Biogeneza peroksizoma mrkog masnog tkiva pacova u hipotiroidizmu indukovanom metimazolom

Abstract

Mrko masno tkivo (eng. BAT, brown adipose tissue) je izuzetno važno za održavanje ukupne metaboličke i energetske homeostaze. Iako su peroksizomi prepoznati kao važni partneri mitohondrija u procesu oksidacije masnih kiselina u mrkim adipocitima, još uvek nema podataka o njihovom nastanku i putevima biogeneze. Na modelu hipotiroidizma indukovanog davanjem metimazola 7, 15 i 21 dan studirali smo remodeliranje peroksizoma i njihovu biogenezu u mrkim adipocitima pacova. Da bismo identifikovali peroksizome i puteve njihove biogeneze koristili smo imunofluorescentno obeležavanje katalaze i dva specifična metoda za njihovu vizualizaciju na ultrastrukturnom nivou, selektivnu citohemijsku tehniku obeležavanja peroksizoma diaminobenzidinom (DAB) i obeležavanje katalaze imunogoldom. Analizom dobijenih rezultata, otkrili smo da peroksizomi u mrkim adipocitima nastaju i kanonskim putem, od već postojećih organela rastom i deobom i de novo: pupljenjem samo sa glatkog endoplazminog retikuluma, samo sa spoljašnje membrane mitohondrija i hibridno – kombinacijom pupljenja sa glatkog endoplazminog retikuluma i sa mitohondrija. Svaki od puteva biogeneze zastupljen je u eutiroidnoj kontroli i intenziviran tokom hipotiroidizma na vremenski zavisan način i karakteriše se specifičnim profilima organela i setovima peroksina. Hipotiroidizam povećava broj peroksizoma za 1.8, 3.6 i 5.8 puta 7, 15. i 21. dana tretmana. Prisustvo peroksizoma, njihova distribucija i stepen sazrevanja su heterogeni u mrkim adipocitima na Harlekinu-sličan način, što odražava razlike u njihovom nastanku. Da bismo otkrili glavne molekulske igrače koji pokreću peroksizomalnu biogenezu, analizirali smo ekspresiju proteina peroksina (Pex) i membranskog transportera PMP70, uključenih u deobu peroksizoma (Pex11β), de novo biogenezu peroksizoma (Pex16 i Pex19) i strukturno/funkcijsko sazrevanje peroksizoma (Pex16, Pex19, PMP70, Pex5, Pex13, Pex26, Pex6). Kanonski put biogeneze, odlikovan brojnim strukturama nalik na tegove i niske bisera, podržan visokim nivoima Pex11β i Drp1, prevladao je 7. dana hipotiroidizma. De novo putevi peroksizomalne biogeneze intenziviraju se 15. dana i postaju dominantni 21. dana hipotiroidizma. Preusmeravanje biogeneze peroksizoma sa kanonskog na de novo put uslovljeno je povišenom ekspresijom Pex19, PMP70, Pex5S, Pex26, a karakteriše se brojnim tubularnim strukturama. Neočekivano, proteinska ekspresija Pex16 i Pex6 je bila snižena od 7. dana do kraja tretmana. Mitohondrijsko poreklo peroksizoma nije afektovano hipotiroidizmom i održava se na nivou eutiroidne kontrole. Proliferacija peroksizoma u hipotiroidizmu se odvija u dva koraka. Prvi korak, kanonskim putem se odvija 7. dana, kao brz odgovor na izmenjeni status tiroidnih hormona, a drugi korak, de novo putem, 21. dana, radi uspostavljanja i održavanja nove metaboličke homeostaze. Interesantno je da 15. dana dolazi do preklapanja ova dva puta. Budući da su ekspresija peroksina i proliferacija peroksizoma pod transkripcionom kontrolom receptora aktiviranih proliferatorima peroksizoma (eng. PPAR, peroxisome proliferator-activated receptor), analizirana je ekspresija proteina PPARα i PPARγ, kao i koaktivatora 1 α (eng. PGC-1α, PPARγ co-activator 1 alpha). Snižena ekspresija PPARα 15. i 21. dana, praćena izrazitim sniženjem ekspresije Pex16 verovatno ima ulogu u kontroli prekomerne proliferacije peroksizoma. Specifično peroksizomalno poreklo od mitohondrija u mrkim adipocitima, nezavisno od tiroidnog statusa, ukazuje na njihovo istovremeno i/ili čak uzajamno regulisanje u mrkim adipocitima pacova...

