ABCB4 missense mutations D243A,K435T,G535D,I490T,R545C,and S978P significantly impair the lipid floppase and likely predispose to secondary pathologies in the human population

Bile salts are natural detergents required to solubilise dietary fat and lipid soluble vitamins. They are synthesised in hepatocytes and secreted into the luminal space of the biliary tree by the bile salt export pump (BSEP), an ATP-binding cassette (ABC) transporter in the canalicular membrane. BSEP deficiency causes cytotoxic accumulation of bile salts in the hepatocyte that results in mild-to-severe forms of cholestasis. The resulting inflammation can also progress to hepatocellular cancer via a novel mechanism involving upregulation of proliferative signalling pathways. A second ABC transporter of the canalicular membrane is also critical for bile formation. ABCB4 flops phosphatidylcholine into the outer leaflet of the membrane to be extracted by bile salts in the canalicular space. These mixed micelles reduce the detergent action of the bile salts and protect the biliary tree from their cytotoxic activity. ABCB4 deficiency also causes cholestasis, and might be expected to cause cholangitis and predispose to liver cancer. Non-synonymous SNPs in ABCB4 have now been described in patients with liver cancer or with inflammatory liver diseases that are known to predispose to cancer, but data showing that the SNPs are sufficiently deleterious to be an etiological factor are lacking. Here, we report the first characterisation at the protein level of six ABCB4 variants (D243A, K435T, G535D, I490T, R545C, and S978P) previously found in patients with inflammatory liver diseases or liver cancer. All significantly impair the transporter with a range of phenotypes exhibited, including low abundance, intracellular retention, and reduced floppase activity, suggesting that ABCB4 deficiency is the root cause of the pathology in these cases.     

Farnesoid X receptor alpha: a molecular link between bile acids and steroid signaling?

Bile acids are cholesterol metabolites that have been extensively studied in recent decades. In addition to having ancestral roles in digestion and fat solubilization, bile acids have recently been described as signaling molecules involved in many physiological functions, such as glucose and energy metabolisms. These signaling pathways involve the activation of the nuclear receptor farnesoid X receptor (FXRα) or of the G protein-coupled receptor TGR5. In this review, we will focus on the emerging role of FXRα, suggesting important functions for the receptor in steroid metabolism. It has been described that FXRα is expressed in the adrenal glands and testes, where it seems to control steroid production. FXRα also participates in steroid catabolism in the liver and interferes with the steroid signaling pathways in target tissues via crosstalk with steroid receptors. In this review, we discuss the potential impacts of bile acid (BA), through its interactions with steroid metabolism, on glucose metabolism, sexual function, and prostate and breast cancers. Although several of the published reports rely on in vitro studies, they highlight the need to understand the interactions that may affect health. This effect is important because BA levels are increased in several pathophysiological conditions related to liver injuries. Additionally, BA receptors are targeted clinically using therapeutics to treat liver diseases, diabetes, and cancers.     

Ascorbic acid and cholesterol: Effect of graded oral intakes on cholesterol conversion to bile acids in guinea-pigs

Summary A significant correlation between liver ascorbic acid (AA) and total bile acids or liver bile acids has been established in guinea-pigs by direct determination of the bile acids, confirming an earlier hypothesis. The oxidation of cholesterol to bile acids is dependent on the AA status, but it cannot be further stimulated by AA when the animals are already on an adequate intake of the vitamin. This suggests that AA has a hypocholesterolaemic effect over a limited range of AA status.     

Circadian rhythm of bile secretion in the rat.

In rats the bile flow and the estimated bile acid independant flow (BAIF) were significantly lower at 17.00 h than at 08.00 and 24.00 h. The decrease in BAIF paralleled the decrease in liver weight. Bile acid excretion was not different.     

Free amino acid composition of the intestinal contents,intestinal cells and hemolymph ofPhilosamia cynthia larvae

Summary Free amino acid composition of the intestinal contents, intestinal cells and hemolymph has been determined in larvae of the mothPhilosamia cynthia. From the hemolymph/lumen concentration ratio, an active transport could be inferred for neutral and basic amino acids. The values of cell/lumen and hemolymph/cell ratios suggested that the active step in the transport mechanism could be localized at the luminal pole of the enterocyte for neutral amino acids (except aromatic amino acids) and at the basolateral pole of the enterocyte for basic amino acids (except arginine).This work was supported by grants from Italian Consiglio Nazionale delle Ricerche and from Ministero della Pubblica Istruzione, Rome. The authors are indebted to Prof. V. Capraro for helpful discussion.     

Reversible impairment of hepatobiliary function induced by streptozotocin in the rat

Summary The effect of streptozotocin (SZ) on hepatobiliary function was studied in rats on the 1st, 7th and 15th days of treatment. Serum glucose increased significantly on the 1st day, and then remained high. Bile flow, bile acids output and BSP biliary excretion were significantly decreased on the 1st day of treatment, whereas serum sorbitol dehydrogenase was increased. All the parameters tested apart from serum glucose tended to normalize with time. The results suggested a transient toxic effect of SZ on the hepatocyte.Acknowledgment. This study was supported by a Research Grant from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), República Argentina. Joaquín V. Rodriguez is gratefully acknowledged for doing the chromatographic analyses of sulfobromophthalein, José M. Pellegrino for performing bile acids determination, and Mr. Raúl A. Trbojevich for surgical assistance.     

