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Objectives: In vivo lipopolysaccharide (LPS) tolerance on bacterial infection was investigated, focusing on liver macrophages., Methods: LPS tolerance was induced by intraperitoneal injections with 5 μg/kg of LPS for 3 consecutive days, and then mice were intravenously infected with Escherichia coli., Results: All LPS-primed mice survived lethal bacterial infection. Drastic enhancement of bactericidal activity of liver macrophages strongly contributed to bacterial clearance. Although LPS-primed mice produced substantial amounts of tumor necrosis factor (TNF) inside the liver, TNF efflux into the systemic circulation was markedly suppressed. These mice showed a dramatic increase in CD11b+ monocyte- derived macrophages in the liver. The CD11b+ macrophages that increased in LPS-primed mice were those with strong phagocytic/bactericidal activity and an upregulated expression of Fcγ receptor I, but the subfraction with a potent TNF-producing capacity and poor phagocytic activity diminished. The adoptive transfer of CD11b+ macrophages from LPS-primed mice to control mice increased survival after bacterial infection and reduced the elevation of plasma TNF. LPS priming did not affect the CD68+ resident Kupffer cells, and CD68+ Kupffer cell-depleted mice still exhibited LPS tolerance with strong resistance to bacteremia., Conclusions: LPS tolerance recruits CD11b+ macrophages to the liver with enhanced bactericidal activity, which plays a central role in resistance to lethal bacteremia., (© 2017 S. Karger AG, Basel.)

Retinol and its derivative, retinoic acid, have pleiotropic functions including vision, immunity, hematopoiesis, reproduction, cell differentiation/growth, and development. Non-alcoholic fatty liver disease (NAFLD) is one of the most common diseases in developed countries and encompasses a broad spectrum of forms, ranging from steatosis to steatohepatitis, which develops further to cirrhosis. Retinol status has an important role in liver homeostasis. The purpose of this study was to evaluate the retinol status and expression of retinol-related proteins, including enzymes and binding proteins, in methionine-choline deficient (MCD) rats as a model of NAFLD. We examined retinol levels in the plasma and liver and gene expression for β-carotene 15,15'-monooxygenase (BCMO), lecithIn: retinol acyltransferase (LRAT), aldehyde dehydrogenase 1A1 (ALDH1A1), ALDH1A2, and cellular retinol binding protein (CRBP)-I in MCD rats. The plasma retinol levels in MCD rats were lower than those in the controls, whereas hepatic retinol levels in MCD rats were higher. BCMO expression in the intestine and liver in MCD rats was lower, whereas that in the testes and the kidneys was higher than in control rats. Expression of LRAT, CRBP-I, ALDH1A1, and ALDH1A2 in the liver of MCD rats was also higher. Altered expression of retinol-related proteins may affect retinol status in NAFLD.

Although ursodeoxycholic acid (UDCA) has long been used for patients with chronic cholestatic liver diseases, particularly primary biliary cirrhosis, it may modulate the host immune response. This study investigated the effect of UDCA feeding on experimental hepatitis, endotoxin shock, and bacterial infection in mice. C57BL/6 mice were fed a diet supplemented with or without 0.3% (wt/vol) UDCA for 4 wk. UDCA improved hepatocyte injury and survival in concanavalin-A (Con-A)-induced hepatitis by suppressing IFN-γ production by liver mononuclear cells (MNC), especially NK and NKT cells. UDCA also increased survival after lipopolysaccharide (LPS)-challenge; however, it increased mortality of mice following Escherichia coli infection due to the worsening of infection. UDCA-fed mice showed suppressed serum IL-18 levels and production of IL-18 from liver Kupffer cells, which together with IL-12 potently induce IFN-γ production. However, unlike normal mice, exogenous IL-18 pretreatment did not increase the serum IFN-γ levels after E. coli, LPS, or Con-A challenge in the UDCA-fed mice. Interestingly, however, glucocorticoid receptor (GR) expression was significantly upregulated in the liver MNC of the UDCA-fed mice but not in their whole liver tissue homogenates. Silencing GR in the liver MNC abrogated the suppressive effect of UDCA on LPS- or Con-A-induced IFN-γ production. Furthermore, RU486, a GR antagonist, restored the serum IFN-γ level in UDCA-fed mice after E. coli, LPS, or Con-A challenge. Taken together, these results suggest that IFN-γ-reducing immunomodulatory property of UDCA is mediated by elevated GR in the liver lymphocytes in an IL-12/18-independent manner.

