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Endogenous synthesis of prostacyclin was positively regulated during the maturation phase of cultured adipocytes

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Abstract

Prostacyclin alternatively called prostaglandin (PG) I2 is an unstable metabolite synthesized by the arachidonate cyclooxygenase pathway. Earlier studies have suggested that prostacyclin analogues can act as a potent effector of adipose differentiation. However, biosynthesis of PGI2 has not been determined comprehensively at different life stages of adipocytes. PGI2 is rapidly hydrolyzed to the stable product, 6-keto-PGF1α, in biological fluids. Therefore, the generation of PGI2 can be quantified as the amount of 6-keto-PGF1α. In this study, we attempted to develop a solid-phase enzyme-linked immunosorbent assay (ELISA) using a mouse antiserum specific for 6-keto-PGF1α. According to the typical calibration curve of our ELISA, 6-keto-PGF1α can be quantified from 0.8 pg to 7.7 ng in an assay. The evaluation of our ELISA revealed the higher specificity of our antiserum without the cross-reaction with other related prostanoids while it exhibited only the cross-reaction of 1.5 % with PGF2α. The resulting ELISA was applied to the quantification of 6-keto-PGF1α generated endogenously by cultured 3T3-L1 cells at different stages. The cultured cells showed the highest capability to generate 6-keto-PGF1α during the maturation phase of 4–6 days, which was consistent with the coordinated changes in the gene expression of PGI synthase and the IP receptor for PGI2. Following these events, the accumulation of fats was continuously promoted up to 14 days. Thus, our immunological assay specific for 6-keto-PGF1α is useful for monitoring the endogenous levels of the unstable parent PGI2 at different life stages of adipogenesis and for further studies on the potential association with the up-regulation of adipogenesis in cultured adipocytes.

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Abbreviations

PG:

Prostaglandin

ELISA:

Enzyme-linked immunosorbent assay

PPARγ:

Peroxisome proliferator-activated receptor γ

COX:

Cyclooxygenase

PGIS:

Prostaglandin I synthase

DME-HEPES:

Dulbecco’s modified Eagle medium with 25 mM HEPES

FBS:

Fetal bovine serum

RT:

Reverse transcriptase

PCR:

Polymerase chain reaction

IBMX:

3-Isobutyl-1-methylxanthine

GM:

Growth medium

DM:

Differentiation medium

MM:

Maturation medium

AA:

Arachidonic acid

TXB2 :

Thromboxane B2

References

  • Antuna-Puente B, Feve B, Fellahi S, Bastard J-P (2008) Adipokines: the missing link between insulin resistance and obesity. Diabetes Metab 34:2–11

    Article  CAS  Google Scholar 

  • Catalioto RM, Gaillard D, Maclouf J, Ailhaud G, Negrel R (1991) Autocrine control of adipose cell differentiation by prostacyclin and PGF2α. Biochim Biophys Acta 1091:364–369

    Article  CAS  Google Scholar 

  • Chawla A, Schwarz EJ, Dimaculangan DD, Lazar MA (1994) Peroxisome proliferator-activated receptor (PPAR) γ: adipose-predominant expression and induction early in adipocyte differentiation. Endocrinology 135:798–800

    CAS  Google Scholar 

  • Chu X, Xu L, Nishimura K, Jisaka M, Nagaya T, Shono F, Yokota K (2009) Suppression of adipogenesis program in cultured preadipocytes transfected stably with cyclooxygenase isoforms. Biochim Biophys Acta 1791:273–280

    Article  CAS  Google Scholar 

  • Fajas L, Miard S, Briggs MR, Auwerx J (2003) Selective cyclo-oxygenase-2 inhibitors impair adipocyte differentiation through inhibition of the clonal expansion phase. J Lipid Res 44:1652–1659

    Article  CAS  Google Scholar 

  • Falcetti E, Flavell DM, Staels B, Tinker A, Haworth SG, Clapp LH (2007) IP receptor-dependent activation of PPARγ by stable prostacyclin analogues. Biochem Biophys Res Commun 360:821–827

    Article  CAS  Google Scholar 

  • Forman BM, Tontonoz P, Chen J, Brun RP, Spiegelman BM, Evans RM (1995) 15-Deoxy-Δ12,14-prostaglandin J2 is a ligand for the adipocyte determination factor PPARγ. Cell 83:803–812

