Toshinori Kamisako

Recent advances in bilirubin metabolism research: the molecular mechanism of hepatocyte bilirubin transport and its clinical relevance

Abstract: Bilirubin is taken up from blood into hepatocytes by sinosuidal membrane transporters and then excreted into bile through the bile canalicular membrane mainly as bilirubin glucuronides. (1) Mechanism of bilirubin uptake into hepatocytes: many organic anions are incorporated into hepatocytes by organic anion transporting polypeptides (rat, oatp 1, oatp2, oatp3; human, OATP), liver-specific transporter (rlst/HLST), and/or by organic anion transporters (OAT2, OAT3). Oatp1 and HLST transport bilirubin monoglucuronide. However, a transporter of unconjugated bilirubin in the sinusoidal membrane has not as yet been identified. Unconjugated bilirubin may also go across the hepatocyte sinusoidal membrane by a diffusion process. (2) Intrahepatic transport and conjugation of bilirubin: ligandin carries bilirubin to the endoplasmic reticulum (ER) of hepatocytes. In the ER, bilirubin is conjugated by bilirubin uridine diphosphate (UDP)-glycosyltransferase (bilirubin UGT; UGT1A1) to form mono- and diglucuronides of bilirubin. (3) Transport mechanism of bilirubin glucuronides across the hepatocyte canalicular membrane: at the canalicular membrane, bilirubin glucuronides are excreted into bile by multidrug resistance-associated protein 2 (MRP2), a member of the ATP-binding cassette transporter family. (4) Regurgitation of bilirubin glucuronides into blood: MRP3, which is located in the lateral membrane, transports bilirubin glucuronides into blood under conditions of impaired biliary bilirubin excretion.

Regulation of biliary cholesterol secretion is associated with abcg5 and abcg8 expressions in the rats: effects of diosgenin and ethinyl estradiol

In this study, we evaluate the effect of diosgenin, ethinyl estradiol and these co-administration on changes of lipoprotein metabolism and expression of hepatic genes those are important for cholesterol metabolism including the recently identified abcg5 and abcg8 in male Wistar rats. Rats were subjected to four experimental groups: (1) control group, (2) diosgenin group, which was fed the diet containing 1% diosgenin for 7 days, (3) ethinyl estradiol group, which received ethinyl estradiol in a dose of 5 mg/kg daily for 5 days, (4) diosgenin-ethinyl estradiol group, which received ethinyl estradiol treatment and was fed the diet containing 1% diosgenin. Diosgenin-feeding induced the hepatic abcg5/abcg8 expressions and biliary cholesterol secretion. Ethinyl estradiol administration reduced hepatic abcg5/abcg8 expressions and biliary cholesterol secretion. There was a positive correlation between hepatic expressions of abcg5/abcg8 and biliary cholesterol secretion. These findings strongly suggest that abcg5 and abcg8 are key proteins for biliary cholesterol excretion. Diosgenin-feeding did not affect the hepatic abcg5/abcg8 expressions and biliary cholesterol excretion in ethinyl estradiol-treated rat. Serum bile acid and bilirubin were higher and biliary bile acid and bilirubin secretions were lower in diosgenin-ethinyl estradiol group than those in ethinyl estradiol group. This finding suggests that diosgenin enhances the cholestatic effect of ethinyl estradiol in the rat. In conclusion, alteration of biliary cholesterol secretion is related to the expressions of hepatic abcg5 and abcg8 in diosgenin- or ethinyl estradiol-treated rat.

Analysis of bilirubin uridine 5'-diphosphate (UDP)-glucuronosyltransferase gene mutations in seven patients with Crigler-Najjar syndrome type II.

AbstractCrigler-Najjar syndrome (CN) type II is caused by a reduction in hepatic bilirubin uridine 5′-diphosphate (UDP)-glucuronosyltransferase activity. Recently, there has been progress in mutation analysis of patients with CN type II. Here, we analyzed both the coding and the promoter regions of the gene in seven Japanese patients with CN type II from five unrelated families. The mutations found in this study were classified into three types. The first type was composed of double homozygous missense mutations (Gly71Arg and Tyr486Asp) in exons 1 and 5. These mutations, which were detected in five patients from three unrelated families, were the commonest. The second type, which was detected in one patient, consisted of a single homozygous missense mutation (Arg209Trp) in exon 1. The third type, which was detected in one patient and was a new type of mutation combination, was composed of a homozygous insertion mutation of the TATAA element and a heterozygous missense mutation (Pro229Gln) in exon 1. Although the first and the second type of mutations are recessive, the third type appears to be dominant with incomplete penetrance, since the allele frequency of the insertion mutation of the TATAA element is very high (40%).

