liver's capacity for fatty acid oxidation (FAO), are of significant therapeutic interest.^{1, 2, 3}

Central to hepatic FAO is mitochondrial function. In MASLD, mitochondrial health is frequently compromised, accelerating disease progression through impaired energy production and increased oxidative stress.^{4, 5} A key driver of this dysfunction is the disruption of mitochondrial dynamics.^{6, 7, 8} This process involves a tightly regulated balance between fusion, orchestrated by mitofusins (MFN1/2) and optic atrophy 1 (OPA1), and fission, primarily driven by dynamin‐related protein 1 (DRP1).^{9, 10, 11} In the steatotic liver, this equilibrium often shifts towards excessive DRP1‐mediated fission, which fragments the mitochondrial network, impairs metabolic efficiency, and contributes directly to lipotoxicity. ¹² The activity of DRP1 is itself regulated, notably via inhibitory phosphorylation at serine 637.^{13, 14, 15} Importantly, preclinical studies have shown that genetically or pharmacologically inhibiting excessive fission or promoting fusion can ameliorate steatosis, validating the restoration of mitochondrial dynamics as a promising therapeutic strategy for MASLD.^{16, 17, 18, 19}

Growth differentiation factor 15 (GDF15), a stress‐responsive hepatokine belonging to the TGF‐β superfamily, has emerged as a potent regulator of systemic metabolism. By signalling through its GDNF family receptor alpha‐like (GFRAL) and rearranged during transfection (RET) heteroreceptor complex, GDF15 modulates key downstream pathways, including MEK‐ERK1/2 and PI3K‐AKT, to exert beneficial effects in various metabolic disorders.^{20, 21} Clinical and experimental evidence indicate that hepatic GDF15 expression is strongly induced during MASLD, where it appears to function as a protective signal against lipid accumulation and inflammation.^{22, 23, 24, 25} However, despite its established role as a hepatoprotective factor, the precise intracellular mechanisms by which GDF15 shields hepatocytes from lipotoxicity remain poorly understood. Specifically, whether GDF15 directly modulates the mitochondrial morphodynamics machinery to preserve metabolic function has not been explored.

Given the critical role of mitochondrial dynamics in MASLD and the emerging protective profile of GDF15, this study was designed to bridge this knowledge gap. We hypothesised that GDF15 mitigates lipotoxic steatosis by directly preserving mitochondrial integrity and function. We employed primary rat hepatocytes and human liver organoids to investigate the impact of GDF15 on mitochondrial morphodynamics, fatty acid oxidation, and lipid metabolism in the context of cellular steatosis induced by distinct fatty acid challenges. Our aim was to elucidate the specific molecular axis connecting GDF15 signalling to the regulation of mitochondrial health in hepatocytes.

2. MATERIALS AND METHODS

2.1. Materials

Recombinant human GDF15 protein (rhGDF15, R&D Systems, Abingdon, United Kingdom) was dissolved in sterile 4 mM hydrochloric acid containing 0.1% bovine serum albumin (BSA) to prepare stock solutions at 100 μg/mL. Mitochondrial division inhibitor 1 (Mdivi‐1, Sigma‐Aldrich, Zwijndrecht, the Netherlands), MEK/ERK1/2 inhibitor U0126 (MedChemExpress, Sollentuna, Sweden), and etomoxir (MedChemExpress, Sollentuna, Sweden) were dissolved in dimethyl sulfoxide (DMSO) to make stock concentrations of 25, 10 and 5 mM, respectively. Sodium palmitate (PA, Sigma‐Aldrich), sodium oleate (OA, Sigma‐Aldrich), and fatty acid‐free BSA (Sigma‐Aldrich) were used to prepare PA and free fatty acid mix (FFA) stock solutions at 20 mM as previously described. ²⁶ The FFA solution consisted of a mixture of OA and PA at a 2:1 ratio.

2.2. Primary rat hepatocytes isolation, culture, and treatment

Male Wistar rats (150–250 g) were purchased from Charles River Laboratories Inc. (Wilmington, MA, USA) and acclimatised for 1 week at 25°C ± 2°C under a 12‐h light–dark cycle with ad libitum access to food and water in the central animal facility of the University Medical Center Groningen. All experimental procedures were conducted in accordance with the guidelines established by the Animal Welfare Body of the University of Groningen (No. 2115139‐01‐001) regarding animal care and utilisation. Animals were initially placed in an isoflurane (5%) inhalation chamber for 5 min to induce a preliminary state of anaesthesia, facilitating subsequent handling. Following this induction phase, complete surgical anaesthesia was achieved via administration of ketamine (100 mg/mL, 60 mg/kg) and medetomidine hydrochloride (1 mg/mL, 0.5 mg/kg) to minimise animal suffering. Primary hepatocytes were isolated using a two‐step collagenase perfusion method as previously described. ²⁷ Cells were cultured in William's E medium (Thermo Fisher Scientific, Waltham, USA) supplemented with 5% fetal bovine serum, 50 nM dexamethasone (Sigma‐Aldrich, St. Louis, USA), 100 units/mL penicillin, 10 μg/mL streptomycin, 250 ng/mL fungizone (1% PSF, Lonza, Basel, Switzerland) and 50 μg/mL gentamicin (Thermo Fisher Scientific, Bleiswijk, the Netherlands) at 37°C in a 5% CO₂‐containing humidified incubator. Only hepatocytes with viability exceeding 80%, as determined by trypan blue exclusion assay, were used for experiments. After 4 h of attachment, the medium was replaced with serum‐ and dexamethasone‐free medium. Cells were pretreated with GDF15 (10, 30, 100 ng/mL) for 30 min, followed by co‐treatment with palmitate (PA, 500 μM) or free fatty acid mixture (FFA, 750 μM) for 16 h. For experiments investigating cellular mechanical properties, cells were pretreated with GDF15 in combination with either Mdivi‐1 or U0126 for 30 min, followed by co‐treatment with PA or FFA for 16 h.

