A Novel Strain Bifidobacterium longum subsp. longum HN001 Ameliorates High-Fat Diet-Induced Obesity in Mice Through Microbiome-Associated Short-Chain Fatty Acids

Abstract

Obesity, characterized by excessive fat accumulation, poses global health risks due to its association with metabolic diseases. Beyond conventional treatments, the gut microbiome has emerged as a promising therapeutic target. Given the critical role of microbiome alterations in obesity, modifying its composition through probiotics, prebiotics, or synbiotics offers a novel strategy for mitigating obesity and related conditions. In this study, we demonstrate the dose-dependent anti-obesity effects of a novel strain, Bifidobacterium longum subsp. longum HN001 (HN001), in a high-fat diet (HFD)-induced mouse model. Among the doses tested (100, 200, and 400 mg/kg), the 200 mg/kg dose showed the most pronounced effect, significantly reducing body weight gain, serum triglyceride (TG), and total cholesterol (TC) levels. These effects were associated with reductions in fat mass and adipocyte hypertrophy in white adipose tissue (WAT), suppression of lipogenesis, and enhanced energy expenditure through WAT browning and thermogenesis in brown adipose tissue (BAT). Microbiome analysis revealed that HN001 increased the abundance of beneficial, SCFA-producing microbes while reducing taxa linked to metabolic dysfunction. Importantly, HN001 restored serum short-chain fatty acid (SCFA) levels, and its lipid-lowering effect in adipocytes was attenuated by a GPR43 antagonist, supporting an SCFA–GPR43–mediated pathway that links microbiome modulation with reduced adipogenesis and enhanced thermogenesis. These findings suggest that HN001 may represent a promising therapeutic strategy for obesity via SCFA-driven modulation of host metabolism and gut microbiome composition.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12602-025-10766-1.

Highlights

• HN001 reduces body weight and lowers serum TG and TC levels in HFD-induced obese mice.

• HN001 attenuates adipocyte hypertrophy in WAT and promotes thermogenesis through browning and BAT activation.

• HN001 modulates the gut microbiome, enriching beneficial taxa and restoring serum SCFA levels.

• The anti-obesity effects of HN001 are partly mediated via SCFA-GPR43 signaling, supporting its therapeutic potential.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12602-025-10766-1.

Introduction

Obesity, characterized by excessive fat accumulation, poses significant global health risks and is primarily caused by an imbalance between energy intake and expenditure. Since 1990, global obesity rates have doubled, with 2.5 billion adults overweight and 890 million classified as obese in 2022 [1]. This epidemic is associated with numerous chronic diseases, including type 2 diabetes mellitus, cardiovascular diseases, metabolic syndromes, and cancers [2], which increase morbidity and mortality worldwide. As obesity prevalence continues to rise, researchers are exploring innovative therapeutic approaches. Beyond conventional pharmacological interventions such as glucagon-like peptide-1 receptor agonists, increasing attention has been directed toward the gut microbiome as a novel and promising target for obesity management [3].

The gut microbiome plays a crucial role in human health and disease, with its composition and diversity influenced by factors such as diet, lifestyle, age, and environment. Among which diet is considered the primary modulator [4]. With diet playing a central role in shaping microbial composition and function, diets high in fat and low in fiber are known to reduce microbial diversity and promote the expansion of pro-inflammatory taxa, thereby contributing to metabolic dysfunction [5]. Moreover, recent studies have linked alterations in the gut microbiota to the onset and progression of various diseases, including neurological disorders, cardiovascular disease, metabolic syndromes, and cancer, making it an important target for therapeutic intervention [6]. Obesity is one of the conditions most affected by gut microbiome changes, which can influence nutrient absorption, energy harvest, and fat storage [7]. Conversely, obesity itself can disrupt gut microbial balance, further contributing to metabolic dysfunction and disease progression [8]. Therefore, modulation of the gut microbiome is pivotal in both the development and treatment of obesity.

Supplementation with probiotics, prebiotics, or synbiotics is an effective approach to modulating the gut microbiome [9]. These interventions have been associated with improvements in various diseases, including metabolic disorders, and have emerged as a promising therapeutic strategies [10]. In the context obesity, Numerous studies have aimed to identify effective microbial strains by comparing the gut microbiota of lean and obese individuals, followed by interventions using candidate microorganisms. Among these, Bifidobacterium and Lactobacillus genus have been extensively studied and are widely recognized for their beneficial effects on obesity [11]. One of the key mechanisms behind these effects is the production of short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, which are known to contribute to metabolic health and play a protective role in various diseases [12]. These strains have demonstrated preventive and ameliorative effects in both preclinical and clinical studies [13]. Despite these advances, further research is needed to identify additional strains with potent anti-obesity activity.

In this study, we investigated the anti-obesity effects of HN001 in HFD-induced mouse model. To assess its impact, we pre-treated mice with HN001 for 2 weeks, followed by 4 weeks of combined HFD feeding and HN001 administration. Body weight, as well as serum TG and TC levels, were monitored throughout the study. Additionally, visceral fat accumulation was evaluated using dual energy X-ray absorptiometry (DEXA) and histological analysis. We further examined lipogenesis-related pathways in WAT to explore underlying mechanisms. Microbiome profiling was performed to identify bacterial taxa that significantly changed in response to HN001 treatment. Given the emerging role of SCFAs as key microbiota-derived metabolites that regulate host lipid metabolism and thermogenesis via receptors GPR43, we also quantified SCFA levels in serum and feces, and assessed the involvement of SCFA-GPR43 signaling in mediating HN001’s metabolic benefits.

