Dietary milk polar lipids ameliorate hepatic lipid accumulation through coordinated regulation of Wnt-PPARγ signaling and remodeling of the gut microbiota

Abstract

This study investigated the protective effects of milk polar lipids (MPL) against non-alcoholic fatty liver disease (NAFLD) and explored the underlying mechanisms using a high-fat high-sucrose (HFHS) diet–induced mouse model. MPL diet significantly reduced body weight gain, adiposity, and hepatic lipid accumulation, in addition to decreasing serum levels of liver injury markers. Mechanistically, MPL diet activated hepatic Wnt/β–catenin signaling, as evidenced by increased expression of low-density lipoprotein receptor–related protein 6 (LRP6), Wnt family member 3 A (Wnt3a), and β-catenin. Concurrently, MPL treatment suppressed peroxisome proliferator–activated receptor gamma (PPARγ) and downstream lipogenic proteins involved in triglyceride synthesis and de novo lipogenesis. In addition, MPL diet markedly reshaped the gut microbiota composition disrupted by HFHS feeding. Notably, MPL group showed a significant increased the abundance of Akkermansia muciniphila and short-chain fatty acid–producing bacteria, including members of Romboutsia and Christensenellaceae. These findings demonstrate that dietary MPL effectively attenuates HFHS diet–induced NAFLD through coordinated regulation of hepatic Wnt–PPARγ signaling and gut microbial ecology.

Introduction

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease worldwide, affecting approximately 38% of the global population (Teng et al., 2022). It co-occurs with other ailments, such as obesity, type 2 diabetes, and metabolic syndrome, and thus represents a substantial public health concern. NAFLD is characterized by excessive lipid accumulation in the liver, occurring in the absence of significant alcohol consumption, and can progress through non-alcoholic steatohepatitis (NASH) and fibrosis to cirrhosis and eventually hepatocellular carcinoma. Although the pathogenesis of NAFLD is multifactorial, emerging evidence suggests that dysregulation of the gut–liver axis plays a central role in its progression (Martin-Mateos & Albillos, 2021).

The gut and liver are functionally connected through the portal circulation and the biliary system, forming the basis of the gut–liver axis. Through this bidirectional communication network, gut microbiota-derived metabolites can directly influence hepatic metabolism and inflammation. These microbial and microbially modified metabolites, including bile acids, short-chain fatty acids (SCFAs), and choline-derived compounds have been shown to regulate lipid metabolism, immune signaling, and insulin sensitivity in the liver (Tripathi et al., 2018). In NAFLD, decreased SCFA production and impaired intestinal mucus barrier integrity are commonly observed, thereby promoting gut microbiota dysbiosis and intestinal hyperpermeability (Kim & Mills, 2024).

Beyond microbiota-associated mechanisms, intracellular signaling pathways involved in lipid metabolism have also been implicated in NAFLD pathogenesis. Among these, Wnt/β-catenin signaling pathway is a pathway that regulates cell proliferation and differentiation, and energy metabolism. Activation of Wnt/β-catenin signaling suppresses adipogenic transcription factors such as peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα), thereby limiting adipogenesis and lipid accumulation (Bennett et al., 2005). In a study by Go et al. (2014), impaired Wnt signaling in low-density lipoprotein receptor–related protein 6 (LRP6) mutant models were found to increase hepatic lipogenesis and plasma lipid levels whereas restoration of the pathway improved metabolic homeostasis. These findings suggest that the Wnt/β-catenin–PPARγ axis can play a regulatory role in hepatic lipid accumulation.

Identification of dietary factors that modulate these pathways therefore represents a promising therapeutic strategy for NAFLD. Bioactive lipids derived from milk have gained attention for their protective effects against metabolic diseases. Milk polar lipids (MPL), are a biologically active class of membrane lipids predominantly localized in the milk fat globule membrane. They mainly comprise phospholipids and sphingolipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM). These lipids can be converted into bioactive metabolites that participate in intracellular signaling pathways (Liu et al., 2025).