2.3. Human liver organoids culture and treatment

Liver specimens were obtained through the TransplantLines biobank in accordance with Dutch legislation and the Code of Conduct for responsible use of human‐derived material in health research. The TransplantLines Biobank and cohort study received approval from the Medical Ethical Committee of the University Medical Center Groningen on August 25, 2014 (approval number METc 2014/077). All procedures complied with the Declaration of Helsinki. The use of coded‐anonymous human tissue specimens waived the requirement for written consent for “further use” of human material. The sex of the liver specimen donors was not an inclusion criterion for this study, and this information was unavailable. Liver organoids were generated and expanded in culture using our previously described method. ²⁸ Briefly, minced liver tissues were digested with a mixture of 2.5 mg/mL collagenase D (Roche, Basel, Switzerland) and 0.1 mg/mL DNase I (Roche) in HBSS containing Ca²⁺ and Mg²⁺ to obtain single liver cells. The cell pellet was resuspended in Matrigel Matrix Basement Membrane (BD Bioscience, San Jose, USA), and the Matrigel/cell suspension mixture was placed in 24‐well plates. Organoids were initially maintained in Expanded Medium (EM+) containing AdDMEM/F12 medium (Gibco, Thermo Fisher Scientific, Waltham, USA) supplemented with 50% in‐house produced Wnt‐3a‐conditioned medium as previously described, 1× Glutamax (Invitrogen, Carlsbad, USA), 1× B27 (Invitrogen, Bleiswijk, the Netherlands), 1× N2 (Thermo Fisher Scientific, Bleiswijk, the Netherlands), 1.25 mM N‐acetylcysteine (Sigma‐Aldrich), 10 mM nicotinamide (Sigma‐Aldrich), 100 ng/mL human FGF10 (PeproTech, London, United Kingdom), 50 ng/mL human HGF (PeproTech), 0.1 μg/mL Rspo‐1 (Stemcell Technologies, Cologne, Germany), 50 ng/mL human EGF (PeproTech), 100 ng/mL Noggin (R&D Systems, Abingdon, United Kingdom), 10 μM Y‐27632 (Sigma‐Aldrich), 5 μM A83‐01 (Tocris Bioscience, Bristol, United Kingdom), 10 μM Forskolin (Sigma‐Aldrich), and 10 nM gastrin (Tocris Bioscience, Bristol, United Kingdom). After 3 days, established organoids were cultured in Expansion Medium (EM‐) without Wnt‐3a‐conditioned medium, Noggin, and Y‐27632. Organoids were passaged every 7–10 days at a ratio of 1:2–1:4 using gentle mechanical pipetting or TrypLE Express enzyme (Gibco, Thermo Fisher Scientific, Bleiswijk, the Netherlands) dissociation. Organoids underwent the identical GDF15, PA, FFA, and U0126 treatment regimens as described for primary rat hepatocytes, except with an extended treatment duration of 72 h.

2.4. Immunofluorescence

Liver organoid sections were deparaffinised, rehydrated, and subjected to antigen retrieval before being blocked with 1% BSA in PBS. Sections were then incubated with primary antibodies in a humidity chamber overnight at 4°C, followed by incubation with Alexa Fluor 488 or 594‐conjugated secondary antibodies.

Hepatocytes cultured on glass coverslips were washed with PBS following treatment. For lipid visualisation, cells were incubated with either BODIPY 493/503 dye (Thermo Fisher Scientific, Waltham, USA) for lipid droplet (LD) staining for 15 min or with BODIPY‐FL C16‐labelled long‐chain free fatty acids (Thermo Fisher Scientific, Waltham, USA) for 30 min. Cells were subsequently fixed with 4% paraformaldehyde for 15 min and washed twice with PBS. Permeabilisation was performed using 0.1% Triton X‐100 (Sigma‐Aldrich) in PBS for 30 min at room temperature. Mitochondria were labelled using MitoTracker Red (Thermo Fisher Scientific, Bleiswijk, the Netherlands) or anti‐TOM20 antibody (Cell Signaling Technology, Leiden, the Netherlands) and visualised with goat anti‐rabbit Alexa Fluor 594 (Thermo Fisher Scientific, Bleiswijk, the Netherlands) at a 1:400 dilution. Slides were mounted using ProLong antifade medium with DAPI (Dako, Agilent Technologies, Amstelveen, the Netherlands). Fluorescence imaging was performed using a confocal microscope (Leica Microsystems, Wetzlar, Germany).

2.5. Western blotting

Hepatocytes and human liver organoids were lysed in buffer containing protease and phosphatase inhibitors. Protein concentrations were determined using the Bio‐Rad DC protein assay kit (Bio‐Rad, Veenendaal, the Netherlands). Equal amounts of protein were separated by SDS‐PAGE and transferred to nitrocellulose membranes (Amersham, Cytiva, Eindhoven, the Netherlands) using a Trans‐Blot Turbo semi‐dry transfer system (Bio‐Rad, Veenendaal, the Netherlands). Membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase‐conjugated secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualised using a ChemiDoc MP Imaging system (Bio‐Rad). Details of all antibodies used in this study are provided in Table S1.

2.6. Oil red O staining

Hepatocytes were fixed in 4% paraformaldehyde for 10 min and subsequently rinsed 2–3 times with 60% isopropanol. Cells were then incubated with Oil Red O solution (Sigma‐Aldrich) for 10 min. Following removal of the staining solution, cells were thoroughly washed with 60% isopropanol followed by tap water until all residual dye was eliminated. Counterstaining was performed with haematoxylin for 1 min. Images were acquired using a slide scanner (Hamamatsu Photonics, Hamamatsu, Japan).

2.7. Mitochondrial morphology assessment

Mitochondrial morphology was quantitatively analysed using a two‐step image processing approach. Initially, images were uniformly adjusted for brightness and contrast using FIJI ImageJ (version 2.16, National Institutes of Health, Bethesda, MD, USA), and regions of interest were isolated from whole‐field images to facilitate visualisation and subsequent analysis. A custom pipeline was then established in CellProfiler (version 4.2.8, Broad Institute, Cambridge, MA, USA), incorporating image preprocessing steps including median filtering to minimise noise and selective enhancement of mitochondrial structures. Mitochondria were identified as primary objects, and morphological parameters were quantified using the “Measure Object Size Shape” module. Two key morphological indices were calculated: form factor (FF) and aspect ratio (AR). FF, calculated as (perimeter^2)/(4π*area), quantifies mitochondrial branching complexity, with higher values indicating more elaborate branching patterns. AR, defined as the ratio of major to minor axis length, represents mitochondrial elongation, where larger values correspond to more elongated mitochondrial structures.

2.8. RNA extraction and quantitative real‐time PCR

Total RNA was extracted from hepatocytes and human liver organoids using TRI reagent (Sigma‐Aldrich). Complementary DNA (cDNA) was synthesised using M‐MLV reverse transcriptase (Thermo Fisher Scientific, Bleiswijk, the Netherlands). RT‐qPCR was performed on a QuantStudio 3 system (Thermo Fisher Scientific, Waltham, MA, USA) using either TaqMan primers and probes or SYBR Green Master Mix (Thermo Fisher Scientific, Bleiswijk, the Netherlands) as detailed in Table S2. Relative gene expression was calculated using the 2^−△△CT method with 18S rRNA serving as the internal reference for normalisation.

