Effects of Lactiplantibacillus plantarum GBCC_F0227 on Lipid Accumulation and Lipid Metabolism in High-Fat Diet-Induced Obese Mice

Abstract

Obesity is characterized by excessive lipid accumulation and chronic inflammation that disrupt metabolic homeostasis. This study investigated the effects of Lactiplantibacillus plantarum GBCC_F0227 on lipid metabolism using differentiated 3T3-L1 adipocytes and a high-fat diet (HFD)-induced obese mouse model. In 3T3-L1 adipocytes, exposure to GBCC_F0227 culture supernatant was associated with reduced intracellular triglyceride accumulation and lower expression of lipid metabolism-related genes. Consistent with the in vitro observations, oral administration of GBCC_F0227 attenuated body weight gain and adipose tissue expansion in HFD-fed mice, together with lower expression of HFD-induced lipid metabolism-related genes (Pparg, Fabp4, Dgat2, and Cs) in white adipose tissue (WAT). GBCC_F0227 treatment also reduced the expression of inflammatory markers (Il-6, Il-1b) as well as Leptin, in epididymal WAT. In the liver, GBCC_F0227 administration lowered the expression of Pparg and Cd36 and reduced hepatic lipid accumulation. Collectively, these findings indicate that GBCC_F0227 administration is associated with coordinated changes in lipid accumulation and lipid metabolism-related gene expression in WAT and liver under HFD conditions. These results highlight the potential of GBCC_F0227 as a probiotic candidate for modulating obesity-associated lipid metabolic alterations.

Introduction

Obesity is a chronic metabolic disorder arising from an imbalance between energy intake and expenditure and is strongly associated with type 2 diabetes, dyslipidemia, nonalcoholic fatty liver disease, and cardiovascular disease [1]. In the obese state, adipocyte hypertrophy and hyperplasia contribute to the establishment of a chronic inflammatory milieu that disrupts adipokine homeostasis and insulin sensitivity, ultimately impairing systemic metabolic regulation [2, 3].

Adipocyte differentiation (adipogenesis) and lipid synthesis (lipogenesis) are governed by a complex network of transcription factors and metabolic enzymes. Among these, peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα) function as master regulators of adipogenesis, driving the expression of downstream adipogenic genes such as fatty acid-binding protein 4 (FABP4), which facilitates intracellular fatty acid trafficking and lipid storage [4]. Lipogenesis is further promoted by fatty acid synthase (FAS), a key enzyme in de novo fatty acid synthesis, and diacylglycerol acyltransferase (DGAT), which catalyzes the terminal step of triglyceride formation [5]. Citrate synthase (CS) supports lipogenic flux by increasing mitochondrial citrate availability for cytosolic lipid synthesis [6]. In parallel, CD36 enhances fatty acid uptake into adipocytes and hepatocytes, thereby promoting triglyceride accumulation and contributing to hepatic steatosis [7]. Coordinated upregulation of these pathways accelerates lipid deposition and drives adipose tissue expansion. In contrast, peroxisome proliferator-activated receptor alpha (PPARα) promotes lipid oxidation and mobilization, particularly in the liver, brown adipose tissue (BAT), and skeletal muscle [8]. Activation of PPARα induces mitochondrial and peroxisomal β-oxidation programs, thereby counteracting triglyceride storage. Lipolytic and oxidative regulators, including lipases and acyl-CoA dehydrogenases (ACADs), act in concert with PPARα to limit excessive lipid accumulation [9]. However, high-fat diet (HFD)-induced metabolic stress frequently suppresses PPARα activity, reducing lipid oxidative capacity and exacerbating adipocyte dysfunction [8].

