Hepcidin is down-regulated in alcohol loading.

BACKGROUND: It is common for alcoholic patients to have excess iron accumulation in the liver, which may contribute to the development of alcoholic liver disease (ALD). However, the mechanism of hepatic iron uptake in ALD is still obscure. Recently, a novel iron-regulatory hormone hepcidin was found that suppresses the absorption of iron from the small intestine and the release of iron from macrophages. To elucidate the contribution of hepcidin toward the hepatic excess iron accumulation in ALD, we examined whether alcohol loading affects hepcidin expression both in ALD patients and in an ethanol-fed mouse model. METHODS: Serum prohepcidin concentration was quantified by enzyme-linked immunosorbent assay. Hepatic hepcidin-1 and hepcidin-2 mRNA expressions in mouse liver were evaluated by quantitative real-time reverse-transcriptase polymerase chain reaction method. The protein expression of prohepcidin in mouse liver was examined immunohistochemically by rabbit antimouse prohepcidin antibody. RESULTS: Serum prohepcidin concentration in ALD was significantly lower than that in healthy subjects (p<0.001). Especially, serum prohepcidin concentrations were decreased in the patients whose serum ferritin value was high. In the ethanol-fed mouse model, hepatic hepcidin-1 mRNA expression was significantly lower than that in control (p=0.04). Prohepcidin was expressed in the cytoplasm of hepatocytes of mice liver tissue sections, and its expression was decreased after ethanol loading. CONCLUSION: Alcohol loading down-regulates hepatic hepcidin expression and leads to the increase of iron absorption from the intestine.

Iron accumulation in alcoholic liver diseases.

Increased hepatic iron is one of the important key factors which contribute alcohol toxicity of liver due to the production of reactive oxygen species. In patients with alcoholic liver diseases (ALD), liver iron is increased and the resulted lipid metabolite 4-hydroxynonenal-protein adduct was also increased. In general, iron is deposited in both parenchymal cells and and Kupffer cells in ALD. However, in patients with mild ALD, the parenchymal iron deposition is dominant rather than reticuloendothelial iron deposition, while the latter iron deposition is domimant in severe ALD, possibly due to endotoxemia and overproduction of inflammatory cytokines. We speculated that a parenchymal iron deposition in mild ALD is an important factor to trigger hepatocytes injury by ethanol, and the possible cause of parencynal iron deposition may be an increase of cellular iron uptake via serum transferrin in hepatocytes after ethanol exposure. By immuno-histochemical study of biopsied liver samples, the expression of transferrin receptor 1 (TfR1), which mediates cellular iron uptake by serum transferrin was increased. This increase of TfR1 by ethanol is confirmed by in vitro experiment using HepG2 cells and primary rat hepatocytes culture. Fe-labeled transferrin incorporation (but not transferrin non-bound iron (NTBI)) into the cells is also increased, suggesting that the increased TfR1 is functional. The increase of TfR1 expression is partially due to the increased activity of iron regulatory protein (IRP) by oxidative stress of ethanol metabolism. Thus, the post-transcriptional regulation of iron uptake by ethanol is involved in the hepatocyte iron accumulation. Another possibility is an increase of intestinal iron absorption. Our recent finding regarding the increase of pro-hepcidin serum in alcoholic patients with high serum ferritin support this assumption.

Increased expression of PPARgamma in high fat diet-induced liver steatosis in mice.

The present study was performed to examine a hypothesis that peroxisome proliferator-activated receptor gamma (PPARgamma) is implicated in high fat diet-induced liver steatosis. Mice were fed with control or high fat diet containing approximately 10% or 80% cholesterol, respectively. Macroscopic and microscopic findings demonstrated that lipid accumulation in the liver was observed as early as 2 weeks after high fat diet and that high fat diet for 12 weeks developed a fatty liver phenotype, establishing a novel model of diet-induced liver steatosis. Gene profiling with microarray and real-time PCR studies demonstrated that among genes involved in lipid metabolism, adipogenesis-related genes, PPARgamma and its targeted gene, CD36 mRNA expression was specifically up-regulated in the liver by high fat diet for 2 weeks. Immunohistochemical study revealed that PPARgamma protein expression is increased in the nuclei of hepatocytes by high fat diet. It was also shown that protein expression of cAMP response element-binding protein (CREB), an upstream molecule of PPARgamma, in the liver was drastically suppressed by high fat diet. All these results suggest for the first time that the CREB-PPARgamma signaling pathway may be involved in the high fat diet-induced liver steatosis.

