Bile Acid Formation Research Articles

Suppression of bile acid synthesis by thyroid hormone in primary human hepatocytes

It is known that thyroid hormones alter the bile acid metabolism in humans, however the effect on individual enzymes has been difficult to elucidate. This is mainly due to the lack of human liver cell lines producing bile acids. We used cultures of primary human hepatocytes to study the effects of triiodothyronine (T(3)) on bile acid synthesis. Primary hepatocytes were isolated from liver tissue obtained from three different patients undergoing liver resection due to underlying malignancy. The hepatocytes were cultured under serum-free conditions and treated from d 1 to d 5 with culture containing 0.1 - 1000 nmol/L of T(3). Bile acid formation and mRNA levels of key enzymes were analysed. The lowest concentration of T(3) decreased cholic acid (CA) formation to 43%-53% of controls and chenodeoxycholic acid (CDCA) to 52%-75% of controls on d 5. The highest dose further decreased CA formation to 16%-48% of controls while CDCA formation remained at 50%-117% of controls. Expression of mRNA levels of cholesterol 7alpha-hydroxylase (CYP7A1) and sterol 12alpha-hydroxylase (CYP8B1) dose-dependently decreased. Sterol 27-hydroxylase (CYP27A1) levels also decreased, but not to the same extent. T(3) dose-dependently decreased total bile acid formation in parallel with decreased expression of CYP7A1 and CYP8B1. CA formation is inhibited to a higher degree than CDCA, resulting in a marked decrease in the CA /CDCA ratio.

Unconjugated faecal bile acids in familial adenomatous polyposis analysed by gas-liquid chromatography and mass spectrometry.

Previous studies have suggested reduced formation of secondary bile acids in patients with familial adenomatous polyposis (FAP). Developments in the collection, extraction and analysis of faecal bile acids as well as in the accurate diagnosis of FAP by DNA markers prompted reinvestigation of this hypothesis. The median (interquartile range (i.q.r.)) faecal bile acid concentration (3.69 (1.66-5.36) mumol per g dry weight) and daily excretion rate (60.5 (29-149) mumol per g per 24 h) in ten patients with FAP were similar to those of nine control subjects (3.31 (0.65-8.38) mumol per g dry weight and 30.1 (7.9-228) mumol per g per 24 h). Although the median (i.q.r.) concentration of only one bile acid (12-oxo-lithocholic acid) was significantly different between patients with FAP and controls (49 (34-70) versus 0 (0-20) nmol per g dry weight, P = 0.006), the derivatives of chenodeoxycholic acid (3.35 (1.76-5.32) versus 0.51 (0.13-2.37) mumol per g dry weight, P = 0.02) and cholic acid (1.63 (0.42-2.34) versus 0.80 (0.13-3.57) mumol per g dry weight, P = 0.006) were increased in those with polyposis. These results show increased bacterial biotransformation of faecal bile acids in patients with FAP.

Structural Stability and Prebiotic Properties of Resistant Starch Type 3 Increase Bile Acid Turnover and Lower Secondary Bile Acid Formation

Microbial metabolism is essential in maintaining a healthy mucosa in the large bowel, preferentially through butyrate specific mechanisms. This system depends on starch supply. Two structurally different resistant starches type 3 (RS3) have been investigated with respect to their resistance to digestion, fermentability, and their effects on the composition and turnover of bile acids in rats. RSA (a mixture of retrograded maltodextrins and branched high molecular weight polymers), which is more resistant than RSB (a retrograded potato starch), increased the rate of fermentation accompanied by a decrease of pH in cecum, colon, and feces. Because they were bound to RS3, less bile acids were reabsorbed, resulting in a higher turnover through the large bowel. Because of the rise of volume, the bile acid level was unchanged and the formation of secondary bile acids was partly suppressed. The results proved a strong relation between RS3, short chain fatty acid production, and microflora. However, butyrate specific benefits are only achieved by an intake of RS3 that result in good fermentation properties, which depend on the kind of the resistant starch structures.