Studies on the in vitro binding of D-penicillamine to cholestyramine

Summary Adsorption of D-penicillamine to cholestyramine depends on the amount of the resin, the pH and the presence of other compounds such as bile salts. In the usual drug to resin ratio (150 mg D-penicillamine and 4–8 g cholestyramine per single dose) the percentage of D-penicillamine adsorbed to cholestyramine was about 10% of the applied dose; Bile salts (10 mmoles/1) inhibited this small adsorption by 87%.     

Lipid sensing and lipid sensors

The field of bile acids has witnessed an impulse in the last two decades. This has been the result of cloning the genes encoding enzymes of bile acid synthesis and their transporters. There is no doubt that the identification of Farnesoid X Receptor (FXR, NR1H4) as the bile acid receptor has contributed substantially to attract the interest of scientists in this area. When FXR was cloned by Forman et al. [1], farnesol metabolites were initially considered the physiological ligands. After identifying FXR and other nuclear receptors as bile acid sensors [2-4], it has become clear that bile acids are involved in the regulation of lipid and glucose metabolism and that these molecules are eclectic regulators of diverse cellular functions. In this review, we will summarize the current knowledge of the functions regulated by bile acids and how their physiological receptors mediate the signaling underlying numerous cellular responses.     

The effect of dietary lactose on the ileal absorption of sodium taurocholate in rats]

When Rats were fed a lactose containing diet, both the absorption rate of sodium taurocholate at the level of ileum and the contents of bile acids of the small intestine were increased. On the contrary, feeding of lactose did not modify the daily fecal excretion of bile acids. It therefore appears that dietary lactose increases the intestinal pool of bile acids by increasing their ileal absorption rate and that this effect of lactose is not subordinated to a modification of bile acid synthesis.     

Reduction of gallstone formation by ascorbic acid in hamsters

Summary The addition of 0.5% of ascorbic acid to the lithogenic diet of golden hamsters whose body pool was labelled with 26-¹⁴C-cholesterol, lowered the formation of gallstones, the cholesterol concentration and half-life in blood plasma and in the liver, and accelerated cholesterol transformation to bile acids.     

The functional role of sodium taurocholate cotransporting polypeptide NTCP in the life cycle of hepatitis B,C and D viruses

Chronic hepatitis B, C and D virus (HBV, HCV and HDV) infections are a major cause of liver disease and cancer worldwide. Despite employing distinct replication strategies, the three viruses are exclusively hepatotropic, and therefore depend on hepatocyte-specific host factors. The sodium taurocholate co-transporting polypeptide (NTCP), a transmembrane protein highly expressed in human hepatocytes that mediates the transport of bile acids, plays a key role in HBV and HDV entry into hepatocytes. Recently, NTCP has been shown to modulate HCV infection of hepatocytes by regulating innate antiviral immune responses in the liver. Here, we review the current knowledge of the functional role and the molecular and cellular biology of NTCP in the life cycle of the three major hepatotropic viruses, highlight the impact of NTCP as an antiviral target and discuss future avenues of research.     

Effect of propylthiouracil on intestinal tumor formation by azoxymethane in rats

Summary Treatment with propylthiouracil (PTU) resulted in a significant decrease in azoxymethane-induced intestinal tumors, total concentration of fecal bile acid as well as the fecal neutral steroids, cholesterol and coprostanol. Thus, a hypothyroid state induced by PTU treatment may affect intestinal carcinogenesis in this animal model by lowering the concentration of fecal bile acids and neutral steroids.We would like to acknowledge the excellent technical assistance of Dale Miller, Kenneth Kohn, Mathew McGee and Michael McLaughlin. We also wish to thank Dr M. S. Pak for the histological work, and Ms Mary Acree and Ms Kathleen Smith for the excellent secretarial assistance. Supported by the Maltida R. Wilson Fund.     

The role of innate immune-stimulated epithelial apoptosis during gastrointestinal inflammatory diseases

The maintenance of mucosal barrier equilibrium in the intestine requires a delicate and dynamic balance between enterocyte loss by apoptosis and the generation of new cells by proliferation from stem cell precursors at the base of the intestinal crypts. When the balance shifts towards either excessive or insufficient apoptosis, a broad range of gastrointestinal diseases can manifest. Recent work from a variety of laboratories has provided evidence in support of a role for receptors of the innate immune system, including Toll-like receptors 2, 4, and 9 as well as the intracellular pathogen recognition receptor NOD2/CARD15, in the initiation of enterocyte apoptosis. The subsequent induction of enterocyte apoptosis in response to the activation of these innate immune receptors plays a key role in the development of various intestinal diseases, including necrotizing enterocolitis, Crohn’s disease, ulcerative colitis, and intestinal cancer. This review will detail the regulatory pathways that govern enterocyte apoptosis, and will explore the role of the innate immune system in the induction of enterocyte apoptosis in gastrointestinal disease.     

