Zebrafish model of palmitic acid induced MAFLD recapitulates pathways conserved in mice and humans

Abstract

Metabolic Dysfunction-Associated Fatty Liver Disease (MAFLD) is a major global health issue, affecting millions, yet its underlying molecular mechanisms remain poorly understood. Here, we propose a novel diet-induced zebrafish model to investigate pathophysiology of MAFLD. To validate the model, we performed comprehensive histological analysis and molecular assessments, including RNA-sequencing, to characterize the disease progression. These approaches enabled us to examine the molecular alterations underlying MAFLD and identify key genes and pathways involved in its development. Our results demonstrate that zebrafish subjected to a high-fat diet exhibit significant weight gain and show prominent fat accumulation in the liver, as confirmed by Oil Red O and BODIPY staining. Quantitative PCR analysis reveals upregulation of key lipogenic genes, including acc, fasn, hmgcs1, and hmgcra, indicating enhanced lipid synthesis. Immunoblotting also shows increased expression of several proteins (SIRT1, SREBP-1c, CEBPA and PGC-1α) involved in lipogenesis and glucose metabolism. Additionally, we observe increased expression of genes associated with endoplasmic reticulum stress, such as atf6, xbp1, gadd45a, and ddit3, along with activation of the mitochondrial unfolded protein response and inflammatory pathways, as indicated by elevated levels of hspd1, hspa9, clpp, lonp1, il1β and il8. These findings point to mitochondrial dysfunction, further supported by the dysregulation of genes involved in oxidative phosphorylation, including uqcrc2, cox4i1, sdha, nd1, and atp5f1b at both mRNA and protein levels. Transcriptomic profiling identifies new candidate markers such as inha, gck, ces2a, id3 and highlights dysregulated pathways involved in metabolism, insulin signaling, and cellular stress, offering insights into MAFLD progression. This study establishes a zebrafish model that recapitulates key features of MAFLD, including histopathological and metabolic alterations. Through transcriptomic and protein analysis, we identify novel biomarkers and pathways, providing new insights into MAFLD pathogenesis and highlighting potential therapeutic targets.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-13154-5.

Introduction

Metabolic dysfunction-associated fatty liver disease is a broad spectrum of hepatic conditions that range from metabolic dysfunction-associated fatty liver (MAFL) to the more advanced and severe metabolic dysfunction-associated steatohepatitis (MASH)^1–3. Patients suffering from simple fatty liver, develop hepatic steatosis wherein there is accumulation of >5% triglycerides in the hepatocytes^4,5. While MAFL can often be reversed through strict calorie restriction and regular exercise, in some cases, it may progress to MASH, a condition marked by inflammation and hepatocellular damage^6,7. This progression can further advance to fibrosis, cirrhosis, and even hepatocellular carcinoma⁸. Referred to as the hepatic manifestation of metabolic syndrome, MAFLD is a multifaceted disorder, that has recently gained attention of the scientific community due to its rising global prevalence⁹. About 32% of the adult population is estimated to be affected by MAFLD worldwide¹⁰. MAFLD is often associated with various other health complications like cardiovascular diseases, coronary artery disease, liver failure, malignancies, and renal failure^1,11. Despite significant research, the pathophysiology and mechanisms driving the progression of this multifactorial and heterogeneous disease remain poorly understood.

Rodent models have traditionally served as the primary means for studying the pathophysiology of MAFLD. These include both genetic (e.g. ob/ob, PTEN null, PPARα knock-out, db/db), nutritional (Methionine-Choline-deficient (MCD) diet, Choline-deficient-L-amino acid defined diet (CDAA), high cholesterol diet) and combination models (db/db mice on MCD diet)^4,12–16. However, none of these models fully capture the complexity of this multifactorial disease and have some inherent limitations, such as high maintenance costs, extended experimental timelines, and physiological differences that limit their relevance to human MAFLD. Owing to its physiological similarity to mammalian systems, zebrafish has gained increased popularity as an excellent model organism in the recent years^17–20. Low maintenance cost, rapid development, and ease of chemical and genetic manipulations, make zebrafish an ideal organism for studying biological processes like development, regeneration as well as various diseases^20,21. Additionally, their metabolic processes, particularly lipid and carbohydrate metabolism, are highly conserved with mammals, making them relevant for metabolic disease research. The liver in zebrafish is fully developed within 4 days post fertilization and comprises of cell types that are functionally equivalent to their mammalian counterparts, including hepatocytes, macrophages, and neutrophils, thus facilitating studies on inflammation and steatosis²². The comparable expression patterns and presence of orthologues of most human lipid and carbohydrate metabolism related genes in zebrafish makes it a physiologically relevant model to study diseases related to dyslipidaemia like MAFLD²³. Several genetic (foie gras, ducttrip, hi559), transgenic (liver specific overexpression of YY1 and Cannabinoid receptor1, heart-specific over expression of IL6), and diet-based models (high cholesterol diet and high fructose diet) of zebrafish have extensively been used to study MAFLD^24–27. Moreover, the similarity in the pathophysiology of zebrafish and mammalian obesity, makes the diet-induced MAFLD models of zebrafish clinically relevant¹⁸.

In this study, we introduce a novel zebrafish model for studying MAFLD, generated by supplementing a palmitic acid-enriched diet combined with overfeeding. Palmitic acid (PA) is a saturated fatty acid known to contribute to hepatic lipid accumulation and inflammation^28,29, making it particularly relevant for modelling steatosis and early MASH-like conditions. PA is already known to be involved in insulin resistance, inflammation, dyslipidaemia, and cellular injury^28–30. This model effectively mimics the lipid accumulation and metabolic disturbances seen in human MAFLD. To gain a comprehensive understanding of the molecular changes associated with this condition, we conducted extensive transcriptomic profiling using RNA sequencing (RNA-seq). Notably, transcriptomic analysis revealed significant similarities between the differentially expressed genes observed in our PA-supplementation model and those reported in human MAFLD datasets, further emphasizing the translational relevance of our approach. We also compared this model with the high fructose diet-induced MAFLD mouse model and observed shared upregulation of processes associated with lipid metabolism, cholesterol biosynthesis, and related enzymatic activities. Moreover, our findings indicate marked disruptions in key regulatory pathways governing hepatic metabolism and mitochondrial function, processes known to be critically involved in MAFLD progression and pathogenesis. These insights enhance the utility of zebrafish as a model for understanding the complex pathogenesis of fatty liver disease. Through this work, we aim to demonstrate the versatility and relevance of zebrafish in investigating MAFLD while providing new insights on the intricate molecular mechanisms that govern the pathogenesis and progression MAFLD.

Results

PA-supplementation leads to lipotoxicity and hepatic injury by dysregulating lipid metabolism, cellular stress pathways and mitochondrial functioning

To establish a diet-induced model of MAFLD, we selected only male zebrafish aged 5–6 months to control potential sex and age-related differences in metabolic response. All fish were matched for weight at the beginning of the experiment to ensure a uniform starting population. Over a 8-week protocol, the PA-fed group received a diet supplemented with 7% palmitic acid (PA) through overfeeding (Fig. 1A). Weight gain was monitored to assess diet-induced obesity in the PA-fed group compared to control fish, with a consistent and significant weight gain observed within 4 weeks of the PA-supplementation protocol, which showed further increase by the end of the study period, indicating successful induction of a steatotic phenotype in adult zebrafish (Fig. 1B and C). We also observed a significant increase in the liver-to-body weight ratio in the PA-fed group as compared to the control (Fig. 1D). Overfeeding with PA-enriched diet also led to an increase in hepatic cell death (data shown in supplementary Figure 1), while only a marginal increase in cell proliferation rates as seen by immunoblotting for cell-proliferation marker PCNA (proliferating cell nuclear antigen) (supplementary Figure 2A, B). To assess morphological changes in liver tissue due to PA supplementation, we performed Hematoxylin-Eosin (H&E) and Oil Red O (ORO) staining on liver sections. H&E-stained liver cross-sections revealed mild morphological changes in the PA-fed group that were indicative of hepatic disorganization and early signs of architectural disruption (Fig. 1E). Oil Red O staining further confirmed extensive lipid droplet accumulation in cryosections of livers from the PA-fed fish, highlighting marked hepatic steatosis compared to the control group (Fig. 1F). Quantification of the lipid droplet area stained in red by ORO also revealed significant increase in the PA-fed tissue sections compared to the control liver sections³¹ (Fig. 1G). Additionally, we also performed BODIPY staining on liver tissue cryosections which revealed significant increase in hepatic lipid deposition in the PA-fed liver in comparison to the control, as is evident from the quantification of the lipid droplet area of the BODIPY stained (green) sections (Fig. 1H and I).These results demonstrate that PA supplementation effectively induces hepatic steatosis and mild tissue disorganization, establishing a clear MAFLD phenotype in adult zebrafish.

