Although the SCFAs have been shown to protect against body weight gain in both the DIO mice and overweight humans, the underlying mechanisms are unclear. In this study, consistent with previous reports17,24,25,26, we demonstrated that dietary supplementation of acetate, propionate, butyrate or their admixture significantly inhibited the body weight gain induced by HFD feeding. Furthermore, these supplemented SCFAs led to significant changes in expressions of GPR43 (an increase in the adipose tissue and a reduction in the colon), as well as bacterial community structure in feces (a reduction in the proportions of Firmicutes and an increase in the proportions of Bacteroidetes) compared to those of the HFD-fed obese mice. SCFAs could modify the structure of the gut microbiota towards a pattern more similar to that of the control mice used in the present study, implying that the alleviation of microbiota disturbance may play a role in SCFA-mediated prevention of obesity.

The production of SCFAs by microbial fermentation of dietary fibers is associated with a wide range of health benefits, including improvements in body composition, energy homeostasis, lipid profiles and reduced body weight, by stimulating multiple hormonal and neural pathways, such as appetite control, enhancement of oxidation and thermogenesis in muscle and liver tissues, and others6,13,14,15,24,25,26. However, no consistent conclusions have been reached on changes of gut SCFA production in obesity. It has been demonstrated that fecal SCFA concentrations in rodent models of genetic obesity and overweight adult humans are higher than those of their lean counterparts, indicating an increased microbial energy harvest in obesity22,23,27. However, in diet-induced obese mice fed a HFD with 45% calories from fat, reduced fecal SCFA (acetate and propionate) concentrations have been reported28. Unfortunately, in the present study, we did not find any changes in the fecal concentrations of the three types of SCFAs in obese mice fed the HFD with 60% calories from fat. This discrepancy may be the result of different shifts in microbial cross-feeding patterns, diet compositions, severity of obesity, genetic background and others. Interestingly, if SCFAs were added to the HFD, more propionate and butyrate were excreted from the feces with no changes in acetate, suggesting that acetate may be more efficiently absorbed into the body and have greatest effects in preventing weight gain. Regarding the roles of acetate in obesity, Lin HV et al. reported less effects of acetate on suppression of excess weight gain compared to propionate and butyrate, and den Besten G et al. found that all three SCFAs attenuated body weight to a similar extent24,26. The inconsistency in the roles of acetate in obesity may be due to differences in mouse species, age, the HFD formula and quantity of SCFAs added. In the former study, three-month-old C57BL/6 N mice were put on HFD (60% of calories from fat) supplemented with molarity-matched sodium salts of butyrate (5% wt wt−1), propionate (4.3% wt wt−1), or acetate (3.7% wt wt−1)24; whereas in the latter study, two-month old C57Bl/6 J mice were fed HFD (45% of calories from fat) with sodium acetate, sodium propionate, or sodium butyrate incorporated into the diet at the same ratio of 5% (wt wt−1). In our study, the HFD (60% of calories from fat), added acetate, propionate or butyrate at 5% (wt wt−1), were fed to three to four week-old C57Bl/6 J mice, and the result showed that all the three SCFAs suppressed the HFD-induced weight gain, with acetate having the greatest effects.

