In this experiment, wild-type AB strain zebrafish were purchased from the National Zebrafish Resource Center (Wuhan, China). L. fermentum E15 was isolated from the feces of healthy infants and preserved at the China General Microbiological Culture Collection Center (CGMCC) with the accession number 22009.

De-Man Rogosa and Sharpe (MRS) medium was purchased from Qingdao Hi-Tech Industrial Park Hope Bio-Technology Co., Ltd. (Qingdao, China). The RNA rapid extraction kit and FastQuant RT Kit (with gDNase) were obtained from Tiangen Biotech Co., Ltd. (Beijing, China). Sodium isovalerate was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Commercial assay kits for total protein (BCA), triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), aspartate aminotransferase (AST), alanine aminotransferase (ALT), superoxide dismutase (SOD) and malondialdehyde (MDA) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Cholesterol, pertussis toxin (PTX), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), and Oil Red O were purchased from Sigma-Aldrich. All reagents of high-performance liquid chromatography (HPLC) grade and short-chain fatty acid metabolite standards were purchased from Sigma-Aldrich.

To observe the macroscopic morphology of L. fermentum E15, the strain was streaked onto MRS solid medium and incubated at 37°C for 48 h in an anaerobic workstation (E500G, GeneScience, United States). The strain was then identified using Gram staining.

Lactobacillus fermentum E15 was inoculated in MRS medium (5 mL liquid medium, autoclaved at 121°C for 15 min) and cultured at 37°C with gentle shaking at 100 rpm for 24 h. Following this, the inoculum was prepared by inoculating 1 mL of the culture to a 50 mL centrifuge tube containing 40 mL of MRS broth medium. The centrifuge tube was then incubated at 37°C and 100 rpm for 24 h. After incubation, bacterial pellet was collected by centrifuging at 10000 rpm for 5 min, followed by two washes with phosphate-buffered saline (PBS) and resuspension in E3 water (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, and 0.33 mM MgSO₄). The bacterial pellet was adjusted to final concentrations of 1 × 10⁴ CFU/mL, 1 × 10⁵ CFU/mL, and 1 × 10⁶ CFU/mL, respectively.

The normal diet (AP100) for zebrafish larvae supplemented with 12% crude fat and 50% crude protein was purchased from Zeigler (Pennsylvania, United States). After the mixture of cholesterol ether solution with the normal diet, the ether was evaporated to obtain the HCD diet and the final concentration of cholesterol in the HCD diet was determined to be 4% (w/w).

Wild-type AB strain adult zebrafish were maintained in a zebrafish breeding system (28.5°C, pH 7.5, conductivity 500–550 μS/cm, 14:10 h light–dark cycle). Afterwards, zebrafish embryos were obtained from those adult zebrafish through natural mating, and then incubated in E3 water (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, and 0.33 mM MgSO₄) and cultured in an incubator at a constant temperature of 28°C. Five days post fertilization (dpf) wild-type AB strain zebrafish larvae were randomly divided into 5 groups with 100 zebrafish larvae in each group, followed by incubation in a 1 L water tank. There were 5 treatments, namely, normal diet (control), 4% HCD (HCD group), 4% HCD + 1 × 10⁴ CFU/mL L. fermentum E15 (1 × 10⁴ CFU/mL L. fermentum E15 group), 4% HCD + 1 × 10⁵ CFU/mL L. fermentum E15 (1 × 10⁵ CFU/mL L. fermentum E15 group), or 4% HCD + 1 × 10⁶ CFU/mL L. fermentum E15 (1 × 10⁶ CFU/mL L. fermentum E15 group). After 5 dpf, the control group was fed with a normal diet. The HCD group and the L. fermentum E15 group were fed with a HCD for a period of 7 days (5–12 dpf). In the following 14-day period (12–26 dpf), the control group continued to be fed with a normal diet, while the HCD group continued to be fed with a HCD. Meanwhile, the L. fermentum E15 group was fed with a HCD supplemented with L. fermentum E15 for 14 days (12–26 dpf). All the groups were fed with the same amount of feed 30 mg/d (twice a day), as described in Figure 1A, and the residual food was removed after 1 h of feeding. All the zebrafish experiments were approved by Animal Welfare and Ethics Committee of Guangdong Human Microecology Engineering Technology Research Center’s Laboratory (approval number: IACUC MC 0329-01-2024).