Materials and Methods

Preparation of HN001

HN001 was provided by the Korea Research Institute of Bioscience and Biotechnology (KRIBB, Daejeon, Republic of Korea). The strain was cultured in a peptone-based medium for 12 h and heat-inactivated at 80 °C for 30 min. After heat treatment, an excipient was added to facilitate spray drying and stabilize the preparation. Spray drying was performed at the GMP facility of Huons Foodience (Gyeonggi-do, Republic of Korea), yielding 0.75 × 10¹⁰ cells/kg in the final product at the administered dose of 100 mg/kg. The final product was used for experimentation.

Animal Study Design

Animal experiments were conducted in accordance with institutional guidelines for the care and use of laboratory animals and were approved by Korea University (IACUC approval number: KUIACUC-2024–0081). Five-week-old male C57BL/6N mice were obtained from Raonbio (Gyeonggi-do, Republic of Korea). The mice were housed in cages under controlled environmental conditions (temperature: 22 ± 2 °C, humidity: 50 ± 5%, with a 12 hours light/dark cycle) and provided with ad libitum access to food and water. After a 7-day acclimatization period, the mice were randomly assigned to five groups: Control, HFD, HFD + HN001 (100 mg/kg), HFD + HN001 (200 mg/kg), and HFD + HN001 (400 mg/kg). Both the HFD (D12492 fat 60%, Research diet, NH, USA) and either HN001 supernatant or vehicle were administered for 4 weeks, following a 2-week oral administration of either the vehicle or HN001 supernatant. Body weight and food intake were monitored twice a week. Rectal temperature was measured with a small-animal rectal thermometer (JD-DT-08G, JEUNGDO Bio&Plant CO., LTD., Seoul, Republic of Korea) during the final week of the experiment. At the end of the experiment, the mice were sacrificed, and biological samples were collected for further analysis. Serum was isolated, and adipose tissues were dissected carefully. WAT was collected from the epididymal fat pad (visceral depot), and BAT was collected from interscapular region.

TG and TC Level Measurement

Serum levels TG and TC were measured using commercial colorimetric assay kits: the Asanset Triglyceride Determination Reagent AsanSet Triglyceride Assay Kit and AsanSet Total Cholesterol Assay Kit (ASAN PHARM. CO., LTD., Seoul, Republic of Korea). The assays were conducted according to the manufacturer’s protocols. Absorbance was measured at 550 nm for TG and 500 nm for TC using a Skanlt microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

DEXA

In the final week of experiment, DEXA images of mice were obtained. Briefly, the mice were anesthetized with isoflurane and scanned using the InAlyzer system (Medikors, Gyeonggi-do, Republic of korea). The InAlyzer automatically quantified the fat mass and lean mass based on these images.

Hematoxylin and Eosin (H&E) Staining

For histological evaluation, WAT tissue samples were fixed overnight in 4% paraformaldehyde solution, then placed in cassettes and dehydrated through a graded ethanol series (70%, 80%, 90%, 95%, and 100%). For paraffin embedding, the tissues were treated with xylene twice for 2 h each, followed by immersion in paraffin in a 60 °C vacuum oven, and then embedded in paraffin blocks. Sections were sliced to a thickness of 3 μm and stored at room temperature. The sections of WAT from mice were stained using hematoxylin and eosin. The paraffin-embedded WAT samples were sliced into 3 μm sections. The slides were deparaffinized with xylene and Histo-Clear (National Diagnostics, Charlotte, NC, USA), and rehydrated through a series of progressively lower alcohol concentrations (100%, 95%, 90%, 80%, and 70%). Gill No. 3 hematoxylin (Sigma Aldrich, Burlington, MA, USA) was applied for 1–2 min, followed by 2 min of eosin staining. After alcohol dehydration, the slides were incubated in Histo-Clear until they were ready for mounting. For quantitative analysis, digital images of H&E-stained sections were captured using a light microscope at 200 × magnification. Using ImageJ software (NIH, USA), the average adipocyte area (μm²) was determined by analyzing more than 50 randomly selected adipocytes per mouse from the epididymal WAT depot. Data were expressed as mean ± SD for each group. All image analyses were conducted in a blinded manner to ensure objectivity.

Nile Red Staining

White adipose tissue (WAT) samples were fixed in 4% paraformaldehyde for 24 h. Subsequently, 10%, 20%, and 30% sucrose in PBS were added. WAT was embedded in OCT compound and sectioned at 10 µm thickness using a Vivo Solutions Cryostat (Gyeonggi, Republic of Korea). Slides were incubated with Nile Red (1 µM in DPBS) for 15 min at room temperature in the dark. After washing with PBS, nuclei were counterstained with DAPI. Images were acquired using a fluorescence microscope (Axiovert 200, Carl Zeiss, Oberkochen, Germany) with appropriate filter settings (excitation/emission: 552/636 nm). Fluorescence intensity was quantified using ImageJ software (National Institute for Health, MD, USA).