White adipose tissue (WAT) also functions as an active endocrine organ [10]. Among its adipokines, leptin plays a central role in regulating appetite and energy expenditure; however, chronic HFD intake induces leptin resistance, leading to compensatory hyperleptinemia and metabolic dysregulation [11]. Persistent lipid overload and inflammation further aggravate adipocyte dysfunction and systemic insulin resistance [2]. Similarly, the liver undergoes progressive lipid accumulation under HFD conditions, developing pathological features characteristic of nonalcoholic fatty liver disease [12]. Collectively, these disturbances underscore the need for interventions that restore endocrine balance, reduce inflammation, and normalize adipose tissue metabolic pathways. Growing evidence indicates that the gut microbiota plays a critical role in regulating host energy metabolism, lipid handling, and immune responses [13]. Microbiota-derived metabolites and signaling pathways, including bile acid modulation, short-chain fatty acid production, and inflammatory signaling regulation, influence adipocyte differentiation, lipid storage, and inflammatory tone [14]. Through these mechanisms, several probiotic strains have been investigated for their potential to modulate lipid metabolism and attenuate obesity, although these effects are highly strain specific.

In our previous work, we identified Lactiplantibacillus plantarum GBCC_F0227 as a promising probiotic candidate with anti-obesity potential in HFD-fed mice. GBCC_F0227 exhibited strong α/β-hydrolase-associated lipase activity and significantly reduced epididymal adipose tissue mass, adipocyte hypertrophy, and circulating triglyceride levels [15]. Notably, while HFD feeding markedly suppressed the expression of adiponectin, a key anti-inflammatory adipokine, GBCC_F0227 administration significantly restored its levels [15]. These findings suggested that GBCC_F0227 modulates host lipid metabolism and adipose tissue homeostasis; however, the molecular mechanisms underlying these effects remained unclear. In particular, it has not been established how GBCC_F0227 regulates adipogenesis, lipogenesis, or lipolysis at the molecular level, nor whether these actions contribute to improvements in adipose inflammation, leptin dysregulation, or hepatic lipid accumulation.

Accordingly, the present study aimed to elucidate the lipid metabolic pathways targeted by GBCC_F0227 using complementary in vitro and in vivo approaches. We investigated whether GBCC_F0227 regulates adipocyte differentiation and lipid synthesis, modulates adipose inflammatory and leptin-related pathways, and attenuates hepatic steatosis under HFD conditions. Through this integrated analysis, we sought to define the mechanistic basis by which GBCC_F0227 mitigates excessive lipid accumulation and contributes to improved metabolic homeostasis in obesity.

Methods

Preparation of Bacterial Samples and Cell-Free Supernatants

Lactiplantibacillus plantarum GBCC_F0227 was isolated from fermented cabbage (sauerkraut) as previously described [15]. For in vivo experiments, GBCC_F0227 cells were cultured and lyophilized by Mediogen (Republic of Korea). The number of viable bacteria was determined as colony-forming units (CFU) to calculate the dosage of the powdered bacteria administered to mice. For in vitro experiments, GBCC_F0227 was cultured in MRS broth at 37°C for 16 h under anaerobic conditions using an anaerobic chamber (Don Whitley Scientific, UK). The culture broth was then centrifuged at 10,000 ×g for 10 min, and the resulting supernatant was filtered through a 0.22-μm syringe filter to obtain the cell-free supernatant. Preparation of the cell-free supernatant was performed by GI Longevity.

3T3-L1 Cell Culture and Differentiation

3T3-L1 preadipocytes were purchased from the Korean Cell Line Bank (KCLB, Republic of Korea) and maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; WELGENE, Republic of Korea) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, USA) and 1% penicillin-streptomycin (P/S; WELGENE) at 37°C in a humidified incubator with 5% CO₂.

For experiments, cells were seeded in 12-well plates at a density of 3 × 10⁴ cells per well and cultured for 2 days. Adipocyte differentiation was induced by culturing the cells for 3 days in differentiation medium, followed by an additional 3 days in insulin medium. The differentiation medium consisted of high-glucose DMEM supplemented with 10% heat-inactivated FBS and 1% P/S and containing 0.5 mM 3-isobutyl-1-methylxanthine (IBMX; Sigma-Aldrich, USA), 1 μM dexamethasone (Sigma-Aldrich), and 5 μg/ml insulin (Sigma-Aldrich). The insulin medium consisted of high-glucose DMEM supplemented with 10% heat-inactivated FBS, 1% P/S, and 5 μg/ml insulin. After differentiation, cells in the experimental group were treated with GBCC_F0227 culture supernatant for 2 days and subsequently maintained in insulin medium for an additional 3 days. In parallel, control cells were maintained in insulin medium for a total of 5 days under identical conditions. Fully differentiated adipocytes were confirmed by Oil Red O staining.

