Although numerous reports indicate that several probiotics improve MS16,17,18,19,20, the properties of probiotics that are involved in these anti-MS effects remain unclear. In the present study, BlaG treatment improved glucose tolerance in HFD-fed mice as well as in ob/ob mice (Fig. 1 and Supplementary Fig. S1). We conducted a comparative analysis using both BlaG and BloJ to investigate the mechanism(s) underlying the anti-MS effect by BlaG. The results showed that only BlaG improved glucose metabolism, concomitantly suppressing the accumulation of body fat and adipocyte hypertrophy (Fig. 2).

Several studies have reported that the anti-obesity effect of probiotics is related to changes in the gut microbiota16,18,25. We thus hypothesised that BlaG exerts an influence on the gut microbiota that differs from that exerted by BloJ because it has a greater proliferative activity in the gut than BloJ22. Interestingly, 16 S rRNA pyrosequencing analysis of caecal contents revealed that BlaG exerted a significantly greater influence on the overall bacterial structure of gut microbiota than BloJ (Fig. 3b and Supplementary Fig. S4b). Analysis of the bacterial composition at genus-level revealed that the relative abundances of the genera Bifidobacterium and Lactobacillus were remarkably enriched in the gut microbiota of the BlaG-treated mice (Fig. 3c). Indeed, qPCR analysis demonstrated that the number of B. lactis in the BlaG-treated group was more than five-fold greater than that of B. longum in the BloJ-treated group following treatment with the same cell numbers of each probiotic strain (see results). Consequently, BlaG treatment resulted in approximately a four-fold increase in the number of Bifidobacterium compared with BloJ treatment (Fig. 3d). These results demonstrated that BlaG proliferated effectively in the gut in HFD-fed mice because there were a greater number of Bifidobacterium cells present in BlaG-treated group, which agrees with previous reports21,22. Therefore, the highly proliferative probiotic strain BlaG altered the overall bacterial structure of the gut microbiota, probably through efficient proliferation, resulting in the improvement of MS, whereas the low numbers of Bifidobacterium strain BloJ had little influence on the microbiota or host metabolism.

The dominant metabolites of Bifidobacterium and Lactobacillus are acetate/lactate and lactate, respectively. Lactate is eventually metabolised to other organic acids including SCFAs by gut microbes26. Hence, we next focused on the changes in levels of SCFAs in the gut and plasma following probiotic treatment. Caecal and plasma acetate levels were significantly elevated by BlaG treatment, whereas the levels were unchanged by BloJ treatment (Fig. 4a,b). Similar elevation of caecal acetate by BlaG treatment was also observed in the ob/ob mice model (Supplementary Fig. S1e). Correlation analysis between the abundances of the gut microbiome and SCFAs revealed that genus Bifidobacterium and genus Lactobacillus correlated positively with SCFAs levels (Fig. 5). These data suggested that the enrichment of Bifidobacterium and Lactobacillus in the BlaG-treated mice might play a major role in accelerating the production of SCFAs in the gut. Because Lactobacillus mostly produces lactate as an end product of carbohydrate metabolism, the increase in Bifidobacterium appeared to have mainly contributed to the elevation of acetate in the BlaG-treated mice.

It has been recently demonstrated that SCFAs regulate energy homoeostasis via GPCRs9,27. GPR41 is activated equally by propionate and butyrate, whereas GPR43 is more responsive to acetate and propionate than to butyrate28,29. In the intestinal epithelium, SCFAs were found to stimulate GPR43, which resulted in the secretion of GLP-1, a gut-derived hormone that plays a significant role in energy metabolism9. In the present study, BlaG treatment significantly enhanced GLP-1 levels in both the colon and plasma, whereas no such effect was observed in the BloJ-treated mice (Fig. 4c,d). In addition, colonic GLP-1 levels correlated positively with luminal acetate levels (Fig. 4e) but not with propionate or butyrate levels (data not shown). These results implied that enhanced luminal acetate levels following BlaG treatment stimulated GLP-1 secretion in the colon, possibly via GPR43.

Kimura et al.9 reported that GPR43 signalling in adipocytes inhibits insulin signalling, and results in the suppression of fat accumulation in adipose tissue and an improvement in systemic insulin resistance. In the present study, BlaG treatment resulted in the elevation of both plasma and caecal acetate levels (Fig. 4a,b) in conjunction with improved glucose tolerance and less body fat accumulation. On the other hand, acetate levels showed a significant negative correlation with the amount of visceral fat and adipocyte size (Fig. 4e,f). These results suggested that the elevation in plasma acetate following BlaG treatment was associated with a reduction in the size of adipocytes, probably through enhanced insulin sensitivity via GPR43 signalling, which resulted in improved glucose tolerance and less fat accumulation. Together with the elevation in GLP-1 expression following BlaG treatment, our findings suggest that the enhanced production of gut acetate probably played a pivotal role in the anti-MS effects observed.

It has been reported that GLP-1 and GPR43 signalling potentiates insulin secretion by beta cells in the pancreas13. In addition, GLP-1 and acetate are reported to control appetite via the central nervous system10,30. However, bifidobacteria treatment did not affect either insulin secretion or food intake in this study (Fig. 1 and Supplementary Figs S2 and 3). Further research is therefore required to investigate why the elevation of both GLP-1 secretion and plasma acetate in this study did not lead to increased insulin secretion or appetite suppression.

Several possible relationships between metabolic parameters and intestine-related parameters, such as intestinal integrity, and between the diversity of the gut microbiota and the Bacteroidetes/Firmicutes ratio have been reported3,31. Some reports have indicated that increases in the number of bifidobacteria following probiotic or prebiotic treatment might restore impaired intestinal integrity4,5. In the present study, there were no changes in intestinal integrity-related parameters (Supplementary Fig. S2c and d) or microbial diversity (Supplementary Fig. S4a). Moreover, phylum-level analysis of the gut microbiota in this study demonstrated that BlaG treatment significantly elevated the abundance of phylum Actinobacteria but did not affect the Bacteroidetes/Firmicutes ratio (Supplementary Fig. S4d). It appears unlikely that intestinal integrity, microbial diversity, or the Bacteroidetes/Firmicutes ratio is involved in the anti-MS effects mediated by BlaG treatment.

In conclusion, B. lactis GCL2505, a highly viable and proliferative probiotic, exerted anti-MS effects, such as improved glucose tolerance and the suppression of visceral fat accumulation, via changes in the overall bacterial structure of the gut microbiota and elevations in the levels of SCFAs, especially acetate.