Fig. 1.

PA-supplementation leads to increase in body length, body weight and lipid accumulation in liver. (A) Schematic representation of the PA-supplementation paradigm. (B) Increased body length of test group upon PA-supplementation in comparison to control group. (C) Assessment of body weight of adult zebrafish fed with control diet or PA supplemented diet (n = 24). (D) Assessment of Liver-to-body weight ratio (n = 10). (E) Hematoxylin and Eosin staining of adult control and PA-fed 4 μm thick paraffin embedded liver tissue sections show increased accumulation of lipid droplets. (F) Oil Red O staining of adult control and PA-fed 8 μm thick liver cryosections (40 × magnification, images captured using DEBRO DIM 150, inverted microscope). Scale Bar: 50 μm. (G) Quantification of Oil Red O staining of control and PA-fed sections (quantified using ImageJ). (H) BODIPY staining of adult control and PA-fed 5 μm thick liver cryosections (63 × magnification, images captured using Confocal Leica DMi microscope). Scale Bar: 50 μm (I) Quantification of BODIPY staining of control and PA-fed sections (quantified using ImageJ).

Next, to understand the transcriptional regulation underlying the observed phenotypes of hepatic injury and steatosis, we examined the relative expression of genes associated with lipid metabolism, ER (Endoplasmic Reticulum) stress, unfolded protein (UPR) response, oxidative stress and mitochondrial functions in the adult model. MAFLD is heavily associated with dyslipidaemia and perturbed cholesterol metabolism^32–34. Disruptions in lipid metabolism can lead to excessive lipid accumulation in hepatocytes, while ER stress and the UPR are critical responses to cellular stress triggered by lipid overload. These pathways are known to be key contributors to hepatocyte dysfunction and cell death, ultimately driving MAFLD progression. Therefore, firstly, to understand the impact of PA-supplementation on hepatic lipogenesis and cholesterol metabolism, we measured the mRNA levels of acetyl-CoA carboxylase (acc), fatty acid synthase (fasn), 3-hydroxy-3-methylglutaryl-CoA synthase 1 (hmgcs1) and 3-hydroxy-3-methylglutaryl-CoA reductase (hmgcra), wherein ACC and FASN is involved in lipogenesis and HMGCS1 and HMGCRA catalyse the first and second reactions of cholesterol biosynthesis pathway. The mRNA levels of all these key enzymes were found to be significantly upregulated in the adult (Fig. 2A).

Fig. 2.

PA-supplementation leads to perturbed expression of genes associated with lipid metabolism, ER stress and unfolded protein response. Relative mRNA expression of (A) Lipid metabolism associated genes (n = 4), (B) ER stress associated genes (n = 4) and (C) Unfolded Protein Response-associated genes (n = 4) and (D) Oxidative phosphorylation genes in adult (n = 5) control and PA-fed groups. (E) Genes associated with fatty acid oxidation (n = 3) and (F) Genes associated with inflammation and antioxidant response system (n = 3). (G) Relative fold change in the intensity of DCFDA fluorescence in PA-Fed group in comparison to control (n = 3). Values are given as mean ± SEM. Student’s t test was performed to determine statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (H) Flowchart showing dysregulation of metabolic and stress pathways upon PA-supplementation resulting in MAFLD progression. acc, acetyl-CoA carboxylase; fasn, fatty acid synthase; hmgcs1, 3-hydroxy-3-methylglutaryl-CoA synthase 1; hmgcra, 3-hydroxy-3-methylglutaryl-CoA reductase; scd, stearoyl- CoA desaturase; atf6, activating transcription factor 6; xbp1, X-box binding protein 1; gadd45a, growth arrest and DNA-damage-inducible gene 45 alpha; ddit3, DNA damage-inducible transcript 3; il1β, interleukin-1β; hspd1, heat shock 60 protein 1; hspa9, heat shock protein 9; clpp, caseinolytic mitochondrial matrix peptidase proteolytic subunit; lonp1, lon peptidase 1; uqcrc2, cytochrome b-c1 complex subunit 2, mitochondrial; cox4i1, cytochrome c oxidase subunit 4 isoform 1, mitochondrial, atp5f1b; ATP synthase subunit beta, mitochondrial, sdha; succinate dehydrogenase, cpt1a; carnitine palmitoyl transferase 1, acadm; medium-chain specific acyl-CoA dehydrogenase, mitochondrial, pparg; peroxisome proliferator-activated receptor gamma, pparab; peroxisome proliferator-activated receptor alpha b, il8; interleukin 8, tgfβ; transforming growth β, gpx1a; glutathione peroxidase 1a.

In conjunction with the severe lipotoxic conditions in the MAFLD livers, malfunctioning of ER and mitochondria leading to activation of mitochondrial unfolded protein response (UPR^mt) pathway further accelerates hepatic injury^35,36. To ascertain if PA-supplementation in our model affects ER and mitochondrial functioning thereby inducing these stress signalling pathways, in turn leading to hepatic injury, we checked for the relative expression levels of genes related to ER stress and UPR^mt pathways. ER stress genes that showed significant upregulation in the PA-fed groups were activating transcription factor 6 (atf6) and its target gene X-box binding protein 1 (xbp1), growth arrest and DNA-damage-inducible gene 45 alpha (gadd45a) and DNA damage-inducible transcript 3(ddit3) (Fig. 2B). Expression levels of mitochondrial chaperone protein (heat shock 60 protein 1 (hspd1)) and proteases (caseinolytic mitochondrial matrix peptidase proteolytic subunit (clpp); lon peptidase 1(lonp1)) associated with the UPR^mt pathway were also seen to be upregulated significantly (Fig. 2C), indicating activation of protective pathways aimed at mitigating ER stress.

An obvious progressive increase in ER lipid content was also seen by staining of 1-month and 2-month PA-fed adult liver paraffin sections with a fluorescent ER specific dye, NBD-Oct that responds environment polarity, indicating that continued intake of PA by adult zebrafish leads to increased lipotoxicity in ER which contributes to activation of ER stress response pathways (Supplementary Figure 3A).

PA-supplementation also led to aberrant expression of genes involved in formation of oxidative phosphorylation (oxphos) complexes, a key function of the mitochondria. Although non-significant, we observed a decreasing trend, in expression of genes encoding mitochondrial oxphos complex I (NADH dehydrogenase subunit 1 (nd1)), Complex III (cytochrome b-c1 complex subunit 2, mitochondrial (uqcrc2)), complex IV (cytochrome c oxidase subunit 4 isoform1, mitochondrial, (cox4i1)) and complex V (ATP synthase subunit beta, mitochondrial, (atp5f1b)). However, significant downregulation was seen in the case of complex II (succinate dehydrogenase, (sdha)) (Fig. 2D).

