Disruption of the histidine triad nucleotide-binding hint2 gene in mice affects glycemic control and mitochondrial function

The histidine triad nucleotide-binding (Hint2) protein is a mitochondrial adenosine phosphoramidase expressed in liver and pancreas. Its physiological function is unknown. To elucidate the role of Hint2 in liver physiology, the Hint2 gene was deleted. Hint2(-/-) and Hint2(+/+) mice were generated in a mixed C57Bl6/J x 129Sv background. At 20 weeks, the phenotypic changes in Hint2(-/-) relative to Hint2(+/+) mice were an accumulation of hepatic triglycerides, decreased tolerance to glucose, a defective counter-regulatory response to insulin-provoked hypoglycaemia, an increase in plasma interprandial insulin but a decrease in glucose stimulated insulin secretion and defective thermoregulation upon fasting. Leptin mRNA in adipose tissue and plasma leptin were elevated. In mitochondria from Hint2(-/-) hepatocytes, state 3 respiration was decreased, a finding confirmed in HepG2 cells where HINT2 mRNA was silenced. The linked complex II to III electron transfer was decreased in Hint2(-/-) mitochondria, which was accompanied by a lower content of coenzyme Q. HIF-2α expression and the generation of reactive oxygen species were increased. Electron microscopy of mitochondria in Hint2(-/-) mice aged 12 months revealed clustered, fused organelles. The hepatic activities of 3-hydroxyacyl-CoA dehydrogenase short chain and glutamate dehydrogenase (GDH) were decreased by 68% and 60%, respectively, without a change in protein expression. GDH activity was similarly decreased in HINT2-silenced HepG2 cells. When measured in the presence of purified sirtuin 3, latent GDH activity was recovered (126% in Hint2(-/-) vs. 83% in Hint2(+/+) ). This suggests a greater extent of acetylation in Hint2(-/-) than in Hint2(+/+) . Conlusions: Hint2 positively regulates mitochondrial lipid metabolism and respiration, and glucose homeostasis. The absence of Hint2 provokes mitochondrial deformities and a change in the pattern of acetylation of selected proteins. (HEPATOLOGY 2012.).

Serum ferritin levels are associated with a distinct phenotype of chronic hepatitis C poorly responding to pegylated interferon-alpha and ribavirin therapy

Elevated serum ferritin levels may reflect a systemic inflammatory state as well as increased iron storage, both of which may contribute to an unfavorable outcome of chronic hepatitis C (CHC). We therefore performed a comprehensive analysis of the role of serum ferritin and its genetic determinants in the pathogenesis and treatment of CHC. To this end, serum ferritin levels at baseline of therapy with pegylated interferon-alpha and ribavirin or before biopsy were correlated with clinical and histological features of chronic hepatitis C virus (HCV) infection, including necroinflammatory activity (N = 970), fibrosis (N = 980), steatosis (N = 886), and response to treatment (N = 876). The association between high serum ferritin levels (> median) and the endpoints was assessed by logistic regression. Moreover, a candidate gene as well as a genome-wide association study of serum ferritin were performed. We found that serum ferritin ≥ the sex-specific median was one of the strongest pretreatment predictors of treatment failure (univariate P < 0.0001, odds ratio [OR] = 0.45, 95% confidence interval [CI] = 0.34-0.60). This association remained highly significant in a multivariate analysis (P = 0.0002, OR = 0.35, 95% CI = 0.20-0.61), with an OR comparable to that of interleukin (IL)28B genotype. When patients with the unfavorable IL28B genotypes were stratified according to high versus low ferritin levels, SVR rates differed by > 30% in both HCV genotype 1- and genotype 3-infected patients (P < 0.001). Serum ferritin levels were also independently associated with severe liver fibrosis (P < 0.0001, OR = 2.67, 95% CI = 1.68-4.25) and steatosis (P = 0.002, OR = 2.29, 95% CI = 1.35-3.91), but not with necroinflammatory activity (P = 0.3). Genetic variations had only a limited impact on serum ferritin levels. Conclusion: In patients with CHC, elevated serum ferritin levels are independently associated with advanced liver fibrosis, hepatic steatosis, and poor response to interferon-alpha-based therapy.

