The Influence of the Gut Microbiome on Host Metabolism Through the Regulation of Gut Hormone Release

1The gastrointestinal (GI) tract is host to a highly complex microbial ecosystem, comprising of bacteria, yeast, fungi, bacteriophages, and other viruses, as well as protozoa and archaea. Commensal bacteria, hereto referred to as gut microbiota, are found along the length of the GI tract and at greatest density within the caecum and colon, and along with their genes and gene products (collectively referred to as the gut microbiome), perform several functions that heavily influence host physiology. Not only does the gut microbiota play a critical role in modulating host immune defense and brain function, it also plays a role in regulating host metabolism. This is well-illustrated by the transfer of microbiota from lean and obese human twins into germ-free (GF) mice lacking a native gut microbiome, resulting in the conveyance of the metabolic phenotype of the host. Microbiota depletion in mice confers significant protection against metabolic dysregulation induced by a high-fat diet such as obesity, glucose intolerance and insulin resistance, all of which are hallmarks of metabolic diseases including type 2 diabetes (T2D). While bacteria-mediated inflammation is associated with detrimental metabolic effects in mice, the underlying mechanisms by which gut microbiota influence metabolism are still not fully understood. The gut microbiome contributes significantly to host energy harvest by converting inaccessible nutrient sources such as plant polysaccharides and other complex carbohydrates, into readily absorbable metabolites. Moreover, a key link has been established between the gut microbiome and the release of several gut hormones that are important regulators of peripheral metabolism.

2Within the mucosal lining of the gut, specialized enteroendocrine (EE) cells synthesize and secrete several hormones that facilitate a range of key physiological processes. Collectively, EE cells constitute the largest endocrine organ in the body, despite making up less than 1% of the total epithelial cell population in the gut. A broad number of EE cell subpopulations have been defined, based largely on their hormone expression profile. EE cells have the capacity to sense the luminal nutrient environment of the gut and are differentially responsive to many dietary compounds and luminal conditions within the intestine. Mounting evidence has highlighted that the gut microbiome influences EE cell hormone release, with downstream consequences for host metabolism and metabolic disease progression (Figure 1). There is also recent evidence that microbial-mediated release of gut hormones may influence other EE cell types, demonstrating the complexity that is the relationship between the gut microbiome, gut hormone release, and host metabolism. This review aims to describe how intestinal microbiota and their metabolites can influence host metabolism through the regulation of gut hormone release.

3The gut microbiota produces an array of metabolites through the breakdown of indigestible carbohydrates (Figure 2). The most abundant of these metabolites are the short chain fatty acids (SCFAs) acetate, propionate and butyrate, which exist at a ratio of approximately 3:1:1 in the human intestinal lumen, respectively, however, this ratio is, at least in part, dependent upon both diet and microbial composition. The fate of these bacteria-derived SCFAs differs substantially: acetate is readily absorbed into the circulation for distribution to peripheral tissues. Propionate, on the hand, is metabolized by the liver upon absorption, while the majority of butyrate is consumed locally by colonocytes as their primary fuel source. While the majority of bacteria-derived SCFAs are present in the colon, lesser amounts have also been detected in the ileum of pigs, as a result of cecoileal reflux, and to a lesser extent, the proximal small intestine. The relative abundance of SCFA is also likely to differ along the length of the gut as a result of the region-specific microbial composition, substrate exposure, and absorption. For example, genes encoding carbohydrate metabolism pathways are enriched in members of the Bacteroidetes phylum, while genes encoding bile acid metabolism pathways are enriched in the bile acid-tolerance Firmicutes.

4Receptors present on EE cells allow these cells to sense luminal and possibly circulating SCFA, which triggers the release of several metabolically active gut hormones. SCFA signal via two predominant mechanisms: (1) inhibition of nuclear histone deacetylase (HDAC) to alter gene transcription and expression, and (2) stimulation of G-protein-coupled free fatty acid receptors 2 and 3 (FFAR3, FFAR3), expressed throughout the length of the GI tract in distinct regional patterns. FFAR2 has equal affinity for acetate, propionate and butyrate, while the affinity of FFAR3 for acetate is substantially lower than for propionate and butyrate. Signaling of SCFA via FFAR2/3 is therefore dependent upon the combination of receptor type and specific metabolite abundance.

