Effects of Bacillus sp. DU-106 on Body Weight, Food, and Water Intake
At the beginning of the intervention, the bodyweight of the MOD group was higher than the NC group with a significantly different. The bodyweight of the MOD group then gradually decreased and became similar to that of the NC group at the end of the experiment. After Bacillus sp. DU-106 treatment, the bodyweight of mice increased when compared to the MOD group (Fig. 1A). At the same time, the food and water intake of the MOD group was markedly higher than that of the NC group. However, oral administrations of Bacillus sp. DU-106 and metformin could reverse these changes, which could decrease the food and water intake in T2D mice (Fig. 1B-C). These data suggest that Bacillus sp. DU-106 treatment could potentially protect against emaciation, polyphagia, and polydipsia in T2D mice.
Effects of Bacillus sp. DU-106 on the Organ Index and Histopathological Assay
The liver and pancreas indexes were markedly increased for the MOD group as compared to the NC group (Fig. 2, p < 0.05 or p < 0.001), while both Bacillus sp. DU-106 and metformin treatments lowered the liver and pancreas index, but there was no significant difference in the pancreas index.
H&E staining indicated that liver histology in the NC group had a well-organized structure, whereas the histological structure of the MOD group was abnormal (Fig. 2A). These lesions mainly include lipid vacuolation, swollen hepatocytes, steatosis, granular degeneration, and large diffuse lipid microvesicles that occupy most of the cytoplasm of hepatocytes; however, oral administrations of Bacillus sp. DU-106 and metformin could reverse these damages to some extent. Histological analysis of the pancreas in the NC group showed that the islets exist as round or oval clusters of cells that were scattered among healthy acinar cells with well-defined borders. In comparison, the pancreatic histology of the MOD group had obvious damage, with characteristics including unclear outer tissues and edges of the islets, irregularly arranged pancreatic islet cells, and atrophied islets infiltrated by inflammatory cells (Fig. 2B). However, the T2D mice treated with Bacillus sp. DU-106 and metformin both showed significant recovery of the pancreatic tissue structure, such as clear edges of the islets. These findings suggest that Bacillus sp. DU-106 can lessen the liver and pancreas damage in T2D mice.
Effects of Bacillus sp. DU-106 on HbA1c、FBG、Insulin Levels, and HOMR-IR
As shown in Fig. 3A, the level of HbA1c in the MOD group was remarkably higher than that of the NC group, while Bacillus sp. DU-106 and metformin treatments lowered the HbA1c levels, especially the Bacillus sp. DU-106 intervention (p < 0.001). The FBG and insulin levels in the MOD group increased significantly compared to those in the NC group, whereas oral administrations of Bacillus sp. DU-106 and metformin both effectively reduced the levels of FBG and insulin (Fig. 3B-C, p < 0.01 or p < 0.001). Notably, the T2D mice treated with Bacillus sp. DU-106 had lowered FBG and insulin levels compared to that received metformin. Consistently, the calculated HOMR-IR index was also higher in the MOD group than the NC group, while treatments with Bacillus sp. DU-106 and metformin led to a significant reduction in the HOMR-IR index (Fig. 3D, p < 0.001).
Effects of Bacillus sp. DU-106 on OGTT and ITT
Upon glucose administration, the blood glucose levels of the MOD group showed a sharp increase at 15 min and its levels at every test time point were significantly higher than those in the NC group, while supplementations of Bacillus sp. DU-106 and metformin effectively inhibited the increase in blood glucose levels (Fig. 3E). The total area under the glucose curve (AUC-glucose) over 120 min was taken as the quantitative results of the OGTT. Although the DC and MC groups of the AUGglucose values were higher than that of the NC group, they were strikingly lower than the AUGglucose value of the MOD group (Fig. 3F, p < 0.001). These results suggest that supplementation with Bacillus sp. DU-106 can repair the impaired glucose tolerance in T2D mice.
As shown in Fig. 3G, the ability of T2D mice to improve insulin resistance is not as good as that of normal mice; however, both Bacillus sp. DU-106 and metformin interventions can improve insulin tolerance of T2D mice. Interestingly, Bacillus sp. DU-106 treatment led to better insulin sensitivity at specific time points than metformin. Similarly, the T2D mice had higher AUCinsulin as compared to the normal mice (Fig. 3H, p < 0.001). As a result of effective treatments with Bacillus sp. DU-106 and metformin, the DC and MC groups had markedly lesser the AUCinsulin values (p < 0.05 or p < 0.001). These findings suggested that Bacillus sp. DU-106 treatment could exhibit a positive effect on insulin resistance in T2D mice.
