Browsing by Author "Rabela, Vuyisane Michael"

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    Obesity, cardiometabolic diseases and aging-the role of Ataxia Telangiectasia Mutated protein kinase: optimization of in vitro lipotoxic H9c2 cardiomyoblasts and rat aortic endothelial cell models for cell signalling analysis
    (Stellenbosch : Stellenbosch University, 2024-02) Rabela, Vuyisane Michael; Huisamen, Barbara; Blignaut, Marguarite; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Medical Physiology.
    ENGLISH ABSTRACT: Background and aim: There has been an interest to establish optimised and standardized protocols for in vitro cell culture models with free fatty acid (FFA) overload to study the effects of obesityinduced cardiovascular diseases (CVDs). However, the disadvantages, such as solvent-induced cytotoxicity in these existing culture models, have been well-established. As such, robust, wellcharacterized cellular models that can fully represent the cardiac and endothelial tissue in vitro are required to study the effects of lipotoxicity on the signalling pathways involved in obesity-induced CVDs. This study aimed to establish and optimise insulin-resistant cardiac (H9c2 cardiomyoblasts) and vascular endothelial (rat aortic endothelial) cell models with palmitic acid (PA) and oleic acid (OA) to determine whether Ataxia Telangiectasia Mutated protein kinase (ATM) is decreased in these models and can contribute to increased markers of senescence as a measure of aging. Experimental design: H9c2 cardiomyoblasts and rat aortic endothelial cells were subjected to 24-hour treatments with increasing amount of PA (100 to 400 µM) and a fixed amount of 100 µM OA (n=3), and in a separate experiment, with 100 nM insulin (n=1 and n=2) for 15 minutes. FFA accumulation in these cell lines were determined with oil red O staining, followed by the assessment of cell viability and oxidative stress using MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and dichloro-dihydrofluorescein (H2DCFDA) for oxidative stress, respectively. Total and phosphorylated ATM, together with antioxidants and other metabolic proteins, were subjected to gel electrophoresis and Western blotting. The mRNA expression of senescence markers (p21Cip1 and p16INK4) together with ATM were determined with RT-qPCR. Nitric oxide was determined with flow cytometry in the endothelial cell model. Results: In H9c2, FFAs induced the upregulation of p16INK4 but not ATM, whereas FFAs did not affect these markers in endothelial cells. Total ATM, but not the phosphorylation (p-ATM-ser1981) was influenced by FFAs in endothelial cells. Metabolic proteins were not influenced by FFAs, whereas antioxidants were decreased under the same conditions in endothelial cells. Our results also showed that FFA treatment attenuate eNOS in endothelial cells, with a concomitant decrease in NO production, indicative of endothelial dysfunction. Our results also showed an attenuated response to insulin in endothelial cells suggesting an insulin resistance model; however, these observations were absent in H9c2 model. Instead, we observed an attenuated mTORC 1 phosphorylation, suggesting that this protein reacted to insulin through other mechanisms other than Akt under these conditions. Conclusion: Collectively, even though we could not confirm insulin resistance in H9c2, we observed an increased p16INK4 but not ATM, suggesting that FFAs induce aging (senescence) independently of https://scholar.sun.ac.za iii | P a g e ATM in this cell lines. As a proof of principle, we observed an attenuated response to insulin stimulation in ROAECs, suggesting insulin resistance. This was also characterised by disrupted metabolism due to a decrease in eNOS phosphorylation together with decrease NO production, indicative of endothelial dysfunction. We also observed a decrease in total ATM in these cell lines, supporting the evidence that ATM is decreased in obese conditions. Altogether, our data suggest that the FFAs overload models have been established