Effects of difructose anhydride III (DFA III) administration on bile acids and growth of DFA III-assimilating bacterium Ruminococcus productus on rat intestine

The growth of DFA III-assimilating bacteria in the intestines of rats fed 3% DFA III for 2 weeks was examined. Sixty-four percent of the DFA III intake had been assimilated on day 3 of ingestion, and almost all of the DFA III was assimilated at the end of the experiment. The DFA III-assimilating bacterium, Ruminococcus productus, in DFA III-fed rats was in the stationary state of 10(8)-10(9) cells/g dry feces within a week from 10(6) cells/g dry feces on day 1 of DFA III ingestion. The number of R. productus cells was associated with the amount of DFA III excreted in the feces. The acetic acid produced from DFA III by R. productus lowered the cecal pH to 5.8. In control-fed rats and DFA III-fed rats, 94% of secondary bile acids and 94% of primary bile acids, respectively, were accounted for in the total bile acids analyzed. DFA III ingestion increased the ratio of primary bile acids and changed the composition of fecal bile acids. In conclusion, R. productus assimilated DFA III, produced short chain fatty acids, and the cecal pH was lowered. The acidification of rat intestine perhaps inhibited secondary bile acid formation and decreased the ratio of secondary bile acids. Therefore, it is expected that DFA III may prevent colorectal cancer and be a new prebiotic candidate.

Cholesterol Accumulation and Liver Cell Death in Mice With Niemann-Pick Type C Disease *

Niemann-Pick type C (NPC) disease develops as a result of mutations in the NPC1 gene that encodes a protein involved in the net movement of unesterified cholesterol from the late endosomal/lysosomal compartment to the metabolically active pool of sterol in the cytosol of virtually every cell in the body. Although early publications emphasized the neurodegeneration occurring in children with this mutation, more recent clinical information suggests that serious liver disease also is an important part of this syndrome. These studies, therefore, were undertaken to characterize the liver dysfunction seen in mice with this same mutation. The NPC mouse develops significant hepatomegaly that reaches 8% of body weight at 5 to 6 weeks of age. This increase in liver size is associated with a linear increase in cholesterol content and with accumulation of amorphous cellular inclusions in both hepatocytes and macrophages. During the few weeks after birth, significant elevation of the plasma alkaline phosphatase level occurs, as also is seen in the human infant with this disease. At 4 to 5 weeks of age, plasma aminotransferase levels also rise abruptly. Histologically, at this time there is apoptosis, but no excess deposition of collagen or glycogen. mRNA expression is elevated for caspase 1, caspase 6, and several enzymes associated with sterol biosynthesis and bile acid formation. In conclusion, the NPC mouse has liver disease similar to that seen in the NPC infant and represents a relevant model for exploring the molecular events occurring in this form of liver disease.

Identification of intermediates in the bile acid synthetic pathway as ligands for the farnesoid X receptor

Bile acid synthesis from cholesterol is tightly regulated via a feedback mechanism mediated by the farnesoid X receptor (FXR), a nuclear receptor activated by bile acids. Synthesis via the classic pathway is initiated by a series of cholesterol ring modifications and followed by the side chain cleavage. Several intermediates accumulate or are excreted as end products of the pathway in diseases involving defective bile acid biosynthesis. In this study, we investigated the ability of these intermediates to activate human FXR. In a cell-based reporter assay and coactivator recruitment assays in vitro, early intermediates possessing an intact cholesterol side chain were inactive, whereas 26- or 25-hydroxylated bile alcohols and C27 bile acids were highly efficacious ligands for FXR at a level comparable to that of the most potent physiological ligand, chenodeoxycholic acid. Treatment of HepG2 cells with these precursors repressed the rate-limiting cholesterol 7alpha-hydroxylase mRNA level and induced the small heterodimer partner and the bile salt export pump mRNA, indicating the ability to regulate bile acid synthesis and excretion. Because 26-hydroxylated bile alcohols and C27 bile acids are known to be evolutionary precursors of bile acids in mammals, our findings suggest that human FXR may have retained affinity to these precursors during evolution.

