**6. Regulation of glucose homeostasis by Gut microbiota**

Gut microbiota has been shown to impact the pancreas directly. Gut microbiota has been proposed to modulate glucose homeostasis through multiple mechanisms [115, 116, 118, 156, 157]. Experimental data support four specific mechanisms through which the gut microbiome influences glucose homeostasis; (1) the β cell modulating effects of metabolites that are formed due to gut anaerobic microbial fermentation [157–159], (2) induction of cytokine activity in the islets of Langerhans via inflammatory cascades [160–164], (3) direct islets signaling affecting insulin and glucagon secretion through incretins modulation [87, 165], (4) alteration in the gut permeability, thus permitting the influx of toxins through intestinal mucosal barrier [166]. Mechanisms 1 and 3 are mainly considered for increased T2DM susceptibility and whereas mechanisms 2 and 4 are particularly implicated in the development of T1DM in early life. As T1DM is characterized by a significant reduction in the number of functional β cells. Cytokine and toxin-induced β cell apoptosis or dedifferentiation are considered major risk factors for T1DM.

Abnormal gut microbiome composition alters the intestinal barrier which favors absorption and increased circulating levels of LPS and BCAA. LPS induces low-grade inflammation and insulin resistance while BCAA is associated with an increased risk of T2DM development. An altered intestinal barrier also reduces the absorption of beneficial SCFAs and secondary bile acids. Metabolically SCFAs are mainly produced as an energy source for the gut epithelium. Butyrate is used by colonic epithelial cells for energy, acetate is used as a fatty acid precursor like cholesterol and propionate is a precursor for the process of hepatic gluconeogenesis [167–169]. Animal data have shown the beneficial impact of acetate supplementation on insulin resistance and glucose tolerance in animals fed with a high-fat diet [170]. Acetate at high intravenous (*i.v*) dose has also been reported to acutely enhance circulating levels of GLP-1 in humans [171]. Butyrate supplementation has been reported to enhance insulin sensitivity in mice fed with a high-fat diet while obesity and insulin resistance fail to develop over the course of 16 weeks [165].

Functional modulation of β cells through secondary metabolites is highly important in maintaining homeostatic glucose levels. SCFA has been highlighted as an important signaling molecule as the recent findings of the presence of functional SCFA cell surface receptors on different tissues including gut and peripheral tissues [172–175]. Gut microbial modulation of the host's metabolism modulated by SCFA production has been demonstrated by the activation of G-protein coupled cell surface receptors (*GPCR*s) also known as free fatty acid receptors (*FFAR*). FFAR includes different GPCRs which bind fatty acids of different chain lengths. GPR40 (FFA1), GPR84, and GPR 120 (FFA4) bind with the medium and long-chain fatty acids. Whereas GPR43 (FFA20), GPR41 (FFA3), and GPR109 bind with SCFAs. Propionate and acetate are found to be the most potent agonists of FFA3/GPR41 while butyrate selectively binds with FFA2. FFARs have been shown to be present in different peripheral tissues including the gut, liver, and pancreas. SCFAs like butyrate and propionate along with secondary bile acids indirectly modulate β cells. SCFAs enhance insulin secretion by activating GLP-1 secretion from the intestines [158, 176]. Butyrate and

propionate bind and activate G-protein coupled receptors (GPR43, GPR119) present on the enteroendocrine L cells and stimulate the release of GLP-1 in humans [177, 178]. Propionate also has been reported to influence β cell activity directly in humans. Propionate inhibits inflammatory cytokine-induced β cell apoptosis in human islets and enhances GSIS response from β cells independent of increased GLP-1 levels [179]. FFARs have been expressed by β cells and reported to modulate β cell activity in terms of GSIS [174, 180]. Apart from β cell modulation in terms of GSIS response, an interesting observation was made in these studies that high-fat diet-induced insulin resistance in a mouse model has shown to influence FFA2 receptor expression in β cells. Apart from these above-mentioned *in vivo* studies a recently published *in vitro* data further extends the notion that acetate, propionate, and butyrate separately enhance insulin secretion along with an increase in the expression levels of insulin genes from rat islets during long-term incubation [181]. Interestingly the authors noted that long-term incubation with butyrate induced a significant downregulation of β cell-specific key transcriptional factors and functional genes involved in the maturity-onset diabetes of the young (MODY). Another interesting finding which was made in this recent study was the significant suppression of the β cell identity genes like *GLUT2*, *GCK*, *Pdx1*, *MafA*, *Nkx-6.1*, and *NeuroD1* after the long-term incubation with butyrate. The global suppression of the β cell identity gene was surprisingly independent of the deacetylase activity of butyrate indicating a non-DNA acetylation mechanism involved. A significant decrease in the gene expression pattern of GLUT2 and GCK in rat islets after the long-term incubation with butyrate indicates that glucose or GSIS was not involved in the increased mRNA levels of *INS1* and secretion of insulin protein. Instead the basal levels of intracellular calcium ions [Ca2+]i was much higher in butyrate-treated islets as compared to the control. The combined effect of acetate, propionate, and butyrate on isolated rat or mouse islets in short- and long-term incubations needs to be examined in future studies. Along with *in vivo* approaches to fully characterize the impact of microbial metabolites on glucose homeostasis in different physiological conditions.