Immunofluorescence Analysis

The cryo-sectioned slides were air-dried at room temperature, then fixed and permeabilized with acetone. Following permeabilization, the sections were blocked with 1% bovine serum albumin and incubated overnight at 4 °C with the appropriate primary antibodies. Primary antibodies used were Perilipin-1 (Cell Signaling Technology, Danvers, MA, USA) and UCP1 (ab10983; Abcam, Cambridge, UK). After washing with PBS, the sections were incubated with fluorophore-conjugated secondary antibodies (e.g., Alexa Fluor 488; Invitrogen, Carlsbad, CA, USA). Stained slides were treated with an anti-photobleaching reagent and sealed with a cover glass. Immunofluorescence images were acquired using a Zeiss Axiovert 200 inverted microscope (Oberkochen, Germany). The average fluorescence intensity was quantified using ImageJ software (NIH, USA).

mRNA Isolation and Quantitative Real-Time PCR (qRT-PCR)

RNA samples from WAT tissues were extracted using TRIzol™ reagent (Thermo fisher scientific, Waltham, MA, USA) according to manufacturer’s instructions. Briefly, WAT tissues were homogenized bead homogenizer in TRIzol, followed by centrifugation at 12,000 × g for 10 min at 4 °C. The upper lipid layer was aspirated, and chloroform was added [14]. Next, isopropanol was used to precipitate the RNA, which was washed with cold 75% ethanol and dried, and subsequently reconstituted in RNase-free water. RNA concentration was measured at 260 nm using SkanIt microplate reader with μdrop plate (Thermo Fisher Scientific, Waltham, MA, USA). cDNA synthesis was performed using Reverse Transcriptase premix (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. qRT-PCR was performed using GoTaq qPCR Master Mix (Promega, WI, USA) on a Quantstudio 3 system (Thermo Fisher Scientific, Waltham, MA, USA). The thermal cycling conditions were as follows: initial denaturation at 94 °C for 2 min, followed by 40 cycles of denaturation at 94 °C for 15 s and annealing/extension at 60 °C for 1 min. The comparative Ct method (ΔΔCt) was used to evaluate RNA expression relative to the control group. The mouse primers were listed in Supplementary Table 1.

Immunoblot Analysis

BAT tissues were lysed in ice-cold RIPA buffer containing a protease inhibitor cocktail and phenylmethylsulfonyl fluoride (PMSF). The tissues were homogenized, sonicated, and centrifuged at 13,000 × g for 20 min to obtain the supernatant. Protein concentration was measured using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Samples were mixed with Laemmli buffer, separated by SDS-PAGE, and transferred onto nitrocellulose membranes. After blocking with 3% bovine serum albumin, the membranes were incubated with primary antibodies overnight at 4 °C. HRP-conjugated secondary antibodies were then incubated for 1 h at room temperature, and the chemiluminescence signal was detected using ECL solution with the ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA) and analyzed using ImageJ. The UCP1 antibody (ab10983) was purchased from Abcam (Cambridge, UK), and the PGC1-alpha antibody (NBP1-04676) was obtained from Novus Biologicals (CO, USA).

Microbiome Analysis

Mouse fecal samples were collected and stored at − 80℃ to preserve microbial DNA, which was then extracted using the Mag-Bind® Universal Pathogen Kit (Omega Bio-Tek, Norcross, GA, USA). The V3-V4 region of the 16S rRNA gene was amplified via PCR using universal primers, and libraries were quantified with a Qubit 4.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing was conducted on an Illumina MiSeq (Illumina, San diego, CA, USA) platform to generate paired-end 300 bp reads. Quality-filtered reads were processed in QIIME2 using DADA2 for denoising and the Silva database for taxonomic assignment.

SCFA Analysis

SCFA concentrations in serum, fecal samples from mice, and HN001 powder solution were measured using a commercial kit (AEFI01346, AssayGenie, Dublin, Ireland) according to the manufacturer’s instructions. Absorbance was read at 450 nm with a SkanIt microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

Culture and Differentiation of 3T3-L1 Cells

3T3-L1 pre-adipocytes were purchased from the Korean Cell Line Bank (Seoul, Republic of Korea). Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (100x , Gibco, USA) at 37 °C in a humidified atmosphere containing 5% CO₂. For differentiation, cells were grown to confluence and then induced with MDI differentiation medium containing 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 μM dexamethasone, and 10 μg/mL insulin (Sigma-Aldrich, USA) for 3 days. Subsequently, cells were cultured in DMEM containing 10% FBS and 10 μg/mL insulin, which was refreshed every 2 days for a total of two treatments. HN001 was administered throughout the entire differentiation period at the indicated concentrations.

WST-8 Assay

To evaluate cell viability, 3T3-L1 adipocytes were seeded into 96-well plates and cultured for 24 h. Differentiation media containing HN001 was then applied in a dose-dependent manner for 7 days. At the endpoint, 10% (v/v) WST-8 solution (QM1000, Biomax, Seoul, Republic of Korea) in differentiation media was added to each well and incubated for 24 h. Absorbance was measured at 450 nm using a SkanIt microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

Oil Red O Staining

To evaluate lipid accumulation, differentiated 3T3-L1 adipocytes were fixed with 10% formalin for 1 h at room temperature, washed with phosphate-buffered saline (PBS), and stained with filtered Oil Red O working solution (0.5% Oil Red O in isopropanol diluted 3:2 with distilled water) for 30 min. Excess stain was removed by rinsing with distilled water, and stained lipid droplets were imaged under a light microscope. For quantification, stained images were analyzed using ImageJ software (NIH, USA) with the Color Transformer plugin, converting images into YIQ color space to accurately measure the stained area corresponding to lipid droplets.

