羟基红花黄色素A对小鼠3T3-L1前脂肪细胞增殖和分化及细胞内脂代谢调节酶作用的研究
|
摘要
目的
肥胖症与心脑血管疾病、2型糖尿病、血脂紊乱、呼吸睡眠暂停综合症等疾病的发生有密切的关系,已经成为危害人类健康的重要疾病。过多的热量摄入并以甘油三脂的形式存储在白色脂肪细胞内是肥胖发生的关键。前脂肪细胞的增殖和分化是成熟脂肪细胞形成的核心,从前脂肪细胞分化成为成熟的脂肪细胞的过程十分复杂,机体内多种细胞因子和激素在脂肪细胞成熟过程中都有调节作用。本课题组前期实验发现红花的主要活性成分羟基红花黄色素A对前脂肪细胞分化到脂肪细胞过程中的关键转录因子PPARγ具调节作用,提示羟基红花黄色素A可能对脂肪细胞的生物学功能发挥影响。本课题旨在研究羟基红花黄色素A对小鼠3T3-L1前脂肪细胞增殖、分化及细胞内脂肪酸合成酶和激素敏感性脂肪酶两种脂代谢调节酶的影响,为进一步探讨HSYA在脂肪细胞内脂质代谢方面新的作用打下基础。
方法
1.以MTT法观察羟基红花黄色素A对小鼠3T3-L1前脂肪细胞增殖的影响。
2.使用油红O染色定量的方法以及甘油三酯GPO-POD酶法测定观察脂肪细胞的成熟,以反映羟基红花黄色素A对小鼠3T3-L1前脂肪细胞分化的影响;
3.应用实时荧光定量PCR和双萤光素酶报告检测系统分别从mRNA水平和启动子活性水平研究羟基红花黄色素对小鼠3T3-L1前脂肪细胞分化过程中脂代谢调节酶脂肪酸合成酶和激素敏感性脂肪酶的影响。
结果
1.羟基红花黄色素A对小鼠3T3-L1前脂肪细胞增殖有抑制和增强两种作用。其对于细胞增殖的显著影响从24小时开始;在24至96小时间,0.01、0.1、1、10mg/L羟基红花黄色素A刺激组均对3T3-L1前脂肪细胞增殖起显著抑制作用,且作用无明显剂量依赖关系,而100 mg/L组仅在24小时对增殖有显著抑制作用,其后的48、72及96小时测定与对照无显著差别。至120小时时,羟基红花黄色素A对细胞增殖均呈促进作用,且促进随浓度增加而增强。
2.羟基红花黄色素A对3T3-L1前脂肪细胞分化为脂肪细胞有抑制和增强两种作用。0.01、1 mg/L羟基红花黄色素A组呈现先抑制后促进的现象,在分化的第4、8、12天,HSYA组细胞内脂质含量显著降低,且降低具剂量依赖关系;而在分化第16天时,细胞内脂质含量较对照组升高,但无统计学差异。而100 mg/L羟基红花黄色素A组细胞内脂质含量则在分化第4天及16天时较对照组显著增多。
3.羟基红花黄色素A对于小鼠3T3-L1前脂肪细胞分化过程中脂代谢调节酶脂肪酸合成酶和激素敏感性脂肪酶主要起促进作用。
结论
1.羟基红花黄色素A对于3T3—L1前脂肪细胞增殖的影响随不同浓度、不同作用时间有所不同。主要趋势为在一定浓度范围内的羟基红花黄色素A刺激作用可在一定时间内抑制前脂肪细胞的增殖,但随着细胞培养时间的延长、细胞密度增加,逐渐表现为促进细胞增殖。
2.羟基红花黄色素A对于3T3—L1前脂肪细胞分化的影响随不同浓度、不同作用时间有所不同。即0.01及1 mg/L羟基红花黄色素A组在3T3—L1前脂肪细胞诱导分化过程中主要起抑制分化作用。而100 mg/L羟基红花黄色素A组则主要表现为促进分化。
3.羟基红花黄色素A对于小鼠3T3-L1前脂肪细胞分化过程中脂代谢调节酶脂肪酸合成酶和激素敏感性脂肪酶都主要起促进作用,但对于激素敏感性脂肪酶的促进作用大于脂肪酸合成酶,可能是导致分化过程中脂质合成少于对照组的原因。
Objective:
Obesity has already been a worldwide epidemic disease and it can lead patients to lots of serious diseases,such as coronary heart disease,stroke,hypertension,type 2 diabetes,hyperlipidemia,sleep apnea,and et al.Excess energy intake and stored in the white adipocytes,as well as the proliferation and differentiation of preadipocytes are the two core factors in the development of obesity.Our previous study showed that the main component of Carthamus tinctorius L.,hydroxysafflor yellow A could modulate the activity of PPARγ,a key transcription factor during preadipocyte differentiation.And the result showed the possible roles of hydroxysafflor yellow A in adipocytes.The aim of this study is to learn the effect of hydroxysafflor yellow A on the proliferation and differentiation of 3T3-L1 preadipocyte as well as the activity of fatty acid synthase and hormone sensitive lipase.
