Hepatocyte nuclear factor-1alpha is an essential regulator of bile acid and plasma cholesterol metabolism.

Maturity-onset diabetes of the young type 3 (MODY3) is caused by haploinsufficiency of hepatocyte nuclear factor-1alpha (encoded by TCF1). Tcf1-/- mice have type 2 diabetes, dwarfism, renal Fanconi syndrome, hepatic dysfunction and hypercholestrolemia. Here we explore the molecular basis for the hypercholesterolemia using oligonucleotide microchip expression analysis. We demonstrate that Tcf1-/- mice have a defect in bile acid transport, increased bile acid and liver cholesterol synthesis, and impaired HDL metabolism. Tcf1-/- liver has decreased expression of the basolateral membrane bile acid transporters Slc10a1, Slc21a3 and Slc21a5, leading to impaired portal bile acid uptake and elevated plasma bile acid concentrations. In intestine and kidneys, Tcf1-/- mice lack expression of the ileal bile acid transporter (Slc10a2), resulting in increased fecal and urinary bile acid excretion. The Tcf1 protein (also known as HNF-1alpha) also regulates transcription of the gene (Nr1h4) encoding the farnesoid X receptor-1 (Fxr-1), thereby leading to reduced expression of small heterodimer partner-1 (Shp-1) and repression of Cyp7a1, the rate-limiting enzyme in the classic bile acid biosynthesis pathway. In addition, hepatocyte bile acid storage protein is absent from Tcf1-/- mice. Increased plasma cholesterol of Tcf1-/- mice resides predominantly in large, buoyant, high-density lipoprotein (HDL) particles. This is most likely due to reduced activity of the HDL-catabolic enzyme hepatic lipase (Lipc) and increased expression of HDL-cholesterol esterifying enzyme lecithin:cholesterol acyl transferase (Lcat). Our studies demonstrate that Tcf1, in addition to being an important regulator of insulin secretion, is an essential transcriptional regulator of bile acid and HDL-cholesterol metabolism.

Cloning and molecular characterization of the ontogeny of a rat ileal sodium-dependent bile acid transporter.

Sodium-dependent bile acid transport in the rat ileum is abruptly expressed at weaning. Degenerate oligonucleotides, based on amino acid sequence identities between the rat liver and hamster ileal transporters, were used to amplify a rat ileal probe. A 1.2-kb cDNA clone, which contains the full coding region (348 amino acids, 38 kD), was isolated by hybridization screening. In vitro translation yielded a 38-kD protein which glycosylated to 48 kD. Sodium-dependent uptake of taurocholate was observed in oocytes injected with cRNA. Northern blot analysis revealed a 5.0-kb mRNA in ileum, kidney, and cecum. A 48-kD protein was detected in ileal brush border membranes and localized to the apical border of villus ileal enterocytes. mRNA and protein expression, which were negligible before weaning, increased dramatically at weaning. Nuclear transcription rates for the transporter increased 15-fold between postnatal days 7 and 28. The apparent molecular weight of the transporter also increased between days 19 and 28. In summary, the developmental regulation of the rat ileal sodium-dependent bile acid cotransporter is characterized by transcriptionally regulated increases in mRNA and protein levels at the time of weaning with changes in apparent molecular weight of the protein after weaning.

Delta 4-3-oxosteroid 5 beta-reductase deficiency described in identical twins with neonatal hepatitis. A new inborn error in bile acid synthesis.

