ATM Expression in peripheral blood mononuclear cells as a biomarker of insulin resistance

Williams, Lois Esther (2019-04)

Thesis (MSc)--Stellenbosch University, 2019.

Thesis

Introduction: The Ataxia Telangiectasia Mutated (ATM) gene codes for the 350 kDa ATM protein kinase. ATM gene mutations cause inactivity/deficiency of the ATM protein, resulting in the autosomal recessive disease Ataxia Telangiectasia (AT). AT patients are predisposed to developing insulin resistance or further progress to type 2 diabetes and are at high risk of developing ischaemic heart disease. Due to the prevalence of insulin resistance in AT patients, we investigated the relationship between ATM protein levels and the degree of insulin resistance and proposed it as a possible early diagnostic technique for insulin resistance. Aim: To determine whether peripheral blood mononuclear cells (PBMCs) can be used to determine ATM levels in insulin resistant subjects and subsequently used as a biomarker of insulin resistance. Objectives: (i) To standardise a protocol for the isolation of PBMCs from rat blood. (ii) To isolate rat PBMCs and determine the ATM levels using Western blotting. (iii) To determine differences between ATM levels in obese rats and compare them to controls. (iv) To analyse PBMCs from a female Black Xhosa population with different degrees of insulin resistance and to determine a relationship with ATM levels. Methods: Male Wistar rats were fed an obesogenic diet (od) for 16 weeks to induce obesity and insulin resistance and compared to age-matched and young controls fed standard rat chow. Body weight and intraperitoneal (IP) fat mass were determined and oral glucose tolerance test (OGTT) was performed. PBMCs were isolated according to the standardised protocol and Western blotted for ATM and P22phox. The Western blotting protocol was repeated with samples collected from patients. Results from the animal model: 1. Effects of obesity/insulin resistance vs. age-matched controls: (i) larger IP fat mass; (ii) increased area under the curve of OGTT’s; (iii) elevated basal glucose levels. (iv) The phospho-(P)/total-(T) ATM ratio was decreased. 2.Effects of age: (i) As expected, older animals weighed more while T-ATM was decreased, P-ATMincreased and the P-/T-ATM ratio increased with age. In PBMC’s from patients, the following were observed: (i) Body mass index (BMI) was significantly higher in obese and pre-diabetic vs. control patients. (ii) The waist-to-hip ratio (WHR) of obese and pre-diabetic women was higher vs. controls. (iii) Trunk-to-limb fat mass (TF/LF) was increased in obese and pre-diabetics vs. controls but (iv) no differences in the lipid profiles were observed except for increased triglyceride levels between young pre-diabetic patients vs. their controls. (v) Fasting blood glucose of obese and pre-diabetic patients was significantly increased vs. controls. (vi) Significantly higher P-ATM levels were seen for obese and pre-diabetic vs. control patients. T-ATM levels increased with the state of insulin resistance. Effects of age: (i) BMI was significantly higher between young (Y) and middle aged (MA) control, obese and pre-diabetic groups vs. their respective controls while (ii) WHR of Y obese and Y pre-diabetic vs. Y controls also increased significantly. (iii) The TF/LF ratio was increased between Y and MA control and Y obese, Y pre-diabetic, MA obese and MA pre-diabetic women vs. their respective controls. Furthermore (iv) the blood glucose levels of Y pre-diabetics were increased vs. Y control. (v) The P-ATM levels was increased in Y pre-diabetic vs. Y control and therefore did not increase with age but the T-ATM levels significantly increased with age. Conclusion: ATM levels can be measured in PBMCs and are affected by the insulin resistant state and age. Unfortunately, due to the variation in ATM levels under different degrees of insulin resistance, it would be difficult to use ATM as a biomarker of insulin resistance.

