Differential impact of high-fructose and ethanol diets on early steatohepatitis and hepatic melanocortin-4 receptor responses in rats

Abstract

BACKGROUND

Metabolic dysfunction-associated steatotic liver disease (MASLD) is increasingly driven by diets high in fructose and fat, often accompanied by alcohol consumption. The melanocortin-4 receptor (MC4R) has been implicated in energy homeostasis, yet its hepatic expression in response to dietary stress remains poorly investigated.

AIM

To investigate the effects of obesogenic diets, high-fat diet (HFD), a high-fructose diet (70% kcal) (HFrD) and HFD supplemented with both ethanol and fructose (HF-EFr) on appetite, metabolic outcomes, serum transaminases, and hepatic MC4R expression in male SD rats over 8-week.

METHODS

Hepatic MC4R expression was assessed by quantitative reverse transcriptase PCR, western blotting and immunostaining. Steatosis and fibrosis were evaluated histologically. Hepatocellular DNA-synthesis was quantified by Ki-67/HepPar-1 immunostaining.

RESULTS

HFrD feeding resulted in hyperphagia, accelerated weight gain, endocrine alterations (leptin and Lepr signaling, impaired insulin clearance, and increased fT3/fT4 ratio), hepatomegaly, and stage 2 fibrosis. HF-EFr-fed rats consumed fewer calories but exhibited pronounced hepatocellular DNA synthesis, elevated aspartate aminotransferase levels, endothelial activation (Pecam-1), increased MC4R expression, and higher relative liver weight. Both diets induced visceral white adipose tissue expansion, hepatic steatosis, and increased expression of lipogenic (Srebp-1c, LXR-α), fructose transporter (Glut5), pro-inflammatory (Il-1β, Cxcl-1), and profibrotic (Pai-1) genes, although the timing and magnitude differed between groups. Quantitative immunofluorescence analysis revealed a diet-induced shift in MC4R subcellular localization, with nuclear-associated recruitment increasing from 69% in controls to > 95% in HFrD and HF-EFr groups (P < 0.001), accompanied by a significant increase in mean fluorescence intensity.

CONCLUSION

High-fructose and ethanol-enriched diets promote early MASLD through complementary (simultaneous) mechanisms. HFrD primarily induces metabolic overload, steatosis, and endocrine dysregulation, whereas HF-EFr enhances hepatocellular stress, inflammatory signaling, and DNA synthesis. Hepatic MC4R expression and localization respond dynamically to these dietary challenges, suggesting a potential adaptive role for peripheral MC4R signaling during early steatohepatitis.

Key Words: Metabolic dysfunction-associated steatotic liver disease; Fatty liver; Inflammation; Fructose; Alcohol

Core Tip: Modern dietary habits often combine high-fructose beverages with alcohol, yet their distinct hepatic effects remain poorly defined. Using controlled rat dietary models, this study demonstrates that a high-fructose diet primarily induces hepatic metabolic substrate overload, endocrine dysregulation, visceral adiposity, and steatosis, whereas fructose combined with ethanol amplifies hepatocellular injury, inflammatory signaling, and regenerative activity. Multi-level analyses quantitative reverse transcriptase PCR, western blot, immunohistochemistry, and immunofluorescence] reveal dynamic hepatic melanocortin-4 receptor (MC4R) responses, including altered glycosylation and increased nuclear localization. These findings provide new insights into a potential peripheral adaptive role of MC4R in early steatohepatitis and highlight how distinct dietary stressors generate different pathogenic trajectories in metabolic dysfunction-associated steatotic liver disease.

INTRODUCTION

The global rise in metabolic dysfunction-associated steatotic liver disease (MASLD) closely parallels shifts in dietary behavior. In particular, the widespread consumption of energy-dense food products rich in saturated fat and refined sugars, most notably high corn fructose syrup (HCFS)[1]. While weight gain and hepatic lipid accumulation is a defining early feature of MASLD, disease progression is driven by additional pathological processes, including inflammation, hepatocyte injury, and DNA-synthesis. The transition from simple steatosis to metabolic dysfunction-associated steatohepatitis (MASH) ultimately increases the risk of fibrosis, chromosomal aberration, and hepatocellular carcinoma (HCC)[2,3].

Fructose has gained particular attention as a potent dietary inducer of metabolic liver injury. Unlike glucose, fructose is known to be primarily metabolized in the liver and bypasses key regulatory steps of glycolysis, thereby promoting unrestrained substrate flux toward de novo lipogenesis and triglyceride (TG) accumulation. Chronic fructose exposure has been shown to induce insulin resistance, oxidative stress, cytokines and chemokines, and inflammatory activation, all of which contribute to MASLD pathogenesis. Importantly, modern dietary patterns in adults rarely involve isolated fructose intake. Instead, fructose-rich processed foods and sugar-sweetened beverages are frequently consumed alongside alcoholic drinks, especially in Westernized societies. Ethanol represents the biologically active component of alcoholic beverages and exerts direct metabolic and hepatotoxic effects through acetaldehyde generation, oxidative stress, and disruption of not only lipid and glucose homeostasis[4], but also protein synthesis[5]. While certain alcoholic beverages may contain bioactive compounds with potential health benefits, ethanol itself accompanied by distillation products.

In this context, combining fructose and ethanol in experimental diets establishes a clinically relevant preclinical experimental model that more closely reflects contemporary dietary behaviors characterized by the concurrent consumption of energy-dense “junk food” and alcohol in real lifestyle. This approach enables investigation of synergistic or additive effects of these nutritional stressors on hepatic metabolism, inflammation, and regenerative responses, while minimizing confounding influences from non-ethanol constituents present in alcoholic beverages. Such combined dietary paradigms are increasingly recognized as informative for studying early pathogenic mechanisms of MASLD that may not be adequately captured by high-fat feeding alone.

Against this metabolic backdrop, the melanocortin system, and particularly the melanocortin-4 receptor (MC4R), has emerged as a central regulator of energy balance and metabolic homeostasis. MC4R has been extensively studied for its role in appetite control and body weight regulation, with genetic disruption of MC4R or its upstream precursor pro-opiomelanocortin (POMC) resulting in severe obesity in both humans and animal models[6]. Melanocortin peptides derived from POMC, including α-, β-, and γ-melanocyte-stimulating hormones (MSH), are produced primarily in the hypothalamus and anterior pituitary and exert their effects through MC4R signaling to tightly regulate food intake and energy expenditure[7].

MC4R activity is closely integrated with peripheral metabolic signals, particularly leptin, the adipocyte-derived hormone that conveys information about energy stores to the central nervous system. Under physiological conditions, leptin-mediated activation of hypothalamic MC4R suppresses appetite and promotes metabolic balance. However, in obesity and metabolic disease, leptin resistance disrupts this regulatory axis, contributing to hyperphagia and weight gain[8]. Beyond its central effects, emerging evidence suggests that melanocortin signaling also exerts anti-inflammatory actions. Notably, melanocortin peptides have been shown to attenuate inflammatory responses in peripheral tissues, including the liver, during acute-phase reactions in experimental models[9]. These observations raise the possibility that MC4R participates in broader metabolic and immunomodulatory processes beyond the central nervous system.

In recent years, attention has shifted toward potential peripheral roles of MC4R in metabolic organs. MC4R expression has been detected in extra-hypothalamic tissues, including the liver, although its functional significance in hepatocytes remains poorly defined[10]. Supporting a possible hepatic role, human genetic studies have linked MC4R variants to increased susceptibility to fatty liver disease, insulin resistance, and components of the metabolic syndrome[11]. Experimental evidence further strengthens this association: MC4R-deficient rodents develop severe hepatic steatosis accompanied by inflammation, fibrosis, and, in advanced stages, hepatocarcinogenesis[12]. These animals display distinctive histopathological features, including macrophage-rich crown-like structures surrounding injured hepatocytes, suggesting a role for MC4R signaling in regulating hepatic inflammation and tissue remodeling.

Diet-induced steatosis activates lipogenic transcriptional programs involving sterol regulatory element-binding proteins (SREBPs) and nuclear receptors such as LXR-α, while simultaneously engaging inflammatory pathways including NF-κB signaling[13,14]. How addition of ethanol to fructose-sweetened diet could influence hepatic MC4R expression is to be explored.

