Significant increases in body weights between mice fed with the control diet and HFHS diet with and without a supplementation of SBp or C3G were detected at ≥ 2 weeks after the start of the intervention (p < 0.05 or 0.01). The trend of body weight increase in mice receiving HFHS diet was continued throughout the experiment. No significant difference was detected among mice fed with HFHS diet with or without a supplementation with SBp or C3G (Fig. 1A). Daily food intake of all mice was measured at onset and every 2 weeks during the dietary experiment until 10 weeks after the start of dietary intervention. No significant difference in food intake was detected among various groups receiving different experimental diets in the present study (Fig. 1B).
Effects of SBp and C3G on glucose and lipid metabolism in HFHS diet-induced obese mice
The HFHS diet significantly increased the levels of fasting plasma glucose (FPG) in mice compared to the control diet (p < 0.01). The supplementation with SBp or C3G in the HFHS diet significantly lowered FPG, compared to that in HFHS alone (p < 0.05 or 0.01). However, the levels of FPG in the SBp or C3G group were still significantly higher than that in the control group. No significant difference in FPG was found between the SBp and C3G group. The HFHS diet also significantly elevated the levels of cholesterol and triglycerides in plasma compared to the control diet (p < 0.01). Plasma cholesterol or triglyceride levels in both the SBp and C3G group were significantly lower than the HFHS group (p < 0.01). Cholesterol and triglyceride levels in the plasma of mice receiving HFHS diet supplemented with SBp or C3G were significantly higher than that in the control group (p < 0.01). No significant difference in plasma cholesterol or triglycerides was detected between the SBp and C3G groups (Fig. 2).
Effects of SBp and C3G on fasting plasma insulin and insulin resistance in mice receiving HFHS diet
Insulin resistance was assessed using HOMA-IR generated from the levels of FPG and insulin in the plasma samples simultaneously withdrawn from mice. The levels of plasma insulin and HOMA-IR in the HFHS group were significantly higher than the control group (p < 0.01). Supplementation with SBp and C3G significantly reduced the levels of insulin and HOMA-IR compared to that in mice fed with the HFHS diet alone (p < 0.01). The levels of insulin and HOMA-IR in the SBp or C3G group were still higher than that in the control group (p < 0.01). No significant difference in insulin or HOMA-IR was detected between the SBp and C3G group (Fig. 3A and 3B).
Effects of HFHS diet and the supplementation of SBp or C3G on circulating inflammatory mediators in mice
The HFHS diet elevated the levels of MCP-1 and PAI-1, two inflammatory markers, in the plasma of mice (p < 0.01). The supplementation of SBp or C3G in the HFHS diet significantly reduced the circulating levels of MCP-1 and PAI-1 compared to the HFHS diet in mice (p < 0.01). The levels of the inflammatory markers in mice receiving SBp or C3G diet were still significantly higher than the control group (p < 0.01). No significant difference in plasma MCP-1 or PAI-1 was detected between mice receiving HFHS diet supplemented with SBp or C3G (Fig. 4A and B).
Impact of supplementation of SBp or C3G on gut microbiota in HFHS diet-fed mice
The results of β-diversity analysis demonstrated that the gut microbial compositions in the stool of mice from the four dietary groups were well separated in principal component analysis (Fig. 5). No significant difference in α-diversity variables was detected in mice receiving different diets (Shannon index, chao1). Table 1 demonstrated that Bacteroidetes and Firmicutes represented the vast majority phylum bacteria in mouse feces. Mice fed with the HFHS diet had lower abundances of Bacteroidetes, Actinobacteria, Proteobacteria and Verrucomicrobia phylum bacteria, but a higher relative abundance of Firmicutes phylum bacteria compared to mice receiving the control diet (p < 0.05 or 0.01). Supplementation with SBp or C3G to the HFHS diet augmented the abundance of Bacteroidetes and reduced that of Firmicutes in mice feces (p < 0.01). The abundance of Actinobacteria in the stool of the C3G group was the only type of phylum bacteria significantly different from that in SBp group (p < 0.05).
Table 1
Effect of different diets on the abundances of gut microbiota on phylum level. Male C57 BL/J6 mice (6 weeks of age) were randomized into 4 groups and received following diets for 11 weeks: control (CTL) group: low-fat diet; HFHS group: HFHS diet; SBp group: SBp (8.0 g/kg/day) supplemented in the HFHS diet; C3G group: C3G (7.2 mg/kg/day) supplemented in the HFHS diet. Values in the tables were expressed in mean ± SD (% of total gut microbiota, n = 8/group). *, **: p < 0.05 or 0.01 versus control group; +, ++: p < 0.05 or 0.01 versus HFHS group; ^: p < 0.05 versus SBp group.