Vitamin E, a critical fat-soluble vitamin antioxidant, is expressed on cell membranes and prevents propagation of lipid peroxidation. α-Tocopherol transfer protein (α-TTP) is a cytosolic protein located in the hepatocytes that acts as the principal regulator of the circulating α-tocopherol levels. Type 2 diabetes is a metabolic disorder characterized by hyperglycemia, caused by insulin resistance. Lipid peroxidation promotes the clinical progression and development of complications in type 2 diabetes. Results of animal and human experiments on the vitamin E status in diabetes are conflicting. The present study was conducted with the objective of investigating the vitamin E status and α-TTP expression in Goto-Kakizaki (GK) rats, a model of type 2 diabetes. In diabetic GK rats, increases of the α-tocopherol levels in the plasma and liver were observed as compared with the levels in the controls. No alternation in the CuZn-superoxide dismutase (SOD) or Mn-SOD gene expression was found in the liver of GK rats as compared with that in the controls. The GK rats showed an increase of the hepatic expression of the α-TTP gene as compared with the level in the controls. It can be suggested that the increased hepatic α-TTP gene expression levels may influence plasma α-tocopherol levels in the diabetic animals. Hence, investigation of the regulatory factors of α-TTP expression may provide important clues to highlighting the antioxidant mechanisms of vitamin E.

Although B cells in vertebrates have been thought to lack phagocytic activity, there has been a recent report of such ability by the B cells of early vertebrates such as fish and frogs. Here, we show for the first time that mouse liver IgM(+) B cells actively phagocytose microsphere beads and Escherichia coli and that they effectively kill bacterial cells. Such phagocytic activity is not observed in other liver MNCs, except for F4/80(+) Kupffer cells. In the presence of fresh mouse serum (but not heat-inactivated serum), the heat-killed E. coli phagocytic activity of liver B cells increased significantly but was inhibited significantly by anticomplement component C3 antibody, suggesting E. coli opsonization by serum factors, including complement components. Upon i.v. injection of FITC-labeled E. coli into mice, a substantial proportion of liver B cells phagocytosed the bacteria, as compared with spleen B cells. Functional phagolysosome formation in liver B cells was supported by several reagents showing an acidic change and lysosomes in the phagocytosed vacuoles. Indeed, mouse liver B cells killed viable E. coli more efficiently than did spleen B cells in vitro. Further, E. coli-phagocytic liver B cells produced a substantial amount of IL-12. These results indicate that liver B cells have phagocytic and bactericidal activities similar to those of dedicated phagocytes and may contribute to bacterial clearance.

Dietary lipids/cholesterol may modulate liver immune function. We have recently found that mouse F4/80 Kupffer cells are classified into phagocytic CD68 Kupffer cells and cytokine-producing CD11b Kupffer cells. We here investigate how a high-fat and/or high-cholesterol diet affects innate immune liver mononuclear cells. For 4 weeks, C57BL/6 mice were fed a high-fat and high-cholesterol diet (HFCD), a high-cholesterol diet (HCD), a high-fat diet (HFD), or a control diet (CD). High-fat and high-cholesterol diet and HCD increased liver cholesterol levels; serum cholesterol levels increased in HFCD and HFD mice but not in HCD mice. The increased proportion of natural killer (NK) cells, downregulated NK1.1 expression of natural killer T cells, and enhanced CD69 and IL-12 receptor β mRNA expression of liver lymphocytes indicate the activation of them by HFCD. IL-12 production from Kupffer cells and interferon γ production from NK/natural killer T cells activated by LPS and/or IL-12 both increased. IL-12 pretreatment more effectively improved the survival of HFCD mice relative to the survival of CD mice upon injections of liver metastatic EL-4 cells. In contrast, HFCD mouse survival decreased after LPS injection and generalized Shwartzman reaction. Consistently in HFCD mice, Toll-like receptor 4 mRNA expression of whole Kupffer cells was upregulated, and CD11b Kupffer cells proportionally increased. Although the proportion of CD68 Kupffer cells decreased in HFCD mice, phagocytic activity of them was enhanced. Mice fed with HCD rather than those fed with HFD showed features closer to HFCD mice. Thus, enhanced function of mouse liver mononuclear cells is likely dependent on the liver cholesterol level, rather than the liver triglyceride level.