Next, we examined the impact of PA-supplementation on mitochondrial fatty acid oxidation. We observed a significant upregulation in mRNA levels of carnitine palmitoyl transferase1a (cpt1a) which is involved in facilitating the entry of long-chain fatty acids into mitochondria. Likewise, other genes like medium-chain specific acyl-CoA dehydrogenase, mitochondrial (acadm), peroxisome proliferator-activated receptor gamma. (pparg) and peroxisome proliferator-activated receptor alpha b (pparab)involved in fatty acid oxidation, were also found to be significantly upregulated in the PA-fed group (Fig. 2E).

Increased and faulty fatty acid oxidation often leads to production of reactive oxygen species (ROS) which eventually leads to activation of oxidative stress pathways. Increased ROS generation is often followed by release of pro-inflammatory cytokines, which subsequently leads to increased cell death and progression of MAFL to more progressed stages of MASH^4,37,38. Therefore, we examined the relative expression levels of gpx1a (gluthathione peroxidase 1a), an enzyme involved in antioxidant system that functions by scavenging ROS. The PA-Fed group showed increased expression of gpx1a as compared to control (Fig. 2F), suggesting a compensatory response to increased oxidative stress. Additionally, we checked for the expression of pro-inflammatory cytokines like il1β, il8 and tgfβ upon PA-supplementation. We observed a significant increase in mRNA levels of il1β and il8 and a non-significant increase in tgfβ upon PA-supplementation (Fig. 2F). Consistently, direct measurement of ROS levels revealed a significant increase in the PA-fed group compared to control (Fig. 2G), reinforcing the presence of heightened oxidative stress and inflammation in this model.

We next examined the expression of key metabolic regulators involved in hepatic lipid and glucose metabolism. Immunoblotting revealed a significant increase in the protein levels of Sirtuin 1 (SIRT1), Sterol Regulatory Element-Binding Protein 1c (SREBP-1c), and Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-Alpha (PGC1α) in the PA-supplemented group compared to control (Fig. 3A–C; Supplementary Figure 3B). We also observed a slight but significant increase in CCAAT/Enhancer-Binding Protein Alpha (CEBPA) levels (Fig. 3D; Supplementary Figure 3B), indicating modulation of transcription factors associated with hepatic lipogenesis and energy homeostasis.

Fig. 3.

PA-supplementation leads to alterations in expression of proteins involved in regulating hepatic lipid and glucose metabolism and mitochondrial oxidative phosphorylation. PA-supplementation leads to significant increase in expression of (A) SIRT1, (B) SREBP-1c, (C) PGC1α, (D) CEBPA and (E) mitochondrial OXPHOS complex protein levels as seen by immunoblotting (n = 3). β-actin used as loading control. Values are given as mean ± SEM. Student’s t test was performed to determine statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Next, we investigated the impact of PA-supplementation on mitochondrial function by assessing the expression of oxidative phosphorylation (OXPHOS) complex proteins, which are central to mitochondrial respiration. In line with our real-time PCR findings, immunoblotting revealed a significant reduction in the expression of OXPHOS Complex II, III, and V proteins, while Complex IV showed a non-significant decrease (n = 3) in the PA-fed group (Fig. 3E; Supplementary Figure 3B). These results suggest that PA-supplementation impairs mitochondrial function, potentially contributing to the metabolic disturbances observed in this model.

Taken together, these results suggest that PA-supplementation in zebrafish leads to increased body weight and pathophysiological changes in overall hepatic architecture and ectopic lipid accumulation by disrupting the hepatic lipid metabolism together with ER and mitochondrial malfunction thus contributing to hepatic steatosis and injury (summarized as a flowchart in Fig. 2H). These changes both at the mRNA and protein levels, collectively point to an integrated response involving lipid metabolic dysregulation, ER stress, UPR activation and dysregulation of mitochondrial functioning as central mechanisms in the progression of hepatic injury and steatosis within our zebrafish MAFLD model.

Calorie restriction after PA-supplementation reverses hepatic steatosis

To further investigate the potential for dietary intervention in reversing or mitigating MAFLD phenotypes, we applied a calorie restriction (CR) regimen in our palmitic acid (PA)-fed zebrafish model. Calorie restriction has been widely studied for its beneficial effects on metabolic health and its ability to reduce hepatic fat accumulation and oxidative stress in both animal models and humans. By limiting caloric intake, we aimed to assess whether a reduced energy input could ameliorate the effects of PA-induced hepatic steatosis and injury, offering insights into possible dietary strategies for MAFLD management. Patients suffering from MAFL are usually prescribed such a calorie restricted diet regime to improve obesity associated steatosis, which however cannot be reversed once progressed beyond steatohepatitis stage^39,40. Therefore, to ascertain if our adult over-feeding model could be reversed to non-steatotic normal conditions, we subjected PA-Fed fish to a calorie restriction (CR) paradigm (Fig. 4A). We fed a subset of PA-fed obese zebrafish with control tetramin diet once daily for 1 month. Any improvement in the liver heath was monitored by measuring body weight, hepatic lipid accumulation and mRNA levels of lipogenic genes after following CR. We observed a reduction in the mean body weight of the CR group, although not significant, when compared to the PA-Fed group. It is interesting to note that after following the CR paradigm for 1 month, the mean body weight of the CR group seemed comparable to the mean body weight in the control group which were given tetramin diet (Fig. 4B). Reversal in hepatic fat overload was observed by Oil red O staining in the CR group (Fig. 4E) which was further quantified to observe a significant decrease in lipid droplet area upon calorie restriction which is in fact comparable to that observed in the control group³¹ (Fig. 4D). This reduction in hepatic lipid accumulation indicates that the CR paradigm may enhance lipid metabolism and improve liver health, potentially offering a protective effect against fatty liver disease. CR paradigm also led to decreased hepatic lipogenesis as seen by reduced expression of DNL pathway genes (Fig. 4C). This downregulation of DNL pathway genes suggests that caloric restriction may lead to a more favorable lipid profile, further supporting its role in promoting metabolic health. Overall, these results demonstrate that calorie restriction can counteract the adverse effects of a PA-supplemented diet, leading to reduced hepatic lipid accumulation and restored liver architecture. This highlights calorie restriction as a promising intervention for mitigating MAFLD symptoms, potentially offering a simple, non-pharmacological strategy for managing MAFLD progression.

Fig. 4.

Calorie restriction improves body weight, lipid accumulation and expression of lipid metabolism related genes. (A) Schematic representation of the calorie restriction paradigm. (B) Body weight analysis of control, PA-fed and calorie restriction groups (n = 3). (C) Calorie restriction affects hepatic expression of genes associated with lipid metabolism (n = 3). Values are given as mean ± SEM. Student’s t test was performed to determine statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not statistically significant. Calorie restriction reverses hepatic lipid accumulation: (D) quantification of Oil Red O staining in control, PA-fed and CR group shows decrease in lipid droplet area upon calorie restriction. (E) Decreased lipid droplet overload shown by Oil Red O staining of 8 μm thick liver cryosections of control, PA-Fed and CR groups (40 × magnification, images captured using DEBRO DIM 150, inverted microscope). Scale bar 50 μm. CR: Control_diet, calorie restriction group given control diet.

