Effect of the bacterial lipopolysaccharide on preadipocyte differentiation: possible contribution of the NO and Rho-kinase

Abstract

Aim: Differentiation of preadipocytes is one of important steps for the adipogenesis. Adipogenesis is a metabolic disorder that accompanied by low grade inflammation and posses a lot of complications. We aimed to investigate effect of the LPS which one of the inflammatory response generating endotoxins on differentiation of 3T3-L1 cells and contribution of NO and Rho/ROCK pathways to this effect. Method: Fibroblast origin 3T3-L1 cells used for the differentiation of preadipocytes to adipocytes. Seeding was performed as 20000 cells on the every well of 24well plates and standard preadipocyte differentiation protocol was applied. In order to inducing differentiation, 0.25 µM dexamethasone, 0.5 mM izobuthylmethylxhantine and 1μM insulin containing 10% FBS/DMEM treated at 0-2^th days. Cells treated with 10% FBS/DMEM containing 1μM insulin on 2-4^th days of the protocol. 10% FBS/DMEM alone applied to wells at 4-8^th days. Incubation was maintained until 8^th day. LPS (10, 100 ng/ml) treatment was performed with or without L-NAME (N^G-nitro-L-arginine methyl esther, 2 and 5x10^-4 M) on certain time points (0-2, 2-4, 4-8, 0-8^th days) of the differentiation protocol. Differentiation evaluated with Oil Red-O staining method performed at 8^th day. Effect of the LPS on iNOS and Rho/Rho-kinase enzyme expressions was evaluated with Western Blot analysis. Besides nitrite levels in cell culture media was measured with Griess method on the presence of LPS and L-NAME. Results: LPS treatment on 3T3-L1 cell culture is significantly supressed the differentiation time points except 0-2^th days. In spite of the pretreatment of the cells with L-NAME did not any effect on the differentiation suppression produced by LPS, L-NAME treatment significantly supressing the differentiation every time points except 0-2^th days. LPS increased both iNOS and ROCK-2 expression. ROCK expression increasing effect of the LPS did not changed by the L-NAME treatment. When treated alone, L-NAME increased the ROCK-2 expression in a similar vein. Conclusion: LPS which is bacterial endotoxin, supressed the differentiation of 3T3-L1 cells. This effect could mediated by inflammatory mediator(s) other than NO. Besides, LPS could supressed the preadipocyte differentiation by a Rho/ROCK dependent mechanism.

Keywords

• Adipocyte,differentiation,LPS,NO,Rho-kinase

Bakteriyel lipopolisakkaridin preadiposit diferensiyasyonu üzerine etkisi: NO’nun ve Rho-kinaz enziminin olası katkısı

Abstract

Amaç: Preadipositlerin diferensiyasyonu adipogenezis için önemli basamaklardan biridir. Adipogenezis, düşük düzeyde inflamasyonun eşlik ettiği ve pek çok komplikasyonu olan metabolik bir hastalıktır. Bu çalışmamızda, inflamatuar yanıt oluşturan bakteriyel endotoksinlerden LPS’nin 3T3-L1 hücrelerinde diferensiyasyon üzerine etkisini ve bu etkiye NO ve Rho/ROCK yolağının katkısını araştırmayı amaçladık. Yöntem: Preadipositlerin adipositlere diferensiyasyonu için fibroblast kökenli 3T3-L1 hücreleri kullanıldı. 24 kuyucuklu pleytlere 20.000 hücre olacak şekilde ekim yapıldı ve standart preadiposit diferensiyasyon protokolü uygulandı. Diferensiyasyonun indüklenmesi için protokolün 0-2. günü 0.25 µM deksametazon, 0.5mM izobutilmetilksantin ve 1μM insülin içeren %10FBS/DMEM uygulandı. Protokolün 2-4. günleri 1μM insülin içeren %10 FBS/DMEM uygulandı. 4-8. gün ise kuyucuklara sadece %10 FBS/DMEM konuldu. İnkübasyon 8. güne kadar sürdürüldü. Diferensiyasyon protokolünün belirli zaman noktalarında (0-2, 2-4, 4-8, 0-8. günler) bakteriyel LPS (10-100 ng/ml), L-NAME (2-5x10^-4 M) varlığında ya da yokluğunda uygulandı. Diferensiyasyon, 8’inci günde Oil Red-O boyaması ile değerlendirildi. LPS’nin iNOS ve Rho/Rho-kinaz ekspresyonları üzerine etkileri de Western-blot analizi ile değerlendirildi. Ayrıca, kültür ortamında nitrit düzeyleri, LPS ve L-NAME varlığında Griess yöntemi ile ölçüldü. Bulgular: LPS uygulaması, 0-2. gün dışındaki zaman aralıklarında diferensiyasyonu anlamlı bir şekilde baskıladı. L-NAME ön uygulaması, bu süpresyonu ortadan kaldırmadı ancak tek başına L-NAME, 0-2. gün dışında tüm zaman aralıklarında diferensiyasyonu süprese etti. LPS hem iNOS hem de ROCK-2 ekspresyonunu arttırdı. LPS’nin ROCK ekspresyonunu arttırıcı etkisi L-NAME tarafından değiştirilmedi. L-NAME tek başına uygulandığında LPS’ye benzer şekilde ROCK-2 ekspresyonunu arttırdı. Sonuç: Bir bakteriyel endotoksin olan LPS, 3T3-L1 hücrelerinde diferensiyasyonu baskılamaktadır. Bu etkiye NO değil ancak onun dışındaki bir inflamatuar mediyatör(ler) aracılık edebilir. Ayrıca LPS, Rho/ROCK bağımlı bir mekanizma ile preadiposit diferensiyasyonunu süprese edebilir. 

Keywords

• Adiposit,diferensiyasyon,LPS,NO,Rho-kinaz

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