2.9. Mitochondrial function measurement

For mitochondrial superoxide detection, hepatocytes cultured in 12‐well plates were stained with 2.5 μM MitoSOX Red (Thermo Fisher Scientific, Bleiswijk, the Netherlands) fluorescent probe at 37°C for 15 min under light protection, then counterstained with Hoechst 33342 (Thermo Fisher Scientific, Bleiswijk, the Netherlands) for 10 min. Cells were subsequently washed 2–3 times with PBS, replenished with fresh medium, and immediately examined by fluorescence microscopy (Leica Microsystems). Mitochondrial membrane potential was determined by incubating hepatocytes with 100 μM JC‐10 (Enzo Life Sciences, Zandhoven, Belgium) dye solution at 37°C for 30 min, followed by direct fluorescence microscopic visualisation without washing.

Mitochondrial respiration was assessed using Seahorse XF96 Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA, USA). Hepatocytes were seeded at a density of 10 000 cells per well 1 day prior to analysis. On the day of the experiment, culture medium was replaced with XF DMEM medium (Agilent Technologies, Amstelveen, the Netherlands) supplemented with 10 mM glucose (Agilent Technologies) and 2 mM glutamine (Agilent Technologies). Cells were then incubated at 37°C in a non‐CO₂ environment for 1 h before measurement. Oxygen consumption rate (OCR) was recorded at baseline and following sequential injections of oligomycin (2.5 μM), dinitrophenol (50 μM), and a mixture of rotenone (2 μM) and antimycin A (4 μM) to evaluate various parameters of mitochondrial function.

2.10. Bioinformatics analysis

Transcriptomic data from 206 liver biopsies of MASL and MASH patients across various fibrosis stages were obtained from the GSE135251 dataset in the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). ²⁹ All analyses were conducted using R software (version 4.4.3, R Foundation for Statistical Computing, Vienna, Austria). Data retrieval was performed with the GEOquery package, followed by normalisation using the limma package. Data processing was carried out with dplyr package in R, while visualisation was accomplished using ggplot2 and ggpubr packages. Gene expression values were normalised to transcripts per million (TPM). To evaluate mitochondrial fusion/fission balance, we calculated the ratios of fusion genes to fission genes (MFN1/DNM1L, MFN2/DNM1L, and OPA1/DNM1L). Relationships between these ratios and MASLD progression were assessed using Spearman correlation analysis. For pairwise comparisons between disease stages, Wilcoxon rank‐sum tests were employed. Overall differences across multiple stages were determined using Kruskal–Wallis tests followed by Dunn's post‐hoc analysis for multiple comparisons. Statistical significance was defined as p < 0.05.

2.11. Statistical analysis

Data are presented as mean ± standard deviation (SD). Statistical comparisons between two groups were performed using two‐tailed Student's t tests. For multiple group comparisons, one‐way analysis of variance (ANOVA) followed by Bonferroni post‐hoc tests was employed. Statistical significance was defined as p < 0.05. All statistical analyses were conducted using GraphPad Prism software (version 9.0, GraphPad Software Inc., San Diego, CA, USA). All authors had access to the study data and had reviewed and approved the final manuscript.

3. RESULTS

3.1. GDF15 ameliorates palmitate and free fatty acid‐induced hepatocyte steatosis and improves lipid metabolism

We first examined the effect of GDF15 on palmitate (PA) and free fatty acid (FFA)‐induced cytotoxicity in hepatocytes. SYTOX Green staining demonstrated that lipotoxic PA (500 μM) induced necrotic cell death, while GDF15 treatment dose‐dependently reduced SYTOX Green‐positive cells. Conversely, non‐lipotoxic FFA (750 μM) and GDF15 alone had no significant impact on cell viability (Figure S1A, B). Using BODIPY 493/503 fluorescent staining, we observed the differential steatotic effects of PA and FFA mixture. While both treatments significantly increased hepatocyte lipid content compared to non‐treated (NT) controls, FFA exhibited a more pronounced inductive effect, resulting in a 1.5‐fold greater LD area compared to PA alone. GDF15 co‐treatment significantly reduced intracellular LD content under both conditions (Figure 1A, B). Oil Red O staining further confirmed these findings, showing that GDF15 concentration‐dependently attenuated PA‐ and FFA‐induced hepatocyte steatosis (Figure 1C). To investigate the underlying mechanisms of GDF15's protective effects, we analysed expression levels of key genes involved in lipid metabolism. RT‐qPCR analysis revealed that in PA‐treated hepatocytes, expression of fatty acid β‐oxidation genes carnitine palmitoyltransferase 1a (Cpt1a), Cpt2, and peroxisome proliferator‐activated receptor alpha (Ppara) decreased by 49.1%, 48.4% and 53.6%, respectively. GDF15 treatment significantly upregulated these genes by 2.2‐fold, 1.9‐fold, and 2.2‐fold, respectively. In FFA‐treated hepatocytes, these genes showed no significant changes (Figure 1D–F). For lipogenesis and fatty acid uptake genes, Srebf1 encoding sterol regulatory element‐binding protein 1 exhibited decreased expression with both PA and FFA treatment. PA treatment did not significantly alter the expression of diacylglycerol O‐acyltransferase 1 (Dgat1), Dgat2, Cd36, or Pparg. FFA induced a 1.5‐fold upregulation of Dgat1 expression, which was reduced by 27.7% following GDF15 treatment. GDF15 showed a trend toward reducing FFA‐induced increases in Dgat2 and Cd36 expression, though these reductions were not statistically significant (Figure S1C–G). To elucidate the upstream mechanism responsible for the suppression of lipogenesis, we investigated the AMP‐activated protein kinase (AMPK) pathway, a known inhibitor of lipid synthesis. Western blot analysis revealed that while FFA treatment alone induced a 1.9‐fold increase in the phosphorylation of AMPK at Thr172 (p‐AMPK), GDF15 co‐treatment led to a further, significant 1.5‐fold potentiation of AMPK activation compared to FFA alone. In contrast, neither PA treatment nor GDF15 co‐treatment with PA significantly altered p‐AMPK levels, underscoring the specificity of this pathway to the non‐lipotoxic FFA model (Figure S1H). These findings suggest that GDF15 may ameliorate lipid accumulation in hepatocytes through distinct mechanisms depending on the lipid challenge: by restoring PA‐suppressed mitochondrial β‐oxidation under lipotoxic conditions, and by attenuating FFA‐enhanced lipogenesis in non‐lipotoxic environments at least in part via augmenting AMPK signalling.

FIGURE 1.

GDF15 alleviates cellular steatosis and upregulates key fatty acid oxidation‐related genes in palmitate (PA)‐treated hepatocytes. Primary rat hepatocytes were pretreated with increasing concentrations of rhGDF15 (10, 30, 100 ng/mL) for 30 min, followed by exposure to PA (500 μM) or FFA (750 μM) for 16 h to induce cellular steatosis. (A) Representative images of BODIPY 493/503 staining (scale bar: 25 μm). (B) Quantification of BODIPY 493/503‐labelled lipid droplet area from nine random fields per group using Image‐Pro Plus software (version 6.0). (C) Representative images of Oil Red O staining (scale bar: 100 μm). (D–F) Relative mRNA expression levels of Cpt1a, Cpt2, and Ppara are presented as fold change compared to NT controls. Data were presented as mean ± SD (n = 4 per group). The statistical significance of differences was analysed by one‐way ANOVA followed by Bonferroni's post‐hoc test. *p < 0.05, **p < 0.01.