To address these gaps, the present study employed a preclinical model incorporating distinct obesogenic dietary regimens such as high fat, high fructose, and combined fructose-ethanol exposure, to examine their differential impact on hepatic metabolism and injury. We systematically assessed hepatic inflammation, hepatocellular injury, steatosis, fibrosis, and proliferative activity, together with MC4R expression and cellular localization. These hepatic outcomes were evaluated alongside endocrine and metabolic readouts, including thyroid hormone profiles and pancreatic markers, to capture integrated organ-level responses to dietary stress. Through this comparative approach, the study aims to clarify how fructose and ethanol differentially modulate hepatic injury and MC4R signaling during the early stages of MASLD.

MATERIALS AND METHODS

Materials

Fructose and skim milk were purchased from AppliChem; the standard chow and Lieber-DeCarli (LDC) diets, ssniff Spezialdiäten GmbH, Germany; Qiagen RNeasy Mini Kit, Qiagen GmbH, Hilden, Germany; Moloney murine leukemia virus reverse transcriptase (M-MLV RT), Promega, Mannheim, Germany; SYBRGreen master mix and stepOne software, AB, Applied Biosystems, Darmstadt, Germany; Complete Protease Inhibitor Cocktail Tablets, Roche, Mannheim, Germany; Hybond-ECL nitrocellulose membranes; Amersham Biosciences, Buckinghamshire, United Kingdom. ECL chemiluminescent solutions A and B western blotting protocol, GE Healthcare, United States; film processor machine, Konica SRX-101A, medical film processor; microtome, Microm HM325; Thermo Scientific, Walldorf, Germany; and 4,6-diamidino-2-phenylindole (DAPI) were from Molecular Probes Europe BV, Leiden, The Netherlands. Rabbit serum, Dako, Glostrup, Denmark; Alexa-555-conjugatedgoat secondary antibody (Molecular Probes, Leiden, The Netherlands), TGs were measured by BioAssay Systems, EnzyChrom^TM Kit, Hayward, United States. Protease inhibitors (Roche, Mannheim, Germany), protein A-agarose (Roche, Mannheim, Germany).

Experimental animals

Eleven-week-old male SD rats (140-150 g) were obtained from Charles River (Sulzfeld, Germany) and housed individually in standard cages. Animals had ad libitum access to chow pellets and water during a one-week acclimatization period. The housing environment was maintained under controlled conditions with a 12:12-hour light/dark cycle and a temperature of 22 °C ± 2 °C in the animal facility of the University Medical Center Göttingen (UMG), Georg August University of Göttingen, Germany. All animal experiments were conducted in accordance with the German Law for the Protection of Animals and institutional guidelines. The experimental protocol was reviewed and approved by the local animal welfare authorities of the UMG, Georg August University of Göttingen, and complied with the regulations of the State of Lower Saxony, Germany.

Animal feeding

Rats were randomly allocated into four experimental groups (n = 5 per group) and maintained on their respective diets ad libitum for four or eight weeks following a 3-day adaptation period. All liquid diets were formulated to be isocaloric (100% total energy), with macronutrient redistribution achieved by substituting carbohydrate-derived calories with fructose and/or ethanol as specified below. Energy calculations were based on standard conversion factors (9 kcal/g for fat, 4 kcal/g for carbohydrates, and 7 kcal/g for ethanol). Detailed macronutrient composition is summarized in Table 1.

Table 1 Diet composition.

Component (% kcal)	Control (chow, solid) (%)	HFD (liquid) (%)	HF-EFr (liquid) (%)	HFrD (liquid) (%)
Fat	13	35	35	18
Carbohydrate	58 (starch/complex)	47% (maltose/dextrin)	30 fructose	70 fructose
Protein	Approximately 29	Approximately 18	Approximately 15	Approximately 12
Ethanol (EtOH)	0	0	Approximately 30	0
Total kcal	100	100	100	100

HFD: High-fat diet; HF-EFr: High-fat plus ethanol and fructose; HFrD: High-fructose diet.

Control: Rats received standard chow pellets (solid diet) providing approximately 13% kcal from fat, 58% kcal from carbohydrate (primarily complex starch), and approximately 29% kcal from protein.

High-fat diet: Rats received the standard liquid LDC formulated diet, providing approximately 35% kcal from fat, 47% kcal from carbohydrate (maltose/dextrin), and approximately 18% kcal from protein[15].

High-fat plus ethanol and fructose: Rats received a modified LDC-based liquid diet in which part of the carbohydrate fraction was replaced with fructose (30% kcal) and ethanol (30% kcal), while maintaining 35% kcal from fat and 15% kcal from protein. Ethanol was incorporated directly into the liquid diet rather than administered separately in drinking water to ensure controlled caloric substitution and precise intake quantification. This formulation models combined metabolic and hepatotoxic stress characteristic of contemporary dietary patterns. The selected ethanol proportion (approximately 30% kcal) follows established experimental paradigms inducing early steatohepatitis without progression to advanced cirrhosis within the study duration.

High-fructose diet: Rats received a modified liquid diet providing 70% kcal from fructose, 18% kcal from fat, and approximately 12% kcal from protein. This group primarily models fructose-driven metabolic overload and hepatic lipogenesis in the absence of ethanol exposure.

For liquid-fed groups [high-fat diet (HFD), high-fat plus ethanol and fructose (HF-EFr), and high-fructose diet (HFrD)], diets were freshly prepared each morning, including weekends, using a mechanical mixer to ensure homogeneity and consistency in nutrient distribution and palatability. The liquid diets were dispensed into specialized feeding bottles, allowing voluntary consumption through drinking spouts. Bottles were weighed prior to placement and again after 24 hours to determine daily intake. Consumed amounts were converted to kilocalories to allow comparison of daily energy intake across groups. The control group received solid chow pellets, which were replenished as needed, and intake was measured by weighing provided and remaining pellets.

Body weight was recorded at baseline and weekly throughout the study period.

Collection and processing of liver tissue and blood samples

At the end of dietary intervention (4 weeks and 8 weeks), animals were weighed and euthanized via intraperitoneal injection of sodium pentobarbital (Narcoren^®; 0.2 mL per 100 g body weight). Blood samples were collected from the inferior vena cava into plain tubes (for serum) and heparinized tubes (for plasma).

Livers were carefully dissected, weighed, and gently rinsed in physiological saline. Three sections from different liver lobes of each rat were immediately fixed in a 4% neutral-buffered formalin and 1% glutaraldehyde solution for formalin-fixed, paraffin-embedded (FFPE) tissue processing. FFPE samples were subsequently used for histopathological analysis. Additional liver tissue samples were flash-frozen in liquid nitrogen and stored at -80 °C for later molecular and biochemical analyses.

To assess liver enlargement (hypertrophy) relative to body size, the relative liver weight (RLW) was calculated as the percentage of liver weight to body weight at the time of sacrifice, using the formula:

RLW (%) = [liver weight (g)/body weight (g)] × 100

RNA extraction and quantitative reverse transcriptase PCR analysis

RNA from liver tissues was isolated by Qiagen RNeasy Mini Kit. 1.0 µg of total RNA was reversed using viral reverse transcriptase. Primers (Supplementary Table 1), which were checked for potential hairpin formation and potential self-annealing, were synthesized by Invitrogen and analyzed using a Fast Platinum SYBR^® Green Universal master mix. Ubiquitin C and β-actin served as housekeeping genes in this model. Each sample was loaded in duplicate in the PCR-96 microplate. The comparative CT method was used to determine the amount of target gene, normalized to the housekeeping genes and relative to a calibrator (2^-ΔΔCT).

Protein isolation and western blot analysis

To investigate protein expression changes in response to dietary interventions, total protein was extracted from both rat liver tissues.

Protein extraction

Tissue samples were homogenized in an ice-cold lysis buffer containing 150 mmol/L NaCl, 20 mmol/L Tris-HCl (pH 7.5), 10% (v/v) glycerol, 1% (v/v) NP-40, 1 mmol/L MgCl₂, and 1 mmol/L CaCl₂. The buffer was freshly supplemented with Complete™ Protease Inhibitor Cocktail Tablets (Roche) and phenylmethanesulfonyl fluoride (100 μg/mL) to prevent degradation of proteins. Protein concentrations were routinely determined in duplicate using the Bradford assay.