Several studies to date have demonstrated that MPL improve lipid metabolism by modulating intestinal lipid digestion and cholesterol absorption. Dietary phospholipids, including those derived from milk, have been shown to interfere with micellar lipid solubilization, thereby reducing intestinal lipid absorption and increasing fecal lipid excretion (Eckhardt et al., 2002). A study involving high-fat-diet-fed animal models revealed that supplementation of phospholipid-rich dairy extracts in the diet led to the attenuation of hepatic lipid accumulation (Wat et al., 2009). Moreover, MPL can alleviate gut dysbiosis by reducing the Bacillota (formerly Firmicutes)-to- Bacteroidota (formerly Bacteroidetes) ratio, a microbial signature frequently associated with metabolic disorders and hepatic steatosis (Millar et al., 2020). Milk-derived SM has been reported to exert beneficial metabolic effects against dyslipidemia, NAFLD, atherosclerosis, and insulin resistance (Park et al., 2025). In another study, milk-derived SM demonstrated stronger protective effects against hepatic steatosis than egg-derived SM did, suggesting that the biological activity of sphingolipids may differ depending on their dietary source (Norris et al., 2016). Despite these findings, the molecular mechanisms underlying the hepatoprotective effects of MPL remain incompletely understood, particularly with respect to gut microbiota modulation and intracellular signaling pathways associated with hepatic lipid metabolism.

This study aims to evaluate the protective effects of MPL diet against hepatic lipid accumulation in a high-fat high-sucrose (HFHS) diet-induced mouse model of NAFLD. Specifically, we investigated whether MPL diet modulates gut microbiota composition and hepatic lipid metabolism through regulation of the Wnt/β-catenin signaling pathway.

Materials and methods

Materials

A whey-derived milk polar lipid concentrate (DS-WPL 25; Solus Biotech, Seoul, Korea) was used in the study. This concentrate contains ≥ 25% total phospholipids, which include SM (≥ 6%), PC (≥ 7%), PE (≥ 4%), and PS (≥ 0.6%). Xenical^®, a commercial formulation of orlistat, was procured from Roche, Basel, Switzerland. Primary antibodies for β-catenin, the Wnt family member 3 A (Wnt3a), fatty acid synthase (FAS), LRP6, PPARγ, acetyl-CoA carboxylase (ACC), stearoyl-CoA desaturase 1 (SCD1), and β-actin were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibody against perilipin 2 (PLIN2) was purchased from Novus Biologicals (Centennial, CO, USA). Antibodies against sterol regulatory element–binding protein 1 (SREBP1) and diacylglycerol acyltransferase 1 (DGAT1) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA), and Thermo Fisher Scientific (Waltham, MA, USA), respectively.

Animals and experimental design

Male C57BL/6J mice (7 weeks old) were obtained from Saeron Bio Inc. (Uiwang, Gyeonggi-do, Korea). The animals were maintained under controlled environmental conditions (22 ± 2 °C, 50 ± 10% relative humidity, and a 12 h light/dark cycle) with free access to food and water. All experimental procedures were approved by the Kookmin University Animal Care and Use Committee (KMU-2024-02). After a one-week acclimation period, the mice were randomly allocated into four experimental groups (n = 6 per group): (i) The normal (NOR) group (control) was fed the AIN-93G standard diet purchased from Saeron Bio Inc.; (ii) the HFHS diet group, fed a diet that provided 43% of its total calories from fats and 24% from sucrose; (iii) the MPL group, fed a modified HFHS diet containing 8% MPL concentrate, in which milk fat was replaced by the MPL concentrate with corresponding adjustments to cellulose.; and (iv) the orlistat group (positive control), fed an HFHS diet supplemented with 0.01% orlistat. Detailed diet formulation is presented in Table 1. The experimental diets were administered for 12 weeks. Body weight and food intake were recorded weekly throughout the experimental period. At the end of the study, the mice were made to fast for 4 h and later euthanized via CO₂ inhalation. Blood and tissues – including liver tissue, adipose tissue, and cecal samples – were collected, immediately frozen in liquid nitrogen, and stored at − 80 °C until analysis.

Table 1.