Cytotoxicity Assay

To determine non-cytotoxic concentrations of GBCC_F0227 culture supernatant, 3T3-L1 preadipocytes were seeded in 96-well plates at a density of 1 × 10⁴ cells per well and treated with GBCC_F0227 culture supernatant at final concentrations of 1%, 5%, 10%, and 20% for 2 days. After treatment, the medium was replaced with high-glucose DMEM containing Cell Counting Kit-8 (CCK-8) solution (Dojindo, Japan), and cells were incubated for 30 min at 37°C in a humidified atmosphere with 5% CO₂. The CCK-8 assay assesses cell viability based on the reduction of a water-soluble tetrazolium salt by dehydrogenases in metabolically active cells, producing a colorimetric signal proportional to the number of viable cells. Absorbance was measured at 450 nm using a SpectraMax iD3 microplate reader (Molecular Devices, USA).

Oil Red O Staining

Differentiated 3T3-L1 cells were washed with phosphate-buffered saline (PBS) and fixed with neutral buffered formalin for 30 min. After fixation, cells were rinsed with 60% (v/v) 2-propanol and stained with 0.4% Oil Red O solution (Sigma-Aldrich) for 10 min in the dark. Excess stain was removed by washing the cells four times with 60% 2-propanol. Images of stained cells were captured using a Nikon ECLIPSE Ts2 microscope equipped with a Digital Sight 1000 camera (Nikon, Japan). For quantitative analysis of lipid accumulation, Oil Red O dye was eluted by incubating the cells with 100% 2-propanol for 5 min, and the absorbance of the extracted solution was measured at 520 nm using a SpectraMax iD3 microplate reader (Molecular Devices, USA). Oil Red O staining of liver tissue was performed by OBEN (Republic of Korea). Image-based quantification of Oil Red O staining was conducted using ImageJ software (National Institutes of Health, USA) according to a previously published protocol [20]. Briefly, images were converted to 8-bit grayscale, threshold values were adjusted, and the stained area and mean gray intensity were quantified.

HFD-Induced Obesity Mouse Model

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC; approval number: GIB-25-06-005) and conducted at the GI Longevity animal facility. Five-week-old male C57BL/6 mice were purchased from Orient Bio (Republic of Korea) and housed under specific pathogen-free (SPF) conditions. After a 1-week acclimatization period, mice were randomly assigned to experimental three groups using a computer-generated randomization method based on bodyweight (n = 10 per group). To minimize potential cage effects, animals from each group were distributed across five independent cages with equal numbers of mice. All analyses were performed using ten mice per group, and no animals were excluded from the study. The experimental groups consisted of the following: (1) a normal chow diet (NCD; Inotiv, USA) group receiving phosphate-buffered saline (PBS), (2) a high-fat diet (HFD; 60% kcal from fat; Research Diets, USA) group receiving PBS, and (3) an HFD group receiving GBCC_F0227 at a dose of 5 × 109 CFU resuspended in PBS. Experimental treatments were administered once daily by oral gavage using a flexible disposable feeding tube (JEUNGDO Bio & Plant, Republic of Korea). Body weight was measured weekly, and body composition, including fat and lean mass, was assessed at weeks 4 and 8 using a Minispec LF50 body composition analyzer (Bruker, USA).

Glucose Tolerance Test (GTT)

The glucose tolerance test (GTT) was performed after 16-h fast. Mice were orally administered glucose at a dose of 1 g/kg body weight, and blood glucose levels were measured from the tail vein at 0, 30, 60, 90, and 120 min using an Auto-Chek Plus glucose meter (i-SENS, Republic of Korea). The area under the curve (AUC) for blood glucose levels over time was calculated using GraphPad Prism version 9 (GraphPad Software, USA).

Measurement of Adipose Tissue Weight and Size

After dissection, adipose tissues were immediately excised and weighed. Representative images of each adipose tissue depot were captured using a Canon EOS M50 camera. The area of each adipose tissue depot was quantified using ImageJ software (National Institutes of Health, USA).