Differential regulation of cytosolic and peroxisomal bile acid amidation by PPARα activation favors the formation of unconjugated bile acids

In human liver, unconjugated bile acids can be formed by the action of bile acid-CoA thioesterases (BACTEs), whereas bile acid conjugation with taurine or glycine (amidation) is catalyzed by bile acid-CoA:amino acid N-acyltransferases (BACATs). Both pathways exist in peroxisomes and cytosol. Bile acid amidation facilitates biliary excretion, whereas the accumulation of unconjugated bile acids may become hepatotoxic. We hypothesized that the formation of unconjugated and conjugated bile acids from their common substrate bile acid-CoA thioesters by BACTE and BACAT is regulated via the peroxisome proliferator-activated receptor alpha (PPARalpha). Livers from wild-type and PPARalpha-null mice either untreated or treated with the PPARalpha activator WY-14,643 were analyzed for BACTE and BACAT expression. The total liver capacity of taurochenodeoxycholate and taurocholate formation was decreased in WY-14,643-treated wild-type mice by 60% and 40%, respectively, but not in PPARalpha-null mice. Suppression of the peroxisomal BACAT activity was responsible for the decrease in liver capacity, whereas cytosolic BACAT activity was essentially unchanged by the treatment. In both cytosol and peroxisomes, the BACTE activities and protein levels were upregulated 5- to 10-fold by the treatment. These effects caused by WY-14,643 treatment were abolished in PPARalpha-null mice. The results from this study suggest that an increased formation of unconjugated bile acids occurs during PPARalpha activation.

Bifidobacterium animalis strain DN-173 010 hydrolyses bile salts in the gastrointestinal tract of pigs

Background: Bile salt hydrolase (BSH) activity is widespread among ingested bifidobacteria and lactobacilli. It is sometimes considered to be beneficial because of its putative lowering effect on cholesterol absorption and sometimes considered to be deleterious because it may compromise normal fat absorption and even promote the formation of secondary cytotoxic bile acids by the resident intestinal flora. However, the true hydrolysis of bile salts in vivo by ingested living bacteria remains unexplored. The aim of the study was to examine whether or not Bifidobacterium animalis DN-173 010 (used in fermented milks), which demonstrates a BSH activity in vitro, was also active in vivo during its transit in the intestine of pigs. Methods: Direct measurement of total and unconjugated bile acids reabsorbed into the portal vein was done, before and after the pigs had been treated for 2 weeks with two daily doses of­≈ 3.5·10[Formula: See Text] colony-forming units of living (6 pigs) or inactivated B. animalis (6 pigs). Results: None of the treatments modified the portal serum concentration of total bile acids over a 6-h postprandial period. Unconjugated bile acids represented up to 44% and 53% of total bile acids after 1 and 2 weeks of treatment with living bacteria, respectively, compared with only 25% (P < 0.05) before treatment or after 1 or 2 weeks of treatment with inactivated bacteria. Conclusions: Living B. animalis DN-173 010 exhibited a BSH activity in the gastrointestinal tract of pigs, most probably in the small bowel. There was no sign of increased formation of secondary bile acids beyond the hydrolysis reaction.

Assay method for mitochondrial sterol 27-hydroxylase with 7α-hydroxy-4-cholesten-3-one as a substrate in the rat liver