Strain-Specific qRT-PCR for HN001 Detection

To detect colonization of HN001 in the intestine, large intestines were collected from control mice and mice administered HN001 at 200 mg/kg. Genomic DNA was extracted from the intestinal contents using a commercial kit, following the manufacturer’s instructions (Takara Bio, Shiga, Japan). Strain-specific qRT-PCR was performed using GoTaq® qPCR Master Mix (Promega, Madison, WI, USA) on a QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). The primer sequences were: forward 5′-AGAGATACGGCTTCCCTTCG-3′ and reverse 5′-CGTGAGTTCCCGGCATAATC-3′, designed with Primer3 based on the NCBI GenBank sequence CP069278.1.

Statistical Analysis

Statistical analysis was performed GraphPad Prism 9 software. Data were presented as mean ± SD for each experimental group (n ≥ 5). Statistical significance was determined by Student’s t-test, with *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 considered significant.

Results and Discussion

Administration of HN001 Ameliorates HFD-Induced Obesity in a Mouse Model

Bifidobacterium longum, a Gram-positive bacterium in the human gastrointestinal tract, is well known for its ability to reduce body weight and fat accumulation [15, 16]. In this study, we identified a novel strain, Bifidobacterium longum subsp. longum HN001, and investigated its potential anti-obesity effects.

To evaluate these effects in an HFD-induced obesity model, mice were divided into five groups: Control, HFD only, HFD + HN001 at 100 mg/kg, HFD + HN001 at 200 mg/kg, and HFD + HN001 at 400 mg/kg. To assess the prophylactic potential of HN001, mice received a daily oral gavage of HN001 for 2 weeks prior to HFD feeding, followed by concurrent HFD feeding and continued HN001 administration for an additional 4 weeks. Under these conditions, body weight was monitored in all groups. Mice in the HFD group showed a significant increase in body weight, whereas administration of HN001 at 200 mg/kg markedly suppressed HFD-induced weight gain (Fig. 1A and B). Unexpectedly, the anti-obesity effect of HN001 peaked at 200 mg/kg, whereas the 400 mg/kg dose seemed to exhibit reduced efficacy. To investigate this phenomenon, we measured daily food consumption. However, there was no significant difference in daily food intake between the HFD-only group and the HFD + HN001 groups across all doses (Fig. 1C). This result suggests that the suppression of weight gain by HN001 is not attributable to reduced food consumption, but rather to metabolic modulation. Furthermore, the attenuated effect at the highest dose implies a potential non-linear dose–response relationship. We hypothesized that this outcome may be partially attributed to biological variation among individual mice, which can influence responsiveness to probiotic interventions, especially at higher doses. Additionally, this non-linear dose–response pattern suggests that higher doses may not always lead to greater benefits, potentially due to saturation of host–microbiota interactions or altered microbial community structure. Although no signs of toxicity were observed, subtle microbiota disruptions or metabolic adaptations at higher concentrations may have attenuated the effect [17].

Fig. 1.

HN001 supplementation reduces body weight gain and improves serum lipid profile in HFD-fed mice. Six-week-old male mice were fed HFD and administered HN001 for 4 weeks following a 2-week pre-treatment with HN001. (A) Body weight gain monitored twice weekly for 1 month. (B) Cumulative body weight gain after 1 month. (C) Daily food intake per mouse. (D) Serum TG levels. (E) Serum TC levels. Data are presented as mean ± SD; N.S, not significant; *p < 0.05, **p < 0.01, ***p < 0.001 (n = 5 per group)

Next, we measured serum TG and TC levels, which are commonly elevated in obese individuals and serve as important indicators of metabolic dysfunction associated with obesity [18]. Consistent with the body weight results, HN001 at 200 mg/kg significantly reduced serum TG levels (Fig. 1D). Moreover, all tested doses of HN001 resulted in significant reductions in serum TC levels (Fig. 1E). Taken together, these results demonstrate that administration of HN001 attenuates HFD-induced obesity.

Administration of HN001 Reduces WAT Mass by Decreasing Lipid Droplet Size in Adipocyte

Obesity is characterized by an excessive accumulation of body fat. WAT functions not only as a major endocrine and secretory organ but also serves as the body’s primary storage site for fat, holding essential energy reserves [19]. Moreover, elevated serum levels of TG and TC have been associated with increased WAT mass [20, 21]. Therefore, we focused on assessing fat accumulation in mice to elucidate the effects of HN001 on body weight reduction.

First, we measured fat mass using DEXA to investigate the relationship between increased body weight and WAT accumulation. As shown in Fig. 2A, administration of HN001 markedly reduced WAT in mice (Fig. 2A). Quantitative analysis revealed significant reductions in WAT mass at 200 mg/kg and 400 mg/kg doses of HN001 (Fig. 2B and C), while lean mass remained unchanged across all groups (Fig. 2D and E). To further validate the fat-reducing effect of HN001, we assessed visceral fat mass post-sacrifice. Visceral fat was specifically selected for analysis due to its strong association with metabolic dysfunction and the elevated risk of obesity-related diseases, including type 2 diabetes and cardiovascular disorders [22, 23]. Unlike subcutaneous fat, visceral fat is more metabolically active and closely linked to systemic inflammation and insulin resistance [24, 25]. Notably, in the DEXA images, the most prominent visual differences between groups were observed in the abdominal region, corresponding to visceral fat, further supporting its selection as a key target. As a result, HN001 exhibited a dose-dependent trend toward visceral fat reduction, with significant decreases observed at the 200 mg/kg and 400 mg/kg doses (Fig. 2F).