A new inborn error in bile acid synthesis, manifest in identical infant twins as severe intrahepatic cholestasis, is described involving the delta 4-3-oxosteroid 5 beta-reductase catalyzed conversion of the key intermediates, 7 alpha-hydroxy-4-cholesten-3-one and 7 alpha,12 alpha-dihydroxy-4-cholesten-3-one for chenodeoxycholic and cholic acid synthesis, to the respective 3 alpha-hydroxy-5 beta (H) products. This defect was detected by fast atom bombardment ionization-mass spectrometry from an elevated excretion and predominance of taurine conjugated unsaturated hydroxy-oxo-bile acids. Gas chromatography-mass spectrometry confirmed these to be 7 alpha-hydroxy-3-oxo-4-cholenoic and 7 alpha,12 alpha-dihydroxy-3-oxo-4-cholenoic acids (75-92% of total). Fasting serum bile acid concentrations were greater than 37 mumol/liter; chenodeoxycholic acid was the major bile acid, but significant amounts of allo(5 alpha-H)-bile acids (approximately 30%) were present. Biliary bile acid concentration was less than 2 mumol/liter and consisted of chenodeoxycholic, allo-chenodeoxycholic, and allo-cholic acids. These biochemical findings, which were identical in both infants, indicate a defect in bile acid synthesis involving the conversion of the delta 4-3-oxo-C27 intermediates into the corresponding 3 alpha-hydroxy-5 beta(H)-structures, a reaction that is catalyzed by a delta 4-3-oxosteroid-5 beta reductase enzyme. This defect resulted in markedly reduced primary bile acid synthesis and concomitant accumulation of delta 4-3-oxo-and allo-bile acids. These findings indicate a pathway in bile acid synthesis whereby side chain oxidation can occur despite incomplete alterations to the steroid nucleus, and lend support for an active delta 4-3-oxosteroid 5 alpha-reductase catalyzing the conversion of the delta 4-3-oxosteroid intermediates to the respective 3 alpha-hydroxy-5 alpha(H)-structures.

Regulation of the rat liver sodium-dependent bile acid cotransporter gene by prolactin. Mediation of transcriptional activation by Stat5.

The intracellular mechanism(s) underlying the upregulation of the hepatic Na+/taurocholate cotransporting polypeptide (ntcp) by prolactin (PRL) are unknown. In this report, we demonstrate a time-dependent increase in nuclear translocation of phosphorylated liver Stat5 (a member of the ignal ransducers and ctivators of ranscription family) that correlated with suckling-induced increases in serum PRL levels. In electrophoretic mobility gel shift assays, nuclear Stat5 exhibited specific DNA-binding ability towards IFN-gamma-activated sequence (GAS)-like elements (GLEs; 5'TTC/A-PyNPu-G/TAA-3') located in the -937 to -904 bp region of the ntcp promoter. Transient cotransfections in HepG2 cells revealed that PRL inducibility (2.5-3-fold) required coexpression of the long form of the PRL receptor (PRLRL) and Stat5. Deletion analysis mapped the PRLinducible region to -1237 to -758 bp of the ntcp promoter. Linking this 0.5-kb region to a heterologous thymidine kinase (tk) promoter, or linking multimerized ntcp GLEs either upstream of the ntcp minimal promoter (-158 to +47 bp) or the heterologous promoter conferred dose-dependent PRL responsiveness. The short form of the PRL receptor failed to transactivate ntcp GLEs. These results indicate that PRL acts via the PRLRL to facilitate Stat5 binding to ntcp-GLEs and to transcriptionally regulate ntcp.

Characterization of hepatic epidermal growth factor receptors in the developing rat.

Binding of 125I-labeled epidermal growth factor (EGF) was characterized in basolateral plasma membranes prepared from the livers of 21-day gestation fetuses, 14-day-old sucklings and adult Sprague-Dawley rats using a self-generating Percoll gradient method. The membrane preparations employed have been previously assayed in terms of plasma membrane protein yield, enrichment of various marker enzymes and sodium-dependent bile acid and amino acid transport. 125I-EGF binding was saturable and time and temperature dependent. Equilibrium analyses showed that the suckling period is characterized by a marked decrease in overall hepatic EGF binding capacity (460 +/- 50 fmol/mg protein) compared to either the fetal period (1290 +/- 160 fmol/mg) or to adults of either sex (males = 1540 +/- 230, females 1010 +/- 130 fmol/mg). Treatment of the suckling rat with parenteral EGF resulted in a 78% reduction in the observed binding capacity when assessed 2 h after growth factor administration. Comparison of binding affinities revealed no significant difference between the suckling and adult preparations (Kd = 0.40 +/- 0.03 vs. 0.39 +/- 0.02 nM, respectively); however, both preparations differed significantly from the fetal group which exhibited a decreased affinity of binding with a higher overall dissociation constant (Kd = 0.68 +/- 0.06 nM). Thus, it appears that major ontogenetic changes occur in the rat hepatic ligand/receptor system for epidermal growth factor. These changes are discussed in the context of transitional events in mammalian development such as birth and weaning.

