Molecular mechanisms of metabolic regulation in hibernation and during cold-acclimation in European ground squirrel (Spermophilus citellus)

Abstract

Fenomen hibernacije predstavlja fascinantan primer plastičnosti kod sisara. Hibernirajući sisari podliježu složenom nizu biohemijskih, fizioloških i bihevioralnih promjena u odgovoru na senzonske energetski-zahtijevne periode kuplovane sa redukovanom dopstupnošću hrane. Cilj disertacije je da se ispitaju promjene ćelijskog metabolizma u tkivima i organima tekunica, ključnim za održanje ukupne energetske homeostaze - mrko i bijelo masno tkivo (engl. brown adipose tissue, BAT i white adipose tissue, WAT), mišići i jetra, tokom perioda aklimacije na nisku temperaturu, kao i u fazi hibernacije. Efekat aklimacije/hibernacije na metaboličko remodeliranje u tkivima i organima tekunica praćen je određivanjem: mitohondrijalnog kapaciteta (genska i/ili proteinska ekspresije komponenti respiratornog lanca i ATP sintaze); termogenog kapaciteta (ekspresija dekuplujućeg proteina 1); ekspresionih profila enzima ključnih metaboličkih puteva: glikolize, β-oksidacije, Krebsovog ciklusa, metabolizama triacilglicerola i glikogena. Ispitivani su i transkripcioni faktori uključeni u metaboličku regulaciju, kao i enzimi antioksidativne odbrane. Takođe, ispitivane su promjene u BAT i depoima WAT na strukturnom i ultrastrukturnom nivou. Mužjaci evropske tekunice Spermophilus citellus su početkom septembra podijeljeni u dvije grupe: kontrolnu, koja je boravila na sobnoj temeraturi (22 ± 1 ºC) i grupu aklimiranu na nisku temperaturu (4 ± 1 ºC). Aktivne, eutermične tekunice, koje nisu ušle u duboku hibernaciju tokom aklimacije, žrtvovane su nakon 1, 3, 7, 12, odnosno 21 dan. Tekunice koje su ušle u hibernaciju žrtvovane su nakon 2-5 dana trajanja hibernacije (kontinuirana rektalna temperatura 4 ºC). BAT, subkutano, retroperitonealno i epididimalno WAT (sWAT, rWAT i eWAT), skeletni mišić (musculus quadriceps) i jetra su uzorkovani odmah po žrtvovanju. Kako bi se ispitali mehanizmi metaboličke regulacije u BAT, specifični za hibernatore, paralelno je rađena komparativna studija aklimacije pacova na nisku temperaturu...The phenomenon of hibernation is a fascinating example of plasticity in mammals. Hibernating mammals are subjected to a complex series of biochemical, physiological and behavioral changes in response to seasonal energy-demanding periods coupled with reduced food availability. The aim of the thesis was to investigate metabolic changes in the key tissues and organs of the ground squirrel, responsible for maintaining overall energy homeostasis - brown and white adipose tissue (BAT and WAT), skeletal muscle and liver, during the acclimation to low temperature, as well as in the hibernation. Effects of acclimation/hibernation on metabolic remodeling in the tissues and organs of the ground squirrel were determined by: mitochondrial capacity (gene and/or protein expression of the components of the respiratory chain and ATP synthase); thermogenic capacity (uncoupling protein 1 content), the expression profile of the key metabolic enzymes involved in: glycolysis, β-oxidation, Krebs cycle, glycogen and triglycerides metabolism. Furthermore, transcription factors involved in the metabolic regulation, as well as antioxidant enzymes, were examined. In parallel, we examined the changes in BAT and WAT depots on the structural and ultrastructural level. Males of the European ground squirrel Spermophilus citellus were divided into two groups in early September: the control group, kept at room temperature (22 ± 1 º C) and a group acclimated to low temperature (4 ± 1 ºC). Active, euthermic ground squirrels, which did not enter into deep hibernation during acclimation, were sacrificed after 1, 3, 7, 12, or 21 days. Ground squirrels that entered hibernation were sacrificed after 2-5 days of hibernation (continuous rectal temperature of 4 ºC). BAT, subcutaneous, retroperitoneal and epididimal WAT (sWAT, rWAT and eWAT), skeletal muscle (musculus quadriceps) and liver were sampled. Parallel, in order to examine the mechanisms of metabolic regulation in the BAT specific for hibernation, comparative study with rats acclimated to low temperatures, was made. Results show that, when hibernating animals are exposed to low temperature, the cellular metabolism in all examined tissues/organs is initially subordinated to maintaining euthermia, i.e. thermogenic process. In the early period of the cold-exposure, shivering and nonshivering thermogenesis is induced in the muscle and BAT, respectively..