Alterations of lipid metabolism in Wilson disease

<p>Abstract</p> <p>Introduction</p> <p>Wilson disease (WD) is an inherited disorder of human copper metabolism, characterised by accumulation of copper predominantly in the liver and brain, leading to severe hepatic and neurological disease. Interesting findings in animal models of WD (Atp7b^-/-and LEC rats) showed altered lipid metabolism with a decrease in the amount of triglycerides and cholesterol in the serum. However, serum lipid profile has not been investigated in large human WD patient cohorts to date.</p> <p>Patients and Methods</p> <p>This cohort study involved 251 patients examined at the Heidelberg and Dresden (Germany) University Hospitals. Patients were analysed on routine follow-up examinations for serum lipid profile, including triglycerides, cholesterol, high density lipoprotein (HDL) and low density lipoprotein (LDL). Data on these parameters at time of diagnosis were retrieved by chart review where available. For statistical testing, patients were subgrouped by sex, manifestation (hepatic, neurological, mixed and asymptomatic) and treatment (D-penicillamine, trientine, zinc or combination).</p> <p>Results</p> <p>A significant difference in total serum cholesterol was found in patients with hepatic symptoms, which diminished under therapy. No alterations were observed for HDL, LDL and triglycerides.</p> <p>Conclusion</p> <p>Contradictory to previous reports using WD animal models (Atp7b^-/-and LEC rats), the most obvious alteration in our cohort was a lower serum cholesterol level in hepatic-affected patients, which might be related to liver injury. Our data suggested unimpaired cholesterol metabolism in Wilson disease under therapy, independent of the applied medical treatment.</p

ATP Release from Vascular Endothelia Occurs Across Cx43 Hemichannels and Is Attenuated during Hypoxia

Background: Extracellular ATP is an important signaling molecule for vascular adaptation to limited oxygen availability (hypoxia). Here, we pursued the contribution of vascular endothelia to extracellular ATP release under hypoxic conditions. Methodology, Principal Findings: We gained first insight from studying ATP release from endothelia (HMEC-1) pre-exposed to hypoxia. Surprisingly, we found that ATP release was significantly attenuated following hypoxia exposure (2 % oxygen, 2263 % after 48 h). In contrast, intracellular ATP was unchanged. Similarly, lactate-dehydrogenase release into the supernatants was similar between normoxic or hypoxic endothelia, suggesting that differences in lytic ATP release between normoxia or hypoxia are minimal. Next, we used pharmacological strategies to study potential mechanisms for endothelialdependent ATP release (eg, verapamil, dipyridamole, 18-alpha-glycyrrhetinic acid, anandamide, connexin-mimetic peptides). These studies revealed that endothelial ATP release occurs – at least in part- through connexin 43 (Cx43) hemichannels. A real-time RT-PCR screen of endothelial connexin expression showed selective repression of Cx43 transcript and additional studies confirmed time-dependent Cx43 mRNA, total and surface protein repression during hypoxia. In addition, hypoxia resulted in Cx43-serine368 phosphorylation, which is known to switch Cx43 hemi-channels from an open to a closed state. Conclusions/Significance: Taken together, these studies implicate endothelial Cx43 in hypoxia-associated repression o

Compositional Changes Among Triglycerides and Phospholipids During FATP4 Sensitization with Palmitate Lead to ER Stress in Cultured Cells

FATP4 expression is upregulated in acquired obesity and polymorphisms in the FATP4 gene are associated with increased blood triglycerides (TG). Since non-alcoholic fatty liver disease (NAFLD) is a manifestation of obesity, it is of interest to understand how FATP4 upon fat overload mediates alterations of lipids that lead to pathogenesis of fatty liver. A model system with FATP4-overexpressed (FATP4) and control Huh-7 cell lines is used to determine FATP4 response to palmitate (Pal). By lipidomics methods, cellular phospholipids (PL), neutral lipids TG, free cholesterol (FC), and cholesterol esters (CE) as well as lipoprotein secretion are determined. Without Pal, FATP4 cells show a significant increase of TG, phosphatidylcholine (PC), and the release of TG-rich and cholesterol-rich low density lipoproteins. Pal-treated FATP4 cells show an increase of all PL subclasses except for phosphatidylserine (PS), TG, FC, and CE. By analyses of ratios among PL subclasses and between PL and neutral lipids, it is determined that FATP4 in response to Pal causes sequentially fatty-acid channeling with a shift from PC and phosphatidylinositol (PI) to neutral lipids and from neutral lipids to PS and phosphatidylethanolamine (PE). This response is concomitant with an activation of CAAT/enhancer binding homologous protein and MAP Kinase JNK. Thus, FATP4 overexpression in response to Pal leads to enhancement of neutral lipids which play a central role in fatty-acid distribution from PC and PI to PE and PS that led to an alteration of ER and mitochondrial membranes and subsequent stress signalling.Practical Applications: Activation of fatty acids by fatty acid transport proteins (FATP) plays an important role in development of obesity and NAFLD. This work highlights functional role of FATP4 on fatty-acid distribution among neutral lipids and phospholipids upon fat overload. These changes may contribute to stress signalling in the pathogenesis of NAFLD. Compositional shift among total PL subclasses and neutral lipids

Influence of hypoxia on Connexin 43 expression.