Mapping of PA-supplemented diet induced pathophysiological changes to alterations in transcriptome

To understand the transcriptional regulation underlying the observed phenotypes of hepatic injury and steatosis, we examined the relative expression of various genes through high throughput sequencing of total RNA isolated from control and PA-supplemented diet fed adult zebrafish livers. By analysing the relative transcriptomic profiles, we aimed to identify key regulatory pathways and molecular mechanisms contributing to the development of hepatic injury and steatosis. Following pre-processing of the raw data, DEGs were identified by the DeSeq2 R package. In total, 1137 differentially expressed (345 down and 792 upregulated) genes based on fold change and 1055 differentially expressed (287 down and 768 upregulated) genes were screened based on log2 fold change. Volcano plot showing significantly differentially expressed genes (p value < 0.05) with their respective log2 fold changes is illustrated in Fig. 5A. Some of the dysregulated genes included cholesterol 7a-hydroxylase (cyp7a1), fatty acid synthase (fasn), CCAT enhancer binding protein alpha (cebpa), glucokinase (gck), retinol dehydrogenase 12 (rdh12) and cholesteryl ester transfer protein (cetp), whose expression patterns were also validated by performing real-time quantitative PCR experiments (Fig. 6). These genes have been shown in various studies to be associated with metabolic dysfunction-associated fatty liver disease, confirming that our findings are consistent with these established links^41–46. The pictorial representation of highly dysregulated genes both upregulated and downregulated with q value < 0.05 and − 1.0 >  = log2 fold changes >  =  + 1.0 is present in Fig. 5B. To explore the potential biological function of DEGs, Gene Set Enrichment analysis (GSEA) was conducted using the ClusterProfiler (v4.10.0) package. As expected, the DEGs showed significant enrichment in biological processes (GO) such as lipid transport, lipid localization, cholesterol metabolic process, insulin secretion, unsaturated fatty acid metabolic process, and lipid biosynthetic process, to name a few. Furthermore, phospholipase activity, lipase activity, glycosaminoglycan binding were notably enriched terms in GO molecular functions as shown in Fig. 5D. According to KEGG enrichment analysis, pathways such as MAPK signaling pathways, calcium signaling pathway, fatty acid biosynthesis, cell cycle, cellular senescence and arachidonic acid metabolism were found to be overrepresented (Fig. 5C). These pathways are integral to the development of MAFLD, as they influence inflammation, cell survival, and lipid metabolism. The presence of these pathways in our analysis underscores the complex interplay between these cellular processes in the progression of hepatic injury and steatosis. The complete list of DEGs and their associated GSEA are included in supplementary data 1.

Fig. 5.

Identification and annotation of differentially expressed genes. (A) Volcano plot of DEGs for MAFLD vs control transcriptomic data. The volcano plot shows the fold-change (x-axis) versus the significance (y-axis) of the identified genes. The significance (p value) and the fold-change were converted to − log10 (p value) and log2 (fold-change), respectively. The vertical and horizontal dotted lines show the cut-off of log2 Fold-change =  ± 1.0, and of p value < 0.05, respectively. There were 792 upregulated genes (upper-right, dots coloured green), and 345 downregulated genes (upper-left, dots coloured red). (B) Hierarchical clustering map of DEGs. The horizontal axis shows the names of each group (control and MAFLD) and the vertical axis shows the clusters of DEGs. The left clustermap shows upregulated DEG’s while the right one shows downregulated DEG’s. Colours towards brown represent high expression values and colours towards yellow represent low expression values. Here q value < 0.05 and log2Fold-change = ± 1.0 is taken as level of significance parameters. Constructed with seaborn package in python (https://seaborn.pydata.org/). (C) GSEA KEGG pathway analysis of DEGs (www.kegg.jp/kegg/kegg1.html). The dot plot represents the KEGG pathways on the y-axis and enrichment scores on the x-axis. The dot represents the gene count, greater gene count corresponds to a greater dot. The colour of the dot varies based on the p value. (D) GSEA Gene Ontology analysis of DEGs. The bar plot shows the gene counts (x-axis). GO terms, biological processes and molecular functions are on the y-axis. The colour of the bar varies based on the significance of p value. Constructed with SRplot (https://www.bioinformatics.com.cn/srplot). (E) STRING analysis showing protein–protein interaction between the proteins encoded by dysregulated genes. Some of these are GPCRs, chemokines, proteases, cell adhesion molecules and signaling receptors. Several are involved in galactose metabolism, lipid metabolism, Notch signaling, the cell cycle and division, developmental processes, as well as cellular maintenance and homeostasis.

Fig. 6.

Relative expression of (A) cyp7a1, cebpa, gck and (B) rdh12 and cetp in adult PA-fed livers in comparison to control (n = 3). Values are given as mean ± SEM. Student’s t test was performed to determine statistical significance. ***p < 0.001, ****p < 0.0001. cyp7a1, cholesterol 7a-hydroxylase; cebpa, CCAT enhancer binding protein alpha; gck, glucokinase; rdh12, retinol dehydrogenase 12; cetp, cholesteryl ester transfer protein.

GSEA analysis did not show a direct association of some of the highly dysregulated genes with the onset or progression of MAFLD. To probe further into the biological role of these prospective candidates, we next mapped the known interactions between all the dysregulated protein-coding genes (-1.0 >= log2 fold changes >= + 1.0 and q value < 0.05) using the STRING database. The cross talk observed between these protein-coding genes is illustrated Fig. 5E. DEG analysis showed inha to be a very highly upregulated gene (Fig. 5B). This gene encodes the alpha subunit of inhibin, a member of the TGF-β superfamily involved in SMAD-mediated signaling. INHA is expressed by various immune cell types including NK cells (CD56⁺ NK cells), dendritic cells, B cells, and T lymphocytes (such as Th17 and regulatory T cells), where it modulates their development, maturation, and function^47–49. Previous studies have demonstrated its involvement in various types of cancer, where it shapes the immune suppressive tumor microenvironment⁴⁸. More specifically its role in hepatocellular carcinoma has also been established⁵⁰. We further looked into the genes that have been reported to interact with inha product. The first primary interactor ID3, encoded by id3 and downregulated in our data, has been shown to be crucial for the proliferation and differentiation of the hepatoblasts during chick liver development⁵¹. Another interactor of INHA, CHRNA4B encoded by chrna4b was also found upregulated in our data. chrna4 gene expresses specifically in hepatocytes and its protein levels has been found increased in mice and patients with metabolic dysfunction-associated steatohepatitis (MASH)⁵². CHRNA4B further interacts with GLT8D1 associated with the glycosyltransferase activity⁵³, which again plays a crucial role in the pathogenesis of MAFLD⁵⁴.

Another candidate among the top downregulated genes in our DEG analysis encodes for GC-Vitamin D-binding protein. Vitamin D-binding protein is highly localized in the liver and pancreatic alpha cells. Studies show that it attenuates liver injury in sepsis and regulates alpha cell function and glucagon secretion⁵⁵. According to the interaction map, GC interacts with proteins coded by various genes. Amongst these interactors, cela1.6, cela1.3, slc4a8, cldni, cpa1, cpb1, c6ast4 and prss59.1 were upregulated, and sb:cb37, ces2a, serpina7, zgc:174259 and proca were downregulated in our data. A recent study has revealed that chymotrypsin-like elastase (CELA) is involved in lipid metabolism⁵⁶. Whereas deletion of carboxylesterase 2a (ces2a) causes hepatic steatosis and insulin resistance in mice by impairing diacylglycerol and lysophosphatidylcholine catabolism⁵⁷. CES2A further interacts with Glucokinase (GCK) which is the primary hexokinase expressed in hepatocytes. Its main function is to phosphorylate glucose that enters via GLUT transporters, converting it into glucose-6-phosphate (G6P). This process ultimately directs glucose into various anabolic and catabolic pathways⁵⁸. This regulation of liver glucose metabolism by enzyme glucokinase is further influenced by various factors such as hormones, metabolites of glucose, and binding proteins (6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK2/FBP2). Changes in glucokinase expression and activity are associated with type 2 diabetes and metabolic dysfunction-associated fatty liver disease⁵⁹. In our data, gck was also found to be highly expressed. Interacting partners gck were found to be pfkma, mat2ab, abcb5 and klb. All of them showed increased expression in our data.

