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. 2006 Sep 29;281(39):28745-54.
doi: 10.1074/jbc.M605815200. Epub 2006 Aug 2.

Insulin regulation of cholesterol 7alpha-hydroxylase expression in human hepatocytes: roles of forkhead box O1 and sterol regulatory element-binding protein 1c

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Insulin regulation of cholesterol 7alpha-hydroxylase expression in human hepatocytes: roles of forkhead box O1 and sterol regulatory element-binding protein 1c

Tiangang Li et al. J Biol Chem. .

"VSports最新版本" 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 7alpha-hydroxylase gene expression in human hepatocytes. Real-time PCR assays showed that physiological concentrations of insulin rapidly stimulated cholesterol 7alpha-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 4alpha and peroxisome proliferator-activated receptor gamma coactivator-1alpha 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 gamma-coactivator-1alpha 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 VSports手机版. .

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Figures

Fig. 1
Fig. 1
Real time PCR assays of insulin effect on CYP7A1 and PEPCK mRNA expressions in human primary hepatocytes. Primary human hepatocytes (HH 1251) were treated with increasing concentrations of insulin for 2 h (A) or 6 h (B). CYP7A1 or PEPCK mRNA expression was assayed in triplicates and expressed as mean ± standard deviation.
Fig. 2
Fig. 2
EMSA of FoxO1 and FoxA1 interaction with the putative rat and human IREs. A. Putative IREs in rat and human CYP7A1 promoter. B. EMSA of in vitro synthesized FoxO1 and FoxA1 binding to rat and human IRE probes. A G6Pase IRE probe was used as a positive control. Un-programmed TNT lysates (T3 and T7) were used as negative controls for non-specific bindings. Sequences of IRE probes used are described in Supplemental data. C. EMSA of in vitro synthesized FoxO1 and FoxA1 with 32P-labeled rat CYP7A1 IRE2 and mutant IRE2 probes. Competition assays were done with 100-fold excess of unlabeled probes. Mutation in rIRE2 probes is shown. D. Effects of FoxO1 on rat CYP7A1 reporter activities. WT: wild type rat CYP7A1 reporter (p-344/luc). IRE2 mutant: rat CYP7A1 reporter (p-344/luc) containing single nucleotide mutation in IRE2 sequence. Reporter (0.2 μg) and indicated expression plasmids (0.1 μg) were transfected into HepG2 cells. Transient transfection and luciferase assays were performed as described in Material and Methods. Mutation in rat IRE2 is shown. Statistical analysis was performed by student’s t-test, “*”, significant, P< 0.05.
Fig. 2
Fig. 2
EMSA of FoxO1 and FoxA1 interaction with the putative rat and human IREs. A. Putative IREs in rat and human CYP7A1 promoter. B. EMSA of in vitro synthesized FoxO1 and FoxA1 binding to rat and human IRE probes. A G6Pase IRE probe was used as a positive control. Un-programmed TNT lysates (T3 and T7) were used as negative controls for non-specific bindings. Sequences of IRE probes used are described in Supplemental data. C. EMSA of in vitro synthesized FoxO1 and FoxA1 with 32P-labeled rat CYP7A1 IRE2 and mutant IRE2 probes. Competition assays were done with 100-fold excess of unlabeled probes. Mutation in rIRE2 probes is shown. D. Effects of FoxO1 on rat CYP7A1 reporter activities. WT: wild type rat CYP7A1 reporter (p-344/luc). IRE2 mutant: rat CYP7A1 reporter (p-344/luc) containing single nucleotide mutation in IRE2 sequence. Reporter (0.2 μg) and indicated expression plasmids (0.1 μg) were transfected into HepG2 cells. Transient transfection and luciferase assays were performed as described in Material and Methods. Mutation in rat IRE2 is shown. Statistical analysis was performed by student’s t-test, “*”, significant, P< 0.05.