Possible dissociation between insulin binding and insulin action in isolated fetal rat hepatocytes.

To directly examine the relationship between insulin receptors and insulin action in fetal tissue, we compared insulin receptor characteristics and insulin-mediated 14C-glucose incorporation into glycogen, as well as glycogen synthase activity, in freshly isolated hepatocytes from 21-day fetal (F) and adult (A) rats. Viability of hepatocytes was documented by trypan blue exclusion (greater than 90%), time-dependent 14C-leucine incorporation into protein, and dose-related incorporation of glucose into glycogen. Percent specific binding of 125I-insulin per unit protein was significantly higher in F than A liver plasma membranes (32.2 +/- 0.3 versus 18 +/- 2.4; P less than 0.01) and Scatchard plots revealed twice the number of receptors in F. Similarly, receptor number per cell surface area was threefold higher in F than in A (150 versus 50 sites/micron2). At a fixed medium glucose concentration of 11.2 mM, insulin stimulated 14C-glucose incorporation into glycogen in a dose-related manner in A with an apparent Km of 1.0 ng/ml and Vmax at 5-10 ng/ml corresponding to 30-40% of total receptor occupancy; no effect was obtained in F with insulin up to 100 ng/ml. Net glucose incorporation into glycogen (nmol/10(6) cells/h) increased progressively with increasing medium glucose concentrations ranging from 1.4 to 27.8 mM; incorporation by F was significantly greater than by A at each glucose concentration. However, whereas insulin at 100 ng/ml significantly augmented net glucose incorporation at each glucose concentration in A, no effect of insulin was apparent in F.(ABSTRACT TRUNCATED AT 250 WORDS)

Biliary tract disease in children.

This article discusses congenital and acquired disorders of the bile ducts and gallbladder in infants and children. Problems, such as extrahepatic biliary atresia, that are unique to infants are covered as well as distinctive aspects of hepatobiliary disease in older children. Biliary tract disease in the fetus and neonate presents an important challenge in that not only is hepatic structure and function disturbed but also the process of normal development may be retarded or altered by the disease process.

Hormonal regulation of hepatic organic anion transporting polypeptides.

Organic anion transporting polypeptides (Oatp) mediate the transport of a wide variety of amphipathic organic substrates. Rat Oatp1b2 and human OATP1B3 are members of a liver-specific subfamily of Oatps/OATPs. We investigated whether prolactin (PRL) and growth hormone (GH) regulated Oatp1b2 and OATP1B3 gene expression via signal transducers and activators of transcription 5 (Stat5). Binding sites for Stat5 transcription factors were located in the promoters of Oatp1b2 and OATP1B3 at -209 to -201 (5'-TTCTGGGAA-3') and -170 to -162 (5'-TTCTGAGAA-3'), respectively. In primary hepatocytes from female and male rats treated with PRL or GH, Oatp1b2 mRNA measured by real-time polymerase chain reaction was significantly induced 2-fold. HepG2 cells were transiently transfected with expression vectors containing Oatp1b2 or OATP1B3 promoter fragments, cDNAs for Stat5a, and the receptors for PRL (PRLR(L)) or GH (GHR), and treated with PRL or GH. PRL and GH induction of Oatp1b2 and OATP1B3 promoter activity required cotransfection of Stat5a and PRLR(L) or GHR. Mutation of the Stat5 binding site in both promoters eliminated hormonal induction. In DNA binding assays, HepG2 cells transfected with cDNAs for Stat5a and PRLR(L) were treated with PRL, and nuclear extracts were probed with a (32)P-labeled oligomer corresponding to -177 to -157 of the OATP1B3 promoter. PRL enhanced the binding of Stat5a to the OATP1B3 promoter and DNA-protein binding was inhibited in competition assays by excess OATP1B3 and Stat5 consensus oligomers but not by mutant Stat5 oligomers. These findings indicate that PRL and GH can regulate Oatp1b2 and OATP1B3 gene expression via the Stat5 signal-transduction pathway.