After the intervention, 20 zebrafish larvae were randomly collected from each group, washed twice with E3 water, and fixed in 4% paraformaldehyde solution for 24 h. These zebrafish larvae were subjected to 25, 50, 75, and 100% 1,2-propanediol gradient dehydration with each gradient for 25 min. Afterwards, these zebrafish were stained with Oil Red O for 48 h and then destained with 1,2-propanediol for 30 min, followed by two washes with PBS. A stereomicroscope (SZ680) was used to capture the images and analyze the lipid accumulation in the zebrafish. The integrated optical density (IOD) values were quantified using ImageJ software to determine the relative quantitative index of lipid accumulation.

After 14 days of treatment with L. fermentum E15, 20 zebrafish larvae were randomly selected from each group and anesthetized using tricaine (0.1 g/L). The body length of zebrafish larvae was measured using a stereomicroscope (SZ680), while the weight of zebrafish larvae was measured using an analytical balance. Subsequently, the BMI value (mg/mm²) was calculated as the ratio of the measured weight to body length.

For each group, 10 zebrafish larvae were randomly selected as one sample, and 3 samples were prepared for subsequent assessments. Briefly, zebrafish larvae were anesthetized in a tricaine solution (0.1 g/L) and subsequently euthanized by immersion in ice-cold E3 water bath as previously described (25). Afterwards, the larvae were washed with pre-chilled PBS and then homogenized in 500 μL of physiological saline. The supernatant was collected by centrifugation at 4°C and 13,000 rpm g for 10 min. The levels of TG, TC, LDL-C, HDL-C, AST activity, ALT activity, SOD activity and MDA were measured using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, China) following the manufacturer’s instructions.

The zebrafish larvae were fixed with 4% paraformaldehyde solution for 24 h before being processed according to standard procedures for H&E staining. Afterwards, the zebrafish larvae were embedded in paraffin then sectioned and stained with H&E. Microscopy was performed to observe the pathological changes in zebrafish liver tissue using a microscope (Nikon Eclipse E100).

The ROS levels in zebrafish larvae were assessed using the fluorescent probe dye DCFH-DA (Sigma-Aldrich). Fourteen days after the treatment with L. fermentum E15, 10 zebrafish larvae were randomly collected from each group and placed in a 6-well cell culture plate with 5 mL DCFH-DA (5 μM) solution into each well. All the samples were incubated in the dark at 28.5°C for 1 h followed by three washes with E3 water and observation under a fluorescence microscope (Leica DMi8). The statistical analysis of fluorescence intensity of individual zebrafish larvae was performed using the ImageJ software.

After the intervention, 20 zebrafish larvae were randomly selected from each group and then subjected to euthanasia for total RNA extraction using Trizol reagent (Invitrogen, United States) after the intervention. Reverse transcription was conducted using HiScript II Q RT SuperMix (Vazyme, China) to synthesize cDNA. The resultant cDNA served as a template for qPCR analysis on a StepOnePlus real-time fluorescence quantitative PCR system using ChamQ Universal SYBR qPCR Master Mix (Vazyme, China). The PCR cycling protocol consisted of pre-denaturation at 95°C for 3 min, 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 15 s, extension at 60°C for 15 s, and a final extension at 60°C for 2 min. The expression levels of target mRNAs were calculated using the 2^-ΔΔCt method by normalizing to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the corresponding primers are listed in Table 1.

Lactobacillus fermentum E15 was inoculated into MRS broth and cultured at 37°C for 24 h, followed by centrifugation (10,000 rpm, 5 min) to collect the supernatant. Afterwards, 10 μL of internal standard (L-2-chlorophenylalanine, 0.3 mg/mL, prepared in methanol) was added into 100 μL of fermentation supernatant and vortexed for 10 s. 300 μL of methanol-acetonitrile (2:1, v/v) was added and vortexed for 1 min. Ultrasonic extraction was performed in ice water bath for 10 min and maintained at −20°C for 30 min. The supernatant was then collected by centrifugation at 13000 rpm, 4°C for 15 min. Next, 200 μL of the supernatant was transferred to an LC–MS/MS injection vial. The concentrations of SCFAs including acetic acid, propionic acid, isobutyric acid, butyric acid, 2-methylbutyric acid, isovaleric acid, valeric acid, 2,2-dimethylbutyric acid, 2-ethylbutyric acid, 3,3-dimethylbutyric acid, 2-methylhexanoic acid, 3-methylhexanoic acid, 4-methylhexanoic acid in the culture supernatant were analyzed using LC - MS/MS.

