Consequently, a greater amount of unabsorbed BAs remain in the intestines where they can be transformed by intestinal microbes into toxic, hydrophobic BAs. Indeed, ASBT KO mice had a 10 – to 20-fold increase in fecal BA excretion and an 80% reduction in BA pool size compared to WT mice despite up-regulated BA synthesis. Paralleling its upstream regulator FXR, ASBTactivity can also be modulated by the gut microbiome. Pharmacological inhibition of ASBT in diabetic fatty rats significantly raised fecal BA concentrations and non-fasting plasma total glucagon-like peptide 1 while decreasing hemoglobin A1c and blood glucose. However, ASBT inhibition also reduced FXR mRNA levels in both the liver and small intestine, likely as compensation for the disrupted BA circulation. Interestingly, ASBT deficiency or inhibition in mice lowered serum glucose, insulin, and TG as a result of diminished sterol regulatory element-binding protein 1 c expression. Based on these results, ASBT inhibition appears as a possible clinical intervention for the management of obesity and diabetes. However, it remains to be determined whether such beneficial effects also occur in humans and whether they outweigh the adverse effects of disrupted BA cycling. An association between ileal ASBT activity and intestinal microbes was established when ampicillin-treated mice showed markedly decreased BA fecal excretion and elevated BA concentrations in hepatic portal blood.
The ampicillin-treated mice displayed significantly higher ileal ASBT mRNA and brush-border membrane protein levels, elevated total BAs, drainage collection pot and reduced intestinal enterobacteria-biotransformed BAs. These observations indicate negative regulation of ileal ASBT expression by the gut microbiome. This modulation of ASBT activity by intestinal microbes was shown to occur in part through a proteasomal degradation pathway since proteasome inhibition attenuated the CA-induced reduction of ileal ASBT protein level in ampicillin-treated mice. Consistent murine findings showed Caco-2 cells infected with E. coli had drastically diminished ASBT activity due to decreased transport Vmax and protein level at the plasma membrane. This suggests that certain species of intestinal microbiome can alter the enterohepatic circulation of BAs by influencing ileal ASBT. Furthermore, hypomorphic genetic variants in human ASBT have also been linked to increased cancer risk of the lower GI tract. One particular human ASBT SNP was associated with a twofold higher risk of colorectal adenomas potentially due to malabsorption of BAs leading to elevated colonic levels of toxic BAs. Another sequencing study revealed that carriers of several minor ASBT variants had significantly reduced ileal ASBT expression at both the mRNA and protein levels. Lastly, three ASBT nonsynonymous SNPs exhibited partially impaired to near complete loss of BA transport compared to WT allele in vitro. Being required for normal enterohepatic BA circulation, ASBT appears to be not only influenced by ileal FXR, cholesterol levels, and microbiota activity but also genetic variation.
Although investigation into the mechanisms through which BAs function in the liver and intestines have been predominantly focused on nuclear receptors such as FXR, there is accumulating evidence for the participation of G-protein coupled BA receptor 1 in mediating the systemic actions of BAs. Highly expressed in the liver, intestine, and brown adipose tissue, TGR5 complements FXR in regulating BA homeostasis, glucose metabolism, and enterohepatic inflammation. Therefore, examination of the regulatory roles of TGR5 and its potential contribution to BA dysregulation, gut dysbiosis and ultimately, liver and colon carcinogenesis is pertinent. TGR5 activation at the plasma membrane initiates the canonical G-protein signal transduction resulting in initiation of downstream pathways that remain poorly defined. Nevertheless, TGR5 was implicated in modulating BA and energy metabolism when TGR5 KO mice on a highfat diet experienced a 21–25% reduction in BA pool size and greater body fat accumulation accompanied by body weight gain relative to WT mice. Intriguingly, TGR5 can stimulate GLP-1 release and suppress hepatic glycogenolysis resulting in improved glucose tolerance, hepatic and pancreatic functions in obese mice. Treatment of enteroendocrine cells with a TGR5 agonist also enhanced GLP-1 secretion. Administration of BA sequestrant to dietinduced obese mice caused markedly higher energy expenditure, body weight reduction, and improved glycemic control. These findings in mice were replicated in monkeys with TGR5 agonist triggering co-secretion of peptide YY and GLP-1 from distal GI L-cells to produce sustained improvements in glucose tolerance. From these studies, TGR5 appears to control BA and glucose metabolism through its up-regulation of PYY and GLP-1. However, it is unknown whether if TGR5 expression or function is inhibited in obese and diabetic individuals. Complementary to FXR, TGR5 can prevent excessive inflammation in the liver and intestines to preserve normal physiology.