SCFAs have been shown to increase the release of the anorectic gut hormones PYY and GLP-1 from enteroendocrine cells and leptin from adipose tissue, leading to a reduction in food intake and body weight gain24,25. Various studies that focused on the elucidation of SCFA target molecules in the host indicated that GPR43 and GPR41 are predominantly activated by the SCFAs, with acetate and propionate preferentially activating GPR43 and propionate and butyrate preferentially activating GPR4115,16. In the present study, the HFD-fed obese mice had higher expressions of GPR43 and GPR41 in the colon, which were positively associated with expressions of PYY and GLP-1. This suggests that higher expressions of GPR 43 and GPR41 in the colon might result in increased expressions of PYY and GLP-1, which may counteract the body weight gain by inhibiting food intake. Dietary supplementation of acetate, propionate, butyrate or their admixture helped to drastically decrease GPR43 and GPR41, thus leading to a reduction in PYY and GLP-1 expressions to keep them within a normal range29. The reduction in these anorectic hormones may have prevented decreases in the food and energy intake after introduction of SCFAs to the HFD. Similarly, in the adipose tissue, higher expression of leptin was induced by the HFD and reversed by supplementation with acetate or the SCFA admixture. Regarding the effects of SCFAs on leptin and their relationships with GPRs, a number of contradictory reports have been published. Some studies reported that the SCFAs, propionate and acetate increase the leptin expression in adipocytes in vitro, whereas butyrate has no effects30,31. However, Frost et al. failed to demonstrate a significant effect of SCFAs on leptin secretion in adipocytes32. Furthermore, the leptin mRNA expression has been found to be up-regulated by GPR43 activation in some studies or by GPR41 activation in others29,30. Here, we found no correlations between GPR43 and the leptin. These different results reported may be due to the different experimental design (in vitro or in vivo) and duration, which change the leptin expression33. Moreover, the lower expressions of GRP43 in the adipose tissue and associated reductions in adiponectin and resistin were found in the HFD-fed obese mice, and dietary SCFA supplementation significantly recovered their expressions to normal ranges or even higher, leading to reductions in body weight. This is consistent with previous reports indicating that activation of GPR43 in the adipose tissue by SCFAs protects against diet-induced obesity by suppressing insulin signaling as well as increasing the consumption of lipids17. The underlying mechanisms may be mediated by SCFA activated expressions of adiponectin and resistin, which have an inverse correlation with severity of insulin resistance34,35.

Nonetheless, it has been shown that dietary SCFA supplementation increased the expressions of adipose cpt1 and cpt2, the rate-limiting enzymes in mitochondrial fatty acid β-oxidation36, which were closely associated with the GPR43 expression herein. Furthermore, we found that SCFA supplementation promoted expressions of the genes associated with mitochondrial biogenesis (PGC-1a, NRF-1, Tfam, β-F1-ATPase, COX IV, cyt-c) and quantity (mtDNA)37, suggesting that the regulation of body weight by SCFAs is mediated by promoting mitochondrial function. Beige adipocytes emerging in white fat depots, similar to classical brown adipocytes, are also involved in fat burning. When animals are exposed to cold or receive chronic ß-adrenergic stimulation, the pre-existing beige adipocytes undergo phenotypic transdifferentiation, and browning will appear morphologically and histochemically38,39. In this study, we found that expressions of several gene markers specific for beige adipocytes were reduced in the HFD-fed obese mice and could be recovered by SCFA supplementation. Furthermore, the expressions of several genes involved in mitochondrial biogenesis and beige adipocytes were positively associated with GPR43 and GPR41 expressions. These data imply that SCFAs may promote beige adipogenesis, resulting in enhanced fat oxidation and energy expenditure, mediated by activation of GPR43 or GPR41. Meanwhile, the increased LPL expression likely resulting from the reduced fiaf expression19 along with the decreased HSL expression in fat indicates the inhibition of TG hydrolysis and lower plasma FFA concentration in obese individuals. Supplementation of SCFAs modified the expressions of LPL, fiaf and HSL to normal levels with no changes in plasma FFA concentration, suggesting that more TG was hydrolyzed to release FFAs, which may be oxidized. Thus, the beneficial effects of SCFAs on body weight induced by HFD feeding may primarily depend on their regulation of energy expenditure, presumably by activating GPR43 or brown fat-mediated thermogenesis in fat tissue17.

During the past ten decades, a large number of studies demonstrated that the pathogenesis of obesity is closely associated with disturbances in the gut microbiota, and variations in the ratio between the two major intestinal microbial phyla (Firmicutes and Bacteroidetes) have been reported21,40. However, comparisons of the gut bacterial communities between obese and lean subjects have produced conflicting results. Several studies involving mouse models and humans have shown an increased proportion of Firmicutes and reduced Bacteroidetes in obesity and significant increases in Bacteroidetes along with weight loss20,21,22,41,42, whereas others have observed no changes in these two phyla or even reported the opposite results23,43,44,45. These phylogenetic differences in different reports may be due to many variables, such as diet, genetic background, sample handing, sequencing techniques, and data analysis tools, among others40. In the current study, a higher abundance of Firmicutes concomitant with a decrease in the abundance of Bacteroidetes was found in the HFD-fed obese mice, which is consistent with several previous reports22,24,41. Interestingly, dietary supplementation of the SCFAs led to a higher abundance of Bacteroidetes and a reduction of Firmicutes accompanied by significant body weight loss.

