小鼠成纤维细胞生长因子-21的细胞代谢研究
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摘要
第一部分小鼠成纤维细胞生长因子-21 shRNA重组载体的构建
目的:构建针对小鼠成纤维细胞因子-21(fibroblast growth factor 21,FGF-21)的短发夹状RNA(short hairpin RNA,shRNA)重组质粒。
方法:设计并构建2条针对小鼠FGF-21基因的shRNA反向重复序列,分别克隆至载体pGenesil-1.2的mU6转录启动子下游,构建pGenesil-FGF21-1和2,然后分别与pDsRed-EGFP-FGF-21共转染BHK工具细胞,运用双荧光报告基因验证其有效性,初步筛选出抑制有效的pGenesil-FGF21重组质粒。
结果:两pGenesil-FGF21组GFP平均荧光强度/RFP平均荧光强度均低于阴性对照组(均P<0.05),且pGenesil-FGF21-1组最低。
结论:成功构建FGF-21-shRNA重组载体,初步筛选出了抑制有效的pGenesil-FGF21重组质粒
第二部分成纤维细胞生长因子-21对小鼠3T3-L1脂肪细胞代谢的影响
目的:分别建立FGF-21表达缺陷以及FGF-21过表达的脂肪细胞模型,探讨FGF-21表达改变对脂肪细胞糖-脂代谢以及脂肪细胞因子表达的影响。
方法:分别将pcDNA-FGF21和pGenesil-FGF21转染3T3-L1脂肪细胞,采用荧光定量PCR和Western Blotting方法检测上述重组质粒对脂肪细胞FGF-21表达的影响,采用2-脱氧-3H-D-葡萄糖(2-deoxy-D-glucose,2-DOG)摄入法测定脂肪细胞葡萄糖转运率(glucose uptake rate,GUR),油红O染色法检测细胞内甘油三酯含量,荧光定量PCR法检测细胞内糖脂代谢相关转录因子的mRNA表达。
结果:在脂肪细胞中,pGenesil-FGF21能有效抑制细胞内FGF-21表达(P<0.05),而转染pcDNA-FGF21的脂肪细胞内FGF-21表达显著升高(P<0.05)。FGF-21表达改变时脂肪细胞内以FGFR1改变为主。FGF-21过表达的脂肪细胞GUR显著升高,细胞内TG降低,FGF-21表达缺陷时则相反(P<0.05);葡萄糖转运蛋白-1(glucose transporter-1,GLUT-1)、胰岛素受体底物-1(insulin receptor substrate-1, IRS-1)、过氧化物酶体增殖物激活受体γ(Peroxisome proliferator-activated receptor-γ, PPARγ)、过氧化物酶体增殖物激活受体α(peroxisome roliferator-activated receptor-α, PPARα)、脂肪甘油三酯脂肪酶(Adipose triglyceride lipase, ATGL)、激素敏感性脂肪酶(Hormone sensitive lipase,HSL)和脂肪细胞脂肪酸结合蛋白2 (adipocyte fatty acid-binding protein, ap2)在FGF-21过表达的脂肪细胞内显著增加,FGF-21表达缺陷时降低(均P<0.05);而对葡萄糖转运蛋白-4 (glucose transporter-4,GLUT-4)表达无显著影响。FGF-21过表达的脂肪细胞内Leptin表达降低,FGF-21表达缺陷时Leptin升高(均P<0.05);而FGF-21表达改变对Visfatin及脂联素的表达无显著影响。
结论:在脂肪细胞中,FGF-21主要通过betaklotho和FGFR1发挥生物学作用,可能通过GLUT-1和IRS-1途径促进脂肪细胞葡萄糖摄取;并促进脂解作用从而降低细胞内脂质含量;还能调控脂肪因子表达。
第三部分成纤维细胞生长因子-21对小鼠Hepa1-6肝细胞代谢的影响
目的:分别建立FGF-21表达缺陷以及FGF-21过表达的小鼠肝细胞模型,探讨FGF-21表达改变对肝细胞糖-脂代谢以及重要转录因子表达的影响。
方法:分别将pcDNA-FGF21和pGenesil-FGF21转染Hepa1-6肝细胞,采用荧光定量PCR和Western Blotting方法检测上述重组质粒对肝细胞FGF-21表达的影响,采用2-脱氧-3H-D-葡萄糖摄入法测定肝葡萄糖转运率(GUR),荧光定量PCR法检测细胞内糖脂代谢相关转录因子的mRNA表达。
结果:pGenesil-FGF21能有效抑制Hepa1-6肝细胞内FGF-21表达(P<0.05),转染pcDNA-FGF21的肝细胞内FGF-21表达显著升高(P<0.05)。betaklotho在过表达FGF-21的肝细胞内呈升高趋势,而在FGF-21缺陷时降低;FGF-21表达改变时肝细胞内以FGFR4改变为主。FGF-21表达改变对肝细胞葡萄糖摄取无显著影响(P>0.05);过表达FGF-21的肝细胞内PPARγ、PPARα、羟甲基戊二酸单酰辅酶A还原酶(3-hydroxy-3-methylglutaryl coenzyme A reductase, HMGR)、磷酸烯醇式丙酮酸羧激酶(phosphoenolpyruvate carboxykinase, PEPCK)表达降低,而低密度脂蛋白受体(low density lipoprotein receptor, LDLr)表达升高;表达缺陷时则相反(均P<0.05);而对肝细胞内GLUT-1、GLUT-4、IRS-1以及葡萄糖激酶(glucokinase, GK)的mRNA表达无显著影响。而FGF-21表达改变对肝细胞内Visfatin的mRNA表达无显著影响。
