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

Yıl 2019, Cilt: 12 Sayı: 2, 257 - 270, 30.08.2019

Ahmet Sencer Yurtsever Kansu Büyükafşar

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

Öz

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.

Anahtar Kelimeler

Adipocyte, differentiation, LPS, NO, Rho-kinase

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

Öz

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. 

Anahtar Kelimeler

Adiposit, diferensiyasyon, LPS, NO, Rho-kinaz

Kaynakça

• Referans1-Flegal KM, Carroll MD, Ogden CL, Curtin LR. Prevalence and trends in obesity among US adults, 1999-2008. JAMA 2010; 303 (3): 235-41.
• Referans2-Finucane M, Stevens GA, Cowan MJ, Danaei G, Lin JK, Paciorek CJ, Singh GM, Gutierrez HR, Lu Y, Bahalim AN, Farzadfar F, Riley LM, Ezzati M. National, regional, and global trends in body-mass index since 1980: systematic analysis of health examination surveys and epidemiological studies with 960 country-years and 9.1 million participants. Lancet 2011; 377 (9765): 557-567.
• Referans3-Obesity and overweight. Who Factsheet No:311, 2015.
• Referans4-Flegal KM, Graubard BI, Williamson DF, Gail MH. Excess deaths associated with underweight, overweight, and obesity. JAMA 2005; 293: 1861-1867.
• Referans5-Peeters A, Barendregt JJ, Willekins F, Mackenbach JP, Al Mamum A, Bonneux L. Obesity in adulthood and its consequences for life expectancy: a life-table analysis. Ann Intern Med 2003; 138: 24-32.
• Referans6-Flegal KM, Carroll MD, Kit BK, Ogden CL. Prevalence of obesity and trends in the distribution of body mass index among US adults, 1999–2010. JAMA 2012; 307 (5): 491–97.
• Referans7-Hotamisligil GS, Shargill NS, Spiegelman BM. Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science 1993; 259: 87–91.
• Referans8-Hotamışlıgil, G. S, Murray, D. L, Choy, L. N, Spiegelman, B. M. Tumor necrosis factor α inhibits signaling from the insulin receptor. Proc Natl Acad Sci USA 1994: 91: 4854–4858.
• Referans9-Shah A, Nehal MN, Reilly MP. Adipose Inflammation, Insulin Resistance, and Cardiovascular Disease. J Parenter Enteral Nutr 2008; 32 (6): 638–644.
• Referans10-McNelis JC, Olefsky JM. Macrophages, immunity, and metabolic disease. Immunity 2014; 41: 36–48.
• Referans11-Crossno JT Jr, Majka SM, Grazia T, Gill RG, Klemm DJ. Rosiglitazone promotes development of a novel adipocyte population from bone. marrow-derived circulating progenitor cells. J Clin Invest 2006; 116: 3220-3228.
• Referans12-Sarjeant K, Stephens JM. Adipogenesis. Cold Spring Harb Perspect Biol 2012; 4 (a008417): 1-19.
• Referans13-Ghorbani A, Abedinzade M. Comparison of In Vitro and In Situ Methods for Studying Lipolysis. ISRN Endocrinology 2013; 205385: 1-6.
• Referans14-Garcia X, Stein F. Nitric Oxide. Semin Pediatr Infect Dis 2006; 17: 55-57.
• Referans15-Denninger JW, Marletta M. A. Guanylate cyclase and the NO/cGMP signaling pathway. Biochim Biophys Acta 1999: 1411, 334-350.
• Referans16-Lago F, Dieguez C, Gomez-Reino J, Gualillo O. Adipokines as emerging mediators of immune response and inflammation. Nat Clin Pract Rheum 2007; 3 (12): 716-724.
• Referans17-Kapur S, Marcotte B, Marette A. Mechanism of adipose tissue iNOS induction in endotoxemia. Am J Physiol Endoc M 1999; 276: 635-641.
