Apoptosis induced by Fas signaling does not alter hepatic hepcidin expression.

AIM: To determine the regulation of human hepcidin (HAMP) and mouse hepcidin (hepcidin-1 and hepcidin-2) gene expression in the liver by apoptosis using in vivo and in vitro experimental models. METHODS: For the induction of the extrinsic apoptotic pathway, HepG2 cells were treated with various concentrations of CH11, an activating antibody for human Fas receptor, for 12 h. Male C57BL/6NCR and C57BL/6J strains of mice were injected intraperitoneally with sublethal doses of an activating antibody for mouse Fas receptor, Jo2. The mice were anesthetized and sacrificed 1 or 6 h after the injection. The level of apoptosis was quantified by caspase-3 activity assay. Liver injury was assessed by measuring the levels of ALT/AST enzymes in the serum. The acute phase reaction in the liver was examined by determining the expression levels of IL-6 and SAA3 genes by SYBR green quantitative real-time PCR (qPCR). The phosphorylation of transcription factors, Stat3, Smad4 and NF-κB was determined by western blotting. Hepcidin gene expression was determined by Taqman qPCR. The binding of transcription factors to hepcidin-1 promoter was studied using chromatin immunoprecipitation (ChIP) assays. RESULTS: The treatment of HepG2 cells with CH11 induced apoptosis, as shown by the significant activation of caspase-3 (P < 0.001), but did not cause any significant changes in HAMP expression. Short-term (1 h) Jo2 treatment (0.2 µg/g b.w.) neither induced apoptosis and acute phase reaction nor altered mRNA expression of mouse hepcidin-1 in the livers of C57BL/6NCR mice. In contrast, 6 h after Jo2 injection, the livers of C57BL/6NCR mice exhibited a significant level of apoptosis (P < 0.001) and an increase in SAA3 (P < 0.023) and IL-6 (P < 0.005) expression in the liver. However, mRNA expression of hepcidin-1 in the liver was not significantly altered. Despite the Jo2-induced phosphorylation of Stat3, no occupancy of hepcidin-1 promoter by Stat3 was observed, as shown by ChIP assays. Compared to C57BL/6NCR mice, Jo2 treatment (0.2 µg/g b.w.) of C57BL/6J strain mice for 6 h induced a more prominent activation of apoptosis, liver injury and acute phase reaction. Similar to C57BL/6NCR mice, the level of liver hepcidin-1 mRNA expression in the livers of C57BL/6J mice injected with a sublethal dose of Jo2 (0.2 µg/g b.w.) remained unchanged. The injection of C57BL/6J mice with a higher dose of Jo2 (0.32 µg/g b.w.) did not also alter hepatic hepcidin expression. CONCLUSION: Our findings suggest that human or mouse hepcidin gene expression is not regulated by apoptosis induced via Fas receptor activation in the liver.

Assessing the distribution and protection status of two types of cool environment to facilitate their conservation under climate change.

Strategies to mitigate climate change can protect different types of cool environments. Two are receiving much attention: protection of ephemeral refuges (i.e., places with low maximum temperatures) and of stable refugia (i.e., places that are cool, have a stable environment, and are isolated). Problematically, they are often treated as equivalents. Careful delineation of their qualities is needed to prevent misdirected conservation initiatives; yet, no one has determined whether protecting one protects the other. We mapped both types of cool environments across a large (∼3.4M ha) mixed-use landscape with a geographic information system and conducted a patch analysis to compare their spatial distributions; examine relations between land use and their size and shape; and assess their current protection status. With a modest, but arbitrary, threshold for demarcating both types of cool environments (i.e., values below the 0.025 quantile) there were 146,523 ha of ephemeral refuge (62,208 ha) and stable refugia (62,319 ha). Ephemeral refuges were generally aggregated at high elevation, and more refuge area occurred in protected areas (55,184 ha) than in unprotected areas (7,024 ha). In contrast, stable refugia were scattered across the landscape, and more stable-refugium area occurred on unprotected (40,135 ha) than on protected land (22,184 ha). Although sensitivity analysis showed that varying the thresholds that define cool environments affected outcomes, it also exposed the challenge of choosing a threshold for strategies to address climate change; there is no single value that is appropriate for all of biodiversity. The degree of overlap between ephemeral refuges and stable refugia revealed that targeting only the former for protection on currently unprotected land would capture ∼17% of stable refugia. Targeting only stable refugia would capture ∼54% of ephemeral refuges. Thus, targeting one type of cool environment did not fully protect the other.

Can volunteers collect data that are comparable to professional scientists? A study of variables used in monitoring the outcomes of ecosystem rehabilitation.

