Showing papers by "Garth J. S. Cooper published in 2014"

Diabetic cardiomyopathy is associated with defective myocellular copper regulation and both defects are rectified by divalent copper chelation.

Abstract: Heart disease is the leading cause of death in diabetic patients, and defective copper metabolism may play important roles in the pathogenesis of diabetic cardiomyopathy (DCM). The present study sought to determine how myocardial copper status and key copper-proteins might become impaired by diabetes, and how they respond to treatment with the Cu (II)-selective chelator triethylenetetramine (TETA) in DCM. Experiments were performed in Wistar rats with streptozotocin (STZ)-induced diabetes with or without TETA treatment. Cardiac function was analyzed in isolated-perfused working hearts, and myocardial total copper content measured by particle-induced x-ray emission spectroscopy (PIXE) coupled with Rutherford backscattering spectrometry (RBS). Quantitative expression (mRNA and protein) and/or activity of key proteins that mediate LV-tissue-copper binding and transport, were analyzed by combined RT-qPCR, western blotting, immunofluorescence microscopy, and enzyme activity assays. Statistical analysis was performed using Student’s t-tests or ANOVA and p-values of < 0.05 have been considered significant. Left-ventricular (LV) copper levels and function were severely depressed in rats following 16-weeks’ diabetes, but both were unexpectedly normalized 8-weeks after treatment with TETA was instituted. Localized myocardial copper deficiency was accompanied by decreased expression and increased polymerization of the copper-responsive transition-metal-binding metallothionein proteins (MT1/MT2), consistent with impaired anti-oxidant defences and elevated susceptibility to pro-oxidant stress. Levels of the high-affinity copper transporter-1 (CTR1) were depressed in diabetes, consistent with impaired membrane copper uptake, and were not modified by TETA which, contrastingly, renormalized myocardial copper and increased levels and cell-membrane localization of the low-affinity copper transporter-2 (CTR2). Diabetes also lowered indexes of intracellular (IC) copper delivery via the copper chaperone for superoxide dismutase (CCS) to its target cuproenzyme, superoxide dismutase-1 (SOD1): this pathway was rectified by TETA treatment, which normalized SOD1 activity with consequent bolstering of anti-oxidant defenses. Furthermore, diabetes depressed levels of additional intracellular copper-transporting proteins, including antioxidant-protein-1 (ATOX1) and copper-transporting-ATPase-2 (ATP7B), whereas TETA elevated copper-transporting-ATPase-1 (ATP7A). Myocardial copper deficiency and defective cellular copper transport/trafficking are revealed as key molecular defects underlying LV impairment in diabetes, and TETA-mediated restoration of copper regulation provides a potential new class of therapeutic molecules for DCM.

Evidence that multiple defects in lipid regulation occur before hyperglycemia during the prodrome of type-2 diabetes

Abstract: Background: Blood-vessel dysfunction arises before overt hyperglycemia in type-2 diabetes (T2DM). We hypothesised that a metabolomic approach might identify metabolites/pathways perturbed in this pre-hyperglycemic phase. To test this hypothesis and for specific metabolite hypothesis generation, serum metabolic profiling was performed in young women at increased, intermediate and low risk of subsequent T2DM. Methods: Participants were stratified by glucose tolerance during a previous index pregnancy into three risk-groups: overt gestational diabetes (GDM; n=18); those with glucose values in the upper quartile but below GDM levels (UQ group; n=45); and controls (n=43, below the median glucose values). Follow-up serum samples were collected at a mean 22 months postnatally. Samples were analysed in a random order using Ultra Performance Liquid Chromatography coupled to an electrospray hybrid LTQ-Orbitrap mass spectrometer. Statistical analysis included principal component (PCA) and multivariate methods. Findings: Significant between-group differences were observed at follow-up in waist circumference (86, 95%CI (79–91) vs 80 (76–84) cm for GDM vs controls, p,0.05), adiponectin (about 33% lower in GDM group, p=0.004), fasting glucose, postprandial glucose and HbA1c, but the latter 3 all remained within the ‘normal’ range. Substantial differences in metabolite profiles were apparent between the 2 ‘at-risk’ groups and controls, particularly in concentrations of phospholipids (4 metabolites with p#0.01), acylcarnitines (3 with p#0.02), short- and long-chain fatty acids (3 with p,=0.03), and diglycerides (4 with p#0.05). Interpretation: Defects in adipocyte function from excess energy storage as relatively hypoxic visceral and hepatic fat, and impaired mitochondrial fatty acid oxidation may initiate the observed perturbations in lipid metabolism. Together with evidence from the failure of glucose-directed treatments to improve cardiovascular outcomes, these data and those of others indicate that a new, quite different definition of type-2 diabetes is required. This definition would incorporate disturbed lipid metabolism prior to hyperglycemia.

The pathogenic mechanism of diabetes varies with the degree of overexpression and oligomerization of human amylin in the pancreatic islet β cells

Abstract: The aggregation of human amylin (hA) to form cytotoxic structures has been closely associated with the causation of type 2 diabetes. We sought to advance understanding of how altered expression and aggregation of hA might link β-cell degeneration with diabetes onset and progression, by comparing phenotypes between homozygous and hemizygous hA-transgenic mice. The homozygous mice displayed elevated islet hA that correlated positively with measures of oligomer formation (r=0.91; P<0.0001). They also developed hyperinsulinemia with transient insulin resistance during the prediabetes stage and then underwent rapid β-cell loss, culminating in severe juvenile-onset diabetes. The prediabetes stage was prolonged in the hemizygous mice, wherein β-cell dysfunction and extensive oligomer formation occurred in adulthood at a much later stage, when hA levels were lower (r=−0.60; P<0.0001). This is the first report to show that hA-evoked diabetes is associated with age, insulin resistance, progressive islet dysfunction...

Expedient synthesis of peptides containing Nε- carboxymethyllysine

Abstract: Accumulation of advanced glycation endproducts ­(AGEs) is responsible for the development and progress of diabetes- and age-related complications. Synthesis of specific chemical probes is key for the detailed understanding of biochemical properties of AGEs and their precise roles in the progression of disease. We herein report the expedient synthesis of such probes in the form of peptides site-specifically glycated by the major lysyl AGE, N e-carboxymethyllysine (CML). The facile and economical incorporation of CML into peptide sequences by using the nosyl group has been achieved in a single step on resin. This new method is a substantial improvement over the existing syntheses of CML-containing peptides in that it does not require the use of expensive reagents or elaborate purification techniques. The impact of CML on the proteolytic stability of the host peptide has been investigated using trypsin digest studies.