THE EFFECT OF SIMVASTATIN AND METFORMIN ON ADIPOGENESIS REGULATION IN VITRO

Cilj istraživanja: Inhibicija adipogeneze potencijalan je mehanizam liječenja pretilosti. Inhibicija proadipogenih regulatora adipogeneze može rezultirati zaustavljanjem procesa adipogeneze, smanjenjem nakupljanja lipida i u konačnici prevencijom povećanja mase masnog tkiva. Cilj istraživanja bio je ispitati učinak metformina i simvastatina na regulaciju adipogeneze te odrediti najučinkovitije koncentracije lijekova i vremenski period djelovanja lijekova u inhibiciji proadipogenih regulatora adipogeneze. Materijal i metode: Istraživanje se provelo na 3T3-L1 staničnoj kulturi preadipocita koji se uz adekvatnu stimulaciju mogu diferencirati u zrele masne stanice. Tijekom procesa adipogeneze stanice su tretirane različitim koncentracijama metformina i simvastatina u različitim vremenskim periodima. Unutarstanično nakupljanje lipida vizualizirano je i Oil Red O bojanjem, ekspresija mRNA ispitivanih čimbenika odredila se PCR-om, a ekspresija proteina Western blot metodom i imunocitokemijom. Rezultati: Tretman metforminom i simvastatinom nije pokazao značajan utjecaj na unutarstanično nakupljanje lipidnih kapljica, iako je ono bilo smanjeno u skupini stanica tretiranih tijekom cjelokupnog trajanja procesa diferencijacije. Tretman je pokazao učinak na smanjenje razina mRNA PPAR-γ, C/EBP-α, SREBP-1C i STAT3 pri većim koncentracijama i primjeni tijekom cjelokupnog trajanja procesa adipogeneze. Učinak tretmana na razine proteina bio je opsežniji, sa značajnim smanjenjem razina PPAR-γ, C/EBP-α, SREBP-1C i STAT3 pri gotovo svim korištenim koncentracijama lijekova u dva vremenska perioda koji obuhvaćaju zadnja četiri dana adipogeneze, te povećanjem razine SMAD3 u dva vremenska perioda koji obuhvaćaju prvih šest dana adipogeneze. Zaključak: Promatrajući rezultate utjecaja metformina i simvastatina na inhibiciju adipogeneze u ovisnosti o obje ispitivane varijable, koncentracije lijekova i vremenskog perioda djelovanja tretmana, možemo zaključiti da su najopsežnije i najznačajnije učinke na mRNA razine ostvarili kombinirani tretmani metforminom i simvastatinom primjenjivani kontinuirano tijekom trajanja procesa diferencijacije. Na razine proteina jednak su učinak kontinuiranoj primjeni lijekovi ostvarili i tijekom kraćeg vremenskog perioda djelovanja od 7. do 9. dana, i to jednako učinkovito u pojedinačnoj i kombiniranoj primjeni u svim ispitivanim koncentracijama.Objectives: Inhibition of adipogenesis is a potential mechanism of obesity treatment. Inhibition of proadipogenic adipogenesis regulators can inhibit adipogenesis, reduce lipid accumulation, and ultimately prevent adipose tissue enlargement. The aim of the study was to examine the effect of metformin and simvastatin on the regulation of adipogenesis and to determine the most effective concentrations and time period for the inhibition of proadipogenic regulators of adipogenesis. Material and Methods: 3T3-L1 cell culture of preadipocytes that can differentiate into mature adipocytes upon adequate stimulation were used in this study. During the adipogenesis, cells were treated with different concentrations of metformin and simvastatin at different time periods. Intracellular lipid accumulation was visualized by Oil Red O staining, mRNA expression of transcription factors was determined by PCR, and protein expression by Western blot and immunocytochemistry. Results: Metformin and simvastatin treatment did not show a significant effect on intracellular accumulation of lipid droplets, although it was reduced in the group of cells treated through the entire duration of the differentiation process. An effect on reducing the mRNA levels of PPAR-γ, C/EBP-α, SREBP-1C and STAT3 was significant with combined treatment at higher concentrations applied through the entire duration of the adipogenesis process. The effect of treatment on protein levels was more extensive, with a significant reduction in protein levels of PPAR-γ, C/EBP-α, SREBP-1C i STAT3 with all drug concentrations used in the two time periods covering the last four days of adipogenesis, and by increasing SMAD3 levels in the two time periods covering the first six days of adipogenesis. Conclusion: Observing the results of the influence of metformin and sivastatin on the inhibition of adipogenesis depending on both examined variables, drug concentrations and time period of treatment, we can conclude that the most comprehensive and significant effects on mRNA levels were achieved by combined metformin and simvastatin treatment used through the whole duration of differentiation process. On protein levels the same effect as continuous drug application was achieved during a shorter period of action from 7th to 9th day, and drugs were equally effective alone as in the combined use