抵抗素(resistin)是體內糖類代謝的重要調控者，與肥胖引起的胰島素阻抗有關。文獻報導isoproterenol (ISO)與TNF-?可抑制3T3-L1脂肪細胞表現resistin mRNA。然而resistin的釋出是否受cAMP訊息傳遞調控目前仍不清楚。環狀核苷酸磷酸雙酯酶(PDE)是細胞內分解cAMP與cGMP的酵素，對調節環狀核苷酸的濃度及其訊息傳導扮演著重要的角色。因此本研究主要目的在探討cAMP-PDE在脂肪細胞內對resistin表現之影響，主要利用ELISA方法測量藥物處理3T3-L1與小鼠脂肪細胞釋放resistin之改變。結果顯示ISO可抑制兩種脂肪細胞釋放resistin量，且cilostazol與rolipram均可增強ISO的抑制作用，然而ISO的抑制作用不會被PKA抑制劑H89回復。以dibutyryl-cAMP (dbcAMP)處理小鼠或3T3-L1脂肪細胞，我們發現僅小鼠脂肪細胞之resistin釋放有顯著下降，至於3T3-L1脂肪細胞，dbcAMP與PDE3抑制劑cilostazol共同處理可明顯抑制resistin釋放，但rolipram則無此協同效果，再者，cilostazol與dbcAMP的抑制作用可被H89完全回復，顯示此抑制作用是藉由抑制PDE3活性進而活化PKA訊息傳導所致。TNF-?亦可降低resistin釋放，但此抑制作用不受cilostazol或cAMP訊息傳導所調控。文獻報導TNF-?抑制resistin表現是由於活化iNOS-NO (nitric oxide)，雖然NO可活化guanylyl cyclase (GC)使cGMP濃度增加，但8-bromo-cGMP對resistin釋放並無影響，表示TNF-?的抑制作用不是藉由增加細胞內cGMP的濃度所致。本研究也利用PKC活化劑phorbol myristate acetate (PMA)與鈣離子增升劑ionomycin處理3T3-L1脂肪細胞，結果顯示PMA或ionomycin單獨或共同處理細胞均可抑制resistin釋放。我們也發現，PI3K抑制劑LY294002與GSK3抑制劑LiCl均可抑制3T3-L1脂肪細胞釋放resistin，綜合以上結果得知，脂肪細胞分泌resistin會被多種訊息傳遞分子調控，包括cAMP/PDE3/PKA、Ca+2/PKC與PI3k等

The adipocyte-secreted hormone resistin has been implicated in regulation of glucose homeostasis, adipogenesis and inflammation, and is linked to obesity-related insulin resistance. Evidence indicates that isoproterenol (ISO) and tumor necrosis factor-? (TNF-?? are pivotal negative mediators in resistin gene expression, but mechanisms underlying these effects remain largely unknown. Cyclic AMP signaling is known to be critical in regulation of lipid and glucose metabolism, and intracellular cAMP concentrations can be regulated by cAMP-hydrolyzing phosphodiesterases (cAMP-PDEs). In this study, we investigated the effects of cAMP-PDE on resistin release in mouse primary and 3T3-L1 adipocytes. By ELISA analysis, we observed that resistin release was significantly reduced in the cells following ISO treatment. The release was further decreased by co-stimulation of the cells with the PDE3 inhibitor cilostazol or PDE4 inhibitor rolipram. Howover, this inhibition was not reversed by the PKA inhibitor H89, indicating that ISO/cAMP-regulated resistin release is not mediated by PKA activity. We have also found that treatment of cells with dibutyryl-cAMP (dbcAMP) in combination with cilostazol, but not rolipram, leads to a marked decrease in resistin release in the adipocytes and this decrease was fully reversed by H89, indicating that the suppression of resistin release by dbcAMP and cilostazol requires PKA activation. The resistin release in the adipocytes was also blocked by TNF-? treatment. However, this effect was not regulated by cilostazol or dbcAMP. We further observed that the resistin release in 3T3-L1 adipcytes was inhibited by the PKC activator phorbol myristate (PMA) as well as the Ca+2 ionophore ionomycin. The PI3K inhibitor LY294002 and the GSK3 inhibitor LiCl also exhibited inhibitory effects on the resistin release in these cells. Taken together, these findings indicate that the resistin release in mouse adipocytes is regulated by several signaling molecules, including cAMP/PDE3/PKA, Ca+2/PKC, PI3K and GSK3.

