Truncating mutations in the Wilson disease gene ATP7B are associated with very low serum ceruloplasmin oxidase activity and an early onset of Wilson disease

<p>Abstract</p> <p>Background</p> <p>Mutations in the gene ATP7B cause Wilson disease, a copper storage disorder with a high phenotypic and genetic heterogeneity. We aimed to evaluate whether 'severe' protein-truncating ATP7B mutations (SMs) are associated with low serum ceruloplasmin oxidase activities and an early age of onset when compared to missense mutations (MMs).</p> <p>Methods</p> <p>The clinical phenotype of 59 genetically confirmed WD patients was analyzed retrospectively. Serum ceruloplasmin was measured by its oxidase activity with <it>o</it>-dianisidine dihydrochloride as substrate and immunologically.</p> <p>Results</p> <p>Thirty-nine patients had two MMs, 15 had the genotype SM/MM, and 5 patients had two SMs on their ATP7B alleles. Enzymatic and immunologic serum ceruloplasmin levels differed significantly between the three groups (P < 0.001 and P < 0.01, respectively). The lowest levels were measured in patients with two SMs (0.0 U/L; IQR, 0.0-0.0 U/L and 0.02 g/L; IQR, 0.01-0.02 g/L, respectively) and the highest in patients with two MMs (17.8 U/L; IQR, 5.8-35.1 U/L and 0.11 g/L; IQR,0.10-0.17 g/L, respectively). The age of onset was also significantly different between the three patient groups (P < 0.05), with SM/SM patients showing the earliest onset (13 years; IQR, 9-13 years) and patients with two MMs showing the latest onset (22 years; IQR, 14-27 years). By ROC curve analysis a ceruloplasmin oxidase level ≤ 5 U/L can predict the presence of at least one SM with a sensitivity of 80% and a specificity of 79.5%.</p> <p>Conclusions</p> <p>In our German study cohort truncating ATP7B mutations were associated with lower ceruloplasmin serum oxidase levels and an earlier age of onset when compared to MMs. Measurement of serum ceruloplasmin oxidase might help to predict presence of truncating ATP7B mutations and might facilitate the mutation analysis.</p

Deficiency of iPLA2β Primes Immune Cells for Proinflammation: Potential Involvement in Age-Related Mesenteric Lymph Node Lymphoma

Proinflammation can predispose the body to autoimmunity and cancer. We have reported that iPLA2β−/− mice are susceptible to autoimmune hepatitis and colitis. Here we determined whether cytokine release by immune cells could be affected by iPLA2β deficiency alone or combined with CD95/FasL-antibody treatment in vivo. We also determined whether cancer risk could be increased in aged mutant mice. Immune cells were isolated from 3-month old male WT and iPLA2β−/− mice, and some were injected with anti-CD95/FasL antibody for 6 h. Kupffer cells (KC) or splenocytes and liver lymphocytes were stimulated in vitro by lipopolysaccharide or concanavalinA, respectively. Whole-body iPLA2β deficiency caused increased apoptosis in liver, spleen, and mesenteric lymph node (MLN). KC from mutant mice showed suppressed release of TNFα and IL-6, while their splenocytes secreted increased levels of IFNγ and IL-17a. Upon CD95/FasL activation, the mutant KC in turn showed exaggerated cytokine release, this was accompanied by an increased release of IFNγ and IL-17a by liver lymphocytes. Aged iPLA2β−/− mice did not show follicular MLN lymphoma commonly seen in aged C57/BL6 mice. Thus, iPLA2β deficiency renders M1- and Th1/Th17-proinflammation potentially leading to a reduction in age-related MLN lymphoma during aging

Ageing sensitized by iPLA 2 β deficiency induces liver fibrosis and intestinal atrophy involving suppression of homeostatic genes and alteration of intestinal lipids and bile acids

