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Leucine-rich α-2 glycoprotein (LRG), one of the acute phase proteins mainly produced by the liver, similar to C-reactive protein, has been recognized as an inflammatory biomarker for rheumatoid arthritis and inflammatory bowel diseases. We recently demonstrated that LRG was also increased in the sera of psoriasis patients and correlated well with disease activity with a sensitivity and specificity much higher than C-reactive protein; however, whether LRG mechanistically contributed to the pathogenesis of psoriasis remained unclear. In this study, we explored the role of LRG in psoriasiform inflammation using LRG-knockout (KO) mice in an imiquimod (IMQ)-mediated model. Following topical treatment with IMQ, serum levels of LRG and its expression in the liver were abruptly elevated. Similarly, an acute surge of proinflammatory cytokines was observed in the liver, including IL-1β, TNF-α, and IL-6, although LRG-KO mice showed delayed responses. LRG-KO mice showed less skin inflammation in the IMQ model than wild-type mice. K5.Stat3C mice developed psoriasis-like lesions following tape stripping, which also abruptly induced LRG expression in the liver. A deficiency of Lrg mitigated tape stripping-induced lesions, similar to the IMQ model. These results indicate that LRG modulates both feed-forward and feedback loops of cytokines in the skin-liver axis involved with psoriasiform inflammation., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Probe electrospray ionization (PESI) is a recently developed ionization technique that enables the direct detection of endogenous compounds like metabolites without sample preparation. In this study, we have demonstrated the first combination use of PESI with triple quadrupole tandem mass spectrometry (MS/MS), which was then applied to intact endogenous metabolite analysis of mice liver, achieving detection of 26 metabolites including amino acids, organic acids, and sugars. To investigate its practicality, metabolic profiles of control and CCl4-induced acute hepatic injury mouse model were measured by the developed method. Results showed clear separation of the two groups in score plots of principal component analysis and identified taurine as the primary contributor to group separation. The results were further validated by the established gas chromatography/MS/MS method, demonstrating the present method's usefulness. In addition, we preliminarily applied the method to real-time analysis of an intact liver of a living mouse. We successfully achieved monitoring of the real-time changes of two tricarboxylic acid cycle intermediates, α-ketoglutaric acid and fumaric acid, in the liver immediately after pyruvic acid injection via a cannulated tube to the portal vein. The present method achieved an intact analysis of metabolites in liver without sample preparation, and it also demonstrates future possibility to establish in vivo real-time metabolome analysis of living animals by PESI/MS/MS.

The oxidative, reductive, and hydrolytic metabolism of methiocarb and the hydrolytic metabolism of carbaryl by liver microsomes and plasma of rats or humans were examined. The effects of the metabolism of methiocarb and carbaryl on their nuclear receptor activities were also examined. When methiocarb was incubated with rat liver microsomes in the presence of NADPH, methiocarb sulfoxide, and a novel metabolite, methiocarb sulfone were detected. Methiocarb sulfoxide was oxidized to the sulfone by liver microsomes and reduced back to methiocarb by liver cytosol. Thus, the interconversion between methiocarb and the sulfoxide was found to be a new metabolic pathway for methiocarb by liver microsomes. The product of methiocarb hydrolysis, which is methylthio-3,5-xylenol (MX), was also oxidized to sulfoxide form by rat liver microsomes. The oxidations were catalyzed by human flavin-containing monooxygenase isoform (FMO1). CYP2C19, which is a human cytochrome P450 (CYP) isoform, catalyzed the sulfoxidations of methiocarb and MX, while CYP1A2 also exhibited oxidase activity toward MX. Methiocarb and carbaryl were not enzymatically hydrolyzed by the liver microsomes, but they were mainly hydrolyzed by plasma and albumin to MX and 1-naphthol, respectively. Both methiocarb and carbaryl exhibited PXR and PPARα agonistic activities; however, methiocarb sulfoxide and sulfone showed markedly reduced activities. In fact, when methiocarb was incubated with liver microsomes, the receptor activities were decreased. In contrast, MX and 1-naphthol showed nuclear receptor activities equivalent to those of their parent carbamates. Thus, the hydrolysis of methiocarb and carbaryl and the oxidation of methiocarb markedly modified their nuclear receptor activities.

The induction of the gene encoding heme oxygenase 1 (Hmox1, HO-1) by Nrf2 is unique compared with other Nrf2 targets. We previously showed that the Nrf2a-mediated induction of zebrafish hmox1a was liver specific and transient. We screened transcription factors that could repress the induction of hmox1a but not other Nrf2a targets and concluded that Bach1b was a prime candidate. In bach1b-knocked-down larvae, the induction of hmox1a was observed ectopically in nonliver tissues and persisted longer than normal fish, suggesting that Bach1 is the only regulator for both the liver-specific and transient induction of hmox1a. Co-knockdown of bach1b with its co-ortholog bach1a enhanced these effects. To determine why Bach1 could not repress the hmox1a induction in the liver, we analyzed the effects of a heme biosynthesis inhibitor, succinylacetone, and a heme precursor, hemin. Succinylacetone decreased the Nrf2a-mediated hmox1a induction, whereas pre-treatment with hemin caused ectopic induction of hmox1a in nonliver tissues, implying that the high heme levels in the liver may release the repressive activity of Bach1. Our results suggested that Bach1 regulates the liver specificity and transience of the Nrf2a-dependent induction of hmox1a and that heme mediates this regulation through Bach1 inhibition based on its level in each tissue., (© 2015 The Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.)