Quantitative Real-Time PCR (qPCR) Analysis

Total RNA was extracted using the easy-spin Total RNA Extraction Kit (iNtRON Biotechnology, Republic of Korea). For complete tissue lysis, adipose tissues were homogenized in lysis buffer for 5 min with stainless steel beads (Qiagen, Netherlands) using a Mixer Mill MM400 (Retsch, Germany).

RNA concentration was measured using a NanoDrop 8000 spectrophotometer (Thermo Fisher Scientific, USA), and complementary DNA (cDNA) was synthesized using the SuPrimeScript cDNA Synthesis Kit (Genetbio, Republic of Korea). qPCR was performed using AccuPower 2× GreenStar qPCR Mix (Bioneer, Republic of Korea) on an Applied Biosystems QuantStudio 3 Real-Time PCR System (Applied Biosystems, USA). Relative gene expression levels were calculated using GAPDH as the housekeeping gene. Primer sequences used for real-time PCR are listed in Table 1.

Table 1.

Primer list used for quantitative real-time PCR.

Hematoxylin and Eosin (H&E) Staining

Hematoxylin and eosin (H&E) staining of liver tissue was performed by OBEN (Republic of Korea). Lipid droplet areas were quantified using ImageJ software (National Institutes of Health). Briefly, image thresholds were adjusted, and the white regions corresponding to lipid droplets were measured and expressed as a percentage of the total liver area.

Statistical Analysis

Statistical analyses were performed using GraphPad Prism version 9 (GraphPad Software, USA). Differences between two groups were assessed using an unpaired two-tailed Student’s t-test. Differences among three groups were assessed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test, or two-way ANOVA followed by Dunnett’s post hoc test, as appropriate. Repeated measurement data, including body weight, were analyzed using two-way ANOVA with time and treatment as factors. All data are presented as mean ± standard error of the mean (SEM). Statistical significance was defined as * P < 0.05, ** P < 0.01, and *** P < 0.001.

Results

GBCC_F0227 Culture Supernatant Reduces Lipid Accumulation and Lipid Metabolism-Related Gene Expression in 3T3-L1 Adipocytes

To investigate the effects of L. plantarum GBCC_F0227 on adipocyte function, differentiated 3T3-L1 adipocytes were treated with culture supernatant derived from GBCC_F0227. Preliminary dose-response analyses revealed cytotoxic effects at high concentrations (Fig. S1); therefore, a non-cytotoxic concentration of 5% was used for all subsequent in vitro experiments. Lipid accumulation was first assessed as an indicator of successful adipocyte differentiation. Differentiated adipocytes exhibited abundant intracellular lipid droplets, whereas treatment with GBCC_F0227 culture supernatant markedly reduced lipid staining intensity (Fig. 1A). Consistently, quantitative analysis of Oil Red O absorbance demonstrated a significant decrease in intracellular lipid content in GBCC_F0227-treated cells compared with untreated differentiated controls (Fig. 1B). To determine whether the observed reduction in lipid accumulation was associated with transcriptional changes, we examined the expression of genes related to lipid metabolism. Treatment with GBCC_F0227 culture supernatant significantly decreased the expression of adipogenesis-related genes, including Pparg, Cebpa, and Fabp4 (Fig. 1C). In addition, the expression of lipogenesis-related genes, such as Fas, Dgat2, and Cs, was also reduced in treated cells (Fig. 1D). In contrast, the expression of genes associated with lipolysis and fatty acid oxidation, including Hsl, Acad, and Ppara, was not significantly altered by GBCC_F0227 treatment (Fig. 1E). Together, these results indicate that exposure to GBCC_F0227 culture supernatant coincides with reduced lipid accumulation and altered expression of genes involved in adipogenesis and lipogenesis, while genes related to lipolysis and fatty acid oxidation remain unchanged.

Fig. 1. Effects of GBCC_F0227 on lipid accumulation and lipid metabolism-related gene expression in 3T3-L1 adipocytes.