Diet composition

Ingredients (g/kg)	NOR	HFHS	MPL	Orlistat
Casein	200	245	245	245
Sucrose	100	287	287	287
Maltodextrin	132	66	66	66
Corn starch	397	47	47	47
Cellulose	50	42	58	42
Corn oil	70	30	30	30
Milk fat	–	96	-	96
Lard	0	115	115	115
Mineral mix	35	43	43	43
Calcium phosphate	–	3.4	3.4	3.4
Vitamin mix	10	19	19	19
L-Cystine	3	3.5	3.5	3.5
Choline bitartrate	2.5	3	3	3
MPLs	–	–	80	–
Orlistat	–	–	–	0.1
Energy density (kcal/g)	4	4.7	4.7	4.7
Carbohydrate (% kcal)	63	34	34	34
Protein (% kcal)	20	21	21	21
Fat (% kcal)	16	43	43	43

MPLs, Milk polar lipids

Body composition

Body composition of the mice was assessed one week before euthanasia using dual-energy X-ray absorptiometry (DEXA; InAlyzer, Medikors Inc., Seongnam, Korea). Mice were anesthetized with isoflurane (Ifran^®, BK Pharm Co., Ltd., Goyang, Korea) during DEXA measurements. Measurements were performed under standardized conditions to determine fat mass, lean mass, and bone mineral content. All scans were conducted according to the manufacturer’s instructions, and data were analyzed using the accompanying InAlyzer software.

Biochemical serum analysis

Blood samples were collected via cardiac puncture, followed immediately by euthanasia and centrifuged at 12,000 × g for 3 min at room temperature to obtain the serum, and the serum samples were stored at − 80 °C until further analysis. Serum triglycerides (TG), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels in the samples were analyzed using a DRI-CHEM NX600 analyzer (FUJIFILM Corporation, Tokyo, Japan). Total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) concentrations were measured using commercial assay kits (BM-CHO-100 and BM-CDL-100; Biomax, Guri, Korea) according to the manufacturer’s instructions.

Histological analysis

For hematoxylin and eosin (H&E) staining, liver and adipose tissues were fixed in 4% formaldehyde, dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin. Paraffin blocks were sectioned into 4 μm-thick slices and stained with hematoxylin and eosin according to a previously described method (Son et al., 2025). For Oil Red O (ORO) staining, liver tissues were embedded in optimal cutting temperature compound and rapidly frozen at − 80 °C. Cryosections (8–10 μm) were fixed in 10% neutral-buffered formalin and incubated with Oil Red O working solution. After staining, sections were counterstained with hematoxylin. All stained sections were examined using a light microscope, and representative images were captured at 20⋅ magnification using the KF Digital Slide Management System (KF-DSMS; KFBIO, Ningbo, China). Adipocyte size was quantified from H&E-stained adipose tissue images using ImageJ software (version 1.54i, National Institutes of Health, Bethesda, MD, USA).

Western blot analysis

Liver tissues were homogenized in RIPA lysis buffer supplemented with a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, USA) using a Bullet Blender tissue homogenizer (BT24M; Next Advance, Troy, NY, USA). The protein concentration in the supernatant was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Equal amounts of protein (30 µg per lane) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). After the electrophoretic procedure was complete, the resolved proteins were transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were then blocked with bovine serum albumin (BSA) and incubated overnight at 4 °C with primary antibodies against β-catenin, Wnt3a, FAS, LRP6, PPARγ, ACC, SCD1, β-actin, PLIN2, SREBP1, and DGAT1. Once incubation was complete, the membranes were washed and, were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Thermo Fisher Scientific, USA). Protein bands were detected using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, USA) and visualized using a ChemiDoc Imaging System (Bio-Rad Laboratories, Hercules, CA, USA). Western blot analysis was performed using liver samples from three biological replicates per group. Band intensities were quantified using ImageJ software and normalized to β-actin as the loading control.