5Bile acids are amphipathic molecules synthesized by hepatocytes from cholesterol and are released into the GI lumen to aid the solubilization, and thus absorption, of dietary lipids. It has long been appreciated that the intestinal microbiota is directly involved in host bile acid metabolism, as effective enterohepatic recycling of bile salts is heavily reliant on deconjugation and dihydroxylation of bile acids by microbial-derived bile salt hydrolases (BSH). This gives rise to secondary bile acids, which are more hydrophobic and can thus be reabsorbed via passive diffusion, limiting bile acid loss through feces. Specific activity of BSH in human gut microbiota differs between different phyla, with BSH in the Firmicutes and Actinobacteria capable of metabolizing all conjugated bile salts and Bacteroidetes BSH activity being specific to tauro-conjugated bile acids. In addition to this role in the GI tract, bile acids are signaling molecules that are implicated in peripheral metabolism. Bile acids have major roles in peripheral metabolism through their action on two bile acid receptors, the G-protein coupled receptor TGR5 (formerly known as Gpbar1), and the nuclear receptor FXR, both of which are expressed in EE cells (Figure 2). Receptor affinity and potency varies substantially between different bile acids. As such, the gut microbiome can exert profound influence on host metabolism by altering the composition of the bile acid pool, through altered bile acid synthesis and re-uptake.

6Structural components of the microbial membrane, such as flagella and membrane-bound lipopolysaccharide (LPS), act as signaling molecules through a number of cellular pattern recognition proteins. LPS is a cell-wall component of Gram-negative bacteria, such as members of the Bacteroidetes phylum, and is a potent ligand for toll-like receptors (TLRs), particularly toll-like receptor 4 (TLR4). In addition to powerful immunity- and inflammation-inducing effects, the expression of TLRs has been demonstrated in a number of EE cells and activation of this receptor triggers secretion of a number of metabolically active hormones such as GLP-1, 5-HT, and PYY (; Figure 2). Clinically, elevated levels of LPS (endotoxemia) are closely associated with obesity and insulin resistance. Mechanisms by which LPS contribute to perturbed glycemic control and adiposity likely involve complex interactions between gut hormone secretion, mucosal barrier integrity, and host inflammation and immune pathways.

7Enterochromaffin (EC) cells are the source of almost all (about 95%) serotonin (5-HT; 5-hydroxytryptamine) within the body. These cells constitute almost half of all EE cells and are dispersed throughout the length of the GI tract in varying densities. EC cells have long been known to be important in many intrinsic gut mechanisms associated with motility and EC cells are able to sense their local nutrient environment and respond by secreting 5-HT in a unique manner. There is now firm evidence that gut-derived 5-HT is a key driver for dysregulation of peripheral metabolism. The absence of gut-derived 5-HT, through pharmacological inhibition or genetic ablation of the rate-limiting enzyme for 5-HT synthesis in the gut, tryptophan hydroxylase 1 (TPH1), conveys protection from diet-induced obesity in mice. Moreover, circulating 5-HT is increased in obese humans and is positively correlated with body mass index and poor glycaemic control.

8The gut microbiome influences 5-HT levels in the host. GF and antibiotic-treated mice have substantially lower levels of EC cell-derived 5-HT when compared to conventionally raised (CONV-R) controls, which are restored by colonization of GF mice with donor gut microbiota. EC cells have the capacity to sense microbial metabolites, as they express both FFAR2 and FFAR3, and a number of olfactory receptors. Acute exposure of mouse primary EC cells to SCFA in culture does not, however, elicit an increase in 5-HT secretion. Rather, the increase in 5-HT observed in the presence of a gut microbiome is likely due to the chronic exposure mediating an increase in the biosynthesis of 5-HT, contributed to by increased EC cell proliferation. In addition, luminal butyrate infusion restores intestinal motility in GF mice, which is blunted in TPH1-KO mice, indicating the effects of butyrate may be mediated by EC cell 5-HT. Acute responses to aromatic metabolites, such as isobutyrate and isovalerate, have been observed in EC cells within intestinal organoid preparations, likely via olfactory receptor activation. However, it is plausible that this is an indirect response, due to the cross-talk with other gut-derived hormones such as GLP-1, which are also increased following exposure to microbial metabolites and have the capacity to signal to EC cells.