Fig. 2.

HN001 decreases fat mass and adipocyte size in HFD-fed mice. (A) DEXA images obtained using InAlyzer system; orange indicates high fat density, green indicates low fat density. (B) Fat mass, (C) fat percentage, (D) lean mass, and (E) lean percentage automatically calculated from DEXA images. (F) Visceral fat weight normalized to body weight. (G) Representative H&E-stained WAT images (200x magnification). (H) Quantification of adipocyte area (> 50 randomly selected adipocytes per mouse). Data are presented as mean ± SD; N.S, not significant. Panel (A–G): n = 5 per group; Panel (H): n = 7 per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Increased WAT mass is typically associated with two key processes: an increase in adipocyte number (hyperplasia) and enlargement of lipid droplets within adipocytes (hypertrophy) [26]. To further investigate the mechanism by which HN001 reduces WAT mass, we performed histological analysis of WAT. H&E staining revealed that HN001 treatment decreased lipid droplet size without affecting adipocyte number (Fig. 2G). Quantification of lipid droplet size showed a pattern consistent with the observed changes in body weight (Fig. 2H). Collectively, these results suggest that HN001 reduces WAT mass in HFD-induced mice primarily by decreasing lipid droplet size.

Administration of HN001 Suppresses HFD-Induced Lipogenesis in WAT

Under physiological conditions, WAT expansion primarily occurs through adipocyte hypertrophy, which involves the enlargement of existing adipocytes. Lipid droplet enlargement results from increased lipid storage or decreased lipid breakdown, leading to changes in fat cell size. In obesity, the pathways of lipogenesis and lipolysis serve as key mediators of adipose tissue remodeling [27]. During lipogenesis, adipocytes convert fatty acids and glucose into TG, which are then stored in lipid droplets. In contrast, lipolysis is often suppressed in obesity, contributing to excessive fat accumulation [28].

To investigate the regulatory pathways underlying lipogenesis and lipolysis, we quantified the mRNA expression levels of key associated genes in WAT of mice. The results showed that the HFD feeding significantly upregulated the expression of lipogenesis-related genes, including fatty acid synthetase (Fasn), acetyl-CoA carboxylase alpha (Acaca), stearoyl-CoA desaturase (Scd1), and sterol regulatory element-binding transcription factor 1 (Srebf1). In contrast, HN001 administration significantly downregulated the expression of these lipogenesis-related genes in a dose-dependent manner (Fig. 3A–D). However, the 400 mg/kg dose did not show a greater reduction in gene expression compared to the 200 mg/kg dose. We believe this finding is consistent with the phenotypic data presented in Fig. 1B and may be attributed to inter-individual variability among mice or potential saturation of host–microbiota interactions, as previously discussed. Nonetheless, these results support the notion that HN001 exerts its anti-obesity effects in HFD-induced mice, at least in part, through the regulation of lipogenesis. Interestingly, the expression of lipolysis-related genes, including lipase E (Lipe), carnitine palmitoyltransferase 1 (Cpt1), Acyl-CoA oxidase 1 (ACOX1), and patatin like phospholipase domain containing 2 (Pnpla2), was not significantly affected by either HFD or HN001 administration (Fig. 3E–H). Under our experimental conditions, only the lipogenesis pathway, rather than lipolysis, appeared responsive in this model. This observation is in line with previous studies reporting that alterations in lipogenesis predominate over changes in lipolysis during the development of obesity [29, 30]. Overall, HN001 suppresses the HFD-induced upregulation of lipogenesis-related gene expression in WAT, thereby contributing to reduced fat mass in mice.

Fig. 3.

HN001 suppresses lipogenesis-related gene expression and enhances lipolysis-related gene expression in WAT. mRNA levels of lipogenesis-related genes (A) Fasn, (B) Acaca, (C) Scd1, and (D) Srebf1. mRNA levels of lipolysis-related genes (E) Lipe, (F) Cpt1, (G) Acox1, and (H) Pnpla2. Data are presented as mean ± SD; N.S, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (n = 5–8 per group)

Administration of HN001 Increases Energy Expenditure by Activating Thermogenesis in WAT and BAT

As aforementioned, obesity results from an imbalance between energy intake and expenditure [31]. Since HN001 did not influence appetite, as evidenced by unchanged food intake (Fig. 1C), we turned our attention to mechanisms involved in increasing energy expenditure. In the absence of physical activity, the browning of WAT into thermogenically active beige adipocytes is a critical process for enhancing energy expenditure [32]. Emerging evidence suggests that gut microbiota can interact with adipose tissue to promote the browning of WAT, in conjunction with physical and chemical stimuli such as cold exposure, diet, hormones, and pharmacological agents [33, 34]. Therefore, we investigated whether HN001 administration induces browning in WAT.

To investigate the browning of WAT, we examined specific markers for beige adipocytes. Specifically, we measured mRNA levels of Cd137 and Cited1, which are established markers of beige adipose tissue [35]. Interestingly, these beige cell markers were downregulated in HFD-induced mice. However, this reduction was significantly reversed by HN001 administration (Fig. 4A and B). Furthermore, we assessed the expression of UCP1 and PGC-1α, key regulators of thermogenesis that function via mitochondrial activation in adipose tissue. Beige adipocytes express these proteins at higher levels than white adipocytes due to their enhanced capacity for energy expenditure [36]. Consistent with the results for beige adipocytes markers, mRNA levels of Ucp1 and Ppargc1a were also significantly restored by HN001 treatment (Fig. 4C and D).