3.2. GDF15 attenuates hepatocyte steatosis by optimising defective mitochondrial fusion processes

To first establish the clinical relevance of mitochondrial dynamics in the context of MASLD, we analysed a public bulk RNA‐sequencing dataset (GSE135251) from 206 MASLD patients spanning the full spectrum of disease severity (MASL to MASH with F0‐F4 fibrosis). ²⁹ By evaluating the ratios of mitochondrial fusion genes (MFN1, MFN2, OPA1) to the mitochondrial fission gene DNM1L, we found that the MFN1/DNM1L, MFN2/DNM1L and OPA1/DNM1L ratios all decreased significantly with increasing disease severity (Figure 2A). Correlation analysis demonstrated that this shift towards mitochondrial fission was significantly and negatively correlated with advancing disease stage (Figure 2B). The persistence of this imbalance across all stages suggests that disrupted mitochondrial dynamics is a fundamental feature of MASLD pathogenesis, likely initiated during the early phase of steatosis. This finding provided the rationale for our subsequent investigation into the role of mitochondrial dynamics in hepatocyte lipid accumulation.

FIGURE 2.

GDF15 improves mitochondrial fusion gene expression in lipotoxic PA‐treated hepatocytes. Analysis of mitochondrial morphodynamic changes using a public bulk RNA‐sequencing dataset (GSE135251) from 206 MASLD patients with varying fibrosis stages (F0–F4). Ratios of mitochondrial fusion genes (MFN1, MFN2, OPA1) to fission gene DNM1L were used as indicators of fusion‐fission balance. (A) Changes in MFN1/DNM1L, MFN2/DNM1L, and OPA1/DNM1L ratios across MASLD disease stages. (B) Correlation analysis between fusion‐fission gene ratios and disease progression. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (C–E) Relative mRNA levels of fusion genes Mfn1, Mfn2, and Opa1 in hepatocytes treated with PA or FFA combined with indicated doses of GDF15 for 16 h (n = 5 per group). (F–G) Relative mRNA levels of fission genes Dnm1l and Fis1 (n = 4 per group). Data were presented as mean ± SD. The statistical significance of differences was analysed by one‐way ANOVA followed by Bonferroni's post‐hoc test. *p < 0.05, **p < 0.01.

RT‐qPCR analysis revealed that GDF15 (100 ng/mL) co‐treatment significantly increased expression levels of Mfn1, Mfn2, and Opa1 genes by 2.0‐fold, 2.0‐fold, and 1.8‐fold, respectively. This intervention effectively counteracted PA‐induced expression decline. Importantly, GDF15's regulatory effect on mitochondrial fusion gene expression was specific to PA‐treated hepatocytes, with no comparable effect observed in FFA‐treated cells (Figure 2C–E). The mitochondrial fission genes Dnm1l and Fis1 showed no significant changes across all experimental groups (Figure 2F, G). At the protein level, PA reduced MFN1 and OPA1 expression by 48.6% and 43.9%, respectively, compared to NT controls. GDF15 treatment dose‐dependently increased these proteins to 2.0‐fold and 1.7‐fold, respectively (Figure 3A, B). Beyond PA's direct inhibition of mitochondrial fusion, we investigated whether PA indirectly regulated mitochondrial dynamics by modulating DRP1 phosphorylation, a key regulator of mitochondrial fission. Phosphorylation at Ser637 inhibits DRP1 activity, causing phosphorylated DRP1‐S637 to remain cytoplasmic and preventing effective binding to the outer mitochondrial membrane, thereby inhibiting mitochondrial fission and indirectly promoting mitochondrial network elongation. ³⁰ Western blot analysis demonstrated that GDF15 upregulated PA‐induced p‐DRP1 (S637) expression 1.9‐fold (Figure 3C). Immunofluorescence staining of TOM20‐labelled mitochondria confirmed that PA induces excessive mitochondrial fission, transforming mitochondrial morphology from elongated rod‐like structures to shorter fragments with reduced connectivity, as evidenced by decreased AR and FF values. GDF15 treatment effectively reversed these morphological alterations (Figure 3D–F). Collectively, these findings indicate that GDF15 enhances impaired mitochondrial fusion, thereby preserving mitochondrial network integrity.

FIGURE 3.

GDF15 corrects mitochondrial morphodynamic imbalance under PA‐induced lipotoxic stress. (A–C) Protein expression levels of MFN1, OPA1, and p‐DRP1 are presented as fold change relative to NT controls (n = 5 per group). (D) Representative images of TOM20‐labelled mitochondria immunofluorescence staining and corresponding binary skeletonised images (scale bar: 20 μm; scale bar of zoom‐in and binary images: 10 μm). (E, F) Quantification of mitochondrial morphological parameters, aspect ratio (AR), and form factor (FF) from eight random fields per group using Fiji ImageJ (version 2.16) and CellProfiler software (version 4.2.8). Data were presented as mean ± SD. The statistical significance of differences was analysed by one‐way ANOVA followed by Bonferroni's post‐hoc test. *p < 0.05, **p < 0.01.

3.3. GDF15 promotes fatty acid translocation into mitochondria and enhances β‐oxidation in PA‐challenged hepatocytes

Emerging evidence suggests that mitochondrial morphological homeostasis is closely linked to the regulation of energy metabolism.^{31, 32} To determine whether GDF15 ameliorates PA‐induced hepatocyte steatosis by enhancing FAO, we evaluated long‐chain fatty acid translocation into mitochondria across treatment groups using dual fluorescence labelling with MitoTracker Red and the green fluorescent PA analogue BODIPY FL C16. Compared to NT controls, PA challenge slightly increased intracellular fatty acid uptake while simultaneously reducing mitochondrial‐fatty acid colocalisation coefficients. GDF15 administration markedly enhanced the colocalisation of fatty acids with mitochondria without substantially affecting overall cellular fatty acid uptake (Figure 4A, B). Conversely, in FFA‐treated hepatocytes, GDF15 significantly attenuated cellular lipid acquisition while maintaining mitochondrial‐fatty acid colocalisation levels. These differential responses reveal a condition‐specific regulatory mechanism through which GDF15 mitigates hepatocyte steatosis: in PA‐induced lipotoxic environments, GDF15 primarily counteracts lipid accumulation by restoring impaired FAO capacity, whereas in FFA‐induced non‐lipotoxic conditions, it predominantly inhibits excessive lipogenesis.

