Potential conflict of interest: Nothing to report.
The gene encoding cholesterol 7α-hydroxylase (CYP7A1) is tightly regulated to control bile acid synthesis and maintain lipid homeostasis. Recent studies in mice suggest that bile acid synthesis is regulated by the fasted-to-fed cycle, and fasting induces CYP7A1 gene expression in parallel to the induction of peroxisome proliferators-activated receptor γ co-activator 1α (PGC-1α) and phosphoenolpyruvate carboxykinase (PEPCK). How glucagon regulates CYP7A1 gene expression in the human liver is not clear. Here we show that glucagon and cyclic adenosine monophosphate (cAMP) strongly repressed CYP7A1 mRNA expression in human primary hepatocytes. Reporter assays confirmed that cAMP and protein kinase A (PKA) inhibited human CYP7A1 gene transcription, in contrast to their stimulation of the PEPCK gene. Mutagenesis analysis identified a PKA-responsive region located within the previously identified HNF4α binding site in the human CYP7A1 promoter. Glucagon and cAMP increased HNF4α phosphorylation and reduced the amount of HNF4α present in CYP7A1 chromatin. Our findings suggest that glucagon inhibited CYP7A1 gene expression via PKA phosphorylation of HNF4α, which lost its ability to bind the CYP7A1 gene and resulted in inhibition of human CYP7A1 gene transcription. In conclusion, this study unveils a species difference in nutrient regulation of the human and mouse CYP7A1 gene and suggests a discordant regulation of bile acid synthesis and gluconeogenesis by glucagon in human livers during fasting. (HEPATOLOGY 2005.)
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The conversion of cholesterol to bile acids in the liver is a major pathway for removing excess cholesterol from the body and plays an important role in maintaining cholesterol homeostasis.1, 2 Imbalance of cholesterol metabolism causes diseases such as atherosclerosis and gallstone disease. CYP7A1 is a liver-specific enzyme that catalyzes the first and rate-limiting step in the bile acid biosynthetic pathway.1 Expression of CYP7A1 is regulated mainly at the gene transcriptional level by many factors, including bile acids, diets, nutrients, cytokines, and hormones.3
Several nuclear receptors, including liver orphan receptor, α-fetoprotein transcription factor (also known as mouse liver–related homolog), hepatocyte nuclear factor 4α (HNF4α), and pregnane X receptor, bind to the CYP7A1 gene and play important roles in regulation of CYP7A1 gene transcription.1, 4 HNF4α is the most abundant orphan nuclear receptor expressed in the liver that binds to a direct repeat with one base spacing (DR1) in the bile acid response element II (BARE-II) (−144/−126) and stimulates CYP7A1 gene transcription.5 Mutation of this HNF4α binding site markedly reduced CYP7A1 promoter activity, and liver-specific conditional disruption of the HNF4α gene in mice drastically repressed CYP7A1 gene expression, indicating that HNF4α is crucial for CYP7A1 gene transcription and regulation.5, 6 Bile acid receptor farnesoid X receptor (FXR) inhibits CYP7A1 gene transcription by inducing a negative nuclear receptor small heterodimer partner (SHP), which then inhibits α-fetoprotein transcription factor and HNF4α transactivation of the CYP7A1 gene.1 FXR regulates a variety of genes involved in bile acid, lipoprotein, glucose, and triglyceride metabolisms.7
Although many studies about physiological responses of CYP7A1 to cholesterol, bile acids, and hormones are conducted in animal models such as mouse and rat, very little is known regarding human CYP7A1 gene regulation. Moreover, recent studies reported that the CYP7A1 gene is differentially regulated in different species. Notably, a high-cholesterol diet induces CYP7A1 gene expression in mice and rats with little change in plasma cholesterol levels, but cholesterol has no significant effect or even suppresses CYP7A1 expression in rabbit, hamster, African green monkeys, and transgenic mice expressing human CYP7A1.8–11 The stimulation of CYP7A1 gene expression by cholesterol is accomplished via liver orphan receptor α, which binds to the CYP7A1 gene promoter in mice and rats, but not in humans.12, 13