Increased expression of multidrug resistance-associated protein 1 (mrp1) in hepatocyte basolateral membrane and renal tubular epithelia after bile duct ligation in rats

Components of the multidrug resistance-associated protein (mrp) family mediate the adenosine triphosphate (ATP)-dependent transport of conjugated organic anions in the liver. Of these, mrp1 and mrp2 have been shown to have similar substrate specificity and nucleotide sequence. The intracellular localization and distribution of mrp1 under normal condition and cholestasis have not been as yet completely elucidated. To clarify this point, in the present study we evaluated the intracellular localization of mrp1 in rat liver and kidney after bile duct ligation (BDL). Bile duct was ligated in Wistar rats. Sequential staining of mrp1 by immunofluorescence was carried out in rat liver and kidneys 1, 3, and 5 days after bile duct ligation using confocal laser scanning microscopy. Weak granular staining of mrp1 was observed in cytoplasm of control rat hepatocytes. In addition to increased cytoplasm staining of mrp1, belt-and granule-like staining of mrp1 in basolateral membrane of hepatocytes was also shown after BDL. Furthermore, mrp1 immunofluorescence increased over time after BDL. No specific immunoflurescence of mrp1 was detected in control rat kidney. However, mrp1-positive staining was observed in epithelia of some renal tubules after BDL. This study showed that mrp1 immunofluorescence increased in hepatocyte basolateral membrane and cytoplasm and epithelia of some renal tubules after BDL. This increased mrp1 expression may be an adaptive response to impairment of hepato-biliary organic anion transport during obstructive cholestasis.

Phytosterol additives increase blood pressure and promote stroke onset in salt-loaded stroke-prone spontaneously hypertensive rats.

1. To assess the effect of dietary phytosterol on stroke and the lifespan of salt‐loaded stroke‐prone spontaneously hypertensive rats (SHRSP), we investigated the effects of the addition of phytosterol to soybean oil (phytosterol content: 0.3%) on stroke onset, lifespan following onset of stroke and overall lifespan compared with canola oil (phytosterol content: 0.9%).

Influenza A virus abrogates IFN-γ response in respiratory epithelial cells by disruption of the Jak/Stat pathway

The innate immunity to viral infections induces a potent antiviral response mediated by interferons (IFN). Although IFN‐γ is detected during the acute stages of illness in the upper respiratory tract secretions and in the serum of influenza A virus‐infected individuals, control of influenza A virus is not dependent upon IFN‐γ as evidenced by studies using anti‐IFN‐γ Ab and IFN‐γ–/– mice. Thus, we hypothesized that IFN‐γ is not critical in host survival because influenza A virus has mechanisms to evade the antiviral activity of IFN‐γ. To test this, A549 cells, an epithelial cell line derived from lung adenocarcinoma, were infected with influenza virus strain A/Aichi/2/68 (H3N2) (Aichi) and/or stimulated with IFN‐γ to detect IFN‐γ‐stimulated MHC class II expression. Influenza A virus infection inhibited IFN‐γ‐induced up‐regulation of HLA‐DRα mRNA and the IFN‐γ induction of class II transactivator (CIITA), an obligate mediator of MHC class II expression. Nuclear translocation of Stat1α upon IFN‐γ stimulation was significantly inhibited in influenza A virus‐infected cells and this was associated with a decrease in Tyr701 and Ser727 phosphorylation of Stat1α. Thus, influenza A virus subverts antiviral host defense mediated by IFN‐γ through effects on the intracellular signaling pathways.

