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

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dc.contributor.advisorHuisamen, Barbaraen_ZA
dc.contributor.advisorBlignaut, Marguariteen_ZA
dc.contributor.authorRabela, Vuyisane Michaelen_ZA
dc.contributor.otherStellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Medical Physiology.en_ZA
dc.date.accessioned2024-02-17T10:08:31Zen_ZA
dc.date.accessioned2024-04-27T00:32:01Zen_ZA
dc.date.available2024-02-17T10:08:31Zen_ZA
dc.date.available2024-04-27T00:32:01Zen_ZA
dc.date.issued2024-02en_ZA
dc.descriptionThesis (MSc)--Stellenbosch University, 2024.en_ZA
dc.description.abstractENGLISH 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 establisheden_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Agtergrond en doelwit: Daar is ‘n behoefte om optimale en gestandardiseerde protokolle te vestig vir in vitro sel kultuur modelle met ‘n oormaat vry vetsure, om die effekte van kardiovaskulêre siektes (KVSs) wat ontwikkel weens vetsug, te bestudeer. Die nadele van bestaande selkultuur modelle is goed beskryf, insluitende die sitotoksisiteit van oplosmiddels. Gevolglik word herhaalbare, bevestigde modelle, wat beide die kardiale sowel as endoteel weefsel verteenwoordig, benodig om sein transduksie padweë wat betrokke is by KVSs tydens vetsug, te ondersoek. Die doelwit van hierdie studie was om optimale insulien-weerstandige kardiale (H9c2 kardiomioblas) en vaskulêre endoteel (rot aortiese endoteel, RAE) selkultuur modelle te vestig met die byvoeging van palmitaat (PS)- en oleaatsuur, (OS), te bepaal of Ataxia Telangiectasia Mutated proteïen kinase (ATM) verminder is en bydra tot verhoogde merkers van veroudering in die vaskulêre sisteem. Eksperimentele ontwerp: H9c2 kardiomioblas en RAO selle was onderhewig aan 24 uur behandeling met ‘n toenemende hoeveelheid PS (100-400 µM) en ‘n vaste hoeveelheid OS (100 µM) (n=3), en in ‘n aparte eksperiment ook met 100 nM insulien (n=1 and 2) vir 15 minute. Die opeenhoping van vetsure is bepaal met olie rooi O verkleuring, en sel lewensvatbaarheid en oksidatiewe stres is bepaal met ‘n dimetiel-difenieltetrazolium bromied (MTT) en di-chloro-dihidrofloresein (DCF) toets. Totale en gefosforileerde ATM, teen-oksidant and metaboliese proteïene was bepaal met die Westerse klad tegniek. Boodskapper RNS uitdrukking van die verouderings merkers, p21 en p16, sowel as ATM, was bepaal met ‘n kwantitatiewe polimerase ketting reaksie (kPKR). Stikstofoksiedis gemeet met behulp van vloeisitometrie in die endoteel sel model. Resultate: Vetsuur behandeling het gelei tot die opregulering van p16INK4, maar nie ATM in die H9c2 selle nie, terwyl vetsuur behandeling nie hierdie merkers in endoteel selle beïnvloed het nie. Totale ATM, maar nie fosforilerig is beïnvloed deur vetsure in die endoteel selle. Die metaboliese proteïene is nie deur die vetsure beïnvloed nie, alhoewel die teen-oksidant proteïene verlaag was in die endoteel selle. Ons resultate dui daarop dat vetsuur behandeling eNOS onderduk in endoteel selle, en dienooreenkomstig NO produksie, wat ‘n aanduiding is van endoteel disfunksie. Daar is ook ‘n onderdrukte reaksie op insulien stimulasie in die endoteel selle wat dui op insulien weerstandigheid, alhoewel hierdie reaksie nie in H9c2 waargeneem is nie. Inteendeel, ‘n afname in mTORC1 fosforilasie is waargeneem, wat aandui dat die proteïen op insulien stimulasie reageer deur alternatiewe meganismes onder hierdie omstandighede. Afleiding: Alhoewel ons nie insulien weestandigheid kon bevestig in die H9c2 selle nie, het ons wel ‘n toename gesien in p16INK4, maar nie ATM, wat daarop dui dat vryvetsure ‘n verouderings fenotipe https://scholar.sun.ac.za v | P a g e kan bewerkstellig, onafhanklik van ATM. Ons het ook ‘n afname in die reaksie op insulien stimulasie waargeneem in die RAE selle, wat dui op insulien weerstandigheid. Dit het gepaard gegaan met ‘n ontwrigte metabolisme weens ‘n afname in eNOS fosforilasie en NO produksie, wat dui op endoteel disfunksie. Ons het ook ‘n afname in totale ATM waargeneem in beide sellyne, wat die waarneming dat ATM afneem onder vetsug kondisies, ondersteun. Alles in ag genome, het ons daarin geslaagoormaat vryvetsuur sel kultuur modelle gevestig.af_ZA
dc.description.versionMastersen_ZA
dc.format.extentxvii, 128 pagesen_ZA
dc.identifier.urihttps://scholar.sun.ac.za/handle/10019.1/130628en_ZA
dc.language.isoen_ZAen_ZA
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subject.lcshCell cultureen_ZA
dc.subject.lcshObesity -- Risk factorsen_ZA
dc.subject.lcshAtaxia telangiectasiaen_ZA
dc.subject.lcshProtein kinasesen_ZA
dc.titleObesity, 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 analysisen_ZA
dc.typeThesisen_ZA
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