The role of melatonin in cardioprotection : an investigation into the mechanisms involved in glucose homeostasis, microvascular endothelial function and mitochondrial function in normal and insulin resistant states

Nduhirabandi, Frederic (2014-04)

Thesis (PhD)-- Stellenbosch University, 2014.

Thesis

ENGLISH ABSTRACT: Introduction: The cardioprotective actions of the hormone melatonin against myocardial ischaemiareperfusion injury (IRI) are well-established. It has recently been shown to prevent the harmful effects of hyperphagia-induced obesity on the susceptibility of the heart to IRI as well as many of the harmful effects of obesity and insulin resistance. However, the exact mechanism whereby it exerts its beneficial action is still unknown. The aims of this study were to determine the effects of relatively short-term melatonin treatment in a rat model of diet-induced obesity on: (i) biometric and metabolic parameters, lipid peroxidation, myocardial IRI and intracellular signalling (ii) mitochondrial oxidative phosphorylation function (iii) cardiomyocyte glucose uptake and intracellular signalling. In addition, the effects of acute melatonin treatment of cardiac microvascular endothelial cells (CMEC) were determined on cell viability, nitric oxide production (NO), TNF- -induced dysfunction and intracellular signalling. Material and Methods: Male Wistar rats were randomly allocated to two groups for 20 weeks feeding with either standard rat chow or a high calorie diet. Each group was subdivided into 3 groups receiving either water throughout or melatonin (4mg/kg/day, in the drinking water) for the last 6 or 3 weeks of the experimental programme. Hearts, perfused in the working mode, were subjected to ischaemia/reperfusion and infarct size determined. Mitochondria and cardiomyocytes were isolated according to standard techniques and oxidative function and glucose uptake respectively determined. CMEC NO production and cell viability were quantified by FACS analysis of the fluorescent probes, DAF-2/DA and propidium iodide/Annexin V respectively. Intracellular signalling was evaluated using Western blot and appropriate antibodies. Results: The high-calorie diet caused significant increases in body weight gain, visceral adiposity, fasting blood glucose, serum insulin, triglycerides, HOMA-IR index and a concomitant reduction in serum adiponectin levels as well as larger myocardial infarct sizes after exposure to IRI compared to the control, indicating increased susceptibility to damage. Three as well as six weeks of melatonin administration to obese and insulin resistant rats reduced serum insulin levels and the HOMA-IR index. Myocardial infarct size was reduced in both control and diet groups. These effects were associated with increased activation of baseline myocardial STAT- 3 and the RISK pathway during reperfusion. The diet had no effect on the oxidative phosphorylation capacity of mitochondria, isolated from non-perfused hearts (baseline), but melatonin administration for 6 weeks induced a reduction in state 3 respiration rate; mitochondria isolated from diet hearts subjected to global ischaemia, exhibited an attenuated oxidative phosphorylation process which was improved by melatonin treatment. Melatonin in vitro enhanced cardiomycyte insulin stimulated glucose uptake of normal young rats but not of insulin resistant rats. In vivo melatonin treatment for 6 weeks increased basal (in diet group) and insulin stimulated glucose uptake in both control and diet groups. Melatonin (1nM) in vitro caused a significant reduction in necrosis and apoptosis of cultured CMEC, associated with a decrease in nitric oxide availability and eNOS activation and a concomitant increase in PKB/Akt, p38MAPK and AMPK activation. The harmful effects of TNF- treatment on signalling in CMEC could be prevented by co-treatment with melatonin. Conclusions: The results suggest that short-term melatonin treatment was able to significantly attenuate the diet-induced increased myocardial susceptibility to ischaemia/reperfusion damage. It may also improve cardiac glucose homeostasis and mitochondrial oxidative phosphorylation in an insulin resistant state. Melatonin in vitro protects CMEC against apoptosis and necrosis and reduces nitric oxide availability. These beneficial effects of melatonin may ultimately be due to its antioxidant capacity or receptor-mediated actions, but this remains to be established.