Another study shows that TGF-β1/p65/MAT2A pathway regulates liver fibrogenesis via intracellular S-adenosylmethionine (SAM)⁶⁰. Also, the genetic variation in abcb5 is associated with the risk of hepatocellular carcinoma⁶¹. The primary interactors of ABCB5 are proteins coded by jag1b, epcam, ccnb1, igsf9ba, itga6b and proca. igsf9ba and proca from this gene pool were downregulated and all others were upregulated in our analysis. CCNB1 promotes the development of hepatocellular carcinoma by mediating DNA replication in the cell cycle⁶². According to the interaction map, CCNB1 is interacting with proteins coded by upregulated genes smcr8b, mid1ip1l, itgb4, cldne, ctrl, r3hdml and downregulated genes diabloa, ccdc141, col5a1, vsx1, foxh1 and cep55l. Midline 1 interacting protein 1 (MID1IP1) promotes cancer metastasis through FOS-like 1-mediated matrix metalloproteinase 9 signaling in hepatocellular carcinoma⁶³. foxh1 exhibits DNA-binding transcription repressor activity, cell growth, and cell migration/invasion, relies on mTOR signaling and plays a role in HCC development⁶⁴. FOXH1 interacts with proteins coded by dysregulated genes ccdc103, mef2b, tfap2c and oplah. In our data, low expression of the col5a1 gene was observed. It is a collagen related gene which plays a role in liver fibrosis⁶⁵. COL5A1 interacts with protein coded by dysregulated genes itgb4, ccnb1, tox3 and slc35c2.

From this analysis, it was observed that pathways previously associated with MAFLD, such as lipid metabolism, cholesterol metabolism, inflammation, and insulin secretion, were dysregulated. This disruption highlights how these processes contribute to hepatic steatosis and injury. The involvement of MAPK signaling, calcium signaling, and arachidonic acid metabolism pathways underscores the complex interplay between inflammation, cell survival, and lipid metabolism in the progression of MAFLD. These pathways are critical for understanding how PA-induced changes lead to cellular stress, lipid accumulation, and liver damage. The identification of key genes like INHA, which interacts with immune signaling pathways, and GC, involved in glucose metabolism and lipid catabolism, illustrates how these molecular networks are disrupted in MAFLD. These findings provide new insights into the mechanisms driving insulin resistance, altered lipid metabolism, and chronic inflammation that are salient hallmarks of MAFLD (Fig. 6). Overall, this study not only highlights the transcriptional changes associated with PA supplementation but also offers a deeper understanding of how these changes contribute to the pathogenesis of MAFLD.

Comparative analysis with murine high fructose diet models and human MAFLD transcriptomic data

Following the findings from our transcriptomic analysis, we performed a cross-species comparative study between our palmitic acid (PA) model and a high fructose diet-induced MAFLD mouse model (accession number PRJNA766592, European Nucleotide Archive). The comparison revealed a substantial overlap in dysregulated pathways such as lipid localization, sterol metabolism, lipid biosynthesis, cholesterol metabolism, oxidoreductase activity, and lipase activity. As illustrated in Fig. 7A–D, these processes were significantly elevated in both the models, highlighting common mechanisms involved in lipid accumulation and liver damage. The supplementary data (Supplementary Figure 4 and Supplementary Data 2) lists the other overlapping processes between the PA-fed zebrafish and the high fructose diet mouse model.

Fig. 7.

Comparison between palmitic acid- and fructose-based model: The dot plots depict the overlap between the categories (A) GO biological process: Lipid metabolism and regulation (B) GO biological process: Other metabolic processes (C) Cellular component and KEGG pathways (D) GO molecular function: Enzyme activity in palmitic acid and fructose based MAFLD model. The vertical items are the names of GO terms, and the length of horizontal graph represents the gene ratio. The depth of the colour represents the adjusted p value. The area of circle in the graph means gene counts. Constructed with ggplot2 in R package tidyverse (https://www.tidyverse.org/). (E) The scatter plot illustrates commonly dysregulated genes with concordant trends in both the high-fructose diet mouse model (x-axis) and palmitic acid (PA)-fed zebrafish model (y-axis). Green dots indicate upregulated genes, while red crosses represent downregulated genes. Constructed with seaborn package (https://seaborn.pydata.org/).

To delve deeper into the shared mechanisms, we focused on 38 genes among the 6,742 commonly expressed genes that showed significant and concordant differential expression (log2FC ≥ 1.0 or ≤ –1.0) across both datasets as depicted in Fig. 7E. Key drivers of de novo lipogenesis (DNL) such as fasn, elovl6, and acaca. were upregulated, supporting their pivotal role in hepatic lipid accumulation⁶⁶. Upregulation of pro-inflammatory and fibrotic mediators such as s100a11,inhbb, and fpr1 underscores chronic inflammation and fibrotic signaling^67–69. Altered expression of cell cycle regulators like cdkn1a, mki67, and cenpf pointed to aberrant hepatocyte proliferation and injury while vldlr and pklr dysregulation points to disrupted lipid and glucose metabolism^70–72. Notably, cyp17a1 and sulf1 have been linked to metabolic dysfunction and oxidative stress, exacerbating steatosis^73,74. Supplementary Data 3 provides full list of overlapping genes.

Together, these findings underscore the conserved molecular landscape underpinning MAFLD pathogenesis in both the PA-fed zebrafish model and the high-fructose diet mouse model. This integrative analysis highlights that despite differing dietary etiologies, both models converge on similar pathways that culminate in dyslipidaemia and liver injury. These findings are relevant because they show how dietary influences on lipid metabolism can lead to liver damage across different species, bridging a critical gap in understanding between human and animal models.

We further compared our PA supplementation model with a human MAFLD transcriptomic dataset (accession number PRJNA558102, European Nucleotide Archive). Our results demonstrated significant similarities between the differentially expressed genes observed in our PA-fed adult zebrafish and those seen in human MAFLD patients (Fig. 8 and supplementary data 4). This comparison revealed upregulation of genes involved in de novo lipogenesis (e.g., hmgcs1 and fasn) and inflammation (e.g., tgfβ1) in both humans and PA-fed zebrafish. Additionally, genes associated with extracellular matrix remodelling and fibrogenesis (e.g., epcam, lamc2, and itgb6) were also upregulated in both models. The observed dysregulation of these genes in both species highlights the clinical relevance of our zebrafish model for studying MAFLD, providing insights into shared mechanisms underlying the disease across different organisms. This cross-species comparison reinforces the utility of our zebrafish model in capturing the complex pathophysiology of MAFLD and suggests that our findings are not only biologically significant but also translatable to human disease.

Fig. 8.

Comparison of human and zebrafish MAFLD models: Genes elevated in both models are highlighted in green, while downregulated genes are highlighted in red. The figure is created using BioRender (https://www.biorender.com/).