In mouse macrophages and Kupffer cells, TGR5 activation suppressed IκBα phosphorylation, p65 translocation, NFκβ DNA binding, and transcriptional regulation activity by stabilizing the interaction between IkBa and β-arrestin 2. Consistently, TGR5 KO mice exhibited more severe LPS-induced liver necrosis and inflammation relative to WT mice whereas TGR5 agonist inhibited LPS-induced NFκβ- mediated expression of pro-inflammatory mediators in WT mouse liver. Loss of TGR5 in mice also increased susceptibility to diethylnitrosamine-induced acute liver injury and cancer due to increased hepatocyte death, compensatory proliferation, and expression of inflammatory cytokines. Moreover, TGR5 KO mice displayed abnormal hydrophobic BA composition, severe hepatocyte necrosis, prolonged cholestasis, exacerbated inflammatory response, and delayed regeneration compared to WT mice post partial hepatectomy. Treatment of non-alcoholic fatty liver disease mice with dual FXR/TGR5 agonist decreased intrahepatic inflammation and improved histological features. Furthermore, TGR5 KO mice developed abnormal colonic mucous cell morphology with an altered molecular architecture of epithelial tight junctions resulting in increased intestinal permeability and microbial translocation. TGR5 stimulation reduces TNFα production by Crohn’s disease-associated macrophages after LPS exposure by inhibiting c-FOS phosphorylation and NFκβ activation. Taken together, these findings implicate TGR5 as another important player in protecting the liver and intestines against excessive inflammation. In addition to regulating energy homeostasis and inflammation, altered TGR5 expression and activity can also influence signaling pathways associated with cancer formation in cells of the liver, intestines, and beyond. Treatment of human gastric carcinoma cells with BAs activated epidermal growth factor receptor and ERK1/2 proliferative signaling in a TGR5-dependent manner. DCA exposure redistributed TGR5 to plasma membrane microdomains to transactivate EGFR resulting in downstream ERK1/2 activation. Low concentrations of CDCA can stimulate human endometrial cancer cell growth by upregulating cyclin D1 expression through TGR5-mediated recruitment of cAMP response element-binding protein to the cyclinD1 gene promoter. In human HCC cells, TGR5 inhibition decreased BA-induced caspase 8 activation by interfering with the JNK-mediated recruitment of caspase 8 to the death-inducing signaling complex. Furthermore, TGR5 stimulation inhibited cell proliferation and migration by suppressing STAT3 phosphorylation, transcription, and DNA binding activity. These results implicate TGR5 as a regulator of survival and growth pathways in liver and GI cells in response to BAs. Thus, within the context of BA dysregulation, over stimulation of TGR5 may encourage carcinogenesis. Indeed, square plastic pot a significant positive correlation was found between BA concentrations and the grades of intestinal atrophy and metaplasia with GI cancer developing more frequently in individuals with high BA concentrations. Strong TGR5 staining was present in 12% of human intestinal metaplasia cases but none in normal gastric epithelium cases and moderate to strong membranous and cytoplasmic TGR5 staining was present in 52% of intestinal but only 25% of diffuse subtype adenocarcinomas. Sequencing of primary sclerosing cholangitis patients identified six TGR5 variants, five of which were found to exhibit reduced or abolished function because of altered localization or activity. Akin to FXR and ASBT, TGR5 appears to be another intersection between BA and glucose homeostasis, inflammation, and cell proliferation in the liver and intestines. Beyond the previously discussed molecular players, BAs also interact with other receptors such as constitutive androstane receptor, pregnane x receptor, and vitamin D receptor to regulate glucose and lipid homeostasis as well as innate immunity. Although BAs are critical for regulating lipid absorption and glucose homeostasis, they can exert harmful effects when their levels become dysregulated.