Differentiated 3T3- L1 adipocytes were treated with culture supernatant from GBCC_F0227, and lipid accumulation and lipid metabolism-related gene expression were evaluated. (A) Representative images of Oil Red O staining. (B) Quantification of Oil Red O staining based on absorbance of the extracted dye. (C-E) Relative mRNA expression levels of genes related to adipocyte differentiation (C), lipid synthesis (D), and lipolysis (E) in 3T3-L1 adipocytes following GBCC_F0227 supernatant treatment (n = 5). Oil Red O staining data were analyzed using one-way ANOVA followed by Tukey’s post hoc test. qPCR data were analyzed using an unpaired two-tailed Student’s t-test. Non-diff., non-differentiated 3T3-L1 cells; Diff., differentiated 3T3-L1 cells; F0227, L. plantarum GBCC_F0227. Data are presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant.

GBCC_F0227 Treatment Attenuates Weight Gain and Improves Glucose Tolerance in HFD-Induced Obese Mice

To evaluate the in vivo effects of GBCC_F0227, obesity was induced in mice by feeding a 60% kcal high-fat diet (HFD) for 8 weeks, during which GBCC_F0227 was administered orally once daily. HFD-fed mice exhibited markedly increased body size relative to normal chow diet (NCD) controls, whereas GBCC_F0227-treated mice displayed visibly reduced adiposity compared with PBS-treated HFD mice (Fig. 2A). Weekly monitoring of body weight revealed that GBCC_F0227 administration progressively attenuated HFD-induced weight gain over the 8-week experimental period (Fig. 2B). Body composition analysis showed that HFD feeding markedly increased both fat mass and lean mass compared with NCD-fed mice (Fig. 2C and 2 D). GBCC_F0227 treatment significantly reduced fat mass at weeks 4 and 8 compared with PBS-treated HFD controls, while lean mass was not significantly altered (Fig. 2C and 2D). Accordingly, the fat-to-lean mass ratio was significantly lower in GBCC_F0227-treated mice at both time points (Fig. 2E). Glucose tolerance was further evaluated to assess whether these changes in body composition were accompanied by alterations in systemic glucose handling. During the glucose tolerance test (GTT), GBCC_F0227-treated mice exhibited a lower peak blood glucose level and a reduced area under the curve (AUC) compared with PBS-treated HFD controls (Fig. 2F). Taken together, these results demonstrate that GBCC_F0227 administration attenuates weight gain and improves glucose tolerance in HFD-fed mice.

Fig. 2. GBCC_F0227 reduces body weight gain and improves glucose tolerance in HFD-induced obese mice.

Mice were fed a normal chow diet (NCD) or a high-fat diet (HFD) and orally administered GBCC_F0227 or PBS daily for 8 weeks (n = 10). Body weight was monitored weekly. (A) Representative images of mice after 8 weeks of treatment. (B) Changes in body weight during the 8-week treatment period and cumulative body weight gain. (C-E) Body composition at weeks 4 and 8, including fat mass (C), lean mass (D), and fat-to-lean mass ratio (E). (F) Glucose tolerance test (GTT) showing blood glucose levels over time and the corresponding area under the curve (AUC). Body weight data were analyzed using two-way ANOVA followed by Dunnett’s post hoc test. Body composition data were analyzed using one-way ANOVA followed by Tukey’s post hoc test. GTT analysis was performed using two-way ANOVA followed by Dunnett’s post hoc test. F0227 denotes L. plantarum GBCC_F0227. Data are presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant.

GBCC_F0227/ Treatment Reduces Fat Accumulation in Adipose Tissues

To further characterize the tissue-specific effects of GBCC_F0227 on adipose tissue expansion, we assessed fat accumulation across three major adipose tissues: epididymal white adipose tissue (eWAT), inguinal white adipose tissue (iWAT), and brown adipose tissue (BAT). HFD feeding induced marked expansion of all adipose tissues compared with NCD controls (Fig. 3A). In particular, BAT from HFD-fed mice exhibited a beige-like appearance reflecting lipid accumulation, whereas BAT from GBCC_F0227-treated mice retained a darker brown coloration (Fig. 3A). Quantitative analysis showed that adipose tissue areas across all three depots were significantly smaller in GBCC_F0227-treated mice compared with PBS-treated HFD controls (Fig. 3B). Similarly, adipose tissue weights were markedly increased by HFD feeding but were significantly reduced following GBCC_F0227 administration (Fig. 3C). These findings are consistent with previous observations showing that GBCC_F0227 decreases adipocyte size in eWAT [15]. Taken together, these data indicate that GBCC_F0227 treatment reduces adipose tissue enlargement and lipid accumulation across multiple adipose depots, consistent with the overall reduction in adiposity observed in GBCC_F0227-treated mice.

