Loss of Enzymes in the Bile Acid Synthesis Pathway Explains Differences in Bile Composition among Mammals.

Bile acids are important for absorbing nutrients. Most mammals produce cholic and chenodeoxycholic bile acids. Here, we investigated genes in the bile acid synthesis pathway in four mammals that deviate from the usual mammalian bile composition. First, we show that naked-mole rats, elephants, and manatees repeatedly inactivated CYP8B1, an enzyme uniquely required for cholic acid synthesis, which explains the absence of cholic acid in these species. Second, no gene-inactivating mutations were found in any pathway gene in the rhinoceros, a species that lacks bile acids, indicating an evolutionarily recent change in its bile composition. Third, elephants and/or manatees that also lack bile acids altogether have lost additional nonessential enzymes (SLC27A5, ACOX2). Apart from uncovering genomic differences explaining deviations in bile composition, our analysis of bile acid enzymes in bile acid-lacking species suggests that essentiality prevents gene loss, while loss of pleiotropic genes is permitted if their other functions are compensated by functionally related proteins.

Genetic testing for familial hypercholesterolemia among survivors of acute coronary syndrome.

BACKGROUND: Familial hypercholesterolemia could be prevalent among patients with acute coronary syndrome. OBJECTIVE: To investigate both the frequency of causative mutations for familial hypercholesterolemia (FH) and the optimal selection of patients for genetic testing among patients with an acute coronary syndrome (ACS). METHODS: One hundred and sixteen patients with an ACS during 2009-2015 were identified through the SWEDEHEART registry. Patients who had either a high total cholesterol level ≥7 mmol L-1 combined with a triglyceride level ≤2.6 mmol L-1 , or were treated with lipid-lowering medication and had a total cholesterol level >4.9 mmol L-1 and a triglyceride level ≤2.6 mmol L-1 were included. Genetic testing was performed first with a regionally designed FH mutation panel (118 mutations), followed by testing with a commercially available FH genetic analysis (Progenika Biopharma). RESULTS: A total of 6.9% (8/116) patients had a FH-causative mutation, all in the LDL-receptor. Five patients were detected on the panel, and further testing of the remaining 111 patients detected an additional 3 FH-causative mutations. Baseline characteristics were similar in FH-positive and FH-negative patients with respect to age, gender, prior ACS and diabetes. Patients with a FH-causative mutation had higher Dutch Lipid Clinical Network (DLCN) score (5.5 (5.0-6.5) vs 3.0 (2.0-5.0), P < 0.001) and a higher low-density lipoprotein level (5.7 (4.7-6.5) vs 4.9 (3.5-5.4), P = 0.030). The Dutch Lipid Clinical Network (DLCN) score had a good discrimination with an area under the curve of 0.856 (95% CI 0.763-0.949). CONCLUSION: Genetic testing for FH should be considered in patients with ACS and high DLCN score.

Impaired Hepatic Uptake by Organic Anion-Transporting Polypeptides Is Associated with Hyperbilirubinemia and Hypercholanemia in Atp11c Mutant Mice.

Biliary excretion of organic anions, such as bile acids (BAs), is the main osmotic driving force for bile formation, and its impairment induces intrahepatic cholestasis. We investigated the involvement of Atp11c in the hepatic transport of organic anions using Atp11c mutant mice, which exhibit hypercholanemia and hyperbilirubinemia. Pharmacokinetic analysis following a constant intravenous infusion in Atp11c mutant mice showed decreased hepatic sinusoidal uptake and intact biliary secretion of [(3)H]17ß estradiol 17ß-d-glucuronide. Consistent with this result, compared with cells and membranes from control mice, isolated hepatocytes, and liver plasma membranes from Atp11c mutant mice had a much lower uptake of [(3)H]17ß estradiol 17ß-d-glucuronide and expression of organic anion-transporting polypeptides, which are transporters responsible for hepatic uptake of unconjugated BAs and organic anions, including bilirubin glucuronides. Uptake of [(3)H]TC into hepatocytes and expression of Na(+)-taurocholate cotransporting polypeptide in liver plasma membranes, which mediates hepatic uptake of conjugated BAs, was also lower in the Atp11c mutant mice. Bile flow rate, biliary BA concentration, and expression of hepatobiliary transporters did not differ between Atp11c mutant mice and control mice. These results suggest that Atp11c mediates the transport of BAs and organic anions across the sinusoidal membrane, but not the canalicular membrane, by regulating the abundance of transporters. Atp11c is a candidate gene for genetically undiagnosed cases of hypercholanemia and hyperbilirubinemia, but not of intrahepatic cholestasis. This gene may influence the pharmacological and adverse effect of drugs because organic anion-transporting polypeptides regulate their systemic exposure.

ATP8B1 and ATP11C: Two Lipid Flippases Important for Hepatocyte Function.

P4 ATPases are lipid flippases and transport phospholipids from the exoplasmic to the cytosolic leaflet of biological membranes. Lipid flipping is important for the biogenesis of transport vesicles. Recently it was shown that loss of the P4 ATPases ATP8B1 and ATP11C are associated with severe Cholestatic liver disease. Mutation of ATP8B1 cause progressive familial Intrahepatic Cholestasis type 1 (PFIC1)and benign recurrent intrahepatic cholestasis type 1 (BRIC 1). From our observations we hypothesized that ATP8B1 deficiency causes a phospholipids randomization at the canalicular membrane, which results in extraction of cholesterol due to increase sensitivity of the canalicular membrane. Deficiency of ATP11C causes conjugated hyperbilirubinemia. In our preliminary result we observed accumulation of unconjugated bile salts in Atp11c deficient mice probably because of regulation in the expression or function of OATP1B2. Similar to ATP8B1, ATP11C have regulation on membrane transporters.