BENEFICIAL EFFECT OF XANTHOANGELOL, A CHALCONE COMPOUND FROM ANGELICA KEISKEI, ON LIPID METABOLISM IN STROKE‐PRONE SPONTANEOUSLY HYPERTENSIVE RATS

1 Recently, we reported that 4‐hydroxyderricin, one of the major chalcones in Angelica keiskei extract (ethyl acetate extract from the yellow liquid of stems), exerted hypotensive and lipid regulatory actions in stroke‐prone spontaneously hypertensive rats (SHRSP). In the present study, we isolated xanthoangelol, another major chalcone in A. keiskei extract, and examined the effect of dietary xanthoangelol on blood pressure and lipid metabolism in SHRSP. 2 Six‐week‐old male SHRSP were fed diets containing 0.02% or 0.1% xanthoangelol (0.02 and 0.10 Xan, respectively) for 7 weeks, with free access to the diet and water. There were no significant changes in daily food intake, bodyweight or systolic blood pressure throughout the experimental period. Serum total cholesterol levels tended to decrease in the two experimental groups (albeit not significantly), which was due to a dose‐dependent decrease in the cholesterol content of the low‐density lipoprotein (LDL) fraction. These results suggest that dietary xanthoangelol decreases serum LDL levels. 3 In the liver, significant dose‐dependent decreases in relative liver liver weight and total triglyceride content were seen in the 0.02 and 0.10 Xan groups. In addition, a significant decrease in total cholesterol content was found in the 0.10 Xan group, which may be due to an elevation of faecal cholesterol excretion in addition to the decrease in liver weight. 4 Investigation of the hepatic mRNA expression of proteins involved in lipid metabolism indicated that there was a significant increase in peroxisome proliferator‐activated receptor (PPAR) α mRNA expression associated with the tendency for increases in acyl‐coenzyme A (CoA) synthetase and acyl‐CoA oxidase mRNA expression in the 0.10 Xan group, which may be responsible, at least in part, for the decrease in hepatic triglyceride content in the xanthoangelol‐treated rats. In additon, a significant increase in LDL receptor mRNA expression in the 0.10 Xan group may be responsible, at least in part, for the decrease in serum LDL levels in the xanthoangelol‐treated rats. 5 In conclusion, dietary xanthoangelol results in a reduction of serum LDL levels and decreases in total cholesterol and triglyceride contents in the liver of SHRSP. These beneficial effects are more effective following consumption of diet containing 0.10% xanthoangelol.

Dysregulated expression of fatty acid oxidation enzymes and iron‐regulatory genes in livers of Nrf2‐null mice

Background and Aim:  Hepatic excessive iron may play a role in the pathogenesis of non‐alcoholic steatohepatitis (NASH). Nrf2 is a master regulator of antioxidative responses. However, the role of Nrf2 in lipid and iron homeostasis remains unclear. Accordingly, it was examined how Nrf2 regulates lipid‐related and iron‐regulatory genes after feeding a high‐fat diet (HFD) with iron.

Alteration of the expression of adenosine triphosphate-binding cassette transporters associated with bile acid and cholesterol transport in the rat liver and intestine during cholestasis.

Background and Aim:  Multidrug resistance protein 2 (Mrp2), Mrp3, adenosine triphosphate‐binding cassette transporter g5 (Abcg5) and adenosine triphosphate‐binding cassette transporter g8 (Abcg8) have been identified as bile acid or cholesterol transporter in the enterocytes as well as hepatocytes. The purpose of the present study was to evaluate intestinal and hepatic adenosine triphosphate‐binding cassette transporter expressions during cholestasis.

Molecular aspects of organic compound transport across the plasma membrane of hepatocytes.

Many organic compounds are taken up from the blood by membrane transporters, taken across the sinosuidal membrane of hepatocytes and then excreted into bile via the bile canalicular membrane. The hepatic uptake of conjugated bile acids is mediated by the sodium taurocholate cotransporting polypeptide. Many organic anions and bulky organic cations are incorporated into hepatocytes by the organic anion transporting polypeptide, while small organic cations are transported by the organic cation transporter. At the canalicular membrane, organic compounds are excreted into bile by ATP‐binding cassette transporters which hydrolyse ATP to ADP. Excretion of monovalent bile acids is mediated by the canalicular bile salt transporter and that of organic anions, including divalent bile acid conjugates, are mediated by the multi‐drug resistance‐associated protein 2, also termed canalicular multi‐specific organic anion transporter. Organic cations are excreted into bile by the multi‐drug resistance gene product (MDR) 1 and phospholipids are excreted by MDR3 (mdr2 in mice and rats). The clinical syndromes associated with alterations of these transporters are also discussed.