Search

Aim: We have recently reported that hyperdynamic state of retinoid metabolism, which may lead to the shortage of retinoid, is observed in patients with non-alcoholic fatty liver disease (NAFLD). Hepatic iron overload, which causes production of reactive oxygen species (ROS), is also frequently seen in NAFLD patients. The aim of the study is to examine iron state and retinoid metabolic state simultaneously, and to clarify the relationship between two disorders. Methods: Thirty-six persons, comprising 17 patients with simple steatosis (SS), 11 with NASH, and 8 normal controls (N), were examined on hepatic expression of iron metabolism-related genes including hemojuvelin (HJV), hepcidin (HEPC), transferrin receptor 1 and 2 (TfR1, TfR2), ferroportin (FPN), neogenin (NEO) and ferritin heavy chain (FtH) and hepatic iron contents in addition to expression 51 genes which is involved in retinoid metabolism and antioxidative action. Results: In patients with NAFLD, expression of HJV, TfR2, FPN, TfR1, FtH, SOD and catalase was increased, compared with that in N. In addition, hepatic iron content, which was increased in NASH, was correlated with expression level of TfR2. Expression of cellular retinoid binding protein (CRBP1), alcohol dehydrogenase 1 (ADH1) and cytochrome P450 26A1(CYP26A1) was significantly correlated with that of HJV, TfR2 and FPN, respectively. Conclusion: The results of the present study suggest that the reasons responsible for iron accumulation in NASH in the present study may partly be due to enhanced expression of TfRs, especially TfR2, and hyperdynamic state of retinoid metabolism is closely related to iron metabolism in the disease.

Aim: The patients with non-alcoholic fatty liver disease (NAFLD) have been reported to be at greater risk for progression to chronic liver disease including liver cirrhosis (LC). To examine the mechanisms for the progression of NAFLD, a genetic analysis of hepatic expression profile in retinoid metabolism in NAFLD was performed since the loss of retinoid signaling is associated with the progression of liver disease via reactive oxygen species (ROS) generation. Methods: Fifty-one genes, which are associated with retinoid metabolism and action, were examined in thirty six subjects including 17 patients with simple steatosis, 11 with non-alcoholic steatohepatitis (NASH) and eight controls were examined by real-time reverse transcriptase polymerase chain reaction. Immunohistochemical study was also done by 3 kinds of antibodies. Results: Higher expression of CRBP1 LRAT, DGT1/2 and CES1 in NAFLD suggests that mutual conversion between retinyl ester and retinal occurs actively. Expression of ADH1/2/3, RDH5/10/11, DHRS3 and RALDH1/3 was increased in NAFLD, suggesting that oxidation process from retinol to all-trans retinoic acid (ATRA) was enhanced. Importantly, greater expression of CYP26A1 indicated that degradation of ATRA was enhanced in NAFLD. Further, expression of SOD1/2, catalase, thioredoxin and uncoupling protein 2 was also enhanced. Conclusion: Hyperdynamic state of retinoid metabolism is present in the liver tissues with NAFLD, which may be a putative mechanism by which NAFLD progresses to chronic liver disease including LC.

Hepatic iron overload has been frequently observed in the liver of patients with chronic liver diseases. In this study, the effect of hepatic fatty acid accumulation on the iron metabolism was investigated. Mice fed a choline-deficient diet developed severe steatosis associated with increased total amount of non-heme iron in the liver. Hepatic lipid contents were well correlated with the iron amount. The choline-deficient diet significantly downregulated hepcidin while increases in hemojuvelin and transferrin receptor 2 and a decrease in Tmprss6 expression were observed. Moreover, ferroportin expression was downregulated in the livers of choline-deficient mice while increases in transferrin receptor 1 and divalent metal transporter 1 and a decrease in ferritin expression were observed in accordance with increased hepatic iron content. The expression of hepcidin and ferroportin mRNA was negatively correlated to hepatic lipid concentrations. These results suggest that enhanced dietary iron intake and reduced hepatic iron efflux occur in the mice fed a choline-deficient diet. In addition, a possible link between hepatic iron and lipid metabolism is also suggested.