    Article  CAS  Google Scholar 

  • Goppelt-Struebe M, Wolter D, Resch K (1989) Glucocorticoids inhibit prostaglandin synthesis not only at the level of phospholipase A2 but also at the level of cyclo-oxygenase/PGE isomerase. Br J Pharmacol 98:1287–1295

    Article  CAS  Google Scholar 

  • Green H, Kehinde O (1974) Sublines of mouse 3T3 cells that accumulate lipid. Cell 1:113–116

    Article  CAS  Google Scholar 

  • Green H, Kehinde O (1975) An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion. Cell 5:19–27

    Article  CAS  Google Scholar 

  • Hopkins N, Gorman RR (1981) Regulation of 3T3-L1 fibroblast differentiation by prostacyclin (prostaglandin I2). Biochim Biophys Acta 663:457–466

    Article  CAS  Google Scholar 

  • Hossain MS, Chowdhury AA, Rahman MS, Nishimura K, Jisaka M, Nagaya T, Shono F, Yokota K (2011) Development of enzyme-linked immunosorbent assay for Δ12-prostaglandin J2 and its application to the measurement of the endogenous product generated by cultured adipocytes during the maturation phase. Prostaglandins Other Lipid Mediat 94:73–80

    Article  CAS  Google Scholar 

  • Hyman BT, Stoll LL, Spector AA (1982) Prostaglandin production by 3T3-L1 cells in culture. Biochim Biophys Acta 713:375–385

    Article  CAS  Google Scholar 

  • Kelton JG, Blajchman MA (1980) Prostaglandin I2 (prostacyclin). Can Med Assoc J 122:175–179

    CAS  Google Scholar 

  • Kliewer SA, Lenhard JM, Willson TM, Patel I, Morris DC, Lehmann JM (1995) A prostaglndin J2 metabolite binds peroxisome proliferator-activated receptor γ and promotes adipocyte differentiation. Cell 83:813–819

    Article  CAS  Google Scholar 

  • Lu S, Nishimura K, Hossain MA, Jisaka M, Nagaya T, Yokota K (2004) Regulation and role of arachidonate cascade during changes in life cycle of adipocytes. Appl Bichem Biotechnol 118:133–153

    Article  CAS  Google Scholar 

  • Massiera F, Saint-Marc P, Seydoux J, Murata T, Kobayahis T, Narumiya S, Guesnet P, Amri EZ, Negrel R, Ailhaud G (2003) Arachidonic acid and prostacyclin signaling promote adipose tissue development: a human health concern? J Lipid Res 44:271–279

    Article  CAS  Google Scholar 

  • Mazid MA, Chowdhury AA, Nagao K, Nishimura K, Jisaka M, Nagaya T, Yokota K (2006) Endogenous 15-deoxy-Δ12,14-PGJ2 synthesized by adipocytes during maturation phase contributes to upregulation of fat storage. FEBS Lett 580:6885–6890

    Article  CAS  Google Scholar 

  • Moncada S, Gryglewski R, Bunting S, Vane JR (1976) An enzyme isolated from arteries transforms prostaglandin endoperoxides to unstable substance that inhibits platelet aggregation. Nature 263:663–665

    Article  CAS  Google Scholar 

  • Narumiya S, Sugimoto Y, Ushikubi F (1999) Prostanoid receptors: structures, properties, and functions. Physiol Rev 79:1193–1226

    CAS  Google Scholar 

  • Négrel R, Gaillard D, Ailhaud G (1989) Prostacyclin as a potent effector of adipose-cell differentiation. Biochem J 257:399–405

    Google Scholar 

  • Permana PA, Menge C, Reaven PD (2006) Macrophage-secreted factors induce adipocyte inflammation and insulin resistance. Biochem Biophys Res Commun 341:507–514

    Article  CAS  Google Scholar 

  • Reginato MJ, Krakow SL, Bailey ST, Lazar MA (1998) Prostaglandins promote and block adipogenesis through opposing effects on PPARγ. J Biol Chem 273:1855–1858

