C57BL mice (17 ± 1.5 g) (n = 40) were obtained from the Experimental Animal Center, Fuzhou Medical College, Nanchang University, Fuzhou, Jiangxi, China. The animals were maintained at a controlled temperature (22°C ± 1°C), humidity (50%), and light (12/12-h light/dark). All animal experiments were approved by the Experimental Animal Ethics Committee of Fuzhou Medical College, Nanchang University. Inulin was obtained from MCE (HY-N7075) and dissolved in drinking water (10% w/w). The blood glucose testing equipment was offered by Sinocare. The TG, low-density lipoprotein (LDL), high-density lipoprotein, cholesterol, free fatty acids, interleukin-6, interleukin-1β, and tumor necrosis factor testing kits were obtained from Nanjing Jiancheng Company. The insulin ELISA kit was purchased from Abcam. The HFD consisted of 20% carbohydrates, 35% proteins, and 45% fats (D12492; Research Diets; 60% of total calories).

After the mice were fasted for 6 h, blood samples were obtained from the orbit of the mice. Then, 5,000 g of the supernatant was centrifuged and stored at −80° until use. According to the manufacturer’s recommendations, the TG, LDL, high-density lipoprotein, cholesterol, free fatty acids, interleukin-6, interleukin-1β, and tumor necrosis factor levels were evaluated. For the fecal lipid test, the mice were placed separately and samples were collected for a 24-h fecal test. Then, the 24-h triglyceride level was calculated, according to the manufacturer’s instructions.

Fresh tissues were ground and crushed, and total RNA was prepared using the TRIzol reagent (Ambion). The amplified products were labeled with SYBR (Yeasen, 10222ES60). RT-PCR was performed using the Bio-rad PCR system. The relative mRNA levels were calculated by comparison using the threshold cycle method. The primer sequences are presented in Table 1 .

Before the glucose tolerance test, the mice were fasted for 16 h and injected with 2 g/kg body weight of glucose. Blood glucose levels in the rat tail blood were measured at 0, 15, 30, 60, and 120 min. Mice were fasted for 6 h before the insulin resistance test. Blood glucose levels were measured at 0, 15, 30, 60, and 120 min after injection of 0.55 µ/kg body weight.

Fresh liver and white adipose tissues were fixed with 4% paraformaldehyde for 24 h and embedded with Oct. Then, the samples were sectioned at a thickness of 4 µm using a Thermo Fisher Scientific Slicer and stored at −80°. For hematoxylin and eosin staining, the cut tissue sections were washed three times with phosphate-buffered saline (PBS), placed into the hematoxylin solution for 1 min, washed three times with PBS, placed into the eosin solution for 1 min, washed three times with PBS, and sealed with xylene transparent and neutral gum. The samples were photographed under a Leica microscope. For oil-red staining, the cut tissue sections were washed three times with PBS, stained with oil red solution for 1 min, washed three times with PBS, washed three times with hematoxylin solution for 1 min, washed three times with PBS, and sealed with glycerol gelatin. The sections were photographed under a Leica microscope. ImageJ software was used to calculate the oil red-stained area.

RIPA lysis buffer was used to extract intestinal epithelial tissue protein. BCA protein assay kit was used to determine the protein concentrations (Beyotime, Shanghai, China). The membrane was sealed with 5% BSA for 2 h. The membrane was subsequently compared with the target Fgf15 (1:1000, ab252922) at 4°C. GAPDH (1:1000) was used as a loading control. Then, the secondary antibody (1:2000) was allowed to bind to the primary antibody for 2 h at room temperature. The signal was captured using an emitter-coupled logic substrate (pierce chemical). ImageJ software was used to quantitatively analyze the band intensity.

Serum and fecal total bile acid levels were determined using total bile acid kits (e003-2-1; Nanjing Jiancheng Co., Ltd.).

Data are expressed as the mean ± standard error of the mean (SEM). Furthermore, significant differences were determined by performing a t-test with least significant difference (LSD) post hoc tests, and statistical significance was set at p < 0.05.

To understand the effects of inulin on glucose and lipid metabolism, we fed inulin to mice on a regular diet for 7 weeks, while the control group was fed with the same quantity of solvent. The results showed that the body weight of inulin-fed mice was significantly lower than that of the control group (p < 0.05; Figure 1A). Inulin significantly improved glucose tolerance and insulin sensitivity in mice fed a normal diet. The area of the glucose tolerance test curve also confirmed that the inulin group significantly improved the glucose tolerance (p < 0.05; Figures 1B, C). To determine the effect of inulin on intestinal lipid synthesis and transport, we examined the genes involved in inulin-fed small intestinal epithelial cells in mice. The results showed that the expressions of some lipid synthesis genes (Srebp-1c, Fasn, Acc, and Scd-1) and lipid transport genes (Cd36 and Apob-48) were significantly lower in inulin-fed mice than those in control mice (Figure 1D). To observe the serum lipid content in inulin-fed mice, we measured the serum levels of TG, LDL, cholesterol, free fatty acids, and high-density lipoprotein. The results showed that the serum levels of TG, LDL, and cholesterol were significantly lower in inulin-fed mice than those in control mice (p < 0.05; Figure 1E). In addition, we did not find any significant changes in the liver, inguinal fat (igWAT), epididymal fat (Epwat), or brown fat (BAT) when the mice were weighed (Figure 1F). To understand the effect of inulin feeding on intestinal bile acid excretion, we examined the total bile acid levels in the serum, portal vein, and feces of mice. The results showed that inulin significantly promoted fecal bile acid excretion (p < 0.05; Figure 1G). To observe the effect of inulin on liver function, serum alanine aminotransferase and aspartate aminotransferase levels were detected; however, no significant differences were observed (Figures 1H, I).

