Critical role of cholic acid for development of hypercholesterolemia and gallstones in diabetic mice

doi:10.1016/j.bbrc.2006.02.108

Biochemical and Biophysical Research Communications

Volume 342, Issue 4, 21 April 2006, Pages 1382-1388

https://doi.org/10.1016/j.bbrc.2006.02.108 Get rights and content

Abstract

We studied bile acid and cholesterol metabolism in insulin-dependent diabetes utilizing genetically modified mice unable to synthesize cholic acid (Cyp8b1−/−). Diabetes was induced in Cyp8b1−/− and wild type animals (Cyp8b1+/+) by alloxan, and the mice were fed normal or cholesterol-enriched diet for 10 weeks. The serum levels of cholesterol were strongly increased in diabetic Cyp8b1+/+ mice fed cholesterol, while diabetic Cyp8b1−/− mice did not show any aberrations regardless of the diet. Diabetic cholesterol-fed Cyp8b1+/+ mice had much higher biliary cholesterol and cholesterol saturation index than all other groups, their bile contained a large number of cholesterol crystals, and their canalicular cholesterol transporter Abcg5/g8 mRNA levels were much higher. Cyp7a1 mRNA levels were similar in all diabetic mice but higher compared to non-diabetic animals. The results indicate a critical role for cholic acid for the development of hypercholesterolemia and gallstones in our animal model.

Section snippets

Materials and methods

Chemicals. The following items were purchased from Sigma–Aldrich Corp., St. Louis, Missouri, USA: Cholesterol (>99% purity), alloxan monohydrate.

Animals and procedures. Male age-matched mice (2–6 months of age) deficient for the Cyp8b1 gene (Cyp8b1−/−) and the corresponding wild type animals (Cyp8b1+/+) were bred from a mixed genetic background (C57BL6/129). Both Cyp8b1−/− and +/+ mice were housed at 22–24 °C at a light circle from 8 a.m. to 8 p.m. in groups of 4–8 animals. Diabetes was induced

Alterations of the plasma glucose levels

Generally, the urine of the alloxan-treated animals started to give positive reactions for glucose 1 week after the injection. A strong hyperglycemia was observed in all 4 groups, with serum glucose values ranging between 20 and 27 mmol/L. Diabetic mice that were subjected to overnight fasting before sacrifice generally displayed slightly lower serum glucose levels but always well over 12 mmol/L (data not shown). Use of lower doses of alloxan (50 mg/kg), as have been suggested previously [18],

Discussion

Formation of cholesterol gallstones can be defined as a failure of the bile constituents to maintain cholesterol in its solubilized form. The prolithogenic state of bile is presently characterized by an excess of bile cholesterol, a rapid nucleation of bile constituents, alterations in the concentrations of biliary proteins and mucin, and finally, a loss of normal gallbladder contractility [19]. However, the most important causative agent for gallstone formation is considered to be the degree

Acknowledgments

We are in great debt for the extremely skilled and professional care of the experimental animals by Mrs. Margareta Hagelin and Mrs. Maj-Britt Alter at the Center for Tumor Biology, Karolinska Institutet. We are also indebted to Miss Maria Olin and Mrs. Lisbet Benthin for the excellent technical assistant. This project was financed by grants from the Swedish Research Council, The Swedish Heart-Lung Foundation, Ruth and Richard Julin’s Foundation, and Astra-Zeneca Inc., Mölndal, Sweden.

References (35)

C.E. Carnovale et al.
Studies on the mechanism of bile salt-independent bile flow impairment in streptozotocin-induced hepatotoxicity
Toxicology
(1991)
B. Verges
Anomalies of lipid metabolism in diabetes mellitus
Rev. Med. Interne.
(1991)
M.C. Hunt et al.
The peroxisome proliferator-activated receptor alpha (PPARalpha) regulates bile acid biosynthesis
J. Biol. Chem.
(2000)
A. Roda et al.
Enzymatic determination of cholesterol in bile
Clin. Chim. Acta
(1975)
M.C. Carey
Critical tables for calculating the cholesterol saturation of native bile
J. Lipid. Res.
(1978)
J. Wang et al.
Studies on LXR- and FXR-mediated effects on cholesterol homeostasis in normal and cholic acid-depleted mice
J. Lipid. Res.
(2006)
J.F. Miquel et al.
Cholesterol saturation, not proteins or cholecystitis, is critical for crystal formation in human gallbladder bile
Gastroenterology
(1998)
J.J. Repa et al.
Regulation of ATP-binding cassette sterol transporters ABCG5 and ABCG8 by the liver X receptors alpha and beta
J. Biol. Chem.
(2002)
J.J. Smit et al.
Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease
Cell
(1993)
W.M. van Waarde et al.
Differential effects of streptozotocin-induced diabetes on expression of hepatic ABC-transporters in rats
Gastroenterology
(2002)

D.Q. Wang et al.