Inleiding: Die Ataxia Telangiectasia gemuteerde (ATM) geen kodeer vir die 350 kDa ATM proteïen kinase. Mutasies in die ATM geen lei tot uitdrukking van geen of onaktiewe proteïen wat die outosomaal resessiewe siekte, Ataxia Telangiectasia (AT), veroorsaak. AT pasiënte is voorbeskik om insulienweerstandigheid of tipe 2 diabetes te ontwikkel en het ‘n hoë risiko vir iskemiese hartsiektes. Na aanleiding van die prevalensie van insulienweerstandigheid in AT pasiënte, het ons die verhouding tussen ATM proteïenvlakke en die graad van insulienweerstandigheid bestudeer en dit voorgestel as ‘n moontlike vroeë diagnostiese tegniek vir insulienweerstandigheid. Doel: Om te bepaal of perifere bloed mononukluêre selle (PBMCs) gebruik kan word om ATM vlakke in insulienweerstandige subjekte te meet en te bepaal of dit as biomerker vir insulienweerstandigheid gebruik kan word. Objektiewes: Om (i) ‘n protokol vir die isolasie van PBMCs uit rot bloed te standardiseer. (ii) Rot PBMCs te isoleer en die ATM vlakke met behulp van Westerse kladtegniede te bepaal. (iii) Verskille tussen ATM vlakke in vetsugtige rotte te vergelyk met kontrole rotte. (iv) PBMCs verkry van Swart vroulike Xhosa pasiënte met verskillende grade van insulienweerstandigheid te analiseer en die verhouding met ATM uitdrukking te bepaal. Metodes: Manlike Wistarrotte is vir 16 weke met ‘n obesogene dieet gevoer en vergelyk met ouderdomsgepaarde en jong kontrole diere op standaard rotkos. Liggaamsgewig en intraperitoneale (IP) vetgewig is bepaal en ‘n orale glukosetoleransie toets (OGTT) uitgevoer. PBMCs is geïsoleer deur die gestandardiseerde metode en ATM en P22phox deur Westerse kladtegnieke bepaal. Die Westerse kladtegniek is op die pasiënte monsters herhaal. Resultate verkry van die diermodel: 1. Effekte van obesiteit/insulienweerstandigheid vs. ouderdomsgepaarde kontroles: (i) groter IP vetmassa; (ii) verhoogde area onder die kurwe van die OGTT’s (iii) verhoogde basale bloedglukosevlakke is gemeet. (iv) Die fosfo-(P)/totale (T) ATM verhouding was verlaag. 2.Effekte van ouderdom: (i) soos verwag, het die ouer diere meer geweeg terwyl T-ATM vlakkeverlaag het, P-ATM verhoog en die P/T verhouding beduidend verhoog het met ouderdom. In PBMC’s van pasiënte is die volgende waargeneem: (i) die liggaamsmassaindeks (LMI) was beduidend hoër in die vetsugtige en pre-diabetiese pasiënte vs. kontroles. (ii) die middel-tot-heup (MTH) verhouding van vetsugtige en pre-diabetiese vroue was hoër as die kontroles. (iii) Die romp-tot-ledemaat (RV/LV) vetmassa was verhoog in die vetsugtige en pre-diabetiese vroue vs. kontroles maar (iv) geen verskille in die lipiedprofiele is waargeneem nie, buiten verhoogde trigliseriedvlakke tussen jong pre-diabetiese pasiënte vs. hulle kontroles. (v) Vastende bloedglukose van vetsugtige en pre-diabetiese pasiënte was beduidend hoër as kontroles. (vi) Beduidend hoër P-ATM vlakke is waargeneem in die vetsugtige en pre-diabetiese vs. kontrole pasiënte. T-ATM vlakke het verhoog met die staat van insulienweerstandigheid. Effekte van ouderdom: (i) LMI was beduidend hoër in die jong (J) en middeljarige (MJ) kontrole, vetsugtige en pre-diabetiese groepe vs. hulle onderskeie kontroles terwyl (ii) die MTH van die J vetsugtige en pre-diabetiese vs. J kontroles ook beduidend hoër was. (iii) Die RV/LV verhouding het toegeneem tussen J en MJ kontrole en J vetsugtig, J pre-diabeties, MJ vetsutgig en MJ pre-diabetiese vroue vs. hulle onderskeie kontroles. Verder (iv) was die bloedglukosevlakke van die J pre-diabetiese pasiënte verhoog teenoor J kontroles. (v) Die P-ATM vlakke was verhoog in die J pre-diabetiese vs. J kontroles en het dus nie met ouderdom verhoog nie maar die totale vlakke van ATM het beduidend toegeneem met ouderdom. Gevolgtrekking: ATM vlakke kan in PBMCs gemeet word en die vlakke word beïnvloed deur die insulienweerstandige staat sowel as ouderdom. Ongelukkig, as gevolg van variasies in ATM vlakke in die verskillende grade van insulienweerstandigheid, sal dit problematies wees om ATM as ‘n biomarker van insulienweerstandigheid te gebruik.

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