Background: Absence of enteral nutrition (EN) reduces hepatic mononuclear cell (MNC) numbers and impairs their functions. However, enteral refeeding (ER) for as little as 12 hours following parenteral nutrition (PN) rapidly restores hepatic MNC numbers. We hypothesized that changes in small intestine and portal vein blood flows related to feeding route might be responsible for this phenomenon., Methods: In experiment 1, mice (n = 19) were randomized to Chow (n = 5), PN (n = 7) or ER (n = 7) groups. The Chow group was given chow ad libitum with intravenous (IV) saline for 5 days. The PN group was fed parenterally for 5 days, while the ER group was re-fed with chow for 12 hours following 5 days of PN. Then, small intestine and portal vein blood flows were monitored and hepatic MNCs were isolated and counted. In experiment 2, the effects of intravenous administration of prostaglandin E(1) (PGE(1)) on hepatic MNC numbers were examined in fasted mice for 12 hours. Mice (n = 28) were randomized to Control (n = 8), PG0 (n = 10), or PG1 (n = 10) groups. The Control group was fed chow ad libitum with IV saline, while the PG0 and PG1 groups were fasted for 12 hours with infusions, respectively, of saline and PGE(1) at 1 microg/kg/minute. Blood flows and hepatic MNC numbers were examined., Results: Experiment 1: ER restored PN-induced reductions in small intestine and portal vein blood flows and hepatic MNC number to the levels in the Chow group. Small intestine and portal vein blood flows correlated positively with hepatic MNC number. Experiment 2: Fasting decreased small intestine and portal vein blood flows and hepatic MNC number. However, PGE(1) restored portal vein blood flow to the level of the Control group, and moderately increased hepatic MNC number. There was a positive correlation between portal blood flow and hepatic MNC number., Conclusions: Reduced small intestine and portal vein blood flows may contribute to impaired hepatic immunity in the absence of EN. ER quickly restores hepatic MNC number through recovery of blood flow in both the small intestine and the portal vein.

Impaired insulin receptor (IR) signaling leads to insulin resistance and type 2 diabetes mellitus. Several inhibitors of the IR tyrosine kinase activity have recently been described and associated with human insulin resistance. Among these negative regulators, protein tyrosine phosphatases (PTPs) are likely to play a pivotal role in IR signaling. Transgenic studies revealed that PTP1B and TCPTP are primary candidates but IR of these animals can be finally dephosphorylated, suggesting that other PTPs are also involved in the dephosphorylation of IR. In this study, we showed that receptor-type PTPepsilon (PTP epsilonM) dephosphorylated IR in rat primary hepatocytes and tyrosines 972, 1158, 1162 and 1163 were primary targets of PTP epsilonM. Wild type as well as substrate-trapping DA forms of PTPepsilonM suppressed phosphorylation of IR downstream enzymes such as Akt, extracellular regulated kinase (ERK) and glycogen synthase kinase 3 (GSK3). It was also demonstrated that PTPepsilonM suppressed insulin-induced glycogen synthesis and inhibited insulin-induced suppression of phosphoenol pyruvate carboxykinase (PEPCK) expression in primary hepatocytes. Furthermore, adenovirally introduced PTPepsilonM also exhibited inhibitory activity against suppression of PEPCK expression in mouse liver. These results suggest that PTPepsilonM is a negative regulator of IR signaling and involved in insulin-induced glucose metabolism mainly through direct dephosphorylation and inactivation of IR in hepatocytes and liver.

A 76-year-old woman admitted to our hospital for jaundice had been treated for diabetes mellitus with insulin for 9 years and voglibose had been added for the 39 months preceding admission. Elevated serum bilirubin and transaminase concentration began to normalize with initiation of corticosteroid treatment 1 week after discontinuation of voglibose. In the in vitro testing of the patient's peripheral blood, lymphocytes showed stimulation by voglibose. In dramatic contrast to an initial liver biopsy specimen that demonstrated cholestasis with submassive and zonal necrosis, the follow-up specimen was normal 1 year later. We believe that patients treated with voglibose should be monitored long-term with monthly determinations of transaminases and bilirubin in serum.

Intrahepatic calculi, highly prevalent in the Far East, including Japan, are characterized clinically by chronic proliferative cholangitis with frequent stone recurrences. Intrahepatic calculi consist of 2 groups, i.e., brown pigment stones, including a high cholesterol content, and cholesterol stones, with the former predominating. To gain insights into the pathogenesis of intrahepatic calculi, cholesterol and bile acid biosynthesis, as well as alterations in intracellular transport and/or canalicular secretion of phospholipid and bile acid were investigated in liver of patients with intrahepatic calculi. Enzyme activities of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase were increased (12.8 +/- 1.9 pmol/min/mg protein, mean +/- SEM vs. 5.5 +/- 0.4 in controls; P < .01) and cholesterol 7 alpha-hydroxylase activities were decreased (1.3 +/- 0.4 vs. 4.9 +/- 0.6; P < .01) in liver specimens of patients with brown pigment stones. In addition, messenger RNA (mRNA) levels of multidrug resistance P-glycoprotein 3 (MDR3 Pgp) and phosphatidylcholine transfer protein (PCTP) were markedly low in the liver specimens compared with the levels in specimens of control subjects, gallbladder stone patients, and patients with obstructive cholestasis. The protein levels and the immunohistochemical staining were decreased for MDR3 Pgp and PCTP in the liver. Consistently, the concentrations of phospholipid were markedly reduced in the hepatic bile from both affected and unaffected hepatic segments. In patients with intrahepatic calculi, biliary cholesterol supersaturation and the formation of cholesterol-rich brown pigment as well as cholesterol stones may be attributed to decreased hepatic transport and biliary secretion of phospholipids, in the setting of increased cholesterogenesis and decreased bile acid synthesis.