9Glucagon-like peptide 1 (GLP-1), a cleavage product of proglucagon, is secreted by L-cells predominantly located in the ileum and colon. GLP-1 is an incretin hormone, released postprandially and in response to nutrients such as glucose to augment insulin and inhibit glucagon secretion from the pancreas. In addition, GLP-1 inhibits gastric emptying and influences satiety and food intake. Together with PYY, GLP-1 is thought to underlie some of the metabolic gains observed following gastric bypass surgery and the action of GLP-1 underlies some of the glucose-lowering ability of the diabetes therapy, metformin. As such, GLP-1-targeted therapeutics including GLP-1 analogs and inhibitors of GLP-1 degradation by the enzyme dipeptidyl peptidase IV (DPP-4) have been extensively exploited for their anti-diabetic properties. Interestingly, the microbiota possess DPP-4-like activity, which in mice is reduced by administration of the DPP-4 inhibitor, vildagliptin, and is accompanied by a shift in microbial composition that is independent of the direct effects of DPP-4 inhibition on microbiota function. Specifically, vildagliptin treatment is associated with a decrease in the abundance of Oscillibacter and increased in the abundance of Lactobacillus, with a reduction in TLR ligands and an increase in propionate. Thus, the DPP-4-like activity of intestinal bacteria can potentially influence the levels of circulating GLP-1 and PYY, which in turn may exist as a feedback loop to influence microbial composition and microbial metabolite abundance.

10A dynamic relationship exists between L-cells and the gut microbiome. Bile acid-mediated activation of TGR5, SCFA signaling, LPS, and other metabolites such as indole are all potent GLP-1 secretagogues. The regulation of GLP-1 by secondary bile acids is dependent upon the receptor-signaling pathway involved, as activation of TGR5 increases GLP-1 secretion while, on the other hand, activation of FXR reduces GLP-1 secretion. This dynamic relationship is made even more complex by TGR5-FXR cross-talk that exists between these two receptors, particularly in the colon. Luminal infusion of the bile acid chenodeoxycholic acid, in rats, triggers release of GLP-1 into the vasculature, in addition to PYY, via TGR5. Indole, another major bacterial metabolite derived from dietary tryptophan, acutely stimulates GLP-1 by prolonging cellular action potential duration. Conversely, chronic exposure to indole dose-dependently decreases GLP-1 secretion in primary murine L cells by inhibiting ATP synthesis pathway. In addition, microbial LPS triggers GLP-1 secretion via TLR4 following mucosal barrier injury, and as such, glucose-stimulated insulin secretion in a mouse model of endotoxemia. Dietary prebiotics such as oligofructose, which increase bacterial SCFA production, are associated with upregulated L-cell differentiation and GLP-1 content in the rat proximal colon, and reduces weight gain when administered before and during a HFD. Increasing L-cell numbers, whereby increasing post-prandial GLP-1 release, are also associated with enhanced satiety and reduced adiposity. The mechanisms by which SCFA increase GLP-1 secretion are region-dependent, as signaling in the small intestine is predominantly via FFAR3, whereas FFAR2-mediated GLP-1 release occurs in the colon.

11Paradoxically, GF and antibiotic-treated mice have higher circulating GLP-1 levels during fasting and reduced mucosal GLP-1 content, compared to genetically identical CONV-R mice. A recent study reported that the gene expression profile of ileal L-cells derived from GF mice differed substantially from CONV-R mice. Notably, many of the genes regulating L-cell functional capacity are upregulated in GF mice and L-cells have a greater number of secretory vesicles in GF mice. What underlies these differences in GF mice is unknown but may be a reflection of GLP-1 resistance, which is observed in diet-induced obesity and associated with altered microbiota composition, particularly in the ileum.

