Ines Sommerer

Diverse Functional Properties of Wilson Disease ATP7B Variants

BACKGROUND & AIMS Wilson disease is a severe disorder of copper metabolism caused by mutations in ATP7B, which encodes a copper-transporting adenosine triphosphatase. The disease presents with a variable phenotype that complicates the diagnostic process and treatment. Little is known about the mechanisms that contribute to the different phenotypes of the disease. METHODS We analyzed 28 variants of ATP7B from patients with Wilson disease that affected different functional domains; the gene products were expressed using the baculovirus expression system in Sf9 cells. Protein function was analyzed by measuring catalytic activity and copper ((64)Cu) transport into vesicles. We studied intracellular localization of variants of ATP7B that had measurable transport activities and were tagged with green fluorescent protein in mammalian cells using confocal laser scanning microscopy. RESULTS Properties of ATP7B variants with pathogenic amino-acid substitution varied greatly even if substitutions were in the same functional domain. Some variants had complete loss of catalytic and transport activity, whereas others lost transport activity but retained phosphor-intermediate formation or had partial losses of activity. In mammalian cells, transport-competent variants differed in stability and subcellular localization. CONCLUSIONS Variants in ATP7B associated with Wilson disease disrupt the proteins transport activity, result in its mislocalization, and reduce its stability. Single assays are insufficient to accurately predict the effects of ATP7B variants the function of its product and development of Wilson disease. These findings will contribute to our understanding of genotype-phenotype correlation and mechanisms of disease pathogenesis.

Circulating adipocyte fatty acid-binding protein induces insulin resistance in mice in vivo

Circulating levels of the adipokine adipocyte fatty acid‐binding protein (AFABP) are increased in obesity. However, the influence of circulating AFABP on insulin sensitivity in vivo remains unclear.

Genetic and biochemical evidence for a functional role of BACE1 in the regulation of insulin mRNA expression

Beta‐site amyloid precursor protein cleaving enzyme (BACE1) is highly expressed in pancreatic β‐cells. The BACE1 gene is located in a region associated with a high diabetes risk in PIMA Indians.

P8 deficiency increases cellular ROS and induces HO-1

The gene p8 encodes for a small cytoprotective protein with no apparent enzymatic activity being proposed to act as co-transcription factor whose expression is increased during inflammation. Recent data from astrocytes demonstrates that p8 suppression leads to induction of heme oxygenase 1 (HO-1). Here, we assessed the cross-talk between p8 and HO-1 in mouse embryonic fibroblasts (MEF) observing an increased expression of HO-1 in p8-deficient (p8(-/-)) MEFs in non-treated and treated conditions. This effect was independent of the cell cycle. Our findings revealed that generation of reactive oxygen species (ROS) was higher in p8(-/-) MEFs. Mitochondria and NADPH oxidases were not the origin of ROS. This observation was not restricted to MEF as suppression of p8 gene transcription in MiaPaCa-2 cells also led to increased intracellular ROS. Additionally, p8 deficiency did not affect the Rac1 dependant NADPH oxidase complex. Our data shows that p8 deficiency increases ROS and subsequently the expression of anti-oxidative enzymes, such as HO-1, suggesting an involvement in the anti-oxidative defense. Moreover, we suggest that the severity of AP observed in p8(-/-) mice is induced by an impaired anti oxidative capacity of the pancreas, which is caused by increased generation of ROS.

p8 Deficiency causes siderosis in spleens and lymphocyte apoptosis in acute pancreatitis.