Development and homeostasis of organs and whole body is critically dependent on the circulatory system. In particular, the circulatory system, the railways shuttling oxygen and nutrients among various organs, is indispensible for inter-organ humoral communication. Since the modern view of the anatomy and mechanics of the circulatory system was established in 17(th) century, it has been assumed that humoral factors are carried to and from organs via vascular branches of the central arteries and veins running along the body axis. Over the past few decades, major advances have been made in understanding molecular and cellular mechanisms underlying the vascularization of organs. However, very little is known about how each organ is linked by vasculature (i.e., inter-organ vascular networks). In fact, the exact anatomy of inter-organ vascular networks has remained obscure. Herein, we report the identification of four distinct vessels, V1(LP), V2(LP), V3(LP) and V4(LP), that bridge between two organs, liver and pancreas in developing zebrafish. We found that these inter-organ vessels can be classified into two types: direct and indirect types. The direct type vessels are those that bridge between two organs via single distinct vessel, to which V1(LP) and V2(LP) vessels belong. The indirect type bridges between two organs via separate branches that emanate from a stem vessel, and V3(LP) and V4(LP) vessels belong to this type. Our finding of V1(LP), V2(LP), V3(LP) and V4(LP) vessels provides the proof of the existence of inter-organ vascular networks. These and other yet-to-be-discovered inter-organ vascular networks may facilitate the direct exchange of humoral factors that are necessary for the coordinated growth, differentiation and homeostasis of the connected organs. It is also possible that the inter-organ vessels serve as tracks for their connected organs to follow during their growth to establish their relative positions and size differences.

Somatic mutation of ten-eleven translocation 2 (TET2) gene is frequently found in human myeloid malignancies. Recent reports showed that loss of Tet2 led to pleiotropic hematopoietic abnormalities including increased competitive repopulating capacity of bone marrow (BM) HSCs and myeloid transformation. However, precise impact of Tet2 loss on the function of fetal liver (FL) HSCs has not been examined. Here we show that disruption of Tet2 results in the expansion of Lin(-)Sca-1(+)c-Kit(+) (LSK) cells in FL. Furthermore, Tet2 loss led to enhanced self-renewal and long-term repopulating capacity of FL-HSCs in in vivo serial transplantation assay. Disruption of Tet2 in FL also led to altered differentiation of mature blood cells, expansion of common myeloid progenitors and increased resistance for hematopoietic progenitor cells (HPCs) to differentiation stimuli in vitro. These results demonstrate that Tet2 plays a critical role in homeostasis of HSCs and HPCs not only in the BM, but also in FL.

Phosphoinositide 3-kinase (PI3K) p85alpha-deficient mice exhibit hypoglycemia as a result of increased insulin sensitivity and glucose uptake in peripheral tissues. Although PI3K is central to the metabolic actions of insulin, its mechanism of action in liver is not well understood. In the present study, we investigated hepatic insulin signaling and glucose homeostasis in p85alpha-deficient and wild-type mice. In the livers of p85alpha-deficient mice, p50alpha played a compensatory role in insulin-stimulated PI3K activation by binding to insulin receptor substrate (IRS)-1/2. In p85alpha-deficient mice, the ratio of p50alpha over p110 catalytic subunit of PI3K in the liver was higher than in the muscles. PI3K activity associated with IRS-1/2 was not affected by the lack of p85alpha in the liver. Insulin-stimulated Akt and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) activities in the liver were similar in p85alpha-deficient and wild-type mice. A hyperinsulinemic-euglycemic clamp study revealed that the glucose infusion rate and the rate of disappearance were higher in p85alpha-deficient mice than in wild-type mice but that endogenous glucose production tended to be higher in p85alpha-deficient mice than in wild-type mice. Consistent with this finding, the expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase in livers after fasting was higher in p85alpha-deficient mice than in wild-type mice. After mice were fasted, the intrahepatic glucose-6-phosphate level was almost completely depleted in p85alpha-deficient mice. The glycogen content fell to nearly zero as a result of glycogenolysis shortly after the initiation of fasting in p85alpha-deficient mice. The absence of an increase in insulin-stimulated PI3K activation in the liver of p85alpha-deficient mice, unlike the muscles, may be associated with the molecular balance between the regulatory subunit and the catalytic subunit of PI3K. Gluconeogenesis was rather elevated in p85alpha-deficient mice, compared with in wild-type mice, and the liver seemed to partially compensate for the increase in glucose uptake in peripheral tissues.

An enormous body of information has been obtained by molecular and cellular biology in the last half century. However, even these powerful approaches are not adequate when it comes to higher-level biological structures, such as tissues, organs, and individual organisms, because of the complexities involved. Thus, accumulation of data at the higher levels supports and broadens the context for that obtained on the molecular and cellular levels. Under such auspices, an attempt to elucidate mesoscopic and macroscopic subjects based on plentiful nanoscopic and microscopic data is of great potential value. On the other hand, fully realistic simulation is impracticable because of the extensive cost entailed and enormous amount of data required. Abstraction and modeling that balance the dual requirements of prediction accuracy and manageable calculation cost are of great importance for systems biology. We have constructed an ammonia metabolism model of the hepatic lobule, a histological component of the liver, based on a single-hepatocyte model that consists of the biochemical kinetics of enzymes and transporters. To bring the calculation cost within reason, the porto-central axis, which is an elemental structure of the lobule, is defined as the systems biological unit of the liver, and is accordingly modeled. A model including both histological structure and position-specific gene expression of major enzymes largely represents the physiological dynamics of the hepatic lobule in nature. In addition, heterogeneous gene expression is suggested to have evolved to optimize the energy efficiency of ammonia detoxification at the macroscopic level, implying that approaches like this may elucidate how properties at the molecular and cellular levels, such as regulated gene expression, modify higher-level phenomena of multicellular tissue, organs, and organisms.

Hepatic ischemia-reperfusion injury remains a significant problem for liver surgery, including transplantation, and apoptosis has been implicated in this type of hepatic injury. Here we found that through the Fas/Fas ligand interaction apoptosis is involved in the late phase of hepatic ischemia-reperfusion injury. The appearance of apoptotic hepatocytes increases significantly after reperfusion, reaching a maximum 12 h after reperfusion. The transcription levels of Fas and Fas ligand are increased after reperfusion. Fas is expressed on hepatocytes, while Fas ligand is expressed on infiltrating immune cells. A close spatial and temporal association of Fas expression and apoptotic cells is demonstrated in the histological observation. These results suggest that infiltrating cells induce apoptosis of hepatocytes through the Fas/Fas ligand interaction, leading to hepatocyte injury. Furthermore, an injection of anti-Fas antibody or neutralizing anti-Fas ligand antibody results in a dramatic decrease in the occurrence of hepatocyte apoptosis and hepatic infiltration of macrophages and natural killer cells as well as liver injury. Our results suggest that blockage of the Fas/Fas ligand interaction is a promising strategy for suppression of hepatic ischemia-reperfusion injury.