Solubilization of unconjugated bilirubin by bile salts.

Freshly precipitated unconjugated bilirubin (UCB) is solubilized rapidly and to a large extent by the sodium salts of di- and trihydroxy bile acids. The solubilization effect depending on bile salt concentration, pH and ionic strength is based on micellar mechanisms.     

Solubilization of unconjugated bilirubin by bile salts

Summary Freshly precipitated uncojugated bilirubin (UCB) is solubilized rapidly and to a large extent by the sodium salts of di- and trihydroxy bile acids. The solubilization effect depending on bile salt concentration, pH and ionic strength is based on micellar mechanisms.     

The use of primary cultures of adult rat hepatocytes to study induction of enzymes and DNA synthesis: effect of nafenopin and electroporation

Primary cultures of adult rat hepatocytes maintained in a well-differentiated state, in a chemically defined medium containing 2% DMSO, have been utilized to study the effect of non-mutagenic hepatocarcinogens such as the peroxisome proliferator nafenopin. The parameters chosen in this in vitro system were those that paralleled the major in vivo effects of nafenopin on the liver, mainly: the proliferation of the endoplasmic reticulum and induction of cytochrome P-452, the proliferation of the peroxisome compartment and the induction of cyanide-insensitive beta-oxidation of fatty acids and the stimulation of liver growth as measured by the DNA synthetic activity of the hepatocytes. In this review, we also describe the morphology of hepatocyte cultures prepared from previously electroporated hepatocytes and the potential for the use of electroporation to introduce growth related genes into hepatocyte cells to study the mechanisms of hepatocyte growth at the molecular level. In addition we describe the formation of endoplasmic reticulum whorls in these cultures as a consequence of nafenopin treatment. 'Whorl formation' by hepatotrophic chemicals has been previously shown to occur in vivo; in this report, it is described for the first time in vitro.     

Bimodal processing of olfactory information in an amphibian nose: odor responses segregate into a medial and a lateral stream

In contrast to the single sensory surface present in teleost fishes, several spatially segregated subsystems with distinct molecular and functional characteristics define the mammalian olfactory system. However, the evolutionary steps of that transition remain unknown. Here we analyzed the olfactory system of an early diverging tetrapod, the amphibian Xenopus laevis, and report for the first time the existence of two odor-processing streams, sharply segregated in the main olfactory bulb and partially segregated in the olfactory epithelium of pre-metamorphic larvae. A lateral odor-processing stream is formed by microvillous receptor neurons and is characterized by amino acid responses and Gα_o/Gα_i as probable signal transducers, whereas a medial stream formed by ciliated receptor neurons is characterized by responses to alcohols, aldehydes, and ketones, and Gα_olf/cAMP as probable signal transducers. To reveal candidates for the olfactory receptors underlying these two streams, the spatial distribution of 12 genes from four olfactory receptor gene families was determined. Several class II and some class I odorant receptors (ORs) mimic the spatial distribution observed for the medial stream, whereas a trace amine-associated receptor closely parallels the spatial pattern of the lateral odor-processing stream. Other olfactory receptors (some class I odorant receptors and vomeronasal type 1 receptors) and odor responses (to bile acids, amines) were not lateralized, the latter not even in the olfactory bulb, suggesting an incomplete segregation. Thus, the olfactory system of X. laevis exhibits an intermediate stage of segregation and as such appears well suited to investigate the molecular driving forces behind olfactory regionalization.     

Reversible impairment of hepatobiliary function induced by streptozotocin in the rat

The effect of streptozotocin (SZ) on hepatobiliary function was studied in rats on the 1st, 7th and 15th days of treatment. Serum glucose increased significantly on the 1st day, and then remained high. Bile flow, bile acids output and BSP biliary excretion were significantly decreased on the 1st day of treatment, whereas serum sorbitol dehydrogenase was increased. All the parameters tested apart from serum glucose tended to normalize with time. The results suggested a transient toxic effect of SZ on the hepatocyte.     

The amino acid composition of rat bile

Summary The pattern of amino acids in the bile of rats differs from the pattern in the serum of these animals, since bile contains significantly greater amounts of acidic and sulphur-containing amino acids and glycine than serum, while the serum contained more basic amino acids than bile, indicating that secretion of amino acids into bile may involve specific transport processes.Acknowledgments. We thank Mr M. Earlam of Pharmaceuticals Division, Imperial Chemical Industries Limited for the aminoacid analyses, and Dr J. S. Morley for helpful discussion. URF gratefully acknowledges a grant (FO 73/2) from the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, Fed. Rep. of Germany. KGW is recipient of a grant from the Scottish Hospital Research Endowments Trust.