1.Trayhurn, P., Endocrine and signaling role of adipose tissue: New perspective on fat, Acta Physiol. Scand. (2005) 184: 285-293
2.Steppan CM, Bailey ST, Bhat S, Brown EJ, BAnerjee RR, Wright CM, Patel HR, Ahima RS, Lazar MA The hormone resistin links obesity to diabetes. Nature (2001) 409:307-312
3.Kee-Hong Kim, Kichoon Lee, Yang Soo Moon, and Hei Sook Sul A Cysteine-rich adipose tissue-specific secretory factor inhibits adipocyte differentiation. J Boil Chem (2001) 276: 11252-6
4.Holocomb IN, Kabakoff RC, Chan B et al. FIZZ1, a novel cysteine-rich secreted protein associated with pulmonary inflammation, defines a new gene family. EMBO J (2000) 19: 4046-55
5.Steppan CM, Lazar MA, et al. A family of tissue-specific resistin-like moleciles. Proc Natl Acad Sci USA (2001) 98:592
6.Mikako Degawa-Yamauchi, Robert V. Considine et al,. Serum Resistin (FIZZ3) Protein Is Increased in Obese Humans J. Clin. Endocrinol. Metab. (2003) 88: 5452
7.Strausberg R. L., Feingold E. A., Grouse L. H., Derge J. G., Klausner R. D., Collins F. S. et al. Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences. Proc. Natl. Acad. Sci. USA (2002) 99: 16899–16903
8.Banerjee R. R. and Lazar M. A. Dimerization of resistin and resistin-like molecules in determined by a single cysteine. J. Biol. Chem. (2001) 276: 25970–25973
9.E.W. Sutherland and T.W. Rall, J. Biol. Chem. (1985) 232:1077-1091.
10.Conti M, Jin SL. The molecular biology of cyclic nucleotide phosphodiesterases. Prog. Nucleic Acid Res. Mol. Biol. (1999) 63:1
11.Francis SH, Turko IV, Corbin JD. Cyclic nucleotide phosphodiesterases: Relating structure and function. Prog. Nucleic Acid Res. Mol. Biol. (2000) 65:1
12.Soderling SH, Beavo JA. Regulation of cAMP and cGMP signaling: new phosphodiesterases and new functions. Curr. Opin. Cell Biol. (2000) 12:174–79
13.Beavo JA, Conti M, Heaslip RJ. Mol. Pharmacol. (1994) 46:399–405
14.Victoria Boswell-Smith, Clive P. Page (2006) Phosphodesterase inhibitors. British Journal of Pharmacology 147:s252
15.Degerman, E., Leroy, M. J., Taira, M., Belfrage, P., and Manganiello, V. C. (1996) in Diabetes Mellitus, A Clinical and Fundamental Textbook (LeRoith, D., Olefsky, J., and Taylor, S., eds) pp. 197–204, Lippincott Raven, Philadelphia, PA
16.Makino, H., Manganiello, V. C., and Kono, T. (1994) Annu. Rev. Physiol. 56,273–295
17.Belfrage, P., Donner, J., Eriksson, H., and Stalfors, P. (1986) in Mechanisms of Insulin Action (Belfrage, P., Donner, J., and Stralfors, P., eds) pp. 323–340, Elsevier Science Publishers B.V., Amsterdam
18.Smith, C. J., and Manganiello, V. C. Mol. Pharmacol. (1988) 35: 381–386
19.Chen J., Wang L., Boeg Y. S., Xia B. and Wang J. Differential dimerization and association among resistin family proteins with implications for functional specificity. J. Endocrinol. (2002) 175: 499–504.
20.Beebe, S. J., Redman, J. B., Blackmore, P. F., and Corbin, J. D. J. Biol. Chem. (1985) 260:15781–15788
21.Shakur. Y., L. Stenson Holst, T. Rahn Landstöm. et al. Regulation and fuction of the cyclic nucleotide phosphodiesterase (PDE3) gene family. Prog Nucleic Acid Res Mol Biol (2001) 66:241
22.Kenan. Y., T. Murata. Y. Shakur. et al. Functions of the N-terminal region of cyclic nucleotide phosphodiestrase 3 (PDE3) isoforms. J Biol Chem (2000) 275:112331
23.Shakur. Y., K. Takeda. Y Kenan. et al. Membrane localization of cyclic nucleotide efficient targeting to, and association of, PDE3 with endoplasmic reticulum. J Biol. Chem (2000) 275:38749.