Fig. 3
Fig. 3
Effect of FoxO1 on human CYP7A1/Luc reporter activity. A. CYP7A1 reporter constructs (0.2 μg) were co-transfected with expression plasmids (0.1 μg) into HepG2 cells and luciferase activity was analyzed 40 h after transfection. B. HNF4α site mutant reporter (mHNF4-ph1887-Luc) was transfected in HepG2 cells. pcDNA3 empty vector or FoxO1 expression plasmid was co-transfected as indicated. C. Mammalian one-hybrid assay. The Gal4/luciferase reporter 5xUAS/TK/Luc (0.2μg) was co-transfected with Gal4 empty vector (−) (0.1μg), Gal4-HNF4α (0.1μg), PGC-1α (0.1μg) and increasing amounts of FoxO1expression plasmid (0.05-0.2μg) as indicated. D. A human CYP7A1 reporter, ph-371-Luc (0.2μg) was co-transfected with FoxO1, HNF4α, and/or PGC-1α (0.1 μg) as indicated into HepG2 cells. Each assay was performed in triplicates and statistical analysis was performed using student’s t-test, “*”, significant, P< 0.05.
Fig. 4
Fig. 4
Effect of SREBP-1c on human CYP7A1 reporter activity. A. Human CYP7A1 reporter constructs (0.2μg) were co-transfected with pcDNA3 empty vector or SREBP-1c expression plasmids (0.1 μg) into HepG2 cells and luciferase activities were analyzed 40 hr after transfection. B. HNF4α site mutant reporter (mHNF4-ph1887/Luc) (0.2μg) was co-transfected with 0.1μg pcDNA3 or SREBP-1c expression plasmid into HepG2 cells. C. Mammalian one-hybrid assay. The Gal4/luciferase reporter, 5xUAS/TK/Luc (0.2μg) was co-transfected with Gal4 empty vector (−)(0.1μg), Gal4-HNF4α (0.1μg), PGC-1α (0.1μg) and increasing amounts of SREBP-1c expression plasmid (0.05-0.2μg) as indicated. D. Human CYP7A1 reporter construct, ph-371-Luc (0.2 μg) was co-transfected with 0.1 μg each of HNF4α and PGC-1α and/or SREBP-1c expression plasmid as indicated. Each assay was performed in triplicates and statistical analysis was performed using student’s t-test, “*”, significant, P< 0.05.
Fig. 5
Fig. 5
Western blotting analysis of FoxO1, SREBP-1c and HNF4α in primary human hepatocytes treated with insulin. Primary human hepatocytes were treated with 10 nM insulin for the time indicated. Total cell lysates or nuclei fractions from donor HH1286, HH1281 and HH1249 were used for detection of FoxO1 (A), SREBP-1c (B) and HNF4α (C) by antibodies, respectively.
Fig. 6
Fig. 6
Chromatin immunoprecipitation assays of insulin effect on FoxO1, SREBP-1c and HNF4α recruitment to CYP7A1 chromatin. A. Human primary hepatocytes (HH1308) were treated with 10 nM insulin for the time indicated. Cells were harvested for ChIP assay as described under Materials and Methods. An antibody against HNF4α, SREBP-1 or FoxO1 was used to immunoprecipitate chromatins for PCR amplification and analysis. Ten % of cell lysate was used as input. Non-immune IgG was used as negative controls. B. ChIP assays of the effect of insulin and T0901317 on HNF4α recruitment of PGC-1α to CYP7A1 chromatin. HepG2 cells in 100 mm dish were transfected with HA-tagged PGC-1α (10 μg). Cells were then treated with insulin (100 nM) or T0901317 (1 μM) for 24 hr. Chromatins were precipitated with an antibody against HA tag or HNF4α for ChIP assays. Ten % of cell lysate was used as input. Non-immune IgG was used as negative controls. C. ChIP assays of HepG2 cells over-expressing SREBP-1c or FoxO1. HepG2 cells in 100 mm dish were transfected with 10 μg of HA-tagged PGC-1α, pcDNA3 empty vector, FoxO1 or SREBP-1c expression plasmid was over-expressed as indicated. Cells were harvested for ChIP assay as described under Experimental procedure. An antibody against HA-tag or HNF4α was used to immunoprecipitate chromatins for PCR amplification and analysis. Ten % of cell lysate was used as input. Non-immune IgG was used as negative controls.

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