Having volunteers collect data can be a cost-effective strategy to complement or replace those collected by scientists. The quality of these data is essential where field-collected data are used to monitor progress against predetermined standards because they provide decision makers with confidence that choices they make will not cause more harm than good. The integrity of volunteer-collected data is often doubted. In this study, we made estimates of seven vegetation attributes and a composite measure of six of those seven, to simulate benchmark values. These attributes are routinely recorded as part of rehabilitation projects in Australia and elsewhere in the world. The degree of agreement in data collected by volunteers was compared with those recorded by professional scientists. Combined results showed that scientists collected data that was in closer agreement with benchmarks than those of volunteers, but when data collected by individuals were analyzed, some volunteers collected data that were in similar or closer agreement, than scientists. Both groups' estimates were in closer agreement for particular attributes than others, suggesting that some attributes are more difficult to estimate than others, or that some are more subjective than others. There are a number of ways in which higher degrees of agreement could be achieved and introducing these will no doubt result in better, more effective programs, to monitor rehabilitation activities. Alternatively, less subjective measures should be sought when developing monitoring protocols. Quality assurance should be part of developing monitoring methods and explicitly budgeted for in project planning to prevent misleading declarations of rehabilitation success.

Regulation of liver hepcidin expression by alcohol in vivo does not involve Kupffer cell activation or TNF-alpha signaling.

Alcohol downregulates hepcidin expression in the liver leading to an increase in intestinal iron transport and liver iron storage. We have previously demonstrated that alcohol-mediated oxidative stress is involved in the inhibition of hepcidin transcription by alcohol in vivo. Kupffer cells and TNF-alpha play a key role in alcohol-induced liver injury. The aim of this study was to define their involvement in the regulation of hepcidin expression by alcohol. Kupffer cells were inactivated or depleted by employing gadolinium chloride and liposomes containing clodronate, respectively. Rats pair fed with the alcohol-Lieber-DeCarli diet for 6 wk and mice fed with 20% ethanol in the drinking water for 1 wk were used as experimental models. Interestingly, alcohol downregulated hepcidin expression in the livers of rats and mice independent of gadolinium chloride or clodronate treatment. One week of alcohol treatment was sufficient to induce a significant increase in TNF-alpha levels and phosphorylation of NF-kappaB subunit p65. The neutralization of TNF-alpha by specific antibodies inhibited p65 phosphorylation. However, neither the neutralization of TNF-alpha nor the lack of TNF-alpha receptor expression reversed alcohol-induced suppression of liver hepcidin expression. The level of alcohol-induced ROS in the liver was also undiminished following Kupffer cell inactivation or depletion. Our results demonstrate that alcohol-induced Kupffer cell activation and TNF-alpha signaling are not involved in the suppression of liver hepcidin expression by alcohol-mediated oxidative stress in vivo. Therefore, these findings suggest that alcohol acts within hepatocytes to suppress hepcidin expression and thereby influences iron homeostasis.

Iron-mediated regulation of liver hepcidin expression in rats and mice is abolished by alcohol.

UNLABELLED: Alcohol reduces and iron increases liver hepcidin synthesis. This study investigates the interaction of alcohol and iron in the regulation of hepcidin messenger RNA (mRNA) expression in animal models. Mice were administered 10% ethanol for 7 days after an iron-overloaded diet. Rats were administered regular or ethanol-Lieber De Carli diets for 7 weeks with or without carbonyl iron. Hfe(-/-) mice were used as a model for genetic iron overload. Hepcidin mRNA expression was determined by real-time polymerase chain reaction (PCR) and northern blotting. Iron elevated and alcohol decreased liver hepcidin expression in mice and rats. Interestingly, despite iron overload, alcohol was capable of suppressing the up-regulation of hepcidin mRNA expression in both models. Liver iron and ferritin protein expression was elevated in alcohol-treated rats, but was not elevated further in rats treated with both iron and alcohol. Duodenal ferroportin protein expression was increased both in alcohol-treated mice and in mice treated with alcohol and iron. Hfe(-/-) mice treated with ethanol for 7 days exhibited a further decrease in hepcidin mRNA expression. The iron-induced increase in DNA-binding activity of the transcription factor CCAAT/enhancer binding protein alpha (C/EBP alpha) was also suppressed by alcohol. CONCLUSION: Alcohol abolishes the iron-induced up-regulation of both liver hepcidin transcription and the DNA-binding activity of C/EBP alpha. Of note, hepcidin protects the body from the harmful effects of iron overload. Our findings therefore suggest that alcohol negates the protective effect of hepcidin, which may have implications for the liver injury observed in alcoholic liver disease and genetic hemochromatosis in combination with alcohol.

Alcohol metabolism-mediated oxidative stress down-regulates hepcidin transcription and leads to increased duodenal iron transporter expression.

