In recent years it has become clear that bile acids are important regulatory molecules capable of activating specific nuclear receptors, G-protein coupled receptors (TGR5), and various cell signaling pathways in the liver and gastrointestinal tract (2
). Bile acids have been reported to activate the AKT pathway (insulin signaling pathway) and the nuclear receptor FXR in hepatocytes (18
). We have previously reported that conjugated (but not unconjugated) bile acids activate the AKT and ERK1/2 signaling pathways via unidentified Gαi
-protein-coupled receptor(s) in primary rat hepatocytes (18
) and in vivo (20
). The activation of ERK1/2 and AKT signaling pathways by bile acids in hepatocytes appears not to involve the GPCR TGR5, because its gene is not highly expressed in hepatocytes, is Gαs
coupled, and is activated by both conjugated and unconjugated bile acids (3
). In the current study, we used the chronic bile fistula rat model and primary rat hepatocytes to study the regulation of the rate-limiting gluconeogenic genes PEPCK and G-6-Pase and the induction of SHP by TCA. We wanted to determine if there is a link between bile acid-activated cell signaling pathways and FXR activation. The SHP promoter has a functional FXR binding site, which allows bile acids to induce this gene. In the chronic bile fistula rat, we observed rapid (1 h) activation of the AKT (~9-fold) and ERK1/2 (3- to 5-fold) signaling pathways following the intraduodenal infusion of TCA at a concentration that has been shown not to be hepatotoxic (29
We measured the effect of TCA on the regulation of PEPCK and G-6-Pase in the chronic bile fistula rat and in primary hepatocytes. The data show a very rapid downregulation of both PEPCK and G-6-Pase mRNA following the infusion of TCA in the chronic bile fistula rat (). Consistent with the in vivo data there was also a rapid (1–2 h) downregulation of PEPCK and G-6-Pase mRNA in primary rat hepatocytes by either TCA or insulin (). Moreover, the downregulation of G-6-Pase and PEPCK mRNA by TCA was inhibited by pretreatment of hepatocytes with PTX (). These data are consistent with our previous results showing that TCA activates the AKT pathway via Gαi
-protein-coupled receptor(s) and suggest that bile acids may function much like insulin in helping to coordinately regulate glucose metabolism in the liver. In this regard, we have previously reported that the addition of insulin or bile acids to primary hepatocytes results in the activation of GS activity (17
). Interestingly, the addition of both a bile acid and insulin resulted in an additive effect on GS activity (20
) and a stronger repression of glucose synthesis (). The addition of TCA or insulin to primary hepatocytes resulted in the rapid phosphorylation of FOX01, a key transcription factor controlling PEPCK and G-6-Pase gene expression (). The phosphorylated form of FOX01 moves from the nucleus to the cytosol and becomes inactive as a transcriptional activator of gluconeogenic genes (43
). Finally, SHP has been reported to physically interact with FOX01, CEBPα, and HNF4α, inhibiting their function. All three of these transcription factors have been reported to be involved in the upregulation of gluconeogenic genes (24
). Hence, the combined effect of activation of the insulin signaling pathway and induction of SHP by TCA may be a rapid and long-lasting repression of the rate-limiting gluconeogenic genes ().
In the chronic bile fistula rat model, we observed a rapid induction of SHP mRNA following the intestinal infusion of TCA (). The kinetics of induction showed that SHP mRNA levels followed closely (~30 min) the activation of the insulin signaling pathway. In primary rat hepatocytes, Wortmannin significantly inhibited the induction of SHP mRNA by TCA (). These results suggest that the insulin signaling pathway must be activated to get optimal induction of SHP mRNA by TCA. There was no significant inhibition of SHP induction when specific chemical inhibitors of the ERK1/2 () or p38 MAPK (data not shown) were added to primary hepatocytes in culture. Moreover, the inhibition of AKT by a specific chemical inhibitor failed to inhibit SHP induction by TCA (). These results suggest that PDK-1, which is upstream of AKT, may be involved in regulating SHP induction by TCA, as PKCζ is known to be activated by PDK-1 (44
). Inhibition of PKCζ by a specific chemical inhibitor or siRNA significantly blocked the induction of SHP by TCA (). In this regard, it has recently been reported that FXR can be phosphorylated by PKCζ, and this may allow the translocation of FXR to the nucleus (36
). Moreover, PKCα and PKCβI have been recently reported to phosphorylate FXR, which promotes transcriptional activity of FXR target genes in plasmids in HepG2 cells, possibly by recruiting the coactivator PGC1α (45
). Our laboratories have previously reported that TCA rapidly activates PKCα and PKCβI in primary rat hepatocytes at relatively low concentrations (13
). However, in primary rat hepatocytes, we did not observe any significant inhibition of SHP mRNA induction by TCA when the Ca2+
-dependent PKC isozyme inhibitor Gö6976 was used at concentrations reported to inhibit FXR-dependent induction in other cells (45
)(). The explanation for these results is not clear, but they could be due to use of different cell types (i.e., primary hepatocytes versus HepG2; FXR promoter constructs versus FXR genes in chromosome; and chenodeoxycholic acid versus TCA as activators of FXR). Additional studies will be required to resolve this issue.
In vitro studies using recombinant FXR show that TCA (EC50
>1 mM) is a very poor activator of FXR, whereas chenodeoxycholic acid (EC50
10 μM) and GW4064 (EC50
70 nM) are excellent activators (37
). In the current study in primary rat hepatocytes, TCA is a very good inducer of SHP at concentrations as low as 5 μM (). Interestingly, inhibition of PI3K or PKCζ markedly reduced the ability of TCA to induce SHP ( and ). In contrast, activation of the insulin signaling pathway significantly reduces the ability of GW4064 to induce SHP in primary hepatocytes (). In total, our current interpretation of these data can be summarized by the model shown in . In this model, TCA activates the insulin signaling pathway via Gαi
-protein-coupled receptor(s). The activation of PI3K and downstream kinases help regulate glucose metabolism in a manner similar to insulin by repressing gluconeogenic genes and activating GS activity (18
). The activation of the insulin signaling pathway and PKCζ appears to be crucial for the optimal induction of SHP by TCA. Currently, it is unclear how PKCζ regulates FXR and SHP induction. It has been reported that FXR might be phosphorylated by different isoforms of PKC (36
); however, we have not been able to confirm this observation by overexpression of the gene encoding FXR in primary rat hepatocytes (unpublished observations). Finally, it has recently been reported that activation of the ERK1/2 plays an important role in regulating the turnover rate of SHP protein (46
). Therefore, the activation of ERK1/2 () by TCA may help stabilize SHP protein. The induction of SHP protein may be involved in the long term (>3 h) repression of gluconeogenic genes.
Fig. 11. Cell signaling model for the regulation of glucose metabolism in hepatocytes by taurocholate. TCA can rapidly activate Gαi-dependent G-protein coupled receptor(s) that activate the AKT (insulin signaling pathway). Activated AKT is known to downregulate (more ...)
There are many examples of cell signaling pathways and nuclear receptors interacting to produce a highly regulated physiological response (47
). Therefore, the activation of cell signaling pathways and specific nuclear receptors by bile acids may be considered a coordinate physiological response to nutrients. Additional studies are needed to determine if different bile acids produce the same or different physiological responses during their enterohepatic circulation. Such studies may determine if the bile acid pool composition plays an important role in regulating the activation of cell signaling pathways and nuclear receptors in the liver and, hence, metabolism.