FIGURE 4.

GDF15 promotes long‐chain fatty acid translocation into mitochondria and enhances FAO capacity in PA‐treated hepatocytes. (A) Representative immunofluorescence images showing colocalisation of MitoTracker Red with BODIPY FL C16‐labelled long‐chain fatty acids. (B) Quantitative analysis of Pearson's coefficient using ImageJ software. (C) Mitochondrial basal, ATP‐linked, and maximal OCR levels in hepatocytes treated with PA with/without indicated doses of GDF15 for 16 h, measured using Seahorse XF96 Extracellular Flux Analyzer with sequential injections of oligomycin (Oligo), dinitrophenol (DNP), and a mixture of rotenone and antimycin (R + A). (D) Etomoxir‐sensitive component of OCR, representing FAO capacity, determined in GDF15‐treated PA‐exposed hepatocytes with/without the potent CPT1α inhibitor. Data were presented as mean ± SD. The statistical significance of differences was analysed by one‐way ANOVA followed by Bonferroni's post‐hoc test. *p < 0.05, **p < 0.01.

Seahorse metabolic flux analyses demonstrated that GDF15 dose‐dependently increased PA‐inhibited mitochondrial basal respiration and maximal respiratory capacity (Figure 4C). To precisely quantify FAO levels in PA‐exposed hepatocytes with or without GDF15 (100 ng/mL) treatment, we measured the etomoxir‐sensitive component of OCR. Results showed that GDF15 significantly upregulated PA‐inhibited FAO (Figure 4D), thereby promoting enhanced fatty acid catabolism. Furthermore, mitochondrial function assessments revealed that GDF15 treatment reduced PA‐induced mitochondrial superoxide levels as measured by MitoSOX fluorescence (Figure S2A, B). Using JC‐10 dye, which forms red fluorescent aggregates at higher mitochondrial membrane potentials and green fluorescent monomers at lower potentials, we observed that GDF15 treatment reversed the PA‐induced decrease in the red/green fluorescence intensity ratio, indicating restoration of mitochondrial membrane potential (Figure S2C, D). Collectively, these findings demonstrate that GDF15 enhances mitochondrial FAO capacity and improves overall mitochondrial function in PA‐challenged hepatocytes.

3.4. GDF15 regulates mitochondrial morphodynamics through suppression of palmitate‐induced ERK1/2 activation

PA‐treated hepatocytes exhibited a 2.1‐fold increase in ERK1/2 phosphorylation, which GDF15 treatment reduced by 54.8% (Figure 5A). To elucidate the role of ERK1/2 signalling in modulating mitochondrial fusion‐fission balance, we employed the MEK/ERK1/2 inhibitor U0126. This inhibitor effectively suppressed ERK1/2 activation while recapitulating GDF15's protective effects. U0126 reversed PA‐induced decreases in fusion proteins OPA1 and MFN1, as well as the fission regulator p‐DRP1, elevating their expression to 1.8‐fold, 1.9‐fold, and 1.9‐fold, respectively, compared to PA treatment alone (Figure 5B). Consistent with these findings, U0126 significantly upregulated Mfn1 and Mfn2 expression at the transcriptional level (Figure 5C–E).

FIGURE 5.

ERK1/2 signalling inhibition is crucial for preservation of mitochondrial morphodynamic homeostasis by GDF15 in lipotoxic cellular steatosis models. (A) Western blot and quantitative analysis of ERK1/2 phosphorylation levels in GDF15‐treated PA‐ or FFA‐exposed hepatocytes (n = 5 per group). (B) Western blot and quantitative analysis of OPA1 (n = 4), MFN1 (n = 4), p‐DRP1/DRP1 (n = 5), and p‐ERK/ERK (n = 5) protein levels in cells pretreated with GDF15 (100 ng/mL) in combination with ERK1/2 inhibitor U0126 for 30 min, followed by co‐treatment with PA for 16 h. (C–E) Relative mRNA levels of Mfn1, Mfn2, and Opa1 determined by RT‐qPCR analysis (n = 4 per group). Data were presented as mean ± SD. The statistical significance of differences was analysed by one‐way ANOVA followed by Bonferroni's post‐hoc test. *p < 0.05, **p < 0.01.

To further define ERK1/2's role in mediating GDF15's enhancement of impaired FAO, RT‐qPCR analysis showed that U0126 potentiated GDF15's upregulation of key FAO genes Cpt1a and Cpt2. In parallel experiments, we utilised Mdivi‐1, a mitochondrial fission inhibitor, as a comparative reference. Mdivi‐1 acted synergistically with GDF15 to reverse PA‐induced suppression of Cpt1a expression (Figure 6A–C), suggesting that alterations in mitochondrial morphodynamics may influence FAO regulatory pathways. Notably, neither U0126 nor Mdivi‐1 significantly altered lipid synthesis‐related gene expression in PA‐treated hepatocytes (Figure S3A–E). Similarly, in FFA‐treated hepatocytes, co‐treatment with either U0126 or Mdivi‐1 did not affect GDF15's downregulation of Dgat1 and Pparg expression (Figure S3F–H). Immunofluorescence staining revealed that U0126 intervention enhanced the translocation of fluorescently labelled PA analogue into TOM20‐labelled mitochondria, evidenced by elevated mitochondrial‐fatty acid colocalisation coefficients (Figure 6D, E). Collectively, these data indicate that GDF15 reinstates mitochondrial morphodynamic homeostasis and enhances compromised FAO, potentially through suppression of ERK1/2 activation, thereby mitigating PA‐induced hepatocyte steatosis.

FIGURE 6.

GDF15 inhibition of the ERK1/2 pathway upregulates key FAO genes and increases long‐chain fatty acid translocation to mitochondria. Hepatocytes were pretreated with GDF15 (100 ng/mL) in combination with either mitochondrial fission inhibitor Mdivi‐1 or U0126 for 30 min, followed by co‐treatment with PA for 16 h. (A–C) Relative mRNA levels of Cpt1a (n = 4), Cpt2 (n = 4), and Ppara (n = 3). (D) Representative images of TOM20‐labelled mitochondria co‐stained with green fluorescent probes for long‐chain fatty acids (scale bar: 20 μm). (E) Quantitative analysis of Pearson's coefficients from nine random fields per group. Data were presented as mean ± SD. The statistical significance of differences was analysed by one‐way ANOVA followed by Bonferroni's post‐hoc test. *p < 0.05, **p < 0.01.