A recent study reported that bile acids impair HNF4α recruitment of peroxisome proliferators-activated receptor γ co-activator 1α (PGC-1α) to both CYP7A1 and PEPCK genes and inhibits their gene transcription14 and suggests that bile acid synthesis and gluconeogenesis are coordinately regulated by bile acid feedback via a mechanism linked to the fasted-to-fed cycle.14 A similar study shows that CYP7A1 mRNA expression increases in parallel with PGC-1α in fasted mice.15 These results are in direct contradiction to the earlier reports that CYP7A1 expression is repressed in piglets16 and rats17–19 during fasting. It appears that nutritional status that alters hormonal levels such as insulin and glucagon may regulate CYP7A1 expression. In the current study, we demonstrated that glucagon and cyclic adenosine monophosphate (cAMP) repressed human CYP7A1 gene expression via protein kinase A (PKA) phosphorylation of HNF4α in primary human hepatocytes and HepG2 cells, the best available models for studying human gene regulation. This result supports a species difference in regulation of human CYP7A1 gene transcription by hormones and underscores the importance of studying human CYP7A1 gene regulation.
HNF4α, hepatocyte nuclear factor 4α; CYP7A1, cholesterol 7α-hydroxylase; Luc, luciferase; BARE II, bile acid response element II; FXR, farnesoid X receptor; SHP, small heterodimer partner; PGC-1α, peroxisome proliferators-activated receptor γ co-activator 1α; cAMP, cyclic adenosine monophosphate; PKA, protein kinase A; PEPCK, phosphoenolpyruvate carboxykinase; 8-Br-cAMP, 8-Bromo-cyclic AMP; PCR, polymerase chain reaction; ChIP, chromatin immunoprecipitation.
Materials and Methods
Primary human hepatocytes were isolated from human donors (HH1183, 54-year-old male; HH1189, 18-year-old male; HH1196, 56-year-old female; HH1201, 69-year-old male; HH1205, 45-year-old male; HH1209, 50-year-old female; HH1210, 35-year-old female; HH1211, 79-year-old male; HH1215, 36-year-old male; HH1220, 40-year-old female) and were obtained from the Liver Tissue Procurement and Distribution System of National Institutes of Health (S. Strom, University of Pittsburgh, Pittsburgh, PA). Cells were maintained in Hank's modified medium modified Williams E medium (Cambrex Bioscience, Inc., Walkersville, MD) supplemented with 0.1% gentamicin sulfate and amphotericin-B without insulin or dexamethasone. The HepG2 and HEK293 cells were obtained from the American Type Culture Collection (Manassas, VA). The cells were maintained in a 1:1 mixture of Dulbecco's modified Eagle's medium and F-12 (50:50; Life Technologies, St. Paul, MN) supplemented with 100 U/mL penicillin G/streptomycin sulfate (Celox, Hopkins, MN) and 10% heat-inactivated fetal bovine serum (Irvine Scientific, Santa Ana, CA).
The mammalian expression plasmids for HNF4α, wild-type PKA catalytic subunit, mutant PKA that lacks the kinase activity, and pHNF4α-tk-Luc reporter that contains 4 copies of the HNF4α binding site in mouse transthyretin gene fused upstream of the thymidine kinase (tk) minimal promoter and luciferase gene were as previously described.4, 20, 21 A series of human CYP7A1 promoter luciferase reporters were previously constructed.22 A human phosphoenolpyruvate carboxykinase (PEPCK) promoter luciferase reporter construct containing 2 kb upstream sequence (pPEPCK−2,000/+73) was provided by Richard Hanson (Case Western Reserve University, Cleveland, OH).23
RNA Isolation and Real-Time Quantitative Polymerase Chain Reaction.