• Referans18-Yan H, Aziz E, Shillabeer G, Wong A, Shanghavi D, Kermouni A, Abdel-Hafez M, Lau DCW. Nitric oxide promotes differentiation of rat white preadipocytes in culture. J Lipid Res 2002: 43, 2123-2129.
• Referans19-Hiroyuki K, Naoko M, Takako K, Shin-Ya T, Megumi W, Tohru M, Teruo K, Hideo Y. Nitric oxide suppresses preadipocyte differentiation in 3T3-L1 culture. Mol Cell Biochem 2007: 300 (1-2), 61-67.
• Referans20-Andersson K, Gaudiot N, Ribiere C, Elizalde M, Giudicelli Y, Arner P. A nitric oxide-mediated mechanism regulates lipolysis in human adipose tissue in vivo. Brit J Pharmacol 1999; 126: 1639–1645.
• Referans21-Penfornis P, Marette A. Inducible Nitric Oxide Synthase Modulates lipolysis in adipocytes. J Lipid Res 2005; 46: 135-142
• Referans22-Hersoug LG, Moller P, Loft S. Role of microbiota-derived lipopolysaccharide in adipose tissue inflammation, adipocyte size and pyroptosis during obesity. Obes Rev 2016; 17(4):297-312.
• Referans23-Sanmiguel C, Gupta A, Mayer EA. Gut Microbiome and Obesity: A Plausible Explanation for Obesity. Curr Obes Rep 2015; 4(2): 250–261.
• Referans24-Cani PD, Bibiloni R, Knauf C, Waget A, Neyrinck AM, Delzenne NM, Burcelin R. Changes in gut microbiota control metabolic endotoxemia-induced inflammation in high-fat diet-induced obesity and diabetes in mice. Diabetes 2008; 57(6):1470–1481.
• Referans25-Cani PD, Amar J, Iglesias MA, Poggi M, Knauf C, Bastelica D, Neyrinck AM, Fava F, Tuohy KM, Chabo C, Waget A, Delmée E, Cousin B, Sulpice T, Chamontin B, Ferrières J, Tanti JF, Gibson GR, Casteilla L, Delzenne NM, Alessi MC, Burcelin R. Metabolic endotoxemia initiates obesity and insulin resistance. Diabetes 2007; 56(7):1761–1772.
• Referans26-Cox AJ, West NP, Cripps AW. Obesity, inflammation, and the gut microbiota. The Lancet Diabetes Endocrinol 2015; 3(3):207-215.
• Referans27-Lowenstein CJ, Alley EW, Raval P, Snowman AM, Snyder SH, Russell SW, Murphy WJ. Macrophage nitric oxide synthase gene: two upstream regions mediate induction by interferon gamma and lipopolysaccharide. Proc Natl Acad Sci USA 1993; 90(20):9730-9734.
• Referans28-Hippenstial S, Soeth S, Kellas B, Fuhrmann O, Seybold J, Krqll M, v. EIchel-StreIber C, Goebeler M, Ludwing S, Suttorp N. Rho proteins and the p38-MAPK pathway are important mediators for LPS-induced interleukin-8 expression in human endothelial cells. Blood 2000; 95: 3044– 3051.
• Referans29-Büyükafşar K, Arıkan O, Ark M, Kubat H, Özveren M.Upregulation of Rho-kinase (ROCK-2) expression and enhanced contraction to endothelin-1 in the mesenteric artery from lipopolysaccharide-treated rats.Eur J Pharmacol 2004; 498: 211–217.
• Referans30-Matsumura S, Abe T, Mabuchi T, Katano T, Takagi T, Okuda-Ashitaka E, Tatsumi S, Nakai Y, Hidaka H, Suzuki M, Yasuharu SY, Minami T, Ito S. Rho-kinase mediates spinal nitric oxide formation by prostaglandin E2 via EP3 subtype. Biochem Biophys Res Commun, 2005, 338, 550–557.
• Referans31-Cetin S, Leaphart CL, Li J, Ischenko I, Hayman M, Upperman J, Zamora R, Watkins S, Ford HR, Wang J, Hackam DJ. Nitric oxide inhibits enterocyte migration through activation of RhoA-GTPase in a SHP-2-dependent manner. Am J Physiol Gastr L Physiol, 2007, 292, G1347-G1358.
