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

Abstract

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...

Activation of Bile Acid Biosynthesis by the p38 Mitogen-activated Protein Kinase (MAPK)

“…7A shows that 7␣-hydroxylase expression increases at the concentrations tested after 6 h but not at shorter times (2 h). Longer incubation (20 h) had either no effect or resulted in a suppression at the highest insulin concentration tested (10 nM), as it has been reported in human hepatocytes (28). HNF-4␣ mRNA remained unchanged at all conditions (Fig.…”

“…After a meal, circulating insulin transiently increases to activate glucose metabolism. It is becoming evident that there is a concomitant increase in 7␣-hydroxylase expression leading to an increase in bile acid synthesis to facilitate fat absorption in the intestine (28). The observation that insulin-induced p38 kinase increases HNF-4␣ protein and phosphorylation, which results in higher 7␣-hydroxylase mRNA levels with no change in HNF-4␣ mRNA (Figs.…”

“…with shorter incubation times, insulin activates 7␣-hydroxylase expression, but with longer times, or at supraphysiological concentrations, insulin suppresses 7␣-hydroxylase expression (Fig. 7A) (28,33). The bile acid pool increases in diabetes both in humans (34) and in other mammals (35).…”

“…Insulin is also a known regulator of bile acid biosynthesis. Thus, recently it has been shown that short term treatment of human primary hepatocytes with physiological concentrations of insulin activates 7␣-hydroxylase expression, whereas longer incubation suppresses it (28). Thus, to investigate whether the p38 kinase pathway could be involved in this regulatory process by increasing nuclear HNF-4␣ protein levels, we incubated primary rat FIGURE 4. p38 chemical inhibitors suppress HNF-4␣ protein, but not mRNA, in primary rat hepatocytes.…”

Disruption of the Murine Protein Kinase Cβ Gene Promotes Gallstone Formation and Alters Biliary Lipid and Hepatic Cholesterol Metabolism

“…Additionally, significantly reduced expression of the sterol 12␣-hydroxylase (CYP8b1) gene is expected to increase the bile hydrophobicity of PKC␤ Ϫ/Ϫ mice (40). The reduction in CYP7A1 expression was not accompanied by any alteration in expression of transcription factors (LXR␣, FXR, and SREBP-1c), which are known to regulate its expression (41)(42)(43). Interestingly, feeding MMLD caused a greater suppression of both CYP7A1 and CYP8b1 genes in the livers of PKC␤ Ϫ/Ϫ mice as compared with controls (Fig.…”