Background: Macrophage migration inhibitory factor (MIF) is involved in inflammatory and immune-mediated diseases but the role of MIF in liver injury has not yet been elucidated., Methods: We investigated biochemically, histologically and immunologically the character of MIF in concanavalin A (Con A)-induced T-cell-mediated liver injury using MIF knockout (KO) mice and wild-type (WT) mice., Results: MIF KO mice showed significantly decreased serum alanine aminotransferase values and suppressed histological change with massive necrosis of the hepatic parenchymal cells and infiltration of inflammatory cells compared with their WT counterparts. This protection was not mediated by either tumor necrosis factor-alpha or interferon-gamma, which are critical mediators of Con A-induced liver injury, as their serum concentrations were shown to be similar in MIF KO and WT mice. On the other hand, a flow cytometric analysis demonstrated that the number of activated hepatic leukocytes decreased more in the MIF KO mice than in the WT mice., Conclusions: A lack of MIF protected the mice from Con A-induced liver injury. Controlling the MIF activity may be a useful therapeutic strategy for treating such T-cell activation-associated liver diseases as autoimmune hepatitis and viral hepatitis.

D-mannose is an essential monosaccharide constituent of glycoproteins and glycolipids. However, it is unknown how plasma mannose is supplied. The aim of this study was to explore the source of plasma mannose. Oral administration of glucose resulted in a significant decrease of plasma mannose concentration after 20 min in fasted normal rats. However, in fasted type 2 diabetes model rats, plasma mannose concentrations that were higher compared with normal rats did not change after the administration of glucose. When insulin was administered intravenously to fed rats, it took longer for plasma mannose concentrations to decrease significantly in diabetic rats than in normal rats (20 and 5 min, respectively). Intravenous administration of epinephrine to fed normal rats increased the plasma mannose concentration, but this effect was negated by fasting or by administration of a glycogen phosphorylase inhibitor. Epinephrine increased mannose output from the perfused liver of fed rats, but this effect was negated in the presence of a glucose-6-phosphatase inhibitor. Epinephrine also increased the hepatic levels of hexose 6-phosphates, including mannose 6-phosphate. When either lactate alone or lactate plus alanine were administered as gluconeogenic substrates to fasted rats, the concentration of plasma mannose did not increase. When lactate was used to perfuse the liver of fasted rats, a decrease, rather than an increase, in mannose output was observed. These findings indicate that hepatic glycogen is a source of plasma mannose.

Regucalcin is a novel calcium ion (Ca(2+)) binding protein that does not contain an EF-hand motif as a Ca(2+)-binding domain and has been demonstrated to play a multi-functional role in many cell types. Human liver regucalcin, consisting of 299 amino-acid residues, was overexpressed in Escherichia coli, purified and crystallized by the vapour-diffusion method in the presence of polyethylene glycol 4000 as a precipitant. A native crystal diffracted to 2.8 A with synchrotron radiation and belongs to space group P2(1), with unit-cell parameters a = 64.87, b = 52.52, c = 86.38 A, beta = 99.86 degrees . Two molecules most probably exist in the asymmetric unit, corresponding to V(M) = 2.2 A(3) Da(-1). Heavy-atom derivative data were collected and the Pb derivative showed one high-occupancy site per molecule.

We report a 19-year-old woman who had a history of type 1 diabetes with recurrent glycogen accumulation in the liver. During her infantile period she presented with no hepatomegaly nor growth retardation. On admission she was diagnosed with diabetic ketoacidosis (DKA). She also had hepatomegaly and elevated transaminase levels, but these abnormalities had resolved after administration of insulin. However, 4 weeks after DKA marked hepatomegaly and elevated transaminases were reappeared with simultaneous hypoglycemia which suggested an impaired glycogenolysis in the extraordinary conditions. We supposed the partial deficiency of liver glycogen phosphorylase activity in this patient and analyzed the liver glycogen phosphorylase gene (PYGL). Deduced amino acid sequence of the PYGL in this patient was completely identical to that reported by Burwinkel et al. (Y15233), however, the nucleotide sequence of PYGL cDNA was heterozygous for substitutions at positions Asp339 (GAT to GAC) on exon 9 and Ala703 (GCT to GCC on exon 17, respectively. These SNPs were also screened in 51 Japanese normal subjects by PCR-based direct sequencing or PCR-RFLP method. The same genotype observed in this patient was detected in 2 of 51(3.9%) normal subjects. These results suggest that the structure of PYGL coding sequence in this patient is unlikely to account for her excessive liver glycogen accumulation. Further studies including genetic analysis on the promoter region of the gene are necessary to clarify the etiology of susceptibility to excessive liver glycogen storage in patients with type 1 diabetes.

It is important to clarify the process by which hypovascular nodules progress to hypervascular hepatocellular carcinoma (HCC). In this study, we used dynamic computed tomography(CT) to retrospectively investigate serial changes in 24 hypovascular nodules of 24 patients until they progressed to hypervascular HCC. The mean time to hypervascular transformation(THT) was 726 days, with a minimum of 208 days and maximum of 2,728 days. Mean nodular diameter at the final examination was 21.2 +/- 8.0 mm, and was significantly larger than that(11.4 +/- 4.8 mm) at the initial time. THT was the shortest(463 days) in a group that had an initial nodular diameter larger than 15 mm. In a group with the low-iso-low contrast enhancement pattern, THT was 361 days, and was shorter than that in the other groups. In a group with the low-low-low contrast enhancement pattern, nodules containing fat were frequently detected(5 patients, 75%). In patients with HCC apart from the nodules, THT for the nodules was 464 days, shorter than that(767.5 days) in other patients. The findings suggest that hypovascular nodules larger than 15 mm in diameter with the low-iso-low contrast enhancement pattern and HCC progress to hypervascular HCC at an early stage.

