The role of Atm in the regulation of cardiomyocyte glucose utilization under normal and insulin resistant conditions
Year: 2016
Type: dissertation
Abstract: Introduction: Ataxia-telangiectasia (A-T) is an autosomal, recessive disorder that progressively affects multiple organs.A-T is caused by mutations in the Atm gene (ataxia-telangiectasia mutated gene), resulting in lack/inactivation of the ATM protein.These patients display a high incidence of insulin resistance or full-blown type 2 diabetes (T2D) and are therefore more prone to develop ischaemic heart disease.According to literature, ATM expression may be altered by obesity and therefore we investigated its expression and impact in the heart in the context of obesity-induced insulin resistance.Aim: To determine the importance of ATM in glucose uptake in cardiomyocytes prepared from control vs. obesity-induced insulin resistant animals.Objectives: (i) To determine ATM levels in rat models of obesity-induced insulin resistance and characterize a suitable model for the study.(ii) To manipulate the ATM protein in isolated cardiomyocytes from both control and insulin resistant animals using suitable activators or inhibitors of ATM followed by the measurement of 2-Deoxy-D-3 [H] glucose (2DG) uptake levels, expression and activation of intermediates of the insulin signalling pathway (IRS1, PI3K, PTEN, PKB, AS160, GSK-3β, AMPK) and GLUT4 expression.Methods: Male Wistar rats were fed a high fat diet (HFD) for 16 weeks to induce obesity and insulin resistance and compared to chow-fed controls.Body weight, intra-peritoneal (IP) fat weight, fasting glucose and insulin levels were determined.Ventricular cardiomyocytes were prepared by collagenase perfusion digestion whereafter 2DG uptake was measured in response to treatment with specific activators/inhibitors of ATM.The specific ATM inhibitor, KU 60019, was used to manipulate the activity of the protein while the expression of proteins in the insulin signalling pathway was determined by Western blotting.Results: (i) No differences in body weight were observed.(ii) HFD animals presented with higher IP fat weight (33.7±2.4 vs. 19.1±1.5 g; p<0.001), basal glucose (5.4±0.16 vs. 4.71±0.05mmol/L; p<0.001) and insulin (14.6±2.0 vs. 6.7±2.2 mlU/ml; p<0.05) levels vs. controls.(iii) KU 60019 inhibited insulin-stimulated 2DG uptake in the control and HFD cardiomyocytes from 26.8±3.93 and 28.38±1.27 to 13.1±2.4(p<0.01) and 18.6±2.8(p<0.05)pmol/mg prot/30min respectively.(iv) HFD downregulated T-ATM expression by 50% (1±0.1 vs. 0.5±0.1 ADU; p<0.001);T-PI3K by 30% (1±0.02 vs. 0.7±0.2ADU; p<0.01);T-PKB by 40% (1±0.03 vs. 0.6±0.08ADU; p<0.01);T-GSK-3β by 10% (1±0.02 vs. 0.9±0.05ADU; p<0.01);T-AMPK by 30% (1±0.03 vs. 0.7±0.09ADU; p<0.05) and P-AS160 by 30% (1±0.08 vs. 0.7±0.2ADU; p<0.001).(v) HFD increased the phosphorylation of IRS1 with 100% at Ser307 (1±0.3 vs. 2.2±0.1 ADU; p<0.05) vs. controls.(vi)
Author: Mignon van Vuuren
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2346625038