Fig. 3. GBCC_F0227 reduces adipose tissue size and weight across white and brown adipose depots.

Epididymal white adipose tissue (eWAT), inguinal white adipose tissue (iWAT), and brown adipose tissue (BAT) were excised from mice fed an NCD or an HFD and orally administered GBCC_F0227 or PBS daily for 8 weeks (n = 10). (A) Representative images of adipose tissues. (B) Quantification of adipose tissue area based on image analysis. (C) Weights of individual adipose tissues. Adipose tissue area (B) and tissue weights (C) were analyzed using one-way ANOVA followed by Tukey’s post hoc test. F0227 denotes L. plantarum GBCC_F0227. Data are presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001.

GBCC_F0227 Alters Lipid Metabolism- and Inflammation-Related Gene Expression in WAT

Previous analyses showed that GBCC_F0227 reduced the size and weight of multiple adipose tissues, including eWAT, iWAT, and BAT (Fig. 3). Because BAT primarily contributes to thermogenesis rather than lipid storage, we examined whether these morphological changes were accompanied by alterations in thermogenic gene expression. Under HFD conditions, the expression of Ucp1 (uncoupling protein 1) and Ppargc1a (peroxisome proliferator-activated receptor gamma coactivator 1 alpha) was increased; however, neither gene was significantly altered by GBCC_F0227 treatment (Fig. S2). In addition, the expression of other key regulators of BAT thermogenic programming, including Prdm16 (PR domain-containing protein 16) and Ppara, was not significantly altered by HFD and remained unchanged following GBCC_F0227 treatment (Fig. S2). These findings suggest that the reduced lipid accumulation observed in BAT following GBCC_F0227 administration is unlikely to be mediated through classical thermogenic gene programs.

Given that WAT is the primary site of triglyceride storage, we next assessed the expression of genes related to lipid metabolism in eWAT and iWAT. Several lipid metabolism-related genes exhibited HFD-induced changes that were attenuated by GBCC_F0227 treatment (Fig. 4A). Among these, Pparg and Fabp4 expressions were increased by HFD, with GBCC_F0227 treatment being associated with lower expression, reaching statistical significance in iWAT. In addition, Dgat2 and Cs, which are involved in triglyceride synthesis pathways, were upregulated by HFD in both tissues and showed reduced expression following GBCC_F0227 treatment. In contrast, the expression of Acad and Ppara, which are related to fatty acid oxidation, was altered by HFD but was not significantly affected by GBCC_F0227 in either eWAT or iWAT (Fig. 4A).

Fig. 4. GBCC_F0227 modulates lipid metabolism- and inflammation-related gene expression in WAT.

eWAT and iWAT were collected from mice fed an NCD or an HFD and orally administered GBCC_F0227 or PBS for 8 weeks (n = 10). Gene expression was analyzed by quantitative real-time PCR (qPCR) and normalized to Gapdh. (A) Relative mRNA expression of lipid metabolism-related genes. (B) Relative mRNA expression of pro-inflammatory cytokines. (C) Relative Leptin mRNA expression. qPCR data were analyzed using one-way ANOVA followed by Tukey’s post hoc test. F0227 denotes L. plantarum GBCC_F0227. Data are presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant.

Because excessive lipid accumulation is commonly associated with inflammatory activation in WAT, we next examined cytokine expression. In eWAT, Il6 and Il1b expressions were significantly elevated by HFD and were reduced following GBCC_F0227 treatment (Fig. 4B). In iWAT, both cytokines were similarly induced by HFD but were not significantly altered by GBCC_F0227 (Fig. 4B). Leptin, which is markedly elevated under HFD feeding, showed reduced expression in both tissues following GBCC_F0227 treatment (Fig. 4C).

Collectively, these results indicate that although GBCC_F0227 administration is associated with reduced lipid accumulation in both WAT and BAT, transcriptional changes were primarily observed in WAT. GBCC_F0227 treatment was associated with coordinated alterations in lipid metabolism- and inflammation-related gene expression in WAT, whereas thermogenesis-related gene expression in BAT remained largely unchanged.

