Abstract
Impaired mitochondrial function may contribute to the onset of contractile dysfunction with insulin resistance/type 2 diabetes. Our aim was therefore to determine alterations in the mitochondrial proteome of a mouse model of obesity/type 2 diabetes. Mitochondrial proteins were isolated from hearts collected from 18- to 20-week-old female db/db mice and compared to matched controls. We performed two-dimensional polyacrylamide gel electrophoresis to determine differentially expressed proteins. Peptides of interest were further analysed by mass spectrometry and Mascot software was employed to identify protein matches. Our data showed that ATP synthase D chain, ubiquinol cytochrome-C reductase core protein 1 and electron transfer flavoprotein subunit alpha peptide levels were altered with obesity. Moreover, we found coordinate downregulation of contractile proteins in the obese heart, i.e. α-smooth muscle actin, α-cardiac actin, myosin heavy-chain α and myosin-binding protein C. We propose that decreased contractile protein levels may contribute to contractile dysfunction of hearts from diabetic mice.
Highlights
MethodsTo investigate our hypothesis we employed 18- to 20-week-old female leptin receptor-deficient (db/db) (BKS.Cg-m+/+Leprdb/J strain) and heterozygous (db/+) mice
Our data showed that ATP synthase D chain, ubiquinol cytochrome-C reductase core protein 1 and electron transfer flavoprotein subunit alpha peptide levels were altered with obesity
We identified changes in several proteins that play a role in mitochondrial energy metabolism, only three, i.e. ATP synthase D chain, ubiquinol cytochrome-C reductase core protein 1 and electron transfer flavoprotein subunit alpha were identified as real changes
Summary
To investigate our hypothesis we employed 18- to 20-week-old female leptin receptor-deficient (db/db) (BKS.Cg-m+/+Leprdb/J strain) and heterozygous (db/+) mice. Mice were obtained from Jackson Laboratory (Bar Harbor, Maine) and exposed for one week to a reverse 12-hour light 12-hour dark cycle with free access to standard mouse chow and water. The reverse cycle was employed since mice are most metabolically active during the night period, and it allowed us to sacrifice mice in the middle of their night phase during our normal laboratory working hours. All animal experiments were approved by the University of Cape Town’s Animal Research Ethics Committee (approval number 03/030) and the investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No 85-23, revised 1996). Louis MO) that allows for rapid isolation of enriched mitochondrial fractions for proteomic studies.[6,7] Approximately 120 mg of left ventricular tissue was dissected out from control and obese mice and weighed, cut into smaller pieces and resuspended in 10 volumes of extraction buffer A (10 mM HEPES: pH 7.5, 200 mM mannitol, 70 mM sucrose, 1 mM EGTA)
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