U svetlu sve većeg broja dokaza koji ukazuju na prisustvo metabolički aktivnog mrkog
masnog tkiva (BAT – brown adipose tissue, engl.) kod odraslih ljudi, kao i na njegovu
potencijalnu ulogu u sprečavanju razvoja gojaznosti, insulinske rezistence i metaboličkog
sindroma uopšte, sve je više studija o rasvetljavanju metaboličkog značaja ovog tkiva, kao i
njegovoj termogenoj stimulaciji u svrhu terapije navedenih poremećaja. Insulin se, kao
važan anabolički hormon, smatra značajnim modulatorom strukturne organizacije i funkcije
BAT. Cilj ovog istraživanja bio je rasvetljavanje molekulskih osnova insulinomindukovanog
strukturnog remodeliranja BAT putem identifikacije mehanizama koji
regulišu insulinom-stimulisanu proliferaciju i diferencijaciju ćelija, kao i identifikacije
mehanizama koji učestvuju u oštećenjima ćelija i njihovom umiranju. Pacovi soja Wistar su
jednom dnevno tretirani fiziološkom (0.4 IU/kg telesne mase) i suprafiziološkom (4 IU/kg
telesne mase) dozom insulina, jedan (akuta...n tretman) ili tri (hroničan tretman) dana. Kao
fiziološka kontrola, dve grupe pacova su u istom trajanju tretirane fiziološkim rastvorom
(akutna i hronična kontrola). Tri sata nakon poslednje doze, životinje su žrtvovane, a
interskapularni depo BAT je izolovan, izmeren i pripremljen za tehnike svetlosne i
elektronske mikroskopije. Analiza tkiva izvršena je korišćenjem metoda histohemijskog i
imunohistohemijskog bojenja, metoda konfokalne i transmisione elektronske mikroskopije
i elektron-disperzivne analize X-zracima (EDX) hemijskog sastava tkiva kao i metoda
stereološke i morfometrijske analize. Rezultati su pokazali postojanje strukturnog i
funkcijskog remodeliranja BAT, gde dominiraju procesi hipertrofije i hiperplazije ćelija,
praćene povećanjem termogenog kapaciteta mrkih adipocita (raste ekspresija dekuplujućeg
proteina 1 (UCP1 – uncoupling protein 1, engl.), udeo mitohondrija, procesi
mitohondriogeneze). S druge strane, primetno je da se, naročito pri hroničnim i tretmanima
visokom dozom insulina, javljaju citotoksični i inhibitorni efekti na pojedine ćelije ili
klastere ćelija: raste učestalost ćelijske smrti, ekstravazacija eritrocita i njihovo uklanjanje od
strane mrkih adipocita i makrofaga, (evidentno toksično po adipocite sudeći po rastu:
ekspresije enzima antioksidativne odbrane; lipidne peroksidacije i nitrozilacije proteina,
učestalosti lipofuscinogeneze). Takođe se pri hronično visokoj hiperinsulinemiji smanjuje
ekspresija regulatora termogeneze (PGC-1α - peroxisome proliferator-activated receptor – γ coactivator - 1α,
engl.) ukazujući da produženo izlaganje visokim dozama insulina, može da inhibira
termogeni odgovor tkiva, o čemu svedoči i povećana učestalost mitohondrijalnih oštećenja.
Povećana ekspresija proinflamatornih citokina (TNF-α - tumor-necrosis factor – α, engl.,
interleukina 6), nukleusnog faktora – kB (NF-kB) i makrofagnog inflamatornog proteina -
3β (MIP-3β) u pojedinačnim zrelim, multilokulusnim adipocitima, preadipocitima i
unilokulusnim adipocitima ukazuje na njihovu potencijalnu ulogu u lokalnom smanjenju
insulinske senzitivnosti. U vezi sa hiperplazijom ćelija, primećeno je da su adipogeneza i
angiogeneza u BAT vremenski i prostorno povezane, tj. da postoji udruženost navedenih
procesa, koja ukazuje na potrebu udruženog nastanka adipocita i endotelskih ćelija, u svrhu
efikasnog strukturno-funkcijskog remodeliranja tkiva. Sumarno, može se zaključiti da
hiperinsulinemija ima značajan efekat na BAT – potvrđeno je anaboličko dejstvo insulina i
važnost njegove uloge u stimulaciji BAT, ali je i pokazana toksičnost visoke doze i
hroničnog izlaganja na ćelije BAT; što je sve doprinelo rasvetljavanju molekulskih
mehanizama u osnovi strukturnog remodeliranja ovog tkiva. Takođe, pokazano je da se
visoka plastičnost BAT ogleda u postojanju funkcionalnih klastera ćelija, u kojima se
odvijaju svi važni tkivni procesi (adipo/angiogeneza, ćelijska smrt, eritrofagocitoza), što
ukazuje na pravilnu uređenost tkivne organizacije i strukturnog remodeliranja, kao
preduslova za pravilno funkcionisanje tkiva, kako u fiziološkim, tako i u uslovima
izmenjene tkivne homeostaze, što je pokazano na modelu hiperinsulinemije.