GBCC_F0227 Treatment Reduces Lipid Accumulation and Alters Lipid Metabolism-Related Gene Expression in HFD-Fed Mouse Liver

Because GBCC_F0227 treatment was associated with reduced lipid accumulation and altered gene expression in eWAT (Fig. 3 and Fig. 4), we next examined whether similar changes were observed in the liver. HFD-fed mice developed prominent hepatic steatosis characterized by abundant lipid droplet accumulation, whereas NCD-fed mice displayed normal hepatic morphology (Fig. 5A). In contrast, livers from GBCC_F0227-treated mice showed visibly reduced lipid deposition (Fig. 5A). Quantitative analysis further demonstrated a significant decrease in lipid droplet area in the GBCC_F0227-treated group (Fig. 5B). Consistent with these findings, Oil Red O staining revealed weaker lipid accumulation in livers from GBCC_F0227-treated mice compared with HFD controls (Fig. S3).

Fig. 5. GBCC_F0227 reduces hepatic lipid accumulation and alters lipid metabolism-related gene expression in HFD-induced obese mice.

Liver tissues were excised from mice fed an NCD or an HFD and orally administered GBCC_F0227 or PBS for 8 weeks (n = 10). Paraffin-embedded liver sections were prepared and subjected to hematoxylin and eosin (H&E) staining. Hepatic gene expression was analyzed by qRT-PCR and normalized to Gapdh. (A) Representative H&E-stained liver sections. Yellow dotted boxes indicate regions shown at higher magnification on the right. Scale bars represent 500 μm (left) and 100 μm (right). (B) Quantification of lipid droplet area expressed as a percentage of total liver area. (C) Relative mRNA expression of genes related to hepatic lipid metabolism. Quantitative image analysis and qPCR data were analyzed using one-way ANOVA followed by Tukey’s post hoc test. F0227 denotes L. plantarum GBCC_F0227. Data are presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant.

To determine whether these histological changes were accompanied by transcriptional alterations, we analyzed the mRNA levels of genes related to hepatic lipid uptake, synthesis, and oxidation. Pparg, which is implicated in hepatic lipid metabolism, was upregulated by HFD and showed significantly lower expression following GBCC_F0227 treatment (Fig. 5C). Similarly, Cd36, a fatty acid transporter involved in hepatic lipid uptake, was reduced in the GBCC_F0227-treated group compared with HFD controls (Fig. 5D). In contrast, Ppara expression was increased by HFD in the liver and was not significantly altered by GBCC_F0227 treatment (Fig. 5E). Collectively, these results indicate that GBCC_F0227 treatment is associated with reduced hepatic lipid accumulation and coordinated changes in the expression of genes related to lipid uptake and storage, whereas the expression of genes involved in fatty acid oxidation remains largely unchanged. These hepatic gene expression patterns are directionally consistent with the changes observed in WAT.

Discussion

Our previous work demonstrated that L. plantarum GBCC_F0227 is associated with reduced weight gain and adiposity in HFD mice [15]. However, the molecular features accompanying these phenotypic changes had not been systematically examined. In the present study, we investigated tissue-level and transcriptional changes associated with GBCC_F0227 administration and evaluated how these changes relate to lipid accumulation in adipose tissue and liver under HFD conditions.

In vitro, exposure of differentiated 3T3-L1 adipocytes to GBCC_F0227 culture supernatant was associated with reduced lipid accumulation and lower expression of several genes commonly used as markers of adipocyte lipid handling, including Pparg, Cebpa, Fabp4, Fas, Dgat2, and Cs (Fig. 1A-1D). Although Pparg, Cebpa, and Fabp4 are well-established regulators of adipocyte differentiation, the cells used in this study were fully differentiated adipocytes. In mature adipocytes, these genes also play important roles in lipid storage, fatty acid trafficking, and triglyceride synthesis, supporting their relevance as indicators of adipocyte lipid metabolic status rather than differentiation per se [16, 17]. In contrast, genes related to lipolysis and fatty acid oxidation (Hsl, Acad, Ppara) were not markedly altered (Fig. 1E). Together, these findings indicate that GBCC_F0227 culture supernatant is associated with transcriptional changes accompanied by reduced lipid deposition in adipocytes. However, because these experiments were performed using cell-free supernatants without condition-matched controls, the present data does not allow definitive attribution of these effects to specific GBCC_F0227-derived metabolites. Nevertheless, consistent with the in vitro observations, oral administration of GBCC_F0227 was associated with attenuated body-weight gain and reduced adipose tissue expansion in HFD-fed mice (Figs. 2A-2E and 3). In iWAT, HFD-induced increases in the expression of several lipid metabolism-related genes, including Pparg, Fabp4, Dgat2, and Cs, were attenuated following GBCC_F0227 treatment (Fig. 4A). Similar trends were also observed in eWAT. Taken together, these findings support an association between GBCC_F0227 administration and altered lipid metabolic gene expression in adipose tissue under HFD conditions.