Fulminant hepatic failure in children.

Fulminant hepatic failure is a clinical syndrome resulting from massive necrosis or severe functional impairment of hepatocytes. This paper reviews the etiology, pathogenesis and management of this condition. Orthotopic liver transplantation may achieve very high survival rates (50-70%).

Extrahepatic biliary atresia.

Extrahepatic biliary atresia is a disease of unknown cause, leading to profound cholestasis and progressive biliary cirrhosis. This paper discusses the diagnosis and management of this condition. It is stressed that bile flow can be established in 80-90% of infants referred to surgery within 60 days after birth. Liver transplantation is essential for infants who are referred late (120 days of age or later); those whose initial portoenterostomy was not successful, and those who develop end-stage liver disease in spite of bile drain-age.

Uptake of taurocholate by hepatocytes isolated from developing rats.

To further define developmental changes in bile acid metabolism, we determined the kinetics of taurocholate uptake by hepatocytes isolated from Sprague-Dawley rats at 7, 14, 21, 28, and 56 days of age. There was a progressive increase in taurocholate uptake with age. The uptake process exhibited saturable kinetics in every age group with a maximum uptake velocity attained above a taurocholate concentration of 200 microM. There were no differences in Km values but Vmax increased progressively between 7 and 56 days of age. These data suggest that the deficit in hepatic excretory function observed in immature mammals of several species may, in part, be related to decreased transport of bile acids.

The intrahepatic cholangiopathies.

The intrahepatic biliary epithelial cells or cholangiocytes are the primary focus of injury in many congenital and acquired liver diseases of childhood. Although cholangiocytes account for only 3 to 5% of the liver cell population, injury and progressive loss of intrahepatic bile ducts can result in considerable morbidity and mortality. Table 1 provides an overview of the various disorders that affect the intrahepatic biliary tree. The more common disorders are discussed in detail below. One of the most important cholangiopathies, biliary atresia, characterized by complete destruction of the extrahepatic biliary tree, with variable involvement of the intrahepatic bile ducts, is discussed elsewhere in this series of articles. There has been considerable progress in our understanding of the embryology and physiology of the intrahepatic biliary system. These topics are also selectively reviewed, with an emphasis on advances that aid in the understanding of the pathophysiology of the disorders which affect the biliary tract in children.

Na(+)-H+ exchange in basolateral plasma membrane vesicles from neonatal rat liver.

Basolateral plasma membrane vesicles (BLMV) were used to determine the activity of the Na(+)-H+ exchanger in neonatal compared with adult rat liver. Imposition of an outwardly directed H+ gradient (pH 5.5in/7.5out) markedly stimulated the uptake of 1 mM 22Na+ by adult and neonatal BLMV above rates observed under pH-equilibrated conditions (pH 7.5in/7.5out) and resulted in a transient uphill accumulation ("overshoot") of Na+ to levels 1.5- to 2-fold higher than at equilibrium. 22Na+ uptake via Na(+)-H+ exchange (identified as the amiloride-sensitive component of total Na+ flux) was greater in BLMV from neonatal compared with adult liver under both pH-gradient and -equilibrated conditions. These age-related differences in Na+ uptake could not be explained by membrane potential effects or by differences in membrane permeability to H+. Kinetic analysis revealed a saturable process with a similar Km for Na+ (8.6 +/- 1.6 vs. 8.1 +/- 1.5 mM) but with a 2.5-fold higher Vmax (8.06 +/- 0.67 vs. 3.20 +/- 0.40 nmol.mg protein-1.min-1; P less than 0.01) in neonatal vs. adult vesicles. We conclude from these studies that basolateral membrane vesicles from neonatal rat liver exhibit enhanced Na(+)-H+ exchange as a result of an increased number or translocation rate of carriers in the plasma membrane.