Primary human hepatocytes were treated with 100 nmol/L glucagon or 1 mmol/L 8-Bromo-cyclic AMP (8-Br-cAMP) for a period from 1 to 24 hours or as indicated. Total RNA was isolated using Tri-reagent (Sigma, St. Louis, MO) according to the manufacturer's instructions. Reverse-transcription reactions were performed using RETROscript kit (Ambion, Austin, TX) following the manufacturer's instructions. Approximately 2 μg total RNA from each sample was reverse-transcribed, and aliquots of the cDNA were subjected to real-time quantitative polymerase chain reaction (PCR) with a Taqman Universal PCR Master Mix (Roche, Branchburg, NJ) following the manufacturer's instructions to detect CYP7A1, PEPCK, PGC-1α, and HNF4α mRNAs. Amplification of ubiquitin C was used in the same reactions as an internal reference gene. Taqman probes for real-time quantitative PCR were ordered from Assay on Demand (Applied Biosystems, Forest City, CA). All PCR reactions were done in triplicate. PCR amplification was performed as follows: 50°C for 2 minutes, 95°C for 10 minutes, 95°C for 15 seconds, and 40 cycles (1 minute each) at 60°C using an ABI PRISM 7500 sequence detector (Applied Biosystems). Amplification data were analyzed using the Sequence Detector version 1.7 software (Applied Biosystems). Relative mRNA expression levels were calculated using the −ΔΔCt method recommended by Applied Biosystems (User Bulletin no. 2, 1997). Statistical analysis of real-time PCR results were done using mean normalized cycle threshold (ΔCt) values and the pooled standard deviation of the mean ΔCt, which were analyzed by one-way ANOVA followed by Tukey's Honestly Significant post hoc test. A P value of less than .05 was considered as a statistically significant difference. Experiments were repeated at least 3 times with different cell preparations.
Luciferase Reporter Assay.
For luciferase reporter assay, HepG2 cells were plated in 24-well plates 24 hours before transfection with reporter or expression plasmids using LipofectAMINE 2000 reagent (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer's instructions. Total DNA used in each transfection was adjusted by adding the appropriate amount of pcDNA3 vector. When studying the effect of 8-Br-cAMP, cells were serum-starved for 24 hours before treatment. Luciferase activities are expressed as relative luciferase unit/β-galactosidase activity as previously described.4
In VivoPhosphorylayion of HNF4α.
Human primary hepatocytes were treated with PKA inducers (100 nmol/L glucagon or 1 mmol/L 8-Br-cAMP) for 6 hours. Cells were washed with phosphate-buffered saline and lysed in ice-cold modified radioimmunoprecipitation buffer (50 mmol/L Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% Na+-deoxycholate, 150 mmol/L NaCl, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L EDTA, 1 mmol/L sodium orthovanadate (Na3VO4), 1 mmol/L NaF), and Complete Protease Inhibitor cocktail (Sigma). Cells debris were centrifuged, and the supernatant diluted with 3 volumes of radioimmunoprecipitation buffer without NaCl was pre-cleared with whole rabbit serum adsorbed on Protein A-Sepharose beads (Amersham Biosciences, Piscataway, NJ) and subsequently subjected to immunoprecipitation with 10 μg anti-HNF4α or non-immune serum overnight at 4°C. The beads were washed several times with radioimmunoprecipitation buffer containing 150 mmol/L NaCl, and the immuno-complexes were subjected to electrophoresis on a 10% sodium dodecyl sulfate polyacrylamide gel and then transferred to a nitrocellulose membrane (Amersham Biosciences). Enhanced chemiluminescence Western blotting (Amersham Biosciences) was performed according to the manufacturer's instructions. Phosphor-HNF4α and HNF4α proteins were detected by incubation of blots with an anti-phosphorprotein antibody (1:2,000 dilution; Zymed, South San Francisco, CA) and anti-HNF4α antibody (1:2,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), respectively.
Chromatin Immunoprecipitation Assay.