结论:在肝细胞中,FGF-21主要通过betaklotho和FGFR4发挥生物学作用,可能通过影响PEPCK表达调控肝细胞糖异生;并主要通过调节肝细胞HMGCR和LDLr表达从而影响胆固醇合成和清除。
PARTⅠCONSTRUCTION OF FIBROBLAT GROWTH FACTOR-21 SHRNA RECOMBINATED VECTOR IN MICE
Objective: To establish fibroblast growth factor-21(FGF-21) shRNA recombinated vector in mice.
Methods: Two reverse repeated motifs targeting of FGF-21 gene were designed and synthesized respectively and inserted into plasmid pGenesil1.2 for generate the recombinated vectors. The effective rate of shRNA-vector (pGenesil-FGF21) was verified by Double-Fluorescence Reporter assay system.
Results: Fluorescence ratio of GFP/RFP in two groups were lower than that in HK group (P<0.05), and the lowest in the pGenesil-FGF21-1 group.
Conclusion: FGF-21 shRNA recombinated vector, pGenesil-FGF21, was established successfully.
PARTⅡTHE EFFECTS OF FIBROBLAT GROWTH FACTOR-21 ON METABOLISM IN 3T3-L1 ADIPOCYTES
Objective: To investigate the effects of FGF-21 on glucose turnover, triglyceride metabolism and the underlying mechanisms in 3T3-L1 adipocytes.
Methods: The two vectors, pGenesil-FGF21 and pcDNA-FGF-21, were transfected into the differentiated 3T3-L1 adipocytes, respectively.The FGF-21 mRNA expressions were measured by real-time quantitative PCR and FGF-21 protein contents were detected by Western Blotting.The uptake rates of 2-Deoxy[3H]-D-glucose (glucose uptake rate, GUR) were evaluated by liquid scintillation counting method (LSCM).The cellular triglyceride (TG) content was measured with a colorimetric assay. The mRNA expressions of betakloth, FGFRs, GLUT-1, GLUT-4, IRS-1,PPAR-α, PPAR-γ, HSL, ATGL, ap2, adiponectin, visfatin and Leptin were detected by real-time quantitative PCR.
Results: In 3T3-L1 adipocytes, pcDNA-FGF21 significantly increased FGF-21 expression (both P<0.05). Moreover, pGenesil-FGF21 caused FGF-21 down-regulation by 77.8% in mRNA expression and 75.3% in protein contents (both P<0.05). The upregulation of FGF-21 markedly increased GUR and decreased intracellular TG content (both P<0.05). The betaknotho and FGFR1 mRNA expressions were increased in FGF-21 upregulated adipocytes (both P<0.05). Meanwhile, the upregulation of GLUT-1, IRS-1, PPARγ, PPARα, ATGL, HSL, ap2 and leptin mRNA expressions were also observed (P<0.05). The reverse changes happened in FGF-21 deficient adipocyte except IRS-1.
Conclusion: In 3T3-L1 adipocytes, the effect of FGF-21 expression change is mainly through betaklotho and FGFR1. FGF-21 may be related to glucose homeostasis and accommodate intracellular fat content in adipocyte.
