Leucine stimulates HGF production by hepatic stellate cells through mTOR pathway

Branched chain amino acids modulate various cellular functions in addition to providing substrates for the production of proteins. We examined the mechanism underlying the stimulation by leucine of hepatocyte growth factor (HGF) production by hepatic stellate cells. Both p70 S6 kinase activity and phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) were up-regulated rapidly after leucine treatment of a rat hepatic stellate cell clone. No such activation was observed following treatment with valine or isoleucine. Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), suppressed leucine-induced activation of p70 S6 kinase and 4E-BP1 and negated the stimulatory effect of leucine on HGF production. An mTOR-dependent signaling pathway mediates the stimulatory effect of leucine on the production of HGF by hepatic stellate cells.     

Sphingosine 1-phosphate enhances portal pressure in isolated perfused liver via S1P2 with Rho activation

Although structural changes are most important to determine vascular resistance in portal hypertension, vasoactive mediators also contribute to its regulation. Hepatic stellate cells (HSCs) are assumed to play a role in modulating intrahepatic vascular resistance based on their residence in the space of Disse and capacity to contract. Because sphingosine 1-phosphate (S1P) has been shown to stimulate HSC contractility, we wondered if S1P could regulate portal pressure. S1P at 0.5-5 microM increased portal pressure in isolated rat perfused liver. This effect was abrogated in the presence of a binding antagonist for S1P2, JTE-013. Perfusion of isolated rat liver with 5 microM S1P increased Rho activity in the liver, and co-perfusion with JTE-013 cancelled S1P-induced Rho activation. Because S1P is present in human plasma at approximately 0.2 microM, S1P might readily regulate portal vascular tone in physiological and pathological status. The antagonist for S1P2 merits consideration for treatment of portal hypertension.     

Complete structure of the carbohydrate moiety of stem bromelain. An application of the almond glycopeptidase for structural studies of glycopeptides.

Asparagine-linked oligosaccharides of stem bromelain glycopeptides were quantitatively released by digestion with the almond glycopeptidase which cleaves beta-aspartylglycosylamine linkage in glycopeptides with oligopeptide moieties. The primary structures of the two oligosaccharide components, (Man)3(Xyl)1(Fuc)1(GlcNAc)2 and (Man)2-(Xyl)1(Fuc)1(GlcNAc)2 were elucidated as Man alpha 1 leads to 6Man alpha 1 leads to 6[Xyl beta 1 leads to 2]Man beta 1 leads to 4GlcNAc beta 1 leads 4[Fuc alpha 1 leads to 3]GlcNAc and Man alpha 1 leads to 6[Xyl beta 1 leads to 2]Man beta 1 leads to 4 GlcNAc beta 1 leads to 4[Fuc alpha 1 leads to 3] GlcNAc, respectively.     

Stimulation by glutamine and proline of HGF production in hepatic stellate cells

Amino acids regulate cellular functions in a variety of cell types. Most notably, leucine stimulates protein production through the mammalian target of rapamycin (mTOR)-dependent signaling pathway. We investigated the effect of amino acids on hepatocyte growth factor (HGF) production. Treatment with glutamine and proline, as well as leucine, increased HGF levels in the culture medium of a rat hepatic stellate cell clone in a dose-dependent manner. Up-regulation of phosphorylation of 70 kDa ribosomal protein S6 kinase and eukaryotic initiation factor 4E-binding protein 1 was not apparent in the cells after treatment with glutamine or proline. When rats received injections of glutamine or proline, hepatic and circulating HGF levels increased and peaked around 12 h after treatment. Glutamine and proline may have the potential to stimulate HGF production but the mechanism underlying this stimulation seems not to be through the mTOR-dependent signaling pathway.     

Plasma lysophosphatidic acid level and serum autotaxin activity are increased in liver injury in rats in relation to its severity

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological actions. We have reported that LPA stimulates hepatic stellate cell proliferation and inhibits DNA synthesis in hepatocytes, suggesting that LPA might play some role in the liver. We have found that plasma LPA level and serum autotaxin (ATX) activity were increased in patients with chronic hepatitis C. However, the clinical significance of LPA and its synthetic enzyme, autotaxin (ATX), is still unclear. To determine whether the increase of plasma LPA level and serum ATX activity might be found generally in liver injury, we examined the possible modulation of them in the blood in rats with various liver injuries. Plasma LPA level and serum ATX activity were increased in carbon tetrachloride-induced liver fibrosis correlatively with fibrosis grade, in dimethylnitrosamine-induced acute liver injury correlatively with serum alanine aminotransferase level or in 70% hepatectomy as early as 3 h after the operation. Plasma LPA level was correlated with serum ATX activity in rats with chronic and acute liver injury. ATX mRNA in the liver was not altered in carbon tetrachloride-induced liver fibrosis. Plasma LPA level and serum ATX activity are increased in various liver injuries in relation to their severity. Whether increased ATX and LPA in the blood in liver injury is simply a result or also a cause of the injury should be further clarified.     

