Twenty-four male C57BL/6J mice, aged 5 weeks, were obtained from Gempharmatech Co., Ltd. (Nanjing, Jiangsu, China) and housed in a controlled, specific pathogen-free environment (21°C ± 2°C, 12-h dark–light cycle) with ad libitum access to food and water. After 1 week of acclimation on a normal diet, as shown in Figure 1A, 24 mice were randomly divided into four groups (n = 6 per group), including the low-fat diet (LFD) group fed with a low-fat diet (D12450J, Research Diets, Inc., United States), the high-fat high-fructose (HFFD) group fed with a high-fat diet (D12492, Research Diets, Inc., United States) plus 10% fructose water, and the low-dose thiamine group (THIL, 50 mg/kg/day, gavage) fed with HFD plus 10% fructose water, and high-dose thiamine group (THIH, 100 mg/kg/day, gavage) fed with HFD plus 10% fructose water. An oral supplement of 50 mg/kg or 100 mg/kg, corresponding to approximately 250 or 500 times the recommended dietary allowances for humans (31), respectively. The LFD group and the HFFD group administered the same volume of vehicle by intragastric gavage. The body weight of each mouse was measured using a calibrated digital scale at the beginning of the feeding period and weekly thereafter until euthanasia. Additionally, overall food intake for each group was monitored weekly throughout the study. The in vivo intervention lasted for 8 weeks (Figure 1A). At the end of the experiment, the mice, aged 14 weeks, were fasted for 12 h and then anesthetized in chambers saturated with isoflurane. The whole blood was collected through retroorbital sampling, after which the mice were sacrificed by cervical dislocation. Tissues, including adipose tissue, colon, and liver, were collected, weighed, and stored at −80°C for further analysis. The liver index was calculated as the ratio of liver weight to the body weight; while the epididymal fat index was calculated as the ratio of epididymal fat weight to the body weight. Animal experiments were conducted in accordance with the Guidelines for Animal Experimentation of China Pharmaceutical University (Nanjing, China), and the protocols were approved by the Animal Ethics Committee of this institution (No. 202407022).

Serum samples were obtained after the blood samples were collected and centrifuged at 3,500 RPM for 15 min. Fasting serum glucose, triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) was measured by biochemistry automatic analyzer (Hitachi 7100, Japan). Serum endotoxin was assessed by commercial colorimetric kits (Xiamen Bioendo Technology, Xiamen, China) according to the manufacturer’s instructions (32).

Fecal thiamine was measured by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) as we previously established (27).

Livers, colon, and epididymal adipose tissues taken from the mice were was fixed with 4% paraformaldehyde overnight and embedded in paraffin wax. Then, the sections were sliced and stained with hematoxylin and eosin (H&E). Frozen liver sections were stained with Oil Red O and counterstained with hematoxylin to visualize the lipid droplets. The images of sections at 300 dpi resolution were captured using Leica Thunder Imaging System (Leica Microsystems). The area of adipocyte in epididymal fat tissue and the area of lipid droplets in liver tissue were determined by Image J software.

The colon sections were incubated with 10% goat serum in phosphate-buffered saline (PBS) for 30 min. Subsequently, the primary antibodies, anti-zonula occludens-1 (anti-ZO-1, Servicebio, GB151981) diluted 1:2,000 and anti-occludin (Servicebio, GB111401) also diluted 1:2,000, were applied to the sections in a blocking solution and incubated overnight at 4°C. Following this, the sections were washed three times with PBS and treated with the specified fluorescein-labeled secondary antibody (Servicebio, GB23303) for 50 min. Cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) for 10 min and then washed three times with PBS. After sealing the slides with an anti-fluorescence quenching agent, images of the sections at 300 dpi resolution were captured using the Leica Thunder Imaging System (Leica Microsystems). The mean fluorescence intensity was determined by Image J software.

Genomic DNA of fecal samples was extracted using MagBeads Fast DNA Kit for Soil (MP Biomedicals, CA, United States) and quantified using Nanodrop One spectrophotometer (Thermo Scientific). DNA was used to generate amplicons using a TruSeq Nano DNA LT Library Prep Kit. V3 and V4 hypervariable regions of prokaryotic 16S rDNA were selected for generating amplicons and following taxonomy analysis (32). DNA libraries were validated by Agilent High Sensitivity DNA Kit (Agilent Technologies, Palo Alto, CA, United States) and quantified by Quant-iT PicoGreen dsDNA Assay Kit. DNA libraries were multiplexed and loaded on an Illumina NovaSeq instrument according to the manufacturer’s instructions (Illumina, San Diego, CA, United States). Sequencing was performed using paired-end configuration; image analysis and base-calling were conducted by the Quantitative Insights Into Microbial Ecology 2 (QIIME2) Software. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) software. 16S rRNA gene sequencing data can be accessed on the Sequence Read Archive (SRA) database, accession number: PRJNA1188793.