A total of 400 wild-type AB strain zebrafish larvae at 5 dpf were randomly divided into four groups (100 larvae in each group) and incubated in 1 L tanks. There are four groups in this experiment: the normal diet group, the normal diet+ L. fermentum E15 group, the HCD group, the HCD + L. fermentum E15 group. Starting from 5 dpf, the normal diet group was fed a normal diet. The normal diet+ L. fermentum E15 group was fed a normal diet together with 1 × 10⁶ CFU/mL L. fermentum E15. The HCD group was fed a HCD. The HCD + L. fermentum E15 group was fed a HCD together with 1 × 10⁶ CFU/mL L. fermentum E15. Each group was fed an equal amount of feed, 30 mg/day (twice daily), and residual food was removed one hour after feeding. After 10 days, 20 zebrafish larvae in each group were randomly selected as one sample, and 3 samples were prepared for assessing SCFAs. Thirty zebrafish were put into a 1.5 mL EP tube with 400 μL of extract solution (methanol-acetonitrile-water = 2:2:1, v/v/v) and forty small steel balls. The EP tube was placed in the refrigerator at −80°C for 5 min, and then the contents of the EP tube were ground in the grinder (60 Hz, 2 min). The sample was centrifuged at low temperature for 15 min (13,000 rpm, 4°C) to collect the supernatant. The acetic acid, propionic acid, butyric acid, and isovaleric acid in the supernatant were detected using LC–MS/MS.

A total of 420 wild-type AB strain zebrafish larvae at 5 dpf were randomly divided into six groups (70 zebrafish larvae in each group) then incubated in 1 L water tanks. These six groups comprised a series of treatments, including the control group (fed a normal diet), the HCD group (fed a HCD), the L. fermentum E15 group (fed a HCD and 1 × 10⁶ CFU/mL L. fermentum E15), the L. fermentum E15+ pertussis toxin (PTX) group (fed a HCD, 1 × 10⁶ CFU/mL L. fermentum E15 and 50 ng/mL PTX), the isovaleric acid group (fed a HCD and 100 μM isovaleric acid), and the isovaleric acid + PTX group (fed a HCD, 100 μM isovaleric acid and 50 ng/mL PTX). After 5 dpf, the control group was fed a normal diet (30 mg/d, twice a day), while the other groups were fed a HCD (30 mg/d, twice a day) for 7 days (5–12 dpf). In the following 14-day period (12–26 dpf), the control group and HCD group continued to be fed as described above. Meanwhile, the L. fermentum E15 group was fed a HCD with the addition of L. fermentum E15 (30 mg/d, twice a day). The L. fermentum E15 + PTX group was fed a HCD, 1 × 10⁶ CFU/mL of L. fermentum E15, and 50 ng/mL of PTX. The isovaleric acid group was fed a HCD and 100 μM of isovaleric acid. The isovaleric acid+PTX group was fed a HCD, 100 μM of isovaleric acid, and 50 ng/mL PTX. All the groups had residual food removed after 1 h of feeding. After the intervention, the lipid accumulation in the zebrafish body was evaluated using Oil Red O staining. Meanwhile, the RT-qPCR was employed to analyze the expression of GPR43 and leptin A mRNA.

All the data were expressed as mean values ± standard deviation (SD), and the statistical analyses were performed using SPSS (version 20.0). One-way ANOVA followed by the Tukey multiple comparisons test was used for comparison between multiple groups. A p value<0.05 was considered as statistically significant. The graphs were obtained using GraphPad Prism 5 software.

Lactobacillus fermentum E15 grew well on MRS medium. After 48 h of incubation, the bacterial colonies appeared circular, with a milky, waxy, and white surface, with entire margin (Figure 1B). Gram staining confirmed that the strain is Gram-positive (Figure 1C).