Discussion

Of late, MAFLD has become one of the most prevailing liver disorders affecting a majority of the global adult as well as paediatric population. Although, several genetic and diet-based animal models have been used for MAFLD research, the pathophysiology of this complex multifactorial disease remains unclear. Zebrafish is emerging as an excellent model organism for MAFLD research, owing to its remarkable functional similarity with mammalian liver. Zebrafish, on exposure to environmental triggers, such as hepatotoxins and fat-enriched diet, develop hepatic steatosis, much like humans, which progresses to MASH and may even culminate in HCC, following pathophysiological pathways similar to that reported in MAFLD/MASH patients^26,75. Our study establishes a diet-induced zebrafish model system, over-fed with high-fat (palmitic acid) diet to recapitulate the various histopathological and metabolic features of this disease. Notably, our studies show that excessive consumption of 7% PA-supplemented diet led to induction of hepatic steatosis in adult zebrafish. The feeding protocol was designed to mimic the high-fat dietary conditions that are prevalent in human MAFLD, thereby providing a relevant physiological context for our experiments. Validation of the model was accomplished through a series of assessments, including weight analysis, histological analysis, Oil Red O and BODIPY staining, cell death/proliferation level assessments, and gene expression and protein analysis, which confirmed the presence of hepatic lipid accumulation, dysregulation of lipid metabolism, activation of stress pathways and mitochondrial malfunctioning, consistent with MAFLD pathogenesis. Increased lipotoxic conditions in the ER of the adult PA-Fed group further established the successful induction of severe steatosis in our model. These findings validated the efficacy of the PA supplementation in inducing steatosis and highlighted the model’s suitability for further investigations into the underlying molecular mechanisms. Obesity associated MAFLD is closely connected with dyslipidaemia which increases the free fatty acid (FFA) flux in the liver thus putting it under considerable metabolic load. In fact, about 26% of the FFA flux in MAFLD is reported to be contributed by hepatic de novo lipogenesis^4,76. Indeed, adult zebrafish fed with PA-supplemented diet shows a significant increase in expression levels of genes related to hepatic DNL and cholesterol synthesis suggesting towards dysregulated hepatic lipid metabolism. This result was further corroborated by immunoblotting analyses that showed increased expression of SIRT1, SREBP-1c and CEBPA, each of which are involved in regulation of lipid metabolism in the liver⁷⁷. SIRT1, an NAD⁺ dependent deacetylase has been extensively implicated in regulating hepatic metabolic homeostasis by deacetylating several transcription factors like PPARα, PGC1α, ChREBP and SREBP-1c, which in turn modulate lipid and glucose metabolism^78,79. While several animal studies have associated decreased SIRT1 activity with MAFLD progression, other evidence suggests that its regulation is highly context-dependent and influenced by dietary status^80–82. In states of nutritional excess, such as in our PA-supplemented model, elevated hepatic fatty acid and cholesterol synthesis may increase the NAD⁺/NADH ratio, thereby enhancing SIRT1 activity^81,82. SIRT1-mediated deacetylation of PGC1α and SREBP-1c enhances their transcriptional activity, promoting gluconeogenesis and de novo lipogenesis, respectively^83,84. Moreover, SREBP-1c is also known to be activated through pathways linked to endoplasmic reticulum (ER) stress and the unfolded protein response (UPR)^46,85–88. Consistent with this, our model showed upregulation of several ER stress-related genes at the mRNA level, along with a marked increase in SREBP-1c protein levels in the PA-fed group. Similarly, we observed elevated expression of PGC1α upon PA overfeeding. Together, these transcriptomic and protein-level changes point to a significant disruption of hepatic metabolic homeostasis, further underscoring the relevance of our model in capturing early pathogenic events in MAFLD.

Moreover, several reports have implicated ER stress as well as mitochondrial malfunctioning to be primary underlying factors in MAFLD pathogenesis^89,90. Disruption of protein transport from ER to Golgi, impaired assembly and secretion of triglyceride as very low-density lipoproteins (VLDLs) and altered calcium homeostasis leads to activation of ER stress pathways, which in turn ensues activation of an adaptive unfolded protein response (UPR) pathway^91–93. Additionally, faulty beta-oxidation of lipids in the mitochondria and generation of reactive oxygen species (ROS) from ώ-oxidation of fatty acids leads to increased oxidative stress, thus triggering inflammation, hepatocyte death and extensive hepatic injury^4,40,94. Our models showed significant activation of inflammation, ER stress, UPR pathways, ROS generation and increased expression of antioxidant system genes upon overconsumption of dietary PA thus explaining the severe hepatic injury observed. We also observed a decreased expression of mitochondrial OXPHOS complexes at both the mRNA and protein levels indicating mitochondrial dysfunction upon PA-supplementation. This result was consistent with previous studies that have reported an adaptive mitochondrial response in MAFLD, which is eventually lost in more progressed stages of MASH^95–97. Several rodent models of MAFLD have also reported impaired oxidative phosphorylation as seen in our model^98–101.

In an attempt to study the reversibility of the steatosis so induced, we followed a calorie restriction paradigm on the adult PA-fed group. This approach was designed to mimic dietary interventions commonly employed in clinical settings for managing metabolic dysfunction-associated fatty liver disease (MAFLD). Interestingly, our adult diet-induced model responded effectively to calorie restriction as suggested by the histological as well as gene expression assessments, thus proving to be an excellent model system to examine the effectiveness of various therapeutic interventions in ameliorating MAFLD. These findings suggest that caloric restriction not only mitigates lipid accumulation but also reinstates a more balanced metabolic state within the liver. This response aligns with existing literature that suggests caloric restriction can improve liver health and reduce fat deposition through various metabolic pathways^6,18,102. By utilizing this palmitic acid-induced zebrafish model, we can explore the underlying mechanisms by which caloric restriction exerts its beneficial effects on liver health. Additionally, this model provides a platform to investigate other dietary interventions, pharmacological treatments, or combinations thereof, enabling a comprehensive evaluation of strategies to prevent or reverse the progression of MAFLD.

Further, to better understand the intricate molecular underpinnings of this heterogeneous disease, we performed transcriptomic profiling of our adult steatotic zebrafish livers. We probed the differential gene expression patterns in the steatotic group in comparison to the healthy control group to uncover candidate genes and signalling pathways that have prominent roles in the pathophysiology of this disease. The dysregulated genes were found to be enriched in various biological processes including lipid transport, localization, cholesterol metabolism, lipid biosynthesis, glucose, and retinoic acid metabolism. Perturbations in lipid, glucose and retinoic acid metabolism, already well-documented in MAFLD research, was also further corroborated by the significantly increased expression of several lipid metabolism related genes (like acc, fasn, cebpa, hmgcs1, hmgcra, and cyp7a1) and decreased expression of retinol dehydrogenase gene (rdh12) in our real-time experiments. Moreover, KEGG pathway analysis revealed MAPK signalling, calcium signalling, cellular senescence and p53 signalling pathways to be overly represented in our dataset. In fact, previous studied have also reported activation of p38 MAPK signalling pathways and perturbations in calcium signalling pathways due to hepatic lipotoxicity, elevated hepatic oxidative stress and ER stress, which eventually leads to increased inflammation, cell death and hepatic fibrogenesis^78,103–105. The KEGG pathway analysis also showed the enrichment of ECM-receptor interaction and cytokine-cytokine receptor interactions in our dataset, which reflect the release of proinflammatory cytokines upon PA-induced injury leading to the activation of hepatic stellate cells (HSCs) thus initiating liver fibrosis. These findings are in parallel with previous reports which suggests that the release of pro-inflammatory cytokines like TGFβ and IL17 and potent chemokine like CXCL10 further results in activation of profibrogenic gene expression and recruitment of inflammatory cells like macrophages and neutrophils exacerbating the liver damage^{104,106–111}. Other significant pathways included steroid biosynthesis, arachidonic acid metabolism, sphingolipid metabolism, and cell cycle. Some of these pathways have also been reported in previous studies to be linked with MAFLD¹¹².

In addition to the revalidation of genes already implicated in the context of MAFLD pathogenesis, our study also unravelled some novel gene candidates which showed marked dysregulation in their expression patterns upon consumption of PA-enriched diet. Highly upregulated genes included—(i) inhibin subunit alpha (inha, log2fold change 19.898), belonging to the TGF-β superfamily of proteins; (ii) ciliary neurotrophic factor receptor (cntfr, log2fold change 4.106), a member of type 1 cytokine receptor family; (iii) cyclin B1 (ccnb1, log2fold change 7.972), known to show increased expression in HCC patients; (iv) transcription factors including LIM homeobox transcription factor 1, beta a (lmx1ba, log2fold change 7.768); and (v) myocyte enhancer factor 2b (mef2b, log2fold change 7.291)¹¹³. Genes that showed prominent downregulation included—(i) procollagen, type V, alpha 1 (col5a1, log2fold change − 4.024); (ii) calcitonin/calcitonin-related polypeptide alpha (calca, log2fold change − 5.031); and (iii) 5-oxoprolinase, ATP hydrolsying (oplah, log2fold change − 1.93), to name a few. Col5a1 has formerly been identified as a basement membrane associated gene involved in the pathogenesis and progression of MAFLD¹¹⁴. Calca that functions in maintaining calcium and chloride ion homeostasis has been implicated in cholestatic liver injury^115,116 while Oplah is involved in glutathione metabolism and shows lower expression in MASH^116,117. Our study therefore provides a list of candidate genes that could be explored further from the perspective of MAFLD pathophysiology. Such investigations will help elucidate the molecular mechanisms that govern the development and progression of this complicated multifaceted disease.