Ageing is a major risk factor for various forms of liver and gastrointestinal (GI) disease and genetic background may contribute to the pathogenesis of these diseases. Group VIA phospholipase A2 or iPLA(2)beta is a homeostatic PLA2 by playing a role in phospholipid metabolism and remodeling. Global iPLA(2)beta(-/-) mice exhibit aged-dependent phenotypes with body weight loss and abnormalities in the bone and brain. We have previously reported the abnormalities in these mutant mice showing susceptibility for chemical-induced liver injury and colitis. We hypothesize that iPLA(2)beta deficiency may sensitize with ageing for an induction of GI injury. Male wild-type and iPLA(2)beta(-/-) mice at 4 and 20-22 months of age were studied. Aged, but not young, iPLA(2)beta(-/-) mice showed increased hepatic fibrosis and biliary ductular expansion as well as severe intestinal atrophy associated with increased apoptosis, pro-inflammation, disrupted tight junction, and reduced number of mucin-containing globlet cells. This damage was associated with decreased expression of intestinal endoplasmic stress XBP1 and its regulator HNF1 alpha, FATP4, ACSL5, bile-acid transport genes as well as nuclear receptors LXR alpha and FXR. By LC/MS-MS profiling, iPLA(2)beta deficiency in aged mice caused an increase of intestinal arachidonate-containing phospholipids concomitant with a decrease in ceramides. By the suppression of intestinal FXR/FGF-15 signaling, hepatic bile-acid synthesis gene expression was increased leading to an elevation of secondary and hydrophobic bile acids in liver, bile, and intestine. In conclusions, ageing sensitized by iPLA(2)beta deficiency caused a decline of key intestinal homeostatic genes resulting in the development of GI disease in a gut-to-liver manner

Group VIA phospholipase A2 deficiency in mice chronically fed with high-fat-diet attenuates hepatic steatosis by correcting a defect of phospholipid remodeling

A defect of hepatic remodeling of phospholipids (PL) is seen in non-alcoholic fatty liver disease and steatohepatitis (NASH) indicating pivotal role of PL metabolism in this disease. The deletion of group VIA calcium independent phospholipase A2 (iPla2 beta) protects ob/ob mice from hepatic steatosis (BBAIip 1861, 2016, 440-461), however its role in high-fat diet (HFD)-induced NASH is still elusive. Here, wild-type and iPla2 beta-null mice were subjected to chronic feeding with HFD for 6 months. We showed that protection was observed in iPla2 beta-null mice with an attenuation of diet-induced body and liver-weight gains, liver enzymes, serum free fatty acids as well as hepatic TG and steatosis scores. iPla2 beta deficiency under HFD attenuated the levels of 1-stearoyl lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), and lysophosphatidylinositol (LPI) as well as elevation of hepatic arachidonate, arachidonate-containing cholesterol esters and prostaglandin E-2. More importantly, this deficiency rescued a defect in PL remodeling and attenuated the ratio of saturated and unsaturated PL. The protection by iPla2 beta deficiency was not observed during short-term HFD feeding of 3 or 5 weeks which showed no PL remodeling defect. In addition to PC/PE, this deficiency reversed the suppression of PC/PI and PE/PI among monounsaturated PL. However, this deficiency did not modulate hepatic PL contents and PL ratios in ER fractions, ER stress, fibrosis, and inflammation markers. Hence, iPla2 beta inactivation protected mice against hepatic steatosis and obesity during chronic dietary NASH by correcting PL remodeling defect and PI composition relative to PC and PE

iPLA2β deficiency attenuates obesity and hepatic steatosis in ob / ob mice through hepatic fatty-acyl phospholipid remodeling