Angiotensin II (Ang II) has been reported to indirectly influence atrial electrical activity and to play a critical role in atrial arrhythmias in hypertensive patients. However, it is unclear whether Ang II has direct effects on the electrophysiological activity of the atrium affected by hypertension. We examined the effects of Ang II on the action potentials of atrial myocytes enzymatically isolated from spontaneous hypertensive rats (SHRs). The action potentials were recorded by the perforated patch-clamp technique and the atrial expression of the receptors AT1a and AT2 was measured by radioimmunoassay. Ang II significantly shortened the action potential durations (APDs) of SHRs without changes in the resting membrane potentials (RMPs). Pretreatment with selective AT1a blockers abolished the Ang II-induced reduction of atrial APDs of SHRs; however, a selective AT2 blocker did not, which was consistent with the results of the receptor assay. Pretreatment with phosphatidylinositol 3 (PI3)-kinase inhibitor, phospholipase C inhibitor, or protein kinase C (PKC) inhibitor abolished the Ang II-induced shortening of atrial APDs, but pertussis toxin and protein kinase A (PKA) inhibitor did not. To study the effects of chronic AT1a inhibition on Ang II-induced shortening of atrial APD, SHRs were treated with AT1a blocker for 4 weeks. AT1a blocker abolished the Ang II-induced reduction of atrial APDs of SHRs and also significantly lowered their blood pressure. In conclusion, Ang II shortened atrial APDs of SHRs via AT1a coupled with the Gq-mediated inositol triphosphate (IP3)-PKC pathway. Our findings indicated that Ang II caused atrial arrhythmias in hypertensive patients by shortening the effective refractory period of the atrium.

Remote reperfusion lung injury following skeletal muscle ischemia and reperfusion accounts for high morbidity and mortality. AMP deaminase (AMPD), a key enzyme for nucleotide cycle, has been implicated in the regulation of this phenomenon. However, the function of Ampd2 and Ampd3 subtype has not been elucidated in remote reperfusion rodent lung injury. We utilized AMPD3 and AMPD2-deficient mice. The two types of AMPD-deficient mice and wild-type (WT) littermates were subjected to ischemia-reperfusion injury. After 3h bilateral hind-limb ischemia and reperfusion, AMPD3 mRNA, AMPD activity and inosine monophosphate (IMP) increased significantly in WT and AMPD2-deficient mice lungs, while they did not show significant alterations in AMPD3-deficient mice lungs. Genetic inactivation of Ampd3 resulted in markedly accelerated myeloperoxidase (MPO) activity along with exaggerated neutrophils infiltration and hemorrhage in the lungs compared to WT and AMPD2-deficient mice, furthermore, IMP treatment significantly attenuated MPO activity and neutrophils infiltration in WT and the two types of AMPD-deficient mice lungs after 3h reperfusion. These findings demonstrate for the first time in AMP-deficient mice models that AMPD3 plays a critical role in remote reperfusion lung injury via generation of IMP and validate the potential to use IMP into the clinical arena to attenuate remote ischemia-reperfusion lung injury.

Changes in the expression rate of epidermal growth factor receptor (EGFR) mRNA with age in the liver of rats were examined to assess the contribution of EGF to the proliferation of hepatocytes in younger animals. Northern blot analyses with a 32P-labeled probe derived from the extracellular domain of EGFR cDNA were evaluated quantitatively by normalizing the densitometric scanning data with the relative amounts of poly(A)+ RNA in the samples. The expression of EGFR mRNA was low during the early period and increased towards the adult level after 21 days of birth. In contrast, the rate of beta-actin mRNA expression was significantly higher immediately after birth and showed a tendency to decrease with time.

To identify gene products involved in castration-induced involution of the rat ventral prostate, we constructed a subtraction cDNA library of the ventral prostate from rats castrated for 48 h. The library was screened with subtracted cDNA probes enriched for sequences with a low copy number expressed in intact or castrated rats. As a result of differential screening, 48 cDNA clones representing 10 different induced mRNAs were isolated. The time course of these mRNA inductions after castration was examined. Within the first 24 h after castration, the level of mRNAs for these cDNA clones was significantly increased and it reached its peak by 48-72 h after castration. Although mRNAs for these cDNA clones were expressed in various tissues from intact rats, an increase in mRNA as a response to castration was observed only in the ventral prostate. Partial sequence analyses of the 10 cDNA clones indicate that three cDNA clones represent rat glutathione S-transferase Yb-1, Yb-2 and Yb-3 subunit mRNA sequences, but for others respective homologues could not be found in a search of the GenBank database (release 67).