Background/aims: Recently, a novel type of cytokeratin (CK) has been added to the classical catalog of CKs as CK20. The aim of the present study was to examine the immunoreactivity for CK20 in normal and developing rat liver and in experimental models of bile ductular and oval cell proliferation., Methods: Eighty-five Fischer rats, subdivided into five groups, were utilized in this study: fetal rats, ranging from day 10 to day 21 of gestation; newborn-neonatal rats, from 2 h to 10 days of age; bile duct ligated (BDL) rats; alpha-naphthyl-isothiocyanate (ANIT)-treated rats; and rats fed a choline-deficient diet containing N-2 Fluorenylacetamide (CD-AAF rats). Frozen sections from each liver were stained with the CK20 specific monoclonal antibody IT-Ks20.10., Results: The present study shows that CK20 is a "bile duct type" CK. In the fetal rat, CK20 appears late during intrahepatic bile duct development, at day 20 of gestation. A marked increase in CK20 expression occurs after birth, suggesting that intrahepatic bile duct maturation continues after birth and that CK20 may be considered as a "maturation" marker of the biliary tree. In BDL rats and in ANIT-treated animals, immunoreactivity of bile ductules for CK20 was strikingly heterogeneous. A variable number of proliferating biliary cells were weakly positive or negative for CK20 and their number increased with the duration of the obstruction or ANIT treatment. In CD-AAF-treated rats, we found a uniform staining of proliferating oval cells for CK20. This finding is in contrast with the observation in BDL and in ANIT groups, and suggests the existence of different mechanisms regulating the proliferation and differentiation of biliary cells under those conditions., Conclusions: In rat liver, CK20 may be added to the list of "bile duct type" cytokeratins. During development, CK20 expression may be related to the maturation stage of the biliary tree. Typical ductular proliferation induced by BDL or ANIT feeding clearly differs from the oval cell proliferation in the CD-AAF model in terms of immunoreactivity for CK20.

The expression of thrombopoietin (TPO) mRNA is observed in several tissues, including liver, kidney, brain, skeletal muscle, intestine, spleen, and bone marrow. Among these organs, the highest expression of TPO mRNA is detected in the liver. We identified cells producing TPO by means of in situ hybridization of adult rat liver using digoxigenin-11-UTP-labeled cRNA probes. We found that the cells expressing TPO mRNA also expressed serum albumin mRNA. TPO mRNA was detected in parenchymal cells (hepatocytes) but not in non-parenchymal cells (including endothelial cells, epithelial cells, and so forth). To determine the location of TPO expression in embryogenesis, sections of fetal mice were further analyzed by in situ hybridization. TPO mRNA was detected only in hepatocytes of fetal liver, which was also the major site of hematopoiesis. The expression of TPO mRNA in fetal liver was observed from 12.5 days postcoitus. Northern blot analysis showed that mouse liver transcribed the same size of TPO mRNA in the fetus and in the adult. These results clearly demonstrate that hepatocytes are the primary site of TPO production in the liver from fetus to adult.

The high prevalence of cholesterol gallstone disease in hypertriglyceridemic patients may be associated with frequent metabolic defects in cholesterol and bile acid syntheses and in the concomitant formation of bile supersaturated with cholesterol. This study had the two aims: 1) to assess whether the defects as well as the degree of biliary cholesterol supersaturation in patients with hyperlipoproteinemia (HLP) can be estimated by the simultaneous determination of plasma mevalonate (MVL) and 7alpha-hydroxy-4-cholesten-3-one (C4); and 2) to assess the possible application of an estimated cholesterol saturation index ([CSI]E) as a means of evaluating the clinical effects of simvastatin on biliary lipid composition. Biliary cholesterol supersaturation was observed in patients with both IIa and IV HLP types. Consistent with the high activity and steady-state messenger RNA level of 3-hydroxy-3 methylglutaryl coenzyme A (HMG-CoA) reductase, plasma MVL was significantly higher in 86 patients with HLP (38 type IIa, 44.1 +/- 2.4 nmol/L and 48 type IV, 56.7 +/- 2.3; P < .01) than in 41 normolipidemic subjects (34.2 +/- 1.5), closely correlating with the molar percentage of cholesterol in bile (r = .61, P = .0001; n = 86). On the other hand, consistent with the high activity and messenger RNA level of cholesterol 7alpha-hydroxylase, plasma C4 was significantly higher in patients with HLP (type IIa, 28.8 +/- 2.3 nmol/L and type IV, 38.3 +/- 2.7; P < .01) than in normolipidemic subjects (17.4 +/- 1.5). Plasma C4 was closely correlated with plasma MVL (r = .40, P = .0001; n = 86), but was inversely correlated with the molar percentage of bile acids in bile (r = .49, P = .0001; n = 86). Assuming that cholesterol supersaturation in patients with HLP may be governed by both an enhanced cholesterol secretion (closely reflected by plasma MVL) and a decreased secretion of bile acids (closely reflected by plasma C4), the multivariate linear regression-analyses revealed that an index defined as estimated CSI ([CSI]E) (%) in patients with HLP was given by the following equation using plasma MVL and C4 (nmol/L): [CSI]E = 1[MVL] + 0.7[C4] + 44.4. Biliary cholesterol supersaturation in patients treated with simvastatin improved in a manner parallel to the time course of decreases in plasma MVL and C4. The [CSI]E before and at the end of treatment were correlated with biliary CSI. These results indicate that defects of hepatic cholesterogenesis, and bile acid synthesis, and the degree of biliary cholesterol supersaturation in patients with HLP can be estimated exactly by the simultaneous determination of plasma MVL and C4; furthermore [CSI]E may be adopted for clinical use as a convenient index of biliary CSI.