Rho-kinase inhibitor prevents hepatocyte damage in acute liver injury induced by carbon tetrachloride in rats

A protective effect of Rho-kinase inhibitor on various organ injuries is gaining attention. Regarding liver injury, Rho-kinase inhibitor is reported to prevent carbon tetrachloride (CCl4)- or dimethylnitrosamine-induced liver fibrosis and hepatic ischemia-reperfusion injury in rats. Because Rho-kinase inhibitor not only improved liver fibrosis but also reduced serum alanine aminotransferase (ALT) level in CCl4-induced liver fibrosis, we wondered whether Rho-kinase inhibitor might exert a direct hepatocyte-protective effect. We examined this possibility in acute CCl4 intoxication in rats. Rho-kinase inhibitor, HA-1077, reduced serum alanine ALT level in rats with acute liver injury induced by CCl4 with the improvement of histological damage and the reduction of the number of apoptotic cells. In cultured rat hepatocytes in serum-free condition, HA-1077 reduced apoptosis evaluated by quantitative determination of cytoplasmic histone-associated DNA oligonucleosome fragments with the reduction of caspase-3 activity and the enhancement of Bcl-2 expression. HA-1077 stimulated phosphorylation of Akt, and wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway, abrogated the reduction of hepatocyte apoptosis by HA-1077 in vitro. Furthermore, wortmannin abrogated the reduction of serum ALT level by HA-1077 in rats with acute liver injury induced by CCl4, suggesting that the activation of PI3-kinase/Akt pathway may be involved in the hepatocyte-protective effect by Rho-kinase inhibitor in vivo. In conclusion, Rho-kinase inhibitor prevented hepatocyte damage in acute liver injury induced by CCl4 in rats and merits consideration as a hepatocyte-protective agent in liver injury, considering its direct antiapoptotic effect on hepatocytes in vitro.     

Comparative structural study of the N-linked oligosaccharides of human normal and pathological immunoglobulin G

The structures of oligosaccharides of normal and pathological immunoglobulin G (IgG) are reported. Asparagine-linked neutral oligosaccharides were released by N-oligosaccharide glycopeptidase (almond) digestion. The reducing ends of the oligosaccharide chains thus obtained were aminated with a fluorescent reagent, 2-aminopyridine, and the mixture of pyridylamino derivatives of the oligosaccharides was separated by reverse-phase high-performance liquid chromatography. It was possible to separate 15 out of the 16 kinds of oligosaccharides that have been suggested to exist in normal human IgG. High-resolution proton nuclear magnetic resonance spectroscopy was used along with chemical methods to determine the structures of the separated oligosaccharides. It has been shown that in normal IgG a biantennary complex-type oligosaccharide with a fucose residue (formula; see text) is predominant and four kinds of oligosaccharides, which are biantennary with bisecting N-acetylglucosamine and without fucose residues, exist only in a very small quantity. The results obtained for normal IgG were compared with those obtained for three myeloma IgG proteins. It has been found that the most abundant species that exist in the pathological proteins analyzed in the present work lack one or two galactose residues at the nonreducing terminal. We show that the fractions of fucose-containing oligosaccharides are markedly decreased in the heavy-chain disease protein Per. It is of particular interest that in this paraprotein the major component is a biantennary complex-type oligosaccharide that lacks a fucose residue and an oligosaccharide with the structure (Formula: see text) exists as one of the most abundant components.     

Structure and location of asparagine-linked oligosaccharides in the Fc region of a human immunoglobulin D

Seven kinds of asparagine-linked oligosaccharides were bound to the Fc region of a human immunoglobulin D(NIG-65). The oligosaccharides quantitatively released from four species of glycopeptides by digestion with almond glycopeptidase, were separated by Bio-Gel p-4 column chromatography and were purified further by thin-layer chromatography. The sugars were identified with GC-MS following the permethylation of respective oligosaccharide. To Asn-68(NIG-65 Fc numbering (1)), two kinds of high-mannose-type oligosaccharides were bonded. To Asn-159, a kind of hybride-type and two kinds of bisected complex-type oligosaccharides were attached. From Asn-210, four kinds of bisected complex-type oligosaccharides were isolated.