Cecal gut microbiome analysis

Genomic DNA was extracted from cecal content using the DNeasy PowerSoil Pro Kit (Qiagen, Hilden, Germany), and 16 S rRNA gene sequencing was performed according to a previously described method (Gwon et al., 2025). DNA concentration and purity were assessed using the VICTOR^® Nivo™ multimode plate reader (PerkinElmer Inc., Waltham, MA, USA) with PicoGreen dsDNA quantification reagents. The V3–V4 hypervariable regions of bacterial 16 S rRNA were amplified using the Illumina 16 S Metagenomic Sequencing Library Preparation protocol. Briefly, 5 ng of input gDNA was used as the template for the first round of PCR amplification using universal primers containing Illumina overhang adapter sequences and Herculase II Fusion DNA Polymerase (Agilent Technologies, Santa Clara, CA, USA). PCR amplicons were purified using AMPure beads (Agencourt Bioscience, Beverly, MA, USA) and subjected to a second PCR step to attach dual indices and sequencing adapters using Nextera XT index primers. The indexed libraries were purified, quantified using PicoGreen reagents, and assessed for fragment size distribution and library quality using TapeStation D1000 ScreenTape (Agilent Technologies, Waldbronn, Germany). Libraries were subsequently normalized, pooled, and quantified by qPCR using the KAPA Library Quantification Kit (Roche, Basel, Switzerland). Sequencing was performed on the Illumina MiSeq™ platform (Illumina, San Diego, CA, USA) according to the manufacturer’s standard paired-end sequencing protocols. Raw 16 S rRNA sequencing reads were processed using Cutadapt (v3.2), DADA2 (v1.18.0), and QIIME (v1.9.0) to generate normalized amplicon sequence variant (ASV) abundance tables, and taxonomic classification was performed against the NCBI_16S database. Alpha diversity was evaluated using the Shannon index and phylogenetic diversity (PD) whole tree, while beta diversity was assessed using UniFrac distances, visualized by principal coordinates analysis, and statistically evaluated by PERMANOVA. LEfSe analysis was used to identify discriminative taxa using p < 0.05 and LDA score > 3.5. Differential abundance between the HFHS and MPL groups was further analyzed using STAMP with Student’s t-test. Alpha diversity indices and selected microbial taxa were analyzed by one-way ANOVA followed by Duncan’s multiple range test, with significance set at p < 0.05.

Statistical analysis

All data are presented as mean ± standard deviation (SD). Statistical analyses were performed using IBM SPSS Statistics software version 29.0 (IBM, Chicago, IL, USA). Inter-group differences were evaluated using one-way analysis of variance (ANOVA). Statistical significance was considered at p < 0.05.

Results and discussion

Dietary MPL reduced body weight and fat mass in HFHS diet-fed mice

Body weight was monitored weekly throughout the experimental period (Fig. 1A). The HFHS diet caused a marked increase in body weight compared with the normal diet. This HFHS diet–induced body weight gain could be significantly attenuated with MPL or orlistat diets, as seen in the body weight outcomes, with the final body weight of the MPL group remaining comparable to that of the NOR group. This outcome suggests that MPL diet effectively prevents excessive weight gain under obesogenic dietary conditions. In addition, relative to the NOR and MPL groups, food and calorie intake in the HFHS group gradually increased throughout the experimental period (Fig. 1B and C). This increase was likely attributable to the increasing body mass of the HFHS diet–fed mice rather than a high appetite. This interpretation is further corroborated by the fact that no significant differences in food intake were observed across groups during the early phase of the experiment (weeks 1 and 2), when body weights were similar.

Fig. 1.

Effects of MPL diet on body weight and body composition. Changes in (A) body weight and (B) food and (C) calorie intake during the 12-week experimental period. Body composition analysis showing (D) fat mass, (E) lean mass, and (F) bone mineral content. Data are presented as mean ± SD. Different letters indicate significant differences among groups (p < 0.05)

The mice in the HFHS group had markedly higher fat mass than mice in the NOR group, with lean mass and bone mineral content being lower (Fig. 1D–F). Notably, mice in the MPL and orlistat groups exhibited significantly lower fat mass than those in the HFHS group. These results demonstrate that MPL or orlistat diets markedly attenuates HFHS-induced fat accumulation. Furthermore, both MPL and orlistat diet partially restored lean mass, while only MPL diet significantly improved bone mineral content. Although only bone mineral content was measured in the present study, these findings suggest that dietary MPL treatment may be associated with the preservation of bone mineral status under HFHS diet-induced metabolic stress. The reduction of fat mass in MPL group may be attributable to specific milk-derived phospholipids and sphingolipids, particularly SM. Previous studies have reported that dietary MPL modulates gut barrier integrity and lipid metabolism, thereby attenuating obesity-related metabolic abnormalities in both LPS-mediated systemic inflammation models and high-fat-diet-induced obese mice (Zhou and Ward, 2024; Norris et al., 2017).

Effects of MPL on serum metabolic parameters

The results of serum biochemical analyses are presented in Table 2. Interestingly, MPL group showed higher serum TG levels than HFHS group. This increase may reflect enhanced mobilization and export of lipids from hepatic and adipose tissue stores, into the circulation rather than impaired lipid metabolism. Previous studies have suggested that dietary phospholipids can influence lipoprotein assembly and lipid transport by altering hepatic lipid handling and very-low-density lipoprotein (VLDL) secretion (Li et al., 2006; Yao and Vance, 1988). These observations suggest that the elevated circulating TG levels observed in the MPL group may be associated with increased lipid turnover and redistribution during the attenuation of hepatic lipid accumulation. However, the relatively short 4 h fasting period before sacrifice may have allowed some postprandial influence on serum TG measurements. Therefore, future studies measuring serum free fatty acids and hepatic VLDL secretion rates are needed to clarify how dietary MPL treatment regulates systemic TG homeostasis.