24.Varnerin, J.P, S.B. Patel, et al. Expression, refolding, and purification of recombinant human phosphodiesterase 3B: Definition of the N-terminus of the catalytic core. Protein Expr Purif (2004) 35 (2):225
25.Nilsson, R., F. Ahmad, K. Swärd, et al. Plasma membrane cyclic nucleotide phosphodieaterase 3B (PDE3B) is associated with caveolae in primary rat adipocyte. Cell Signal (2006) 18 (10):1713
26.Faiyaz Ahmad, V. Manganiello, et al. Insulin-induced formation of macromolecular complexes involved in activation of cyclic nucleotide phosphodiesterase 3B (PDE3B) and its interaction with PKB. Biochem. J. (2007) 404:257–268
27.Rondinone, C. M., E. Carvalho, T. Rahn, et al. Phosphorylation of PDE3B by phosphatidylinositol 3-kinase associated with the insulin receptor. J. Biol. Chem (2000) 275:10093
28.Kitamura, T., Y. Kitamura, S. Kuroda, et al. Insulin-induced phosphorylation and activation of cyclic nucleotide phosphodiesterase 3B by the serine-threonine kinase. Akt Mol Cell Biol. (1999) 9:6286
29.Zmusa-Trzebiatowska, E., A. Oknianska, V. Manganiello, et al. Role of PDE3B in insulin-induced glucose uptake, GLUT-4 translocation and lipogenesis in primary rat adipocyte. Cell Signal (2006) 3:382.
30.Rahn, T., M. Ridderstråle, H. Tornqvist, et al. Essential role of phosphatidylinositol 3-kinase in insulin-induced activation and phosphorylation of the cGMP-inhibited cAMP phosphodiesterase in rat adipocytes. Studies using the selective inhibitor wortmannin. FEBS Lett (1994) 350:314.
31.Wijkander. J., T. Rahn Landstöm, V. Manganiello, et al Insulin-induced phosphorylation and activation of phosphdiesterase 3B in rat adipocyte: Possible role for protein kinase B but not mitogen-activated protein kinase or p70 S6 kinase. Endorcinology (1998) 139:219
32.Ahmad, F., L. N. Cong, L. Stenson Holst, et al. Cyclic nucleotide phosphodiesterase 3B is a down stream target of protein kinase B and may be involved in regulation of effects of protein kinase B on thymidine incorporation in FDCP2 cells. J Immounol (2000) 164 (9):4678.
33.Smith. C. J., V. Vasta., E. Degerman, et al. Hormine-sensitive cyclic GMP-inhibited cyclic AMP phosphodiesterase in rat adipocytes. Regulation of insulin- and cAMP-dependent activation by phosphorylation. J Biol Chem (1991) 266:13385
34.Bilal Omar, Eva Degerman, et al. Regulation of AMP-activated protein kinase by cAMP in adipocytes: Roles for phosphodiesterases, protein kinase B, protein kinase A, Epac and lipolysis. Cellular Signalling (2009) 21:760
35.Boden, G. Role of fatty acids in the pathogenesis of insulin resistance and NIDDM. Diabetes (1997) 46:3
36.Frayn, K. N., C. M. Willians, and P. Arner. Are increased plasma non-esterified fatty acid concentrations a risk marker for corconary heart disease and other chronic disease? Clin Sci (Lond) (1996) 90:243.
37.Engfeldt P, Ostman J., et al. Phosphodiesterase activity in human subcutaneous adipose tissue in insulin- and noninsulin-dependent diabetes mellitus. J Clin Endocrinol Metab. (1982) Nov;55(5):983-8
38.Hun Choi, Y., S. Park, S. Hockman, et al. Alterations in regulation of energy homeostasis in cyclic nucleotide phosphodiesterase 3B (mPDE3B)-null mice. J. Clin Invest (2006) 116:3240
39.Hotamisligil GS, Arner P, Caro JF, Atkinson RL, Spiegelman BM. Increased adipose tissue expression of tumor necrosis factor-alpha in human obesity and insulin resistance. J Clin Invest (1995) 95:2409–2415
40.Vassalli P. The pathophysiology of tumor necrosis factors. Annu Rev Immunol (1992) 10:411–452.
41.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
42.Hotamisligil GS, Murray DL, Choy LN, Spiegelman BM. Tumor necrosis factor alpha inhibits signaling from the insulin receptor. Proc Natl Acad Sci USA (1994) 91:4854–4858.