Up-regulation of transferrin receptor 1 in chronic hepatitis C: Implication in excess hepatic iron accumulation.

BACKGROUND/AIMS: : To clarify the mechanism of excess hepatic iron accumulation in chronic hepatitis C, we investigated the expressions of transferrin receptor 1 and divalent metal transporter 1 in hepatocytes, both of which are involved in cellular iron uptake, in relation to the degree of hepatic iron accumulation and hepatic fibrosis by immunohistochemistrical study. METHODS: : Forty-six hepatic tissues with chronic hepatitis C and five normal hepatic tissues were examined. Chemical detection of hepatic iron accumulation was performed by Perl's Prussian blue stain. The immunohistochemistrical study was performed by avidin-biotin complex method with alkaline phosphatase. RESULTS: : In chronic hepatitis C: (1) Hepatic iron accumulation was significantly increased in relation to the advance of the fibrosis. (2) Divalent metal transporter 1 decreased significantly in relation to the advance of hepatic fibrosis. (3) Transferrin receptor 1 expression was always detected, although not in normal hepatic tissues; there was no relation between expression levels and the degree of hepatic fibrosis. CONCLUSIONS: : These data demonstrated that the transferrin receptor 1 expression was up-regulated irrespective of the degree of hepatic iron accumulation, suggesting that the up-regulation of transferrin receptor 1 might act as one of the key mechanisms implicated in the accumulation of hepatic iron in chronic hepatitis C.

Decreased susceptibility to renovascular hypertension in mice lacking the prostaglandin I2 receptor IP.

Persistent reduction of renal perfusion pressure induces renovascular hypertension by activating the renin-angiotensin-aldosterone system; however, the sensing mechanism remains elusive. Here we investigated the role of PGI2 in renovascular hypertension in vivo, employing mice lacking the PGI2 receptor (IP-/- mice). In WT mice with a two-kidney, one-clip model of renovascular hypertension, the BP was significantly elevated. The increase in BP in IP-/- mice, however, was significantly lower than that in WT mice. Similarly, the increases in plasma renin activity, renal renin mRNA, and plasma aldosterone in response to renal artery stenosis were all significantly lower in IP-/- mice than in WT mice. All these parameters were measured in mice lacking the four PGE2 receptor subtypes individually, and we found that these mice had similar responses to WT mice. PGI2 is produced by COX-2 and a selective inhibitor of this enzyme, SC-58125, also significantly reduced the increases in plasma renin activity and renin mRNA expression in WT mice with renal artery stenosis, but these effects were absent in IP-/- mice. When the renin-angiotensin-aldosterone system was activated by salt depletion, SC-58125 blunted the response in WT mice but not in IP-/- mice. These results indicate that PGI2 derived from COX-2 plays a critical role in regulating the release of renin and consequently renovascular hypertension in vivo.

Induction of transferrin receptor by ethanol in rat primary hepatocyte culture.

BACKGROUND: It is not uncommon for alcoholics to have iron accumulation in the liver, a condition that may contribute to the development of alcoholic liver disease. Recently, we reported that the expression of transferrin receptor, which mediates cellular iron uptake, was increased in hepatocytes in patients with alcoholic liver disease. To elucidate the mechanism of the iron accumulation in hepatocytes in such disease, we examined whether ethanol exposure induced the transferrin receptor expression and increased the cellular iron uptake. METHODS: Rat primary hepatocytes were isolated and cultured in the presence of 20 micromol/liter of iron and 25 mmol/liter of ethanol. RESULTS: Ethanol exposure to the hepatocytes demonstrated an ~2-fold increase in transferrin receptor expression for 24 hr, shown by Western blot analysis and S-methionine metabolic labeling, 19% increase in Fe-transferrin uptake by hepatocytes, and 20% increase in activity of iron regulatory protein examined by band shift assay. CONCLUSION: Ethanol exposure induced the transferrin receptor expression, partially through the activation of iron regulatory protein, and increased the transferrin-bound iron uptake in rat hepatocyte cultures. The induction of transferrin receptor by ethanol might be one of the mechanisms of iron accumulation in the hepatocytes in alcoholic liver disease.

Usefulness of contrast-enhanced wide-band Doppler ultrasonograpy to diagnose alveolar echinococcosis of the liver and evaluate the effect of the treatment.