12Peptide tyrosine-tyrosine (PYY) is synthesized and secreted by L-cells, in addition to GLP-1, and is predominantly expressed in the lower small intestine and colon. PYY regulates food intake and satiety through activation of central G protein-coupled Y2 receptors on neuropeptide Y (NPY) and AgRP neurons in the hypothalamic arcuate nucleus. This initiates a signaling cascade whereby appetite-stimulating NPY neurons are suppressed, allowing for the disinhibition of the satiety-inducing proopiomelanocortin (POMC)/α-MSH pathway. Obese humans have reduced circulating PYY, as a result of attenuated colonic PYY secretion, rather than PYY-resistance. Circulating PYY exists as two forms: PYY1-36 and the DPP-4-cleaved PYY3-36, with the latter being the most dominant postprandial circulating form and the most biologically potent with respect to its anorectic effects.

13The ability of gut microbiota to influence PYY secretion therefore has significant implications for the development of obesity and metabolic disease. Microbial SCFAs, particularly butyrate, cause a dose- and time-dependent increase in PYY gene expression in two EE model cell lines and in primary human colonic cell cultures. In addition, oral administration of butyrate moderately increases circulating PYY. The mechanisms by which SCFA increase the biosynthesis of PYY appear to be via a combination of FFAR2/3 signaling by all SCFA, and inhibition of HCAD by propionate and butyrate. Although, these mechanisms appear to be species-specific and were not accompanied by an increase in GLP-1 secretion that is seen following exposure of primary mouse colonic cultures to propionate. The use of a FFAR2 knockout mouse demonstrates the involvement of SCFA signaling in increasing the number of PYY-containing cells, particularly in mice exposed to a diet rich in the SCFA-precursor, inulin. Alteration of the human gut microbiota through a 4-day broad-spectrum antibiotic regimen, acutely and reversibly increased postprandial plasma PYY. However, the precise alterations in microbial metabolites and bacterial species that underlie this change are unknown. Secondary bile acids are also potent stimuli for PYY secretion and the mechanisms by which this occurs are consistent with those for GLP-1 secretion. Luminal perfusion of a mixture of both primary and secondary bile acids into a vascularly perfused rat lumen increases venous effluent PYY levels in a TGR5-dependent manner, while the same effect was observed with infusion of the secondary bile acid CDCA alone.

14Glucose-dependent insulinotropic peptide (GIP), also known as gastric inhibitory peptide, is an incretin hormone released postprandially in the small intestine from classically defined K cells. The activity of GIP is conveyed through GIP receptors (GIPR) expressed in pancreatic β-cells, adipocytes, bone cells, and in neurons of the CNS. Similar to GLP-1, the biological activity of GIP is rapidly attenuated by enzymatic breakdown by DPP-IV. Within the pancreas, GIP contributes significantly to postprandial insulin secretion, through increased insulin biosynthesis and upregulated β-cell proliferation. Defective GIP-signaling is believed to underlie, at least in part, the attenuated glucose-stimulated insulin secretion seen in T2D individuals. GIP is also widely considered an adipogenic hormone as it promotes lipid uptake and storage in adipocytes.

15Elevated GIP level was associated with the observed increased adiposity induced by a sub-therapeutic antibiotic regimen administered to mice at weaning for 7-weeks, as the treatment did not alter plasma levels of other gut hormones. The treatment significantly increased Firmicutes/Bacteroidetes ratio and caecal SCFA levels, which could potentially underlie the increased GIP level and thus, the increased adiposity. However, recent contradictory evidence demonstrates that carbohydrates within the lumen inhibit GIP secretion, via the microbial SCFA-FFAR3 signaling pathway. Whether the discrepancy seen across these studies is due to differential signaling via FFAR2 and FFAR3 is unknown. Consistent with specific receptor pathways for SCFA-mediated gut hormone release, oral administration of sodium butyrate into mice has been shown to transiently increase GIP and GLP-1 secretion, while sodium pyruvate and a SCFA cocktail are selective for increased GIP, but not GLP-1 or PYY.