No obvious lipid accumulation can be observed in the liver and blood vessels of the control group, whereas a substantial accumulation of lipid droplets can be detected in those of the HCD group (Figure 2A). Notably, the accumulation of red lipid droplets in the liver and blood vessels of zebrafish larvae treated with L. fermentum E15 was significantly reduced compared with the HCD group. Meanwhile, the DOI values in the L. fermentum E15 group (1 × 10⁴ CFU/mL group: 2955.18 ± 418.74; 1 × 10⁵ CFU/mL group: 1984.73 ± 344.58; 1 × 10⁶ CFU/mL group: 1536.17 ± 354.87) were significantly lower than those in the HCD group (3881.41 ± 397.32) (p < 0.001; Figure 2B). Additionally, the BMI was increased by HCD diet (0.029 ± 0.002 mg/mm²) as compared with the control group (0.023 ± 0.004 mg/mm²) (p < 0.001), suggesting that obesity was induced (Figure 2C, Table S1). The L. fermentum E15 treatment (1 × 10⁵ CFU/mL group: 0.027 ± 0.002 mg/mm²; 1 × 10⁶ CFU/mL group: 0.027 ± 0.001 mg/mm²) significantly inhibited the increase of BMI as compared to the HCD group (p < 0.05). The liver histopathological observations showed that the liver tissue of the control group was intact with dense arrangement of liver cells (Figure 2D). Meanwhile, many lipid vacuoles, deformed hepatocytes, and irregular arrangements in the liver tissue were observed in the HCD group, and the liver cells lost normal features with irregular arrangement. In contrast, there was a significant amelioration in the L. fermentum E15 (1 × 10⁶ CFU/mL) group, which showed a significant reduction of lipid vacuoles in the liver issue with normal morphology and arrangement of liver cells. These results suggested that L. fermentum E15 can remarkably improve the HCD diet-induced lipid accumulation in the liver and blood vessels.

The lipid-lowering effect of L. fermentum E15 was further evaluated by assessing the levels of TG, TC, LDL-C and HDL-C. As compared with the control group (TG: 1.910 ± 0.113 mmol/g prot; TC: 0.594 ± 0.100 mmol/g prot; LDL-C: 0.095 ± 0.016 mmol/g prot), higher levels of TG (4.916 ± 0.427 mmol/g prot), TC (1.260 ± 0.042 mmol/g prot) and LDL-C (0.186 ± 0.005 mmol/g prot) were detected in the HCD group (p < 0.001), while the level of HDL-C (0.009 ± 0.002 mmol/g prot) was significantly lower than in the control group (0.023 ± 0.004 mmol/g prot) (p < 0.01) (Figures 3A–D). After the intervention of L. fermentum E15, compared with the HCD group, the levels of TG (1 × 10⁵ CFU/mL group: 3.182 ± 0.446 mmol/g prot; 1 × 10⁶ CFU/mL group: 2.753 ± 0.186 mmol/g prot), TC (1 × 10⁴ CFU/mL group: 1.008 ± 0.041 mmol/g prot; 1 × 10⁵ CFU/mL group: 0.941 ± 0.076 mmol/g prot; 1 × 10⁶ CFU/mL group: 0.682 ± 0.083 mmol/g prot), and LDL-C (1 × 10⁴ CFU/mL group: 0.155 ± 0.011 mmol/g prot; 1 × 10⁵ CFU/mL group: 0.143 ± 0.014 mmol/g prot; 1 × 10⁶ CFU/mL group: 0.116 ± 0.013 mmol/g prot) were significantly reduced (p < 0.05), and the level of HDL-C (1 × 10⁵ CFU/mL group: 0.017 ± 0.002 mmol/g prot; 1 × 10⁶ CFU/mL group: 0.019 ± 0.003 mmol/g prot) was increased significantly (p < 0.05). Meanwhile, the biochemical indicators ALT and AST were also assessed to reflect liver function. The activity of ALT (31.972 ± 2.379 U/g prot) and AST (57.963 ± 4.214 U/g prot) in zebrafish larvae was significantly increased by feeding HCD as compared to the control group (ALT: 11.522 ± 0.737 U/g prot; AST: 37.432 ± 3.618 U/g prot) (p < 0.05). As compared to the HCD group, the ALT (1 × 10⁴ CFU/mL group: 27.405 ± 1.340 U/g prot; 1 × 10⁵ CFU/mL group: 19.764 ± 0.889 U/g prot; 1 × 10⁶ CFU/mL group: 16.508 ± 1.289 U/g prot) and AST (1 × 10⁵ CFU/mL group: 45.659 ± 2.560 U/g prot; 1 × 10⁶ CFU/mL group: 43.881 ± 2.971 U/g prot) activities of zebrafish larvae in L. fermentum E15 group were significantly decreased (p < 0.05), suggesting that L. fermentum E15 can ameliorate lipid metabolic disorder and liver function abnormalities (Figures 3E,F).