In the present study, we investigated the role of the spleen in experimental hepatic ischemia/reperfusion in the rat. After a 90-min period of ischemia in the left and middle hepatic lobes, the ischemia was released and the liver was reperfused for up to 24 h. Plasma alanine aminotransferase reached a peak 3 h after the onset of reperfusion, and gradually decreased thereafter. A histological examination revealed evidence of hepatocellular necrosis and degeneration, especially 24 h after the onset of reperfusion. In addition, there was a noticeable accumulation of polymorphonuclear cells in the liver following ischemia/reperfusion. A splenectomy performed just prior to ischemia/reperfusion reduced both biochemical and histological hepatocellular injury. The number of polymorphonuclear cells in the liver following ischemia/reperfusion was significantly reduced in rats subjected to splenectomy, suggesting that the increase in polymorphonuclear cells may contribute to liver injury. The number of mononuclear cells also increased in the marginal zones of the spleen following ischemia/reperfusion, and appeared to be derived from the splenic monocyte/macrophage population, based on immunohistochemical studies. The spleen plays an important role in the pathogenesis of hepatic ischemia/reperfusion injury and the splenic monocyte/ macrophage population contributes to liver damage.

A total of 100 nonobese and normolipidemic subjects (29 control subjects, 49 patients with cholesterol stones [CSs], and 22 patients with brown pigment stones) were studied to elucidate the pathogenetic contributions of deoxycholate (DC) to supersaturated bile formation with special reference to de novo syntheses of cholesterol and bile acids in the liver. A higher proportion of DC was observed in gallbladder bile from patients with CSs (CSs; 21.7 +/- 1.4%, mean +/- SEM, vs. control subjects; 10.2 +/- 0.9%). Cholesterol saturation in bile was elevated parallel to the increase of DC (r = .48; P = .0002), irrespective of the existence of stones. In a comparison between the 52 subjects with increased DC in bile (> 10% of biliary bile acids) and the 20 subjects without the increase (< 10%), the molar percentage of cholesterol in bile was significantly higher in the former (9.4 +/- 0.5%) than in the latter (6.7 +/- 0.4%) (P < .001). Consistent with the decrease in steady-state level of low-density lipoprotein (LDL) receptor-messenger RNA (mRNA), the catalytic activity and mRNA level of microsomal hepatic 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme for de novo cholesterol synthesis, were significantly lower in the former (2.9 +/- 0.3 pmol/min/mg protein) than in the latter (5.1 +/- 0.6) (P < .0001). Biliary molar percentage of bile acids was significantly lower in the former (69.8 +/- 1.1%) than in the latter (75.2 +/- 1.5%) (P < .01). However, contrary to expectations, the catalytic activity and mRNA level of cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme for bile acid synthesis, were significantly higher in the former (5.8 +/- 0.4 pmol/min/mg protein) than in the latter (3.7 +/- 0.6) (P < .01). The magnitude of the impaired gallbladder emptying (control subjects; 78.4 +/- 4% vs. CSs; 58 +/- 3%; P < .0005) together with the prolonged small intestinal transit (control subjects; 126 +/- 9 minutes vs. CSs; 198 +/- 9 minutes; P < .01) correlated significantly with the increased percentage of DC in bile. It is concluded that in cholesterol gallstone disease an increase of DC in bile, linked to an impaired gallbladder emptying together with a prolonged small intestinal transit, may play a significant role in downregulating de novo cholesterol synthesis but not bile acid synthesis in the liver.(ABSTRACT TRUNCATED AT 400 WORDS)

Recently, we showed that a 59 kDa non-phosphorylated sialoprotein purified from rat bone matrix is the rat counterpart of bovine fetuin and human alpha 2-HS glycoprotein and that fetuin synthesized and secreted by adult rat hepatocytes in primary culture is mostly phosphorylated (phosphofetuin), though fetuin is known to contain no phosphorus. Here we report that the rate of synthesis of phosphofetuin by hepatocytes in culture was reduced by inflammatory cytokines such as human interleukin (hIL)-6, human tumor necrosis factor-alpha and hIL-1 alpha, but dose-dependently stimulated by growth factors of hepatocytes, such as hepatocyte growth factor (HGF)/scatter factor (SF), epidermal growth factor and insulin, as determined by metabolic labeling and Northern blot analysis using cDNA for rat fetuin as a probe. We also showed that administration of HGF/SF stimulated gene expression of rat fetuin in vivo.