Objectives The gene p8 was initially described in pancreatic tissue during acute experimental pancreatitis, a disease that is characterized by a systemic immune response. Although early reports suggested that p8 affects leukocyte migration during acute pancreatitis (AP), no studies revealing its immune-modulatory effects have been performed. Methods We investigated the composition of the cellular immune system in naive p8 knockout (p8 −/−) mice and compared with matched wild-type mice during pancreatitis. Results In young mice, there were no relevant differences in the composition of peripheral and splenic CD3+, CD3+CD4+, CD3+CD8+, CD11b+Gr-1−, and Gr-1+ cells. In mature p8 −/− mice, increased splenic CD4+CD25+FoxP3+ cells, spleen siderosis, and increased marginal zones in the splenic white pulp were found. During AP, peripheral and splenic CD3+ and CD3+CD4+ declined stronger in the p8 −/− mice. The spleen of the p8 −/− mice showed severe hypoplasia of the white pulp and mild hyperplasia of the red pulp. This was associated with a significantly increased rate of apoptosis. Conclusions We conclude that p8 has no impact on the cellular composition of the adaptive and innate immune systems in noninflammatory conditions. However, it may limit apoptosis and maintain homeostasis of the immune reaction during AP.

p8 deficiency leads to elevated pancreatic beta cell mass but does not contribute to insulin resistance in mice fed with high-fat diet

Background p8 was initially described as being overexpressed in acute pancreatitis and encoding a ubiquitous stress protein. Analysis of insulin sensitivity and glucose tolerance in p8-knockout and haplodeficient mice revealed counterintuitive results. Thus, we determined glycemic control of p8 in mice fed with standard (SD) and high-fat diet (HFD). Methods p8-/- and wild type (p8+/+) mice were used for analysis of glucagon (immunohistochemistry), insulin levels (ELISA) and beta cell mass. Hyperinsulinemic- euglycemic glucose clamp technique, i.p. glucose tolerance test (ipGTT), i.p. insulin tolerance test (ipITT) and metabolic chamber analysis were performed in SD (4% fat) and HFD (55% fat) groups. Results p8-/- mice showed no differences in glucagon or insulin content but higher insulin secretion from pancreatic islets upon glucose stimulation. p8 deficiency resulted in elevated beta cell mass but was not associated with increased insulin resistance in ipGTT or ipITT. Glucose clamp tests also revealed no evidence of association of p8 deficiency with insulin resistance. Metabolic chamber analysis showed equal energy expenditure in p8-/- mice and wild type animals. Conclusion p8 depletion may contribute to glucose metabolism via stress-induced insulin production and elevated beta cell mass. Nevertheless, p8 knockout showed no impact on insulin resistance in SD and HFD-fed mice.

Loss of Bace1 in Mice Does Not Alter the Severity of Caerulein Induced Pancreatitis

Context Beta-site alpha-amyloid protein cleaving enzyme1 (BACE1) plays a key role in the pathogenesis of Alzheimer’s disease. Additional to its moderate expression in the brain, high levels of BACE1 mRNA were found in the pancreas. Murine Bace1 has been immunohistochemicaly detected at the apical pole of acinar cells within the exocrine pancreas of mice and Bace1 activity was observed in pancreatic juice. In vitro experiments revealed enteropeptidase as a putative substrate for Bace1 suggesting a role in acute pancreatitis. Objective The aim of this study was to address a protective mechanism of Bace1 in acute experimental pancreatitis in mice. Methods Acute experimental pancreatitis was induced by intraperitoneal injection of caerulein in homozygote Bace1 -/- mice and wild type mice. Serum and tissue analyses were carried out after 4 h, 8 h and 24 h. Measurement of plasma amylase and lipase was performed to confirm pancreatitis induction. In order to assess the severity of pancreatitis H&E stained pancreatic sections were examined regarding edema, inflammation and apoptosis. Immunohistochemical detection of myeloperoxidase (MPO) positive cells was carried out to further quantify the extent of inflammation. Expression of Bace2 within the pancreas was analyzed by immunohistochemistry and RT-qPCR. Results We demonstrate that total loss of Bace1 in mice leads to no alterations in the course of acute experimental caerulein-pancreatitis. Bace1-/- mice develop a moderate pancreatitis that is comparable in histomorphological and serological features with those seen in wild type mice. Discussion We discuss the results in the context of the applied caerulein induced edematous pancreatitis model and possible compensatory mechanisms via Bace2 that might be responsible for the observed results.