Molekulske osnove strukturnog remodeliranja mitohondrija indukovanog kalcijumom i insulinom u mrkim adipocitima pacova

Abstract

Osnovna uloga mrkog masnog tkiva je održanje tjelesne temperature i održanje energetske homeostaze organizma. Izlaganje hladnoći, a i povećan unos hrane, dovodi do stimulacije, proliferacije i diferencijacije mrkih adipocita, što je praćeno remodeliranjem mitohondrija, termogenih organela mrkih adipocita. U okviru strukturnog remodeliranja mitohondrije pokazuju širok dijapazon morfoloških promjena (broj mitohondrija u ćeliji, srednji dijametar, fuzija i fisija mitohondrija, volumenska gustina kristi) što je povezano sa prisustvom pojedinih kompleksa elektron transportnog lanca, ATP sintaze, familije UCP proteina, posebno UCP1, a i drugih proteina unutrašnje membrane mitohondrija. Kalcijum igra veoma važnu ulogu u termogenezi, njegova koncentracija u citoplazmi i organelama mrkih adipocita je hormonski regulisana, prije svega noradrenalinom. Mitohondrije, s jedne strane, imaju važnu ulogu u oblikovanju signalnih puteva kalcijuma, a kalcijum, s druge strane, u fiziološkim koncentracijama stimuliše mitohondrijalni metabolizam i povećava produkciju energije, dok u visokim koncentracijama indukuje apoptozu. Insulin je jedan od glavnih modulatora termogene funkcije mrkih adipocita, gdje stimuliše sintezu i deponovanje lipida u mrkim adipocitima, proliferaciju mrkih adipocita, angiogenezu i vazodilataciju. Pokazano je da insulin utiče na aktivnost elektron transportnog lanca, a i da mitohondrije pojačavaju insulinsku osjetljivost putem redoks regulacije proteina tirozin kinaze i insulinskog receptora. Cilj ove doktorske disertacije je rasvjetljavanje kako kalcijum, s jedne strane, a insulin, s druge strane, utiču na molekulske mehanizme koje su uključene u strukturno remodeliranje mitohondrija mrkih adipocita pacova. U eksperimentu su korišćeni Wistar pacovi, starosti dva mjeseca. U eksperimentu sa Ca-SANDOZ, životinje su podijeljene u dve grupe, jedna je pila Ca-SANDOZ rastvoren u vodi, a druga, kontrolna, česmensku vodu, tri dana. U eksperimentu sa insulinom, pacovi su podijeljeni u šest grupa, četiri grupe su primale nisku (0.4 IU) ili visoku (4 IU) dozu insulina i to jedan (akutno) ili tri dana (hronično). Ostale dvije grupe, kontrolne, su primale fiziološki rastvor akutno ili hronično. Trećeg dana tretmana Ca-SANDOZ, odnosno tri sata poslije posljednjeg tretmana insulinom, životinje su žrtvovane dekapitacijom, a interskapularni depo BAT je izolovan i pripremljen za biohemijske, mikroskopske i molekularno-biološke analize, i za izolaciju mitohondrijalnih frakcija. BAT i mitohondrijama bogata frakcija su analizirane Western Blot analizom, imunohistohemijskom, imunofluorescentnom i imunogold analizama, ultrastukturnim i stereološkim analizama, a analizirana je i aktivnost ATP sintaze...

Essential role of brown adipose tissue is maintaining body temperature and energy homeostasis of organism. Exposure to cold and increased diet intake lead to stimulation, proliferation and differentiation of brown adipocytes, what is followed by remodeling of mitochondria, thermogenic organelles of brown adipocytes. In the context of structural remodeling, mitochondria show wide repertoire of morphological changes (e.g. mitochondria number in cell, mean diameter, fusion and fission of mitochondria, volume density of mitochondrial cristae) what is connected with abundance of components of electron transport chain (ETC), ATP synthase, UCP proteins - especially UCP1 protein, and other proteins of inner mitochondrial membrane. Calcium plays important role in thermogenesis, and its concentration in cytosol and organelles of brown adipocytes is hormone regulated, particularly by noradrenaline. Mitochondria has essential role in shaping calcium signaling pathways, and calcium, on other hand, stimulates mitochondrial metabolism and increases energy production, but in high concentrations induces apoptosis. Insulin is one of major modulators of thermogenic function of brown adipocytes, and stimulates synthesis and storage of lipids in brown adipocytes, proliferation of brown adipocytes, angiogenesis and vasodilatation. It is showed that insulin has effect on electron transport chain activity, but also mitochondria enhances insulin sensitivity via redox regulation of tyrosine kinases and insulin receptor. The aim of this doctoral dissertation is elucidation how calcium and insulin effect on molecular mechanisms involved in structural remodeling of mitochondria of rat brown adipocytes. Two-month-old male Wistar rats were used. In Ca-SANDOZ study, they were divided into two groups - Ca-SANDOZ drinking or tap water drinking for three days. In insulin experiment, rats were divided into six groups, where four groups treated with low (0.4 IU) or high (4 IU) dose of insulin, for one (acutely) or three (chronically) days. Another two groups served as control, treated with 0.9% saline solution for one or three days. On third day of Ca-SANDOZ treatment, or three hours after last administration of insulin, they were sacrificed by decapitation, and interscapular portion of BAT was isolated and prepared for biochemical, microscopic and molecular biology analyses, also for isolation of mitochondria-enriched BAT fraction. BAT and mitochondria-enriched fraction were analyzed by Western blot analysis, imunohistochemical, immunofluorescent, immunogold, ultrastructural and stereological analyses. Also, ATP synthase activity was measured...