A detailed comparison was made of the bile acid composition in gallstones (brown pigment stones) and paired bile and liver from both affected and unaffected lobes by gallstones, which were taken at operation from 16 patients with hepatolithiasis, with the aim of elucidating whether stone formation is derived from possible local disturbances limited to intrahepatic bile ducts. Brown pigment stones in the intrahepatic bile ducts, most of which were accompanied by bile with high cholesterol saturation, had significantly more cholesterol, and less calcium bilirubinate and bile acid than those found in the extrahepatic bile ducts. Intrahepatic gallstones had significantly lower amounts of secondary and unconjugated bile acids, the bile acids modified by bacterial intervention, than extrahepatic stones. Bile specimens from both affected and unaffected lobes showed significantly increased molar percentages of cholesterol and decreased percentages of bile acids than bile from controls. In contrast, liver specimens from both lobes showed significantly higher concentrations of total bile acids. Secondary bile acids were present in a much lower proportion in bile and liver from both lobes than in bile and liver from controls. On the other hand, unconjugated bile acids were present in a much higher proportion in bile and liver from patients and only in negligible amounts in bile from controls. Furthermore, the plasma levels of mevalonate and those of 7 alpha-hydroxy-4-cholestene-3-one were found to be significantly higher and lower in patients than in controls, respectively, indicating that in hepatolithiasis cholesterol synthesis might increase and bile acid synthesis might decrease in the liver. These findings suggested that alterations of bile acid composition in gallstones, bile, and liver of patients with hepatolithiasis may be attributed to not only secondary changes resulting from local disturbances limited to intrahepatic bile ducts but also possible primary alterations of hepatocyte metabolism, such as bile acid conjugation and primary defects in cholesterol and bile acid synthesis.

We have investigated (by use of intact and saponinpermeabilized canine hepatocytes) the roles of Mg2+ and guanyl nucleotides in regulating glucagon-receptor interactions. In contrast to intact cells, saponinpermeabilized hepatocytes bind [[125I]iodo-Tyr10]glucagon according to a single first-order process and exhibit a single apparent dissociation constant for glucagon binding during steady-state incubations. Further analysis of the permeabilized cell system demonstrated (a) the temperature-sensitive action of Mg2+ to enhance the extent and affinity of glucagon-receptor interactions at steady-state, (b) the conversion of Mg(2+)-independent hormone-receptor complexes to Mg(2+)-dependent complexes, (c) the effect of guanyl nucleotides to inhibit specifically the Mg(2+)-dependent component of glucagon-receptor interactions, (d) the more rapid association of glucagon with receptor during cell incubations occurring in the presence of guanyl nucleotides or in the absence of Mg2+, and (e) the ability of guanyl nucleotides to induce both high and low affinity states of glucagon-receptor interactions. Additional experiments identified an effect of cell incubations in the presence of glucagon to limit the subsequent binding of hormone, the ability of GDP, GTP, or guanosine-5'-3-O-(thio)triphosphate (GTP gamma S) to dissociate previously bound glucagon, and a specific requirement for GDP to re-activate the glucagon receptor for additional cycles of hormone binding. A model is presented in which (a) glucagon binds to receptor in a Mg(2+)-independent fashion, (b) glucagon-receptor complexes are converted to a Mg(2+)-dependent state, (c) guanyl nucleotide exchange initiates both an alteration in glucagon-receptor affinity and the subsequent dissociation of hormone, and (d) in the context of the intact cell, G protein-mediated hydrolysis of GTP to GDP is required to reinitialize the system.