3.5. Human liver organoids validate GDF15's protective mechanism against lipid accumulation through restoration of mitochondrial morphodynamics via ERK1/2 inhibition

To establish translational relevance of our findings, we employed human liver organoids as a physiologically relevant model system that more accurately recapitulates human hepatic physiology.^{33, 34, 35} Both PA and FFA exposure induced lipid accumulation in these organoids, with FFA demonstrating higher potency. GDF15 administration markedly attenuated steatosis severity in both conditions (Figure 7A, B). Transcriptional analysis revealed that GDF15 normalised the PA‐induced suppression of CPT1A and reversed FFA‐mediated upregulation of DGAT1. Interestingly, while FFA treatment reduced SREBF1 expression, neither PA, FFA, nor GDF15 significantly altered expression of DGAT2, stearoyl‐CoA desaturase (SCD1), or fatty acid synthase (FASN), suggesting selective modulation of lipid metabolism pathways (Figure 7C–H).

FIGURE 7.

GDF15 alleviates lipid accumulation in PA‐ and FFA‐induced 3D steatotic liver organoid models. Human liver organoids were pretreated with GDF15 (100 ng/mL) for 30 min followed by PA or FFA for 72 h to model MASLD. (A) Representative bright field and immunofluorescence images of BODIPY 493/503‐labelled lipid droplets (scale bars: 100 μm for bright field and 500 μm for immunofluorescence). (B) Quantification of BODIPY 493/503‐labelled lipid droplet area from 11 random fields per group. (C–H) Relative mRNA levels of key lipid metabolism and synthesis genes CPT1A, DGAT1, DGAT2, SREBF1, SCD1, and FASN (n = 3 healthy donors per group). Data were presented as mean ± SD. The statistical significance of differences was analysed by one‐way ANOVA followed by Bonferroni's post‐hoc test. *p < 0.05, **p < 0.01.

We next sought to validate our mechanistic findings in this advanced model. Consistent with our observation in primary hepatocytes, GDF15 activated the AMPK pathway in the context of non‐lipotoxic steatosis. While FFA treatment itself caused a non‐significant 1.9‐fold increase in p‐AMPK, co‐treatment with GDF15 resulted in a significant 2.1‐fold elevation in p‐AMPK levels compared to the FFA‐treated group. Again, this effect was specific, as no significant changes in AMPK phosphorylation were observed in the PA‐induced steatosis model (Figure 8A).

FIGURE 8.

GDF15 ameliorates steatosis in human liver organoids by modulating distinct AMPK and ERK1/2 signalling pathways. (A) Representative Western blots and corresponding analysis of AMPK phosphorylation in GDF15‐treated liver organoids subjected to PA‐ and FFA‐induced steatosis (n = 3 per group). (B) Western blot and quantitative analysis of OPA1, MFN1, p‐DRP1/DRP1, and p‐ERK/ERK levels in PA‐induced steatotic liver organoids treated with GDF15 and U0126 (n = 3 per group). (C) Relative mRNA levels of CPT1A determined by RT‐qPCR (n = 3 per group). Data were presented as mean ± SD. The statistical significance of differences was analysed by one‐way ANOVA followed by Bonferroni's post‐hoc test. *p < 0.05, **p < 0.01.

Mechanistic investigations using U0126 confirmed that ERK1/2 signalling mediates the protective effects of GDF15 on mitochondrial dynamics and lipid homeostasis in human liver organoids. Immunoblot analyses demonstrated that GDF15 suppressed PA‐induced ERK1/2 hyperactivation while concurrently enhancing expression of mitochondrial fusion proteins OPA1 and increasing phosphorylation of the fission protein DRP1. Although MFN1 showed a trend toward increased expression with GDF15 treatment, these changes did not reach statistical significance. Notably, U0126 treatment potentiated these effects, further correcting mitochondrial fusion‐fission imbalance (Figure 8B). Consistent with these observations, RT‐qPCR results showed that pharmacological inhibition of ERK1/2 enhanced GDF15‐mediated upregulation of CPT1A expression (Figure 8C). Collectively, these findings in human liver organoids corroborate our observations in primary rat hepatocytes, validating the conservation of the GDF15‐ERK1/2‐mitochondrial axis in ameliorating hepatic steatosis across species.

4. DISCUSSION

Lipotoxicity serves as a critical initiating factor in the pathogenesis of MASLD.^{3, 36} The multifaceted dysregulation of hepatic lipid metabolism encompasses increased lipid uptake, enhanced lipid synthesis, reduced fatty acid oxidation, and decreased very low‐density lipoprotein secretion, collectively leading to hepatic fat accumulation. This metabolic imbalance subsequently triggers liver dysfunction, inflammation, and fibrosis. ³⁷ Our study elucidates a novel mechanism through which GDF15 alleviates lipid accumulation under distinct metabolic conditions. Notably, in lipotoxic environments induced by PA, GDF15 preserves mitochondrial morphodynamic homeostasis and enhances FAO capacity via inhibition of the ERK1/2 signalling pathway. In contrast, under non‐lipotoxic FFA exposure, GDF15 reduces steatosis primarily by suppressing lipogenic pathways rather than influencing β‐oxidation processes.

GDF15, a stress‐induced cytokine with pleiotropic effects across various tissues, has emerged as a promising therapeutic target for metabolic disorders. Previous studies have demonstrated its beneficial effects in reducing food intake, attenuating β‐cell apoptosis, improving glycaemic control, mitigating oxidative stress, and exhibiting cardioprotective properties.^{38, 39, 40} Clinical observations reveal elevated plasma GDF15 concentrations correlating with the severity of glucolipid disorders and MASLD progression, suggesting a compensatory response to metabolic stress.^{41, 42} This compensatory role is further substantiated by in vivo genetic models. Loss‐of‐function studies using mice with liver‐specific GDF15 deletion have shown that these animals exhibit exacerbated steatohepatitis, hepatomegaly, and impaired insulin sensitivity with metabolic stressors.^{24, 43, 44} Conversely, complementary gain‐of‐function approaches, including both liver‐targeted transgenic overexpression and the exogenous administration of GDF15, effectively attenuate hepatic steatosis and inflammation in rodent models.^{25, 45} While the consistent demonstration of GDF15's hepatoprotective effects across these diverse whole‐organism models provides a compelling rationale for our study to dissect the direct cellular mechanisms, it is important to contextualise our findings. As a systemic hormone, GDF15's primary metabolic effects in vivo, such as appetite suppression, are mediated through its canonical receptor GFRAL in the central nervous system. ⁴⁶ Our in vitro system, while essential for isolating the direct, cell‐autonomous actions of GDF15 on hepatocytes, cannot predict the net outcomes of GDF15 administration in a whole organism, where the hepatoprotective actions we observed may be synergistic with or potentially overshadowed by its potent central effects. Our work provides a candidate cellular mechanism for the direct hepatic benefits of GDF15, which warrants further validation in sophisticated in vivo systems that can dissect its liver‐autonomous versus central effects. A key question arising from our observation of direct hepatocyte‐autonomous effects is therefore the identity of the mediating receptor. The canonical GDF15 receptor, GFRAL, is known to be expressed almost exclusively in the hindbrain and is largely absent in peripheral tissues, including the liver. ⁴⁷ In line with this, our own RT‐qPCR analysis confirmed that GFRAL mRNA was undetectable in both our primary rat hepatocytes and human liver organoids. This finding suggests that the hepatoprotective effects of GDF15 observed in our study are mediated through a non‐canonical, GFRAL‐independent signalling system. This conclusion is supported by a growing body of evidence for GFRAL‐independent actions of GDF15. GDF15 has been shown to inhibit integrin activation and neutrophil recruitment via an ALK5‐TGFβRII heterodimer, and to improve liver fibrosis by reprogramming macrophage metabolism and exerting direct anti‐fibrotic effects on hepatic stellate cells.^{24, 48, 49} Collectively, these findings indicate that GDF15 engages in complex, tissue‐specific signalling and that the precise identity of the receptor responsible for its direct effects on hepatocytes remains a critical area for future investigation. Therefore, comprehensive in vivo experiments will be essential to confirm these direct mechanisms within a complete physiological system.