In conclusion, the development of the palmitic acid (PA)-supplemented zebrafish model represents a significant advancement in our ability to study MAFLD in a relevant in vivo system. The transcriptomic profiling performed on this model not only reinforces the complex nature of hepatic lipid metabolism but also opens avenues for future research aimed at unravelling the molecular underpinnings of this increasingly prevalent disease. This study’s cross-species comparison with high fructose diet-induced MAFLD mouse models and human MAFLD transcriptomics demonstrates the conserved mechanisms of lipid metabolism and liver damage across species. These findings validate the clinical relevance of our zebrafish model and suggest its utility in studying the impact of dietary interventions and therapeutic strategies for MAFLD. By highlighting the similarities between the human condition and the PA-fed zebrafish model, our research emphasizes the importance of cross-species comparisons in translating preclinical findings to human treatments, ultimately advancing our understanding and management of MAFLD.

Methods

Zebrafish care and feeding regime

The animal studies were conducted in compliance with the guidelines and protocols approved by the Institutional Animal Ethics Committee (IAEC) of IISER Mohali. All procedures followed relevant guidelines and regulations and efforts were made to minimize suffering. This study adheres to the ARRIVE guidelines. Wild type adult zebrafish (6–8 months old) were bought from the local vendor and maintained at 28.5 ℃ with a 14-h light/ 10-h dark cycle, with the fish being fed during the day cycle. After a 3-week acclimatization period, the fish were weighed, sex-matched and randomly separated into 2 groups (n = 20*2). Approximately 10 fish were housed in 3L tanks containing system water. The control group was given tetramin flakes (46% crude protein, 11% crude fat and 3% crude fibre; Tetra GmbH, Germany). A PA-supplemented tetramin diet was prepared for the test group by dissolving 7% (w/w) palmitic acid (Cat No. 57-10-3; SRL) in diethyl ether (Cat No. 60-29-7; SRL), which was then completely evaporated in a fume hood. The control group was fed with 21.6 mg tetramin diet/feed/fish twice daily, while the test group was overfed with 20 mg PA-rich tetramin diet/feed/fish four times daily. This over-feeding protocol was followed for a period of 8 weeks.

For all body weight measurements, the fish were first anesthetized for 45-60 seconds using 0.02% (w/v) MS222 (3-amino benzoic acid ethyl ester, also called ethyl 3-aminobenzoate), commonly referred to as tricaine (cat no. TRIC-M-GR-0010). The body weight measurements were taken before the start and after the completion of the protocol.

Annexin V staining

Degree of apoptosis in liver tissue samples upon PA-induced hepatic injury was evaluated by staining with annexin and propidium iodide (PI) dyes followed by flow cytometry. Annexin is known to selectively stain early apoptotic cells, while propidium iodide stains late apoptotic or necrotic cells based on plasma membrane integrity and permeability. Liver tissue dissected from adult fish was initially incubated in trypsin (Cat. No. 25-200-056, Gibco) containing cell culture media (Dulbecco’s modified eagle media, DMEM, cat. No. 11965092, Gibco) without fetal bovine serum (FBS, Cat, No. 10270106, Gibco) for 15 min at 37 ℃. Trypsin was then neutralized by adding 1.5 mL of DMEM with 10% FBS, followed by vigorous pipetting to ensure complete homogenization of the tissue sample. The cell-suspension was then centrifuged at 1500 rpm for 5 min followed by resuspension in sterile phosphate-buffered saline (1 × PBS). The resuspended cell suspension was then washed twice in 1 × PBS followed by staining with Annexin and PI staining with the Annexin V apoptosis detection kit (Cat No. 88-8005-72; Invitrogen). A control liver sample resuspended in the provided binding buffer was used as the unstained control for this assay.

ROS assay

DCFH-DA (2′,7′-Dichlorodihydrofluorescein diacetate, Cat No: D6883-50 mg, Sigma) is a cell permeable fluorescent dye, which when acted upon by intracellular ROS is oxidized to fluorescent DCF. The fluorescent intensity, which is proportional to intracellular ROS levels, was measured at 484 Ex/535Em. For this, liver tissue dissected from adult fish was initially incubated in trypsin (Cat. No. 25-200-056, Gibco) containing cell culture media (Dulbecco’s modified eagle media, DMEM, cat. No. 11965092, Gibco) without fetal bovine serum (FBS, Cat, No. 10270106, Gibco) for 15 min at 37 ℃. Trypsin was then neutralized by adding 1.5 mL of DMEM with 10% FBS, followed by vigorous pipetting to ensure complete homogenization of the tissue sample. The cell-suspension was then centrifuged at 1500 rpm for 5 min followed by resuspension in sterile phosphate-buffered saline (1 × PBS) thrice. This was followed by incubation with 20 μM solution of DCFHDA dye for 30 min in dark. The cell suspension was then centrifuged and the pellet so obtained was resuspended in 1 × PBS. 100 μl of this cell suspension was plated on a 96-well black bottom plate followed by measurement of fluorescence reading.

Immunoblotting

Dissected livers were lysed in commercial Cell Lysis Buffer II (Cat. No. FNN0021; Invitrogen) containing protease inhibitor, followed by two consecutive centrifugations at 14000 rpm for 20 min and 10 min respectively. Equal amount of protein (70 μg) was then mixed with 1 × Laemelli buffer and heated at 95 ℃ for 7 min. Following SDS PAGE , the proteins were transferred onto nitrocellulose membrane at 70 V for 150 min. Blocking was performed with 5% BSA + 5%FBS solution in 1X tris-buffered saline (TBS) containing 0.1% (v/v) tween-20 (TBST) for 2 h. Blots were probed with primary antibody overnight at 4 ℃ on a shaker. Primary antibodies used were mouse anti-PCNA (1:2000 dilution, Cat. No. sc25280, santa cruz), rabbit anti-Sirt1 (1:2000 dilution, Cat No: sc-15404, santa cruz), mouse anti-SREBP1c (1:1000 dilution, Cat No:), rabbit anti-PGC1α(1:1000 dilution, Cat No: PA1-31,202, Invitrogen), mouse anti-OXPHOS (1:5000 dilution, Cat No:45-8099, Invitrogen) rabbit anti-C/EBPA (1:1000 dilution, Cat. No:22955, Cell Signalling Technology) mouse anti-β-actin (1:5000 dilution, Cat. No. 01171591, Invitrogen)-.The following day, blots were washed with 1 × TBST thrice and incubated with HRP- conjugated secondary antibody (goat anti-mouse (Cat. No. E-AB-1001, Elabscience), goat anti-rabbit (Cat. No:A120-201P, Bethyl Laboratories)) at room temperature for 2 h at a dilution of 1:5000. The blots were then developed using the SuperSignal West Pico PLUS Chemiluminescent Substrate (Cat. No. 34580, Thermo Scientific) in Vilber Gel Documentation System. Densitometric analysis was done using Quantity One (Bio-Rad, v.4.6.9) software.

