An investigation into the importance of the ATM protein in the myocardial pathology associated with insulin resistance and type 2 diabetes

Espach, Yolandi (2017-03)

Thesis (PhD)--Stellenbosch University, 2017

Thesis

ENGLISH ABSTRACT : Introduction: Ataxia-telangiectasia (A-T) is an autosomal, recessive disorder that is caused by mutations in the ataxia-telangiectasia mutated (ATM) gene. The gene product, ATM, is a 350 kDa serine/threonine protein kinase with a large number of substrates in various pathways. Patients suffering from A-T display a high incidence of insulin resistance or type 2 diabetes mellitus (T2DM) and are more susceptible to ischaemic heart disease. Although it is known that the ATM protein is expressed in the heart and that structural and functional changes are observed in the hearts of ATM knock-out mice, very little research has been done on ATM and its role in insulin signalling in the cardiovascular context. This project aimed to identify and characterise an obese, insulin resistant animal model in which myocardial ATM expression is altered; to use this model to determine the effect of ATM manipulation on myocardial function and response to ischaemia/reperfusion injury (IRI); and to determine the effect of ATM manipulation on insulin signalling networks. Methodology: Male Wistar rats received a high caloric diet (HCD) or standard rat chow (control) for 16 weeks after which biometric data was collected. ATM was manipulated in perfusion studies prior to ischaemia using insulin (activator) and KU-60019 (inhibitor). Hearts perfused using the Langendorff balloon model were subjected to global ischaemia followed by reperfusion and Western blots were performed to evaluate insulin signalling intermediates. Hearts were also perfused using the working mode and subjected to regional ischaemia. Following reperfusion, infarct size (IFS) was determined. Aortic ring isometric tension studies were performed to determine the effect of KU-60019 on vasodilation. Results: The HCD resulted in significantly increased body mass, visceral fat mass, glucose levels, insulin levels and HOMA-IR index compared to the control diet and ATM expression was reduced in the HCD hearts. Cardiac function and IFS were comparable in the control and HCD hearts. In control hearts, insulin administration activated the insulin signalling network prior to ischaemia and cardiac function was improved during reperfusion. Insulin had no effect on the insulin signalling network or cardiac function in the insulin resistant HCD hearts. High concentrations of insulin increased IFS in both the control and HCD hearts. ATM inhibition improved cardiac function in control and HCD hearts during early reperfusion but had no effect on cardiac function during later reperfusion. ATM phosphorylation was increased by insulin and decreased by KU-60019 in control hearts, but could not be manipulated in HCD hearts. Insulin-stimulated PKB/Akt activation is not ATM-dependent in the heart. However, ATM inhibition appears to down-regulate insulin signalling via PI3K, PTEN and GSK-3β. ATM inhibition caused NO-dependent vasodilation in control hearts, suggesting a role for ATM in vasoconstriction. Conclusion: ATM is a complex signalling regulator with numerous substrates. In our study, we found that acute cardiac ATM inhibition did not result in significant cardiac dysfunction or complete abrogation of insulin signalling. However, we found that ATM possibly plays a significant role in vasoconstriction. More research needs to be done to fully understand the cardiac role of ATM in insulin signalling.

AFRIKAANSE OPSOMMING : Inleiding: Ataxia-telangiectasia (A-T) is ‘n autosomale, resesiewe siekte wat deur mutasies in die ATM geen veroorsaak word. Die geenproduk, ATM, is ‘n 350 kDa serien/treonien proteϊenkinase met ‘n groot aantal substrate wat in verskeie molekulêre paaie aktief is. Daar is ʼn hoë insidensie van insulienweerstandigheid en tipe 2 diabetes mellitus onder A-T pasiënte en hulle ontwikkel ook meer iskemiese hartsiektes. Alhoewel dit bekend is dat ATM in die hart uitgedruk word en dat struktruele en funksionele veranderinge gesien kan word in die harte van muise wat nie die ATM geen het nie, is daar baie min bekend oor ATM se rol in insulien-seintransduksie in die hart. Hierdie projek het beoog om ‘n vetsugtige, insulienweerstandige rotmodel te identifiseer waarin ATM vlakke verander is; om te bepaal hoe die hart reageer op iskemie/herperfusie wanneer ATM gemanipuleer word, en om te bepaal hoe ATM manipulasie insulien-seintransduksie beϊnvloed. Metodiek: Manlike Wistar rotte wat vir 16 weke ‘n hoë-kalorie dieet (HCD) of standaard rotkos (kontrole) gevoer is, is gebruik. ATM uitdrukking en/of aktivering is in die harte gemanipuleer deur hulle voor iskemie met insulien (aktiveerder) of KU-60019 (inhibitor) te perfuseer. Harte is met die Langendorff ballonmodel aan globale iskemie blootgestel en na herperfusie is proteϊene van die insulien-seintransduksiepaaie met behulp van die Western-klad tegniek ge-analiseer. Harte is ook deur middel van die werkharttegniek aan streeksiskemie/herperfusie blootgestel waarna infarktgrootte bepaal is. Isometriese spanningsondersoeke is op aortaringe uitgevoer om die effek van KU-60019 op verslapping te bepaal. Resultate: HCD diere het beduidende hoër liggaamsmassas en visserale vet massas; hoër glukosevlakke en insulienvlakke; ‘n verhoogde HOMA-IR indeks en laer ATM vlakke gehad as die kontrole diere. Miokardiale funksie en infarktgroottes het nie verskil tussen kontrole en HCD harte nie. Insulien kon insulien-seintransduksie voor iskemie in kontrole harte stimuleer en het hartfunksie tydens herperfusie verbeter. Daarteenoor het insulien geen effek op hartfunksie of insulien-seintransduksie in die HCD harte gehad nie. In beide die kontrole en HCD harte het hoë konsentrasies insulien infarkte vergroot. In kontrole en HCD harte het ATM inhibisie hartfunksie vroeg tydens herperfusie verbeter, maar het geen effek op hartfunksie later in herperfusie getoon nie. Alhoewel ATM fosforilering deur insulien verhoog is en deur KU-60019 verlaag is in kontrole harte, was daar geen verandering in ATM fosforilering in HCD harte nie. Insulien-gestimuleerde aktivering van PKB/Akt was nie afhanklik van ATM aktivering in die hart nie. ATM inhibisie het wel gelei tot onderdrukking van insulien-seintransduksie deur effekte op PI3K, PTEN en GSK-3β. ATM inhibisie het ook verslapping in aortaringe op ʼn stikstofoksied-afhanklike manier veroorsaak wat ‘n aanduiding is dat ATM betrokke is by vasokonstriksie. Gevolgtrekkings: Deur ‘n groot aantal substrate te beïnvloed, speel ATM ‘n belangrike rol in die regulering van seintranduksiepaaie. Ons het bevind dat akute inhibisie van ATM in die hart nie ‘n beduidende negatiewe effek op hartfunksie gehad het of insulien-seintransduksie onderdruk het nie. Ons het wel bevind dat ATM moontlik ‘n beduidende rol speel in vasokonstriksie. Verdere studies is nodig om die kardiovaskulêre rol van ATM in insulien-seintransduksie ten volle te verstaan.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/100837
This item appears in the following collections: