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 (64Cu) 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 protein’s 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.
Objective: 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. Methods: C57BL/6NTac mice (10 weeks) were treated over 8 weeks i.p. with saline (control) or recombinant AFABP (0.5 mg/kg/d). A comprehensive characterization of metabolic parameters, body composition, and energy expenditure was performed. Furthermore, the effect of AFABP on pancreatic b-cell responsiveness, hepatic glycogen content, and peroxisome proliferator-activated receptor (PPAR) c protein expression was elucidated. Results: In male mice, AFABP treatment induced insulin resistance with significantly increased fasting insulin, C-peptide, and homeostasis model assessment of insulin resistance. In female animals, a similar trend was observed. In both genders, no difference in body weight, lipid parameters, body composition, or energy expenditure could be detected between AFABP-treated and control mice. Insulin resistance in male AFABP-treated mice was accompanied by decreased PPARc protein content in perigonadal adipose tissue and diminished circulating adiponectin. AFABP treatment did not affect pancreatic b-cell responsiveness and hepatic glycogen content. Conclusions: Circulating AFABP induces insulin resistance in male mice. AFABP-mediated degradation of PPARc in adipose tissue and subsequently decreased expression of insulin-sensitizing adiponectin are potential mechanisms for this effect.
Obesity is associated with dyslipidemia and subclinical inflammation that promotes metabolic disturbances including insulin resistance and pancreatic β-cell dysfunction. The nuclear protein, transcriptional regulator 1 (NUPR1) responds to cellular stresses and features tissue protective properties. To characterise the role of NUPR1 in endocrine pancreatic islets during inflammatory stress, we generated transgenic mice with β-cell-specific Nupr1 overexpression (βNUPR1). Under normal conditions, βNUPR1 mice did not differ from wild type (WT) littermates and display normal glucose homeostasis and β-cell mass. For induction of inflammatory conditions, mice were treated with multiple low-dose streptozotocin (mld-STZ) and/or fed a high fat diet (HFD). All treatments significantly worsened glycaemia in WT mice, while βNUPR1 mice substantially preserved insulin secretion and glucose tolerance. HFD increased β-cell mass in all animals, with β-NUPR1 mice tending to show higher values. The improved outcome of βNUPR1 mice was accompanied by decreased NF-kB activation and lymphocyte infiltration in response to mld-STZ. In vitro, isolated βNUPR1 islets preserved insulin secretion and content with insignificantly low apoptosis during culture stress and IL-1β exposure. These findings suggest that NUPR1 plays a vital role in the protection of β-cells from apoptosis, related degradation of insulin storages and subsequent secretion during inflammatory and obesity-related tissue stress.
Objective: Beta-site amyloid precursor protein cleaving enzyme (BACE1) is highly expressed in pancreatic b-cells. The BACE1 gene is located in a region associated with a high diabetes risk in PIMA Indians. Design and Methods: INS-1E cells were used to study the impact of siRNA-mediated BACE1 knockdown and glucose metabolism was characterized in Bace1 -/-mice. BACE1 gene was sequenced in DNA samples from 48 subjects and 13 representative single nucleotide polymorphisms (SNPs) were then genotyped for association studies in 1,527 Caucasians.Results: Reduction of Bace1 expression results in a significant decrease in insulin mRNA expression in INS-1E cells. Bace1 -/-mice display significantly lower body weight, lower plasma insulin concentrations, but normal glucose tolerance and insulin sensitivity. In a case-control study including 538 healthy controls and 989 patients with type 2 diabetes (T2D), one SNP (rs535860) was significantly associated with T2D (P < 3.5 3 10 25, adjusted for age, sex, and BMI). Conclusions: Reduced Bace1 expression causes impaired insulin expression in pancreatic b-cells of Bace1 -/-mice, suggesting that BACE1 plays a role in the regulation of insulin biogenesis. The functionally relevant rs535860 SNP may decrease BACE1 expression by creating a new miR-661 binding site and could therefore contribute to T2D development.Obesity (2013) 21, E626-E633.
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