Alveolar echinococcosis is a rare parasitic disease caused by Echinococcus multicularis and most commonly involves the liver. Early diagnosis and accurate evaluation of the effect of the treatment are essential to improve the prognosis of patients with alveolar echinococcosis of the liver (AEL). The aim of this study was to demonstrate the characteristic imaging of AEL by contrast-enhanced Dynamic Flow (CE-DF) employing a wide-band Doppler technique for the diagnosis and the accurate evaluation of the effect of the treatment. Four patients with five AEL lesions before treatment or during medication were examined by CE-DF. All of the five AEL lesions examined by CE-DF revealed a defect with an irregular and distinct margin like a worm-eaten defect appearance, which was never observed on other hepatic lesions, in liver perfusion image during post-vascular phase. In addition, CE-DF made it possible to measure the size of AEL lesions accurately because the margin was detected distinctly. These data suggest that CE-DF is clinically useful for the diagnosis of AEL and the evaluation of the effect of the treatment.

Clinical utility of sequential imaging of hepatocellular carcinoma by contrast-enhanced power Doppler ultrasonograpy.

The aim of this study was to investigate the clinical utility of sequential imaging of hepatocellular carcinoma (HCC) by contrast-enhanced power Doppler ultrasonograpy (CE-PDUS) to differentiate hepatocellular carcinoma from adenomatous hyperplasia (AH) and regenerated nodule (RN) and to predict the degree of differentiation of HCC. Fifty-one patients with 62 hepatic lesions including 33 moderately and poorly differentiated HCCs, 19 well-differentiated HCCs, seven AHs and three large RNs were examined by CE-PDUS. The imaging patterns during early arterial phase (tumor vessel image), late vascular phase (tumor perfusion image) and post-vascular phase (liver perfusion image) were classified as diffuse, basket, peripheral, central and no enhancement; as whole tumor, partial tumor and no enhancement; as whole tumor, partial tumor and no defect, respectively. The diffuse pattern in the tumor vessel image, the whole enhancement pattern in the tumor perfusion image and the whole defect pattern in the liver perfusion image were observed in moderately and poorly differentiated HCCs only. The basket pattern in the tumor vessel image and the partial defect pattern in the tumor perfusion image were observed in HCCs only. All AH/RNs showed no defect pattern in the liver perfusion image. The sequential imaging of HCC during early arterial, late vascular and post-vascular phases by CE-PDUS is clinically useful to differentiate HCC from AH/RN and to predict the degree of differentiation of HCC.

Up-regulation of transferrin receptor expression in hepatocytes by habitual alcohol drinking is implicated in hepatic iron overload in alcoholic liver disease.

BACKGROUND: Hepatic iron overload is often seen in alcoholic liver disease (ALD). We previously reported that the expression of 4-hydroxy-2-nonenal-protein adducts, which is a lipid peroxidative product and can be used as a marker of radical-mediated cellular damage, was increased in iron-overloaded hepatocytes with ALD. However, the mechanism of hepatic iron overload in ALD has not been clarified. In this study, to elucidate the mechanism of hepatic iron overload in ALD, we immunohistochemically investigated the expression of transferrin receptor (TfR), which mainly acts for cellular iron uptake. METHODS: Hepatic tissues were obtained from 31 patients with ALD and 5 normal livers by percutaneous needle biopsy under laparoscopy or ultrasound guidance. Chemical detection of hepatic iron accumulation was performed by Perls' Prussian blue stain. Immunohistochemical detection of TfR expression was done using human monoclonal anti-TfR antibody (TR104) according to the avidin-biotin complex method with alkaline phosphatase. RESULTS: Excess iron accumulation was found in 22 hepatic tissues with ALD but not in any normal hepatic tissues. TfR expression was increased in hepatocytes of 18 hepatic tissues with ALD but was not detected in any normal hepatic tissues. The mean duration of abstinence of patients who demonstrated positive TfR expression in hepatocytes was significantly shorter than that of patients who demonstrated negative TfR expression (positive: 14 days; negative: 30 days). However, total ethanol consumption, daily ethanol intake, and serum aspartate aminotransferase and gamma-glutamyl transpeptidase values on admission were not significantly correlated with TfR expression in hepatocytes. CONCLUSIONS: The up-regulation of TfR expression in hepatocytes is implicated in hepatic iron overload in ALD, and habitual alcohol drinking is an important factor for the induction of TfR expression.