Effects of Bacillus sp. DU-106 on Serum Lipids
As shown in Fig. 4A-D, the levels of TC, TG, and LDL-C in serum of the MOD group were markedly higher, while HDL-C levels were lower than that of the NC group (p < 0.01 or p < 0.001), suggesting that T2D mice may suffer from abnormal lipid metabolism and hyperlipidemia. Bacillus sp. DU-106 and metformin treatments adjusted dyslipidemia by reducing TC, TG, and LDL-C levels and by increasing HDL-C levels (p < 0.01, p < 0.05, or p < 0.001); however, as compared to metformin, treatment with Bacillus sp. DU-106 resulted in a more desirable change in these parameters
Effects of Bacillus sp. DU-106 on the activities of SOD and MDA
To study the antioxidant effect of Bacillus sp. DU-106 on the serum lipids in T2D mice, we investigated the antioxidant capacity by analyzing the activities of relevant enzymes, MDA and SOD (Fig. 4E-F). In comparison with the NC group, MDA levels in the MOD group increased, while SOD activities were markedly reduced (p < 0.01 or p < 0.001); however, administrations of Bacillus sp. DU-106 and metformin showed strong antioxidant activity, as evidenced by declined MDA levels and elevated SOD activities (p < 0.05 or p < 0.01). Again, the mice that were administered Bacillus sp. DU-106 showed better antioxidant activity than those with metformin. These findings were supportive that Bacillus sp. DU-106 ameliorated oxidative stress in T2D mice.
Effects of Bacillus sp. DU-106 on Gut Microbiota in T2D Mice
The composition and abundance of gut microbiota in mice feces were analyzed by the bacterial 16S rRNA genes pyrosequencing to evaluate the impact of Bacillus sp. DU-106 on T2D mice. A total of 232,135 clean reads were obtained from 28 samples after quality filtration, and an average of 6,273 ± 852 reads were generated per sample. Based on the operational taxonomic unit (OTU), we found that NC, MOD, DC, and MC had 28 common OTUs, whereas unique OTUs were detected by each group (38 for the NC group, 26 for the MOD group, 380 for the DC group, and 49 for the MC group, respectively); these unique OTUs indicated that treatment with Bacillus sp. DU-106 increased the functional and microbial diversity in T2D mice (Fig. 5A). We profiled the gut bacterial communities of the groups by measuring Chao1 and Ace indexes as an indicator of richness and Shannon and Simpson indexes as markers of diversity. Chao1, Ace, and Shannon indexes decreased, while the Simpson index increased in the MOD group as compared to the baseline level (p < 0.001). Bacillus sp. DU-106 administration restored the microbiota community diversity and richness, suggesting it can enhance the α-diversity of fecal microbiota in T2D mice (Fig. 5B-E, p < 0.01, p < 0.05 or p < 0.001). The principal coordinates analysis (PCoA) was used to analyze β-diversity, which showed the structural differences of the intestinal microbiota among four test groups (Fig. 5F). The NC group was separately clustered from the MOD group. And, the MOD and DC groups also showed a unique clustering of microbial community structure. These results suggest that treatment with Bacillus sp. DU-106 can reshape the gut microbial structure in T2D mice.
Effects of Bacillus sp. DU-106 on the Taxonomic Composition of Gut Microbiota
To assess the changes in the intestinal microbiota of the Bacillus sp. DU-106 and metformin treatments, we compared their relative abundance by sequencing the four groups at phylum and genus levels (Fig. 6A-C). At the phylum level, the fecal microbiota of all mice mainly consisted of Firmicutes, Bacteroidetes, Proteobacteria, Tenericutes, Actinobacteria, Cyanobacteria, and Verrucomicrobia. The MOD group showed higher abundance in Firmicutes and lower abundance in Bacteroidetes compared with the NC group, thereby increasing the ratio of Firmicutes to Bacteroidetes. Contrarily, Bacillus sp. DU-106 intervention reversed this trend to some extent (p < 0.001). At the genus level, the abundances of Ileibacterium and Faecalibaculum were decreased, while that of Romboutsia, Lactobacillus, and Staphylococcus were increased in the MOD group compared with the NC group. However, the genera of Blautia, Ileibacterium, Faecalibaculum, Faecalibacterium, and unidentified_Lachnospiraceae were markedly more abundant in the DC group than those in the MOD group, whereas metformin elevated the abundant of Akkermansia and Allobaculum. In a word, these data show that Bacillus sp. DU-106 and metformin can reshape the intestinal microbiome and improve gut dysbiosis in T2D mice.
Correlation Between Gut Microbiota And Biochemical Parameters
To investigate the associations of the gut microbiota with T2D, we run a Spearman’s correlation test between the 35 genera and biochemical parameters. As shown in Fig. 6D, Romboutsia, Staphylococcus, and Lactobacillus were significantly and positively correlated with HbA1c, FBG, insulin, AUC-insulin, MDA, TG, TC, and LDL-C levels, as well as the HOMR-IR index, whereas Ileibacterium and Faecalibaculum exhibited an inverse correlation. Additionally, Blautia and unidentified_Lachnospiraceae were strongly negatively associated with HDL-C. Taken together, increased the abundance of Ileibacterium, Faecalibaculum, Blautia, and unidentified_Lachnospiraceae could be beneficial for the alleviation of T2D, but Romboutsia, Staphylococcus, and Lactobacillus might be bad for the T2D treatment.