Obesity was associated with divergent regulation of fatty acid oxidation-related genes in WAT, characterized by reduced Ppara expression and increased Acad expression (Fig. 4A). Downregulation of Ppara under obese conditions has been widely reported and is considered a hallmark of impaired transcriptional control of lipid oxidation during chronic nutrient excess [18]. In contrast, increased Acad expression in WAT and liver has also been observed in obesity [19, 20] and is generally interpreted as a compensatory response to elevated fatty acid availability rather than an indication of fully restored oxidative capacity. Notably, GBCC_F0227 treatment did not significantly alter the expression of these genes, suggesting that its effects are unlikely to involve direct modulation of fatty acid oxidation pathways in adipose tissue. In the liver, HFD feeding induced prominent lipid accumulation accompanied by increased expression of genes related to lipid uptake and storage, including Pparg and Cd36 (Fig. 5C and 5D). In contrast to WAT, hepatic Ppara expression was increased by HFD (Fig. 5E), consistent with previous reports [21, 22]. GBCC_F0227 treatment was associated with reduced hepatic lipid deposition and lower expression of lipid uptake- and storage-related genes, including Pparg and Cd36, whereas Ppara expression remained unchanged (Fig. 5C-5E). These findings suggest that GBCC_F0227 administration is linked to changes in hepatic lipid handling that favor reduced lipid influx and storage. In BAT, HFD feeding increased both Ucp1 and Ppargc1a without concomitant changes in upstream thermogenic regulators such as Prdm16 and Ppara, suggesting partial or compensatory activation of thermogenic gene expression. GBCC_F0227 treatment did not further alter thermogenesis-related gene expression (Fig. S2).

In addition to lipid-related changes, GBCC_F0227 treatment was associated with reduced expression of pro-inflammatory cytokines (Il-6 and Il-1b) in eWAT, whereas similar effects were not observed in iWAT (Fig. 4B). This tissue-specific pattern underscores the heterogeneity of inflammatory responses within adipose depots. GBCC_F0227 treatment also significantly reduced Leptin expression in both WAT depots (Fig. 4C), suggesting modulation of obesity-associated leptin dysregulation. Collectively, these results demonstrate that GBCC_F0227 administration is associated with coordinated changes in lipid accumulation and lipid metabolism-related gene expression across WAT and liver in HFD-fed mice. Rather than indicating activation of lipid oxidation, the observed patterns are consistent with a reduction in lipid overload at the tissue level. While these findings provide insight into host-microbe associations linked to adiposity in a mouse model, further studies incorporating functional metabolic measurements and controlled mechanistic analyses will be required to define the molecular mediators involved and to determine whether similar processes are conserved in humans.

Conclusion

This study demonstrates that Lactiplantibacillus plantarum GBCC_F0227 administration is associated with reduced lipid accumulation and altered lipid metabolism-related gene expression in WAT and liver under HFD conditions. Rather than being linked to enhanced lipolysis, the observed effects were characterized by coordinated changes in gene related to lipid storage and uptake, accompanied by reduced inflammatory and leptin gene expression in eWAT. Collectively, these findings suggest that GBCC_F0227 is associated with modulation of obesity-related lipid metabolic patterns in a diet-induced obese mouse model. supporting its potential as a food-derived probiotic candidate for further investigation.

Supplemental Materials

Supplementary data for this paper are available on-line only at http://jmb.or.kr.