Publication: In vitro nonalkolik yağlı karaciğer modelinde mitokondriyal kökenli mots-c peptidinin hastalık etyopatogenezi üzerine etkileri
Abstract
Amaç: 2015 yılında mitokondriyal DNA’ya ait yeni bir gen keşfedilmiştir. Bu genin fonksiyonel ürünü olan 16 amino asitlik 12S rRNA tip-c mitokondriyal açık okuma çerçevesi (MOTS-c) peptidinin deneysel hayvan modellerinde AMPK yolağı üzerinden insülin direnci ve obeziteyi azalttığı bildirilmiştir. Ancak bu peptidin NAFLD üzerine etkisi ile ilişkili olarak literatürde bilgimiz dahilinde herhangi bir çalışma yoktur. Bu araştırmadaki amacımız in vitro NAFLD modelinde, MOTS-c peptidinin yağlanmayı azaltan bir etkisinin olup olmadığını tespit etmektir. Yöntem:Çalışmamızda, öncelikle 75 cm2’lik flasklara ekimi yapılan HepG2 hücreleri 24 saatlik düşük glikoz ortamında aç bırakılmıştır. Ardından 1mM konsantrasyonunda palmitik ve oleik asit uygulanarak in vitro NAFLD modeli oluşturulmuştur. Yağlanma Oil Red O (ORO) yöntemi ile kantifiye edilmiştir. Hücre canlılığı Cell Count and Viability Kit (MUSE) ile tespit edilmiştir. Hücrelere 10 μM MOTS-c peptidi 24 saat süre ile uygulanmıştır. Bulgular: Yağlanma modeli oluşturulan HepG2 hücrelerinde yapılan duplike çalışmalar sonucu kontrol gruba göre anlamlı bir yağ birikimi görülmüş ve model oluşturulmuştur (p<0,05). MOTS-c peptidinin hücreler üzerine herhangi bir toksik etkisi olmadığı hücre canlılığı analizi ile saptanmıştır. MOTS-c peptidinin yağlanmayı azalttığı tespit edilmiştir (p<0,05).Sonuçlar: MOTS-c peptidinin yağlanmayı azaltıcı etkisi ile NAFLD etyopatogenezi ilişkili olduğu gözlemlenmiştir. Bu etkiyi gerçekten AMPK yolağını hedef alarak gerçekleştirdiğini göstermek amacıyla AMPK yolağında bulunan AMPK ve ACC proteinlerine ve aktif formları olan p-AMPK ve p-ACC proteinlerine Western Blot yöntemi ile bakılmasını planlamaktayız. Bu çalışma ile NAFLD tedavisi için MOTSc peptidinin tedavi adaylarından biri olabileceğini düşünmekteyiz.
Aim: In 2015, a novel gene belonging to mitochondrial genome was discovered. It has been identified in studies on cell lines and animal models that this gene’s functional product which is a 16-amino-acid peptide named mitochondrial open reading frame of the 12S rRNA-c (MOTS-c) regulates insulin sensitivity and metabolic homeostasis by AMPK pathway. However, according to our literature search, there are not any studies investigating MOTS-c effects on NAFLD etiopathogenesis. For this reason, we aim to examine MOTS-c effects on the pathogenesis of the disease in in vitro NAFLD model. Methods: In our study, first of all, HepG2 cells cultured in T75 flasks were starved in low glucose medium. After that, palmitic and oleic acid mix in 1 mM concentration was given to cells in order to generate in vitro NAFLD model. Lipid droplets were confirmed by using Oil Red O method. Cell viability was confirmed by using Cell Count and Viability Kit (MUSE). Then, MOTS-c in 10 μM concentration was applied to cells in the time with 24 hours. Results: As a result of duplicate experiments on fatty HepG2 cells, compared to control group cells, it has been considered to be very statistically significant and in vitro NAFLD model was created (p<0,05). According to cell viability analysis, the MOTS-c peptid is not toxic on cells. Finally, the MOTS-c peptide decreases the lipid droplets (p<0,05). Conclusion: The MOTS-c peptide is associated with NAFLDetiopathogenesis via a reducing effect of lipid droplets. With the aim of showing that MOTS-c targets AMPK pathway, we have planned to examine AMPK and ACC and also active forms p-AMPK and p-ACC by Western Blot. With this study, we think that MOTS-c could be a treatment candidate for NAFLD.
Aim: In 2015, a novel gene belonging to mitochondrial genome was discovered. It has been identified in studies on cell lines and animal models that this gene’s functional product which is a 16-amino-acid peptide named mitochondrial open reading frame of the 12S rRNA-c (MOTS-c) regulates insulin sensitivity and metabolic homeostasis by AMPK pathway. However, according to our literature search, there are not any studies investigating MOTS-c effects on NAFLD etiopathogenesis. For this reason, we aim to examine MOTS-c effects on the pathogenesis of the disease in in vitro NAFLD model. Methods: In our study, first of all, HepG2 cells cultured in T75 flasks were starved in low glucose medium. After that, palmitic and oleic acid mix in 1 mM concentration was given to cells in order to generate in vitro NAFLD model. Lipid droplets were confirmed by using Oil Red O method. Cell viability was confirmed by using Cell Count and Viability Kit (MUSE). Then, MOTS-c in 10 μM concentration was applied to cells in the time with 24 hours. Results: As a result of duplicate experiments on fatty HepG2 cells, compared to control group cells, it has been considered to be very statistically significant and in vitro NAFLD model was created (p<0,05). According to cell viability analysis, the MOTS-c peptid is not toxic on cells. Finally, the MOTS-c peptide decreases the lipid droplets (p<0,05). Conclusion: The MOTS-c peptide is associated with NAFLDetiopathogenesis via a reducing effect of lipid droplets. With the aim of showing that MOTS-c targets AMPK pathway, we have planned to examine AMPK and ACC and also active forms p-AMPK and p-ACC by Western Blot. With this study, we think that MOTS-c could be a treatment candidate for NAFLD.
Description
Keywords
Cell Culture, Genetics, Genetik, HepG2, Hücre Kültürü, Insulin resistance, İnsüline direnç, Karaciğer Hastalıkları, Liver Diseases, Mitochondrial MOTS-c Peptide, MitochondrialDNA, Mitokondriyal DNA, Mitokondriyal MOTS-c Peptidi HepG2, Non-Alcoholic Fatty Liver Disease, Non-Alkolik Yağlı Karaciğer Hastalığı