The effects of three of the most widely used histamine H2-receptor antagonists, cimetidine, ranitidine and famotidine, on liver cell growth were studied in vitro using adult rat hepatocytes in primary culture, because these antagonists are commonly given to patients with hepatic cirrhosis or fulminant hepatic failure for protection against peptic ulcers and gastrointestinal hemorrhage. At their clinically effective concentrations in the blood (0.5-5 micrograms/ml cimetidine, 0.25-2.5 micrograms/ml ranitidine and 0.05-0.5 microgram/ml famotidine), these three antagonists did not have any effect on replicative DNA synthesis either in the presence or absence of insulin plus epidermal growth factor (EGF). However, unexpectedly DNA synthesis stimulated by insulin and EGF was found to be enhanced by 0.05-0.5 mg/ml cimetidine, although it was unaffected or inhibited by ranitidine and famotidine at the concentrations tested. Cimetidine caused maximal enhancement of 1.5-2 times the control level of DNA synthesis at a concentration of 0.25 mg/ml. Cimetidine also had an enhancing effect at submaximal concentrations of insulin and EGF, but neither cimetidine nor the other antagonists had any stimulatory effect on DNA synthesis in the absence of insulin plus EGF. This enhancement of DNA synthesis by cimetidine resulted in significant increase in the total DNA content of the hepatocytes in culture. Under the conditions used, cimetidine had the lowest toxicity of these three antagonists and ranitidine the highest, as judged from data on DNA synthesis and the total protein content of cultured hepatocytes, leakage of aminotransferases from the cells and morphological observations.

Whole body autoradiography revealed that the distribution pattern of [14C]dehydrocorydaline in the mouse and rat liver was heterogeneous (or reticular) regardless of time after intravenous administration of the labeled agent. Microautoradiography by dry-mounting method revealed that the macroscopic heterogeneous pattern was due to the periportal localization of the radioactive compound in the hepatic lobule. By comparison with [14C]salicylid acid, [14C]diphenylhydantoin and [14C]p-chlorophenoxyacetic acid whose distribution pattern are homogeneous in the liver, the present studies indicated that the existence and persistence of heterogeneous distribution of [14C]dehydrocorydaline in the liver had the following causes: 1. Shortly after intravenous administration, the amount of [14C]dehydrocorydaline circulated to the liver was greatly restricted by its significant distribution in non-hepatic tissues. This was shown by the whole body autoradiography, radiometry of tissues and quantitative comparison of volumes of distribution in non-hepatic tissues. Therefore, 2. perilobular hepatocytes alone could take up [14C]dehydrocorydaline and consequently, centrilobular cells were unavailable to it: heterogeneous distribution pattern is formed. This was shown by microautoradiography as described above, and by the rapid and significant uptake of [14C]dehydrocorydaline by isolated hepatocytes in vitro and by the liver to which the labeled agents were continuously administered in situ. It was also substantiated by the more homogeneous distribution pattern in the liver of the rat to which greater amount of [14C]dehydrocorydaline was gradually given into the portal vein and of the mouse with allyl formate-induced perilobular damage. 3. Redistribution of [14C]dehydrocorydaline scarcely occurred in the whole body and therefore radioactive substance was not significantly supplied to the liver: the distribution pattern remained unchanged. This was shown by the whole body autoradiography and radiometry of tissues.

Whole body autoradiography revealed that the distribution pattern of [35S]chlorpromazine and [14C]imipramine in the mouse and rat liver was heterogeneous (or reticular) shortly after intravenous administration of the labeled agents and then became homogeneous. Microautoradiography by dry-mounting method revealed that the macroscopic heterogeneous pattern of [35S]chlorpromazine was due to its periportal localization in the hepatic lobule. The present studies indicated that the heterogeneous distribution was re-arranged to a homogeneous one in the following way: 1. The amount of [35S]chlorpromazine and [14C]imipramine circulated to the liver was greatly restricted by their significant distribution in non-hepatic tissues shortly after administration. This was shown by whole body autoradiography, radiometry of tissues and volumes of distribution in non-hepatic tissues. Therefore, 2. perilobular hepatocytes alone could take up the agents and consequently, centrilobular cells were unavailable to them: heterogeneous distribution pattern is formed. This was shown by microautoradiography described above, and by the rapid and significant uptake of the agents by isolated hepatocytes in vitro and of [35S]chlorpromazine by the liver to which the agent was continuously administered in situ. However, 3. re-distribution of [35S]chlorpromazine and [14C]imipramine occurred thereafter. Therefore, the radioactive compounds were significantly supplied to the liver late after administration: the pattern became homogeneous. This was shown by the whole body autoradiography and radiometry.

Monohydroxy bile acids in liver tissue may be of importance because of their hepatotoxicity and strong cholestatic effects. Recently, the existence of lithocholate in liver tissue in two forms was suggested by Nair et al. (Lipids. 1977. 12: 922-929) i.e., either in free form or as so-called tissue-bound lithocholate released exclusively by cholylglycine hydrolase treatment. The presence of the latter aroused much interest in relation to its hepatotoxicity and possible role in tumor induction. In the present investigation lithocholyl-epsilon-L-lysine, proposed as the predominant tissue-bound bile acid, was synthesized and its metabolic behavior was tested. Lithocholyl-epsilon-lysine was not deconjugated by cholylglycine hydrolase treatment but only by alkaline hydrolysis. Bile acids in seven cirrhotic and three noncirrhotic liver samples were extracted with 95% ethanol-0.1% ammonium hydroxide. The bile acids in the extract and residue were quantified by glass capillary gas-liquid chromatography using selected ion monitoring. The presence of so-called tissue-bound lithocholate could not be substantiated in either cirrhotic or noncirrhotic liver tissues. Nearly complete extraction of lithocholate was achieved by the use of organic solvent alone. Therefore, tissue-bound lithocholate, if it exists at all, may be attached to tissue by a physical linkage which can be disrupted by the use of conventional organic solvent.