Complete mapping of crystallization pathways during cholesterol precipitation from model bile: influence of physical-chemical variables of pathophysiologic relevance and identification of a stable liquid crystalline state in cold, dilute and hydrophilic bile salt-containing systems

J. Lipid. Res.

(1996)

H.N. Ginsberg et al.

Regulation of plasma triglycerides in insulin resistance and diabetes

Arch. Med. Res.

(2005)

E. Windler

What is the consequence of an abnormal lipid profile in patients with type 2 diabetes or the metabolic syndrome?

Atheroscler Suppl.

(2005)

F. Milliat et al.

Short and long-term effects of streptozotocin on dietary cholesterol absorption, plasma lipoproteins and liver lipoprotein receptors in RICO rats

Exp. Clin. Endocrinol. Diabetes

(2000)

A. De Santis et al.

Gallstones and diabetes: a case-control study in a free-living population sample

Hepatology

(1997)

C.E. Carnovale et al.

Reversible impairment of hepatobiliary function induced by streptozotocin in the rat

Experientia

(1984)

K. Uchida et al.

Altered bile acid metabolism related to atherosclerosis in alloxan diabetic rats

J. Atheroscler. Thromb.

(1996)

Cited by (16)

Addition of Dexamethasone Alters the Bile Acid Composition by Inducing CYP8B1 in Primary Cultures of Human Hepatocytes
2016, Journal of Clinical and Experimental Hepatology
Citation Excerpt :
This is in line with previous data, where transplanted patients that undergo rejection treatment, with methylprednisolone, have a decrease in total bile acids.29 Furthermore, previous studies have shown that CA facilitates intestinal absorption of lipids, especially cholesterol, and may have a role in gallstone formation.5,6 Bile composition may also be of importance for development of atherosclerosis as studies on double knockout animals of Cyp8b1 and ApoE have a 50% reduction of atherosclerotic lesions compared to ApoE knockout mice.4
Primary human hepatocytes offer the best human in vitro model for studies on human liver cell metabolism. Investigators use a variety of different media supplements and matrix biocoatings and the type of culture system used may influence the outcome.
To optimize in vitro conditions for primary human hepatocytes with regard to bile acid synthesis.
Human hepatocytes were isolated and cultured on collagen type I or EHS matrigel in cell media with or without dexamethasone. The glucocorticoid receptor (GR) antagonist RU486 was used to elucidate the involvement of GR.
Hepatocytes cultured on EHS matrigel produced more bile acids and expressed higher levels of cholesterol 7α-hydroxylase (CYP7A1) than cells cultured on rat tail collagen. Supplementation with dexamethasone increased the formation of cholic acid (CA) and decreased chenodeoxycholic acid formation. In line with these results, the mRNA expression of sterol 12α-hydroxylase (CYP8B1) increased following dexamethasone treatment. Surprisingly, the mRNA expression of CYP7A1 and CYP27A1 was not increased to the same extent. By using the GR antagonist RU486, we concluded that CYP8B1 induction is mediated via a GR-independent pathway. An altered expression of retinoid-related orphan receptor (ROR) α and ROR α target gene Glucose-6-phosphatase (G6Pase) suggests that ROR α signaling may regulate CYP8B1 expression.
Primary human hepatocytes have an increased bile acid synthesis rate when cultured on matrigel as compared to collagen. Exposure to glucocorticoid hormones stimulates the expression of CYP8B1, leading to an increased formation of CA and alteration of the bile acid composition. The effect is most likely mediated through a GR-independent pathway, possibly through ROR α.
Retinoic acid-related orphan receptor α regulates diurnal rhythm and fasting induction of sterol 12α-hydroxylase in bile acid synthesis
2013, Journal of Biological Chemistry
Citation Excerpt :
For that reason, CA (0.5%) is included in lithogenic diets to accelerate hypercholesterolemia, cholesterol gallstones, and atherosclerosis in C57BL6 mice (5, 6). Ablation of the CYP8B1 gene reduces dietary cholesterol absorption and prevents hypercholesterolemia and gallstone formation in diabetic mice (7–9) 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).
Sterol 12α-hydroxylase (CYP8B1) is required for cholic acid synthesis and plays a critical role in intestinal cholesterol absorption and pathogenesis of cholesterol gallstone, dyslipidemia, and diabetes. In this study we investigated the underlying mechanism of fasting induction and circadian rhythm of CYP8B1 by a cholesterol-activated nuclear receptor and core clock gene retinoic acid-related orphan receptor α (RORα). Fasting stimulated, whereas restricted-feeding reduced expression of CYP8B1 mRNA and protein. However, fasting and feeding had little effect on the diurnal rhythm of RORα mRNA expression, but fasting increased RORα protein levels by cAMP-activated protein kinase A-mediated phosphorylation and stabilization of the protein. Adenovirus-mediated gene transduction of RORα to mice strongly induced CYP8B1 expression, and increased liver cholesterol and 12α-hydroxylated bile acids in the bile acid pool and serum. A reporter assay identified a functional RORα response element in the CYP8B1 promoter. RORα recruited cAMP response element-binding protein-binding protein (CBP) to stimulate histone acetylation on the CYP8B1 gene promoter. In conclusion, RORα is a key regulator of diurnal rhythm and fasting induction of CYP8B1, which regulates bile acid composition and serum and liver cholesterol levels. Antagonizing RORα activity may be a therapeutic strategy for treating inflammatory diseases such as non-alcoholic fatty liver disease and type 2 diabetes.