Patients with alcoholic liver disease frequently exhibit iron overload in association with increased hepatic fibrosis. Even moderate alcohol consumption elevates body iron stores; however, the underlying molecular mechanisms are unknown. Hepcidin, a circulatory peptide synthesized in the liver, is a key mediator of iron metabolism. Ethanol metabolism significantly down-regulated both in vitro and in vivo hepcidin mRNA and protein expression. 4-Methylpyrazole, a specific inhibitor of the alcohol-metabolizing enzymes, abolished the effects of ethanol on hepcidin. However, ethanol did not alter the expression of transferrin receptor1 and ferritin or the activation of iron regulatory RNA-binding proteins, IRP1 and IRP2. Mice maintained on 10-20% ethanol for 7 days displayed down-regulation of liver hepcidin expression without changes in liver triglycerides or histology. This was accompanied by elevated duodenal divalent metal transporter1 and ferroportin protein expression. Injection of hepcidin peptide negated the effect of ethanol on duodenal iron transporters. Ethanol down-regulated hepcidin promoter activity and the DNA binding activity of CCAAT/enhancer-binding protein alpha (C/EBPalpha) but not beta. Interestingly, the antioxidants vitamin E and N-acetylcysteine abolished both the alcohol-mediated down-regulation of C/EBPalpha binding activity and hepcidin expression in the liver and the up-regulation of duodenal divalent metal transporter 1. Collectively, these findings indicate that alcohol metabolism-mediated oxidative stress regulates hepcidin transcription via C/EBPalpha, which in turn leads to increased duodenal iron transport.

Emerging therapeutics for chronic hepatitis B.

Hepatitis B is a global health problem. Patients with chronic hepatitis B (CHB) carry a significant risk to eventually develop cirrhotic liver disease. Recent therapeutic advances against CHB offer excellent potential for long-term suppression of hepatitis B virus (HBV) replication during antiviral therapy, and occasionally a durable remission off medication. Selection of appropriate patients for antiviral therapy depends on identification of HBV replication and an elevated alanine aminotransferase level or histologic liver injury. Pegylated interferon alpha offers potent immunomodulatory and antiviral activity with the potential for durability, but also with adverse effects and significant cost. The nucleoside or nucleotide analogs, lamivudine, adefovir, and entecavir, suppress HBV replication and are extremely well-tolerated, but long-term or even lifelong therapy is required. Most experience has been gained with lamivudine, but viral resistance occurs frequently. Newer analogs appear to be relatively free of this problem. Approaches using a combination of agents have promise, but have yet to be proven superior to individual drugs alone.

Recent developments in the pathophysiology of cholestasis.

The past decade has brought tremendous growth in the under-standing of the pathophysiologic mechanisms involved in cholestasis, both at the genetic and acquired levels. The discovery and characterization of an array of hepatobiliary transport proteins, the nuclear receptors that regulate them, and the potential clinical implications of these defective, altered, or variably expressed proteins are the key elements of our current understanding of cholestasis. It is hoped that future studies will enhance therapeutic options and the ability to care for patients with cholestatic disorders.

Gastrointestinal disorders of the critically ill. Cholestasis of sepsis.

Cholestasis of sepsis is a form of hepatocellular cholestasis that occurs as a result of sepsis. Usually, prior to the development of cholestasis, the manifestations of sepsis dominate the clinical picture. The occurrence of cholestasis is without direct bacterial involvement of the biliary system and appears to be mediated systemically by pro-inflammatory cytokines. These cytokines are released in response to the vigorous inflammatory reaction mediated by endotoxinaemia and bacterial wall lipopolysaccharides. The principal cytokines involved are the pro-inflammatory tumour necrosis factor-alpha (TNF-alpha), interleukin (IL) 1-beta and IL-6. Interplay between these cytokines and a series of hepatocyte membrane transporters appears to result in the cholestasis. Management principles focus upon the control of sepsis.

Metabolic liver disease in the young adult.

This chapter describes the gene mutations, phenotypes, diagnosis and therapy of the common metabolic liver diseases in young adulthood: haemochromatosis, Wilson disease, alpha(1)-anti-trypsin deficiency and cystic fibrosis. The remarkable variability of the phenotypical expression of the mutated genotypes makes screening recommendations and the establishment of prognosis for these liver disorders in young adults problematical. The diagnosis and therapy of the young adult with metabolic liver disease is discussed, with an emphasis on maintaining quality-of-life and balancing the importance of early intervention with the stigmatization of the diagnosis of potentially life-threatening liver disease. There is a critical need for the development of biochemical markers that would predict the risk of expression of clinical phenotypes and prognosis.