43.Stephens JM, Pekala PH. Transcriptional repression of the GLUT4 and C/EBP genes in 3T3-L1 adipocytes by tumor necrosis factor-alpha. J Biol Chem (1991) 266:21839–21845
44.Mathias Fasshauer, Ralf Paschke, et al. Hormonal regulation of adiponectin gene expression in 3T3-L1 adipocytes. Biochem. Biophys. Res. Commun. (2002) 290:1084-1089
45.Mathias Fasshauer, Ralf Paschke. et al, Tumor necrosis factor alpha is a negative regulator of resistin gene expression and secretion in 3T3-L1 adipocytes. Biochem. Biophys. Res. Commun. (2001) 288:1027
46.Chi-Chang Juan, Low-Tone Ho, et al. Involvement of iNOS and NO in TNF-?-downregulated resistin gene expression in 3T3-L1 adipocyte. Obesity (2008) 16:1219
47.Mathias Fasshauer, Ralf Paschke, et al. Isoproterenol inhibits resistin gene expression through a Gs-protein-coupled pathway in 3T3-L1 adipocytes. FEBS letters (2001) 500: 60-63
48.Dan Cassel, Thomas Pfeuffer, et al. Mechanism of cholera toxin action: Covalent modification of the guanyl nucleotide-binding protein of the adenylate cyclase system. Proc. Nati. Acad. Sci (1987) 75:2669
49.Kenneth B. Seamon, John W. Daly, et al. Forskolin: Unique diterpene activator of adenylate cyclase in membranes and in intact cells. Proc. Nati. Acad. Sci (1981) 78:3363
50.Charlotte K. Billington, Ian P. Hall, et al. A major functional role for phosphodiesterase 4D5 in human airway smooth muscle cells. Am J Respir Cell Mol. Biol. (2008) 38:1
51.Alina Oknianska, Eva Degerman. et al, Long-term regulation of cyclic nucleotide phosphodiesterase type 3B and 4 in 3T3-L1 adipocyte. Biochem. Biophys. Res. Commun. (2007) 353:1080-1085
52.Bradford, M. M.. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. (1976) 72: 248-254
53.Jin SL, Conti M. Induction of the cyclic nucleotide phosphodiesterase PDE4B is essential for LPS-activated TNF-alpha responses. Proc. Natl. Acad. Sci. (2002) 28;99(11):7628-33.
54.Jin SL, Lan L, Zoudilova M, Conti M. Specific role of phosphodiesterase 4B in lipopolysaccharide-induced signaling in mouse macrophages. J Immunol. (2005) Aug 1;175(3):1523-31.
55.Peter J. Havel et al, Effects of inhibiting transcription and protein synthesis on basal and insulin-stimulated leptin gene expression and leptin secretion in cultured rat adipocytes. Biochem. Biophys. Res. Commun (2003) 307:907-914
56.Rupert C. Honnor, Constantine Londos, et al. cAMP-dependent protein kinase and lipolysis in rat adipocytes. J. Biol. Chem (1985) 260:15122
57.Jiro Nakamura, Naomichi Okamura, and Yasushi Kawakami. Augmentation of lipolysis in adipocytes from fed rats, but not from starved rats, by inhibition of rolipram-sensitive phosphodiesterase 4 Archives of Biochemistry and Biophysics (2004) 425:106–114
58. Rebecka Lindh, Eva Degerman, et al. Multisite phosphorylation of adipocyte and hepatocyte phosphodiesterase 3B. Biochimica et Biophysica Acta (2007) 1773:584–592
59.Tova Rahn Landström, Eva Degerman, et al. Down-regulation of cyclic-nucleotide phosphodiesterase 3B in 3T3-L1 adipocytes induced by tumour necrosis factor ? and cAMP. Biochem. J. (2000) 346:337-343.
60. William P. Arnold, Ferid Murad, et al. Nitric oxide activates guanylate cyclase and increases guanosine 3′:5′-cyclic monophosphate levels in various tissue preparations. Proc. Natl. Acad. Sci (1977) 47:3203
61. Mary L. Standaert, Robert V. Farese, et al Protein Kinase C-ζ as a downstream effector of phosphatidylinositol 3-kinase during insulin stimulation in rat adipocytes potential role in glucose transport. J Biol Chem (1997) 273:30075-30082.
62. Nobuhiro Shojima, Tomoichiro Asano, et al. Humoral regulation of resistin expression in 3T3-L1 and mouse adipose cells. Diabetes (2002) 51:1737
63. Haiyan Song, Tomoichiro Asano, et al Resistin is regulated by C/EBPs, PPARs, and signal-transducing molecules. Biochem. Biophys. Res. Commun (2002) 299:291-298.
64.Yen-Hang Ch, Yung-Hsi Kao, et al 17?-Estradiol stimulates resistin gene expression in 3T3-L1 adipocytes via the estrogen receptor, extracellularly regulated kinase, and CCAAT/enhancer binding protein-??pathways. Endocrinology (2006) 147(9):4496-4504
65. Stephen J. Oreña, Robert S. Garofalo, et al. Inhibition of glycogen-synthase kinase 3 stimulates glycogen synthase and glucose transport by distinct mechanisms in 3T3-L1 adipocytes. J Biol Chem (2000) 275:15765
66.Ronadip R. Banerjee, Mitchell A. Lazar, et al. Regulation of fasted blood glucose by resistin. Science (2004) 303: 1195
67. Michael Blüher, Ralf Paschke, et al. Improvement of insulin sensitivity after adrenalectomy in patients with pheochromocytoma. Diabetes care (2000) 23, 1591-1592
68. Patti, M.E. Ann. NY Acad. Sci. (1999) 892: 187-203
69. Christopher J. Hupfeld, Jerrold M. Olefsky, et al. ?-Arrestin 1 down-regulation after insulin treatment is associated with supersensitization of ?2 adrenergic receptor G?s signaling in 3T3-L1 adipocytes. Proc. Natl. Acad. Sci (2003) 100:161
70. So Youn Park, Ki Whan Hong, et al. Cilostazol ameliorates metabolic abnormalities with suppression of proinflammatory markers in a db/db mouse model of type 2 diabetes via activation of peroxisome proliferator-activated receptor ??transcription. JPET (2009) 329:571–579
71. S.-L.Catherine Jin, Macro Conti, et al. Specific role of phosphodiesterase 4 B in lipopolysacharride-induced signaling in mouse macrophages. The Journal of Immunology. (2005) 175: 1523-1531
72. Kimberly L. Doge-Kafka, Jonh D. Scott, et al. The protein kinase A anchoring protein mAKAP coordinates two integrated cAMP effector pathways. Nature (2005) 437:574
73. Debbie Willoughby, Dermot MF Cooper, et al. An anchored PKA and PDE4 complex regulates subplasmalemmal cAMP dynamics. The EMBO Journal (2006) 25:2051-2061
74. Tao Xie, Lee S. Weinstein, et al. The alternative stimulatory G protein ?-subunit XL?s is a critical regulator of energy and glucose metabolism and sympathetic nerve activity in adult mice. J Biol Chem (2006) 281: 18989–18999
75. Gokhan S. Hotamisligil, Bruce M. Spiegelman, et al. Adipose expression of tumor necrosis factor-?:direct role in obesity-linked insulin resistance. Science (1993) 259:87
76. Gokhan S. Hotamisligil, Bruce M. Spiegelmant, et al. IRS-1-mediated inhibition of insulin receptor tyrosine kinase activity in TNF-? and obesity-induced insulin resistance. Science (1996) 271:665
77. Gautam Bandyopadhyay, Robert V. Farese, et al. Activation of protein Kinase C (?????and ?? by insulin in 3T3-L1 cells. J Biol Chem (1997) 272:2551
78. Teddy T. C. Yang, Gerald R. Crabtree, et al. Role of transcription factor NFAT in glucose ans insulin homeostasis. Molecular and Cellular Biology (2006) 26:7372
79. Takuya Tomaru, Mitchell A. Lazar, et al. Adipocyte-specific expression of murine resistin is mediated by synergism between peroxisome proliferate-activated receptor ? and CCAAT/Enhancer-binding proteins. J Biol Chem (2009) 284:6116
80. Haiyan Song, Tomoichiro Asano, et al. Resistin is regulated by C/EBPs, PPARs, and signal-transducing molecules. Biochem. Biophys. Res. Commun (2002) 299:291
81. Sarah E. Ross, Ormond A. MacDougald, et al. Glycogen synthase kinase 3 is an insulin-regulated C/EBP? kinase. Molecular and Cellular Biology (1999) 19:8433
82. Darren A.E. Cross, Philip Cohen, et al. Insulin activates protein kinase B, inhibits glycogen synthase kinase-3 and activates glycogen synthase by rapamycin-insensitive pathways in skeletal muscle and adipose tissue. FEBS Letters (1997) 406:211