Human liver glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purified and crystallized using PEG 3350 as a precipitant. However, the crystals were extremely fragile towards osmotic shock. A 1% change in PEG 3350 content causes destruction of the crystals. After many trials for freezing the crystals, X-ray diffraction data from a native crystal were collected at 2.8 A resolution using as a cryoprotectant a mixture consisting of paraffin oil and Paratone-N in a 3:1 ratio and a cryoloop covered with Formver film. Crystals belong to space group P2(1), with unit-cell parameters a = 63.23, b = 97.84, c = 84.23 A, beta = 104.1 degrees. Molecular replacement with a starting model consisting of a homology model based on the low-resolution structure of human skeletal muscle GAPDH, which has 90% identical residues with the liver protein, led to a solution. Most of the current model was assigned properly in the electron-density map, but the map corresponding to some important regions containing the phosphate-binding loop was ambiguous. It is planned to crystallize human liver GAPDH in the presence of phosphate ions and/or some kind of inhibitor in order to fix the flexible region.

The hepatitis B virus (HBV) gene has been detected in hepatocellular carcinoma (HCC) tissue negative for the hepatitis B surface antigen and positive for the hepatitis C virus (HCV) antibody, but the precise role of the HBV gene in hepatocarcinogenesis has yet to be clarified. We studied the HBV gene in liver tissue several years before the emergence of HCC. Eleven patients diagnosed with HCV-positive chronic liver disease and who developed HCC were assigned to group A. HBV DNA was detected in 8 of the 11 patients (73%). Twenty-five patients, who did not develop HCC, were selected as group B. Six of the group B patients were classified as DNA-positive (24%). The HBV DNA in liver tissue was found to be significantly related to HCC development (P < 0.01). Thus, the presence of the HBV gene in patients with chronic HCV associated-liver injury appears to promote hepatocarcinogenesis, although prospective studies are needed to confirm this result.

The Fas/Fas ligand interaction plays a crucial role in various liver diseases, and administration of agonistic anti-Fas antibody to mice causes massive hepatic apoptosis and fulminant hepatic failure. Several growth factors have recently been found to function in preventing apoptosis. In this study, we demonstrated that overexpression of transforming growth factor alpha (TGFalpha) has a dramatic protective effect on Fas-mediated hepatic apoptosis at the biochemical and histological levels. Moreover, 85.7% (six out of seven) of TGFalpha transgenic mice survived the lethal liver damage, whereas all wild-type mice died. Expression of Bcl-xL, an anti-apoptotic protein, was greatly increased in the transgenic mice. Taken together, our findings suggest that TGFalpha protects against Fas-mediated liver apoptosis in vivo and up-regulation of Bcl-xL may participate in protective effect of TGFalpha.

SOCS3 (CIS3/JAB2) is an SH2-containing protein that binds to the activation loop of Janus kinases, inhibiting kinase activity, and thereby suppressing cytokine signaling. During embryonic development, SOCS3 is highly expressed in erythroid lineage cells and is Epo independent. Transgene-mediated expression blocks fetal erythropoiesis, resulting in embryonic lethality. SOCS3 deletion results in an embryonic lethality at 12-16 days associated with marked erythrocytosis. Moreover, the in vitro proliferative capacity of progenitors is greatly increased. SOCS3-deficient fetal liver stem cells can reconstitute hematopoiesis in lethally irradiated adults, indicating that its absence does not disturb bone marrow erythropoiesis. Reconstitution of lymphoid lineages in JAK3-deficient mice also occurs normally. The results demonstrate that SOCS3 is critical in negatively regulating fetal liver hematopoiesis.

To investigate the effect of pivalate on carnitine import and carnitine synthesis in the liver, we measured carnitine uptake in isolated rat hepatocytes with L-[(14)C] carnitine and concentrations of free carnitine, gamma-butyrobetaine and acylcarnitines using tandem mass spectrometry. Hepatocytes from rats treated with 20 mmol/L of pivalate for 4 wk had greater L-[(14)C] carnitine uptake than those of unsupplemented rats after 5, 10, 30 and 90 min. Addition of 1 mmol/L of pivalate or 1 mmol/L of pivaloylcarnitine to control cell suspensions did not affect L-[(14)C] carnitine uptake. The K(m) values for L-[(14)C] carnitine uptake for pivalate-treated rats were significantly lower than control (2.9 +/- 0.7 mmol/L for pivalate-treated rats, 6.2 +/- 1.1 mmol/L for controls). The concentration of free carnitine was not reduced in the liver of pivalate-treated rats, whereas the concentrations of acetylcarnitine and gamma-butyrobetaine were significantly lower than controls. In the heart and muscle the concentration of free carnitine was significantly lower and that of gamma-butyrobetaine was higher than controls. These results suggest that carnitine transport from plasma into the liver and synthesis in the liver are accelerated in rats with secondary carnitine deficiency induced by the administration of pivalate.

A mouse liver homogenate was shown to contain enzymatic activities catalyzing the sulfation of 3,4-dihydroxyphenylalanine (Dopa) and tyrosine isomers with a pH optimum of 8.25. Western blot analysis revealed a 34 kDa protein exhibiting immunologic cross-reactivity to antiserum against rat liver SULT1B1 sulfotransferase. By employing the reverse transcriptase-polymerase chain reaction (RT-PCR) technique, a 910-base pair product encoding the putative mouse liver SULT1B1 sulfotransferase was obtained. Using this PCR product as a probe, a cDNA containing the entire open reading frame of the mouse liver SULT1B1 sulfotransferase was cloned from a mouse liver Lambda ZAP cDNA library. The nucleotide sequence indicated it is a new enzyme. The deduced amino acid sequence exhibited 87.6, 72.3, 55.9, 54.2, 52.8, 51.1, and 49.4% identity to the amino acid sequences of the rat liver SULT1B1 sulfotransferase, human thyroid hormone sulfotransferase, mouse phenol sulfotransferase, rat liver phenol sulfotransferase, rat liver hydroxyarylamine sulfotransferase, mouse estrogen sulfotransferase, and rat estrogen sulfotransferase. Upon transfection of COS-7 cells with an expression vector (pcDNA3) harboring the cDNA encoding this new enzyme, a 34 kDa protein exhibiting immunologic cross-reactivity to antiserum against the rat liver SULT1B1 sulfotransferase was expressed. The recombinant sulfotransferase exhibited enzymatic activities toward Dopa and tyrosine isomers, as well as dopamine and 3,3',5-triiodo-L-thyronine. Northern blot analyses indicated the SULT1B1 sulfotransferase was predominantly expressed in liver, but not in the other ten mouse organs examined. Furthermore, the enzyme was found to be expressed in a developmental stage-dependent manner, being at a very low level in liver samples from 1-day-old mice and then gradually increasing to the maximum level in liver samples from 4-week-old mice.