Chromatin immunoprecipitation (ChIP) assays were performed using a ChIP Assay kit (Upstate Cell Signaling Solutions, Lake Placid, NY) according to the manufacturer's instructions. Human primary hepatocytes were treated with 100 nmol/L glucagon or 1 mmol/L 8-Br-cAMP for various times, and chromatin was cross-linked in 1% formaldehyde and sonicated as previously reported.4 Cell lysate solution (5%) in ChIP dilution buffer was kept aside as “input.” Ten micrograms HNF4α antibody (Santa Cruz Biotechnology) was added to precipitate DNA–protein complexes and non-immune IgG was used as a control. A 391-bp DNA fragment (−432 to −41) containing the BARE-I and BARE-II of the CYP7A1 promoter was PCR amplified for 30 cycles using 5 μL DNA as template and analyzed on a 1.5% agarose gel. PCR primers for amplifying were as follows: 5′-ATCACCGTCTCTCTGGCAAAGCAC-3′; reverse primer: 5′-CCATTAACTTGAGCTTGGTTGACAAAG-3′.
Glucagon and cAMP Repressed CYP7A1 mRNA Expression in Human Primary Hepatocytes.
To understand the nutrient regulation of human CYP7A1 gene expression, we first investigated the effect of glucagon on CYP7A1 gene expression in primary cultures of human hepatocytes. Treatment of human primary hepatocytes with glucagon (100 nmol/L) caused a time-dependent decrease in CYP7A1 mRNA levels to 10% of the control in 24 hours (Fig. 1A). As expected, glucagon rapidly increased PEPCK mRNA levels by 10- to 20-fold, but did not significantly change HNF4α mRNA levels. Interestingly, glucagon induced PGC-1α mRNA levels by 10-fold in 3 hours and 3-fold in 24 hours.
That glucagon induces cAMP, which acts as the second messenger that activates protein kinase A (PKA), which phosphorylates the downstream targets of glucagon, has been established. We hypothesized that the cAMP pathway might play a role in the glucagon-mediated repression of CYP7A1. Therefore, human primary hepatocytes were treated with 1 mmol/L 8-Br-cAMP, a stable analog of cAMP, and analyzed for CYP7A1 mRNA levels. As shown in Fig. 1B, CYP7A1 mRNA levels were strongly suppressed by 8-Br-cAMP, similar to glucagon treatment. As a positive control experiment, 8-Br-cAMP inducted PEPCK and PGC-1α mRNA levels by approximately 15- to 20-fold. This compound had no significant effect on HNF4α mRNA expression.
Because time-course study showed that glucagon and 8-Br-cAMP strongly repressed CYP7A1 but induced PEPCK and PGC-1α mRNA levels after 6 hours' treatment, we analyzed the dose response of glucagon and 8-Br-cAMP at this point of treatment. Treatment of human primary hepatocytes with glucagon (1-100 nmol/L) or 8-Br-cAMP (0.01-1 mmol/L) showed a dose-dependent suppression of CYP7A1 mRNA levels; in contrast, these 2 compounds dose-dependently induced PEPCK and PGC-1α mRNA levels. HNF4α mRNA levels were not affected by these 2 compounds (Fig. 1C-D).
To confirm the specificity of the PKA signaling pathway in mediating glucagon inhibition of CYP7A1 gene expression, several specific inhibitors of signaling pathways were used to treat human primary hepatocytes 1 hour before addition of glucagon (100 nmol/L). Real-time PCR analysis indicated that pretreatment of a PKA inhibitor, H-89 (10 μmol/L) prevented glucagon inhibition of CYP7A1 mRNA expression (Fig. 1E). However, there was no significant effect of Ro 31-8220 (an inhibitor of protein kinase C), PD98059 (an inhibitor of mitogen-activated protein kinase, MAPK), SB-203580 (an inhibitor of stress-activated protein kinase), and SP600125 (an inhibitor of c-jun N-terminal kinase) on CYP7A1 mRNA expression. These results indicated that the PKA pathway activated by glucagon specifically inhibited CYP7A1 mRNA expression in human primary hepatocytes.