Our research advances the mechanistic understanding of GDF15's hepatoprotective actions by revealing condition‐specific regulation of lipid metabolism. In hepatocytes treated with high‐concentration PA (500 μM) for 16 h, we observed marked suppression of key FAO genes (Cpt1a, Cpt2, and Ppara), which GDF15 co‐treatment effectively restored. This contrasts with previous studies employing lower PA concentrations or shorter exposure times, wherein PA typically upregulates FAO markers as an adaptive response. ⁵⁰ Our findings suggest that when PA concentrations exceed cellular metabolic buffering capacity, the adaptive response fails, resulting in suppressed FAO capacity. This pathological state more accurately reflects MASLD pathophysiology than models using milder lipid challenges.^{51, 52} Nevertheless, a further consideration is that our study employs an acute lipotoxic challenge, which primarily illuminates GDF15's role in modulating short‐term metabolic injury. These findings may not be fully translatable to the long‐term therapeutic management of chronic liver disease, which involves complex progressive feedback mechanisms, systemic inflammation, and tissue remodelling such as fibrosis. Future studies using chronic disease models are thus needed to assess the sustained efficacy and therapeutic potential of GDF15. Interestingly, in FFA‐treated hepatocytes, GDF15 primarily downregulated lipogenic genes, particularly Dgat1, which governs TG esterification. Our findings provide a direct upstream mechanism for this observation. We demonstrated that in the FFA model, but not the PA model, GDF15 enhanced the phosphorylation and activation of AMPK. This provides a mechanistic link, as activated AMPK is a well‐established suppressor of de novo lipogenesis. A consequence of AMPK activation is the suppression of lipogenesis via the inhibition of the master transcriptional regulator, SREBP‐1c. ⁵³ Activated AMPK is known to prevent the proteolytic processing and nuclear translocation of SREBP‐1c, thereby reducing its ability to drive the expression of target genes, including Dgat1. We hypothesise that the GDF15‐AMPK‐SREBP‐1c axis is the most probable pathway mediating the suppression of lipogenesis. Future studies designed to directly quantify nuclear SREBP‐1c protein and its binding to the Dgat1 promoter will be essential to definitively validate this proposed cascade. This is noteworthy as TG storage within LDs is a key cytoprotective mechanism, sequestering excess fatty acids to prevent lipotoxicity and ER stress. ⁵⁴ Therefore, GDF15's action likely transcends simple inhibition, orchestrating a broader metabolic shift. We propose this involves a multi‐pronged strategy that shifts the metabolic fate of fatty acids from storage toward catabolism. This may involve tempering fatty acid uptake, as suggested by the trend in Cd36 expression, and promoting their flux into FAO via post‐transcriptional regulation, given the stable Cpt1a mRNA levels. Additionally, accelerating the turnover of stored lipids through lipolysis represents another plausible mechanism.^{55, 56} These differential effects under varying lipid environments suggest context‐dependent metabolic regulation by GDF15, highlighting its sophisticated role in hepatic lipid homeostasis.

Mitochondrial dysfunction represents a central nexus in MASLD pathogenesis. ⁵⁷ Our research demonstrated that PA‐induced lipotoxicity disrupted mitochondrial morphodynamic balance, manifested as decreased expression of fusion molecules (MFN1, OPA1) and altered phosphorylation of the fission regulator DRP1, resulting in excessive fission. GDF15 treatment restored this balance, enhanced long‐chain fatty acid translocation to mitochondria, and improved FAO capacity. While it was previously demonstrated that mitochondrial fission under physiological conditions may enhance FAO by altering membrane curvature and reducing CPT1 sensitivity to malonyl‐CoA, ³² our findings highlight a distinct phenomenon: under lipotoxic stress, excessive fission coincides with downregulation of key FAO enzymes, marking pathological injury rather than adaptation. This distinction underscores the importance of context in interpreting mitochondrial dynamics and metabolic regulations.

Mechanistically, we identified the ERK1/2 signalling pathway as a critical mediator of GDF15's therapeutic effects. GDF15 ameliorated PA‐induced lipotoxicity by suppressing ERK1/2 hyperactivation, restoring mitochondrial morphodynamic homeostasis, and enhancing compromised FAO. The MEK/ERK1/2 inhibitor U0126 phenocopied GDF15's beneficial effects, while the fission inhibitor Mdivi‐1 acted synergistically with GDF15 to restore Cpt1a expression, confirming the intimate link between mitochondrial dynamics and metabolic gene regulation. Interestingly, we observed a slight, though not statistically significant, trend toward increased ERK1/2 activation by GDF15 in the non‐lipotoxic FFA model. The precise significance of this observation remains to be determined, but it suggests that the signalling output of the ERK1/2 pathway may be highly contingent on the specific metabolic context. This is consistent with the established view that the role of ERK1/2 in regulating mitochondrial dynamics and lipid metabolism is complex and context‐dependent, potentially involving intermediate molecules like Bcl‐2 interacting protein 3 that modulate both OPA1 and DRP1 activity.^{58, 59, 60, 61, 62} Our observations align with a negative regulatory role for ERK1/2 in FAO under lipotoxic conditions, where its inhibition by GDF15 correlates with increased Cpt1a expression and enhanced FAO capacity. The validation of these mechanisms in human liver organoids significantly enhances the translational relevance of our findings. These 3D structures more faithfully recapitulate human hepatic physiology compared to conventional cell cultures, providing a robust platform for modelling human disease and predicting therapeutic responses. The conservation of the GDF15‐ERK1/2‐mitochondrial axis in this advanced model strongly supports its physiological significance. At the same time, the generalisability of these preclinical findings warrants careful consideration. Our experiments were conducted exclusively in male rats, and the sex of the donors for the human liver organoids was unavailable. This is a notable constraint for several key reasons. First, MASLD exhibits well‐documented, sex‐dependent differences in its epidemiology, prevalence, and progression. ⁶³ Second, these clinical observations are rooted in the fundamental biological underpinnings of hepatic sexual dimorphism, which involves distinct hormonal milieus and sex‐specific genetic factors that regulate liver metabolism and inflammatory responses. ⁶⁴ Third, and of specific relevance to our work, emerging evidence indicates that GDF15 biology itself is subject to sexual dimorphism, meaning its expression, regulation, and downstream signalling pathways could be influenced by these sex‐specific factors. ⁶⁵ Consequently, the protective effects we observed may not be directly applicable to females. Furthermore, the relatively small number of human samples analysed limits the statistical power of these findings, underscoring the need for further validation in larger, sex‐balanced cohorts.

