The role of Atm in the regulation of cardiomyocyte glucose utilization under normal and insulin resistant conditions

Van Vuuren, Mignon (2016-03)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Introduction: Ataxia-telangiectasia (A-T) is an autosomal, recessive disorder that progressively affects multiple organs. A-T is caused by mutations in the Atm gene (ataxia-telangiectasia mutated gene), resulting in lack/inactivation of the ATM protein. These patients display a high incidence of insulin resistance or full-blown type 2 diabetes (T2D) and are therefore more prone to develop ischaemic heart disease. According to literature, ATM expression may be altered by obesity and therefore we investigated its expression and impact in the heart in the context of obesity-induced insulin resistance. Aim: To determine the importance of ATM in glucose uptake in cardiomyocytes prepared from control vs. obesity-induced insulin resistant animals. Objectives: (i) To determine ATM levels in rat models of obesity-induced insulin resistance and characterize a suitable model for the study. (ii) To manipulate the ATM protein in isolated cardiomyocytes from both control and insulin resistant animals using suitable activators or inhibitors of ATM followed by the measurement of 2-Deoxy-D-3[H] glucose (2DG) uptake levels, expression and activation of intermediates of the insulin signalling pathway (IRS1, PI3K, PTEN, PKB, AS160, GSK-3β, AMPK) and GLUT4 expression. Methods: Male Wistar rats were fed a high fat diet (HFD) for 16 weeks to induce obesity and insulin resistance and compared to chow-fed controls. Body weight, intra-peritoneal (IP) fat weight, fasting glucose and insulin levels were determined. Ventricular cardiomyocytes were prepared by collagenase perfusion digestion whereafter 2DG uptake was measured in response to treatment with specific activators/inhibitors of ATM. The specific ATM inhibitor, KU 60019, was used to manipulate the activity of the protein while the expression of proteins in the insulin signalling pathway was determined by Western blotting. Results: (i) No differences in body weight were observed. (ii) HFD animals presented with higher IP fat weight (33.7±2.4 vs. 19.1±1.5 g; p<0.001), basal glucose (5.4±0.16 vs. 4.71±0.05 mmol/L; p<0.001) and insulin (14.6±2.0 vs. 6.7±2.2 mlU/ml; p<0.05) levels vs. controls. (iii) KU 60019 inhibited insulin-stimulated 2DG uptake in the control and HFD cardiomyocytes from 26.8±3.93 and 28.38±1.27 to 13.1±2.4 (p<0.01) and 18.6±2.8 (p<0.05) pmol/mg prot/30min respectively. (iv) HFD downregulated T-ATM expression by 50% (1±0.1 vs. 0.5±0.1 ADU; p<0.001); T-PI3K by 30% (1±0.02 vs. 0.7±0.2 ADU; p<0.01); T-PKB by 40% (1±0.03 vs. 0.6±0.08 ADU; p<0.01); T-GSK-3β by 10% (1±0.02 vs. 0.9±0.05 ADU; p<0.01); T-AMPK by 30% (1±0.03 vs. 0.7±0.09 ADU; p<0.05) and P-AS160 by 30% (1±0.08 vs. 0.7±0.2 ADU; p<0.001). (v) HFD increased the phosphorylation of IRS1 with 100% at Ser307 (1±0.3 vs. 2.2±0.1 ADU; p<0.05) vs. controls. (vi) KU 60019 inhibited insulin-stimulated phosphorylation of ATM in the HFD animals (p<0.05). (vii) The inhibitory effect of KU 60019 on P-PI3K (p85) and P-PKB in both the control and HFD animals was significant (p<0.05 & p=0.001 respectively). (viii) In addition, by comparing results with younger animals, we observed age-related effects in insulin-sensitivity of cardiomyocytes. Conclusion: HFD was coupled to the development of insulin resistance, as these animals presented with higher IP fat weight, basal glucose and insulin levels and also downregulation of multiple metabolic signalling proteins and insulin sensitivity in the heart. Older control animals were also more resistant to the action of insulin than young animals. KU 60019 inhibited insulin-stimulated 2DG uptake, placing ATM in the pathway whereby insulin stimulates glucose uptake in the heart. KU 60019 significantly attenuated baseline PI3K activity and insulin-stimulated activation of ATM and PKB but did not affect AMPK/GLUT4 levels. Thus downregulation of ATM expression in obesity may be central to the development of myocardial insulin resistance.

