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...