Statistical analysis was performed using GraphPad Prism software (version 8). The results of biological assay are presented as mean ± standard deviation (SD). The differences between two groups were analyzed by Student’s t-test. Datasets that involved more than two groups were assessed by one way analysis of variance (ANOVA) followed by Tukey’s multiple comparison’s test. Permutational multivariate ANOVA (PERMANOVA) and principal-coordinate analysis (PCoA) were performed to analyze microbial β-diversity. The correlation between the microbiota and phenotypic characteristics was assessed using the multivariate linear regression test (MAASLIN). Additionally, correlations among the microbes were examined with Sparse Correlations for Compositional Data (SparCC). The statistical significance was set at p < 0.05.

The effects of thiamine in preventing HFFD-induced obesity and the associated metabolic disorders were investigated by treating mice with high-dose thiamine for 8 weeks. As shown in Figures 1B,C, within 8 weeks of HFFD feeding, the body weight of mice in the HFFD group increased significantly compared with the LFD group, while oral administration of high-dose thiamine significantly inhibited HFFD-induced body weight gain after 4 weeks of thiamine administration. However, the weight loss effect was not observed to be dose-dependent between the THIL and THIH groups. No significant difference in food intake was observed among groups fed with HFFD, suggesting that the effects of thiamine were not due to reduced food consumption (Figure 1D and Supplementary Table S1). The epididymal fat index in HFFD-fed mice increased significantly than those in LFD-fed mice, and a trend toward a decreased epididymal fat index was observed in the THIH group, but not in the THIL group (Figure 1E and Supplementary Table S1). The histological analysis corroborated that thiamine intervention decreased epididymal fat deposits and adipocyte area (Figure 1F and Supplementary Table S1). In addition, thiamine intervention increased the liver index and alleviated the hepatic steatosis in liver tissues (Figures 1G,H and Supplementary Table S1). Furthermore, we found that the HFFD-induced hyperglycemia was attenuated by thiamine intervention, evidenced by a reduction of serum fasting glucose levels in thiamine-treat groups (Figure 1I and Supplementary Table S1).

Compared to LFD feeding, HFFD feeding significantly altered lipid metabolism in mice, accompanied by adipose deposition, lipid accumulation in liver. However, these shifts were observed to be reversed by thiamine intervention. As shown in Figures 2A–D and Supplementary Table S1, the serum levels of TC and LDL-C were significantly reduced in the thiamine-treat groups compared with HFFD group. While, no significant changes in the serum levels of serum TG and serum HDL-C were observed in the thiamine-treated groups. Oil Red O staining revealed that lipid accumulation within the liver was reduced in thiamine-treat groups (Figure 2E and Supplementary Table S1).

Endotoxemia is a pivotal contributor to the onset and development of obesity. as shown in Figure 3A and Supplementary Table S1, we observed a higher serum endotoxin in HFFD group, whereas this increase was significantly reversed by thiamine intervention. The impairment of intestinal barrier is one of the important causes of endotoxemia; to further explore the effect of thiamine intervention on the histological damage in HFFD-induced obese mice, H&E staining was performed for histological examination of colon sections. The colon sections of healthy mice in LFD group exhibited a natural structure with a balanced distribution of goblet cells. However, compared to LFD group, HFFD feeding resulted in the destruction of crypt structures in the mucosa, edema in the submucosa, a reduction in goblet cells, and infiltration of inflammatory cells (Figure 3B). Notably, high-dose thiamine supplementation significantly alleviated these colonic structural damage and infiltration degree of inflammatory cells in HFFD-fed mice (Figure 3B).

Studies have shown that the integrity of the intestinal epithelial barrier is compromised in obesity, which is characterized by dysfunction of tight junctions. Tight junction proteins (e.g., ZO-1 and occludin) of epithelial cells are vital for the intestinal barrier integrity. To further explore the effect of thiamine intervention on intestinal barrier integrity, immunofluorescence staining analyses were performed to analyze the levels of intestinal tight junction proteins. As shown in Figures 3C–E, the levels of ZO-1 and occludin were significantly reduced in the HFFD-treated group compared to LFD group. Conversely, high-dose thiamine supplementation mitigated the intestinal barrier damage induced by HFFD feeding. These results suggest that thiamine intervention may positively regulate intestinal barrier permeability by preventing the decrease of tight junction protein levels in HFFD-induced obese mice.