Sodium taurocholate cotransporting polypeptide (SLC10A1) deficiency: conjugated hypercholanemia without a clear clinical phenotype.

UNLABELLED: The enterohepatic circulation of bile salts is an important physiological route to recycle bile salts and ensure intestinal absorption of dietary lipids. The Na(+)-taurocholate cotransporting polypeptide SLC10A1 (NTCP) plays a key role in this process as the major transporter of conjugated bile salts from the plasma compartment into the hepatocyte. Here we present the first patient with NTCP deficiency, who was clinically characterized by mild hypotonia, growth retardation, and delayed motor milestones. Total bile salts in plasma were extremely elevated (up to 1,500 µM, ref. <16.3) but there were no clinical signs of cholestatic jaundice, pruritis, or liver dysfunction. Bile salt synthesis and intestinal bile salt signaling were not affected, as evidenced by normal plasma 7α-hydroxy-4-cholesten-3-one (C4) and FGF19 levels. Importantly, the presence of secondary bile salts in the circulation suggested residual enterohepatic cycling of bile salts. Sequencing of the SLC10A1 gene revealed a single homozygous nonsynonymous point mutation in the coding sequence of the gene, resulting in an arginine to histidine substitution at position 252. Functional studies showed that this mutation resulted in a markedly reduced uptake activity of taurocholic acid. Immunofluorescence studies and surface biotinylation experiments demonstrated that the mutant protein is virtually absent from the plasma membrane. CONCLUSION: We describe the identification of NTCP deficiency as a new inborn error of metabolism with a relatively mild clinical phenotype. The identification of NTCP deficiency confirms that this transporter is the main import system for conjugated bile salts into the liver but also indicates that auxiliary transporters are able to sustain the enterohepatic cycle in its absence.

Common and rare single nucleotide polymorphisms in the LDLR gene are present in a black South African population and associate with low-density lipoprotein cholesterol levels.

The LDL receptor has an essential role in regulating plasma LDL-C levels. Genetic variation in the LDLR gene can be associated with either lower or moderately raised plasma levels of LDL-C, or may cause familial hypercholesterolemia. The prevalence of single-nucleotide polymorphisms (SNPs) in the LDLR in the black South African population is not known and therefore, we aimed to determine the genotypic variation of the LDLR in the study population as well as to define the association of the different genotypes with plasma LDL-C levels. A random selection of 1860 apparently healthy black South African volunteers aged 35-60 years was made in a cross-sectional study. Novel SNPs were identified in a subset of 30 individuals by means of automated sequencing before screening the entire cohort by means of the Illumina VeraCode GoldenGate Genotyping Assay on a BeadXpress Reader system. Twenty-five SNPs were genotyped, two of which were novel. A very rare SNP, rs17249141, in the promoter region was significantly associated with lower levels of LDL-C. Four other SNPs (rs2738447, rs14158, rs2738465 and rs3180023) were significantly associated with increased levels of LDL-C. We can conclude that some of the various SNPs identified do indeed associate with LDL-C levels.

The emerging role of acyl-CoA thioesterases and acyltransferases in regulating peroxisomal lipid metabolism.

The importance of peroxisomes in lipid metabolism is now well established and peroxisomes contain approximately 60 enzymes involved in these lipid metabolic pathways. Several acyl-CoA thioesterase enzymes (ACOTs) have been identified in peroxisomes that catalyze the hydrolysis of acyl-CoAs (short-, medium-, long- and very long-chain), bile acid-CoAs, and methyl branched-CoAs, to the free fatty acid and coenzyme A. A number of acyltransferase enzymes, which are structurally and functionally related to ACOTs, have also been identified in peroxisomes, which conjugate (or amidate) bile acid-CoAs and acyl-CoAs to amino acids, resulting in the production of amidated bile acids and fatty acids. The function of ACOTs is to act as auxiliary enzymes in the α- and ß-oxidation of various lipids in peroxisomes. Human peroxisomes contain at least two ACOTs (ACOT4 and ACOT8) whereas mouse peroxisomes contain six ACOTs (ACOT3, 4, 5, 6, 8 and 12). Similarly, human peroxisomes contain one bile acid-CoA:amino acid N-acyltransferase (BAAT), whereas mouse peroxisomes contain three acyltransferases (BAAT and acyl-CoA:amino acid N-acyltransferases 1 and 2: ACNAT1 and ACNAT2). This review will focus on the human and mouse peroxisomal ACOT and acyltransferase enzymes identified to date and discuss their cellular localizations, emerging structural information and functions as auxiliary enzymes in peroxisomal metabolic pathways.

Exopolysaccharide sugars contribute to biofilm formation by Salmonella enterica serovar typhimurium on HEp-2 cells and chicken intestinal epithelium.

Recently, we demonstrated that Salmonella enterica serovar Typhimurium can form biofilm on HEp-2 cells in a type 1 fimbria-dependent manner. Previous work on Salmonella exopolysaccharide (EPS) in biofilm indicated that the EPS composition can vary based upon the substratum on which the bacterial biofilm forms. We have investigated the role of genes important in the production of colanic acid and cellulose, common components of EPS. A mutation in the colanic acid biosynthetic gene, wcaM, was introduced into S. enterica serovar Typhimurium strain BJ2710 and was found to disrupt biofilm formation on HEp-2 cells and chicken intestinal tissue, although biofilm formation on a plastic surface was unaffected. Complementation of the wcaM mutant with the functional gene restored the biofilm phenotype observed in the parent strain. A mutation in the putative cellulose biosynthetic gene, yhjN, was found to disrupt biofilm formation on HEp-2 cells and chicken intestinal epithelium, as well as on a plastic surface. Our data indicate that Salmonella attachment to, and growth on, eukaryotic cells represent complex interactions that are facilitated by species of EPS.