Fig. 4.

HN001 promotes WAT browning, increases rectal temperature, and upregulates thermogenesis-related proteins. (A–D) mRNA levels of browning-related genes Cd137, Cited1, Ucp1, and Ppargc1a. (E) Rectal temperatures during the final week of the experiment. (F) Immunoblot analysis of thermogenesis-related proteins UCP1 and PGC-1α. (G, H) Quantification of UCP1 and PGC-1α protein levels. Data are presented as mean ± SD; N.S, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Panel (A–D, F–H): n = 5–8 per group; Panel (E): n = 4 per group

Thermogenesis is a key component of total energy expenditure in mammals [37]. To assess thermogenesis activity in mice, we measured rectal temperature. Rectal thermometry is widely regarded as the gold standard for assessing core body temperature in small animals [38], offering greater accuracy than non-invasive methods. Initially, we attempted to monitor body temperature using an infrared thermal camera; however, this approach failed to detect significant differences between groups (data not shown), likely due to its limited ability to measure internal temperature and its susceptibility to external environmental conditions. Thus, we employed rectal thermometry to obtain more reliable and physiologically relevant readings, which revealed a significant increase in body temperature at both the 100 mg/kg and 400 mg/kg doses (Fig. 4E). We hypothesized that this thermogenic effect might be more pronounced under cold-stress conditions. However, we chose to exclude cold-stress exposure to isolate the effect of HN001, as thermogenesis could otherwise be confounded by the combined effects of environmental stimuli and probiotic treatment. Nonetheless, HN001 administration alone was sufficient to elevate body temperature. Furthermore, to explore the underlying mechanisms, we analyzed the expression of thermogenesis-related proteins in BAT. While browning of WAT contributes to thermogenesis, BAT is the principal tissue responsible for heat production in mammals. Therefore, we focused on BAT to further evaluate energy expenditure in HN001-treated mice.

To this end, we examined the expression of the thermogenesis-related proteins UCP1 and PGC-1α in BAT. HN001 administration led to increased UCP1 protein levels, with significant upregulation observed at 200 mg/kg and 400 mg/kg doses. Similarly, PGC-1α protein levels were significantly elevated at the 100 mg/kg and 200 mg/kg doses (Fig. 4F–H). These findings indicate that HN001 enhances energy expenditure in HFD-induced mice by upregulating thermogenesis-related proteins in BAT. Taken together, our results suggest that HN001 administration promotes total energy expenditure by activating thermogenic pathways in both WAT and BAT.

HN001 Modulates the Gut Microbiota in HFD-Induced Obese Mice

Obesity is closely associated with alterations in gut microbiome, and conversely, changes in the microbiome can independently contribute to the development and progression of obesity [39]. Moreover, interventions targeting the gut microbiota such as probiotics and prebiotics, have demonstrated therapeutic potential. These insights highlight the critical role of microbiome modulation in obesity management. Based on this, we hypothesized that the anti-obesity effects of HN001, including its ability to enhance total energy expenditure, may be mediated through gut microbiota alterations. To test this hypothesis, we performed a comprehensive microbiome analysis.

Alpha diversity analysis, assessed using observed operational taxonomic units and Shannon diversity index, revealed significant differences in microbial richness and evenness among the groups (Fig. 5A). HFD feeding significantly reduced diversity, whereas HN001 supplementation markedly restored diversity compared to the HFD group. To assess compositional differences in microbial communities, beta diversity analysis was conducted using the Bray–Curtis and Jaccard dissimilarity indices. Both indices demonstrated distinct clustering between the ND and HFD groups (Fig. 5B). Importantly, the microbial composition in the HN001-treated group diverged significantly from the HFD group, indicating that HN001 modulates microbial community structure.

Fig. 5.

HN001 modulates gut microbiota composition and enriches SCFA-producing taxa in HFD-fed mice. (A) Alpha diversity assessed using observed OTUs and Shannon diversity index. (B) Beta diversity analyzed using Bray–Curtis and Jaccard dissimilarity indices. (C) Relative taxonomic abundance at the phylum level. (D–E) LEfSe analysis showing significantly enriched microbial clades between groups, visualized as a cladogram (D) and a bar chart of LDA scores (E). (F) XOR analysis showing genera uniquely detected in HFD or HN001 groups. Inflammatory/dysbiosis-associated taxa were enriched in HFD, while SCFA-producing and metabolically beneficial taxa were enriched in HN001. Data are presented as mean ± SD; N.S, not significant; *p < 0.05, **p < 0.01, ***p < 0.001 (n = 4–5 per group)

The microbiome taxonomy results are presented in Fig. 5C–F. At the phylum level, the ND and HFD groups showed significantly different microbial compositions. However, no significant differences were observed between the HFD-only and HFD plus HN001 200 mg/kg groups (Fig. 5C). Despite this, LEfSe analysis revealed distinct shifts in bacterial composition between the HFD and HN001-treated groups. The resulting cladogram highlights significantly enriched clades using color-coded annotations. In the HFD group, taxa such as Erysipelotrichales (e), Streptococcaceae (f), and Negativicutes (k) were significantly overrepresented. These taxa are commonly associated with pro-inflammatory responses and HFD-induced dysbiosis [40–42]. In contrast, the HN001 group showed increased abundance of beneficial microbial taxa, including Bifidobacteriaceae (a), Bifidobacteriales (b), Actinobacteria (c), and Tannerellaceae (d). Additional enriched clades in the HN001 group included Butyricicoccaceae (g), Peptococcaceae (i), and Peptococcales (j) (Fig. 5D and E). These microbial taxa have been reported to be negatively associated with obesity and metabolic dysfunction or positively associated with healthy individuals, suggesting that HN001 may exert its anti-obesity effects in part through favorable modulation of the gut microbiota [43–47].