Intestinal bacterial translocation in rats with cirrhosis is related to compromised Paneth cell antimicrobial host defense

Liver cirrhosis is associated with bacterial translocation (BT) and endotoxemia. Most translocating bacteria belong to the common intestinal microbiota, suggesting a breakdown of intestinal barrier function. We hypothesized that diminished mucosal antimicrobial host defense could predispose to BT. Two rodent models of portal hypertension with increased BT were used, CCl(4)-induced ascitic cirrhosis and 2-day portal vein-ligated (PVL) animals. BT was assessed by standard microbiological techniques on mesenteric lymph nodes. Total RNA was isolated systematically throughout the intestinal tract, and expression of Paneth cell α-cryptdins and β-defensins was determined by real-time quantitative polymerase chain reaction (qPCR). To determine functional consequences, mucosal antimicrobial activity was assessed with a fluorescence-activated cell sorting assay. BT was detectable in 40% of rats with cirrhosis. Compared with the group without BT, these animals exhibited diminished intestinal Paneth cell α-cryptdin 5 and 7 expression. In contrast, PVL was associated with BT in all animals but did not affect antimicrobial peptides. The decrease in Paneth cell antimicrobials was most pronounced in the ileum and the coecum. Other antimicrobials showed no changes or even an induction in the case of BT at different sites. Antimicrobial activity toward different commensal strains was reduced, especially in the distal ileum and the cecum in experimental cirrhosis with BT (excluding PVL). Conclusion: Compromised Paneth cell antimicrobial host defense seems to predispose to BT in experimental cirrhosis. Understanding this liver-gut axis including the underlying mechanisms could help us to find new treatment avenues.

Keratin variants associate with progression of fibrosis during chronic hepatitis C infection

Keratins 8 and 18 (K8/K18) protect the liver from various forms of injury. Studies of liver explants from a large cohort of U.S. patients showed that K8/K18 mutations confer a risk to developing end-stage liver diseases, though which diseases are preferentially involved is unknown. We tested the hypothesis that K8/K18 variants are associated with chronic hepatitis C (CHC) and that their presence correlates with progression of fibrosis. Genomic DNA was isolated from peripheral blood of a well-characterized German cohort of 329 patients with CHC infection. Exonic regions were PCR-amplified and analyzed using denaturing high-performance liquid chromatography and DNA sequencing. Our findings showed: (1) amino acid altering keratin heterozygous variants in 24 of 329 CHC patients (7.3%) and non-coding heterozygous variants in 26 patients (7.8%), and (2) 3 new exonic K8 variants (T26R/G55A/A359T); 6 novel non-coding variants and one K18 coding variant (K18 S230T; 2 patients). The most common variants were K8 R341H (10 patients), K8 G62C (6 patients) and K8 I63V (4 patients). A novel and exclusive association of an intronic KRT8 IVS7+10delC deletion in all 10 patients with K8 R341H was observed. Notably, there was a significant association of exonic, but not of intronic K8 variants with increased fibrosis. In conclusion, previously described and novel K8 variants are present in a German population and collectively associate with progression of fibrosis in CHC infection. The unique 100% segregation of the most common K8 variant, R341H, with an intronic deletion suggests that one of these two genetic changes might lead to the other.

Interferon-inducible expression of APOBEC3 editing enzymes in human hepatocytes and inhibition of hepatitis B virus replication

Hypermutations in hepatitis B virus (HBV) DNA by APOBEC3 cytidine deaminases have been detected in vitro and in vivo, and APOBEC3G (A3G) and APOBEC3F (A3F) have been shown to inhibit the replication of HBV in vitro, but the presumably low or even absent hepatic expression of these enzymes has raised the question as to their physiological impact on HBV replication. We show that normal human liver expresses the mRNAs of APOBEC3B (A3B), APOBEC3C (A3C), A3F, and A3G. In primary human hepatocytes, interferon alpha (IFN-alpha) stimulated the expression of these cytidine deaminases up to 14-fold, and the mRNAs of A3G, A3F, and A3B reached expression levels of 10%, 3%, and 3%, respectively, relative to GAPDH mRNA abundance. On transfection, the full-length protein A3B(L) inhibited HBV replication in vitro as efficiently as A3G or A3F, whereas the truncated splice variant A3B(S) and A3C had no effect. A3B(L) and A3B(S) were detected predominantly in the nucleus of uninfected cells; however, in HBV-expressing cells both proteins were found also in the cytoplasm and were associated with HBV viral particles, similarly to A3G and A3F. Moreover, A3G, A3F, and A3B(L), but not A3B(S), induced extensive G-to-A hypermutations in a fraction of the replicated HBV genomes. In conclusion, the editing enzymes A3B(L), A3F, and most markedly A3G, which are expressed in liver and up-regulated by IFN-alpha in hepatocytes, are candidates to contribute to the noncytolytic clearance of HBV.

Impaired 11 beta-hydroxysteroid dehydrogenase contributes to renal sodium avidity in cirrhosis: hypothesis or fact?

Exaggerated renal sodium retention with concomitant potassium loss is a hallmark of cirrhosis and contributes to the accumulation of fluid as ascites, pleural effusion, or edema. This apparent mineralocorticoid effect is only partially explained by increased aldosterone concentrations. I present evidence supporting the hypothesis that cortisol confers mineralocorticoid action in cirrhosis. The underlying molecular pathology for this mineralocorticoid receptor (MR) activation by cortisol is a reduced activity of the 11 beta-hydroxysteroid dehydrogenase type 2, an enzyme protecting the MR from promiscuous activation by cortisol in healthy mammalians.