Mitochondrial sterol 27-hydroxylase (EC 1.14.13.15) is an important enzyme, not only in the formation of bile acids from cholesterol intermediates in the liver but also in the removal of cholesterol by side chain hydroxylation in extrahepatic tissues. The enzyme has been assayed by complicated methods using radiolabeled substrates or deuterium-labeled tracers. These methods may be inaccurate for measuring enzyme activity, because the amount of electron-transferring proteins may be insufficient for maximal velocity. To solve this problem, after solubilization of the enzyme from rat liver mitochondria with n-octyl-beta-d-glucopyranoside (OGP), we measured the enzyme activity by incubating the solubilized enzyme with saturated amounts of electron-transferring proteins. In our assay system, using 7alpha-hydroxy-4-cholesten-3-one (HCO) as a substrate, we could easily measure the product, 7alpha,27-dihydroxy-4-cholesten-3-one, with HPLC monitoring absorbance at 240 nm. The product formation was proportionate to the time up to 5 min and the protein concentration up to 0.5 mg of protein/ml. The maximal velocity of the enzyme was 1.1 nmol/min/mg of protein, which was 4- to 16-fold higher than previously reported values. A simple and accurate assay method for sterol 27-hydroxylase in rat liver mitochondria is herein described.

Feedback Regulation of Bile Acid Synthesis in Primary Human Hepatocytes: Evidence That Cdca Is the Strongest Inhibitor

Primary human hepatocytes were used to elucidate the effect of individual bile acids on bile acid formation in human liver. Hepatocytes were treated with free as well as glycine–conjugated bile acids. Bile acid formation and messenger RNA (mRNA) levels of key enzymes and the nuclear receptor short heterodimer partner (SHP) were measured after 24 hours. Glycochenodeoxycholic acid (GCDCA; 100 μmol/L) significantly decreased formation of cholic acid (CA) to 44% ± 4% of controls and glycodeoxycholic acid (GDCA) decreased formation of CA to 67% ± 11% of controls. Glycoursodeoxycholic acid (GUDCA; 100 μmol/L) had no effect. GDCA or glycocholic acid (GCA) had no significant effect on chenodeoxycholic acid (CDCA) synthesis. Free bile acids had a similar effect as glycine–conjugated bile acids. Addition of GCDCA, GDCA, and GCA (100 μmol/L) markedly decreased cholesterol 7α–hydroxylase (CYP7A1) mRNA levels to 2% ± 1%, 2% ± 1%, and 29% ± 11% of controls, respectively, whereas GUDCA had no effect. Addition of GDCA and GCDCA (100 μmol/L) significantly decreased sterol 12α–hydroxylase (CYP8B1) mRNA levels to 48% ± 5% and 61% ± 4% of controls, respectively, whereas GCA and GUDCA had no effect. Addition of GCDCA and GDCA (100 μmol/L) significantly decreased sterol 27–hydroxylase (CYP27A1) mRNA levels to 59% ± 3% and 60% ± 7% of controls, respectively, whereas GUDCA and GCA had no significant effect. Addition of both GCDCA and GDCA markedly increased the mRNA levels of SHP to 298% ± 43% and 273% ± 30% of controls, respectively. In conclusion, glycine–conjugated and free bile acids suppress bile acid synthesis and mRNA levels of CYP7A1 in the order CDCA &gt; DCA &gt; CA &gt; UDCA. mRNA levels of CYP8B1 and CYP27A1 are suppressed to a much lower degree than CYP7A1. (Hepatology 2003;38:930–938).

Feedback regulation of bile acid synthesis in primary human hepatocytes: Evidence that CDCA is the strongest inhibitor