Table 2.

Effects of MPL diet on serum biochemical markers in HFHS diet-fed mice

	NOR	HFHS	MPL	Orlistat
TG (mg/dL)	90.9 ± 10.5c	87.7 ± 12.7c	122.4 ± 18.0a	100.8 ± 25.0bc
TC (mg/dL)	129.4 ± 19.0b	209.9 ± 36.4a	166.8 ± 62.3ab	196.6 ± 23.5a
HDL-C (mg/dL)	43.5 ± 10.3b	68.2 ± 19.7a	59.9 ± 19.0ab	63.6 ± 10.6a
LDL-C (mg/dL)	8.5 ± 3.7b	17.3 ± 6.1a	13.3 ± 6.4ab	14.8 ± 2.9ab
ALT (U/L)	22.5 ± 9.1b	61.1 ± 67.4a	33.4 ± 15.8ab	44.5 ± 41.8ab
AST (U/L)	65.0 ± 16.3b	87.3 ± 47.5a	50.3 ± 10.7b	71.5 ± 35.3b

Data are presented as mean ± SD. Different superscript letters within the same row indicate significant differences among groups (p < 0.05). Data were analyzed by one-way ANOVA followed by Duncan’s multiple range test, except for ALT, which did not meet the assumptions of normality and homogeneity of variance and was therefore additionally evaluated using the Kruskal–Wallis test with Bonferroni-adjusted pairwise comparisons. TG, triglyceride; TC, total cholesterol; HDL-C, high density lipoprotein cholesterol, LDL-C, low density lipoprotein cholesterol; ALT, alanine aminotransferase; AST, aspartate aminotransferase

In contrast with the results for TG, the HFHS group exhibited significantly elevated levels of TC and LDL-C compared with the NOR group. This result indicates impaired systemic lipid metabolism induced by HFHS diet consumption. MPL diet ameliorated these alterations, with TC and LDL-C levels tending to return toward those observed in the NOR group. ALT and AST, the widely recognized biomarkers of hepatic injury, were also reduced by MPL diet. This suggests a protective effect against HFHS diet-induced hepatic dysfunction and hepatocellular damage.

Dietary MPL inhibited lipid accumulation in adipose and liver tissues

Histological analysis of adipose tissue revealed significant adipocyte hypertrophy in the HFHS group, relative to the NOR group. This could be owing to excessive lipid storage and adipose tissue expansion induced by chronic HFHS-diet consumption (Fig. 2A and F). MPL and orlistat groups exhibited significantly reduced adipocyte sizes relative to the HFHS group. Consistent with these histological findings, HFHS feeding led to a significant increase in both inguinal white adipose tissue (iWAT) and mesenteric white adipose tissue (mWAT). However, these increases were significantly suppressed by MPL or orlistat groups (Fig. 2D and E). Excessive adipocyte expansion is closely associated with adipose tissue dysfunction, chronic low-grade inflammation, and increased free fatty acid flux to peripheral tissues, all of which contribute to the progression of obesity-associated metabolic disorders and NAFLD (Fabbrini et al., 2010). By extension, the reduction in adipocyte size and adipose tissue mass following dietary MPL treatment may contribute to improved systemic lipid homeostasis and reduced ectopic lipid deposition.

Fig. 2.

Effects of MPL diet on adipose and hepatic lipid accumulation. Representative hematoxylin and eosin (H & E) stained sections of (A) epididymal white adipose tissue and (B) liver tissue. (C) Representative Oil Red O–stained liver sections. Weights of (D) inguinal white adipose tissue (iWAT) and (E) mesenteric white adipose tissue (mWAT). (F) Quantification of adipocyte size, (G) Liver weight. (H) Hepatic triglyceride content. Data are presented as mean ± SD. Different letters indicate statistically significant differences among groups (p < 0.05)

Histological examination of liver tissues via H&E staining and Oil Red O staining further revealed substantial hepatic lipid accumulation in HFHS-fed mice (Fig. 2B and C). In contrast, hepatic steatosis was markedly reduced in the MPL and orlistat groups. Similarly, HFHS feeding significantly increased liver weight compared with the NOR group, whereas MPL or orlistat diets effectively reduced liver weight (Fig. 2G). The MPL group also exhibited significantly reduced hepatic TG levels (Fig. 2H).