To determine the association between the alterations in the aforementioned biochemical parameters and the alterations in gene expression, the mRNA expression levels of SREBP-1, PPAR-γ, Fasn, and PPAR-α lipid metabolism-related molecules were detected. The RT-qPCR analysis showed that compared with the control group, mRNA expression levels of SREBP-1 (2.81 fold), PPAR-γ (3.16 fold) and Fasn (3.07 fold) in HCD-induced zebrafish larvae were significantly increased (p < 0.001) (Figures 4A–C), while those of PPAR-α (0.52 fold) were significantly decreased (p < 0.001) (Figure 4D). In addition, the zebrafish in 1 × 10⁴ CFU/mL L. fermentum E15 group (SREBP-1: 2.24 fold; PPAR-γ: 2.66 fold), 1 × 10⁵ CFU/mL L. fermentum E15 group (SREBP-1: 2.04 fold; PPAR-γ: 1.89 fold), and 1 × 10⁶ CFU/mL L. fermentum E15 group (SREBP-1: 1.36 fold; PPAR-γ: 1.48 fold) showed a significant reduction in mRNA expression levels of SREBP-1 and PPAR-γ as compared to the HCD group (p < 0.01; Figures 4A,B). The mRNA expression levels of Fasn showed a significant reduction in the L. fermentum E15 group (1 × 10⁵ CFU/mL group: 2.41 fold; 1 × 10⁶ CFU/mL group: 1.78 fold) compared with the HCD group (p < 0.01) (Figure 4C). Moreover, the L. fermentum E15 groups had a significantly higher mRNA expression level of PPAR-α (1 × 10⁴ CFU/mL group: 0.67 fold; 1 × 10⁵ CFU/mL group: 0.83 fold; 1 × 10⁶ CFU/mL group: 0.90 fold) compared with the HCD group (p < 0.05; Figure 4D). These results showed that L. fermentum E15 probably alleviated HCD-induced hyperlipidemia by regulating lipid metabolism disorder.

High-fat dietary intake is highly associated with oxidative stress, including increased ROS production and severe oxidative damage to liver tissue (26). The fluorescence intensity of DCFH-DA staining showed that the ROS levels in zebrafish larvae in the HCD group (470.30 ± 49.57%) were significantly increased as compared to the control group (100.00 ± 23.98%) (p < 0.001; Figures 5A,B). The abdominal and liver tissues of zebrafish larvae in the HCD group showed an high ROS level, which was possibly attributed to the lipid accumulation in these regions. However, L. fermentum E15 significantly reduced the ROS level (1 × 10⁴ CFU/mL group: 169.10 ± 10.66%; 1 × 10⁵ CFU/mL group: 155.66 ± 26.03%; 1 × 10⁶ CFU/mL group: 114.14 ± 24.78%) in the HCD-fed zebrafish larvae as compared to the HCD group (p < 0.001; Figures 5A,B). Previous study showed that SOD belongs to an essential antioxidant enzyme that plays a critical role in mitigating the negative effects of free radicals in the body. Conversely, MDA is a cytotoxic molecule that induces cross-linking and polymerization of macromolecules including proteins (24). The effects of L. fermentum E15 on SOD activity and MDA levels in the HCD-fed zebrafish larvae were further investigated. As compared to the control group (SOD: 45.42 ± 1.31 U/mg prot; MDA: 6.69 ± 1.38 nmol/mg prot), a dramatic reduction of SOD activity (14.89 ± 2.09 U/mg prot) was detected in the HCD group (p < 0.001), while the MDA level (27.21 ± 2.65 nmol/mg prot) was significantly higher (p < 0.001; Figures 5C,D). The L. fermentum E15 group showed a significantly higher SOD activity (1 × 10⁴ CFU/mL group: 21.48 ± 3.54 U/mg prot; 1 × 10⁵ CFU/mL group: 32.54 ± 1.63 U/mg prot; 1 × 10⁶ CFU/mL group: 36.17 ± 3.06 U/mg prot) and a lower MDA level (1 × 10⁴ CFU/mL group: 118.34 ± 2.86 nmol/mg prot; 1 × 10⁵ CFU/mL group: 12.98 ± 2.04 nmol/mg prot; 1 × 10⁶ CFU/mL group: 10.54 ± 0.93 nmol/mg prot) as compared to the HCD group (p < 0.05). These results suggested that L. fermentum E15 can mitigate the oxidative damage in the HCD-fed zebrafish larvae.