Hepatic taurine transport: a Na+-dependent carrier on the basolateral plasma membrane.

Highly purified rat basolateral liver plasma membrane vesicles were used to examine the mechanism and the driving forces for hepatic uptake of the beta-amino acid, taurine. An inwardly directed 100 mM NaCl gradient stimulated the initial rate of taurine uptake and energized a transient twofold accumulation of taurine above equilibrium ("overshoot"). In contrast, uptake was slower and no overshoot was detected in the presence of a KCl gradient. A negative intravesicular electrical potential generated by the presence of permeant anions or an outwardly directed K+ gradient with valinomycin increased Na+-stimulated taurine uptake. External Cl- stimulated Na+-dependent taurine uptake independent of effects on the transmembrane electrical potential difference. Na+-dependent taurine uptake showed a sigmoidal dependence on extravesicular Na+ concentration, suggesting multiple Na+ ions are involved in the translocation of each taurine molecule. Na+-dependent taurine uptake demonstrated Michaelis-Menten kinetics with a maximum velocity of 0.537 nmol.mg protein-1.min-1 and an apparent Km of 174 microM. [3H]taurine uptake was inhibited by the presence of excess unlabeled taurine, beta-alanine, or hypotaurine but not by L-glutamine or L-alanine. In summary, using basolateral liver plasma membrane vesicles, we have shown that hepatic uptake of taurine occurs by a carrier-mediated, secondary active transport process specific for beta-amino acids. Uptake is electrogenic, stimulated by external Cl-, and requires multiple Na+ ions for the translocation of each taurine molecule.

Enhanced uptake of taurine by basolateral plasma membrane vesicles isolated from developing rat liver.

Inasmuch as taurine biosynthesis is decreased during early postnatal life, an efficient mechanism for taurine uptake by the liver must be present to maintain intracellular stores of this beta-amino acid. Therefore, basolateral liver plasma vesicles prepared from 14-day and adult rats were used to examine taurine transport during development. For both age groups, the presence of an inwardly directed Na+ gradient stimulated the initial rate of taurine uptake and caused a transient accumulation of taurine above equilibrium. For all time points before equilibrium, taurine uptake was significantly greater with membrane vesicles from 14-day compared to adult rat livers. In contrast, no age-related differences in Na+-independent uptake as measured with a K+ gradient were detected. With equal intravesicular and extravesicular Na+ concentrations, taurine uptake remained significantly greater in the younger age group. For both age groups, Na+-dependent taurine uptake was saturable but the apparent Km and Vmax for Na+-dependent taurine uptake were significantly greater in membrane vesicles from 14-day compared to adult rats. These findings suggest that an increased number of functional carriers for taurine are present in developing compared to adult basolateral plasma membrane perhaps reflecting the needs of the immature liver for this essential nutrient.

Determinants of bile formation during development: ontogeny of hepatic bile acid metabolism and transport.