Kamili et al. (2010) reported that supplementation with phospholipid-rich milk extract attenuated hepatic lipid accumulation, partly through the inhibition of intestinal cholesterol absorption and promotion of fecal excretion of lipids. Although lipid excretion was considerably increased, only small quantities of dietary phospholipids (2.4%, 3.7%, 0.1%, and 0.2% of dietary PC, SM, PE, and phosphatidylinositol, respectively) were detected in the feces, indicating that the phospholipid supplements themselves had been efficiently absorbed. These findings suggest that dietary MPL, especially SM, may not only influence hepatic lipid metabolism after absorption but also exert local effects within the intestine before being absorbed. In particular, milk-derived SM has been reported to exhibit stronger inhibitory effects on hepatic lipid accumulation than egg-derived SM, suggesting the potential importance of the dietary source and composition of the lipid in regulating metabolic responses (Norris et al., 2016).

MPL diet modulates the hepatic Wnt-PPARγ signaling pathway

To investigate the molecular mechanisms underlying the hepatoprotective effects of MPL, we further examined the hepatic Wnt–PPARγ signaling pathway and the expression of lipid metabolism–related proteins in HFHS diet–fed mice. HFHS feeding significantly suppressed key components of the canonical Wnt/β-catenin signaling pathway, as evidenced by reduced hepatic expression of LRP6 and Wnt3a compared with the NOR group (Fig. 3A–C). On the other hand, MPL or orlistat diets significantly increased the expression of LRP6 and Wnt3a relative to the HFHS group. Notably, only MPL diet significantly upregulated total β-catenin expression, which is consistent with increased expression of components of the Wnt/β-catenin signaling in the liver.

Fig. 3.

Effects of MPL diet on hepatic Wnt-PPARγ lipogenic protein expression. Protein expression levels of (A) LRP6, (B) Wnt3a, (C) β-catenin, (D) PPARγ, (E) DGAT1, (F) PLIN2, (G) SREBP1, (H) ACC, (I) SCD1, and (J) FAS. β-actin was used as a loading control. Data are presented as mean ± SD. Different letters indicate significant differences among groups (p < 0.05). LRP6, low-density lipoprotein receptor–related protein 6; Wnt3a, wingless-related integration site 3 A; PPARγ, peroxisome proliferator-activated receptor gamma; DGAT1, diacylglycerol acyltransferase 1; PLIN2, perilipin 2; SREBP1, sterol regulatory element-binding protein 1; ACC, acetyl-CoA carboxylase; SCD1, stearoyl-CoA desaturase 1; FAS, fatty acid synthase

Activation of the LRP6/Wnt signaling cascade stabilizes β-catenin, allowing its translocation into the nucleus, where it interacts with transcriptional regulators involved in lipid metabolism, including PPARγ. Previous studies have demonstrated that activation of Wnt/β–catenin signaling suppresses adipogenic transcriptional programs mediated by PPARγ and C/EBPα, thereby inhibiting adipogenesis and lipid accumulation (Ahmad et al., 2020). The increased expression of LRP6, Wnt3a, and total β-catenin in the MPL group is consistent with a partial restoration of Wnt/β-catenin signaling. These changes may be associated with the suppression of PPARγ-related lipid storage and the attenuation of hepatic lipid accumulation. Although increasing evidence implicates Wnt signaling in metabolic regulation, its precise role in the pathogenesis and progression of NAFLD remains incompletely understood. MPL or orlistat diets significantly downregulated the expression of key TG synthesis and lipid droplet formation regulators, including PPARγ, DGAT1, and PLIN2, compared with the HFHS group (Fig. 3D–F). In addition, dietary MPL significantly reduced the expression of the lipogenic transcription factor SREBP1 and the downstream lipogenic enzymes involved in de novo lipogenesis, including ACC, SCD1, and FAS (Fig. 3G–J).

