A potential link between adiposity and the heart: Investigating a posttranscriptional mechanism of cardiac ATM protein regulation in obesity

Botha, Danelle (2021-03)

Thesis (MSc)--Stellenbosch University, 2021.

Thesis

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.

Agtergrond:Vetsug is ‘n risikofaktor vir metaboliese abnormaliteite, insluitend insulienweerstandigheid, wat lei tot tipe II diabetes en kardiovaskulêre siektes. Wit vetweefsel (WV) is ‘n endokriene orgaan verantwoordelik vir die instandhouding van sistemiese energie homeostase. Vetsug lei tot groot veranderinge in WV funksie, insluitend die ektopiese versameling van vet rondom organe en wangereguleerde adiposietmetabolisme en -sekresie, wat perifere organe nadelig kan beïnvloed. Ataxia-telangiectasia gemuteerde proteïenkinase (ATM) reguleer glukose-en redoks homeostase en dra sodoende bytot normale sellulêre metabolisme. ATM proteïenvlakke word egter onderdruk in spier-en hartweefsel van vetsugtige en insulienweerstandige diere en verteenwoordig dus a moontlike skakel tussen vetsug enperifere metaboliese disfunksie. Die onderliggende meganisme wat lei tot die onderdrukking van ATM in die hart tydens vetsug is egter onbekend. ATM proteïenvlakke word onderdruk deur spesifieke mikroRNAs (miRNAs) in kankerselle. MiRNAs is kort molekules wat proteïentranslasie onder normale en siektestoestande beheer. Die huidige studie ondersoek dus of adiposietsekresies moontlik ATM proteïenvlakke deur middel van miRNAs in kardiomioblaste tydens vetsug beheer. Metodes:1) H9c2 kardiomioblaste is behandel met kombinasies van höe vrye vetsure (VV; palmitaat en oleaat), glukose en/of insulien (n=3) om vas te stel of kardiomioblaste metabolies sensitief is vir kondisies wat vetsug naboots. ATM vlakke en aktivering (fosforilering), asook ander metabolies-verwante proteïene, is bepaal deur middel van Westerse-klad tegniek. Twee miRNAs wat ATM teiken in kanker (miRNA-421 en miRNA-18a) se uitdrukkingsvlakke is met qRT-PCR bepaal. 2) H9c2 kardiomioblaste is behandel met gekondisioneerde media afkomstig van primêre adiposiete om metaboliese sensitiwiteit vir adiposietsekresies te bepaal. Die adiposiete is gedifferensieer vanaf adiposiet stamselle afkomstig van die subkutane en visserale vetkompartemente van kontrole (n=4) en hoë-vet dieet (HVD; n=4) manlike Wistar rotte. Die basale metaboliese proteïenprofiele van die adiposiete is deur middel van Westerse-klad tegniek bepaal en die VV samestelling van die gekondisioneerde media is met GC-FID bepaal. ATM vlakke en aktivering, asook ander relevante metaboliese proteïene, is ook deur middel van Westerse-klad tegniek bepaal. Die uitdrukkingsvlakke van miRNA-421 en miRNA-18a, asook vier addisionele metaboliese miRNAs wat ATM in silicoteiken(miRNA-194, miRNA-210, miRNA-322, miRNA-181b), is met qRT-PCR bepaal. Resultate:In H9c2 selle is ATM aktiwiteit (fosfo/totaal-verhouding), maar nie proteïenvlakke nie, i) afgereguleer deur ‘n kombinasie van VV en insulien in vergelyking met VV alleen en insulien alleen en ii) opgereguleer (p<0.05) deur gekondisioneerde media van adiposiete afkomstig van kontrole en HVD rotte in vergelyking met onbehandelde selle. Die basale metaboliese proteïenprofiele enVV sekresieprofiele van adiposiete is nie beïnvloed deur die vetkompartement of dieet nie, maar deur die adiposiet-spesifieke differensiasieproses. Die miRNA-181b vlakke is afgereguleer (p<0.01) in H9c2 selle deur visserale en subkutane adiposietsekresies onder HVD kondisies.Bespreking en gevolgtrekking:Hierdie studie toon vir die eerste keer dat kardiomioblast i) ATM aktiwiteit deur hoë insulien, VV en adiposietsekresies beïnvloed word en ii) miRNA-181b uitdrukkingsvlakke in ‘n HVD omgewing onderdruk word. Onderdrukte miRNA-181b vlakke is voorheen in die miokardium van vetsugtige muise waargeneem en assosieer met inflammasie, insulienweerstandigheid en kardiomiopatie. Ek spekuleer dat HVD-afkomstige adiposietsekresies moontlik miRNA-181b in kardiomioblaste onderdruk voor die aanvang van metaboliese afwykings.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/110021
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