Molekulski mehanizmi metaboličke regulacije u hibernaciji i tokom aklimacije na hladnoću kod tekunice (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. Rezultati pokazuju da je, prilikom izlaganja hibernirajućih životinja niskoj temperaturi, ćelijski metabolizam u svim ispitivanim tkivima/organima inicijalno podređen održanju eutermije, tj. termogenom procesu koji se odvija u skeletnim mišićima i BAT. U ranom periodu izlaganja hladnoći, u skeletnim mišićima i BAT, dolazi do indukcije termogeneze (drhteće i nedrhteće), koja je metabolički podržana razlaganjem šećera. Međutim, produkcija toplote u oba termogena organa asocirana je sa energetskim disbalansom, koji je tokom produžene aklimacije najvjerovatnije odgovoran za supresiju puteva potrošnje energije (primarno termogeneze) i pripremu tekunica za ulazak u stanje hipotermije/hipometabolizma. Najveći dio goriva za termogenezu, u ranoj fazi aklimacije, obezbijeđuje se iz visceralnih depoa WAT (posebno eWAT), u kojima je detektovano smanjenje površine adipocitnog profila, usled intenzivne lipolize. Kasnije tokom izlaganja hladnoći, kada se termogeni kapacitet BAT suprimira, u sva tri ispitivana depoa WAT indukovana je aktivnost AMP-aktivirane protein kinase (AMPK), „blokirana” lipoliza i putevi potrošnje energije, a stimulisan oksidativni metabolizam. Jetra tokom aklimacije ima centralno mjesto koordinacije lipidnog i ugljenohidratnog metabolizma. Rezultati disertacije pokazuju da je u fazi duboke hibernacije termogeni kapacitet BAT i skeletnih mišića značajno suprimiran. Takođe, hipoksija-inducibilni factor-1 (HIF-1) ima centralnu ulogu u metaboličkom remodeliranju termogenih organa u fazi hibernacije. Osim suprimiranja energetski-zahtijevnih termogenih procesa, HIF-1 u uslovima hipometabolizma ima ulogu i u regulaciji, tj. indukciji glikolitičkog puta, važne komponente energetske homeostaze tokom hibernacije. Slično reprogramiranje metabolizma dešava se i u visceralnim depoima WAT. Jetra u hibernaciji ostvaruje veoma sličnu metaboličku strategiju kao pri uslovima gladovanja: indukcija katabolizma lipida i supresija razlaganja glukoze, uz istovremenu indukciju glukoneogeneze. Rezultati disertacije ukazuju da je u hibernaciji kapacitet za oksidaciju lipida povećan u svim ispitivanim tkivima/organima, osim u visceralnim depoima WAT. U BAT, intenziviranje puteva oksidacije masnih kiselina primarno služi održanju neophodnog nivoa termogeneze, dok je u skeletnim mišićima i jetri povezano sa adaptacijom na hipotermične/hipometaboličke uslove hibernacije. Uočljuv manji stepen interorganske komunikacije kada je lipidni metabolizam u pitanju, sugeriše da se tokom hibernacije uspostavlja određeni stepen tkivne autonomnosti. Generalno se može zaključiti da karakteristično tkivno-zavisno remodelovanje energetskog metabolizma predstavlja centralnu osovinu i uslov fiziološke plastičnosti hibernatora koja im omogućava brojne prednosti u odnosu na nehibernirajuće srodnike i preživljavanje u nepovoljnim uslovima sredine, bilo da su aktivni i eutermični ili letargični i hipotemični.

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. Although these processes are metabolically supported by the breakdown of glucose, the production of heat in both thermogenic organs is associated with energy imbalance. This is likely responsible for suppression of energy consumption pathways (primarly thermogenesis) during the extended acclimation period, and for preparation of ground squirrel to a state of hypothermia/hypometabolism. In the early period of acclimation, the fuel for thermogenesis is preferentially provided from visceral WAT depots (particularly eWAT), wherein decreased adipocyte surface profile, a result of intensive lipolysis, is observed. Later, during exposure to cold, when the thermogenic capacity in BAT is suppressed, activity of AMP-activated protein kinase is induced in all three WAT depots, lipolysis and energy-consuming pathways are "blocked" and oxidative metabolism is stimulated. Liver is central coordinator of the lipid and carbohydrate metabolism during cold acclimation. In the deep hibernation, thermogenic capacity of BAT and skeletal muscle is significantly suppressed. It seems that hypoxia-inducible factor-1 (HIF-1) plays a central role in the metabolic remodeling of the thermogenic organs in hibernation, by both suppressing the energy-demanding, thermogenic processes, and also, by inducing glycolytic pathway, an important component of energy homeostasis in hypometabolic conditions. Similar metabolic reprogramming occurs in the visceral WAT depots. In hibernation, liver shows analogous metabolic strategy as in fasting conditions: induction of lipid catabolism and suppression of glucose degradation, with the simultaneous induction of gluconeogenesis. Results indicate that capacity for lipid oxidation is increased in all tested tissues/organs, except in visceral WAT depots, during hibernation. In BAT, the intensification of fatty acid oxidation pathways primarily serves to maintain the necessary level of thermogenesis, whereas in skeletal muscle and liver this is associated with adaptation to hypothermic/hypometabolic conditions of hibernation. Noticeable lower level of interorgan communication, when it comes to lipid metabolism, suggests that tissues establish a certain degree of autonomy during hibernation. In general, it can be concluded that characteristic tissue-dependent remodeling of the energy metabolism is the central axis and the precause of physiological plasticity of hibernators that provides them many advantages over the nonhibernating counterparts, and the survival under adverse environmental conditions, whether they are active and euthermic or lethargic and hypothermic.