SCFAs are the main metabolic products of gut microbiota that serve as an important energy source for colonic epithelial cells (27) and play a critical role in inhibiting the occurrence of obesity by regulating host energy metabolism and gut homeostasis (27, 28). Thus, the content of SCFAs in the culture supernatant of L. fermentum E15 was assessed using LC–MS/MS targeted metabolomics technology. The levels of acetic acid (1711.97 ± 72.02 ng/mL), propionic acid (79.68 ± 4.83 ng/mL), butyric acid (26.00 ± 0.80 ng/mL) and isovaleric acid (31.92 ± 1.57 ng/mL) in the culture supernatant of L. fermentum E15 were significantly increased as compared to the MRS medium group (acetic acid: 865.35 ± 7.09 ng/mL; propionic acid: 29.86 ± 0.83 ng/mL; butyric acid: 0 ng/mL; isovaleric acid: 0 ng/mL) (Figures 6A–D; p < 0.001). Additionally, we assessed the levels of acetic acid, propionic acid, butyric acid, and isovaleric acid in zebrafish larvae fed either a normal diet or a HCD. Our results showed that supplementation with L. fermentum E15 significantly increased the levels of acetic acid, propionic acid, butyric acid, and isovaleric acid both in zebrafish larvae fed either a normal diet or a HCD (Figures 6E–H) (p < 0.05).

A recent study has reported that Lactobacillus acidophilus inhibits non-alcoholic fatty liver disease (NAFLD)-associated hepatocellular carcinoma through producing valeric acid (29). To shed light on the mechanisms by which L. fermentum E15 improves hyperlipidemia, the effects of isovaleric acid on lipid accumulation in HCD-fed zebrafish larvae were further investigated. The GPR43 receptor has been reported to be the primary receptor for SCFAs in intestine and liver (7, 29). To investigate whether the anti-hyperlipidemic effects of L. fermentum E15 are mediated by SCFAs, PTX was used to block the GPR43 receptor (29). A significantly lower accumulation of red lipid droplets was detected in the liver and blood vessels of zebrafish larvae from both the L. fermentum E15 group and the isovaleric acid group as compared to the HCD group (Figure 7A). In contrast, the effects of L. fermentum E15 and isovaleric acid on reducing lipid accumulation in HCD-fed zebrafish larvae were eliminated when the GPRs inhibitor (PTX) was used (Figures 7A,B). These findings suggested that isovaleric acid metabolized by L. fermentum E15 can reduce lipid accumulation in HCD-fed zebrafish larvae.

In order to investigate the mechanism behind the effects of L. fermentum E15 on HCD-induced hyperlipidemia in zebrafish larvae, the mRNA expression levels of GPR43 and leptin A were detected. Significantly increased mRNA expression levels of GPR43 and leptin A were observed in the L. fermentum E15 group (GPR43: 1.98 fold; leptin A: 0.87 fold) and isovaleric acid group (GPR43: 1.51 fold; leptin A: 0.73 fold) as compared to the HCD group (GPR43: 0.83 fold; leptin A: 0.42 fold) (p < 0.01; Figures 7C,D). In addition, the GPRs inhibitor PTX effectively inhibited the promoting effects of L. fermentum E15 and isovaleric acid on mRNA expression levels of GPR43 and leptin A (p < 0.01). These results suggested that L. fermentum E15 can alleviate HCD-induced lipid accumulation by activating the GPR43 receptor through the metabolism of SCFAs.