Western immunoblotting

Equal amounts of total protein (75 μg per sample) were separated by SDS-PAGE using 4%-12% polyacrylamide gradient gels, then electrotransferred onto Hybond-ECL nitrocellulose membranes (GE Healthcare). Equal loading and transfer efficiency were initially verified/normalized by Ponceau S staining. Membranes were blocked for 2 hours at room temperature (RT) in 5% (w/v) skim milk prepared in phosphate buffered saline (PBS) with 0.1% (v/v) Tween-20 (PBST).

Subsequently, membranes were incubated overnight at 4 °C with the appropriate primary rabbit antibody under gentle agitation in a humidified chamber. Following three washes with PBST, membranes were incubated for 1 hours at with HRP-conjugated secondary antibodies. After additional washing steps, immunoreactive bands were visualized using enhanced chemiluminescence detection reagents (Solutions A and B, Thermo Fisher), according to the manufacturer’s protocol.

Protein bands were visualized on X-ray films and quantified by densitometric analysis using ImageJ software (version 1.46). β-actin was used as the loading control for normalization of target protein expression. Target protein signals were normalized to the corresponding β-actin band intensity for each lane to control for loading variability. All Western blot experiments were independently performed several times.

The polyclonal rabbit anti-rat MC4R antibody (Abcam) used in this study has been previously validated in rat liver tissue by independent groups demonstrating specific detection of hepatic MC4R at both mRNA and protein levels, including cytoplasmic and nuclear localization during acute-phase response and liver regeneration models[9,16]. The observed bands at approximately 55 kDa and 37 kDa correspond to previously reported glycosylated and core unglycosylated forms of MC4R in rodent tissues.

Plasma/serum study

After week 4 and week 8 of feeding, fasting blood samples were collected from rats and centrifuged at 3500 × g for 15 minutes at 4 °C. Heparinized blood was used for the plasma study the activities of transaminases and pancreatic amylase, and determination of glucose, uric acid, total cholesterol, TG, and high-density lipoprotein-cholesterol (HDL-C) levels according to clinical manufacturer protocol. Fasting serum levels (ng/mL) of leptin, insulin, T3, and T4 were measured by radioimmunoassay. Serum C-peptide levels were determined by ELISA. The Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) was calculated according to the formula:

[fasting glucose (mg/dL) × fasting insulin (μIU/L)]/405

Determination of TG content in the liver

Frozen liver samples (100 mg) were homogenized in ice-cold 2 × PBS using a Tissuelyser. Tissue lipids were extracted with 2:1 methanol/chloroform, vacuum dried, and resuspended in 5% fat-free bovine serum albumin. TG contents were colorimetrically determined using a commercially available kit. Values were normalized to the initial wet weight of liver portion and expressed as mg TG/g liver.

TG content (mg/g) = [measured conc (mg/mL) × homogenate volume (mL)]/tissue weight (g).

Histological studies

Nile red staining for hepatic TG: To visualize intracellular neutral lipid droplets, enabling assessment of lipid accumulation in 5 µm thick hepatic cryosections. After fixation with paraformaldehyde and washing in PBS, nuclei were counterstained using DAPI (5 minutes). Fat droplets were then stained by immersing the sections in a warm mixture of Kaiser’s glycerol gelatin, ddH₂O, and Nile Red solution. After cover-slipping and briefly drying the slides in the dark, stained lipids were viewed using an epifluorescence microscope (excitation 450-560 nm, emission 590 nm), and representative images were acquired.

Masson’s Trichrome staining: Masson’s trichrome was applied to 5 µm-thick liver sections, following the manufacturer’s instructions. This technique allowed us to evaluate collagen deposition (fibrosis), overall tissue architecture, and the presence of fat vesicles which appear as variably sized, empty white spaces. The degree of fibrosis was evaluated according to the established Desmet & Scheuer system (0-4): 0 = none, 1 = minimal portal fibrosis, 2 = mild with early lobular extension, 3 = moderate with incomplete/complete septa, 4 = numerous septa, indicating transition to cirrhosis.

Immunofluorescent staining: We performed dual immunofluorescence (IF) to accurately localize the MC4R within hepatocytes, using the cytoplasmic marker HepPar-1 for identifying hepatocytes.

Liver cryosections (4 μm) prepared from flash-frozen samples were fixed in cold methanol for 9 minutes followed by acetone for 1 minute at -20 °C. To block non-specific binding and permeabilize the membranes, sections were incubated for 1 hour at RT with a blocking solution (5% normal goat serum in 0.05% PBST).

The primary antibodies, a polyclonal rabbit anti-MC4R antibody and a mouse anti-HepPar-1 antibody, were then applied, and the sections were incubated overnight at 4 °C. After washing five times in PBST (5 minutes each at RT), sections were incubated for 1 hours at RT in the dark with the corresponding fluorophore-conjugated secondary antibodies: Alexa Fluor^® 555 goat anti-rabbit IgG (red) for MC4R and Alexa Fluor^®488 goat anti-mouse IgG (green) for HepPar-1. Nuclei were counterstained with DAPI (5 minute, RT, dark), followed by three washes in PBS.

Slides were mounted using Fluoromount-G (Southern Biotech, anti-fade) and coverslipped. Images were captured using an Axiovert 200M epifluorescence microscope (Zeiss, Jena, Germany). The HepPar-1 staining delineated the hepatocyte cytoplasm, facilitating the visualization of MC4R subcellular localization, particularly in relation to the diet-induced cytoplasmic lipid accumulation.

Quantification of MC4R expression

For semi-quantitative analysis of MC4R subcellular localization, digital fluorescence images were analyzed using automated segmentation protocols. Hepatocyte nuclei were identified based on DAPI staining, and the total hepatocyte population per image was determined by automated nuclear counting. To evaluate nuclear recruitment of MC4R, the percentage of MC4R-positive nuclei was calculated by identifying hepatocytes in which the MC4R signal (Alexa Fluor^® 555) overlapped with or was immediately adjacent to the nuclear compartment.

Mean fluorescence intensity (MFI) was measured to estimate relative receptor density. To exclude non-parenchymal areas (e.g., vascular lumina or clear vacuolar spaces resulting from lipid extraction during tissue processing), a tissue mask was applied during image analysis. MFI was quantified specifically within MC4R-positive puncta using a fixed intensity threshold (40 arbitrary units, A.U.) to distinguish specific immunoreactivity from background signal.

Assessment of hepatocellular DNA-synthesis

Hepatocellular proliferative activity was assessed by immunohistochemical staining for nuclear proliferation marker Ki-67). Ki-67 Labelling index was quantified across eight non-overlapping high-power fields HPF) for each animal. The result was expressed as the percentage of positive nuclei Ki-67 positive nuclei/total nuclei × 100). Crucially, quantification was performed by two independent scientists blinded to the treatment groups.

Immunohistochemistry for detecting MC4R

Immunohistochemical localization of MC4R was performed on 5 μm FFPE liver sections using a standard peroxidase-based detection method. We employed this technique to obtain a high-resolution, conventional brightfield overview of MC4R expression throughout the entire tissue and to clearly visualize its relationship with the large, unstained macrovesicular lipid accumulation evident in the cytoplasm of the treated animals. 5 μm FFPE sections were first deparaffinized in xylene and rehydrated through graded ethanol washes. Antigen retrieval was performed by microwaving the slides 10 mmol/L citrate buffer (pH 6.0) for 5 minutes at 95 °C.

Endogenous peroxidase activity was quenched using a freshly prepared glucose oxidase-peroxidase solution (0.18 g glucose, 0.005 g glucose oxidase, 100 μL 1M sodium azide in 100 mL PBS) for 10 minutes at 37 °C. The sections were then incubated in a commercial peroxidase blocking solution for 30 minutes at RT in the dark. After three washes with PBS, non-specific binding sites were blocked with goat serum for 1 hour at RT. Sections were incubated overnight at 4 °C with a rabbit polyclonal anti-rat MC4R antibody (1:50, diluted in PBS containing 5% Triton X-100. The following day, slides were incubated for 1 hour at 37 °C with the HRP-conjugated goat anti-rabbit secondary antibody, supplemented with (5 μL rat serum and 300 μL γ-globulin-free serum). The signal was visualized after washing by applying DAB (50 mg/100 mL PBS with 33 μL H₂O₂) until a brown reaction product developed. Slides were rinsed in PBS, counterstained with hematoxylin for 45 seconds, washed in tap water, and coverslipped using pre-warmed Kaiser’s glycerin gelatin mountinga medium (65 °C).