PKA Repressed Human CYP7A1Promoter Activity.
To further study the effect of the PKA pathway in repressing CYP7A1 gene transcription, transient transfection assay of human CYP7A1-luciferase reporter (CYP7A1-Luc) activity was performed in HepG2 cells. Treatment of 8-Br-cAMP (1 mmol/L) repressed the CYP7A1 reporter activity by approximately 60%, but stimulated a human PEPCK reporter (PEPCK-Luc) activity by approximately 10-fold (Fig. 2A -B). This PEPCK reporter has all important response elements (i.e., glucocorticoid receptor, CCAAT Response Element Binding protein, HNF4, and forkhead transcription factor Foxo1) that are known to regulate PEPCK gene transcription. We next co-transfected an expression plasmid for PKA catalytic subunits to test the role of PKA in mediating glucagon and cAMP effect on human CYP7A1 reporter activity. As shown in Fig. 2C, addition of increasing amounts of wild-type PKA expression plasmids markedly inhibited CYP7A1 reporter activity, whereas an expression plasmid for mutant PKA20 did not affect the reporter activity. As a positive control, wild-type PKA stimulated PEPCK-Luc activity, whereas the mutant PKA had no effect (Fig. 2D). These data demonstrated that activation of PKA repressed CYP7A1 but stimulated PEPCK gene transcription, consistent with the effect of cAMP on CYP7A1 and PEPCK mRNA expression (Fig. 1).
PKA Repression of Human CYP7A1Is Liver Specific.
CYP7A1 is only expressed in the hepatocytes. To test whether glucagon/cAMP/PKA-mediated repression of CYP7A1 is liver specific, we studied the effect of PKA on CYP7A-Luc reporter activity in the liver-derived HepG2 cells and kidney-derived HEK293 cells. In HepG2 cells, co-transfection of the wild-type PKA expression plasmid strongly repressed CYP7A1-Luc reporter activity, but the mutant PKA that lacks the kinase activity did not have any effect on CYP7A1 reporter activity (Fig. 3A). In HEK293 cells, the basal reporter activity is much lower than in HepG2 cells. Co-transfection of either a wild-type or mutant PKA expression plasmid did not significantly modulate CYP7A1 reporter activity (Fig. 3B). In contrast, PEPCK reporter activity was strongly stimulated by wild-type PKA in both cell types, and mutant PKA had no effect (Fig. 3C-D). These data indicated that PKA-mediated repression of CYP7A1 expression was liver specific, whereas PEPCK, which is expressed in both liver and kidney, was regulated by PKA in both liver and kidney cells.
Identification of a Region Conferring cAMP Inhibition of the Human CYP7A1Gene.
We next attempted to identify potential sequences conferring glucagon/cAMP inhibition of the CYP7A1 gene. A series of 5′-deletion constructs of human CYP7A1-Luc reporter were used in transfection assay. Deletion of human CYP7A1 promoter sequence from −1178 to −150 did not affect 8-Br-cAMP inhibition of reporter activity (Fig. 4A). However, further deletion to −135 abolished the repressive effect of 8-Br-cAMP, suggesting that the region between −150 and −135 conferred the negative effect of cAMP. Interestingly, this region was previously identified as a BARE-II, which contains a DR-1 sequence for binding of HNF4α.24
To further confirm that this DR-1 site mediates the cAMP response, mutations were introduced into the DR-1 sequence of the ph-298CYP7A1-Luc reporter (WT-298, Fig. 4B). This mutant reporter (MT-298) did not respond to cAMP. These results suggested that glucagon/cAMP-activated pathway repressed CYP7A1 gene transcription through the HNF4α binding site.
PKA Represses HNF4αTranscriptional Activity.