PARTⅢTHE EFFECTS OF FIBROBLAT GROWTH FACTOR-21 ON METABOLISM IN HEPA1-6 HEPATOCYTES
Objective: To investigate the roles of FGF-21 on glucose metabolism, cholesterol metabolism and the underlying mechanisms in Hepa1-6 hepatocytes.
Methods: The two vectors, pGenesil-FGF21 and pcDNA-FGF-21, were transfected into Hepa1-6 hepatocytes, respectively. The FGF-21 mRNA expressions were measured by real-time quantitative PCR and FGF-21 protein contents were detected by Western Blotting.The uptake rates of 2-deoxy-[3H]-D-glucose (GUR) were evaluated by liquid scintillation counting method. The mRNA expressions of betaklotho, FGFRs, GLUT-1, GLUT-4, IRS-1,PPARα, PPARγ, PEPCK, HMGR, LDLr and visfatin were detected by real-time quantitative PCR.
Results: In Hepa1-6 hepatocytes, pcDNA-FGF21 significantly increased FGF-21 expression (both P<0.05). Moreover, pGenesil-FGF21 caused FGF-21 down-regulation by 86.3% in mRNA expression and 77.3% in protein contents (both P<0.05). FGF-21 has no effect on GUR. The betaknotho and FGFR4 mRNA expressions were increased in FGF-21 upregulated hepatocytes (both P<0.05). Meanwhile, FGF-21 over-expression reduced PPARγ, PPARα, HMGR and PEPCK mRNA expression and increased LDLr expression (P<0.05), but had no effect on GLUT-1, GLUT-4, IRS-1, GK and visfatin expression in Hepa1-6 hepatocytes.
Conclusion: The effect of FGF-21 expression change is mainly through betaklotho and FGFR4 in hepa1-6 hepatocytes. FGF-21 maybe plays an important role in cholesterol metabolism and liver gluconeogenesis, but no glucose uptake.
目的:构建针对小鼠成纤维细胞因子-21(fibroblast growth factor 21,FGF-21)的短发夹状RNA(short hairpin RNA,shRNA)重组质粒。
方法:设计并构建2条针对小鼠FGF-21基因的shRNA反向重复序列,分别克隆至载体pGenesil-1.2的mU6转录启动子下游,构建pGenesil-FGF21-1和2,然后分别与pDsRed-EGFP-FGF-21共转染BHK工具细胞,运用双荧光报告基因验证其有效性,初步筛选出抑制有效的pGenesil-FGF21重组质粒。
结果:两pGenesil-FGF21组GFP平均荧光强度/RFP平均荧光强度均低于阴性对照组(均P<0.05),且pGenesil-FGF21-1组最低。
结论:成功构建FGF-21-shRNA重组载体,初步筛选出了抑制有效的pGenesil-FGF21重组质粒
第二部分成纤维细胞生长因子-21对小鼠3T3-L1脂肪细胞代谢的影响
目的:分别建立FGF-21表达缺陷以及FGF-21过表达的脂肪细胞模型,探讨FGF-21表达改变对脂肪细胞糖-脂代谢以及脂肪细胞因子表达的影响。
方法:分别将pcDNA-FGF21和pGenesil-FGF21转染3T3-L1脂肪细胞,采用荧光定量PCR和Western Blotting方法检测上述重组质粒对脂肪细胞FGF-21表达的影响,采用2-脱氧-3H-D-葡萄糖(2-deoxy-D-glucose,2-DOG)摄入法测定脂肪细胞葡萄糖转运率(glucose uptake rate,GUR),油红O染色法检测细胞内甘油三酯含量,荧光定量PCR法检测细胞内糖脂代谢相关转录因子的mRNA表达。
