Insulin Is a Dominant Suppressor of Sterol 12 -Hydroxylase P450 (CYP8B) Expression in Rat Liver: Possible Role of Insulin in Circadian Rhythm of CYP8B

217

Sterol 12␣-hydroxylase catalyzes the synthesis of cholic acid and controls the ratio of cholic acid over chenodeoxycholic acid in the bile. Transcription of CYP8B1 is inhibited by bile acids, cholesterol, and insulin. To study the mechanism of CYP8B1 transcription by bile acids, we have cloned and determined 3389 base pairs of the 5-upstream nucleotide sequences of the human CYP8B1. Deletion analysis of CYP8B1/luciferase reporter activity in HepG2 cells revealed that the sequences from ؊57 to ؉300 were important for basal and liver-specific promoter activities. Hepatocyte nuclear factor 4␣ (HNF4␣) strongly activated human CYP8B1 promoter activities, whereas cholesterol 7␣-hydroxylase promoter factor (CPF), an NR5A2 family of nuclear receptors, had much less effect. Electrophoretic mobility shift assay identified an overlapping HNF4␣-and CPFbinding site in the ؉198/؉227 region. The human CYP8B1 promoter activities were strongly repressed by bile acids, and the bile acid response element was localized between ؉137 and ؉220. Site-directed mutagenesis of the HNF4␣-binding site markedly reduced promoter activity and its response to bile acid repression. On the other hand, mutation of the CPF-binding site had little effect on promoter activity and bile acid inhibition. A negative nuclear receptor, small heterodimer partner markedly inhibited transactivation of CYP8B1 by HNF4␣. Mammalian two-hybrid assay confirmed that HNF4␣ interacted with small heterodimer partner. Furthermore, bile acids and farnesoid X receptor reduced the expression of nuclear HNF4␣ in HepG2 cells and rat livers and its binding to DNA. Bile acids and farnesoid X receptor also inhibited mouse HNF4␣ gene transcription. In summary, our data revealed the critical roles HNF4␣ play on CYP8B1 transcription and its repression by bile acids. Bile acids repress human CYP8B1 transcription by reducing the transactivation activity of HNF4␣ through interaction of HNF4␣ with SHP and reduction of HNF4␣ expression in the liver.

Section: Discussionsupporting

confidence: 92%

Transcriptional Regulation of the Human Sterol 12α-Hydroxylase Gene (CYP8B1)

Zhang

Chiang

2001

217

“…FoxO1 also can act as a co-repressor of PGC-1␣ and hepatocyte nuclear factor 4␣ (HNF4␣), thus inhibiting transcription of PGC-1␣ and HNF4␣-regulated genes, including CYP7A1 and CYP8B1 (40,41). Insulin is known to inhibit CYP8B1 (11). Thus, insulin action via FoxO1 should inhibit, not induce, CYP8B1 as suggested from a liver-specific FoxO1 knock-out mouse study (37).…”

Section: Discussionmentioning

confidence: 99%

“…Ablation of the CYP8B1 gene reduces dietary cholesterol absorption and prevents hypercholesterolemia and gallstone formation in diabetic mice (7)(8)(9) and atherosclerosis in ApoE knock-out mice (10). It has been reported that CYP8B1 expression is elevated in streptozocin-induced diabetic rats, and insulin inhibits CYP8B1 and regulates circadian rhythm of CYP8B1 (11). Bile acid synthesis and CYP7A1 expression exhibit a strong circadian rhythm, peaking at the onset of dark and decreasing in the light cycle (12)(13)(14)(15).…”

mentioning

confidence: 99%

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Retinoic Acid-related Orphan Receptor α Regulates Diurnal Rhythm and Fasting Induction of Sterol 12α-Hydroxylase in Bile Acid Synthesis

Pathak

Chiang

2013

Background: Sterol 12␣-hydroxylase catalyzes cholic acid synthesis. ROR␣ is a cholesterol-activated and fasting-induced nuclear receptor and core clock gene. Results: Upon fasting, glucagon/PKA phosphorylated and stabilized ROR␣ protein to induce CYP8B1 and diurnal rhythm. Conclusion: ROR␣ is a key regulator of CYP8B1 that regulates bile acid and cholesterol homeostasis. Significance: Antagonizing ROR␣ activity may be a therapeutic strategy for treating non-alcoholic fatty liver disease and diabetes.