<p>A, B. Confluent HMEC-1 or HUVEC monolayers were exposed to normoxia or hypoxia (2% oxygen) over indicated time periods. Total RNA was isolated and transcriptional responses were assessed by real-time reverse-transcriptase polymerase chain reaction. Data were calculated relative to an internal housekeeping gene (β-actin) and are expressed as fold change over normoxia at each time point. Results are derived from 3 experiments in each condition. C. Confluent HMEC-1 monolayers were exposed to hypoxia over indicated time periods. Cells were lysed and proteins were resolved by SDS-PAGE and transferred to PVDF-membrane. Membranes were probed with a connexin 43 antibody, proteins were detected by chemiluminescene. The same blot was reprobed for β-actin as a control for protein loading. A representative experiment of 3 is shown. D. Confluent HMEC-1 monolayers were exposed to hypoxia over indicated time periods. Monolayers were washed, surface proteins were biotinylated, and cells were lysed. Connexin 43 was immunoprecipitated, followed by addition of Protein G Microbeads. Proteins were resolved by SDS-PAGE and resultant Western blots were probed with Streptavidin. A representative experiment of 3 is shown. E. Confluent HMEC-1 monolayers were exposed to normoxia or hypoxia over indicated time periods. Cells were lysed and proteins were resolved by SDS-PAGE and transferred to PVDF-Membrane. Membranes were probed with phospho-connexin 43 antibody specific for phosphorylated ser368, and proteins were detected by chemiluminescene. The same blot was probed for β-actin as a control for protein loading. A representative of 3 is shown. In subsets of experiments, cells were pretreated with the protein kinase C inhibitor bisindolylmaleimide (10 µM; +BIM).</p

Endothelial ATP release during hypoxia.

<p>A, To study extracellular ATP release from normoxic endothelia, monolayers of confluent HMEC-1 were washed and the culture media was replace with calcium containing HBSS. ATP content from their supernatant was sampled at indicated time points and quantified using a luminometric ATP detection assay. B, To measure extracellular ATP release under hypoxic conditions, confluent HMEC-1 monolayers were exposed to hypoxia (2% oxygen) over indicated time periods. Culture media was replaced with calcium containing HBSS and the ATP content within the supernatant was measured after 30 min incubation time. C, For intracellular ATP measurement, confluent HMEC-1 monolayers were exposed to hypoxia over indicated time periods, culture medium was discarded and cells were lysed by adding ice-cold water. ATP concentrations were measured as above. D, To measure the influence of different oxygen concentrations on endothelial ATP release, HMEC-1 were exposed to indicated degrees of hypoxia (21–2% of oxygen) over 24 h. Culture media was replaced with calcium containing HBSS and the ATP content within the supernatant was measured after 30 min incubation time. E, Confluent HMEC-1 monolayers were exposed to hypoxia as indicated. To assess lytic ATP release, LDH concentrations within the supernatant were measured by an LDH detection kit. In control experiments, cells were lysed with Triton X-100 (*p<0.01, n = 6 for all experiments).</p

Endothelial connexin expression.

<p>Confluent HMEC-1 monolayers were exposed to normoxia or hypoxia (12 h). Total RNA was isolated and real-time reverse-transcriptase polymerase chain reaction was employed to screen for transcriptional modulation of connexin expression. Data were calculated relative to an internal control gene (β-actin) and are expressed as fold change over normoxia at each time point. Results are derived from 3 experiments in each condition.</p

Connexin-mimetic peptides in endothelial ATP release.

<p>A, B, Confluent HMEC-1 monolayers were washed and treated with connexin-mimetic peptides (A: Cx40 peptide, SRPTEKNVFIV, 50 µmol; B: Cx43 peptide, SRPTEKTIFII, 50 µmol). ATP content within the supernatant was measured by a luminometric ATP detection assay after an incubation period of 20 min and compared with control HMEC-1 treated with 50 µM bovine albumin (n = 6).</p

ATP release from HMEC treated with the protein kinase C inhibitor bisindolylmaleimide (BIM).

<p>To study the role of Cx43 ser398 phosphorylation status in ATP release from endothelia, monolayers of confluent HMEC-1 were treated with BIM (+BIM, 10 µM) or vehicle control (-BIM), exposed to normoxia or hypoxia (24 h, 2% oxygen), washed and the culture media was replace with calcium containing HBSS. ATP content from the supernatant was sampled at indicated time points and quantified using a luminometric ATP detection assay (*p<0.05 compared to Normoxia – BIM; n = 6).</p

Human primer pairs used for real-time RT-PCR.

<p>Human primer pairs used for real-time RT-PCR.</p