**5. Transcriptional Regulation of G5G8**

*ABCG5 ABCG8* is effectively a single gene with a common promoter that regulates expression of both transcripts encoding each half of the transporter. To the best of our knowledge, there are no reports of differential regulation of *Abcg5* and *Abcg8* transcripts. Transcriptional regulation of *Abcg5 Abcg8* by a small molecule LXR agonist (T0901317) precipitated its discovery as the defective gene in sitosterolemia [22]. Expression of both mRNA and protein increases in liver and intestine in response to small molecule LXR agonists and dietary cholesterol, which promotes the accumulation of endogenous LXR agonists, oxysterols (Figure 1 (7)) [22,29–31,49,90]. Liver receptor homolog 1 (LRH1), GATA binding factor 4 (GATA-4), and hepatocyte nuclear factor 4α (HNF4α) binding sites map to the 374 base pair intergenic promoter that separates the initiation codons for each protein, the latter of which synergize with LXREs located in distal regions of the gene, to activate the promoter and increase expression of both transcripts [91,92]

Hepatic expression of *Abcg5 Abcg8* is also induced by FXR agonists and bile acids, and where examined, in an FXR-dependent fashion (Figure 1 (7)) [54,93–95]. However, regulation by bile acid FXR agonists is far more complicated. FXR-mediated activation of *Abcg5 Abcg8* requires fibroblast growth factor 15/19 (FGF15/19), which is itself an FXR target gene that is secreted from the ileum in response to bile acids and promotes Src-mediated phosphorylation of hepatic FXR, and FXR binding to the *Abcg5 Abcg8* promoter [96]. *FNDC5*/Irisin is an FXR target gene that increases *Abcg5 Abcg8* mRNA in both the livers and intestines of transgenic mice, but the extent to which the endogenous gene plays a role in *Abcg5 Abcg8* regulation and sterol homeostasis mice or humans is not known [97]. Whereas cholesterol and its metabolites tend to increase expression of *Abcg5 Abcg8* mRNA, agonists for the constitutive androstane receptor (CAR) repress expression of the transporter under conditions of elevated exogenous or endogenously-derived FXR agonists [98,99].

Transcriptional regulation of *Abcg5 Abcg8* by LXR and FXR fits well with its central role in opposing the accumulation of excess cholesterol. Expansion of whole body neutral and/or acid sterol pools increases expression [31,54,84,93,94,100,101]. Conversely, blocking absorption of cholesterol or bile acids reduces expression in either liver or intestine [37,101–103]. Less clear is the physiological benefit, if any, for alterations in *Abcg5 Abcg8* expression by regulators of metabolism. Thyroid hormone increases *Abcg5 Abcg8* mRNA, biliary cholesterol secretion, and fecal sterol excretion in both intact and hypophysectomized rats [104]. Hepatic *Abcg5 Abcg8* mRNA and protein are upregulated in the absence of insulin signaling in mice, an effect attributed to disinhibition of Forkhead box protein O1 (FOXO-1) [105,106]. The opposite was observed in a type 1 diabetic model in rats [107]. The insulin-sensitizing drug, metformin, increased *Abcg5 Abcg8* mRNA and protein, an effect attributed to reduced period 2 occupancy of the *ABCG5 ABCG8* promoter and disinhibition of gene expression [108]. Indeed, *Abcg5 Abcg8* mRNA exhibits a robust circadian rhythm at the transcriptional level (not observed for protein level, unpublished observation) and hepatic *Abcg5 Abcg8* mRNA and biliary cholesterol are reduced in *Bmal1*-deficient mice [108,109]

Alterations in *Abcg5 Abcg8* expression have been reported by a variety of nutritional cues, including upregulation in response elevated n-3 polyunsaturated fatty acids [110–112]. While upregulation of *ABCG5 ABCG8* is generally observed in high fat diets containing cholesterol and cholesterol-free, high fat diets, a single oral gavage of triacylglycerols robustly repressed intestinal *Abcg5 Abcg8* mRNA in mice [113]. In an independent study, suppression of *Abcg5 Abcg8* mRNA following high fat, high sucrose feeding was not observed, but the diet used in this study contained both added cholesterol and cholate, and thus hepatic cholesterol was increased five-fold compared to mice fed the control diet [114]. Diets containing high levels of sucrose robustly repressed expression of hepatic, but not intestinal *Abcg5 Abcg8* in rats [115]. Collectively, the data suggests that dietary repression of *ABCG5 ABCG8* may reflect reductions in the abundance of LXR and FXR agonists rather than active repression by sucrose or triglyceride. Alternatively, differences across studies may be associated with species and strain differences, both of which have been reported for various ligands [116,117]. Diets supplemented with soy protein increased hepatic *Abcg5 Abcg8* mRNA in rats [118]. The mechanisms for such an effect is not known but may include modulation of the intestinal microbiota. Germ free mice exhibited elevations in both intestinal and hepatic *Abcg5 Abcg8* mRNA relative to specific pathogen free mice in the absence and presence of ezetimibe [119]. Depletion of dietary iron upregulated hepatic *Abcg5 Abcg8* mRNA and promotes increased biliary cholesterol secretion [120]. Dietary calcium supplementation was shown to increased intestinal *Abcg5 Abcg8* mRNA and fecal neutral sterol excretion in a hamster model of menopause [121].

Female biological sex has long been associated with increased biliary cholesterol. Female mice had modest, but significant increases in biliary cholesterol and *ABCG5 ABCG8* mRNA in the human transgenic strain [49]. Ovariectomy was subsequently shown to reduce, and estrogen replacement to increase *Abcg5 Abcg8* mRNA across the intestine in independent strains of mice [116,122]. Diosgenin is a choleretic compound with estrogenic properties that increases biliary cholesterol secretion. Its ability to increase biliary cholesterol is largely G5G8-dependent, but reports on its impact on *Abcg5 Abcg8* expression are conflicting, showing no change in mice but an increase in both liver and intestine in rats [54,123]