Sodium retention and ascites formation in a cholestatic mice model: role of aldosterone and mineralocorticoid receptor?

Renal sodium retention in experimental liver cirrhosis originates from the distal nephron sensitive to aldosterone. The aims of this study were to (1) determine the exact site of sodium retention along the aldosterone-sensitive distal nephron, and (2) to evaluate the role of aldosterone and mineralocorticoid receptor activation in this process. Liver cirrhosis was induced by bile duct ligation in either adrenal-intact or corticosteroid-clamped mice. Corticosteroid-clamp was achieved through adrenalectomy and corticosteroid supplementation with aldosterone and dexamethasone via osmotic minipumps. 24-hours renal sodium balance was evaluated in metabolic cages. Activity and expression of sodium- and potassium-dependent adenosine triphosphatase were determined in microdissected segments of nephron. Within 4-5 weeks, cirrhosis induced sodium retention in adrenal-intact mice and formation of ascites in 50% of mice. At that time, sodium- and potassium-dependent adenosine triphosphatase activity increased specifically in cortical collecting ducts. Hyperaldosteronemia was indicated by increases in urinary aldosterone excretion and in sgk1 (serum- and glucocorticoid-regulated kinase 1) mRNA expression in collecting ducts. Corticosteroid-clamp prevented induction of sgk1 but not cirrhosis-induced sodium retention, formation of ascites and stimulation of sodium- and potassium-dependent adenosine triphosphatase activity and expression (mRNA and protein) in collecting duct. These findings demonstrate that sodium retention in cirrhosis is independent of hyperaldosteronemia and of the activation of mineralocorticoid receptor. CONCLUSION: Bile duct ligation in mice induces cirrhosis which, within 4-5 weeks, leads to the induction of sodium- and potassium-dependent adenosine triphosphatase in cortical collecting ducts, to renal sodium retention and to the formation of ascites. Sodium retention, ascites formation and induction of sodium- and potassium-dependent adenosine triphosphatase are independent of the activation of mineralocorticoid receptors by either aldosterone or glucocorticoids.

Regulation of human liver delta-aminolevulinic acid synthase by bile acids

Aminolevulinic acid synthase 1 (ALAS1) is the rate-limiting enzyme of heme synthesis in the liver and is highly regulated to adapt to the metabolic demand of the hepatocyte. In the present study, we describe human hepatic ALAS1 as a new direct target of the bile acid-activated nuclear receptor farnesoid X receptor (FXR). Experiments in primary human hepatocytes and in human liver slices showed that ALAS1 messenger RNA (mRNA) and activity is increased upon exposure to chenodeoxycholic acid (CDCA), the most potent natural FXR ligand, or the synthetic FXR-specific agonist GW4064. Moreover, overexpression of a constitutively active form of FXR further increased ALAS1 mRNA expression. In agreement with these observations, an FXR response element was identified in the 5' flanking region of human ALAS1 and characterized in reporter gene assays. A highly conserved FXR binding site (IR1) within a 175-bp fragment at -13 kilobases upstream of the transcriptional start site was able to trigger an FXR-specific increase in luciferase activity upon CDCA treatment. Site-directed mutagenesis of IR1 abolished this effect. Binding of FXR/retinoid acid X receptor heterodimers was demonstrated by mobility gel shift experiments. Conclusion: These data strongly support a role of bile acid-activated FXR in the regulation of human ALAS1 and, consequently, hepatic porphyrin and heme synthesis. These data also suggest that elevated endogenous bile acids may precipitate neuropsychiatric attacks in patients with acute hepatic porphyrias.

Self-reactivity in the dimeric intravenous immunoglobulin fraction

Therapeutic intravenous immunoglobulin (IVIg) preparations contain antibodies reflecting the cumulative antigen experience of the donor population. IVIg contains variable amounts of monomeric and dimeric IgG, but there is little information available on their comparative antibody specificities. We have isolated highly purified fractions of monomeric and dimeric IgG by size-exclusion chromatography. Following treatment of all fractions at pH4, analyses by immunodot and immunocytology on human cell lines showed a preferential recognition of autoantigens in the dimeric IgG fraction. Investigation of the HEp-2 cytoplasmic proteome by 2D-PAGE, Western blot, and subsequent identification of IVIg reactive spots by mass spectrometry (LC-MS/MS) showed that IVIg recognized only a restricted set of the total proteins. Similar experiments showed that more antigens were recognized by the dimeric IgG fraction, especially when the dissociated dimer fraction was used, as compared to its monomeric counterpart. These observations are consistent with idiotype-anti-idiotype masking of auto-specific Abs in the dimeric fraction of IVIg.