    Article  CAS  Google Scholar 

  • Sartipy P, Loskutoff DJ (2003) Monocyte chemoattractant protein 1 in obesity and insulin resistance. Proc Natl Acad Sci USA 100:7265–7270

    Article  CAS  Google Scholar 

  • Sethi JK, Hotamisligil GS (1999) The role of TNFα in adipocyte metabolism. Semin Cell Dev Biol 10:19–29

    Article  CAS  Google Scholar 

  • Shono F, Yokota K, Horie S, Yamamoto K, Yamashita K, Watanabe H, Miyazaki H (1988) A heterologous enzyme immunoasssay of prostaglandin E2 using a stable enzyme-labeled hapten mimic. Anal Biochem 168:284–291

    Article  CAS  Google Scholar 

  • Stehle RG (1982) Physical chemistry, stability, and handling of prostaglandins E2, F2α, D2, and I2: a critical summary. Methods Enzymol 86:436–458

    Article  CAS  Google Scholar 

  • Syeda PK, Hossain MS, Chowdhury AA, Rahman MS, Nishimura K, Jisaka M, Nagaya T, Shono F, Yokota K (2012) Generation of monoclonal antibody for 15-deoxy-Δ12,14-PGJ2 and development of enzyme-linked immunosorbent assay for its quantification in culture medium of adipocytes. Appl Biochem Biotechnol 167:1107–1118

    Article  CAS  Google Scholar 

  • Tontonoz P, Hu E, Spiegelman BM (1994) Stimulation of adipogenesis in fibroblasts by PPARγ2, a ligand-activated transcription factor. Cell 79:1147–1156

    Article  CAS  Google Scholar 

  • Tsuboi H, Sugimoto Y, Kainoh T, Ichikawa A (2004) Prostanoid EP4 receptor is involved in suppression of 3T3-L1 adipocyte differentiation. Biochem Biophys Res Commun 322:1066–1072

    Article  CAS  Google Scholar 

  • Wise H (2003) Multiple signaling options for prostacyclin. Acta Pharmacol Sin 24:625–630

    CAS  Google Scholar 

  • Xie Y, Kang X, Ackerman WE IV, Belury MA, Koster C, Rovin BH, Landon MB, Kniss DA (2006) Differentiation-dependent regulation of the cyclooxygenase cascade during adipogenesis suggests a complex role for prostaglandins. Diabetes Obes Metab 8:83–93

    Article  CAS  Google Scholar 

  • Xu L, Nishimura K, Jisaka M, Nagaya T, Yokota K (2006) Gene expression of arachidonate cyclooxygenase pathway leading to the delayed synthesis of prostaglandins E2 and F2α in response to phorbol 12-myristate 13-acetate and action of these prostanoids during life cycle of adipocytes. Biochim Biophys Acta 1761:434–444

    Article  CAS  Google Scholar 

  • Yokota K, Morishima T, Naagya M, Jisaka M, Takinami K (1996) Modification of cultured Madin-Darby canine kidney cells with dietary unsaturated fatty acids and regulation of arachidonate cascade reaction. Biosci Biotechnol Biochem 60:1096–1103

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by JSPS KAKENHI Grant Numbers 25450128 and 25-03091.

Conflict of interest

The authors declare that there are no conflicts of interest.

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Authors and Affiliations

  1. Department of Life Science and Biotechnology, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane, 690-8504, Japan

    Mohammad Sharifur Rahman, Ferdous Khan, Pinky Karim Syeda, Mitsuo Jisaka, Tsutomu Nagaya & Kazushige Yokota

  2. Department of Molecular and Functional Genomics, Center for Integrated Research in Science, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane, 690-8504, Japan

    Kohji Nishimura

  3. Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Yamashiro-cho, Tokushima-shi, Tokushima, 770-8514, Japan

    Fumiaki Shono

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  1. Mohammad Sharifur Rahman
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  2. Ferdous Khan
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  4. Kohji Nishimura
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Correspondence to Kazushige Yokota.

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Rahman, M.S., Khan, F., Syeda, P.K. et al. Endogenous synthesis of prostacyclin was positively regulated during the maturation phase of cultured adipocytes. Cytotechnology 66, 635–646 (2014). https://doi.org/10.1007/s10616-013-9616-9

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