结果:在脂肪细胞中,pGenesil-FGF21能有效抑制细胞内FGF-21表达(P<0.05),而转染pcDNA-FGF21的脂肪细胞内FGF-21表达显著升高(P<0.05)。FGF-21表达改变时脂肪细胞内以FGFR1改变为主。FGF-21过表达的脂肪细胞GUR显著升高,细胞内TG降低,FGF-21表达缺陷时则相反(P<0.05);葡萄糖转运蛋白-1(glucose transporter-1,GLUT-1)、胰岛素受体底物-1(insulin receptor substrate-1, IRS-1)、过氧化物酶体增殖物激活受体γ(Peroxisome proliferator-activated receptor-γ, PPARγ)、过氧化物酶体增殖物激活受体α(peroxisome roliferator-activated receptor-α, PPARα)、脂肪甘油三酯脂肪酶(Adipose triglyceride lipase, ATGL)、激素敏感性脂肪酶(Hormone sensitive lipase,HSL)和脂肪细胞脂肪酸结合蛋白2 (adipocyte fatty acid-binding protein, ap2)在FGF-21过表达的脂肪细胞内显著增加,FGF-21表达缺陷时降低(均P<0.05);而对葡萄糖转运蛋白-4 (glucose transporter-4,GLUT-4)表达无显著影响。FGF-21过表达的脂肪细胞内Leptin表达降低,FGF-21表达缺陷时Leptin升高(均P<0.05);而FGF-21表达改变对Visfatin及脂联素的表达无显著影响。
结论:在脂肪细胞中,FGF-21主要通过betaklotho和FGFR1发挥生物学作用,可能通过GLUT-1和IRS-1途径促进脂肪细胞葡萄糖摄取;并促进脂解作用从而降低细胞内脂质含量;还能调控脂肪因子表达。
第三部分成纤维细胞生长因子-21对小鼠Hepa1-6肝细胞代谢的影响
目的:分别建立FGF-21表达缺陷以及FGF-21过表达的小鼠肝细胞模型,探讨FGF-21表达改变对肝细胞糖-脂代谢以及重要转录因子表达的影响。
方法:分别将pcDNA-FGF21和pGenesil-FGF21转染Hepa1-6肝细胞,采用荧光定量PCR和Western Blotting方法检测上述重组质粒对肝细胞FGF-21表达的影响,采用2-脱氧-3H-D-葡萄糖摄入法测定肝葡萄糖转运率(GUR),荧光定量PCR法检测细胞内糖脂代谢相关转录因子的mRNA表达。
结果:pGenesil-FGF21能有效抑制Hepa1-6肝细胞内FGF-21表达(P<0.05),转染pcDNA-FGF21的肝细胞内FGF-21表达显著升高(P<0.05)。betaklotho在过表达FGF-21的肝细胞内呈升高趋势,而在FGF-21缺陷时降低;FGF-21表达改变时肝细胞内以FGFR4改变为主。FGF-21表达改变对肝细胞葡萄糖摄取无显著影响(P>0.05);过表达FGF-21的肝细胞内PPARγ、PPARα、羟甲基戊二酸单酰辅酶A还原酶(3-hydroxy-3-methylglutaryl coenzyme A reductase, HMGR)、磷酸烯醇式丙酮酸羧激酶(phosphoenolpyruvate carboxykinase, PEPCK)表达降低,而低密度脂蛋白受体(low density lipoprotein receptor, LDLr)表达升高;表达缺陷时则相反(均P<0.05);而对肝细胞内GLUT-1、GLUT-4、IRS-1以及葡萄糖激酶(glucokinase, GK)的mRNA表达无显著影响。而FGF-21表达改变对肝细胞内Visfatin的mRNA表达无显著影响。
结论:在肝细胞中,FGF-21主要通过betaklotho和FGFR4发挥生物学作用,可能通过影响PEPCK表达调控肝细胞糖异生;并主要通过调节肝细胞HMGCR和LDLr表达从而影响胆固醇合成和清除。
PARTⅠCONSTRUCTION OF FIBROBLAT GROWTH FACTOR-21 SHRNA RECOMBINATED VECTOR IN MICE
Objective: To establish fibroblast growth factor-21(FGF-21) shRNA recombinated vector in mice.
Methods: Two reverse repeated motifs targeting of FGF-21 gene were designed and synthesized respectively and inserted into plasmid pGenesil1.2 for generate the recombinated vectors. The effective rate of shRNA-vector (pGenesil-FGF21) was verified by Double-Fluorescence Reporter assay system.
Results: Fluorescence ratio of GFP/RFP in two groups were lower than that in HK group (P<0.05), and the lowest in the pGenesil-FGF21-1 group.
Conclusion: FGF-21 shRNA recombinated vector, pGenesil-FGF21, was established successfully.
PARTⅡTHE EFFECTS OF FIBROBLAT GROWTH FACTOR-21 ON METABOLISM IN 3T3-L1 ADIPOCYTES
Objective: To investigate the effects of FGF-21 on glucose turnover, triglyceride metabolism and the underlying mechanisms in 3T3-L1 adipocytes.