Statistical analysis

Data from all groups were compared with control using ANOVA, followed by post Tukey’s post-hoc test. The values were presented as mean ± SEM, and were considered statistically significant when P < 0.05.

RESULTS

Diet-induced changes in energy intake, growth dynamics, and liver weights

Rats were fed chow pellets (control), HFD which is also called LDC diet, HF-EFr, or a modified LDC with high-fructose (70%) diet (HFrD) up to 8 weeks.

Macroscopic intra-abdominal appearance of visceral organs and adipose tissue following dietary intervention

Control animals exhibited a normal abdominal organ arrangement with minimal visceral adipose tissue and preserved intestinal translucency (Figure 1A). HFD-fed rats showed increased visceral fat mass, predominantly within storage-associated depots, accompanied by moderate hepatomegaly. In contrast, HF-EFr animals displayed relatively less overall adiposity but a conspicuous expansion of mesenteric and perivisceral fat with increased vascular prominence. HFrD-fed rats demonstrated marked visceral adiposity, particularly involving epididymal and mesenteric fat depots, together with evident hepatomegaly. Liver color was pale and glossy.

Figure 1 Differential effects of fructose-enriched diets on energy intake, growth dynamics, and liver hypertrophy. A: Representative gross photographs of the abdominal cavity from male rats after 8 weeks of dietary intervention. Yellow arrows point at the liver, black arrows point at the mesenteric white adipose tissue (WAT), and the blue arrows point at the epidydimal WAT; B: Cumulative weekly caloric intake (kcal/rat/week) measured over the 8-week feeding period, highlighting the hyperphagia in the modified liquid Lieber-DeCarli (LDC) diet with high 70% kcal fructose (HFrD) group; C: Absolute body weight (g) measured weekly, illustrating the delayed growth but accelerated weight gain trajectory of the HFrD group, and the attenuated body weight gain of the high fat plus ethanol 30% and 30% fructose group; D: Absolute liver weight (gram); E: Relative liver weight (RLW, %), reflecting diet-associated hepatomegaly, measured at weeks 4 and 8. The control group was given chow pellets; other groups received lipid diet in bottles, high-fat diet (standard LDC diet). ^aP < 0.05 vs control, ^bP < 0.05 vs HFrD. Statistical analysis was performed using two-way ANOVA with Tukey’s post hoc test unless otherwise indicated. Data are expressed as mean ± SEM (n = 5/group). HFD: High-fat diet; HF-EFr: High-fat plus ethanol and fructose; HFrD: High-fructose diet.

Energy intake and body weight progression

Cumulative caloric intake differed markedly among groups (Figure 1B). HFrD rats consumed the highest total calories (6160 ± 340 kcal), particularly between weeks 4-8, whereas HF-EFr rats showed the lowest intake (4850 ± 160 kcal), comparable to controls (4950 ± 85 kcal). Baseline body weights were similar across groups (161-175 g). By week 8, control and HFD rats reached comparable final weights (488 g and 482 g), while HF-EFr rats exhibited the slowest growth and lowest final weight (432 g; P < 0.05 vs all groups) Figure 1C. In contrast, HFrD rats showed marked hyperphagia-driven growth, with the greatest interval weight gain between weeks 4 and 8 (163 ± 5 g), reaching 465 g. Interval analysis (Table 2) confirmed that HF-EFr rats gained the least total weight over 8 weeks (257 ± 6 g; P < 0.05 vs all groups).

Table 2 Cumulative food intake (kcal) and body weight gain (g) during different experimental phases.

Group	Food intake (kcal)			Body weight gain (g)
	Week 0-4	Week 4-8	Total (0-8 week)	Week 0-4	Week 4-8	Total (0-8 week)
Control	2240 ± 40	2720 ± 42	4950 ± 85	226 ± 4	98 ± 3	324 ± 6
HFD	2612 ± 75	2710 ± 70	5320 ± 120	205 ± 4	104 ± 2	309 ± 4.5
HF-EFr	2340 ± 140	2540 ± 80	4850 ± 160c	173 ± 3a	84 ± 7a	257 ± 6a,b,c
HFrD	2790 ± 160	3370± 180	6160 ± 340a	141 ± 5a	163 ± 5a	304 ± 8

^aP < 0.05 vs corresponding period of the control group.

^bP < 0.05 vs high-fat diet group.

^cP < 0.05 vs corresponding period of the high-fat plus ethanol and fructose group.

This table summarizes total caloric intake and net body weight gain for each diet group during three intervals: Weeks 0-4, 4-8, and the full 8-week period (0-8 weeks). Values should be expressed as mean ± SEM (n = 5). HFD: High-fat diet; HF-EFr: High-fat plus ethanol and fructose; HFrD: High-fructose diet.

Absolute and relative liver ratios

Absolute liver weights increased from week 4 to week 8 in all groups (Figure 1D). At week 8, HF-EFr (17.5 g) and HFrD (18.2 g) rats exhibited significantly heavier livers than control (14.3 g) and HFD (15.5 g) rats (P < 0.05). Consistently, relative liver weight (RLW; Figure 1E) was already elevated at week 4 in HF-EFr (4.0%) and HFrD (4.4%) rats compared with control (3.3%) and HFD (3.6%), indicating early hepatomegaly. By week 8, RLW remained high in HF-EFr rats (4.1%), whereas it modestly declined in HFrD rats (3.9%), likely reflecting dilution by rapid body weight gain. RLW decreased to 2.9% and 3.2% in control and HFD rats, respectively.

These findings demonstrate distinct patterns of visceral fat distribution and liver enlargement across dietary groups.

Serum lipid profile

As shown in Supplementary Figure 1, fructose- and ethanol-containing diets induced pronounced dyslipidemia. At week 4, HF-EFr rats exhibited marked elevations (231 mg/dL vs control 35 mg/dL) in serum TGs (Supplementary Figure 1A) and total cholesterol levels (77 mg/dL vs control 55 mg/dL, Supplementary Figure 1B) compared with all other groups (P < 0.05). By week 8, TG levels remained elevated in both HF-EFr and HFrD groups, with the HFrD group showing a significant time-dependent increase.

HDL-C levels (Supplementary Figure 1C) were unchanged across groups at week 4; however, by week 8, HFrD rats exhibited a significant reduction (36 mg/dL) compared with all other groups (P < 0.05), indicating impaired lipid quality rather than isolated hyperlipidemia.

Serum markers for organ ‘‘stress’’

Serum alanine aminotransferase (ALT) activity was comparable among all experimental groups at both assessed time points, with the exception of the HF-EFr group at week 4 (Supplementary Figure 1D). At this time point, ALT levels were significantly elevated in HF-EFr rats (61 U/L) compared with control animals (41 U/L) and all other dietary groups (P < 0.05), indicating an early hepatocellular response to combined fructose and ethanol exposure.

In contrast, serum aspartate aminotransferase (AST) levels showed a delayed elevation. At week 8, AST activity was significantly higher in the HF-EFr group (87 U/L) compared with controls (73 U/L) (P < 0.05; Supplementary Figure 1E), whereas no significant differences were observed at earlier time points. At week 8, the AST/ALT ratio was higher in HF-EFr fed rats (1.75) compared with controls (1.50).

Chronic fructose loading (HFrD) induced a significant increase in serum pancreatic amylase activity by week 8 (2425 U/L) compared to the control group (1482 U/L). This elevation exceeded all other experimental groups (P < 0.05; Supplementary Figure 1F), indicating altered pancreatic enzyme activity under high-fructose load.

Histopathological assessment of hepatic lipid accumulation and fibrosis

To evaluate the morphological consequences of the dietary interventions, liver sections were subjected to Nile Red staining for neutral lipids and Masson’s trichrome staining for collagen deposition and overall tissue architecture at weeks 4 and 8 (Figure 2).

