Medical Physiology

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Browsing Medical Physiology by browse.metadata.advisor "Blignaut, Marguerite"

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    A potential link between adiposity and the heart: Investigating a posttranscriptional mechanism of cardiac ATM protein regulation in obesity
    (Stellenbosch : Stellenbosch University, 2021-03) Botha, Danelle; Huisamen, Barbara; Blignaut, Marguerite; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.
    Background:Obesity isarisk factor for metabolic abnormalities,includinginsulin resistance, which precedes type II diabetes and cardiovascular diseases.White adipose tissue(WAT)isan endocrineorgan responsible for maintaining systemic energy homeostasis. Obesity results in dramatic changes in WAT behaviour, including ectopic visceral accumulationand dysregulated adipocyte metabolismand secretions, which may adversely affect peripheral organs.Ataxia-telangiectasia mutated kinase (ATM) regulates glucose and redox homeostasis, thereby contributing to normal cellular metabolism. However, ATM protein levels are down-regulated in muscle and cardiac tissues of obese and insulin resistant animals, and therefore represents a potential link between obesity and peripheral metabolic dysfunction. The underlying mechanism of cardiac ATM suppression in obesity, however, remains unknown. ATM protein levels are suppressed by specific microRNAs (miRNAs) in cancer cells. MiRNAs are short molecules that govern protein translation in health and disease. Therefore, this study investigates whether adipocyte secretions potentially regulate cardiomyoblast ATM protein levels through a miRNA-mediated mechanism in obesity.Methods:1) H9c2 cardiomyoblasts were treated with combinations of high free fatty acids (FFAs; palmitic-and oleic acid), glucose and/or insulin (n=3) to establish whether cardiomyoblasts are metabolically sensitive to obese-simulating conditions. ATM levels and activation (phosphorylation), together with other relevant metabolic proteins, were determined through Western blotting.The expression of two miRNAs that target ATM in cancer (miRNA-421 and miRNA-18a) were determined with qRT-PCR. 2) H9c2 cardiomyoblasts were treated with primary adipocyte-derived conditioned media to establish whether cardiomyoblasts are metabolically sensitive to adipocyte secretions. The adipocytes were differentiated from adipose stem cells (ASCs) originating from the subcutaneous and visceral fat depots of lean (control; n=4) and high-fat diet (HFD; n=4) male Wistar rats. The basal metabolic protein profiles of adipocytes were determined through Western blotting and the FFA composition of the conditioned media were determined through GC-FID. Cardiomyoblast ATM levels and activation, together with other relevant metabolic proteins, were determined through Western blotting. The expression of miRNA-421 and miRNA-18a, and four additional metabolic miRNAs that target ATM in silico(miRNA-194, miRNA-210, miRNA-322, miRNA-181b), were determined with qRT-PCR. Results:In H9c2 cells, ATM activity (phospho/total-ratio), but not protein levels, werei) decreased by a combination of highFFAs and insulin compared to FFAs alone and insulin alone, and ii) increased (p<0.05) in response to control-and HFD-derived adipocyte secretions compared to untreated cells. The basal metabolic protein profiles and FFA secretory profiles of adipocytes were not influenced by fat depot or diet, but by the adipocytic differentiation process. Lastly, visceral and subcutaneous adiposesecretions down-regulated(p<0.01)miRNA-181b levels in H9c2 cells under HFD conditions.Discussion and conclusion:This study shows, for the first time, that cardiomyoblast i) ATM activity is influenced by high insulin, FFAs and adiposesecretions, and ii) miRNA-181b expression is down-regulated in response to HFD-derived adiposesecretions. Reduced miRNA-181b levels were previously observed in the myocardium of obese mice and associates with inflammation, insulin resistance and cardiomyopathy. We speculate that cardiomyoblast miRNA-181b down-regulation in response to HFD-derived adipocyte secretions maypotentiallybean early indicator of metabolic dysfunction.