Phylum bacteria | CTL (%) | HFHS (%) | SBp (%) | C3G (%) |
Actinobacteria | 5.66 ± 2.75 | 0.26 ± 0.11** | 0.24 ± 0.15** | 2.85 ± 1.83*,+,^ |
Bacteroidetes | 54.28 ± 6.91 | 34.25 ± 7.36** | 55.80 ± 6.95++ | 48.36 ± 4.31++ |
Firmicutes | 23.59 ± 6.41 | 59.53 ± 8.00** | 36.01 ± 5.73**,++ | 42.34 ± 4.63**,++ |
Proteobacteria | 2.36 ± 0.71 | 0.68 ± 0.20** | 0.81 ± 0.21** | 0.63 ± 0.26** |
Tenericutes | 0.21 ± 0.23 | 0.20 ± 0.14 | 0.24 ± 0.16 | 0.46 ± 0.34 |
Verrucomicrobia | 13.88 ± 3.81 | 4.96 ± 3.23** | 6.78 ± 2.27** | 5.29 ± 1.81** |
Others | 0.02 ± 0.02 | 0.12 ± 0.09* | 0.11 ± 0.09 | 0.08 ± 0.03 |
The relative abundance of Bacteroidetes phylum bacteria in mice with HFHS diets was significantly lower than that in mice fed with the other three types of diets (p < 0.01, Fig. 6A). The relative abundance of Firmicutes phylum bacteria in the HFHS group was significantly higher compared to that in the other three groups (p < 0.01, Fig. 6B). Besides, the HFHS group also had a significantly lower ratio of Bacteroidetes/Firmicutes (B/F) and higher in Firmicutes/Bacteroidetes ratio compared to that in the control, SBp or C3G group (p < 0.05 or 0.01). The SBp group had a significantly higher B/F ratio and lower F/B ratio compared to the HFHS group. C3G supplementation induced a significantly lower F/B ratio (p < 0.01), but not in B/F ratio, compared to the HFHS group (Fig. 6C and 6D).
Statistical differences in the relative abundances of all types of gut family bacteria, except that of Bifidobacteriaceae, were detected among mice receiving various diets (p < 0.01, Fig. 7A). Mice fed with the HFHS diet had evidently higher relative abundances in Erysipelotrichaceae or Lachnospiraceae and lower abundance in Muribaculaceae family bacteria compared to mice in the other groups (heatmap in Fig. 7A). Supplementation of SBp or C3G decreased the abundance of Lachinospiraceae, and increased that of Muribacculaceae compared to HFHS diet (p < 0.05). However, the relative abundances of several family bacteria in the SBp group were different from that in the C3G group (see open triangles in Fig. 7A). Heatmap (Fig. 7B, right) demonstrated correlations between the relative abundance of family bacteria and some diabetes-related biochemical parameters. The abundances of Defluviitaleaceae, Eggerthellaceae, Erysipelotrichaceae, Family XIII, Lachnospiraceae, Peptpcoccaceae, Peptostreptococcaeae, Rununococcaceae and Streptococcaceae family bacteria were positively correlated with body weights, glucose/lipid metabolism and inflammatory markers in the mice, while Clostridiaceae 1, Clostridiales vadinBB60 group, Muribaculaceae and Lactobacillaceae were negatively correlated with the physical, metabolic and inflammatory variables. The relative abundance of Muribaculaceae in mice in the HFHS group was the lowest among all groups. Both SBp and C3G supplementations significantly increased the abundances of Muribaculaceae family bacteria compared to the HFHS group.
The greatest difference in the mean proporation of family bacteria between the HFHS group and SBp or C3G group was Muribaculaceae family bacteria (Fig. 7C and 7D). The mean proportion of Muribaculaceae in the HFHS group was significantly lower than that in the SBp or C3G group (p < 0.001). The mean proportion of Lachnospiraceae was significantly higher in the HFHS group and lower in the SBp and C3G group (p < 0.05 or 0.001). Similar pattern of changes were detected in the mean proportion of Erysipelotrichaceae, which was higher in the HFHS group but lower in the SBp and C3G group. The mean proportion of Ruminococcaceae was significantly higher in the HFHS group compared to the SBp group (p < 0.001, Fig. 7C); while, no significant difference in the mean proportion of Ruminococcaceae was detected between the HFHS and C3G groups (Fig. 7D).
Functional predication of the changes in gut microbial genes induced by HFHS diet with and without the supplementation of SBp or C3G
Function prediction using PICRUSt analysis demonstrated that the impact of the HFHS diet and that supplemented with SBp or C3G on the abundances of genes of gut microbes in 15 classes of predicted functions in mice (p < 0.01, ANOVA, Fig. 8). Microbial genes related to membrane transport were the most abundant in the stool of mice in the present study. At least two types of ASV (operational taxonomic unit or OTUs) distribution among the cellular functions in mice receiving different dietary intervention were found: 1) the HFHS diet resulted in the highest microbial abundances compared to that in the control group, and the supplementation with SBp or C3G attenuated the effect of the HFHS diet; 2) mice in the control group had more abundant ASV than in the HFHS group, and the supplementation of SBp or C3G substantially increased ASV compared to the HFHS diet, but still relatively less than that in the control group. Examples for the first type of cellular functions include membrane transport and cell motility, which may be implicated in inflammation. The second type of cellular functions included the metabolism for energy, carbohydrates, amino acids, lipids, cofactors and vitamins, replication and repair, which may potentially contribute to metabolism. It is noticed that transcription was activated, and the translation pathway was suppressed in mice receiving the HFHS diet. SBp or C3G supplementation attenuated the effect of the HFHS diet in transcription or translation in mice. The findings suggest that SBp and C3G may neutralize the effect of the HFHS diet in inflammation and promote metabolism in mice via the modulation of gut microbiota.