Because HNF4α is involved in mediating PKA suppression and PKA-dependent phosphorylation of HNF4α is known to reduce its DNA-binding and transactivation activity,25 we studied the effect of PKA on HNF4α transcriptional activity of a heterologous luciferase reporter containing 4 copies of a HNF4α binding sequence (from mouse transthyretin promoter) fused to a minimal TK promoter (pHNF4-tk-Luc). As shown in Fig. 5A, co-transfection of HNF4α drastically stimulated this heterologous reporter activity. Addition of increasing amounts of the wild-type PKA plasmid drastically reduced reporter activity. In contrast, a mutant PKA, which has no kinase activity, did not have any effect on the reporter activity. Co-transfection of HNF4α stimulated human CYP7A1-Luc reporter activity by 2-fold. Wild-type but not mutant PKA strongly inhibited the reporter activity stimulated by HNF4α. Interestingly, increasing the amount of wild type PKA repressed HNF4α-stimulated CYP7A1 promoter reporter activity below the basal activity, suggesting that PKA abolished the activity stimulated by the endogenous HNF4α present in HepG2 cells (Fig. 5B). These data suggested that PKA might phosphorylate HNF4α DNA binding domain or activation function domains (AF1 and AF2), and resulted in inhibition of HNF4α transactivation of the CYP7A1 gene.
Glucagon and cAMP Increase Phosphorylayion of HNF4α.
To determine whether glucagon and 8-Br-cAMP affect phosphorylation of HNF4α, primary human hepatocytes were treated with glucagon or 8-Br-cAMP for 6 hours, and cellular extracts were immunoprecipitated with an anti-HNF4α antibody. HNF4α and phosphorylated-HNF4α in immunoprecipitants were analyzed by Western blot with an anti-HNF4α antibody and anti-phosphorprotein antibody, respectively. As shown in Fig. 6, treatment of primary hepatocytes with glucagon or 8-Br-cAMP resulted in a marked increase in the amount of phosphorylated HNF4α in the immunoprecipitants detected by an anti-phosphor antibody, but did not affect the amount of HNF4α in the precipitants. This result suggests that glucagon and 8-Br-cAMP strongly stimulated HNF4α phosphorylation, but did not affect the expression of HNF4α protein in primary human hepatocytes.
Glucagon and cAMP Dissociate HNF4α From Human CYP7A1 Chromatin.
We then studied the effect of glucagon and 8-Br-cAMP on HNF4α binding to CYP7A1 chromatin using primary human hepatocytes for ChIP assay. As shown in Fig. 7A, glucagon treatment rapidly decreased the amounts of CYP7A1 chromatin precipitated by an antibody against HNF4α in a time-dependent manner (Fig. 7A). No signal was detected when non-immune immunoglobulin G was used for immunoprecipitation. Moreover, 8-Br-cAMP also rapidly decreased the amount of human CYP7A1 chromatin precipitated by an anti-HNF4α antibody (Fig. 7B). Taken together, these results indicated that glucagon and cAMP decreased HNF4α binding to human CYP7A1 chromatin in human primary hepatocytes.
In the current study, we demonstrated that glucagon and cAMP repressed CYP7A1 gene expression levels in a time- and dose-dependent manner in human primary hepatocytes, and the PKA signaling pathway mediated the inhibitory effect of glucagon and cAMP. We provided the strong evidences that the glucagon/cAMP/PKA signaling increased the phosphorylation state of HNF4α and reduced its DNA binding and transactivation of the CYP7A1 gene.