Figure 2 Hepatic steatosis and progressive fibrosis characterization by Nile red (left panel), Masson’s trichrome (right panel) at weeks 4 and 8, respectively, and quantification of hepatic triglyceride content. A and B: Control rats micrographs show preserved hepatic architecture without lipid accumulation or fibrosis; C and D: High-fat diet group micrographs display diffuse micro- and medio-vesicular steatosis with minimal collagen deposition; E and F: High-fat plus ethanol and fructose (HF-EFr) group micrographs exhibit abundant lipid droplets of variable sizes, with increased portal and perisinusoidal collagen deposition extending into adjacent lobular regions, consistent with stage 2 fibrosis; G and H: Lieber-DeCarli high-fructose diet (HFrD) group micrographs demonstrate marked macro-vesicular steatosis, mainly in the periportal (portal vein) region accompanied by stage 2 fibrosis; Micrographs were acquired at × 20 magnification; I: × 5 magnification of Nile red stain of HFrD rats fed for 2 weeks; J: Biochemical quantification of hepatic triglyceride (TG) content (mg TG/g liver homogenate) at weeks 4 and 8. ^aP < 0.05 vs control. TG content in the liver HF-EFr at week 8 is significant over all groups. HFD: High-fat diet; HF-EFr: High-fat plus ethanol and fructose; HFrD: High-fructose diet.

At week 4, control rat livers maintained normal architecture with well-organized hepatocyte cords and lacked detectable lipid droplets (Figure 2A). In contrast, HFD-fed rats demonstrated diffuse microvesicular-to-mediovesicular steatosis, predominantly located in periportal regions and extending toward zone III (Figure 2C). In the HF-EFr group, the combined dietary regimen resulted in widespread lipid droplets of varying sizes scattered across both periportal and pericentral zones (Figure 2E). The HFrD produced the most pronounced early injury, characterized by predominantly macrovesicular steatosis with scattered microvesicles, mainly localized to zones I–II and detectable as early as week 2 (Figure 2G and I).

Assessment of collagen deposition revealed progressive architectural remodeling by week 8 in fructose-exposed groups, occurring in parallel with persistent steatosis. While control and HFD livers displayed minimal collagen deposition with no evidence of fibrosis (Figure 2B and D), both HF–EFr and HFrD livers exhibited stage 2 fibrosis, characterized by increased perisinusoidal and portal collagen deposition with early extension into adjacent lobular areas (Figure 2F and H). This pattern is consistent with early fibrotic remodeling observed during the progression of MASH.

Does dyslipidemia mirror hepatic lipid burden?

By week 8, the HF-EFr group displayed the most severe lipid accumulation, reaching approximately a 3-fold increase in hepatic TG content relative to controls (Figure 2J). Notably, the HFrD group exhibited a significant decline in hepatic TG levels at week 8 compared with week 4 (P < 0.05).

Glucose homeostasis and pancreatic β-cell-related indices

Fasting serum glucose levels increased over time in all dietary groups (Figure 3A). At week 4, glucose concentrations were highest in fructose-fed rats (HF-EFr: 209 mg/dL; HFrD: 245 mg/dL) compared with control (125 mg/dL) and HFD (174 mg/dL) groups. By week 8, hyperglycemia was further exacerbated in fructose-enriched diets, with HF-EFr (261 mg/dL) and HFrD (292 mg/dL) rats remaining significantly elevated relative to control (212 mg/dL) and HFD (245 mg/dL) animals.

Figure 3 Fructose diets induce systemic dysregulation of glucose level, β-cell function, leptin, and the thyroid hormone homeostasis. A: Fasting serum glucose levels; B: C-peptide level; C: C-peptide-to-insulin ratio (index of hepatic insulin clearance); D: Homeostatic Model Assessment for Insulin Resistance (HOMA-IR); E: Serum free triiodothyronine (fT3); F: Serum free thyroxine (fT4); G: FT3/fT4 ratio (index of T4-to-T3 peripheral conversion); H: Fasting circulating leptin levels, as a surrogate marker of adiposity, positively correlating with total body fat mass. ^aP < 0.05 vs control, ^bP < 0.05 vs HFrD. HFD: High-fat diet; HF-EFr: High-fat plus ethanol and fructose; HFrD: High-fructose diet.

To delineate pancreatic and hepatic contributions to this dysregulation, serum C-peptide levels, the C-peptide/insulin ratio, and pancreatic amylase activity were assessed (Figure 3B and C; Supplementary Figure 1F). In HFD rats, C-peptide levels were elevated at week 4 and accompanied by a high C-peptide/insulin ratio, indicating preserved hepatic insulin clearance; however, this ratio declined markedly by week 8 despite only a modest reduction in C-peptide, suggesting progressive impairment of clearance.

In contrast, the HFrD rats exhibited a significant approximately 4-fold increase in C-peptide between weeks 4 (749 pM) and week 8 (2984 pM), reflecting a robust compensatory β-cell secretory response. Notably, the C-peptide/insulin ratio remained relatively preserved, indicating sustained hepatic insulin handling despite escalating metabolic stress. The HF-EFr group showed comparatively stable C-peptide levels, modest changes in the ratio, and no marked amylase induction, consistent with a more constrained pancreatic response.

Insulin resistance, assessed by HOMA-IR (Figure 3D), revealed distinct temporal patterns. The HF-EFr rats displayed early insulin resistance at week 4 (1.98), which partially normalized by week 8 (1.60). Conversely, HFrD rats showed a low HOMA-IR at week 4 (0.60) followed by a sharp progressive increase by week 8 (2.61), paralleling both the marked rise in C-peptide and the elevation in pancreatic amylase activity. HFD rats followed a similar, albeit less pronounced, progressive pattern.

Endocrine and neuroendocrine integration

Thyroid hormone homeostasis and peripheral conversion: Assessment of thyroid hormone parameters revealed distinct adaptive endocrine responses, particularly in HFrD rats (Figure 3E-G). At week 4, HFrD rats exhibited significantly elevated serum free triiodothyronine (fT3, 4.5 ng/L), an effect that persisted through week 8, despite concomitant reductions in free thyroxine (fT4) levels in both fructose-fed groups. The lowest fT4 concentrations were observed in HFrD rats at week 4 (17.7 ng/L vs 26.7 ng/L in controls; P < 0.001).

The fT3/fT4 ratio, serving as an index of peripheral T4-to-T3 conversion, was markedly elevated in HFrD rats at week 4 (25.4) and remained significantly higher than control and HFD groups at week 8. This pattern indicates altered peripheral T4-to-T3 conversion under high-fructose conditions.

Leptin and adiposity signaling: Fasting circulating leptin levels increased over time in all groups, with the most pronounced elevations observed in fructose-fed rats (Figure 3H). In HFrD rats, leptin levels rose sharply from week 4 (3.9 ng/mL) to week 8 (13.8 ng/mL), reaching concentrations significantly higher than all other groups. The HF-EFr group also showed a substantial increase (9.2 ng/mL), exceeding both control (4.2 ng/mL) and HFD (4.7 ng/mL) rats at week 8. These sustained leptin elevations reflect fructose-driven adiposity expansion and are paralleled the increased adiposity in fructose-fed groups.

Synergistic ethanol and fructose intake induces differential upregulation of glycosylated and unglycosylated hepatic MC4R in rats

Our molecular analyses confirmed that hepatic MC4R expression is highly responsive to dietary manipulation. At week 4, the HF-EFr group exhibited a robust approximately 4-fold increase in MC4R mRNA expression compared with controls (P < 0.05), comparable to the HFrD group but exceeding the response observed in HFD rats (Figure 4A). By week 8, this transcriptional upregulation remained pronounced, reaching a maximum of approximately 6.7-fold in the HF-EFr group (P < 0.01).

Figure 4 MC4R mRNA and protein expression dynamics exhibit diet- and time-dependent differences in glycosylation status. A: MC4R mRNA levels determined by real-time reverse transcription PCR; B: Densitometric analysis of the mature (glycosylated) MC4R protein (55 kDa); C: Densitometric analysis of the immature (unglycosylated) MC4R protein (37 kDa). Data in panels A-C are expressed as fold change relative to the control group (Y-axis) at weeks 4 and 8 (X-axis); D and E: Representative western blots of MC4R (55 kDa and 37 kDa) and β-actin (42 kDa, loading control) in hepatic lysates (75 µg protein per lane resolved by SDS-PAGE) at week 4 and week 8 from control, high-fat diet, high-fat plus ethanol and fructose, and modified Lieber-DeCarli high-fructose diet groups. Band intensities were quantified by densitometry using ImageJ. MC4R intensities were normalized to β-actin, and fold changes were calculated relative to the mean of the corresponding control group at each time point. Data represent mean ± SEM of independent samples. ^aP < 0.05 vs control, ^bP < 0.05 vs HFD. HFD: High-fat diet; HF-EFr: High-fat plus ethanol and fructose; HFrD: High-fructose diet.