In patients receiving interferon therapy for chronic hepatitis C, serum hepatitis C virus (HCV) RNA often reverts from an undetectable to a detectable form after completion of treatment. Detection of the negative strand of HCV-RNA in liver tissue is regarded as an index of viral proliferation. Therefore, we investigated changes in the hepatic negative-strand HCV-RNA following interferon therapy to determine whether this parameter could predict the long-term response to treatment. The subjects of this study were 27 patients with chronic active hepatitis C. Serum positive-strand and hepatic tissue negative-strand HCV-RNA were detected using polymerase chain reaction. At the completion of interferon treatment, serum HCV-RNA was not detected in 21 patients. One year following treatment it remained undetectable in 14 of these patients but it had reverted to a detectable form in seven. The 14 patients in whom hepatic negative-strand RNA was not detected between 2 weeks and 12 months after treatment, had not relapsed after another year. In the 13 remaining patients, negative-strand RNA was found in liver tissue and serum RNA either reverted to a detectable form or remained detectable throughout. From these findings, we conclude that the detection of negative-strand HCV-RNA in liver tissue 2 weeks after the completion of interferon therapy is useful for predicting the long-term effect of therapy.

The anti-oxidative effect of bilirubin was investigated in an ischemia-reperfusion model of rat liver. The rat portal vessel and liver artery were ligated with a vascular clip, and after reperfusion the urine was collected at intervals. The amount of biopyrrins, the oxidative metabolites of bilirubin, in rat urine reached a maximum 4 hours after reperfusion. Biotripyrrin-a and -b which are biopyrrins isolated from human urine were included in urinary bilirubin oxidative metabolites of rats after reperfusion. The hepatic mRNA level of heme oxygenase-1 (HO-1), the rate limiting enzyme of bilirubin biosynthesis, reached a maximum after 4 hours. Furthermore, the hepatic activity of HO began to rise 4 hours after treatment and remained high until 24 hours posttreatment. These findings suggest that bilirubin acts as a physiological antioxidant in vivo in ischemia-reperfusion and that bilirubin biosynthesis is evoked by oxidative stress.

The role of taurocholate, one of the major conjugated bile acids present in portal blood, in excretion of bilirubin from liver parenchyma to biliary and vascular compartments was studied in isolated perfused rat liver. Contents of bilirubin and carbon monoxide (CO) in the bile or venous effluents were determined by enzyme-linked immunosorbent microassay with the use of antibilirubin monoclonal antibody 24G7 and myoglobin-assisted spectrophotometry, respectively. In the presence of taurocholate, bilirubin excreted into the biliary compartment constituted greater than 90% of the total bilirubin excreted from the liver (0.26 nmol.min-1.g liver-1), corresponding to 60% of the outflow of CO into the venous effluents. In its absence, however, the total amount of bilirubin excreted into extrahepatic compartments was reduced to 27% of CO flux, and more than 90% of the excreted bilirubin was in the venous effluent. Thus a choleretic bile acid such as taurocholate is necessary for directional transport of bilirubin into bile in the perfused liver preparation.

To investigate the carnitine deficiency induced by pivalate, rats had free access to drinking water with or without pivalate. Consumption of 20 mmol/L pivalate for 1 wk decreased the levels of both free and total carnitine in plasma to approximately 20% of levels before treatment. After 4 wk, the concentrations of free carnitine in the liver, heart and muscle of pivalate-treated rats were approximately 60-80% of the control, and in the kidney, 26% of the control. Fractional excretion of free carnitine (FEFC) in pivalate-treated rats was measured; however, the treatment for 3 or 8 d did not affect the values relative to those obtained before treatment. Treatment with pivalate for 4 wk did not affect plasma concentrations of glucose, ammonia and free fatty acids (FFA) in the rats; however, the concentration of 3-hydroxybutyrate (3-OHB) was higher, and the FFA/3-OHB ratio was lower than those of controls. In a liver perfusion study, ketogenesis from oleate and gluconeogenesis from lactate and pyruvate in rats treated with pivalate for 4 wk were not different from controls. These results suggest that administration of pivalate did not induce the excessive excretion of free carnitine in urine, and secondary carnitine deficiency induced by intake of 20 mmol/L pivalate for 4 wk did not cause severe metabolic changes in rat liver.

We found that simple serum-free medium containing sodium selenite (Se) is effective for long-term maintenance of functional hepatocytes cultured on a pepsin-digested collagen gel (DC-gel). The concentration of Se was important for maintenance of hepatocytes, and its optimal concentration was 10(-7) approximately 10(-6)M. The effect of Se was specific, as other metals did not have the same effect. The effect was equal to that of fetal bovine serum for maintenance of functional hepatocytes. Using this medium, we could obtained a high level of hepatocellular function including the production of albumin and transferrin, and activity of p450 throughout a long-term culture. Matrigel was almost equal to DC-gel for albumin secretion, but less effective for transferrin secretion, and P450 activity in long-term cultures. The growth of cells on DC-gel or matrigel was not observed, and cell morphology of a round-shaped form was similar on both substrata. These results indicate that serum-free medium containing Se in a DC-gel culture system provides a simple method for long-term culture of hepatocytes.

A quantitative study of the effect of carnitine deficiency on expression of glycolytic and gluconeogenic enzymes was performed using juvenile visceral steatosis mice which are systemically deficient in carnitine. The amounts of glucokinase and L-type pyruvate kinase mRNA were reduced in homozygotes, compared to heterozygotes and normal controls at 2 and 8 weeks. Liver-type phosphofructokinase, however, did not differ significantly. The abundance of fructose 1,6-bisphosphatase mRNA was unchanged at 2 and 8 weeks. The level of phosphoenolpyruvate carboxykinase mRNA was increased slightly at 2 weeks, but not at 8 weeks. A part of these changes could not be explained by the plasma glucose or insulin level. Carnitine administration restored the mRNA of these enzymes to normal levels. These results suggest that carnitine deficiency affects the expression of these liver enzymes.