Methods: The two vectors, pGenesil-FGF21 and pcDNA-FGF-21, were transfected into the differentiated 3T3-L1 adipocytes, respectively.The FGF-21 mRNA expressions were measured by real-time quantitative PCR and FGF-21 protein contents were detected by Western Blotting.The uptake rates of 2-Deoxy[3H]-D-glucose (glucose uptake rate, GUR) were evaluated by liquid scintillation counting method (LSCM).The cellular triglyceride (TG) content was measured with a colorimetric assay. The mRNA expressions of betakloth, FGFRs, GLUT-1, GLUT-4, IRS-1,PPAR-α, PPAR-γ, HSL, ATGL, ap2, adiponectin, visfatin and Leptin were detected by real-time quantitative PCR.
Results: In 3T3-L1 adipocytes, pcDNA-FGF21 significantly increased FGF-21 expression (both P<0.05). Moreover, pGenesil-FGF21 caused FGF-21 down-regulation by 77.8% in mRNA expression and 75.3% in protein contents (both P<0.05). The upregulation of FGF-21 markedly increased GUR and decreased intracellular TG content (both P<0.05). The betaknotho and FGFR1 mRNA expressions were increased in FGF-21 upregulated adipocytes (both P<0.05). Meanwhile, the upregulation of GLUT-1, IRS-1, PPARγ, PPARα, ATGL, HSL, ap2 and leptin mRNA expressions were also observed (P<0.05). The reverse changes happened in FGF-21 deficient adipocyte except IRS-1.
Conclusion: In 3T3-L1 adipocytes, the effect of FGF-21 expression change is mainly through betaklotho and FGFR1. FGF-21 may be related to glucose homeostasis and accommodate intracellular fat content in adipocyte.
PARTⅢTHE EFFECTS OF FIBROBLAT GROWTH FACTOR-21 ON METABOLISM IN HEPA1-6 HEPATOCYTES
Objective: To investigate the roles of FGF-21 on glucose metabolism, cholesterol metabolism and the underlying mechanisms in Hepa1-6 hepatocytes.
Methods: The two vectors, pGenesil-FGF21 and pcDNA-FGF-21, were transfected into Hepa1-6 hepatocytes, respectively. The FGF-21 mRNA expressions were measured by real-time quantitative PCR and FGF-21 protein contents were detected by Western Blotting.The uptake rates of 2-deoxy-[3H]-D-glucose (GUR) were evaluated by liquid scintillation counting method. The mRNA expressions of betaklotho, FGFRs, GLUT-1, GLUT-4, IRS-1,PPARα, PPARγ, PEPCK, HMGR, LDLr and visfatin were detected by real-time quantitative PCR.
Results: In Hepa1-6 hepatocytes, pcDNA-FGF21 significantly increased FGF-21 expression (both P<0.05). Moreover, pGenesil-FGF21 caused FGF-21 down-regulation by 86.3% in mRNA expression and 77.3% in protein contents (both P<0.05). FGF-21 has no effect on GUR. The betaknotho and FGFR4 mRNA expressions were increased in FGF-21 upregulated hepatocytes (both P<0.05). Meanwhile, FGF-21 over-expression reduced PPARγ, PPARα, HMGR and PEPCK mRNA expression and increased LDLr expression (P<0.05), but had no effect on GLUT-1, GLUT-4, IRS-1, GK and visfatin expression in Hepa1-6 hepatocytes.
Conclusion: The effect of FGF-21 expression change is mainly through betaklotho and FGFR4 in hepa1-6 hepatocytes. FGF-21 maybe plays an important role in cholesterol metabolism and liver gluconeogenesis, but no glucose uptake.
引文
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[22]杨刚毅.代谢紊乱和脂肪细胞因子与胰岛素抵抗[J].中国糖尿病杂志.2007,15(3):129-13.
[23] Ogawa Y, Kurosu H, Yamamoto M, et al.βKlotho is required for metabolic activity of fibroblast growth factor 21[J]. Proc Natl Acad Sci U.S.A.2007,104(18): 7432~7.
[24] Kurosu H, Ogawa Y, Miyoshi M, Yamamoto M, Nandi A, Rosenblatt KP, Baum MG, Schiavi S, Hu MC, Moe OW, Kuro-o M.Regulation of fibroblast growth factor-23 signaling by klotho[J].J Biol Chem. 2006, 281(10):6120~3.