Critically, MC4R protein expression, assessed by western blotting, revealed distinct temporal regulation of the glycosylated (approximately 55 kDa) and unglycosylated (approximately 37 kDa) receptor forms (Figure 4B-E). The mature, glycosylated MC4R was significantly increased in both HF-EFr and HFrD groups at weeks 4 and 8, peaking at approximately threefold above control levels by week 8. In contrast, the unglycosylated MC4R form showed a pronounced and selective induction in the HF–EFr group at week 4, reaching a 5.2-fold increase relative to all other groups (P < 0.05), and remained significantly elevated at week 8 (3.6-fold). The HFrD group displayed a moderate increase in unglycosylated MC4R at week 4 (2.5-fold vs control and HFD); however, levels of this immature receptor form returned to baseline by week 8.

Dietary modulation of MC4R subcellular localization in rat hepatocytes

We investigated the specific subcellular localization of MC4R within hepatocytes using two complementary approaches: IF and immunohistochemistry (IHC) (Figure 5).

Figure 5 Fructose-enriched diets are associated with prominent nuclear MC4R immunoreactivity in hepatocytes as demonstrated by dual immunofluorescence staining and immunohistochemistry. A-C: Row represents the control group showing some small, and mainly nuclear positive MC4R red dots [immunofluorescence (IF)]; D: Immunohistochemistry (IHC) at week 8 shows faint cytoplasmic MC4R (brown) in some hepatocytes of formalin-fixed, paraffin-embedded following antigen retrieval. Nuclei are counterstained with hematoxylin (× 400, scale = 50 μm); E-G: High-fat diet (HFD) group micrographs show MC4R (red) and DAPI-stained nuclei (blue); H: Higher magnification (× 400) micrograph at week 8 highlights nuclear MC4R dots; I-K: Represent hepatic micrographs of high-fat plus ethanol and fructose group; MC4R (red) and DAPI (blue) reveal rounded nuclear MC4R positivity; L: Higher magnification (× 400) confirms nuclear localization at week 8; M-O: Micrographs of Lieber-DeCarli high-fructose diet group display the strongest MC4R nuclear positivity and accumulation (IF); P: IHC shows MC4R in both nuclear and cytoplasmic compartments, with numerous clear vacuoles due to lipid droplet loss (steatosis), nuclei were counterstained by hematoxylin (× 400); Q: Percentage of MC4R-positive nuclei at week 4; R: Mean fluorescence intensity (MFI) of nuclear-associated MC4R at week 4. ^aP < 0.05 vs control, ^bP < 0.05 vs HFD (one-way ANOVA with Tukey post-hoc test). n = 5 rats per group; about 15 images analyzed per group. Nuclear MC4R immunoreactivity is described at the level of protein localization by IF and IHC; no functional inference regarding nuclear MC4R signaling is made here. HFD: High-fat diet; HF-EFr: High-fat plus ethanol and fructose; HFrD: High-fructose diet; PV: Portal vein.

IF staining

In the control group, MC4R appeared as faint, punctate signals predominantly localized within the nuclear compartment (Figure 5A, C and D). In contrast, the HFD (Figure 5E-G) and HFrD (Figure 5M-O) groups exhibited markedly stronger nuclear MC4R immunoreactivity, appearing as discrete intranuclear puncta within hepatocytes. The HF-EFr group displayed the most intense MC4R signal, characterized by larger and more numerous nuclear-positive foci than any other group (Figure 5I-K).

Higher-magnification images confirmed the apparent nuclear localization of MC4R by colocalization with DAPI at week 8 (Figure 5N, HFD group). Notably, hepatocytes in the HF-EFr group (Figure 5L) exhibited rounded nuclear MC4R-positive structures, accompanied by numerous clear cytoplasmic vacuoles, likely representing lipid droplets removed during tissue processing. This feature was also observed, though to a lesser extent, in the HFrD group, consistent with diet-induced steatosis. HepPar1, used as a cytoplasmic counterstain (green), clearly delineated hepatocyte boundaries and confirmed cellular specificity of the MC4R signal.

IHC

In control liver sections, MC4R immunoreactivity (brown DAB signal) was minimal, with occasional weak cytoplasmic or perinuclear staining in scattered hepatocytes (Figure 5D), consistent with low basal hepatic MC4R expression. In contrast, HFrD liver sections exhibited markedly increased MC4R immunopositivity in a large proportion of hepatocytes (Figure 5P).

MC4R staining was detected in both cytoplasmic and nuclear compartments, with cytoplasmic signals displaying a granular or vesicular pattern. Notably, several hepatocyte nuclei exhibited clear DAB positivity, consistent with MC4R nuclear immunoreactivity observed in fructose-fed groups. The frequent presence of cytoplasmic vacuoles further corroborated the histopathological evidence of steatosis in this group.

Quantitative assessment of nuclear recruitment and intensity

To strengthen these visual observations, semi-quantitative image analysis was performed (Figure 5Q and R). In the control group, 69% of hepatocyte nuclei were MC4R-positive, with a MFI of 54 ± 3. All dietary interventions increased nuclear MC4R localization, although to different extents. In the HFD group, the percentage of MC4R-positive nuclei increased to 85%, accompanied by a moderate rise in MFI to 60 ± 3, which remained lower than that observed in the fructose-containing groups.

In contrast, fructose-containing diets produced a marked increase in nuclear MC4R localization, with the HFrD (97%) and HF-EFr (96%) groups showing the highest proportion of MC4R-positive nuclei (Figure 5Q). In addition, MFI values were significantly elevated in these groups compared with both controls and the HFD group. The HFrD group exhibited the highest receptor density (84 ± 4 MFI, P < 0.001), followed by the HF-EFr group (76 ± 5) (Figure 5R).

Of note, no obvious hepatic zonation of MC4R immunoreactivity was observed across the hepatic lobule in any dietary group. MC4R-positive hepatocytes were present in both periportal and pericentral regions, indicating that receptor distribution was not confined to a specific lobular zone under the examined dietary conditions.

Relative expression of metabolic, inflammatory, and hormonal genes in rat liver tissue

To characterize hepatic transcriptional responses to the dietary interventions, gene expression profiling was performed in liver tissue for selected metabolic, hormonal, and inflammatory targets (Figure 6). Genes were grouped according to their primary functional pathways.

Figure 6 Relative gene expression in rat liver tissue following dietary intervention for 4 and 8 weeks, expressed as fold change to the control group. A: Lepr; B: Gcr; C: Irs-2; D: Srebp-1c; E: Lxr-α; F: Glut5 fructose transporter; G: Il-1β; H: Cxcl-1 (chemokine); I: Pai-1; J: Pecam-1, highlighting differential metabolic, inflammatory, and endothelial responses across dietary interventions. ^aP < 0.05 vs control, ^bP < 0.05 vs HFD. Statistical analysis was performed using two-way ANOVA with Tukey’s post hoc test unless otherwise indicated. Data are expressed as mean ± SEM (n = 5/group). HFD: High-fat diet; HF-EFr: High-fat plus ethanol and fructose; HFrD: High-fructose diet.

Lepr: At week 4, hepatic Lepr mRNA levels showed minimal differences between the HFD and the combined high-fat, ethanol, and fructose (HF-EFr) groups relative to controls. By week 8, however, Lepr expression was significantly increased in fructose-containing diets, with a approximately 5.5-fold upregulation in the HFrD group and a approximately 2.5-fold increase in the HF-EFr group compared with controls (Figure 6A).

Gcr: Hepatic Gcr expression was most prominently increased in the HFrD group, with a 2.8-fold elevation at week 4 and a 2.5-fold elevation at week 8 relative to controls (Figure 6B). Other dietary groups exhibited more modest changes.

Irs-2: In contrast, Irs-2 mRNA levels were reduced across all experimental groups compared with controls. The greatest downregulation was observed in the HFrD group at week 8 (Figure 6C).

Lipogenic transcription factors (Srebp-1c and Lxr-α): At week 4, both Srebp-1c and Lxr-α were significantly upregulated specifically in the HFrD group (Figure 6D and E). By week 8, increased expression of both genes was observed across all dietary groups, with the highest levels consistently detected in fructose-containing diets (HF-EFr and HFrD).