PLA2G6 or GVIA calcium-independent PLA2 (iPLA2 beta) is identified as one of the NAFLD modifier genes in humans, and thought to be a target for NAFLD therapy. iPLA2 beta is known to play a house-keeping role in phospholipid metabolism and remodeling. However, its role in NAFLD pathogenesis has not been supported by results obtained from high-fat feeding of iPLA2 beta-null (PKO) mice. Unlike livers of human NAFLD and genetically obese rodents, fatty liver induced by high-fat diet is not associated with depletion of hepatic phospholipids. We therefore tested whether iPLA2 beta could regulate obesity and hepatic steatosis in leptin-deficient mice by cross-breeding PKO with ob/ob mice to generate ob/ob-PKO mice. Here we observed an improvement in ob/ob-PKO mice with significant reduction in serum enzymes, lipids, glucose, insulin as well as improved glucose tolerance, and reduction in islet hyperplasia. The improvement in hepatic steatosis measured by liver triglycerides, fatty acids and cholesterol esters was associated with decreased expression of PPAR gamma and de novo lipogenesis genes, and the reversal of beta-oxidation gene expression. Notably, ob/ob livers contained depleted levels of lysophospholipids and phospholipids, and iPLA2 beta deficiency in ob/ob-PKO livers lowers the former, but replenished the latter particularly phosphatidylethanolamine (PE) and phosphatidylcholine (PC) that contained arachidonic (AA) and docosahexaenoic (DHA) acids. Compared with WT livers, PKO livers also contained increased PE and PC containing AA and DHA. Thus, iPLA2 beta deficiency protected against obesity and ob/ob fatty liver which was associated with hepatic fatty-acyl phospholipid remodeling. Our results support the deleterious role of iPLA2 beta in severe obesity associated NAFLD. (C) 2016 Elsevier B.V. All rights reserved

Constitutive oxidants from hepatocytes of male iPLA2β-null mice increases the externalization of phosphatidylethanolamine on plasma membrane

We have found that group VIA calcium-independent phospholipase A2 (iPLA2 beta) has specificity for hydrolysis of phosphatidylethanolamine (PE) in mouse livers. Phospholipids (PLs) are transported to plasma membrane and some PLs including PE are externalized to maintain membrane PL asymmetry. Here we demonstrated that hepatocytes of iPLA2 beta-null (KO) mice showed an increase in PE containing palmitate and oleate. We aimed to examine whether externalization of PE on the outer leaflets could be affected by iPLA2 beta deficiency and its modulation by reactive oxygen species (ROS) or apoptosis. As duramycin has high affinity to PE, we used duramycin conjugated with biotin (DLB) and streptavidin 488 as a probe for detection of externalized PE. Compared to WT, naive KO hepatocytes showed an increase in both PE externalization and ROS generation. These events were observed in male but not in female KO mice. Hydrogen peroxide or menadione treatment enhanced PE externalization to the same extent for both male/female WT and KO hepatocytes. By indirect immunofluorescence, DLB-streptavidin staining was observed as small punctuated spots on the cell surface of menadione-treated KO hepatocytes. Unlike the reported PS externalization, CD95/FasL treatment did not lead to any increase in PE externalization, and iPLA2 beta deficiency-dependent PE externalization was also not correlated with apoptosis. Thus, constitutive (but not induced) ROS generation in iPLA2 beta-deficient hepatocytes leads to PE externalization observed only in male mice. Such PE externalization may imply detrimental effects regarding further oxidation of PE fatty acids and the binding with pathogens on the outer leaflets of hepatocyte plasma membrane

iPla2β deficiency in mice fed with MCD diet does not correct the defect of phospholipid remodeling but attenuates hepatocellular injury via an inhibition of lipid uptake genes

Group VIA calcium-independent phospholipase A2 (iPla2 beta) is among modifier genes of non-alcoholic fatty liver disease which leads to non-alcoholic steatohepatitis (NASH). Consistently, iPla2 beta deletion protects hepatic steatosis and obesity in genetic ob/ob and obese mice chronically fed with high-fat diet by replenishing the loss of hepatic phospholipids (PL). As mouse feeding with methionine- and choline-deficient (MCD) diet is a model of lean NASH, we tested whether iPla2 beta-null mice could still be protected since PL syntheses are disturbed. MCD-diet feeding of female wild-type for 5 weeks induced hepatic steatosis with a severe reduction of body and visceral fat weights concomitant with a decrease of hepatic phosphatidylcholine. These parameters were not altered in MCD-fed iPla2 beta-null mice. However, iPla2 beta deficiency attenuated MCD-induced elevation of serum transaminase activities and hepatic expression of fatty-acid translocase Cd36, fatty-acid binding protein-4, peroxisome-proliferator activated receptor gamma, and HDL-uptake scavenger receptor B type 1. The reduction of lipid uptake genes was consistent with a decrease of hepatic esterified and unesterified fatty acids and cholesterol esters. On the contrary, iPla2 beta deficiency under MCD did not have any effects on inflammasomes and proinflammatory markers but exacerbated hepatic expression of myofibroblast a-smooth muscle actin and vimentin. Thus, without any rescue of PL loss, iPla2 beta inactivation attenuated hepatocellular injury in MCD-induced NASH with a novel mechanism of lipid uptake inhibition. Taken together, we have shown that iPla2 beta mediates hepatic steatosis and lipotoxicity in hepatocytes in both obese and lean NASH, but elicits exacerbated liver fibrosis in lean NASH likely by affecting other cell types