The studies cited in this brief review stress that the development of hepatic transport processes is extraordinarily complex. Important changes in hepatic morphology and synthetic capacity are required before maturation of membrane carriers for bile acids. Transport systems at both poles of the hepatocyte develop independently. An increase in bile acid synthesis at several stages during the development appears to be an ontogenic event that is programmed to occur in concert with functional maturation of the enterohepatic circulation. Expression of specific membrane transporters for bile acids can be observed in fetal liver and postnatal ileum during periods of expansion of the bile acid pool. It is likely that specific defects, such as congenitally absent or defective bile acid transport proteins, will eventually be discovered in rare patients with undefined cholestatic syndromes. The absence of active ileal bile acid transport has recently been demonstrated in several children with congenital bile acid malabsorption. Whether bile acids can actually induce or regulate production of their own carriers during development has not been determined, but an increase in bile acid pool through feeding of exogenous bile acid has been shown to stimulate an increase in plasma membrane carriers for bile acids in adult rat liver. Thus, a number of factors, including available driving forces for transport, bile acid pool size and composition, effectiveness of intracellular compartmentation and transfer, and the function of membrane carriers, can all contribute to low rates of bile flow and bile acid secretion, depending on the stage of development.

Differential ontogenic regulation of basolateral and canalicular bile acid transport proteins in rat liver.

The hepatic transport systems mediating bile acid uptake and excretion undergo independent, stage-specific expression during development in the rat. In this study, the mechanisms underlying ontogenic regulation of both the Na(+)-dependent basolateral bile acid transporter and canalicular bile acid transporter/ecto-ATPase were examined. Steady state mRNA levels for the basolateral transporter were less than 20% of adult values prior to birth, increased to 35% on the first postnatal day, and reached adult levels by 1 week of age. This was paralleled by transcription rates, which were low prior to birth, reached 47% by day 1, and were maximal by 1 week of age. Steady state mRNA levels for ecto-ATPase were 12% of adult values prior to birth and showed a 2-fold increase by the first day of life. Thereafter, there was a gradual increase in mRNA for this transporter, with adult levels being reached at 4 weeks of age. Transcription rates paralleled this increment, although adult levels were reached earlier. Surprisingly, for both transporters, the full complement of protein was present well before adult levels of mRNA were reached. The basolateral protein was expressed at 82% of adult levels on the first day of life but was of lower apparent molecular mass (39 kDa), a difference that persisted until 4 weeks of age. N-Glycanase digestion suggested that this difference could be fully accounted for by N-linked glycosylation. The ecto-ATPase protein was present at 33% of adult levels prior to birth, 77% by 1 day, and 84% of adult levels by 1 week of age. Unlike the basolateral transporter, the apparent molecular weight of this protein did not change during development. In summary, the ontogeny of bile acid transporters on the plasma membrane of the hepatocyte is complex and appears to be regulated at transcriptional, translational, and post-translational levels.

Bile salt metabolism in the first year of life.

Bile salt metabolism was examined in 12 infants (ages 7 weeks to 10 months) who had recovered from protracted infantile diarrhea. Cholic acid kinetics were measured with the isotopic dilution technique. Cholate pool size was 938 +/- 89 mg/m2 (means +/- S.E.), synthetic rate was 478 +/- 52 mg/m2/day, and the fractional turnover rate was 0.537 +/- 0.060 day -1. Estimated chenodeoxycholate pool size was 607 +/- 103 mg/m2. similar cholic acid pool sizes were previously observed in older children; however, the synthetic and turnover rates were significantly lower, p less than 0.05 and 0.025, respectively. A significant inverse relationship between age and fractional turnover rate (r = -0.577, p less than 0.05) was observed in the first 10 months of life. Fasting serum cholylglycine measured by radioimmunoassay was significantly higher (p less than 0.01) in infants than in children. Peak postprandial concentrations were higher in infants than in older children. Accelerated hepatic bile salt synthesis rapidly increases the size of small pools at birth. Intraluminal bile salt concentrations concurrently increase, thereby normalizing fat solubilzation. Slow maturation of intestinal transport mechanisms may result in normal cycling of bile salts by 3 to 7 months of age. Persistent immaturity of hepatic uptake and/or excretion of bile salts leads to intrahepatic retention of a portion of the pool, with resultant regurgitation into the serum during the first months of life.