Beyond these experimental design considerations, the therapeutic application of GDF15 requires navigating its complex, paradoxical biology. While our study highlights acute hepatoprotective effects, it is crucial to acknowledge that GDF15 is also classified as a “gerokine”. The chronic elevation of GDF15 is strongly associated with detrimental conditions such as inflammaging, sarcopenia, cachexia, and all‐cause mortality.^{66, 67} This duality suggests that GDF15's effects are highly context‐dependent, with a likely transition from protective to pathological based on the duration and magnitude of exposure. The supraphysiological concentrations used in our study (10–100 ng/mL), while mirroring levels in severe acute disease and being necessary for mechanistic dissection, stand in contrast to the lower, chronic levels implicated in aging‐related pathologies. ⁶⁸ This underscores a critical future research avenue: the definition of a therapeutic window. Future translational studies must aim to determine the optimal concentration, dosing frequency, and treatment duration that can maximise the beneficial metabolic effects of GDF15 while avoiding the activation of pathways leading to its detrimental systemic consequences.

In summary, our findings demonstrate that GDF15 ameliorates lipid accumulation through distinct context‐dependent pathways. Under lipotoxic PA exposure, GDF15 restores mitochondrial morphodynamic homeostasis and enhances fatty acid oxidation via ERK1/2 inhibition, whereas in non‐lipotoxic conditions, it primarily downregulates lipogenic pathways. We identified the ERK1/2 signalling cascade as a critical mediator that regulates mitochondrial fusion‐fission balance and metabolic gene expression, with pharmacological inhibition of this pathway recapitulating GDF15's beneficial effects. The validation of these mechanisms in human liver organoids enhances the translational relevance of our discoveries. These insights not only elucidate novel regulatory pathways in hepatic lipid metabolism but also strengthen the rationale for exploring GDF15 as a therapeutic target for MASLD. Our work provides a foundational, mechanistic rationale that justifies future, more complex investigations required for clinical translation. While our pharmacological data, particularly the phenocopy by U0126 and the non‐additive effects in co‐treatment experiments, provide evidence for GDF15 acting via ERK1/2 inhibition, we acknowledge the correlative nature of this link. A key future objective is therefore to establish definitive causality through a rescue experiment demonstrating that a constitutively active form of MEK/ERK can abolish GDF15's protective effects, although performing such genetic manipulations in primary hepatocytes and complex 3D organoids presents formidable technical challenges. This goal, along with identifying the specific downstream molecular effectors of ERK1/2 and the upstream non‐GFRAL receptor mediating GDF15's direct actions, will be crucial for a comprehensive understanding of GDF15's role across the spectrum of metabolic diseases.

AUTHOR CONTRIBUTIONS

Design: Jia Li and Han Moshage conceived and designed the study. Conduct/Data Collection: Jia Li and Qi Zhou conducted experiments and collected data. Mengmeng Xia, Yakun Li, Junyu Wang, Manon Buist‐Homan, Vincent E. de Meijer, Hans Blokzijl, and Klaas Nico Faber provided essential resources and materials. Analysis: Jia Li and Qi Zhou analysed and interpreted the data. Writing Manuscript: Jia Li wrote the original draft. Jia Li, Mengmeng Xia, Junyu Wang, Manon Buist‐Homan, Vincent E. de Meijer, Hans Blokzijl, Klaas Nico Faber, and Han Moshage reviewed and edited the manuscript. Han Moshage supervised the project and acquired funding. All authors read and approved the final manuscript.

CONFLICT OF INTEREST STATEMENT

The authors declare no competing interests.

Supporting information

Figure S1. GDF15 reduces PA‐induced necrotic cell death and FFA‐induced lipid synthesis. (A, B) Representative images and quantitative analysis of SYTOX Green‐positive cells (scale bar: 25 μm). (C–G) Relative mRNA expression levels of Dgat1, Dgat2, Cd36, Srebf1, and Pparg (n = 3–4 independent experiments per group). (H) Representative Western blots and quantitative analysis of AMPK phosphorylation in GDF15‐treated hepatocytes subjected to PA‐ and FFA‐induced steatosis (n = 3 per group). Data were presented as mean ± SD. The statistical significance of differences was analysed by one‐way ANOVA followed by Bonferroni's post‐hoc test. *p < 0.05, **p < 0.01.

Figure S2. GDF15 ameliorates mitochondrial dysfunction in hepatocytes under lipotoxic PA stress. (A, B) Representative images of MitoSOX staining and corresponding fluorescence intensity in each group (scale bar: 25 μm) from seven random fields per group. (C, D) Representative images of JC‐10 staining and relative red/green fluorescence intensity ratio values (scale bar: 25 μm) from seven random fields per group. Data were presented as mean ± SD. The statistical significance of differences was analysed by one‐way ANOVA followed by Bonferroni's post‐hoc test. *p < 0.05, **p < 0.01.

Figure S3. GDF15 does not significantly affect lipid uptake and synthesis genes in PA‐treated hepatocytes, while ERK1/2 signalling is dispensable for GDF15‐mediated reduction of lipogenic gene expression in FFA‐treated cells. Cells were pretreated with GDF15 (100 ng/mL) in combination with either U0126 or Mdivi‐1 for 30 min, followed by co‐treatment with PA or FFA for 16 h. (A–E) Relative mRNA levels of Dgat1, Dgat2, Srebf1, Pparg, and Cd36 under PA‐induced lipotoxic conditions. (F–H) Relative gene expression levels of Dgat1, Dgat2, and Pparg under FFA‐induced non‐lipotoxic conditions (n = 3 per group). Data were presented as mean ± SD. The statistical significance of differences was analysed by one‐way ANOVA followed by Bonferroni's post‐hoc test. *p < 0.05, **p < 0.01.

Table S1. Primary antibodies used in this study.

Table S2. Primers and probes for RT‐qPCR analysis.

DATA AVAILABILITY STATEMENT

Research data supporting this publication can be assessed by contacting the corresponding author with a valid request.