“…Insulin treatment restores bile acid pool and synthesis to the normal levels. It has been reported that physiological concentrations of insulin (1.4 -14 nM) inhibit bile acid synthesis by down-regulation of CYP7A1 (7)(8)(9), sterol 12␣-hydroxylase (CYP8B1) (10), and sterol 27-hydroxylase (CYP27A1) gene transcription (9). We have previously reported that insulin plays a dominant role in the inhibition of CYP7A1 gene transcription (7,8).…”

mentioning

confidence: 99%

Insulin Regulation of Cholesterol 7α-Hydroxylase Expression in Human Hepatocytes

Kong

Owsley

et al. 2006

Bile acid synthesis and pool size increases in diabetes, whereas insulin inhibits bile acid synthesis. The objective of this study is to elucidate the mechanism of insulin regulation of cholesterol 7␣-hydroxylase gene expression in human hepatocytes. Real-time PCR assays showed that physiological concentrations of insulin rapidly stimulated cholesterol 7␣-hydroxylase (CYP7A1) mRNA expression in primary human hepatocytes but inhibited CYP7A1 expression after extended treatment. The insulin-regulated forkhead box O1 (FoxO1) and steroid regulatory element-binding protein-1c (SREBP-1c) strongly inhibited hepatocyte nuclear factor 4␣ and peroxisome proliferator-activated receptor ␥ coactivator-1␣ trans-activation of the CYP7A1 gene. FoxO1 binds to an insulin response element in the rat CYP7A1 promoter, which is not present in the human CYP7A1 gene. Insulin rapidly phosphorylates and inactivates FoxO1, whereas insulin induces nuclear SREBP-1c expression in human primary hepatocytes. Chromatin immunoprecipitation assay shows that insulin reduced FoxO1 and peroxisome proliferators-activated receptor ␥-coactivator-1␣ but increased SREBP-1c recruitment to CYP7A1 chromatin. We conclude that insulin has dual effects on human CYP7A1 gene transcription; physiological concentrations of insulin rapidly inhibit FoxO1 activity leading to stimulation of the human CYP7A1 gene, whereas prolonged insulin treatment induces SREBP-1c, which inhibits human CYP7A1 gene transcription. Insulin may play a major role in the regulation of bile acid synthesis and dyslipidemia in diabetes.The liver plays a central role in lipid metabolism and maintaining whole body lipid homeostasis, which is dysregulated in metabolic syndrome (syndrome X), obesity, and diabetes (1). Bile acid synthesis in the liver is the predominant pathway for cholesterol catabolism and is regulated by cholesterol 7␣-hydroxylase (CYP7A1) 2 (2). Bile acids are physiological agents that facilitate biliary cholesterol excretion, intestinal absorption of nutrients, and disposal of toxic metabolites. Bile acids are also signaling molecules that activate bile acid receptors to regulate bile acid synthesis and glucose metabolism (2). In vivo studies show that bile acid pool and excretion increase in diabetic human patients (3, 4) and in experimental diabetic animals (5, 6). Insulin treatment restores bile acid pool and synthesis to the normal levels. It has been reported that physiological concentrations of insulin (1.4 -14 nM) inhibit bile acid synthesis by down-regulation of CYP7A1 (7-9), sterol 12␣-hydroxylase (CYP8B1) (10), and sterol 27-hydroxylase (CYP27A1) gene transcription (9). We have previously reported that insulin plays a dominant role in the inhibition of CYP7A1 gene transcription (7,8). How insulin regulates human CYP7A1 and what factors mediate the insulin effects remain unknown.Insulin is known to induce more than 100 genes but inhibit only a few hepatic genes involved in glucose metabolism (11). The consensus sequence of negative insulin response element (IRE), T(G...