HepG2 human hepatoma cells, labelled with [35S]sulphate in media containing L-3,4-dihydroxyphenylalanine (L-dopa), (D-dopa), DL-m-tyrosine or D-p-tyrosine, were found to produce the [35S]sulphated forms of these compounds. Addition to the labelling media of m-hydroxybenzylhydrazine, an aromatic amino acid decarboxylase inhibitor, greatly enhanced the production of L-dopa O-[35S]sulphate and DL-m-tyrosine O-[35S]sulphate, with a concomitant decrease in the formation of dopamine O-[35S]sulphate and m-tyramine O-[35S]sulphate. With 3'-phosphoadenosine 5'-phospho[35S]sulphate as the sulphate donor., HepG2-cell cytosol was shown to contain enzymic activity catalysing the sulphation of L-dopa, D-dopa, L-m-tyrosine, D-m-tyrosine, L-p-tyrosine and D-p-tyrosine. The pH optimum of the enzyme, designated dopa/tyrosine sulphotransferase, was determined to be 8.75 with D-m-tyrosine as the substrate. The enzyme exhibited stereoselectivity for the D-form of dopa or tyrosine isomers. Addition of 10mM MnCl2 to the reaction mixture resulted in a remarkable stimulation of dopa/tyrosine sulphotransferase activity, being as high as 267.8 times with D-p-tyrosine as the substrate. Quantitative assays revealed L-dopa, D-dopa and D-m-tyrosine to be better substrates than L-p-tyrosine. When the HepG2-cell cytosol was subjected to DEAE Bio-Gel and hydroxyapatite column chromatography, dopa/tyrosine sulphotransferase was co-eluted with the thermolabile 'M-form' phenol sulphotransferase. Furthermore dopa/tyrosine sulphotransferase displayed properties similar to that of the M-form phenol sulphotransferase with respect to thermostability and sensitivity to 2,6-dichloro-4-nitrophenol. Whether the M-form phenol sulphotransferase is truly (solely) responsible for the dopa/tyrosine sulphotransferase activity present in HepG2 cells remains to be clarified.

We conducted a quantitative study of the effect of carnitine deficiency on the mRNA level of carnitine palmitoyltransferase II in the liver, muscle and heart of mice with juvenile visceral steatosis, a strain that is systematically deficient in carnitine. The amount of carnitine palmitoyltransferase II mRNA was increased in liver and muscle of homozygotes, as compared with heterozygotes and normal controls, at 2, 4, and 8 wk of age. The mRNA levels of this enzyme were normalized after carnitine administration. The mRNA level of carnitine palmitoyltransferase II in the heart was increased only at 8 wk, and was not affected by carnitine administration. These results suggest that carnitine displays some effect on the mRNA level of the carnitine palmitoyltransferase II gene in liver and muscle, probably through fatty acid metabolic change.

Expression of key regulatory enzymes involved in glucose metabolism was studied in the livers of Otsuka Long-Evans Tokushima fatty (OLETF) rats, a model of non-insulin dependent diabetes mellitus. The activity and mRNA levels of glucokinase and L-type pyruvate kinase was increased in the liver of OLETF rats compared with control rats. There was no such remarkable change in liver-type phosphofructokinase. The activities of glucose-6-phosphatase and fructose-1,6-biphosphatase also increase despite high plasma levels of glucose and insulin. The activity of phosphoenolpyruvate carboxykinase did not show any significant change. The mRNA levels for fructose-1,6-biphosphatase, and phosphoenolpyruvate carboxykinase exhibited no marked changes. These results suggest that the expression of glucose-6-phosphatase and fructose-1,6-biphosphatase is disordered in OLETF rats.

A novel sulfotransferase was purified from the rat liver cytosol to electrophoretic homogeneity via five column chromatography steps (hydroxylapatite I, DEAE Bio-Gel, ATP-agarose I, hydroxylapatite II, and ATP-agarose II). The minimum molecular weight of the purified enzyme was determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis to be approximately 33,000. Gel filtration chromatography revealed a native molecular weight of approximately 34,000, indicating the enzyme being present in the monomeric form. The purified sulfotransferase displayed enzymatic activities, with a pH optimum of 9.25, toward various tyrosine and 3,4-dihydroxyphenylalanine (Dopa) isomers, except DL-ortho-tyrosine. Thyroid hormones, as well as dopamine and p-nitrophenol, could also be used as substrates. The apparent Km value of the enzyme (designated the Dopa/tyrosine sulfotransferase) for L-Dopa, determined at a constant 14 microM of 3'-phosphoadenosine 5'-phosphosulfate, was 0.76 mM. The intact enzyme was found to be N-blocked when subjected to N-terminal sequencing. Three internal partial amino acid sequences, obtained by analyzing its proteolytic fragments, were found to be distinct from the homologous sequences of other known rat liver sulfotransferases. The deduced amino acid sequence of a full-length cDNA isolated from a rat liver cDNA library confirmed the identity of the Dopa/tyrosine sulfotransferase as a new type of aryl sulfotransferase. Upon transfection of COS-7 cells with an expression vector (pMSG-CMV) harboring the full-length cDNA, a 33-kDa protein displaying enzymatic and immunological properties similar to those of the purified Dopa/tyrosine sulfotransferase was expressed.

Novel erythrocyte pyruvate kinase gene defects were found in a patient without a family history of consanguinity. The polymerase chain reaction products of the R-type pyruvate kinase cDNA from the propositus contained two point mutations of Ser80 (TCC)-->Pro (CCC) and Arg490 (CGG)-->Trp (TGG). Allele-specific polymerase chain reaction of the genomic DNA revealed that this patient was a compound heterozygote. The mobilities of the patient's L- and R-type pyruvate kinase by thin-layer polyacrylamide gel electrophoresis were abnormal. The results are consistent with the fact that these mutations are within exons common to the hepatic and erythrocyte isozymes.