AFRIKAANSE OPSOMMING: Inleiding: Ataxia-telangiectasia (A-T) is ‘n outosomale resessiewe siektetoestand wat verskeie organe progressief affekteer. Hierdie siektetoestand ontstaan weens mutasies in die Atm geen (ataxia-telangiectasia ge-muteerde geen), wat veroorsaak dat die ATM proteïen baie laag uitgedruk word of onaktief bly. A-T individue is meer vatbaar vir die ontwikkeling van insulien-weerstandigheid asook diabetes tipe 2 (T2D) wat gevolglik hulle risiko vir iskemiese hartsiektes verhoog. Volgens vorige studies kan vetsug tot verlaagde ATM uitdrukking bydra. Weens laasgenoemde, het ons die uitdrukking/aktivering van ATM in die konteks van ‘n vetsug-geïnduseerde insulien-weerstandige model in die hart ondersoek. Doelstelling: Om die belang van ATM in glukose opname te bepaal in kontrole en vetsug-geïnduseerde insulin-weerstandige kardiomiosiete. Objektiewes: (i) Om ATM vlakke te bepaal in vetsug-geïnduseerde insulien-weerstandige rotte en sodoende ‘n geskikte model vir die studie te kies. (ii) Om ATM in geïsoleerde ventrikulêre kardiomiosiete van beide kontrole en vet diere met geskikte aktiveerders/inhibitore van ATM te manipuleer, waarna 2-Deoksi-D-3[H] glukose (2DG) opnames, die uitdrukking/aktivering van proteïene van die insulien-seintransduksie-pad (IRS1, PI3K, PTEN, PKB, AS160, GSK-3β, AMPK) en GLUT4 uitdrukking, bepaal sal word. Metodes: Manlike Wistar rotte is gebruik. Die helfte van hierdie rotte is ‘n hoë vet dieet (HFD) vir 16 weke gevoer om vetsug/insulien-weerstandigheid te induseer, teenoor die kontrole diere wat slegs standaard rotkos ontvang het. Die liggaamsgewig, intra-peritoneale (IP) vet, vastende glukose- en insulien-vlakke is bepaal. Ventrikulêre kardiomiosiete van beide diergroepe is voorberei deur die harte met kollagenase te perfuseer waarna 2DG opname gemeet is t.o.v die behandeling met spesifieke aktiveerders/inhibitore van ATM. Die spesifieke ATM inhibitor, KU 60019 is gebruik om ATM aktiwiteit te inhibeer. Die uitdrukking/aktivering van insulien-seintransduksie proteïene is deur Westerse klad tegnieke bepaal. Resultate: (i) Daar was geen verskille in die liggaamsgewig tussen die diergroepe nie. (ii) HFD diere het hoër IP vet-massa (33.7±2.4 vs. 19.1±1.5 g; p<0.001), basale glukose vlakke (5.4±0.16 vs. 4.71±0.05 mmol/L; p<0.001) en insulien-vlakke (14.6±2.0 vs. 6.7±2.2 mlU/ml; p<0.05) teenoor die kontrole diere gehad. (iii) KU 60019 het die insulien-gemedieerde 2DG opname respons in die kontrole en HFD diere onderskeidelik onderdruk vanaf 26.8±3.93 en 28.38±1.27 tot 13.1±2.4 (p<0.01) en 18.6±2.8 (p<0.05) pmol/mg prot/30min. (iv) HFD het T-ATM uitdrukking met 50% verlaag (1±0.1 vs. 0.5±0.1 ADU; p<0.001); T-PI3K met 30% (1±0.02 vs. 0.7±0.2 ADU; p<0.01), T-PKB met 40% (1±0.03 vs. 0.6±0.08 ADU; p<0.01), T-GSK-3β met 10% (1±0.02 vs. 0.9±0.05 ADU; p<0.01), T-AMPK met 30% (1±0.03 vs. 0.7±0.09 ADU; p<0.05 en P-AS160 met 30% (1±0.08 vs. 0.7±0.2; p<0.001) teenoor die kontroles. (v) HFD het IRS1 fosforilering(Ser307) met 100% verhoog (1±0.3 vs. 2.2±0.1 ADU; p<0.05) vs. die kontroles. (vi) KU 60019 het die insulien-gestimuleerde fosforilering van ATM in die HFD diere onderdruk (p<0.05). (vii) Die inhibitoriese effek van KU 60019 op P-PI3K en P-PKB in beide diergroepe was beduidend (p<0.05 & p=0.001 onderskeidelik). (viii) Deur resultate met jonger diere te vergelyk, is duidelike ouderdomsgeïnduseerde effekte op die insuliensensitiwiteit van kardiomiosiete waargeneem. Gevolgtrekking: Die HFD het IP vet, basale glukose en insulien vlakke verhoog, asook verskeie metaboliese seintransduksie proteïne en insulien sensitiwiteit in die hart afgereguleer. KU 60019 het insulien-gestimuleerde 2DG opname geïnhibeer, wat bewys dat ATM deel vorm van die pad waardeur insulien glukose-opname in die hart stimuleer. KU 60019 het die aktivering van basale PI3K afgereguleer sowel as die insulien-gestimuleerde aktivering van ATM en PKB, met geen effek op AMPK en GLUT4 vlakke nie. Dus mag die afregulering van ATM uitdrukking in vetsugtigheid sentraal staan in die ontwikkeling van miokardiale insulienweerstandigheid.

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