It is important to note that most of the above observations were found division-wide, and the development of a microbiome favored by high-fat diets promotes energy harvest and storage-promoting obesity and metabolic diseases. The underlying mechanisms include suppression of the fiaf expression, leading to increased triglyceride storage in adipose tissues by promoting LPL expression19,46,47, and more bacterial lipopolysaccharide (LPS) transferred from the intestinal lumen to the blood, contributing to the low-grade inflammation, reduction in glucose tolerance, body weight gain and fat mass development, and oxidative stress that characterize the metabolic syndrome47,48. Similar to a previous report47, our results showed a significant increase in Proteobacteria, which includes Gram-negative bacteria; this results in increased LPS in the gut, which can act as an inflammatory factor to trigger the onset of insulin resistance, obesity, and diabetes49. Accordingly, in this study, the HFD-fed mice had elevated plasma glucose and insulin concentrations as well as increased expressions of plasma IL-1β, IL-6 and MCP-1, which were reduced by dietary SCFA supplementation. Furthermore, SCFAs along with monosaccharides produced by bacterial fermentation in the colon are absorbed and used as substrates for hepatic lipogenesis and gluconeogenesis20,22, as well as to activate GPR43 and GPR41 in tissues such as the colon and adipose tissue15,16,50. This is supported by our results indicating that the GPR43 expression in the colon was positively correlated with the ratio of Firmicutes to Bacteroidetes, and interestingly, dietary supplementation of the SCFAs down-regulated the ratio of Firmicutes to Bacteroidetes as well as the colonic GPR43 expression. In addition, the reduced ratio of Firmicutes to Bacteroidetes after SCFA supplementation may promote adipose HSL expression, causing increases in the fat hydrolyzed and oxidized. However, these findings need to be corroborated by causative studies in the future.

The shifts in gut microbiota based on obesity at the division level do not always correlate with the shifts at lower taxonomic levels. Sequencing in this current study showed that the HFD-fed obese mice had higher abundances of the genera Bacteroides, Odoribacter and Alistipes, and dietary SCFA supplementation did not change their abundance or increase them further. In addition, differences in taxa abundance at the phylum level have little relevance when considering all their individual groups21,40. Thus, many bacterial groups at lower taxonomic levels deserve attention, such as the genera Desulfovibrio in the Proteobacteria phylum, which may be linked to obesity51 and can be reduced by glycomacropeptide application52. Accordingly, in this current study we showed that the HFD-fed obese mice had a higher Desulfovibrio abundance, and adding acetate and propionate to diets reduced its abundance, but this did not reach statistical significance. Additionally, the abundance of Ruminococcaceae in the Firmicutes phylum, a family containing several butyrate-producing bacteria, was found to be higher53 or lower54 in obese mice. In post-surgery individuals, the fecal microbiota was characterized by an overall decrease of bacterial diversity, with a significant reduction in Ruminococcaceae, Lachnospiraceae, Clostridiaceae, Eubacteriaceae, and Coriobacteriaceae55. Intestinal colonization by Lachnospiraceae, which belongs to Firmicutes, isolated from the feces of hyperglycemic obese mice contributes to the development of diabetes in obese mice56. With regard to these bacteria, we found higher abundances of Ruminococcaceae_incertae_sedis and _unclassified, Lachnospiraceae_incertae_sedis and _unclassified in the DIO mice, and their reduction was associated with SCFA supplementation to some extent. The genera Lactobacillus and Bifidobacterium containing probiotic strains are linked to body weight. In the HFD-fed obese mice, the level of Bifidobacterium is reduced, whereas prebiotics promote a selective increase of this genus, and this increase is correlated with reduced adiposity and microbe-derived inflammation57. In contrast, higher concentrations of Lactobacillus species belonging to the Firmicutes phylum were found in obesity both in rodents and humans in comparison with lean subjects58,59. Consistent with these reports, we found that Lactobacillus abundance in the HFD-fed obese mice as well as obesity in preschool-age children (data not shown) was increased and that addition of acetate or butyrate reduced the level of Lactobacillus in mice fed a HFD. The changes in Bifidobacterium among different groups are difficult to explain because of very low abundance of Bifidobacterium detected in our study, and this needs to be investigated further.