Regulation of cholesterol and bile acid homeostasis by the cholesterol 7α-hydroxylase/steroid response element-binding protein 2/microRNA-33a axis in mice

Abstract

Bile acid synthesis not only produces physiological detergents required for intestinal nutrient absorption, but also plays a critical role in regulating hepatic and whole-body metabolic homeostasis. We recently reported that overexpression of cholesterol 7α-hydroxylase (CYP7A1) in the liver resulted in improved metabolic homeostasis in Cyp7a1 transgenic (Cyp7a1-tg) mice. This study further investigated the molecular links between bile acid metabolism and lipid homeostasis. Microarray gene profiling revealed that CYP7A1 overexpression led to marked activation of the steroid response element-binding protein 2 (SREBP2)-regulated cholesterol metabolic network and absence of bile acid repression of lipogenic gene expression in livers of Cyp7a1-tg mice. Interestingly, Cyp7a1-tg mice showed significantly elevated hepatic cholesterol synthesis rates, but reduced hepatic fatty acid synthesis rates, which was accompanied by increased (14) C-glucose-derived acetyl-coenzyme A incorporation into sterols for fecal excretion. Induction of SREBP2 also coinduces intronic microRNA-33a (miR-33a) in the SREBP2 gene in Cyp7a1-tg mice. Overexpression of miR-33a in the liver resulted in decreased bile acid pool, increased hepatic cholesterol content, and lowered serum cholesterol in mice.

Conclusion: This study suggests that a CYP7A1/SREBP2/miR-33a axis plays a critical role in regulation of hepatic cholesterol, bile acid, and fatty acid synthesis. Antagonism of miR-33a may be a potential strategy to increase bile acid synthesis to maintain lipid homeostasis and prevent nonalcoholic fatty liver disease, diabetes, and obesity.

Figure 1. Cyp7a1-tg mice have increased cholesterol synthesis and decreased fatty acid synthesis in the liver

A. Hepatic cholesterol and fatty acid synthesis rates were measured as described in Materials and Methods. The activities of ³H and ¹⁴C were determined in a scintillation counter. The cholesterol and fatty acid synthesis rates were expressed as ¹⁴C radioactivity derived from ¹⁴C-acetate (adjusted by internal recovery standard ³H-cholesterol radioactivity) and incorporated into cholesterol or fatty acids per minute per gram liver tissue. B. Wild type and Cyp7a1-tg mice were ip. injected with a single dose of glucose (8 g/kg) containing 5 µCi [1-¹⁴C] glucose, and fecal ¹⁴C sterol amount was estimated as described in Materials and Methods. The fecal acidic sterols (bile acids) and neutral sterols (cholesterol) derived from ¹⁴C-glucose were estimated by measuring the radioactivity of ¹⁴C incorporated, adjusted by internal standard ³H-cholesterol radioactivity, and expressed as percentage of total administrated ¹⁴C activity. Results are expressed as mean ± SE; “*” indicates p < 0.05 vs. WT. “**” indicates p < 0.05 vs. ¹⁴C incorporation into cholesterol in WT mice, n = 4-5.

Figure 2. Induction of CYP7A1 induces SREBP2 and miR-33a expression in Cyp7a1-tg mice

Wild type and Cyp7a1-tg mice were fed a standard chow diet or Western diet for 4 months. The mRNA expression of (A) SREBP2 and (B) miR-33a was measured by real-time PCR. Results are expressed as mean ± S.E. “*” indicates p < 0.05 vs. WT of same diet, n=4.

Figure 3. Effects of hepatic miR-33a over-expression on bile acid and cholesterol metabolism in mice

Wild type C57BL6J mice were administered adenovirus expressing miR-33a (Ad-miR-33a) or control adenovirus (Ad-null) via tail vein injection and were sacrificed after 7 days. A. Hepatic mRNA expression was measured by real-time PCR. B. Hepatic CYP7A1 enzyme activity. C. Total bile acid pool size. D. Serum cholesterol. E. Hepatic cholesterol. Results are expressed as mean ± S.E. “*” indicates p < 0.05 vs. Ad-null controls, n=4.

Figure 4. Regulation of CYP7A1 mRNA by miR-33a in HepG2 cells

HepG2 cells were transfected with miRIDIAN miR-33a mimic (A-C) or miRIDIAN miR-33a hairpin inhibitor (D-F) at 50nM and 100nM for 48 hr. Respective controls were used to equally adjust the total amount transfected among samples. mRNA expression was measured by real-time PCR, 48 hr after transfection. Assays were performed in triplicate and expressed as mean ± S.D. “*” indicates p < 0.05 vs. controls.

Figure 5. Identification of a putative miR-33a target site in human CYP7A1 3’-UTR region

A. Effect of miR-33a mimic on wild type (pMir-hCYP7A1 (1–200)) and mutant (pMir-hCYP7A1 (1–200)) reporter activity in HepG2 cells. B. Effect of miR-33a mimic on human pMir-hCYP7A1 (203–982) reporter activity in HepG2 cells. C. Putative miR-33a target site in the human CYP7A1 3’-UTR. D. Effect of miR-33a mimic on human ABCA1 3’-UTR reporter activity in HepG2 cells. Assays were performed in triplicate and expressed as mean ± S.D. “*” indicates p > 0.05 vs. controls.

Fig 6

Regulation of cholesterol and bile acid metabolism by the CYP7A1/SREBP2/miR-33a axis. This figure illustrates the coordinated regulation of cholesterol and bile acid metabolism based on this study. Under conditions when cellular cholesterol decreases or cholesterol catabolism increases, SREBP2 is activated. To prevent cellular cholesterol from decreasing, SREBP2 induces LDLR-mediated LDL-C uptake into hepatocytes. In addition, SREBP2 induces a number of key genes to stimulate de novo cholesterol synthesis. On the other hand, SREBP2 binds to its own gene promoter to induce SREBP2 gene transcription, which also results in co-induction of miR-33a. Increased miR-33a down-regulates ABCA1 and ABCG1 to reduce cellular cholesterol efflux to HDL. Meanwhile, miR-33a inhibits CYP7A1 and bile acid synthesis to inhibit cholesterol catabolism. Under conditions when cellular cholesterol increases, cholesterol represses these SREBP2-miR-33a regulated pathways, while cholesterol feed-forward induces CYP7A1 (in mouse) to convert cholesterol into bile acids and to stimulate biliary cholesterol secretion via ABCG5/G8 heterodimers. This feed-forward activation of CYP7A enzyme activity by cholesterol and feedback inhibition of CYP7A1 translation by miR-33a provide a rapid post-transcriptional mechanism for regulation of bile acid synthesis to maintain hepatic lipid homeostasis.