Fructose transporter (Glut5/Slc2a5): Expression of Glut5 was markedly induced in fructose-fed groups as early as week 4. By week 8, the HFrD group demonstrated the strongest upregulation, reaching approximately an 11-fold increase compared with controls (Figure 6F).

Inflammatory and pro-fibrotic markers: Hepatic expression of Il-1β and Cxcl-1 was notably elevated in the HFrD group (Figure 6G and H). Among inflammatory and remodelling markers, Pai-1 mRNA exhibited the most pronounced response, with an approximately 15-fold increase at week 4 in the HFrD group relative to controls (Figure 6I).

Endothelial and vascular marker (Pecam-1/CD31): Pecam-1 gene expression was highest in the HF-EFr group (approximately 3.5-fold vs control), followed by the HFrD group (approximately 2.1-fold vs control) (Figure 6J).

Fructose-enriched diets significantly promoted hepatocellular proliferation

Assessment of hepatocellular proliferation by Ki-67 immunostaining revealed a clear, diet-dependent increase in proliferative activity (Figure 7A). Liver sections from the control and HFD groups displayed low baseline Ki-67 positivity, consistent with minimal hepatocyte turnover. In contrast, both fructose-containing dietary groups, the high-fat, ethanol, and fructose (HF-EFr) group and the HFrD group, showed a marked increase in Ki-67-positive nuclei.

Figure 7 Indirect immunofluorescence staining for the proliferation marker Ki-67. A: Representative liver sections show Ki-67-positive nuclei at week 4. DAPI was used for nuclear counterstaining (× 20); B: Indices of hepatocellular DNA synthesis were quantified as the percentage of Ki-67-positive cells across eight high-power fields per liver sample. Statistical analysis was performed using one-way ANOVA. Data are expressed as mean ± SEM (n = 5/group). ^aP < 0.05 vs control, ^bP < 0.05 vs HFD. HFD: High-fat diet; HF-EFr: High-fat plus ethanol and fructose; HFrD: High-fructose diet.

Quantitative analysis of the proliferation index confirmed a significant increase in Ki-67 positivity in both fructose-fed groups compared with control and HFD groups (P < 0.05; Figure 7B). The HF-EFr group exhibited the highest proliferative response, with a 3.8-fold increase relative to controls, exceeding the 3.1-fold increase observed in the HFrD group.

DISCUSSION

In this study, we demonstrate that fructose-enriched dietary regimens elicit a coordinated spectrum of metabolic, inflammatory, and proliferative hepatic responses that extend beyond simple steatosis, with distinct phenotypic patterns emerging depending on whether fructose is consumed alone or in combination with ethanol. Using a controlled dietary framework, we show that high-fructose intake, either alone (HFrD) or combined with ethanol (HF-EFr), drives progressive disturbances in glucose homeostasis, hepatic insulin signalling, lipogenic transcriptional programs, inflammatory activation, and hepatocellular proliferation. These alterations occur alongside modulation of MC4R-associated pathways, suggesting engagement of integrated metabolic sensing mechanisms within the liver. Importantly, HFrD predominantly promotes metabolic overload and endocrine dysregulation, involving pancreatic, thyroid, and leptin-related pathways, whereas HF-EFr preferentially amplifies inflammatory injury and regenerative responses, indicating complementary yet non-identical pathogenic trajectories.

Gross examination of the abdominal cavity revealed diet-dependent differences in intra-abdominal white adipose tissue (WAT) distribution, with preferential expansion of mesenteric and epididymal depots in animals exposed to fructose-based diets, particularly when combined with ethanol. Such qualitative differences are consistent with the recognized heterogeneity among visceral WAT depots, which differ in metabolic activity, endocrine output, and sensitivity to nutritional stress[17]. Importantly, adipose expansion under dietary challenge is also governed by upstream neuroendocrine pathways, including melanocortin signalling; disruption of prohormone processing enzymes such as PCSK2 reduces adiposity and protects against diet-induced weight gain in mice[18].

These multisystemic alterations are best understood through the “Multiple Parallel Hits Hypothesis”, which posits that MASH progression is not a linear sequence but rather the result of simultaneous metabolic insults[19,20]. In our model, the concurrent expansion of dysfunctional adipose tissue, gut-derived metabolic stress from fructose, and direct ethanol-induced lipotoxicity represent these “parallel hits”. Unlike the outdated two-hit model, this framework explains how inflammatory signaling and fibrogenic priming (e.g., Pai-1 induction) can occur nearly simultaneously with lipid accumulation, rather than as a delayed secondary consequence.

However, the addition of ethanol and fructose (HF-EFr) acts as a potent “synergistic hit”, accelerating the transition from simple steatosis to MASH. Clinically, this highlights that individuals with underlying fatty liver are at a significantly higher risk for rapid disease progression and fibrotic remodelling if they consume even moderate amounts of alcohol and refined sugars.

A central finding of this work is the pronounced impairment of glucose homeostasis in fructose-containing diets. While fasting glucose levels increased over time across all experimental groups, the magnitude and persistence of hyperglycemia were most striking in the HFrD and HF-EFr groups, evident as early as week 4 and further exacerbated by week 8. This acceleration of dysglycemia compared with HFD alone is consistent with the well-established metabolic behavior of fructose, which bypasses key glycolytic control points and enhances hepatic gluconeogenesis[21,22]. The resulting hyperglycemia parallels early stages of type 2 diabetes, where pancreatic β-cells compensate for peripheral insulin resistance by increasing insulin secretion, a pattern reflected in the observed elevations of serum C-peptide and HOMA-IR. Interestingly, chronic high-fructose intake (HFrD) drove a unique phenotype of “exocrine pancreatic overactivation” (elevated amylase) and extreme hyperleptinemia. This indicates that even in the absence of ethanol, high fructose load creates a systemic hypermetabolic stress that targets the pancreas and drives leptin resistance. This is clinically relevant for patients consuming high-sugar diets, as it suggests that pancreatic exhaustion may precede or parallel liver failure[23].

At the hepatic level, molecular markers of insulin resistance reinforced this systemic phenotype. Irs-2 was selectively downregulated in fructose-fed animals, consistent with its central role in hepatocyte insulin signalling and its association with compensatory hyperinsulinemia[24]. In contrast, Irs-1 expression remained largely unchanged, probably indicating pathway-specific vulnerability to fructose-induced stress. We found that a concurrent upregulation of hepatic Lepr transcripts, together with elevated circulating leptin levels, increased calories intake, body weight and mesentery fat suggest a compensatory yet ineffective attempt to restore leptin-mediated metabolic control, consistent with leptin resistance observed in obesity and metabolic syndrome[25,26]. Collectively, these findings illustrate how fructose overload disrupts the coordinated hormonal regulation of energy and glucose homeostasis.

Fructose-driven lipogenesis emerged as a dominant feature of hepatic metabolic remodelling, particularly in HFrD-fed animals. Both Srebp-1c and Lxr-α were markedly upregulated, consistent with fructose’s established capacity to stimulate de novo lipogenesis[27,28]. The substantial induction of Glut5 (Slc2a5), reaching approximately 11-fold in HFrD livers, underscores the liver’s adaptive capacity to import and metabolize excess fructose. Because fructose metabolism bypasses phosphofructokinase-mediated regulation, enhanced Glut5 expression likely amplifies substrate flux into lipogenic (e.g., Lxr-α)[29] and gluconeogenic pathways[30], mechanistically linking dietary fructose intake to hepatic fat accumulation and metabolic stress.

In parallel, fructose-rich diets triggered early inflammatory and fibrogenic signalling. Hepatic Il-1β and Cxcl-1 transcripts were elevated, reflecting activation of innate immune pathways and chemokine-mediated immune cell recruitment, key processes in the transition from steatosis to steatohepatitis[31]. Notably, Pai-1 expression increased approximately 15-fold in HFrD livers by week 4, suggesting early extracellular matrix remodelling and fibrogenic priming prior to overt histological fibrosis[32]. These findings indicate that consumption of high fructose-derived energy alone is sufficient to initiate inflammatory and profibrotic signalling in the liver, even in the absence of ethanol.