Impairment of intestinal barrier and endotoxemia is closely linked to gut microbiota disruption or dysbiosis. Notably, we observed that fecal thiamine levels in the HFFD group were significantly lower than those in the LFD group. In contrast, oral administration of high-dose thiamine resulted in a significant increase in fecal thiamine levels (Figure 4A). We next analyzed the composition and structure of fecal microbiota by 16S rRNA gene sequencing method to study the effect of thiamine on the gut microbiota of obese mice. As shown in Figure 4B, Chao1 index gradually reached a saturation plateau as the number of sequences increased, indicating that the amount of sequencing data is sufficient to reflect the most of the microbial information in the samples. Alpha diversity indices reflect the intrasample diversity of gut microbiota. As shown in Figures 4C–E and Supplementary Table S1, the gut microbiota structure of HFFD-feeding mice was characterized by increased Shannon index and Simpson index compared to the LFD group. Notably, a trend toward a decreased Shannon index was observed in the THIH group, but not in the THIL group. Furthermore, the Chao1 index was altered by thiamine intervention when compared to the HFFD group. Beta diversity reflects differences in species diversity among samples. The results of PCoA indicated a distinct separation gut microbiota community among the four groups (Figure 4F).

To identify the key gut microbes that most likely explain differences between HFFD group and LFD group, Linear Discriminant Analysis Effect Size (LEfSe) analysis was performed. Multiple taxonomic differences between two groups were identified (LDA score >2) (Figure 4G), indicating HFFD feeding had a dramatic influence on composition on the gut microbiota. We next examined the abundance of the aforementioned bacteria in the gut microbiota of each group at the phylum, genus, and species levels to further investigate the impact of thiamine intervention on the alterations of microbial composition induced by HFFD feeding. At the phylum level, gut microbiota in the four groups was mainly composed of Firmicutes, Actinobacteria, Proteobacteria, Bacteroidetes, Deferribacteres, Verrucomicrobia, Tenericutes, TM7, Acidobacteria and Chloroflexi (Figure 5A). Among them, we observed that the relative abundance of Proteobacteria was significantly boosted in the HFFD group, whereas thiamine intervention prominently reversed the relative abundance of that caused by HFFD feeding (Figure 5C and Supplementary Table S1). Thiamine intervention also exhibited a trend toward a decreased Bacteroidetes (Figure 5D and Supplementary Table S1). On the contrary, the relative abundance of Actinobacteria was significantly depleted in the HFFD group, whereas thiamine intervention prominently increased the relative abundance of that caused by HFFD feeding (Figure 5B and Supplementary Table S1). At the genus level (Figures 5E–J and Supplementary Table S1), we observed that the relative abundance of Oscillospira, Ruminococcus and AF12 were significantly boosted in the HFFD group, whereas thiamine intervention prominently reversed the relative abundance of them caused by HFFD feeding. Notably, Adlercreutzia enriched in HFFD group only exhibited a significant reduction in THIH group, but not in the THIL group. On the contrary, the relative abundance of Bifidobacterium was significantly depleted in the HFFD group, while thiamine intervention exhibited a tendency to restore the relative abundance affected by HFFD feeding. At the species level (Figures 5K–M and Supplementary Table S1), we observed that the relative abundance of Ruminococcus gnavus was significantly boosted, whereas Bifidobacterium pseudolongum was significantly depleted in the HFFD group. Notably, thiamine intervention significantly reversed these changes.

We also investigated the potential alterations in functional pathways. As shown in Supplementary Figure S1, the PICRUSt analysis revealed several alterations in KEGG pathways between the LFD and HFFD groups. Notably, “fatty acid biosynthesis” and “beta-alanine metabolism” were enriched, while “protein digestion and absorption” and “other glycan degradation” were diminished in the HFFD group compared to the LFD group.

Correlation analyses were conducted to identify the relationships between gut microbiota and obesity-related phenotypes, as well as among the gut microbes. As shown in Figure 6A and Supplementary Table S2, the abundance of Actinobacteria, Bifidobacterium, and Bifidobacterium pseudolongum were negatively correlated with BMI, TC, LDL-C, and fasting serum glucose. In contrast, Proteobacteria, Oscillospira, and Ruminococcus gnavus exhibited positive correlations with BMI, TC, LDL-C, and fasting serum glucose. Additionally, as shown in Figure 6B and Supplementary Table S3, the abundance of Proteobacteria was positively correlated with Ruminococcus gnavus. Collectively, these findings suggest that high-dose thiamine mitigates HFFD-induced obesity by modulating specific gut microbiota.