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

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Spontaneous diabetes in hemizygous human amylin transgenic mice that developed neither islet amyloid nor peripheral insulin resistance.

OBJECTIVES: We sought to 1) Determine whether soluble-misfolded amylin or insoluble-fibrillar amylin may cause or result from diabetes in human amylin transgenic mice and 2) determine the role, if any, that insulin resistance might play in these processes. RESEARCH DESIGN AND METHODS: We characterized the phenotypes of independent transgenic mouse lines that display pancreas-specific expression of human amylin or a nonaggregating homolog, [(25,28,29)Pro]human amylin, in an FVB/n background. RESULTS: Diabetes occurred in hemizygous human amylin transgenic mice from 6 weeks after birth. Glucose tolerance was impaired during the mid- and end-diabetic phases, in which progressive beta-cell loss paralleled decreasing pancreatic and plasma insulin and amylin. Peripheral insulin resistance was absent because glucose uptake rates were equivalent in isolated soleus muscles from transgenic and control animals. Even in advanced diabetes, islets lacked amyloid deposits. In islets from nontransgenic mice, glucagon and somatostatin cells were present mainly at the periphery and insulin cells were mainly in the core; in contrast, all three cell types were distributed throughout the islet in transgenic animals. [(25,28,29)Pro]human amylin transgenic mice developed neither beta-cell degeneration nor glucose intolerance. CONCLUSIONS: Overexpression of fibrillogenic human amylin in these human amylin transgenic mice caused beta-cell degeneration and diabetes through mechanisms independent from both peripheral insulin resistance and islet amyloid. These findings are consistent with beta-cell death evoked by misfolded but soluble cytotoxic species, such as those formed by human amylin in vitro.

Reversal of diabetes-evoked changes in mitochondrial protein expression of cardiac left ventricle by treatment with a copper(II)-selective chelator.

Cardiac disease is the commonest cause of death amongst diabetic patients. Diabetic cardiomyopathy, which has a poor prognosis, is characterized by left ventricular hypertrophy and impaired cardiac function and mitochondrial damage is said to contribute to its development. We recently showed that treatment with the Cu(II) -selective chelator, triethylenetetramine (TETA), improved cardiac structure, and function in diabetic subjects without modifying hyperglycemia. Thus, TETA has potential utility for the treatment of heart disease. To further understand the molecular mechanism by which it causes these effects, we have conducted the first study of the effect of oral TETA on protein abundance in the cardiac left ventricle of rats with severe streptozotocin-induced diabetes. Proteomic methods showed that of 211 proteins changed in diabetes, 33 recovered after treatment. Through MS, 16 proteins were identified which may constitute major targets of drug action. Remarkably, most of these were mitochondrial proteins with roles in energy metabolism. In addition to components of the mitochondrial respiratory chain and enzymes involved in fatty acid oxidation, TETA treatment normalized both myocardial expression and enzymatic activity of carnitine palmitoyltransferase 2. These findings indicate that mitochondria constitute major targets in the mechanism by which TETA restores cardiac structure and function in diabetes.