Liver histology and staining

Liver tissue samples were fixed in 4% paraformaldehyde (PAF) solution for 4 h at 4 ℃ followed by paraffin embedding. Histological assessments of the liver tissue were done by hematoxylin and eosin (H&E) staining on 4 μm thick liver paraffin sections. Dewaxing of the paraffin sections were done by successive heat and xylene treatments. The sections were then rehydrated by gradient ethanol washes, followed by washing in ultrapure water. This was followed by staining with hematoxylin for 5 min. Subsequently, the sections were washed first with water, then with acid-alcohol and then again with water before being counterstained with 0.25% eosin for 90 s. Further dehydration was performed by washing with increasing concentrations of ethanol. Lastly the sections were washed in xylene and then mounted for imaging.

Oil Red O (ORO) staining and BODIPY staining

Hepatic lipid accumulation was examined by Oil Red O and BODIPY staining, both of which selectively stains only neutral lipids. For adult model, the liver tissue was fixed overnight in 4% paraformaldehyde solution at 4℃, following which, the liver tissue samples were subjected to gradient washes with 5% and 20% sucrose solutions, and OCT embedding. The samples were stored in -80℃ until sectioning. For Oil Red O staining, the cryosections of 8 μm thickness were first air-dried, fixed in 4% paraformaldehyde for 10 min at room temperature and then subjected to quick washes with distilled water and 60% isopropanol for 1 min each. The sections were then stained with freshly prepared Oil Red O solution (SRL Cat. No. 23576) for 20 min. This was followed by quick rinses with 60% isopropanol and water and then co-staining with hematoxylin for 1 min. Finally, the sections were washed with water to remove excess stain and mounted for imaging. This stain colours the lipid droplets in red and the nuclei in blue. Images were captured using the DEBRO DIM 150, inverted microscope. For BODIPY staining, cryosections of 5 μm thickness, were air dried, followed by fixation with 4% paraformaldehyde for 10 min at room temperature. The slides were then washed with 1 × PBS thrice for 5 min each and then stained with 10 μM BODIPY stain (diluted in PBS) for 30 min in dark. Excess stain was washed away with 1 × PBS (thrice, 5 min each) followed by counterstaining with DAPI (1 μg/mL in 1 × PBS) in dark for 5 min. Finally, the sections were washed with PBS and mounted in Mowiol. Imaging was done using Confocal Leica DMi Microscope. The lipid droplets appeared in green while the nucleus was stained in blue. Quantification of Oil Red O stained and BODIPY stained control and PA-fed sections were done using ImageJ software³¹.

Quantitative real-time PCR

Total RNA was isolated from liver tissue using Trizol method with the RNeasy lipid tissue mini kit (Cat. No. 74804; Qiagen). 1 μg of the isolated RNA was used for cDNA synthesis using the PrimeScript 1^st strand cDNA synthesis kit (Cat. No. 6110A, TaKaRa). All quantitative real-time PCR (qPCR) reactions were performed in the thermal cycler (CFX96 real-Time system; Biorad) using 100 ng cDNA and iTaq Universal SYBR® Green Supermix (Cat. No. 1725124; Biorad). For all the qPCR reactions, Glyceraldehyde 3-phosphate dehydrogenase (gapdh) was used as the housekeeping gene and the relative fold change was calculated using the delta-delta Ct method.

Staining for ER stress

4 μm thick paraffin embedded tissue samples were used for staining. 5 μM NBD-Oct dye solution was prepared in pH 7.4 PBS for staining¹¹⁸. The formalin fixed paraffin embedded (FFPE) tissue sections were subjected to gradient dewaxing, followed by staining with 200 μl of the dye solution. The sections were incubated for 30 min at room temperature and were subsequently washed with PBS and then mounted. Images were captured using an Olympus FV3000 confocal laser scanning microscope with PLAPON40XO (1.25 NA, 0.3 mm WD) objective. The image processing was done with the help of cellSens Dimension v 3.2 (Olympus) and ImageJ 1.54f. For fluorescence imaging, a 488 nm excitation laser was used and the emission window was kept at 500–540 nm. The confocal aperture was kept at 1.0 Airy Disk (AU) while the dwell time was 4 μs/pixel. The laser power, gain, and offset were kept the same for all image acquisitions.

Calorie restriction paradigm

Fish were subjected to calorie restriction for a period of 4 weeks after overfeeding with PA-supplemented diet for 8 weeks. The calorie restriction group (n = 10) was given 21.6 mg Tetramin diet/feed/fish once every day. Effects of calorie restriction on body weight, hepatic fat accumulation, and gene expression patterns were evaluated.

Whole genome transcriptomic profiling

Total RNA was isolated from two control and three PA-supplemented diet fed livers following the manufacturer’s protocol using the RNeasy lipid tissue mini kit (Cat. No. 74804; Qiagen). The quality of the RNA so extracted was assessed by agarose gel electrophoresis and evaluating their RNA integrity number (RIN) scores. Only samples with RIN values above 7 were processed further with Illumina NovaSeq 6000 sequencing platform. Raw data were cleaned using Trim Galore (v0.6.4) (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and were checked for quality using FastQC (v0.11.8) (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). STAR aligner (v2.7.9a) was deployed to align the reads to the zebrafish reference genome GRCz11 (danRer11) once they had passed quality control. After mapping, transcripts were counted with the feature Counts (v2.0.3) tool^119,120. Normalization of the read counts, as well as differential expression analysis, were performed by using DESeq2 package (v1.42.0) of Bioconductor. To find differentially expressed genes (DEGs), Wald tests were performed on DESeq2 for the MAFLD vs control comparison. Genes were marked as upregulated for the log2 fold change > = 1.0 and downregulated for the log2fold change <  = − 1.0 with q value < 0.05. R package clusterProfiler (v4.10.0) was used for Gene set enrichment analysis (GSEA) GO as well as for KEGG pathway enrichment analyses (www.kegg.jp/kegg/kegg1.html)^121,122 . For protein–protein interaction (PPI) analysis database STRING was utilised (https://string-db.org/)¹²³.

Statistical analysis

All experiments have been performed with at least 3 biological replicates. Data have been represented as mean ± SEM and p values have been calculated using two-tailed Student’s t-test using GraphPad Prism Software (https://www.graphpad.com/quickcalcs/ttest1/), wherein p < 0.05 is statistically significant.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Abbreviations

CR

Calorie restriction

DEG

Dysregulated genes

ECM

Extracellular matrix

ER

Endoplasmic reticulum

GSEA

Gene set enrichment analysis

GO

Gene ontology

MAFL

Metabolic dysfunction-associated fatty liver

MASH

Metabolic dysfunction-associated steatohepatitis

PA

Palmitic acid

UPR

Unfolded protein response

Author contributions

The experiments were conceived and designed by S.M., and J.K.D. The experiments were performed by D.B., Sh.K., B.C., A.R., T.D., H.S., and A.L.K. Sa.K., J.K.D., and S.M., analysed the data. S.M. and J.K.D. wrote the paper. All authors participated in the discussion of the data and in production of the final version of the manuscript.

Funding

SM was supported by a Start-Up Research Grant from the Science and Engineering Board (SRG/2021/000341), a Ramalingaswami re-entry fellowship from the Department of Biotechnology (BT/RLF/Re-entry/70/217), and IFCPAR/CEFIPRA (Indo-French Centre for Promotion of Advanced Research/Centre Franco-Indien pour la Promotion de la Recherche Avancée) grant no. 6503-J. J.K.D. was supported by Har Govind Khorana-Innovative Young Biotechnologist Award from the Department of Biotechnology (BT/13/IYBA/2020/07).

Data availability

Sequence data that support the findings of this study have been deposited in the INSDC with the primary accession code PRJEB83101 provided by EMBL-EBI European Nucleotide Archive (ENA).

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

The animal studies were conducted in compliance with the guidelines and protocols approved by the Institutional Animal Ethics Committee (IAEC) of IISER Mohali. All procedures followed relevant guidelines and regulations and efforts were made to minimize suffering. This study adheres to the ARRIVE guidelines.