Primary human hepatocytes were used to elucidate the effect of individual bile acids on bile acid formation in human liver. Hepatocytes were treated with free as well as glycine-conjugated bile acids. Bile acid formation and messenger RNA (mRNA) levels of key enzymes and the nuclear receptor short heterodimer partner (SHP) were measured after 24 hours. Glycochenodeoxycholic acid (GCDCA; 100 μmol/L) significantly decreased formation of cholic acid (CA) to 44% ± 4% of controls and glycodeoxycholic acid (GDCA) decreased formation of CA to 67% ± 11% of controls. Glycoursodeoxycholic acid (GUDCA; 100 μmol/L) had no effect. GDCA or glycocholic acid (GCA) had no significant effect on chenodeoxycholic acid (CDCA) synthesis. Free bile acids had a similar effect as glycine-conjugated bile acids. Addition of GCDCA, GDCA, and GCA (100 μmol/L) markedly decreased cholesterol 7α-hydroxylase (CYP7A1) mRNA levels to 2% ± 1%, 2% ± 1%, and 29% ± 11% of controls, respectively, whereas GUDCA had no effect. Addition of GDCA and GCDCA (100 μmol/L) significantly decreased sterol 12α-hydroxylase (CYP8B1) mRNA levels to 48% ± 5% and 61% ± 4% of controls, respectively, whereas GCA and GUDCA had no effect. Addition of GCDCA and GDCA (100 μmol/L) significantly decreased sterol 27-hydroxylase (CYP27A1) mRNA levels to 59% ± 3% and 60% ± 7% of controls, respectively, whereas GUDCA and GCA had no significant effect. Addition of both GCDCA and GDCA markedly increased the mRNA levels of SHP to 298% ± 43% and 273% ± 30% of controls, respectively. In conclusion, glycine-conjugated and free bile acids suppress bile acid synthesis and mRNA levels of CYP7A1 in the order CDCA > DCA > CA > UDCA. mRNA levels of CYP8B1 and CYP27A1 are suppressed to a much lower degree than CYP7A1. (H epatology 2003;38:930-938.)

The Human Bile Acid-CoA:Amino Acid N-Acyltransferase Functions in the Conjugation of Fatty Acids to Glycine

Bile acid-CoA:amino acid N-acyltransferase (BACAT) catalyzes the conjugation of bile acids to glycine and taurine for excretion into bile. By use of site-directed mutagenesis and sequence comparisons, we have identified Cys-235, Asp-328, and His-362 as constituting a catalytic triad in human BACAT (hBACAT) and identifying BACAT as a member of the type I acyl-CoA thioesterase gene family. We therefore hypothesized that hBACAT may also hydrolyze fatty acyl-CoAs and/or conjugate fatty acids to glycine. We show here that recombinant hBACAT also can hydrolyze long- and very long-chain saturated acyl-CoAs (mainly C16:0-C26:0) and by mass spectrometry verified that hBACAT also conjugates fatty acids to glycine. Tissue expression studies showed strong expression of BACAT in liver, gallbladder, and the proximal and distal intestine. However, BACAT is also expressed in a variety of tissues unrelated to bile acid formation and transport, suggesting important functions also in the regulation of intracellular levels of very long-chain fatty acids. Green fluorescent protein localization experiments in human skin fibroblasts showed that the hBACAT enzyme is mainly cytosolic. Therefore, the cytosolic BACAT enzyme may play important roles in protection against toxicity by accumulation of unconjugated bile acids and non-esterified very long-chain fatty acids.

On the substrate specificity of human CYP27A1: implications for bile acid and cholestanol formation

The mitochondrial sterol 27-hydroxylase (CYP27A1) is required for degradation of the C27-sterol side chain in bile acid biosynthesis. CYP27A1 seems, however, to have roles beyond this, as illustrated by patients with a deficient sterol 27-hydroxylase due to mutations of the CYP27A1 gene [cerebrotendinous xanthomatosis (CTX)]. These subjects have symptoms ranging from accumulation of bile alcohols and cholestanol to accelerated atherosclerosis and progressive neurologic impairment. The present work describes a detailed investigation on the substrate specificity of recombinant human CYP27A1. In accordance with some previous work with rat liver mitochondria, the activity in general increased with the polarity of the substrate. An obvious example was the finding that cholesterol was 27-hydroxylated more efficiently than cholesterol oleate but less efficiently than cholesterol sulfate. The oxysterols 24S-hydroxycholesterol and 25-hydroxycholesterol were 27-hydroxylated less efficiently than cholesterol, possibly due to steric hindrance. Surprisingly, sterols with a 3-oxo-Delta4 structure were found to be hydroxylated at a much higher rate than the corresponding sterols with a 3beta-hydroxy-Delta5 structure. The rates of hydroxylation of the sterols were: 7alpha-hydroxy-4-cholesten-3-one>4-cholesten-3-one>7alpha-hydroxycholesterol>24-hydroxy-4-cholesten-3-one> cholesterol>25-hydroxy-4-cholesten-3-one>24-hydroxycholesterol>or=25-hydroxycholesterol. The possibility is discussed that the findings may have implications for oxysterol-mediated regulation of gene expression. The very high activity of CYP27A1 towards the cholestanol precursor 4-cholesten-3-one may be of importance in connection with the accumulation of cholestanol in patients with CTX.