This report describes for the first time the identification of four forms of biliverdin reductase including two biliverdin-IX beta reductases and two biliverdin-IX alpha reductases, designated isozymes I and II and isozymes III and IV, respectively, in human liver cytosolic fractions. The four forms of biliverdin reductase were purified to homogeneity. There was a 7,800-15,000-fold increase in specific activity when compared with the crude preparation, and the recovery was 8-26%. The purified enzymes were monomers with a molecular weight of about 21,000 (isozymes I and II) and 34,000 (isozymes III and IV). The enzymes were strictly specific for biliverdin, and no other oxidoreductase activities were detected in the purified preparations. The purified enzymes used NADPH and NADH as electron donors for the reduction of biliverdin. The apparent Km values of isozymes I, II, III, and IV for NADPH were 35.9, 13.1, 10.9, and 34.1 microM, respectively, whereas those for NADH were 5.6, 8.2, 7.9, and 23.4 mM, respectively. It was assumed that NADPH rather than NADH was the physiological electron donor in the intracellular reduction of biliverdin. The apparent Km value of isozymes I and II for biliverdin-IX beta in the NADPH system was 0.3 microM whereas those of isozymes III and IV for biliverdin-IX alpha were 1.0 and 0.8 microM, respectively. Isozymes I and II used biliverdin-IX beta, -IX gamma, and -IX delta as substrates but not biliverdin-IX alpha, and isozymes III and IV preferred biliverdin-IX alpha as the most effective substrate among the four biliverdin isomers. The NADPH-dependent enzyme activities were inhibited by substrate concentrations in excess of 3-4 microM. The NADPH-dependent enzyme activities, especially isozymes III and IV, were sensitive to SH reagents including iodoacetamide, p-chloromercuribenzoic acid, and N-ethylmaleimide. The optimum pH of the reaction with NADPH for isozymes I and II was 8.2 whereas that for isozymes III and IV was 7.4. The proportion of the total activity of isozymes I and II to that of isozymes III and IV was considerably higher in the fetal than in the adult liver.

The amino acid sequence of biliverdin-IX beta reductase (EC1.3.1.24) from human liver was determined by automated Edman degradation of peptides generated by enzymatic and chemical cleavages. The enzyme was a single polypeptide chain of 204 amino acid residues, and its amino acid sequence had no significant homology to that of rat liver biliverdin-IX alpha reductase. Biliverdin-IX alpha reductase from human liver had intense homology to the rat enzyme. Cysteinyl residues are essential for the enzymatic activity of biliverdin-IX alpha, but nonessential for that of biliverdin-IX beta reductase. The results strongly indicate that the two enzymes, biliverdin-IX alpha reductase and biliverdin-IX beta reductase, are distinct in enzymatic action mechanisms as well as ancient origins of gene.

The diuretic activity of a quinolinone oxime diuretic, M12285, was examined after renal arterial, i.v. and portal injection in rats. M12285 injected into the renal artery at a dose of 1 mg/kg caused no diuretic effect, whereas i.v. and portal injections induced marked diuresis dose dependently. The minimum effective dose with portal injection was lower (1 mg/kg) than that with i.v. injection (3 mg/kg) and the start of the effect was faster with portal injection. These results indicated that some metabolic modification in the liver is necessary for the diuretic activity to appear. Accordingly, we performed in situ rat liver perfusion with M12285 and obtained several metabolites. Renal arterial injection of each fractionated metabolite of M12285 revealed that all the diuretic activity derived from one of these metabolites. From IR and 1H-nuclear magnetic resonance (1HNMR) measurements, the chemical structure of this active metabolite was assumed to be a sulfate-conjugated form of M12285 at the oxime moiety. Based on this tentative chemical structure, we synthesized the oxime sulfate of M12285 (potassium salt, M17000) and confirmed the identity of IR and 1HNMR spectra. Administration of M17000 into the renal artery induced apparent diuresis in a dose-dependent manner in both rats and dogs. These results indicate that the oxime sulfate of M12285 is responsible for the diuretic activity of M12285. Therefore, we synthesized several derivatives of M17000 and confirmed their possible therapeutic value as a novel family of diuretics, namely quinolinone oxime sulfonic acids.

We have cloned a full-length cDNA for rat-liver-type phosphofructokinase. The similarities of the rat liver-type phosphofructokinase mRNA to the human and mouse counterparts were 94% and 99% in their amino acid sequences and 88% and 94% in the nucleotide sequences of their coding regions, respectively. Rat liver-type phosphofructokinase mRNA was expressed in all tissues examined, but its level was regulated tissue-specifically. The nutritional and hormonal regulations of the mRNA in the liver were examined in comparison with those of two other key glycolytic enzymes, glucokinase and L-type pyruvate kinase. The level of liver-type phosphofructokinase mRNA was essentially unchanged by starvation (72 h) or diabetes. The mRNA level also did not change significantly on refeeding starved rats on a high carbohydrate diet, or treating diabetic ones with insulin. These results suggested that rat liver-type phosphofructokinase mRNA in the liver was not under control of diet or insulin, in contrast to glucokinase and L-type pyruvate kinase.

The messages for LF-B1, which interacts with the cis-acting element of PKL-I to play an essential role in expression of L-type pyruvate kinase (PK) in the liver, and L-type PK were found to be present in RIN-m5F insulinoma cells as well as the liver, kidney and small intestine, although the levels of the two mRNAs in these tissues were not correlated. Gel retardation assay suggested that similar nuclear proteins bound to two other cis-acting elements, PKL-II and PKL-III, were expressed in both liver and insulinoma cells, and that additional PKL-III-binding proteins were present only in RIN-m5F cells. Thus, we suggest that the mechanism of L-type PK expression in pancreatic B cells is similar to that in the liver.