• Referans32-Riento K, Ridley AJ. Rocks: multifunctional kinases in cell behaviour. Nat Rev Mol Cell Biol 2003; 4: 446–456.
• Referans33-Furukawa N, Ongusaha P, Jahng WJ, Araki K, Choi CS, Kim HJ, Lee YH, Kaibuchi K, Kahn BB, Masuzaki H, Kim JK, Lee SW, Kim YB. Role of Rho-kinase in regulation of insulin action and glucose homeostasis. Cell Metab 2005; 2: 119–129.
• Referans34-Noguchi M, Hosoda K, Fujikura J, Fujimoto M, Iwakura H, Tomita T, Ishii T, Arai N, Hirata M, Ebihara K, Masuzaki H, Itoh H, Narumiya S, Nakao K. Genetic and pharmacological inhibition of Rho-Associated Kinase II enhances adipogenesis. J Biochem 2007; 282 (40): 29574-29583.
• Referans35-Ribiere, C, Jaubert AM, Gaudiot N, Sabourault D, Marcus ML, Boucher JL, Denis-Henriot D, Giudicelli Y. White adipose tissue nitric oxide synthase: a potential source for NO production. BBRC 1996; 222, 706–712.
• Referans36-Elizalde M, Rydén M, Harmelen VV, Eneroth P, Gyllenhammar H, Holm C, Ramel S, Ölund A, Arner P, Andersson K. Expression of nitric oxide synthases in subcutaneous adipose tissue of nonobese and obese humans. J Lipid Res 2000; 41, 1244-1251.
• Referans37-Yamada Y, Eto M, Ito Y, Mochizuki S, Son BK, Ogawa S, Lijima K, Kaneki M, Kozaki K, Toba K, Akishita M, Ouchi Y. Suppressive Role of PPARγ-Regulated Endothelial Nitric Oxide Synthase in Adipocyte Lipolysis. PLoS ONE 10(8): e0136597
• Referans38-Andrukhov O, Haririan H, Bertl K, Rausch WD, Bantleon HP, Moritz A, Rausch-Fan X. Nitric oxide production, systemic inflammation and lipid metabolism in periodontitis patients: possible gender aspect. J Clin Periodontol 2013; 40: 916–923.
• Referans39-Cintra LTA, Samuel RO, Azuma MM, de Queiróz AOS, Ervolino E, Sumida DH, de Lima VMF, Gomes-Filho JE. Multiple Apical Periodontitis Influences Serum Levels of Cytokines and Nitric Oxide. JOE 2016; 42 (5): 747–751.
• Referans40-Andersson J, Nagy S, Bjork L, Abrams J, Holm S, Andersson U. Bacterial toxin-induced cytokine production studied at the single-cell level. Immunol Rev,1992, 127, 69-96.
• Referans41-Roberts AB. Molecular and cell biology of TGF-beta. Miner Electrolyte Metab 1998; 24, 2-3, 111-119.
• Referans42-Vardouli L, Moustakas A, Stournaras C. LIM-kinase 2 and cofilin phosphorylation mediate actin cytoskeleton reorganization induced by transforming growth factor. J Biol Chem 2005; 280 (12), 11448–11457.
• Referans43-Pei Z, Lin D, Song X, Li H, Yao H. TLR4 signaling promotes the expression of VEGF and TGFb1 in human prostate epithelial PC3 cells induced by lipopolysaccharide. Cell Immunol 2008; 254, 20–27.
• Referans44-Kopp A, Buechler C, Neumeier M, Weigert J, Aslanidis C, Schölmerich J. Schäffler A. Innate Immunity and Adipocyte Function: Ligand-specific Activation of Multiple Toll-like Receptors Modulates Cytokine, Adipokine, and Chemokine Secretion in Adipocytes. Obesity 2009; 17: 648–656.
• Referans45-Palsson-Mcdermott EM, O’neill LAJ. Signal transduction by the lipopolysaccharide receptor, Toll-like receptor-4. Immunology 2004; 113: 153–162.
• Referans46-Ahmad R. Al-Mass A, Atizado V, Al-Hubail A, Al-Ghimlas F, Al-Arouj M, Bennakhi A, Dermime S, Behbehani K. Elevated expression of the toll like receptors 2 and 4 in obese individuals: its significance for obesity-induced inflammation. J Inflamm 2012; 9 (48): 1-11.