Bile acids induce the expression of the human peroxisome proliferator-activated receptor alpha gene via activation of the farnesoid X receptor.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor that controls lipid and glucose metabolism and exerts antiinflammatory activities. PPARalpha is also reported to influence bile acid formation and bile composition. Farnesoid X receptor (FXR) is a bile acid-activated nuclear receptor that mediates the effects of bile acids on gene expression and plays a major role in bile acid and possibly also in lipid metabolism. Thus, both PPARalpha and FXR appear to act on common metabolic pathways. To determine the existence of a molecular cross-talk between these two nuclear receptors, the regulation of PPARalpha expression by bile acids was investigated. Incubation of human hepatoma HepG2 cells with the natural FXR ligand chenodeoxycholic acid (CDCA) as well as with the nonsteroidal FXR agonist GW4064 resulted in a significant induction of PPARalpha mRNA levels. In addition, hPPARalpha gene expression was up-regulated by taurocholic acid in human primary hepatocytes. Cotransfection of FXR/retinoid X receptor in the presence of CDCA led to up to a 3-fold induction of human PPARalpha promoter activity in HepG2 cells. Mutation analysis identified a FXR response element in the human PPARalpha promoter (alpha-FXR response element (alphaFXRE)] that mediates bile acid regulation of this promoter. FXR bound the alphaFXRE site as demonstrated by gel shift analysis, and CDCA specifically increased the activity of a heterologous promoter driven by four copies of the alphaFXRE. In contrast, neither the murine PPARalpha promoter, in which the alphaFXRE is not conserved, nor a mouse alphaFXRE-driven heterologous reporter, were responsive to CDCA treatment. Moreover, PPARalpha expression was not regulated in taurocholic acid-fed mice. Finally, induction of hPPARalpha mRNA levels by CDCA resulted in an enhanced induction of the expression of the PPARalpha target gene carnitine palmitoyltransferase I by PPARalpha ligands. In concert, these results demonstrate that bile acids stimulate PPARalpha expression in a species-specific manner via a FXRE located within the human PPARalpha promoter. These results provide molecular evidence for a cross-talk between the FXR and PPARalpha pathways in humans.

Human Sterol 27-Hydroxylase (CYP27) Overexpressor Transgenic Mouse Model: EVIDENCE AGAINST 27-HYDROXYCHOLESTEROL AS A CRITICAL REGULATOR OF CHOLESTEROL HOMEOSTASIS

CYP27-overexpressed transgenic mice were generated with the use of a human full-length CYP27 coding region cloned into a ubiquitous expression vector. Positive transgenic mice were identified by tail DNA genotyping and high fecal 27-hydroxycholesterol content. The levels of 27-hydroxycholesterol were found to be 3-5 times higher in the circulation and the tissues of the overexpressed mice when compared with littermate controls. There were no gross morphological differences between the overexpressed mice and their controls. Total cholesterol and triglyceride levels were not affected by overexpression of CYP27. Serum lathosterol was also normal, suggesting a normal rate of cholesterol synthesis. Serum levels of 7alpha-hydroxycholesterol were unaffected, suggesting a normal rate of bile acid formation in the pathway involving cholesterol 7alpha-hydroxylase. Biliary bile acid composition was slightly affected by CYP27 overexpression in female but not in male mice. Fecal levels of neutral steroids were slightly but significantly increased in overexpressor female mice but not in male mice. Levels of 24-hydroxycholesterol in the circulation were significantly reduced in the overexpressed mice, probably as a consequence of a recently described catabolic pathway involving CYP27. Combined with the results of our previous work on mice with a disruption of the CYP27 gene, the present results suggest that the levels of 27-hydroxycholesterol are not of critical importance for cholesterol homeostasis in mice.