Methionine- and Choline-Deficient Diet Enhances AdiposeLipolysis and Leptin Release in aP2-Cre Fatp4-Knockout Mice

Scope Inadequate intake of choline commonly leads to liver diseases. Methionine‐ and choline‐deficient diets (MCDD) induce fatty liver in mice which is partly mediated by triglyceride (TG) lipolysis in white adipose tissues (WATs). Because Fatp4 knockdown has been shown to increase adipocyte lipolysis in vitro, here, the effects of MCDD on WAT lipolysis in aP2‐Cre Fatp4‐knockout (Fatp4A−/−) mice are determined. Methods and Results Isolated WATs of Fatp4A−/− mice exposed to MCD medium show an increase in lipolysis, and the strongest effect is noted on glycerol release from subcutaneous fat. Fatp4A−/− mice fed with MCDD for 4 weeks show an increase in serum glycerol, TG, and leptin levels associated with the activation of hormone‐sensitive lipase in subcutaneous fat. Chow‐fed Fatp4A−/− mice also show an increase in serum leptin and very‐low‐density lipoproteins as well as liver phosphatidylcholine and sphingomyelin levels. Both chow‐ and MCDD‐fed Fatp4A−/− mice show a decrease in serum ketone and WAT sphingomyelin levels which supports a metabolic shift to TG for subsequent WAT lipolysis Conclusions Adipose Fatp4 deficiency leads to TG lipolysis and leptin release, which are exaggerated by MCDD. The data imply hyperlipidemia risk by a low dietary choline intake and gene mutations that increase adipose TG levels

Elevation of blood lipids in hepatocyte-specific fatty acid transport 4-deficient mice fed with high glucose diets

Fatty acid transport protein4 (FATP4) is upregulated in acquired and central obesity and its polymorphisms are associated with blood lipids and insulin resistance. Patients with FATP4 mutations and mice with global FATP4 deletion exhibit skin abnormalities characterized as ischthyosis prematurity syndrome (IPS). Cumulating data have shown that an absence of FATP4 increases the levels of cellular triglycerides (TG). However, FATP4 role and consequent lipid and TG metabolism in the hepatocyte is still elusive. Here, hepatocyte-specific FATP4 deficient (Fatp4(L-/-)) mice were generated. When fed with chow, these mutant mice displayed no phenotypes regarding blood lipids. However when fed low-fat/high-sugar (HS) or high-fat/high-sugar (HFS) for 12 weeks, Fatp4(L-/-) mice showed a significant increase of plasma TG, free fatty acids and glycerol when compared with diet-fed control mice. Interestingly, Fatp4(L-/-) mice under HS diet had lower body and liver weights and they were not protected from HFS-induced body weight gain and hepatic steatosis. Male mutant mice were more sensitive to HFS diet than female mutant mice. Glucose intolerance was observed only in female Fatp4(L-/-) mice fed with HS diet. Lipidomics analyses revealed that hepatic phospholipids were not disturbed in mutant mice under both diets. Thus, hepatic FATP4 deletion rendered an increase of blood lipids including glycerol indicating a preferential fatty-acid channeling to TG pools that are specifically available for lipolysis. Our results imply a possible risk of hyperlipidemia as a result of abnormal metabolism in liver in IPS patients with FATP4 mutations who consume high-sugar diets