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

Year 2019, Volume: 12 Issue: 2, 257 - 270, 30.08.2019

Ahmet Sencer Yurtsever Kansu Büyükafşar

https://doi.org/10.26559/mersinsbd.538541

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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