Differential Effects of Sterol Regulatory Binding Proteins 1 and 2 on Sterol 12α-Hydroxylase: SREBP-2 SUPPRESSES THE STEROL 12α-HYDROXYLASE PROMOTER

The most important pathway for the catabolism and excretion of cholesterol in mammals is the formation of bile acids. Improper regulation of this pathway has implications for atherosclerosis, cholesterol gallstone formation, and some lipid storage diseases. Sterol 12 alpha-hydroxylase (12 alpha-hydroxylase) is required for cholic acid biosynthesis. The alpha(1)-fetoprotein transcription factor FTF is crucial for the expression and the bile acid-mediated down-regulation of 12 alpha-hydroxylase. Cholesterol, on the other hand, down-regulates expression of the 12 alpha-hydroxylase gene. In this study, we show that the two sterol regulatory binding proteins (SREBPs) have opposite effects on the 12 alpha-hydroxylase promoter. SREBP-1 activated the 12 alpha-hydroxylase promoter, as it does with many other cholesterol-regulated genes. In contrast, SREBP-2 suppressed 12 alpha-hydroxylase promoter activity. SREBP-1 mediates the cholesterol-down-regulation of 12 alpha-hydroxylase promoter by binding to two inverted sterol regulatory elements found approximately 300 nucleotides from the transcriptional initiation site. SREBP-2 mediated suppression of 12 alpha-hydroxylase without binding to its promoter. Data are presented suggesting that SREBP-2 suppresses the 12 alpha-hydroxylase promoter by interacting with FTF. This is the first report of a promoter responding oppositely to two members of the SREBP family of transcription factors. These studies provide a novel function and mode of action of a SREBP protein.

Dual Mechanisms for Repression of the Monomeric Orphan Receptor Liver Receptor Homologous Protein-1 by the Orphan Small Heterodimer Partner

The orphan nuclear hormone receptor liver receptor homologous protein-1 (LRH-1; NR5A2, also known as FTF), an unusual receptor that binds DNA as a monomer, is an essential regulator of expression of a rate-limiting enzyme in bile acid formation, cholesterol 7-alpha-hydroxylase. In a classic negative feedback loop that is a crucial component of the complex regulation of cholesterol metabolism, cholesterol 7-alpha-hydroxylase expression is decreased when bile acid levels are high. This repression is thought to be based on the bile acid-dependent induction of expression of the orphan receptor small heterodimer partner (SHP) NR0B2, which inhibits the activity of LRH-1. We have explored the molecular basis for this important regulatory effect by characterizing the mechanisms by which mouse and human SHP inhibit LRH-1-mediated transactivation. Both SHP proteins specifically interact with the AF-2 transactivation domain of LRH-1 both in vivo and in vitro. This domain is a common target for coactivator interaction, and the SHP proteins can compete with p160 coactivators for binding to LRH-1. In addition to the N-terminal receptor interaction domain, SHP includes a C-terminal domain with autonomous repression function. Neither a deletion nor a point mutation specifically affecting this domain blocked the ability to interact with LRH-1 to compete for coactivator binding or to repress LRH-1 transactivation. However, the relative ability of these mutants to inhibit LRH-1-mediated transactivation was markedly decreased. We conclude that the proposed central role of SHP in cholesterol metabolism is based on a two-step mechanism that is dependent on both coactivator competition and direct transcriptional repression.