AIM Bone marrow-derived mesenchymal stem cells (MSC) are expected to be an excellent source of cells for transplantation. We aimed to study the culture conditions and involved genes to differentiate MSC into hepatocytes. METHODS The culture conditions to induce the efficient differentiation of human bone marrow-derived UE7T-13 cells were examined using cytokines, hormones, 5-azacytidine and type IV collagen. RESULTS We found that combination of acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF) with type IV collagen coating induced hepatic differentiation of UE7T-13 cells at over 30% frequency, where expression of albumin mRNA was increased over 20-fold. The differentiated cells had functions of albumin production, glycogen synthesis and urea secretion as well as expressing hepatocyte-specific genes. In addition, these cellshave binuclear and cuboidal morphology, which is a characteristic feature of hepatocytes. During hepatic differentiation, UE7T-13 cells showed depressed expression of WISP1 and WISP2 genes, members of the CCN family. Conversely, knockdown of WISP1 or WISP2 gene by siRNA stimulated hepatic differentiation. The effect of aFGF/bFGF/HGF/type IV collagen coating and WISP1-siRNA on hepatic differentiation was additive. CONCLUSION The present study suggests that aFGF/bFGF/HGF/type IV collagen coating is the efficient condition for hepatic differentiation of UE7T-13 cells, and that WISP1 and WISP2 play an important role in hepatic transdifferentiation of these cells.

The mechanisms of prevention of the development of liver cancer by NIK-333, an acyclic retinoid (ACR), were investigated. The transgenic mice expressing the dominant negative form of retinoic acid receptor alpha (RARE mice), that produce reactive oxygen species and lead to development of liver tumor were used. The effect of NIK-333 on hepatocarcinogenesis in RARE mice was studied. The RARE mice were examined after feeding 0.03% and 0.06% NIK-333 diets at 12 months of age. In the mice fed 0.06% NIK-333 diet, tumor incidence was greatly suppressed, compared to that of wild type mice (0/9 versus 5/9, P0.05), but not in the mice fed 0.03% NIK-333 diet. In addition, expression of cytochrome p450 4a14 and acyl-CoA oxidase was normalized, and the percentages of positive cells for 8-hydroxy-2'-deoxyguanosine, 4-hydroxy-2-nonenal and proliferating cell nuclear antigen were decreased. Furthermore, expression of beta-catenin and cyclin D1 was also depressed. These data suggest that NIK-333 suppressed liver tumor in association with repression of oxidative stress.

Retinoids have several biological functions including cell growth, differentiation and apoptosis. In liver, retinoids are known to be associated with regeneration, fibrosis and carcinogenesis. The facts that retinoid droplets in stellate cells are lost with progression of liver disease and that effectiveness of an acyclic retinoid on second primary liver cancer suggest the importance of liver as a target organ of retinoids. Our recent work has indicated that retinoids have antioxidant effects in association with regulation of fatty acid metabolism. In this review article, we discussed the important function of retinoids in liver, mainly from molecular aspects.

Truncated and full-length epidermal growth factor (EGF) receptors are produced in rat liver cells. The truncated EGF receptor mRNA is almost identical to the full-length EGF receptor mRNA except for the lack of a 3' region of the full-length receptor mRNA. To understand the stability of rat EGF receptor mRNAs, we analyzed the expression of EGF receptor mRNAs in the hepatoma cell line, AH66 and liver cells. Ten, 7 and 5 kb full-length and 2.7 kb truncated EGF receptor mRNAs were detected in both of them. The half-lives of the 10 and 2.7 kb EGF receptor mRNAs were determined in AH66 cells using a transcriptional inhibitor, 5,6-dichlororibofuranosylbenzimidazole. The half-lives of the 10 and 2.7 kb mRNAs were 1.2 and 11 h, respectively. These results indicated that the truncated mRNA is 4 times more stable than the full-length mRNA in rat cells. As for the stability, the role of a sequence of 3'-untranslated region of the EGF receptor mRNA was discussed.