To further identify unique microbial signatures, XOR analysis was performed to determine genera exclusively present in either the HFD or HN001 group. A total of 25 genera were uniquely detected in the HFD group, including Prevotella, Megamonas, Faecalibacterium, Erysipelatoclostridiaceae, and members of the Eubacterium and Clostridium genera. These taxa have been linked to inflammation, metabolic dysfunction, HFD-induced microbial imbalance [45, 48–50]. Conversely, 15 genera were found exclusively in the HN001 group, such as such as Lachnospiraceae_UCG-010, Subdoligranulum, Micromonospora, Bacillus, and Caldicoprobacter (Fig. 5F). These microbes are associated with gut homeostasis, SCFAs production, and improved metabolic outcomes [51–57]. These results emphasize the group-specific microbial shifts induced by HN001 intervention, potentially contributing to metabolic recovery and anti-obesity effects.

Overall, HN001 administration modulates the gut microbiome toward a profile associated with metabolic health. The enrichment of specific beneficial microbial taxa, many of which are known for anti-inflammatory properties and SCFA production, supports a mechanistic link between microbiota changes and the observed physiological improvements in the HFD-induced obese mice.

HN001’s Anti-obesity Effects Involve SCFA-Mediated GPR43 Signaling That Suppresses Lipogenesis and Promotes Thermogenesis

SCFAs are the principal metabolites generated through the fermentation of non-digestible carbohydrates that escape digestion and absorption in the small intestine. Their production arises from a complex interplay between dietary components and the gut microbial ecosystem within the colon. The identification of specific host receptors that recognize SCFAs as natural ligands across various tissues has heightened interest in their role as signaling molecules mediating communication between the gut microbiota and the host. Importantly, alterations in microbial composition—such as those induced by probiotic supplementation—can enhance SCFA production, thereby helping to reduce the risk or severity of various human diseases, particularly metabolic disorders [58]. Consistent with our findings, several beneficial microbial taxa enriched by HN001 treatment, including Bifidobacteriaceae, Actinobacteria, Tannerellaceae, Butyricicoccaceae, Subdoligranulum, Bacillus, and Caldicoprobacter, are well recognized for their capacity to promote host health via SCFA production [51, 56, 57, 59–61]. This suggests that the observed metabolic benefits of HN001 may be mediated, at least in part, through SCFA-associated pathways. Therefore, we focused on evaluating the role of SCFAs in contributing to the anti-obesity effects of HN001.

First, we measured SCFA concentrations in mice following HN001 administration in the HFD-induced obesity model. In serum, HFD-induced obese mice showed decreased SCFA concentrations compared to the ND group, whereas HN001 treatment restored these levels (Fig. 6A). However, in feces, SCFA concentrations were reduced in HFD-fed mice compared to the ND group, but no significant changes were observed following HN001 treatment (Fig. 6B). Since fecal SCFAs primarily reflect microbial production in the gut, this discrepancy between serum and fecal SCFA concentrations is notable and warrants further investigation. Several clinical studies have reported similar discrepancies, suggesting that serum SCFAs are more directly associated with host metabolic health than fecal SCFAs, as evidenced by stronger correlations between serum SCFA patterns and various human diseases [62, 63]. Although we did not directly quantify primary SCFA production in the gut, we measured SCFA content in the HN001 powder itself. HN001 was found to contain 5.528 ± 0.37 pg of SCFAs per 1 mg of powder (data not shown). Taken together, these findings suggest that HN001 supplementation may increase serum SCFA concentrations, at least in part, through direct SCFA provision in addition to microbiota-mediated effects.

Fig. 6.

HN001’s anti-obesity effects are partly mediated via SCFA-associated signaling in vivo and in vitro. (A, B) SCFA concentrations in serum (A) and feces (B) of HFD-fed mice with or without HN001 supplementation. (C) Representative Oil Red O staining images of 3T3-L1 adipocytes and (D) quantification of lipid accumulation. (E) Immunoblot analysis and quantification of lipogenesis-related proteins. (F) Immunoblot analysis and quantification of thermogenesis-related proteins. For in vitro analysis, 3T3-L1 adipocytes were differentiated with MDI for 3 days followed by insulin treatment for 4 days (two cycles) in the presence or absence of HN001 (100 μg/mL) and the GPR43 antagonist GLPG0974 (0.1 µM, applied throughout the differentiation period). Data are presented as mean ± SD; N.S, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Panel (A, B): n = 4 per group; Panel (D): n = 6 per group; Panel (E, F): n = 4 per group

Next, to investigate whether HN001 exerts a preventive effect on HFD-induced obesity through SCFA-mediated mechanisms, we conducted in vitro experiments. SCFAs are known to act via several receptors, including GPR41 (G protein-coupled receptor 41), GPR43, GPR109a, and Olfr78 (Olfactory receptor 78). Among these, GPR41 and GPR43 are the most well-characterized SCFA receptors. [64]. To validate the involvement of SCFA signaling, we employed GLPG0974, a selective antagonist of GPR43. The concentration of HN001 powder was first optimized by assessing cytotoxicity (Fig. S3). Subsequently, 3T3-L1 adipocytes were differentiated in the presence of HN001, with or without GLPG0974, and then stained with Oil Red O. HN001 treatment markedly suppressed lipid droplet formation, whereas co-treatment with GLPG0974 reversed this effect (Fig. 6C). Quantitative analysis of Oil Red O staining further confirmed the lipid-lowering effect of HN001, which was attenuated by GLPG0974 co-treatment (Fig. 6D). These findings suggest that the anti-obesity effects of HN001 may be mediated, at least in part, through SCFA-GPR43 signaling pathways.