Short-chain fatty acid formation at fermentation of indigestible carbohydrates

Short chain fatty acids (SCFAs; acetic, propionic and butyric acid) are formed during bacterial fermentation of carbohydrates in the colon. The interest in SCFA production is related to an increasing body of knowledge of the physiological effects of these acids. SCFAs are important anions in the colonic lumen and serve locally as nutrients for the mucosa cells, stimulating mucosal proliferation and blood flow. Especially butyric acid has been emphasized. It is the main energy substrate for the colonocytes and has been suggested to play a role in the prevention and treatment of diseases of the colonic mucosa, such as distal ulcerative colitis and cancer. SCFA production decreases the luminal pH, and may thereby stimulate mineral absorption and reduce secondary bile acid formation in the colon. Colonic generation of SCFAs has also been related to systematic and metabolic effects, e.g. SCFAs may influence the motility along the gastrointestinal tract and propionic acid has been suggested to inhibit the cholesterol synthesis from acetic acid in the liver. The SCFA formed at fermentation is quantitatively and qualitatively influenced by the type and amount of carbohydrate substrate. Further, certain combinations of carbohydrates may have synergistic effects on the SCFA pattern and may also shift the site of fermentation. This opens possibilities to design foods with tailored features regarding SCFA release in the human colon with potential health implications. There is a potential that in the future it will be possible to control SCFA production in the colon regarding pattern and place for release. Keywords: Carbohydrates, dietary fibre, fermentation, SCFA, resistant starch

Identification of unusual 7-oxygenated bile acid sulfates in a patient with Niemann-Pick disease, type C

Niemann-Pick disease, type C, was diagnosed in a 3-month-old boy with hepatosplenomegaly, mild signs of cholestasis, hepatic inflammation and extramedullary erythropoesis, together with chronic airway disease. He developed muscular hypotonia, psychomotor retardation, rickets, and signs of peripheral neuropathy. The patient was found to excrete abnormal amounts of unusual bile acids in urine at 3 and 5 months of age. These acids were shown to have a 3β-hydroxy-Δ5 structure and to carry an oxo or hydroxy group at C-7. They were sulfated at C-3 and nonamidated or conjugated with glycine or taurine at C-24. Part of the 7-hydroxy acids, presumably the 7β-hydroxylated one, was also conjugated with N-acetylhexosamine, probably N-acetylglucosamine, at the 7-hydroxy group. Possible metabolic pathways for the formation of the 7-oxo and 7β-hydroxycholenoic acids are discussed. Based on previous data concerning the effects of 3β-hydroxy-Δ5 bile acids on bile acid transport, it is suggested that the formation of such bile acids is responsible for the cholestasis in this patient.—Alvelius, G., O. Hjalmarson, W. J. Griffiths, I. Björkhem, and J. Sjövall. Identification of unusual 7-oxygenated bile acid sulfates in a patient with Niemann-Pick disease, type C. J. Lipid Res. 2001. 42: 1571-1577.

The role of taurine in diabetes and the development of diabetic complications.

The ubiquitously found beta-amino acid taurine has several physiological functions, e.g. in bile acid formation, as an osmolyte by cell volume regulation, in the heart, in the retina, in the formation of N-chlorotaurine by reaction with hypochlorous acid in leucocytes, and possibly for intracellular scavenging of carbonyl groups. Some animals, such as the cat and the C57BL/6 mouse, have disturbances in taurine homeostasis. The C57BL/6 mouse strain is widely used in diabetic and atherosclerotic animal models. In diabetes, the high extracellular levels of glucose disturb the cellular osmoregulation and sorbitol is formed intracellularly due to the intracellular polyol pathway, which is suspected to be one of the key processes in the development of diabetic late complications and associated cellular dysfunctions. Intracellular accumulation of sorbitol is most likely to cause depletion of other intracellular compounds including osmolytes such as myo-inositol and taurine. When considering the clinical complications in diabetes, several links can be established between altered taurine metabolism and the development of cellular dysfunctions in diabetes which cause the clinical complications observed in diabetes, e.g. retinopathy, neuropathy, nephropathy, cardiomyopathy, platelet aggregation, endothelial dysfunction and atherosclerosis. Possible therapeutic perspectives could be a supplementation with taurine and other osmolytes and low-molecular compounds, perhaps in a combinational therapy with aldose reductase inhibitors.