PKA has been reported to phosphorylate the A-box of the HNF4α DNA-binding domain and decrease its DNA-binding activity.25 AMP kinase phosphorylated HNF4α on serine 304 and reduced HNF4α transactivation activity by inhibiting its homodimerization and stability.26 Phosphorylation of tyrosine residues on HNF4α affected its nuclear localization and transactivation.27 This study further supports our hypothesis that HNF4α plays a critical role in regulating CYP7A1 gene transcription and that reducing HNF4α binding and trans-activating activity by physiological regulators such as bile acids, cytokines, and glucagon results in inhibition of the CYP7A1 gene.28 Diverse intracellular signaling pathways have been shown to mediate bile acid feedback inhibition of the CYP7A1 gene. Bile acids are able to activate the protein kinase C and cytokine signaling pathways, which activate the MAPK/c-jun N-terminal kinase pathway and inhibit the CYP7A1 gene.7 This study adds the glucagon/cAMP/PKA signaling pathway to a network of signaling pathways that inhibits the CYP7A1 gene. These signaling mechanisms are independent of the FXR/SHP pathway in bile acid feedback regulation of bile acid synthesis.7 These signaling pathways converge to regulate HNF4α, the most important regulator of the CYP7A1 gene.
Our results that glucagon and cAMP inhibit human CYP7A1 gene expression are consistent with the earlier reports that glucagon or cAMP decreased CYP7A1 expression in rat primary hepatocytes29 and fasting represses CYP7A1 expression in rats and piglets.16–19 Reduction of bile acid synthesis by fasting could result in increasing hepatic cholesterol levels, which is consistent with the fasting-induced serum total cholesterol levels in healthy non-obese humans.30 However, these results are in contradiction to two recent reports that fasting and cAMP induce both CYP7A1 and PEPCK expression14, 15 and the hypothesis that bile acid synthesis and gluconeogenesis are coordinately regulated by bile acids in response to the fasting-to-fed cycle.14 These two studies were conducted in mice, and the species differences between mice and humans in regulation of CYP7A1 and bile acid synthesis are well documented.7 A most recent study reported that CDCA and an FXR agonist GW4064 induce PEPCK mRNA expression in mouse livers, rat liver cell lines, and rat and human primary hepatocytes.31 This is also in complete opposite to bile acid inhibition of PEPCK reported by De Fabiani et al.14 The previous results of glucagon/cAMP effect on CYP7A1 mRNA expression in rat primary hepatocytes are consistent with this study in human primary hepatocytes, suggesting that primary hepatocytes are suitable for studying CYP7A1 regulation.
According to this study, we propose that glucagon released from the pancreas during fasting may inhibit CYP7A1 gene transcription and reduce bile acid synthesis and bile acid pool size in human liver. This should result in an inactivation of the FXR/SHP pathway during fasting. Glucagon markedly induces PGC-1α, which is a potent co-activator of CCAAT response element binding protein and glucocorticoid receptor that strongly upregulate PEPCK gene transcription during fasting to maintain postprandial glucose levels and prevent hypoglycemia.32 Because bile acid synthesis requires adenosine triphosphate and bile acids are not needed during fasting, there is no physiological benefit for upregulating bile acid synthesis during fasting as suggested by De Fabiani et al.14 On feeding, the glucagon level is reduced and CYP7A1 expression is de-repressed to produce bile acids for fat absorption in the intestine. Bile acids are quantitatively re-absorbed in the ileum and transported to the liver to activate the FXR/SHP pathway and other signaling pathways that inhibit bile acid synthesis. The FXR/SHP pathway also may inhibit PEPCK and prevent hyperglycemia and diabetes during the postprandial period. The enterohepatic recirculation of bile acids is an important physiological process that tightly controls the bile acid pool size to protect hepatocytes from bile acid toxicity during the fasting and fed states. Similarly, the fasting and feeding cycle regulates glucose metabolism and homeostasis. By virtue of their physiological roles in nutrient absorption and distribution, bile acids may play a role in control glucose and lipid metabolism to maintain metabolic homeostasis.
In summary, our current study showed a mechanism of transcriptional regulation of bile acid synthesis by glucagon in humans. Inhibition of CYP7A1 gene expression and bile acid synthesis during fasting by glucagon/cAMP/PKA signaling may ensure the maximal activation of gluconeogenesis to maintain glucose homeostasis and energy metabolism. This study establishes an important role for glucagon regulation of bile acid synthesis.