Cytochrome P450s and cholesterol homeostasis
2006, Pharmacology and Therapeutics
By participating in pathways of cholesterol biosynthesis and elimination, different cytochrome P450 (P450 or CYP) enzymes play an important role in maintenance of cholesterol homeostasis. CYP51 is involved in cholesterol biosynthesis, whereas CYP 7A1, 27A1, 46A1, 7B1, 39A1, and 8B1 are the key enzymes in cholesterol catabolism to bile acids, the major route of cholesterol elimination in mammals. Cholesterol transformations to steroid hormones are also initiated by the P450 enzyme CYP11A1. Finally, one of the major drug-metabolizing P450s CYP3A4 seems to contribute to bile acid biosynthesis as well. The 9 P450s will be the focus of this review and assessed as drug targets for cholesterol lowering.
Cholesterol Gallstone Susceptibility Loci: A Mouse Map, Candidate Gene Evaluation, and Guide to Human LITH Genes
2006, Gastroenterology
Citation Excerpt :
In the absence of 12α-hydroxylation, chenodeoxycholic (3α,7α-dihydroxy) acid is formed. Importantly, comparison of diabetic wild-type mice (mixed B6;129 background) with diabetic Cyp8b1 knockout mice confirmed the importance of cholic acid to cholesterol absorption and in the precipitation gallbladder.130 In hamsters fed a lithogenic diet, insulin injections augmented cholesterol gallstone formation.128
Cholesterol gallstones are prevalent and costly, chiefly among developed countries. In addition to numerous environmental risk factors, a complex genetic basis determines the risk for developing cholesterol gallstones. Rather than monogenic mutations that are rare and occur in limited populations, genetic predisposition to gallstone susceptibility in general populations arises from polymorphisms in multiple genes, each making a small contribution to overall risk. Because mouse and human genomes are conserved and only a critical subset of homologous genes appears rate limiting for gallstone susceptibility in these species, a genetic map of mouse lithogenic (Lith) loci provides a “roadmap” for the discovery of human LITH genes. Quantitative trait locus (QTL) mapping was employed to identify Lith loci in mice. Repeated detection of colocalizing QTLs among 9 crosses of 12 genetically diverse progenitor strains suggests identification of most major Lith QTLs, including Lith1–Lith23. Therefore, using this knowledge, our priority is to predict human LITH genes. To date, predominantly, prior knowledge of gene-product function was invoked to postulate causal roles for genes in gallstone susceptibility. Unfortunately, few such genes colocalized with empirical Lith loci, suggesting absence of causality or very limited contributions. Consequently, we present a systematic and comprehensive analysis of literature to support or refute the contributions to gallstone susceptibility by genes located within critical regions of empirical QTLs. We envisage that identification of human LITH genes based on our translational approach will provide targets for the development of new means of prevention and nonsurgical management of cholelithiasis.
Dietary diosgenin transcriptionally down-regulated intestinal NPC1L1 expression to prevent cholesterol gallstone formation in mice
2023, Journal of Biomedical Science
Effect of Linoleic Acid on Cholesterol Levels in a High-Fat Diet-Induced Hypercholesterolemia Rat Model
2023, Metabolites

View all citing articles on Scopus

View full text

Critical role of cholic acid for development of hypercholesterolemia and gallstones in diabetic mice

Abstract

Section snippets

Materials and methods

Alterations of the plasma glucose levels

Discussion

Acknowledgments

Toxicology

Rev. Med. Interne.

J. Biol. Chem.

Clin. Chim. Acta

J. Lipid. Res.

J. Lipid. Res.

Gastroenterology

J. Biol. Chem.

Cell

Gastroenterology

J. Lipid. Res.

Arch. Med. Res.

Atheroscler Suppl.

Short and long-term effects of streptozotocin on dietary cholesterol absorption, plasma lipoproteins and liver lipoprotein receptors in RICO rats

Exp. Clin. Endocrinol. Diabetes

Gallstones and diabetes: a case-control study in a free-living population sample

Hepatology

Reversible impairment of hepatobiliary function induced by streptozotocin in the rat

Experientia

Altered bile acid metabolism related to atherosclerosis in alloxan diabetic rats

J. Atheroscler. Thromb.