Previous studies have reported that MPL attenuates hepatic steatosis by suppressing lipogenic factors, particularly SCD1 (Wat et al., 2009). However, the upstream molecular mechanisms responsible for these metabolic effects have remained largely unclear. Emerging evidence suggests that polar lipids can alter membrane lipid composition and influence membrane-associated signaling proteins. Baliyan et al. (2023) reported that MFGM supplementation significantly modified membrane lipidomic profiles, particularly by increasing phospholipids such as PC and PE. In addition, membrane phosphoinositide metabolism has been shown to play an essential role in activation of the membrane-associated LRP6/Wnt signaling pathway (Pan et al., 2008). Therefore, it is plausible that MPL-derived phospholipids modulate membrane lipid composition and influence membrane-associated proteins such as LRP6 and Wnt, thereby activating the Wnt–PPARγ signaling axis and suppressing hepatic lipid accumulation.

Effect of dietary MPL on gut microbiota diversity and composition

To investigate whether the beneficial effects of MPL supplementation on hepatic lipid metabolism were linked to modulation of the gut microbiota, 16 S rRNA sequencing analysis was performed using cecal samples collected from the mice. Alpha diversity analysis demonstrated that HFHS diet feeding significantly reduced gut microbial diversity compared with the NOR group, as evidenced by decreases in both the Shannon diversity index and the phylogenetic diversity (PD whole tree) (Fig. 4A and B). MPL diet slightly increased these diversity indices relative to the HFHS group, but no significant differences were observed, indicating that MPL treatment did not fully restore alpha diversity to the NOR group level. Principal coordinate analysis (PCoA) based on β-diversity indicated that the HFHS group formed a distinct cluster from the NOR group, suggesting that HFHS feeding markedly altered the structure of the gut microbial community (Fig. 4C). The MPL group, on the other hand, formed a separate cluster from both the HFHS group and the orlistat group, suggesting that MPL diet ameliorates the HFHS diet–induced dysbiosis in the gut microbiota, establishing a unique microbial configuration. PERMANOVA further supported this separation, with a significant overall group effect (R² = 0.309, p = 0.001) and significant separation between the HFHS and MPL groups (R² = 0.310, p = 0.004). Because the microbial profile of the orlistat group remained relatively similar to that of the HFHS group, it can be inferred that the microbiota-modulating effects of MPL may extend beyond simple inhibition of intestinal lipid absorption.

Fig. 4.

Effects of MPL diet on gut microbiota diversity and composition. Alpha diversity of gut microbiota assessed using the (A) Shannon diversity index and (B) phylogenetic diversity (PD whole tree). (C) Principal coordinates analysis (PCoA) plot based on unweighted UniFrac distances showing β-diversity among experimental groups and statistical significance of community separation was evaluated by PERMANOVA. (D) Relative abundance of gut microbiota at the phylum level. (E) Heatmap showing taxonomic composition at the family level. (F) Linear discriminant analysis effect size (LEfSe) identifying significantly enriched genus-level taxa among groups. (G) Differential abundance analysis between HFHS and MPL groups using Student’s t-test. Relative abundance of representative taxa associated with metabolic health, including (H) Akkermansia muciniphila, (I) Lactobacillus, (J) Romboutsia, and (K) Christensenellaceae. Data are presented as mean ± SD. Different letters indicate statistically significant differences among groups (p < 0.05)

At the phylum level, HFHS diet supplementation substantially altered the relative abundance of major bacterial taxa relative to normal diet supplementation. Specifically, there was an increase in the abundance of Bacillota, but a reduction in the abundance of Bacteroidota (Fig. 4D). MPL diet partially reversed these HFHS-induced changes, particularly by reducing the abundance levels of Bacillota. Furthermore, it significantly increased the abundance of bacteria belonging to Verrucomicrobiota, a phylum that includes Akkermansia muciniphila – a bacterium strongly associated with gut barrier integrity and metabolic health.

Heatmap analysis at the family level further demonstrated distinct differences in gut microbial composition among experimental groups (Fig. 4E). The HFHS and orlistat groups exhibited broadly similar gut microbial patterns, whereas MPL diet induced a clearly distinct microbial profile. These observations support the possibility that MPL directly modulates the microbial ecology of the gut through mechanisms independent of pancreatic lipase inhibition. Linear discriminant analysis effect size (LefSe) analysis at the genus level identified several discriminative bacterial taxa among the experimental groups (Fig. 4F). HFHS feeding was associated with the enrichment of Lactobacillus and Limosilactobacillus, whereas MPL diet selectively increased the abundance of Akkermansia and Romboutsia. Differential abundance analysis further confirmed significant differences in several key microbial genera between the HFHS and MPL groups (Fig. 4G).