There is an increasing amount of evidence that indicates presence of brown adipose tissue
(BAT) in adult humans, and its potential role in prevention of obesity, insulin resistance
and metabolic syndrom, in general. That is why number of studies on elucidation of the
metabolic importance of this tissue, along with its thermogenic stimulation in order to treat
these disorders, increases. As an important anabolic hormone, insulin is considered as a
major modulator of BAT structure and function. The aim of this study was to elucidate the
molecular basis of insulin-induced structural remodeling of BAT through the identification
of mechanisms that regulate insulin-stimulated proliferation and differentiation and
through the identification of mechanisms involved in cell damage and death. Wistar strain
rats were treated intraperitoneally with physiological (0.4 IU/kg BW) or supraphysiological
(4 IU/kg BW ) dose of insulin for one (acute treatment) or three days (chronic treatment),
respective...ly. Two groups of rats served as physiological controls, and were treated with
saline (acute and chronic control). Three hours after the last injection the animals were
sacrificed and the interscapular portion of BAT was removed, its weight was measured and
it was processed for electron and light microscopic examinations. Methods of
histochemical staining, immunohistochemical labeling, confocal and transmission electron
microscopy, tissue electron-dispersive X-ray microanalysis (EDX) and stereological and
morphometric analyses were performed. The results showed the existence of structural and
functional remodeling of BAT, based on adipocyte hypertrophy and hyperplasia which are
followed by increasing of thermogenic capacity (expression of uncoupling protein 1
(UCP1), cellular proportion of mitochondria, mitochondrial biogenesis). On the other
hand, it is obvious that, especially after chronic and high-dose treatments, insulin cytotoxic
and inhibitory effects on individual cells or cell clusters occur: incidence of cell death
increases, as well as red blood cell extravasation and their removal by brown adipocytes and
macrophages (which is evidently toxic for the adipocyte, since expression of antioxidative
defense enzymes, lipid peroxidation, protein nitrosilation and lipofuscin formation were
increased). Also, during chronically high hyperinsulinemia, expression of peroxisome
proliferator-activated receptor - γ coactivator - 1α (PGC-1α), regulator of thermogenesis
decreases, indicating that prolonged exposure to high doses of insulin can inhibit
thermogenic response of BAT, as evidenced by the increased frequency of mitochondrial
damage in brown adipocytes. Increased expression of proinflammatory cytokines (tumornecrosis
factor - α (TNF-α), interleukin 6), nuclear factor – kB (NF-kB) and macrophage
inflammatory protein - 3β (MIP-3β) in individual mature, multilocular adipocytes,
preadipocytes and unilocular adipocytes, demonstrates their potential role in local decrease
of insulin sensitivity. Regarding hyperplasia of cells, it was noticed spaciotemporal
association between processes of adipogenesis and angiogenesis, which means that there is
a connection between these processes, demonstrating the need for the coupled
differentiation of adipocytes and endothelial cells, for the purpose of efficient structural
and functional remodeling of the tissue. In summary, it can be concluded that
hyperinsulinemia has a significant impact on BAT – we confirmed the anabolic effect of
insulin, and the importance of its role in BAT stimulation, but also demonstrated the
toxicity of high dose and chronic exposure to insulin on BAT cells, all of which contribute
to understanding the molecular mechanisms underlying structural remodeling of the tissue.
In addition, it was shown that the high plasticity of BAT is reflected in the existence of
functional clusters of cells, which take place in all the important processes in the tissue
(adipo/angiogenesis, cell death, erythrophagocytosis), indicating the proper arrangement of
tissue organization and structural remodeling as a prerequisite for the proper functioning of
the tissue under physiological conditions and during altered tissue homeostasis, as shown
on the model of hyperinsulinemia.