16Cholecystokinin (CCK) is derived from the classically named “I cells” predominantly localized to the upper small intestine. CCK is released in response to dietary fat and protein intake. CCK has well-defined roles in appetite regulation, gastric emptying and motility and the release of bile acids and pancreatic enzymes that are important for digestion, through activation of CCK receptors. Less is known about gut microbial regulation of CCK compared to other gut hormones, largely due to the exposure of CCK-containing cells to microbiota limited to the small intestine. In pigs, ileal infusion of the SCFAs acetate, propionate and butyrate during feeding increased plasma CCK levels and paradoxically inhibits pancreatic secretion. Limited investigations have been undertaken into microbial regulation of CCK in humans, however. One report from Roux-en-Y gastric bypass patients has revealed no changes in circulating CCK levels across normal weight and obese individuals pre- and post-surgery, despite a significant shift in microbial composition following surgery. However, reduced CCK protein expression is observed in dissociated cells from the proximal small intestine of GF mice, which was not due to reduced numbers of EE cells.

17The diversity of gut microbiota and relative abundance of microbial metabolites (metabolomic profile) is heavily dependent on specific dietary components, as evidenced by the substantial difference in microbial communities with consumption of plant-rich or protein-rich diets. This is due to the nutrient-induced selective pressures placed on microbiota, favoring bacterial species enrichened in the genes required for specific substrate metabolism. For example, plant-based diets and intake of probiotics increases luminal fiber and complex carbohydrate content, whereby selecting for species enriched in carbohydrate-active enzymes. Animal-based diets rich in fats and proteins and low in fiber increase luminal bile acid content, favoring bile acid-resistant microbes enriched with genes for bile acid metabolism, such as bile acid hydrosases and sulfite reductase. Dietary fiber is also a major influence on gut transit (reviewed in detail by ), which, in turn, is an important determinant of fecal microbiome composition and metabolism. In the absence of dietary fiber, however, a compensatory shift in the gut microbiome has been observed, with an increase in populations expressing mucin-degrading enzymes, suggesting an overall microbial preference for fiber-based substrates.

18The increased consumption of non-nutritive sweeteners (NNS), such as saccharin, sucralose and aspartame, while being acutely beneficial for reducing caloric intake and blood glucose excursions, also has long-term consequences for microbiome composition and glucose intolerance. Notably, consumption of common NNS has been demonstrated in mice to exacerbate the development of glucose intolerance and weight gain in males, which is mediated by distinct functional alterations to the gut microbiome. Specifically, the NNS saccharin and acesulfame-K increased the abundance of members of Bacteroidetes and reduced abundance of Firmicutes. The effects of NNS on metabolism and microbial composition in humans is largely dictated by the native microbial composition prior to NNS exposure, while in mice, the response also appears to be gender-specific. Nevertheless, the NNS-induced changes in microbial composition observed in these studies is consistent with the microbial composition seen with obesity and metabolic disease.

19The richness and diversity of the human gut microbiome correlates with metabolic function and a core obesogenic microbiome has been established, characterized by a high ratio of Firmicutes to Bacteroidetes. Shifting the composition of the microbiome to reduce the abundance of Bacteroidetes, as seen with short-term caloric restriction, conveys improved metabolic outcomes in mice as a result of decreased LPS production and reduced TLR4 signaling. There are also large-scale observational studies that linked antibiotic use to risk of type 2 diabetes, although caution should be excised in interpreting these results, as hyperglycaemia is a risk factor for infection and may thus warrant increased antibiotic use. In addition, specific clinically used antibiotics, particularly vancomycin-imipenem and ciprofloxacin, induce differential effects on microbiome composition and microbial metabolite abundance following regrowth, which may have downstream implications for antibiotic-specific effects on metabolism. Ingestion of xenobiotics, such as pharmaceuticals and environmental chemicals, has the potential to modify gut microbial composition with downstream consequences for metabolism. This is evidenced by the diabetes drug, metformin, for which a shift in gut microbial composition is in part responsible for its therapeutic effects. The metabolism of xenobiotics by gut microbiota is also chemically distinct, which highlights the gut microbiome as a possible tool for targeted drug design and delivery. Recent work by has elegantly shown that migration from a non-Westernized culture to a Westernized culture rapidly and inter-generationally impacts the diversity of gut microbiota. This loss of microbial complexity and biodiversity resulted in a loss of key microbial enzymes required for plant fiber digestion, partly attributed to altered dietary composition and reduced food diversity, and may predispose individuals to metabolic disease. As such, interventions to shift gut microbiota composition may be a powerful therapeutic tool for the treatment of obesity and metabolic disorders.

20All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

21The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a past collaboration with one of the authors DK.