Publication: Hepatositlerde fruktoz ile indüklenen lipit birikiminde rol oynayan faktörlerin aydınlatılması
Abstract
Amaç: Sterol düzenleyici element bağlayıcı protein 1c (SREBP-1c), yağ asitlerinin biyosentezinde rol oynayan bir transkripsiyon faktörüdür. Lipit birikimini ve metabolik düzensizliği teşvik ederek metabolik disfonksiyon ilişkili yağlı karaciğer hastalığına (MAFLD) yol açabilmektedir. Beslenme alışkanlıklarında fruktozun artan yaygınlığı, asetil-CoA üretiminde artışa sebep olarak SREBP-1c aktivasyonunu sağlamaktadır. Çalışmanın amacı hepatositlerde fruktoz ile indüklenen lipit birikiminde rol oynayan faktörlerin araştırılması ve bu doğrultuda SREBP-1c’ nin, lipit metabolizması ve lipofaji ile ilişkilendirilerek aydınlatılmasıdır. Gereç ve Yöntem: Çalışma, lipit metabolizması ve lipofaji mekanizmalarını aydınlatmayı planlandığı için hücre kültürü ile gerçekleştirildi. Bu kapsamda öncelikle, fruktoz ve SREBP-1c siRNA uygulanan hücreler üzerinde oluşan lipit damlacığı (LD) sayı/ boyutu BODIPY boyaması ile, lipit metabolizmasında görevli mRNA ekspresyonları RT- PCR yöntemi ile incelendi. Devamında, LD’ larının otofaji ile yıkımı (lipofaji) konfokal mikroskobu ile belirlendi. Çalışmanın ikinci bölümünde ise, Bafilomycin A1 (BafA1) uygulaması ile otofajik akış inhibe edildi ve değişen lipit birikimi, lipit metabolizması, lipofaji aktivitesi ve otofajiden sorumlu protein ekspresyonları incelenerek SREBP-1c ile ilişkilendirildi. Bulgular: Fruktoz uygulaması, SREBP-1c seviyeleri ile birlikte LD sayısını ve boyutunu arttırdı. SREBP-1c susturulması LD sayısını azaltırken, BafA1 bu etkiyi kısmen hafifletti. Ayrıca, SREBP-1c susturulması stearoil koenzim A desatüraz-1 (SCD-1) dışında diğer lipojenik mRNA ekspresyonları üzerinde etki göstermedi. İlaveten, BafA1 uygulanan ve fruktoz ile lipit birikiminin indüklendiği hücrelerde LD/ LAMP1 ko-lokalizasyonu artmasına rağmen Beclin1 ve p62 ekspresyonu ile LC3II/ I oranı değişmedi. Sonuç: Fruktoz ile lipit birikiminin indüklendiği hepatositlerde SREBP-1c' nin susturulması, lipofajiye bağlı bir şekilde LD birikimini azaltarak yararlı etkisini göstermiştir.
Objective: Sterol regulatory element binding protein 1c (SREBP-1c) is a transcription factor involved in the biosynthesis of fatty acids. It promotes lipid accumulation and metabolic dysregulation, leading to metabolic dysfunction fatty liver disease (MAFLD). Increasing prevalence of fructose in dietary habits leads to an increase in acetyl-CoA production and activation of SREBP-1c. The aim of the study was to investigate the factors involved in fructose-induced lipid accumulation in hepatocytes and to elucidate SREBP-1c protein in relation to lipid metabolism and lipophagy. Materials and Methods: The study was carried out with cell culture since it was planned to elucidate the mechanisms of lipid metabolism and lipophagy. In this context, firstly, the number/ size of lipid droplet (LD) formed on fructose and SREBP-1c siRNA treated cells were analysed by BODIPY staining and mRNA expressions involved in lipid metabolism were examined by RT-PCR method. Subsequently, the degradation of LDs by autophagy (lipophagy) was determined by confocal microscopy. In the second part of the study, autophagic flow was inhibited by Bafilomycin A1 (BafA1) treatment. and altered lipid accumulation, lipid metabolism, lipophagy activity and protein expressions responsible for autophagy were examined and associated with SREBP-1c. Results: Fructose administration resulted an increase in the number and size of lipid droplets (LDs) along with the levels of SREBP-1c. The silencing of SREBP-1c resulted a reduction in the number of LDs, while the partial attenuation of this effect was noted with the administration of BafA1. Moreover, the silencing of SREBP-1c had no impact on the expression of lipogenic mRNAs other than stearoyl-coA desaturase-1 (SCD-1). Additionally, despite the increase in LD/ LAMP1 co-localization in BafA1-treated cells, in which the lipid accumulation was induced by fructose, Beclin1 and p62 expression, together with LC3II/ I ratio, did not changed. Conclusion: SREBP-1c silencing in hepatocytes shows its beneficial effect by decreasing fructose-induced LD accumulation in a lipophagy dependent manner.
Objective: Sterol regulatory element binding protein 1c (SREBP-1c) is a transcription factor involved in the biosynthesis of fatty acids. It promotes lipid accumulation and metabolic dysregulation, leading to metabolic dysfunction fatty liver disease (MAFLD). Increasing prevalence of fructose in dietary habits leads to an increase in acetyl-CoA production and activation of SREBP-1c. The aim of the study was to investigate the factors involved in fructose-induced lipid accumulation in hepatocytes and to elucidate SREBP-1c protein in relation to lipid metabolism and lipophagy. Materials and Methods: The study was carried out with cell culture since it was planned to elucidate the mechanisms of lipid metabolism and lipophagy. In this context, firstly, the number/ size of lipid droplet (LD) formed on fructose and SREBP-1c siRNA treated cells were analysed by BODIPY staining and mRNA expressions involved in lipid metabolism were examined by RT-PCR method. Subsequently, the degradation of LDs by autophagy (lipophagy) was determined by confocal microscopy. In the second part of the study, autophagic flow was inhibited by Bafilomycin A1 (BafA1) treatment. and altered lipid accumulation, lipid metabolism, lipophagy activity and protein expressions responsible for autophagy were examined and associated with SREBP-1c. Results: Fructose administration resulted an increase in the number and size of lipid droplets (LDs) along with the levels of SREBP-1c. The silencing of SREBP-1c resulted a reduction in the number of LDs, while the partial attenuation of this effect was noted with the administration of BafA1. Moreover, the silencing of SREBP-1c had no impact on the expression of lipogenic mRNAs other than stearoyl-coA desaturase-1 (SCD-1). Additionally, despite the increase in LD/ LAMP1 co-localization in BafA1-treated cells, in which the lipid accumulation was induced by fructose, Beclin1 and p62 expression, together with LC3II/ I ratio, did not changed. Conclusion: SREBP-1c silencing in hepatocytes shows its beneficial effect by decreasing fructose-induced LD accumulation in a lipophagy dependent manner.