125I-Hemoglobin.haptoglobin injected intravenously into rats was incorporated into liver parenchymal cells as evidenced by a cell separation technique. A mixture of freshly isolated liver parenchymal and nonparenchymal cells failed to internalize and degrade the 125I-hemoglobin.haptoglobin added, although it retained the ability to bind the molecule. The liver parenchymal cells in primary culture also lacked the ability to degrade 125I-hemoglobin.haptoglobin, although they bound the molecule more extensively as compared with the freshly isolated liver cells. It was confirmed that the 125I-hemoglobin.haptoglobin which was bound to the freshly isolated liver parenchymal cells localized on the outer surface of liver plasma membranes. Scatchard plots revealed the existence of two binding sites for 125I-hemoglobin-haptoglobin on the isolated liver plasma membrane: an apparent high affinity binding site (Kd = 1.3 X 10(-7) M) and an apparent low affinity binding site (Kd = 4.0 X 10(-6) M) at 37 degrees C. In contrast, freshly isolated liver parenchymal cells had only an apparent low affinity binding site (Kd = 1.4 X 10(-6) M) at 37 degrees C. Impairment of the apparent high affinity binding site during the isolation procedure with collagenase seemed to be related to loss of the ability to internalize and degrade the 125I-hemoglobin.haptoglobin molecules into the freshly isolated liver parenchymal cells or liver parenchymal cells in primary culture.

Male C3H, BALB/c and DBA/2 mice were injected subcutaneously with a single dose of 30 mumol CdCl2/kg. Most of the C3H mice died within 15 h, while all of DBA/2 survived for at least 48 h. At 6 h after Cd-injection, histology revealed severe hemorrhage, fatty change, zonal hepatocyte necrosis and congestion of C3H livers, while these findings were rare in the other strains even at 48 h post-injection. The rate of metallothionein (MT) induction at 6 h was significantly lower in C3H mice than in the other two strains.

1. Glycoprotein synthesis was investigated with [1-14C]glucosamine in vivo. [14C]Glucosamine was administered intravenously 24h after hepatectomy to rats. 2. Incorporation into the acid-soluble fraction was maximum at 15 min after injection both in sham-operated and hepatectomized rats. 3. Enhancement of incorporation into UDP-N-acetylhexosamine in regenerating liver was observed. However, its specific activity was lower, because of a greater enhancement of synthesis de novo of the amino sugar. 4. In the liver acid-insoluble fraction, maximum incorporation of [14C]glucosamine was at 30 min in sham-operated rats and 2 h in hepatectomized rats respectively. 5. In sham-operated rats, incorporation into the plasma acid-insoluble fraction followed that of the liver acid-insoluble fraction, but hepatectomy resulted in a rapid enchancement of incorporation into plasma. 6. It is concluded that synthesis of liver glycoproteins is stimulated after partial hepatectomy and that glycoproteins synthesized are released rapidly into the plasma.

The intracellular site of incorporation and degradation of heme and globin moiety of hemoglobin-haptoglobin in rat liver cells was investigated in vivo. Hemoglobin-haptoglobin, administered intravenously to rats, is cleared from the circulation and incorporated exclusively into liver parenchymal cells through the receptor specific for the molecule (Kino, K., Tsunoo, H., Higa, Y., Takami, M., Hamaguchi, H., and Nakajima, H. (1980) J. Biol. Chem. 255, 9616-9620). Intracellular distribution of radioactivity was determined after intravenous administration of [3H-Heme,14C-Globin]hemoglobin-haptoglobin to rats. The doubly labeled hemoglobin-haptoglobin was incorporated first in organelles of lower anodic mobility in carrier-free electrophoresis and of low density (density range, 1.05-1.07 g/ml) in Percoll density gradient centrifugation recovered in Golgi subfractions of the liver cells in a substantially intact form. In the subsequent stages, these organelles progressively acquired a higher anodic mobility as well as higher density, presumably through fusion with other organelles. In the resulting organelles of higher anodic mobility in electrophoresis and high density (density range, 1.07-1.15 g/ml) in Percoll, the hemoglobin-haptoglobin first dissociated symmetrically into two 82,000-dalton subunits having intact heme, and then the organelles containing only 3H radioactivity but no 14C radioactivity were separated by electrophoresis. Most of the 3H radioactive materials in these organelles are identified as intact [3H]heme. These investigations suggest that the heme moiety of hemoglobin-haptoglobin in the organelles is detached from globin-haptoglobin and binds to another carrier protein prior to conversion of heme to bilirubin.

The intracellular site of uptake and degradation of globin-haptoglobin, the protein moiety of hemoglobin-haptoglobin, in rat liver cells was investigated in vivo. Hemoglobin-haptoglobin, administered intravenously to rats, is cleared from circulation and incorporated exclusively into liver parenchymal cells through the receptor specific for the molecule (Kino, K., Tsunoo, H., Higa, Y., Takami, M., Hamaguchi, H., and Nakajima, H. (1980) J. Biol. Chem. 255, 9616-9620). Intrahepatocellular distribution of radioactivity was determined after intravenous administration of (125I-hemoglobin)-haptoglobin or hemoglobin-(125I-haptoglobin) to rats. The 125I-labeled hemoglobin-haptoglobin was incorporated first in organelles of low density (density range, 1.05-1.07 g/ml) recovered in Golgi subfractions of the liver cells in a substantially intact form. The organelles progressively acquired a higher density, presumably through fusion with primary lysosomes. In the resulting organelles of high density (density range, 1.07-1.15 g/ml), which are probably secondary lysosomes, hemoglobin-haptoglobin first dissociated symmetrically to yield two 82,000-dalton subunits by a limited proteolysis, and further digestion of the constituent polypeptide chains seemed to proceed thereafter in the organelles during the transport process across the cells.

The presence of a receptor specific for the hemoglobin . haptoglobin complex is demonstrated in rat liver plasma membranes. Hemoglobin . haptoglobin complex, administered intravenously to rats, was cleared from the circulation at a constant rate with exclusive incorporation of the molecule into hepatocytes. This incorporation was unaffected by the simultaneous injection of asialoglycoprotein or heme . hemopexin complex. In vitro experiments with isolated liver plasma membranes indicated the absence of competitive binding of these molecules to the membrane and suggested that this receptor might recognize an altered conformation of the haptoglobin moiety of the complex resulting from the binding with hemoglobin. These observations suggest that the mechanism of recognition and binding of hemoglobin . haptoglobin complex by the receptor is different from that of the asialoglycoprotein receptor or heme . hemopexin receptor.