Background & Aims We previously reported that impaired retinoid signaling in the liver causes steatohepatitis and hepatocellular carcinoma. Recently, oxidative stress induced by hepatic iron overload has emerged as an important factor for the progression of liver disease in patients with chronic hepatitis C, alcoholic liver disease, and nonalcoholic steatohepatitis. In this study, the relationship between retinoid signaling and iron metabolism in the liver was investigated. Methods The effect of retinoids on the iron metabolism was examined in HuH7 cells treated with all- trans retinoic acid and acyclic retinoid NIK-333. In in vivo experiments, we used the mice expressing the dominant negative form of retinoic acid receptor α gene under the control of albumin enhancer/promoter (RAR-E Tg) and iron-overloaded wild mice fed with retinoid-deficient and retinoid-excess diets. Results Hepatic iron accumulation and increased expression of hemojuvelin were observed in RAR-E Tg mouse liver. Retinoid treatment significantly suppressed expression of hemojuvelin and mildly suppressed expression of transferrin receptor type 2 and hepcidin, accompanied by decreased hepatic iron content and iron-induced oxidative stress in vitro and in vivo. Overexpression of hemojuvelin in HuH7 hepatoma cells led to a significant increase in cellular iron content. Conclusions Our results suggest that retinoids are involved in hepatic iron metabolism through transcriptional regulation of hemojuvelin. This study demonstrated a novel functional role of retinoids in preventing iron-induced oxidative stress in the liver.

Epidermal growth factor (EGF) receptor mRNA expression in livers of newborn, adult control and partially hepatectomized rats 1 and 3 days post-operation was examined by Northern blot analysis. A human EGF receptor cDNA (P 64-1, Ullrich et al., 1984) was hybridized to RNA species of 10, 6 and 3 kb. These mRNAs were profoundly decreased in livers from newborn rats and relatively decreased in regenerating rat livers. beta-Actin mRNA in these tissues showed a tendency to increase over that of the adult controls.

Binding sites for mouse epidermal growth factor (EGF) were observed in purified nuclear fraction from rat liver. The binding data yielded a curvilinear Scatchard plot which was decomposed into two binding components with different binding affinities. The binding component with higher affinity (Kd approximately 10(-10) M) represented approximately 15% of the total binding, while the high affinity binding sites were less than 5% of the total in the microsomes. The ligand-dependent autophosphorylation, one of the characteristic features of EGF receptor in the plasma membrane, was not observed at the site of the receptor (170 KDa) in the nuclear fraction. The binding characteristics for EGF fluctuated during the course of liver regeneration after partial hepatectomy; the binding capacity in the nuclear fraction increased in contrast to the decrease in the microsomes. However, the binding sites in the nuclear fraction obtained in the early period after partial hepatectomy consisted only of low affinity ones.

To elucidate the molecular mechanism of rat thymus involution induced by administration of glucocorticoid, we screened a cDNA library for polysomal poly (A) +RNA from adrenalectomized rat thymi by a differential colony hybridization method. Labeled cDNAs for mRNAs isolated from thymi of adrenalectomized rats and rats receiving dexamethasone (Dex) treatment were used as probes. Eight cDNA clones for mRNAs which had a diminished response to administration of Dex were isolated. The relative concentration of cloned mRNAs in the thymic polysomal RNA was significantly decreased 3 h after hormone administration, while changes in the nuclear RNA were not significant after Dex administration. One of the selected cDNA clones designated pRTGR-8 corresponded to mRNA of about 3, 000 nucleotides, and a nuclear run-off transcription assay indicated that the rate of transcription of pRTGR-8 RNA was repressed by Dex administration. The cloned cDNAs obtained in this experiment may provide useful probes for studying the negative regulation mechanism of gene expression by glucocorticoids.

3H-Dexamethasone (Dex)-receptor complexes prepared from the rat liver cytosol efficiently bound to the nuclear matrix from the same tissue. The binding was increased with the concentration of the 3H-Dex-receptor complex added and reached a maximum plateau. However, when the partially purified 3H-Dex-receptor complex was used, saturation of the binding sites in the nuclear matrix was not observed in the range of concentration of 3H-Dex-receptor complex used. Therefore, it was considered that the apparent saturability observed in the binding of the unpurified receptor complexes is caused by the translocation inhibitor(s) in the cytosol. When the binding capacity was expressed on the basis of unit weight of DNA, the nuclear matrix exhibited 20 times more of that of the unfractionated nuclei. However, no line of evidence of enrichment of the binding sites in the DNA isolated from the nuclear matrix was observed. These observations show that the role of the nuclear matrix in the action of glucocorticoid is quite uncertain.