Brown adipose tissue is especially important for maintaining overall metabolic and energy homeostasis. Even though peroxisomes are recognized as important partners of mitochondria carrying out fatty acid oxidation in the brown adipocytes (BA), there is still no evidence about their origin and pathway(s) of biogenesis. At the model of hypothyroidism induced by methimazole for 7, 15, and 21 days we studied peroxisomal remodeling and origin in the rat’s brown adipocytes. To identified peroxisome and pathways of their biogenesis, we employ immunofluorescent labeling of catalase and two specific methods for their visualization at the ultrastructural level, a selective cytochemical diaminobenzidine (DAB) technique and catalase immunogold labeling. We found that peroxisomes originated by both, canonic, from pre-existing organelle by growth and division and de novo pathways: only from smooth endoplasmic reticulum by budding, only from the outer mitochondria membrane, and by combining budding from smooth endoplasmic reticulum and mitochondria as a hybrid in nature. Each pathway is operative in euthyroid control and intensified over the course of hypothyroidism in a time-dependent manner, and characterized by specific organellar profiles and sets of peroxins. Hypothyroidism increases the peroxisomal number by 1.8-, 3.6- and 5.8-time on days 7, 15, and 21 of treatment, respectively. Peroxisomal presence, distribution, and degree of maturation are heterogeneous in brown adipocytes in a Harlequin-like manner, reflecting the differences in their origin. To revealed the main molecular players that drive peroxisomal biogenesis we determined protein expression of peroxins (Pex) and peroxisomal membrane transporter PMP70, involved in peroxisome division (Pex11β), de novo peroxisome biogenesis (Pex16 and Pex19), and peroxisome structural/functional maturation (Pex16, Pex19, PMP70, Pex5, Pex13, Pex26, Pex6). The canonic pathway, through numerous dumbbell-like and pearls on strings structures, and supported by high levels of Pex11β and Drp1, prevailed on the 7th day of hypothyroidism. De novo pathways of peroxisomal biogenesis were intensified on the 15th day and become dominant on the 21st day. This redirecting of peroxisome biogenesis from canonic to de novo pathway is driven by increased protein expression of Pex19, PMP70, Pex5S, Pex26, and characterized by numerous tubular structures. Unexpected, Pex16 and Pex6 expression was decreased from day 7 until the end of the experiment. The mitochondrial origin of peroxisomes is not affected by hypothyroidism and is maintained at the level of euthyroid control. Hence, peroxisomal proliferation in hypothyroidism takes place in two steps. The first step, by the canonic pathway, occurred on the 7th day, as a fast response to the altered thyroid status, and the second step, by de novo pathway, occurred on the 21st day, to establish and maintain new metabolic homeostasis. It is interesting that on the 15th day, these two pathways overlap. Since Pex expression and peroxisomal proliferation are under transcriptional control of the peroxisome proliferator-activated receptors (PPAR), the protein expression of PPARα and PPARγ, but also PGC-1α (PPARγ co-activator 1 α), were analyzed. Decreased PPARα expression on the 15th and 21st day along with Pex16 strong downregulation, could be served to inhibit peroxisomal overproduction. The specific peroxisomal origin from mitochondria in brown adipocytes, regardless of thyroid status, indicates their simultaneous and/or even mutual regulation in rat’s brown adipocytes...