To further understand the effect of inulin on the gut microbiota, we performed 16sRNA sequencing of intestinal contents in mice fed a normal diet. We found that inulin increased the α-diversity of the gut microbiota in mice (Figure 2A). At the same time, the principal component analysis showed that inulin increased the β-diversity of the gut microbes in mice (Figure 2B). To further evaluate the microbial changes, we analyzed the microbial “Phylum” levels and found that inulin increased the proportion of the Bacteroidetes enteritidis and decreased the proportion of Firmicutes. Furthermore, the ratio of bacteroides to Firmicutes was significantly increased (Figures 2C, D).

To further explore the effects of inulin on glycolipid metabolism, we used mice fed a HFD and inulin or solvent as experimental treatments. The results showed that inulin significantly reduced the weight in mice fed a HFD, which was significantly lower at 7 weeks than the control group (Figure 3A). Glucose tolerance and insulin resistance tests were performed 20 weeks after feeding a HFD. The results showed that inulin significantly improved the glucose tolerance and insulin resistance symptoms associated with a HFD (Figures 3B–D). To further evaluate the effects of inulin on intestinal absorption in mice fed a HFD, we examined the lipid and bile acid reabsorption genes in small intestinal epithelial cells. The results showed that inulin significantly decreased intestinal lipid absorption (Srebp-1c, Fasn, ACC, CD36, NPC1L1, and APOB48) and the expression of inflammatory genes (TNF-α, IL-6, and IL-1β) (p < 0.05). In addition, inulin reduced the expression of the bile acid reabsorption gene (FGF15) (p < 0.05; Figure 3E). To determine the effect of inulin on serum lipids and inflammation in mice fed a HFD, we measured serum levels of lipids, total bile acids, and inflammatory markers. The results showed that inulin decreased the serum levels of alanine aminotransferase, aspartate aminotransferase, IL-6, IL-1β, and TNF-α (p < 0.05) in mice fed with HFD (Figures 4A–C). Furthermore, inulin improved the insulin resistance index (p < 0.05) in mice fed a HFD (Figure 4D). The levels of TG, LDL, and FFA were significantly lower in the inulin group than those in the control group (p < 0.05) (Figure 4E). Interestingly, inulin-fed mice on a HFD had lower levels of serum total bile acids, whereas fecal excretion of bile acids was higher (p < 0.05) (Figure 4F). To this end, we examined the fecal TG content and found that the fecal TG content was significantly higher in inulin-fed mice fed a HFD (p < 0.05) (Figure 4G). We further weighed the liver and fat of mice on a HFD and found that inulin reduced the weight of liver, inguinal fat (igWAT), and epididymal fat (EPWAT) (p < 0.05) (Figure 4H). To further explore the beneficial effect of inulin on the liver, we examined liver-associated genes for lipid absorption and inflammation. Compared with the control group, inulin reduced the steatosis lipid synthesis genes (CHREBP, Srebp-1c, FASN, and Scd-1) in mice fed a HFD and also significantly decreased the expressions of IL-6, IL-1β, and TNF-1α in the liver. In addition, inulin reduced the expression of FGF15, a rate-limiting gene for bile acid synthesis in the liver (p < 0.05; Figure 4I). These results suggest that inulin improves the glycolipid metabolic phenotype of mice fed a HFD by reducing the reabsorption of intestinal bile acids and TG excretion from the feces, reducing the metabolic syndrome induced by a HFD.

To further explore the benefit of inulin on lipid accumulation in the liver, we performed oil-red staining of liver samples of inulin- and HFD-fed mice and analyzed the liver lipid proportion. The results showed that inulin significantly reduced the liver lipid accumulation (p < 0.05; Figures 5A, B) in mice fed a HFD. To observe the maturity of white fat, we performed hematoxylin and eosin staining of inguinal fat and found that inulin significantly reduced the diameter of inguinal fat in mice fed a HFD. The inguinal fat size of inulin-fed mice on a HFD was 40% smaller than that of controls (p < 0.05; Figures 5C, D). To further determine the mechanism of bile acid reabsorption in the gut, we examined the expression level of FGF15, a gut bile acid reabsorption protein. We found that inulin significantly reduced the expression of intestinal bile acid reabsorption protein FFG15 in mice fed a high-fat diet. These results suggest that the inhibitory effect of inulin on the intestinal bile acid reabsorption protein FGF15 leads to a reduction in intestinal lipid absorption, which in turn affects lipid production and accumulation of liver and white fat (p < 0.05; Figures 5E, F).