The addition of ethanol produced a qualitatively distinct hepatic phenotype, characterized by amplified injury and regenerative responses. Despite comparable caloric intake, HF-EFr animals exhibited greater hepatomegaly, enhanced vascular inflammation, and stronger hepatocyte proliferative signals than other regimens. Upregulation of Pecam-1 in the HF-EFr group is consistent with endothelial activation and vascular remodelling, processes increasingly recognized as contributors to fatty liver disease progression[33]. Intrahepatic responses to combined dietary stress likely reflect complex interactions among hepatocytes, macrophages, and endothelial cells, where cytokine release from liver macrophages induces chemokine production in hepatocytes and alters adhesion molecule expression[34]. This framework provides a plausible explanation for the patchy inflammatory patterns and differential PECAM-1 responses observed across hepatic zones, where increased expression may preferentially reflect larger vessel endothelium rather than sinusoidal cells.

Within this inflammatory milieu, hepatocellular proliferation was increased in fructose-containing diets, as indicated by Ki-67 immunostaining. Although both HFrD and HF-EFr groups showed higher proliferation than controls, the response was most pronounced in HF-EFr animals, suggesting that ethanol amplifies regenerative signalling under fructose-induced stress. This proliferation likely represents an acute-phase-like adaptive response to metabolic and inflammatory injury rather than a primary mitogenic drive, consistent with cytokine-linked hepatocyte cell-cycle activation[35]. Prolonged exposure to combined dietary insults, however, may shift this response toward maladaptive remodelling and fibrosis.

The observed elevation in the AST/ALT ratio specifically in the HF-EFr group (1.75) mirrors the clinical signature of alcoholic liver disease and advanced fibrosis. This suggests that ethanol exposure disrupts the liver's enzymatic balance and accelerates structural damage (stage 2 fibrosis) far more aggressively than fat accumulation alone. From a public health perspective, this emphasizes that liver protection strategies must focus on abstinence from ethanol in the context of metabolic syndrome to prevent the 'early hepatocellular response’ we observed as early as week 4.

MC4R-related pathways appear to participate in this integrative hepatic response. Although classically characterized as a hypothalamic regulator of appetite and energy balance, MC4R is increasingly recognized as a peripheral metabolic modulator with pleiotropic signaling capacity[36]. Previous studies in rat models of acute-phase response and liver regeneration have demonstrated hepatic MC4R upregulation and nuclear translocation, supporting a functional role beyond central neuroendocrine regulation[9]. In the present study, fructose-enriched diets were associated with increased hepatic MC4R mRNA and protein expression, most prominently in the HF-EFr group. These changes occurred in parallel with metabolic disturbance, inflammatory activation, and enhanced Ki-67 immunoreactivity, but this temporal association should not be interpreted as direct evidence of receptor-driven proliferative signalling. Rather, MC4R upregulation may reflect an adaptive response to sustained nutritional and inflammatory stress, potentially integrating neuroendocrine cues with intrahepatic metabolic regulation.

At the mechanistic level, MC4R is a G protein-coupled receptor capable of engaging multiple downstream pathways, including Gs-cAMP signaling, Gq/11 activation, β-arrestin recruitment, and ERK phosphorylation. Differential signaling profiles have been demonstrated for distinct MC4R variants and ligands, underscoring the receptor’s capacity for pathway bias and context-dependent signalling. ERK activation, in particular, plays a central role in hepatocyte proliferation and liver regeneration. In regenerative models such as partial hepatectomy, ERK phosphorylation precedes hepatocyte cell-cycle entry, supporting the concept that stress-associated MC4R upregulation could modulate hepatocellular responsiveness to proliferative cues through ERK-dependent mechanisms. Within this framework, MC4R is more appropriately interpreted as a modulatory component within a broader regenerative and inflammatory signalling network rather than a primary mitogenic stimulus[16].

Thyroid hormone axis alterations further underscore distinct adaptive responses across dietary conditions. HFrD-fed rats exhibited elevated serum level of fT3, reduced fT4, and an increased fT3/fT4 ratio. Although this profile is frequently interpreted as reflecting enhanced peripheral T4-to-T3 conversion via D1 deiodinase activity under conditions of increased metabolic demand[37], alternative mechanisms warrant consideration. Variations in thyroid hormone–binding proteins, including thyroxine-binding globulin and transthyretin (prealbumin), both synthesized in part by hepatocytes, as well as changes in hepatic hormone clearance could influence circulating hormone dynamics. However, because free hormone fractions (fT3 and fT4) were measured rather than total concentrations, fluctuations in binding capacity are unlikely to fully account for the selective elevation of the fT3/fT4 ratio. Moreover, inflammatory and hepatic stress states typically suppress D1 deiodinase activity through cytokine (IL-6, TNF-α)-mediated pathways, leading to reduced fT3 availability rather than an increased ratio[38]. Thus, the endocrine profile observed in HFrD animals more plausibly reflects adaptive upregulation of peripheral thyroid hormone activation in response to fructose-driven metabolic stress.

In contrast, HF-EFr animals demonstrated reductions in both serum fT3 and fT4 levels without a compensatory increase in the fT3/fT4 ratio, a pattern consistent with impaired peripheral conversion and features of non-thyroidal illness syndrome under combined metabolic and inflammatory burden[39]. Such suppression of thyroid hormone activation parallels the hepatic acute-phase response, during which cytokine signaling alters thyroid hormone receptor expression and deiodinase pathways in liver tissue[35].

Our findings of pronounced visceral adipose tissue expansion in the HFrD group alongside elevated hepatic Gcr expression parallel clinical observations by Misra et al[40], who reported that enhanced glucocorticoid action is associated with visceral adiposity and insulin resistance in humans. In this context, fructose-associated hepatic Gcr upregulation may contribute to preferential lipid sequestration within the liver and mesenteric fat depots, favoring a phenotype characterized by metabolically driven visceral fat accumulation rather than inflammation-dominant adipose remodeling.

Collectively, these data underscore the integrated hepatic consequences of dietary patterns combining fructose-rich foods with alcohol. Fructose appears to play a central role in promoting metabolic overload, lipogenesis, obesity-associated changes, and endocrine dysregulation, whereas ethanol may exacerbate hepatocellular injury, vascular inflammation, and regenerative proliferation. Interactions among hepatocytes, macrophages, and endothelial cells, together with proliferative responses resembling early acute-phase adaptation, provide a framework for integrating the molecular and histological findings observed in this model. These early metabolic and inflammatory changes are clinically relevant, as steatotic livers exhibit increased vulnerability to injury and impaired functional outcomes in transplantation settings, a limitation that has recently been explored experimentally through donor preconditioning strategies[41]. The combination of visceral adiposity, hypertriglyceridemia, and reduced HDL-C observed particularly in fructose-fed rats resembles a cardiometabolic risk profile associated with accelerated atherosclerosis in humans, supporting the broader systemic implications of hepatic metabolic dysfunction.

Several limitations warrant consideration. The relatively small sample size (n = 5 per group) may limit statistical robustness. While gene expression and immunohistochemical analyses provide mechanistic context, they do not establish functional causality. Direct interrogation of MC4R and Ki-67 signalling pathways through pharmacological modulation would strengthen causal inference. The experimental design captures early and intermediate stages; longer-term studies are required to determine progression toward advanced fibrosis or HCC.

CONCLUSION

HFrD, alone or combined with ethanol, induce a coordinated cascade of hepatic metabolic dysregulation, inflammatory activation, and proliferative remodeling through distinct but interacting pathways. HFrD predominantly promotes hepatic nutrient excess, visceral adiposity, endocrine perturbation, and pancreatic stress, whereas HF-EFr amplifies hepatocellular injury, vascular activation, and regenerative proliferation, accompanied by delayed AST elevation, increased AST/ALT ratio, and enhanced MC4R expression. Fructose functions as a primary metabolic stressor, while ethanol intensifies intrahepatic inflammatory and structural responses. Alterations in MC4R, leptin, glucocorticoid, and thyroid hormone pathways likely reflect adaptive hepatic responses integrating metabolic and inflammatory cues. These findings provide mechanistic insight into how combined nutritional exposures accelerate early liver pathology and highlight the importance of targeting synergistic dietary factors in early disease prevention.

ACKNOWLEDGEMENTS

Authors acknowledge the invaluable help of Dr F. Schultze, Mrs D. Fey, Mrs C. Wultrout, C. Donaski, and Mrs S. Zachmann for their expert assistance and guides.