[25] Urakawa I, Yamazaki Y, Shimada T, Iijima K, Hasegawa H, Okawa K, Fujita T, Fukumoto S, Yamashita T.Klotho converts canonical FGF receptor into a specific receptor for FGF23[J].Nature. 2006,444(7120):770~4.
[26] Ito S, Kinoshia S, Shiraishi N, rt al. Molecular cloning and expression analyses of mouseβklotho, which encodes a novel Klotho family protein[J]. Mech Dev.2000,98(1-2): 115~119
[27] Wang H, Qiang L, Farmer SR. Identification of a domain within peroxisome proliferator-activated receptor gamma regulating expression of a group of genes containing fibroblast growth factor 21 that are selectively repressed by SIRT1 in adipocytes[J].Mol Cell Biol. 2008,28(1):188~200.
[28] Moyers JS, Shiyanova TL, Mehrbod F, Dunbar JD, Noblitt TW, Otto KA, Reifel-Miller A, Kharitonenkov A. Molecular determinants of FGF-21 activity-synergy and cross-talk with PPARgamma signaling[J].J Cell Physiol. 2007,210(1):1~6.
[29] Xu J, Lloyd DJ, Hale C, Stanislaus S, Chen M, Sivits G, Vonderfecht S, Hecht R, Li YS, Lindberg RA, Chen JL, Jung DY, Zhang Z, Ko HJ, Kim JK, Véniant MM.Fibroblast growth factor 21 reverses hepatic steatosis, increases energy expenditure, and improves insulin sensitivity in diet-induced obese mice[J]. Diabetes. 2009,58(1):250~9.
[30] Hotta Y, Nakamura H, Konishi M, Murata Y, Takagi H, Matsumura S, Inoue K, Fushiki T, Itoh N. Fibroblast growth factor 21 regulates lipolysis in white adipose tissue but is not required for ketogenesis and triglyceride clearance in liver[J]. Endocrinology. 2009,150(10):4625~33.
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[32] Kurosu H, Choi M, Ogawa Y, et al. Tissue-specific expression of betaKlotho and fibroblast growth factor rceptor isoforms determines metabolic activity of FGF-19 and FGF-21. J Biol Chem.2007,282(37):26687-95.
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[20] Gulcelik NE, Karakaya J, Gedik A, Usman A, Gurlek A. Serum vaspin levels in type 2 diabetic women in relation to microvascular complications[J]. Eur J Endocrinol.2009,160:65~70.
[21] Ye Y, Hou XH, Pan XP, Lu JX, Jia WP. Serum vaspin level in relation to postprandial plasma glucose concentration in subjects with diabetes[J]. Chin Med J (Engl).2009,122:2530~3.
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[25] Aust G, Richter O, Rohm S, et al.Vaspin serum concentrations in patients withcarotid stenosis[J]. Atherosclerosis.2009,204:262~6.
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[1] Frayn KN. Visceral fat and insulin resistance-causative or correlative[J]? Br J Nutr.2000,83:S71~S77.
[2] Wajchenberg BL. Subcutaneous and visceral adipose tissue: their relation to the metabolic syndrome[J]. Endocr Rev. 2000,21:697~738.
[3] Van Harmelen V, Reynisdottir S, Eriksson P, et al. Leptin secretion from subcutaneous and visceral adipose tissue in women[J]. Diabetes.1998,47:913~917.
[4] Hara K, Yamauchi T, Kadowaki T. Adiponectin: an adipokine linking adipocytes and type 2 diabetes in humans[J]. Curr Diab Rep.2005,5:136~40.
[5] Fried SK, Bunkin DA, Greenberg AS. Omental and subcutaneous adipose tissue of obese subjects release interleukin 6: depot difference and regulation by glucocorticoid[J]. J Cli. Endocrinol Metab. 1998,83:847~850.
[6] Li Q, Chen R, Moriya J, et al. A novel adipocytokine, visceral adipose tissue-derived serine protease inhibitor (vaspin), and obesity[J]. J Int Med Res.2008,36:625~629.
[7] Wada J. Vaspin: a novel serpin with insulin-sensitizing effects[J]. Expert Opin Investig Drugs.2008,17:327~333.
[8] Hida K, Wada J, Zhang H, et al. Identification of genes specifically expressed in the accumulated visceral adipose tissue of OLETF rats[J]. J Lipid Res.2000,41:1615~22.