In abnormally high concentrations, hydrophobic secondary BAs are cytotoxic, leading to DNA damage and cell death. BAs also have antimicrobial and amphipathic properties that are regarded as important regulators for the gut microbe environment with DCA being the most potent of all BAs at physiological concentrations in the human colon. DCA at a concentration of 0.5mM can effectively inhibit intestinal bacteria growth in cell culture, indicating that BA can regulate intestinal microbe composition through environmental stress. The human intestines house 10–100 trillion microbes, providing efficient metabolic capability to process indigestible dietary sources. Since the gut microbiome profile is largely dependent on the host diet and surrounding intestinal environment, there is a degree of variability among individuals, even in twins. However, shared microbial genes are identifiable to construct a “core microbiome” at the genetic level. Disturbances to the gut microbiome with phylum-level changes can result in altered nutrient acquisition, energy extraction, and metabolism. The crosstalk between BAs and the gut microbiome was established in the mid-1960s and continues to be a major research focus. A direct relationship was observed between the intestinal microbiota and BA levels using nuclear magnetic resonance spectroscopy after microbial colonization in germ-free mice. These intestinal microbes stimulated multiple pathways in xenobiotic metabolism and pharmacokinetics in the liver, kidney, plasma, urine, and colon. CYP8B1, required for the production of CA, helps to control the hydrophobicity of the BA pool by regulating the CA/CDCA ratio in conjunction with CYP7A1 and CYP27A1. Together, these enzymes are regulated by FXR, in a negative feedback manner. However, overexpression of FXR in germ-free mice did not lead to downregulation of Cyp8b1, Cyp7a1, or Cyp27a1. Therefore, the regulation of these genes by FXR is more complex than previously thought with possible participation by the gut microbiome. In addition to regulating intestinal Fgf15, gut microbes also regulated hepatic Cyp7a1 through intestinal FXR as well as endogenous FXR antagonists, tauro-conjugated alpha, and beta-muricholic acid as demonstrated in a mice feeding study. Conventionally raised mice had reduced expression level of FXR antagonists and Cyp7a1 and increased expression of Cyp8b1 allowing for greater intestinal FXR activity compared to germ-free mice. Since gut microbe colonization did not affect liver FXR, it is postulated that the intestinal microbiome acts mainly in the ileum to create a more hydrophobic BA profile by suppressing Cyp7a1 expression through increased induction of intestinal Fgf15. Previous studies have shown that consumption of a high-fat diet increased BA secretion and altered the gut microbial profiles in obese animal models and in patients with type 2 diabetes. The human gut contains a variety of species with different segments of the GI tract varying in bacterial density and diversity. For instance, the ileum contains around 107 colony-forming units per gram of bacteria with a dominance of gram-negative aerobes and obligate anaerobes while the colon houses 1012 colony-forming units per gram of bacteria with a dominance of anaerobes. Gram-positive firmicutes and actinobacteria and gramnegative bacteroidetes are the most prominent phyla with each taxon differing in their ability to extract and utilize dietary and host-derived resources, leading to variations in host lipid, glucose, and amino acid metabolism. It was found that differences in body composition between obese twins and lean twins were attributable in part to variations in bacterial fermentation of short-chain fatty acids, metabolism of branched-chain amino acids, and transformation of BAs. Co-housing ln mice with ob mice prevented diet-induced obesity in the latter by facilitating an ingression of bacteroidetes from the ln to ob mouse gut and altered several BA concentrations to resemble those in ln cage mates and ln control. Notably, a high fat diet with minimal fruits and vegetables selected against microbial profiles associated with leanness and prevented successful ingression by lnmicrobes. The association between the intestinal microbiota and diet-induced weight gain in both mice and human models mentioned earlier suggests that BA may be interacting with the intestinal microbes in the pathogenesis of obesity and diabetes. Studies have found that increasing levels of the primary BA cause a shift toward firmicutes, particularly genus Clostridium cluster XIVa, which raises production of hydrophobic secondary BAs. Firmicutes increased significantly from 54.1% in the control group to 93.4% in the high CA group at the expense of bacteroidetes and other minority bacterial populations. Potentially pathogenic proteobacteria, specifically gammaproteobacteria E. coli, also expanded in the gut of rats fed the high CA diet, decreasing bacterial densities and diversity. Metagenome sequencing of the fecal microbiome of obese individuals was consistent with these murine study findings.