Among the microbial alterations observed across the groups, the significant increase in the abundance of A. muciniphila noted in the MPL group warrants further discussion (Fig. 4H). A. muciniphila is a mucin-degrading bacterium that plays a critical role in maintaining intestinal barrier integrity and regulating metabolic homeostasis in the host. Reduced abundance of Akkermansia has been consistently observed in patients with NAFLD and obesity-associated metabolic disorders (Lee et al., 2020). Previous studies have demonstrated that A. muciniphila supplementation attenuates high-fat diet-induced hepatic steatosis, inflammation, and metabolic dysfunction by improving gut barrier integrity and suppressing endotoxin-mediated inflammation (Kim et al., 2020). Furthermore, administration of A. muciniphila in NAFLD mouse models reduced body weight gain, hepatic lipid accumulation, and liver injury while regulating bile acid metabolism through the intestinal FXR–FGF15 signaling axis (Wu et al., 2023). In addition, A. muciniphila has been reported to enhance mitochondrial fatty acid oxidation and gut–liver metabolic communication through modulation of L-aspartate metabolism and bile acid signaling (Rao et al., 2021). Therefore, the marked enrichment of A. muciniphila observed in the MPL group was associated with the attenuation of hepatic steatosis in the present study. However, the present study shows an association, rather than a causal relationship. Further studies are needed to determine whether A. muciniphila directly contributes to the metabolic benefits of dietary MPL treatment.

Another noteworthy difference in microbial population variation across groups is the significant increase in the abundance of Lactobacillus in the HFHS group – an increase not observed in the MPL-treated group (Fig. 4I). Several previous studies have reported increases in the abundance of bile acid–resistant Lactobacillus species in NAFLD and obesity models, potentially reflecting altered intestinal bile acid composition under high-fat dietary conditions (Tripathi et al., 2018; Zeng et al., 2013). By extension, the suppression of Lactobacillus enrichment in the MPL group, together with the increased abundance of A. muciniphila, may support improved intestinal bile acid homeostasis.

In addition to Akkermansia, MPL diet significantly increased the abundance of Romboutsia and members of the Christensenellaceae family compared with the HFHS and orlistat groups (Fig. 4J and K). Romboutsia lituseburensis and Christensenella minuta have been reported to improve lipid metabolism and attenuate hepatic steatosis, partly through enhanced production of short-chain fatty acids (SCFAs) and modulation of host energy metabolism (Mazier et al., 2021; Yin et al., 2023).

The findings of this study therefore demonstrate that dietary MPL beneficially reshapes the gut microbiota composition in HFHS-fed mice. In particular, the increased abundance of A. muciniphila and taxa reported to produce SCFAs may be associated with the improvement of NAFLD through modulation of intestinal barrier integrity, bile acid metabolism, inflammatory signaling, and hepatic lipid metabolism. However, future studies measuring microbial metabolites, including SCFAs and bile acids, are needed to clarify the functional links between MPL-induced microbial alterations and hepatic metabolic outcomes.

Conclusion

MPL diet effectively attenuated HFHS diet–induced metabolic abnormalities, including body weight gain, adiposity, and hepatic lipid accumulation, and liver injury. These beneficial effects were accompanied by activation of hepatic Wnt/β–catenin signaling, along with suppression of PPARγ-mediated lipid storage and SREBP1-driven de novo lipogenesis. In addition to regulating hepatic signaling, MPL diet markedly reshaped gut microbiota composition by increasing the abundance of A. muciniphila and SCFA-producing taxa such as Romboutsia and Christensenellaceae. These microbial alterations may be associated with improved intestinal barrier integrity, bile acid metabolism, and gut–liver metabolic communication, thereby further alleviating hepatic lipid accumulation and metabolic dysfunction. The present findings therefore suggest that MPL exerts protective effects against HFHS diet-induced NAFLD through coordinated regulation of hepatic lipid metabolism and gut microbial ecology.

Author contributions

Conceptualization: Kim H, Imm JY. Data curation: Kim H. Investigation: Kim H, Park D, Kwon YJ. Writing - original draft: Kim H. Writing - review & editing: Kim H, Imm JY.

Funding

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT and Future Planning (RS-2020-NR048142).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Conflict of interest

The authors declare no competing interests.