[9] Kawano K, Hirashima T, Mori S, Saitoh Y, Kurosumi M, Natori T. Spontaneous long-term hyperglycemic rat with diabetic complications, Otsuka Long-Evans Tokushima Fatty (OLETF) strain[J]. Diabetes.1992,41:1422~1428.
[10] Hida K, Wada J, Eguchi J, et al. Visceral adipose tissue-derived serine protease inhibitor: a unique insulin-sensitizing adipocytokine in obesity[J]. Proc Natl Acad Sci U S A.2005,102:10610~5.
[11] Kl?ting N, Berndt J, Kralisch S, et al. Vaspin gene expression in human adiposetissue: association with obesity and type 2 diabetes[J]. Biochem Biophys Res Commun. 2006,339:430~436.
[12] Tan BK, Heutling D, Chen J, et al. Metformin decreases the adipokine vaspin in overweight wonlen with polycystic ovary syndrome concomitant with improvement in insulin sensitivity and a decrease in insulin resistance[J]. Diabetes.2008,57:1501~1507.
[13] Youn BS, Kl?ting N, Kratzsch J, et al. Serum vaspin concentrations in human obesity and type 2 diabetes[J]. Diabetes.2008,57:372~377.
[14] Suleymanoglu S, Tascilar E, Pirgon O, Tapan S, Meral C, Abaci A. Vaspin and its correlation with insulin sensitivity indices in obese children[J]. Diabetes Res Clin Pract.2009,84:325~8.
[15] Lee MK, Jekal Y, Im JA, et al. Reduced serum vaspin concentrations in obese children following short-term intensive lifestyle modification[J]. Clin Chim Acta. 2010,411:381~5.
[16] Handisurya A, Riedl M, Vila G, et al. Serum vaspin concentrations in relation to insulin sensitivity following RYGB-induced weight loss[J]. Obes Surg.2010,20:198~203.
[17] Kempf K, Rose B, Illig T, et al. Vaspin (SERPINA12) genotypes and risk of type 2 diabetes: Results from the MONICA/KORA studies[J]. Exp Clin Endocrinol Diabetes.2010,118:184~9.
[18] von Loeffelholz C, M?hlig M, Arafat AM, et al.Circulating vaspin is unrelated to insulin sensitivity in a cohort of nondiabetic humans[J]. Eur J Endocrinol.2010,162:507~13.
[19] Seeger J, Ziegelmeier M, Bachman A, et al. Serum levels of the adipokine vaspin in relation to metabolic and renal parameters[J]. J Cli. Endocrinol Metab.2008,93:247~251.
[20] Gulcelik NE, Karakaya J, Gedik A, Usman A, Gurlek A. Serum vaspin levels in type 2 diabetic women in relation to microvascular complications[J]. Eur J Endocrinol.2009,160:65~70.
[21] Ye Y, Hou XH, Pan XP, Lu JX, Jia WP. Serum vaspin level in relation to postprandial plasma glucose concentration in subjects with diabetes[J]. Chin Med J (Engl).2009,122:2530~3.
[22] Ma Z, Gingerich RL, Santiago JV, et al. Radioimmunoassay of leptin in human plasma[J]. Clin Chem.1996,42:942~946.
[23] Nishizawa H, Shimomura I, Kishida K, et al. Androgens decrease plasma adiponectin, an insulin-sensitizing adipocyte-derived protein[J]. Diabetes.2002,51:2734~2741.
[24] Kapoor D, Clarke S, Stanworth R, Channer KS, Jones TH. The effect of testosterone replacement therapy on adipocytokines and C-reactive protein in hypogonadal men with type 2 diabetes[J]. Eur J Endocrinol.2007,156:595~602.
[25] Aust G, Richter O, Rohm S, et al.Vaspin serum concentrations in patients withcarotid stenosis[J]. Atherosclerosis.2009,204:262~6.
[26] Kukla M, Zwirska-Korczala K, Hartleb M, et al.Serum chemerin and vaspin in non-alcoholic fatty liver disease[J]. Scand J Gastroenterol.2010,45:235~42.
[27] Febbraio MA, Steensberg A, Starkie RL, McConell GK, Kingwell BA. Skeletal muscle interleukin-6 and tumor necrosis factor-alpha release in healthy subjects and patients with type 2 diabetes at rest and during exercise[J]. Metabolism.2003,52:939~944.