To further investigate the mechanisms by which HN001 suppresses lipid droplet formation in 3T3-L1 adipocytes, we examined the expression levels of genes involved in lipogenesis, lipolysis, and thermogenesis. Among lipogenesis markers, FASN and SREBP1 were significantly reduced in the HN001 treatment group (Fig. 6E), whereas SCD1 and p-ACC showed no notable changes in this in vitro model (data not shown). Similarly, lipolysis marker including HSL, CPT1, and ATGL also were not significantly altered (data not shown), consistent with the observation in the animal model. Finally, thermogenesis related protein UCP1 and CIDEA (Cell Death Inducing DFFA Like Effector A) were upregulated by HN001 treatment, while PGC-1α remained unchanged (data not shown). These in vitro findings are consistent with our in vivo results, suggesting that HN001 primarily attenuates obesity by suppressing lipogenesis and enhancing thermogenic pathways rather than stimulating lipolysis.

Importantly, the reversal of HN001’s lipid-lowering effect by the GPR43 antagonist GLPG0974 suggests that these changes are at least partly mediated through SCFA–GPR43 signaling. This observation complements our in vivo data showing restoration of serum SCFA levels after HN001 administration. Taken together, these findings indicate that HN001 exerts its anti-obesity effects primarily through suppression of lipogenesis and activation of thermogenic pathways via SCFA-mediated GPR43 signaling, rather than through lipolysis. The strong concordance between the 3T3-L1 cell model and the animal study supports the hypothesis that SCFA-driven modulation of adipocyte metabolism plays a pivotal role in the anti-obesity effects of HN001.

Clinical Implications and Limitations

Our findings indicate that HN001 exerts marked anti-obesity effects in an HFD-induced mouse model by modulating the gut microbiome (Fig. 5), suppressing adipose lipogenesis (Fig. 3), and enhancing thermogenic pathways (Fig. 4). These effects are in line with reports for other B. longum strain, such as DS0950, which reduces obesity through up-regulation of thermogenesis-related genes [65]. Moreover, in a clinical study, B. longum BB536 reduced visceral fat and total fat in normal-weight and overweight adults, supporting translational relevance of our findings [15]. In the present study, our results further suggest that these effects are partly mediated through SCFA-associated signaling. Notably, the restoration of serum SCFA levels by HN001 provides a mechanism link of translational significance, aligning with clinical evidence that probiotic supplementation can beneficially modulate host metabolism.

However, several limitations should be acknowledged. While our targeted qPCR and SCFA analyses provide valuable mechanistic insights, more comprehensive approaches such as untargeted metabolomics and RNA sequencing are needed to fully elucidate the pathways involved. We detected an HN001-specific signal in intestine of mice receiving 200 mg/kg HN001, whereas signals in controls were at or below the detection limit (Fig. S4), indicating HN001’s presence in the intestine during dosing and supporting its direct contribution to the observed microbial shifts. Nevertheless, qPCR cannot distinguish live from dead cells or confirm mucosal adherence or persistence after dosing cessation; future work employing culture-based recovery, strain-resolved metagenomics, and wash-out designs will be important. The discrepancy between serum and fecal SCFA levels also warrants further investigation, as it may reflect complex host–microbiota interactions. Finally, although these findings are promising, validation in human clinical trials is essential to determine the therapeutic potential, optimal dosage, and long-term safety of HN001.

Conclusion

This study demonstrated that the novel strain Bifidobacterium longum HN001 supplementation significantly attenuates HFD-induced obesity by reducing body weight gain, serum TG and TC levels, and fat mass, confirmed by DEXA imaging and histological analysis. Mechanistically, HN001 suppressed lipogenesis in WAT and promoted thermogenesis in BAT, thereby enhancing energy expenditure. Microbiome profiling further revealed that HN001 enriched beneficial taxa associated with improved metabolic health. Complementary in vitro studies suggested that SCFA–GPR43 signaling plays a pivotal role in mediating these effects. Together, these results highlight HN001 as a promising candidate for obesity management, with strong translational potential for future clinical applications.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contribution

Chae Hwan Lee: Conceptualization, Methodology, Formal analysis, Investigation, Validation, Visualization, Data curation. Youngji Han: Visualization, Writing-original draft, Data curation. Joo Young Ryu: Methodology. Investigation. Minseo Jung: Methodology, Investigation. Chae Rin Park: Conceptualization, Resources. Mi Ran Jang: Conceptualization, Resources. Youn Gil Kwak: Conceptualization, Resources. Hanvit Cha: Conceptualization, Methodology, Visualization, Investigation, Writing-original draft, review, and editing, Supervision, Project administration, Funding acquisition. Jin Hyup Lee: Conceptualization, Supervision, Project administration, Resources, Funding acquisition.

Data Availability

Data will be made available on request.

Declarations

Competing interests

The authors declare no competing interests.