Bile acid analysis of mild and severe cirrhotic liver showed that with the advancement of cirrhosis the concentration of chenodeoxycholic acid in liver tissue becomes higher, resulting in the lower ratio of cholic to chenodeoxycholic acid probably due to the progressive alteration of cholic and chenodeoxycholic acid synthesis with the advancement of liver cirrhosis. Bile acid analysis of paired liver and bile of severe cirrhosis showed that the ratio of cholic to chenodeoxycholic acid in liver was lower than that in bile or even with that in bile. This can be explained by postulating the impaired hepatic secretion of bile acids, especially chenodeoxycholic acid. The impaired secretion together with the relatively well preserved chenodeoxycholic acid synthesis results in the accumulation of chenodeoxycholic acid in liver tissue with cirrhosis.

Chain elongation of fusaric acid and related compounds in the presence of rat liver preparations was investigated by gas chromatography-mass spectrometry. The mitochondrial fraction catalyzed the elongation of the CoA esters of fusaric acid and 5-butyl-2-pyrimidinecarboxylic acid utilizing acetyl-CoA as a C2 donor. The microsomal fraction failed to afford elongation products. However, when the CoA ester of 3-(5-butyl-2-pyrimidinyl)-3-hydroxypropionic acid was incubated in the presence of the mitochondrial or the microsomal fraction, the corresponding alpha beta-unsaturated and saturated metabolites were identified in both cases, suggesting that the microsomal fraction could not catalyze the condensation or the keto-reduction of these heteroaromatic carboxylic acids.

Whole body autoradiography revealed that the distribution pattern of [14C]aminotriazole in the mouse liver was homogeneous after intravenous administration of the labeled agent and then became heterogeneous (or reticular). Microautoradiography by dry-mounting method revealed that the macroscopic heterogeneous pattern was due to the central localization of the radioactive compound in the hepatic lobule. The present studies indicated that the heterogeneous distribution could be explained as follows. The amount of [14C]aminotriazole circulated to the liver was large since the compound was not so significantly distributed in non-hepatic tissues: distribution pattern was homogeneous in the liver. This was shown by whole body autoradiography and radiometry of tissues. A part of [14C]aminotriazole radioactivity present in the liver was gradually bound covalently to hepatic macromolecules. This was shown by whole body autoradiography after whole body sections of the mouse were extracted by acid, and by the biochemical fractionation of the liver. The covalently bound radioactivity alone became apparent in centrilobular hepatocytes: the distribution was heterogeneous. This was shown by microautoradiography and by the finding that the elimination rate of the bound radioactivity was slower than that of unbound radioactivity.

We have recently found the presence of human hepatocyte growth factor in sera of patients with fulminant hepatic failure and have purified human hepatocyte growth factor from plasma of a patient with fulminant hepatic failure. In this paper, we report the clinical significance of human hepatocyte growth factor in blood from patients with fulminant hepatic failure. The effect of sera or plasma from 17 patients with fulminant hepatic failure on liver cell growth was examined by use of adult rat hepatocytes in primary cultures. Sera or plasma from 16 of the 17 patients with fulminant hepatic failure stimulated DNA synthesis in hepatocytes more effectively than normal human serum. The mean growth-promoting activity for the 17 patients with fulminant hepatic failure was about 16 times higher than that obtained for normal human serum. This growth-promoting activity of the patients' blood was not related to sex, age, clinical outcome of the patients or type of fulminant hepatic failure, but was intimately related to the clinical grade of hepatic coma. Sera or plasma with Grade III and IV coma showed stimulatory activity on DNA synthesis more markedly than sera or plasma from patients with coma of less than Grade II. In the surviving group, this activity decreased as the hepatic coma of patients improved. In fact, this activity of sera from patients at the recovery stage showed no significant increase compared with that of normal human serum. In the group of terminal patients, this activity increased as the coma developed.(ABSTRACT TRUNCATED AT 250 WORDS)

Bile acid composition of ten paired human livers and bile specimens were compared with gas chromatography-selected ion monitoring. The bile acid composition in liver and in bile was found to be similar but not identical. This difference seems to be a reflection of bile acid synthesis in liver tissue. It is suggested that analysis of bile acid composition in liver tissue is useful to evaluate abnormality of bile acid synthesis in several pathological states.