AFRIKAANSE OPSOMMING: Inleiding: Die vermoë van die hormoon, melatonien, om die hart teen iskemie/ herperfusiebesering (IHB) te beskerm, is welbekend. Onlangs is ook getoon dat melatonien IHB en verskeie van die nadelige effekte van vetsug en insulienweerstandigheid in hiperfagiegeïnduseerde vetsug kan voorkom. Die meganisme(s) betrokke by hierdie voordelige prosesse is egter grootliks onbekend. Die doel van hierdie studie was om die gevolge van korttermyn melatonienbehandeling in ‘n model van hiperfagiegeïnduseerde vetsug te ondersoek op (i) biometriese en metaboliese parameters, lipiedperoksidasie, miokardiale IHB en intrasellulêre seintransduksie, (ii) mitochondriale oksidatiewe fosforilasie, (iii) glukoseopname en intrasellulêre seintransduksie in kardiomiosiete en aanvullend, (iv) die invloed van akute melatonienbehandeling van kardiale mikrovaskulêre endoteelselle op sellulêre oorlewing, stikstofoksiedproduksie, TNF- - geïnduseerde disfunksie en seintransduksie. Metodiek: Manlike Wistarrotte is ewekansig in twee groep verdeel en vir 20 weke met standaard-rotkos of ‘n hoëkaloriedieet gevoer. Elke groep is in 3 subgroepe verdeel, wat deurgaans water of melatonien (4mg/kg/dag in die drinkwater) vir 3 of 6 weke voor die beëindiging van die eksperiment ontvang het. Harte is geperfuseer volgens die werkharttegniek, blootgestel aan iskemie/herperfusie en die infarktgrootte bepaal. Mitochondria en kardiomiosiete is volgens standaardtegnieke geïsoleer vir bepaling van oksidatiewe funksie en glukoseopname respektiewelik. NO produksie en sellewensvatbaarheid was gekwantifiseer deur vloeisitometriese analises (FACS) van die fluoresserende agense, DAF-2/DA en propidium jodied/Annexin V onderskeidelik. Intrasellulêre seintransduksie is evalueer met behulp van die Western kladtegniek en geskikte antiliggame. Resultate: Die hoëkaloriedieet het ‘n beduidende toename in liggaamsgewig, visserale vet, vastende bloedglukose, seruminsulienvlakke, trigliseriede, HOMA-IR-indeks en ‘n gepaardgaande verlaging in serumadiponektienvlakke tot gevolg gehad, sowel as groter miokardiale infarkte na iskemie/herperfusie. Laasgenoemde dui op ‘n groter vatbaarheid vir iskemiese beskadiging in harte van vetsugtige diere. Drie sowel as ses weke van melatonienbehandeling het die seruminsulienvlakke en HOMAindeks in vetsugtige diere beduidend verlaag, vergeleke met die kontroles. Miokardiale infarktgroottes was verminder in beide kontrole- en vetsuggroepe. Hierdie effekte het met ‘n verhoogde aktivering van basislyn STAT-3 en PKB/Akt en ERKp44/p42 tydens herperfusie gepaard gegaan. Die dieet het geen invloed op die oksidatiewe fosforilasiekapasiteit van mitochondria, geïsoleer uit harte van ongeperfuseerde harte, gehad nie (basislyn), maar melatonienbehandeling vir 6 weke het Staat 3 respirasie verlaag. Mitochondria, geïsoleer uit harte van vetsugtige rotte wat aan globale iskemie onderwerp was, het ‘n onderdrukte oksidatiewe fosforilasieproses gehad, wat egter deur melatonienbehandeling verbeter is. Melatonien in vitro het insuliengestimuleerde glukoseopname deur kardiomiosiete van jong, maar nie vetsugtige rotte nie, verhoog. In vivo melatonientoediening vir 6 weke het egter basale (in die dieetgroep) en insuliengestimuleerde glukoseopname in beide kontrole- en vetsuggroepe verhoog. Toediening van melatonien in vitro aan mikrovaskulêre endoteelselkulture het ‘n beduidende afname in nekrose, apoptose, stikstofoksied- beskikbaarheid en eNOS aktivering teweeggebring, tesame met ‘n verhoogde aktivering van PKB/Akt, p38MAPK en AMPK. Die nadelige effekte van TNF- toediening op seintransduksie in die mikrovaskulêre endoteelselle is deur melatonien voorkom. Gevogtrekkings: Die resultate toon dat melatonien ‘n merkwaardige beskermende effek op die toename in vatbaarheid vir iskemiese beskadiging in vetsugtige rotte gehad het. Dit mag ook miokardiale glukose-homeostase en mitochondriale oksidatiewe funksie in insulienweerstandigheid verbeter. Melatonien in vitro beskerm mikrovaskulêre endoteelselle teen nekrose asook apoptose en verminder die beskikbaarheid van stikstofoksied. Hierdie voordelige effekte van melatonien mag aan sy anti-oksidantvermoëns of stimulasie van die melatonienreseptor toegeskryf word, maar bewyse daarvoor ontbreek nog.

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