The inulin-supplemented diet can improve metabolic disorder related symptoms in high fat-sucrose diet-induced gestational diabetes mice
To investigate the effect of inulin treatment on glycolipid metabolism disorders in high- fat/sucrose diet induced gestational diabetes mice, we examined the body weight, daily food intake and glycolipid metabolism related parameters. The body weight, FBG, FINS, TG, TC, LDL-C and the area under the curve (AUC) of the oral glucose tolerance test (OGTT) of the HFDG group mice were significantly elevated compared with those of NCDG group mice (Figure 1A-G, Figure 2A-D), indicating severe glucose intolerance and dyslipidemia.
In contrast, ITFG group mice fed the ITF-supplemented diet showed improved metabolic parameters (Figure 1A-G, Figure 2A-D). After ITF intervention, body weight, serum TG, TC and LDL-C on GD18 reduced significantly by 4.54 g, 0.48 mmol/l, 1.04 mmol/l and 0.494 mmol/l (p< 0.05, vs HFDG group) (Figure 1C, Figure 2A, B, D). Additionally, the AUC of OGTT on GD 14, FBG and serum insulin on GD 18 were lowered by 7.95 mmol/L/h, 2.04 mmol/l and 3.46 mIU/L (p< 0.05, vs HFDG group), indicating a significant improvement in glucose tolerance (Figure 1G, D, E). According to hepatic and adipose tissues staining (Figure 2E), HFDG group mice exhibited severe hepatic lipid droplets and adipocyte hypertrophy, which were alleviated after ITF treatment. Overall, the above results indicate that ITF have a beneficial effect that ameliorates glycolipid metabolism disorders in HFD mice.
Changes of Fecal Microbial Diversity
We used the 16S rRNA gene amplicon sequencing method (V4 region) and generated 2,131,728 reads for a total of 25 samples, with an average of 85269± 22,171 reads per sample. At each stage, NCD-HFD-ITF and NCDG-HFDG-ITFG pairs shared less common OTUs with each other, respectively. The Venn graph exhibited common OTUs for NCD-HFD-ITF and NCDG-HFDG-ITFG pairs decreased from 579 before mating to 438 on GD18 of gestation, respectively (Figures 3A).
To assess fecal microbial community structure, richness (Chao 1 index) and diversity (Simpson index) were calculated (Figure 3B, C). For the Chao 1 index, the data of ITF group exhibited significantly higher than NCD and HFDG groups (p<0.05, p<0.01). A remarkable increment in Simpson index with ITF supplementation was found compared with HFD and HFDG groups in the present study (p<0.05). All the results above provide the view that ITF addition could effectively improve the decline of Chao 1 index and Simpson index induced by high fat addition.
We then used Principal Co-ordinate Analysis to categorize the Operational Taxonomic Units (OTU) data into two main factors that explained 64.42% of the variance (Figure 3E), which showed that the microbiome in NCD (NCD and NCDG), HFD (HFD and HFDG) and ITF (ITF and ITFG) treatment groups significantly differed from one another while the two groups of the same treatment shared some overlapping regions before and after conception, which indicated that the overall gut microbial community had been significantly modified. The four groups exhibited significant, tight clustering according to NCD or ITF diet. Independent biological replicates were generally consistent, but more variable among mice fed by HFD (Figure 3D).
Changes of the Relative Abundance at Phylum Level
The phylum Bacteroidetes was dominant among the 9 phyla (>1% in at least one sample) present in the gut microbiota from the six groups of mice, and the ratio of Firmicutes/ Bacteroidetes was increased in HFD and HFDG mice over NCD and NCDG groups, but lower in the ITF and ITFG groups compared with HFD and HFDG mice (Figure 4). The gut microbiota in obese individuals has usually shown an increased Firmicutes/Bacteroidetes ratio . Therefore, the decreased Firmicutes/Bacteroidetes ratios of ITF and ITFG mean that this feature in obesity could be reversed by the ITF-supplemented diet. High fat treatment decreased the relative abundance of Proteobacteria before mating (p<0.01). ITF supplementation increased the relative abundance of Verrucomicrobia compared with HFD before mating and on GD18 of gestation (p<0.01). Relative abundances of Deferribacteres group of HFD and Cyanobacteria group of NCD were not detected in fecal samples on GD18 of gestation. Moreover, relative abundances of Actinobacteria decreased in HFD before mating, while increased substantially when reaching the perinatal period. The majority of genera were affected by gestation stage indicating that their relative abundances changed greatly over the pregnancy progress.
Changes of the Relative Abundance at Genus Level
The relative abundances at genus level (>1% in at least one sample) were present in Figure 5. Fat addition (HFD and HFDG) increased the relative abundances of Dubosiella and Lactobacillus and decreased that of Romboutsia and Alloprevotella compared to the NCD (NCD and NCDG). The abundance of Bifidobacterium increased, whereas that of Dubosiella decreased with the intervention of ITF before and after conception. Our results also indicated that the abundance of Akkermansia was significantly higher in the ITF-treated (ITF and ITFG) groups than any other group. The heat map analysis of microbial community composition at the family level confirmed that the abundance of Dubosiella that cause obesity and metabolic syndrome-related inflammation were reduced (Figure 6).
Next, to identify the changes in specific bacterial taxa after the inulin-supplemented diet intervention before and after conception, we utilized the linear discriminant analysis (LDA) effect size (LEfSe) to compare the cecal microbiota composition between the NCD, HFD and ITF groups, LDA score was selected to discriminate specific taxa in different groups. Compared with the HFD group, the ITF mice had a higher abundance of f-Ruminococcaceae, f-prevotellaceae, o-Verrucomicrobiales, g-Akkermansia, c-Verrucomicrobiae, p-Verrucomicrobia and f-Akkermansiaceae but lower abundance of g-Unidentified clostridiales, f-Unidengtified Clostridiales, g-Dubosiella, c-Erysipelotrichia, o-Erysipelotrichales and f-Erysipelotrichaceae (Figure 7A-E). Correspondingly, g-Bacteroides, f-Ruminococcaceae and f-Bacteroidaceae were enriched in ITFG group on GD18 of gestation (Figure 8A-C).
Correlations between glycolipid metabolism indicator and bacterial abundance
At phylum level, we analyzed the correlations between significant glycolipid metabolism indicator and gut microbiota on GD18 of gestation. Bacteroidetes abundance was negatively correlated with FBG, FINS, TG and TC, whereas Firmicutes abundance was positively correlated with FBG, FINS and TG (Figure 9A). Moreover, Actinobacteria abundance was positively correlated with FINS and TC (Figure 9A).
At genus level, the relative abundance of Dubosiella showed positively correlated with FBG, FINS and TC (Figure 9B). Romboutsia abundance was positively correlated with FBG (Figure 9B).