Methods:
1.To study the effect of hydroxysafflor yellow A on the proliferation of 3T3-L1 preadipocytes by MTT detection.
2.To study the effect of hydroxysafflor yellow A on the differentiation of 3T3-L1 preadipocytes by the means of Oil Red O staining and Triglyceride GPO-POD enzymatic detection.
3.To study the effects of hydroxysafflor yellow A on the activity of fatty acid synthase and hormone sensitive lipase by real time PCR and Dual luciferase Reporter Assay system.
Results:
1.Hydroxysafflor yellow A could enhance or inhibit the proliferation of 3T3-L1 preadipocytes.During 24 to 96 hours,0.01、0.1、1、10mg/L hydroxysafflor yellow A could significantly inhibit the proliferation of 3T3-L1 preadipocytes.And 100 mg/L hydroxysafflor yellow A could only inhibit the proliferation in the 24-hour action time significantly.After 120 hours,hydroxysafflor yellow A could stimulate the growth of 3T3-L1 preadipocytes in a dose-dependent manner.
2.Hydroxysafflor yellow A could stimulate or repress the differentiation of 3T3-L1 preadipocytes.0.01、1 mg/L hydroxysafflor yellow A decreased the lipid content in the differentiation day 4,8 and 12,while increased it in day 16.100 mg/L hydroxysafflor yellow A mainly stimulated the lipid formation in the day 4 and 16 during the differentiation of 3T3-L1 preadipocytes.
3.Hydroxysafflor yellow A enhanced the activity of fatty acid synthase and hormone sensitive lipase during the differentiation of 3T3-L1 preadipocytes.
Conclusions
1.Hydroxysafflor yellow A could enhance or inhibit the proliferation of 3T3-L1 preadipocytes in specific proliferation stages with specific concentration。
2.Hydroxysafflor yellow A could stimulate or repress the differentiation of 3T3-L1 preadipocytes in specific stages with specific concentration.
3.Hydroxysafflor yellow A enhanced the activity of fatty acid synthase and hormone sensitive lipase during the differentiation of 3T3-L1 preadipocytes.The stronger stimulation effect of hormone sensitive lipase than fatty acid synthase may the main cause lead to the reduction of lipid formation during differentiation of 3T3-L1 preadipocytes.
肥胖症与心脑血管疾病、2型糖尿病、血脂紊乱、呼吸睡眠暂停综合症等疾病的发生有密切的关系,已经成为危害人类健康的重要疾病。过多的热量摄入并以甘油三脂的形式存储在白色脂肪细胞内是肥胖发生的关键。前脂肪细胞的增殖和分化是成熟脂肪细胞形成的核心,从前脂肪细胞分化成为成熟的脂肪细胞的过程十分复杂,机体内多种细胞因子和激素在脂肪细胞成熟过程中都有调节作用。本课题组前期实验发现红花的主要活性成分羟基红花黄色素A对前脂肪细胞分化到脂肪细胞过程中的关键转录因子PPARγ具调节作用,提示羟基红花黄色素A可能对脂肪细胞的生物学功能发挥影响。本课题旨在研究羟基红花黄色素A对小鼠3T3-L1前脂肪细胞增殖、分化及细胞内脂肪酸合成酶和激素敏感性脂肪酶两种脂代谢调节酶的影响,为进一步探讨HSYA在脂肪细胞内脂质代谢方面新的作用打下基础。
方法
1.以MTT法观察羟基红花黄色素A对小鼠3T3-L1前脂肪细胞增殖的影响。
2.使用油红O染色定量的方法以及甘油三酯GPO-POD酶法测定观察脂肪细胞的成熟,以反映羟基红花黄色素A对小鼠3T3-L1前脂肪细胞分化的影响;
3.应用实时荧光定量PCR和双萤光素酶报告检测系统分别从mRNA水平和启动子活性水平研究羟基红花黄色素对小鼠3T3-L1前脂肪细胞分化过程中脂代谢调节酶脂肪酸合成酶和激素敏感性脂肪酶的影响。
结果
1.羟基红花黄色素A对小鼠3T3-L1前脂肪细胞增殖有抑制和增强两种作用。其对于细胞增殖的显著影响从24小时开始;在24至96小时间,0.01、0.1、1、10mg/L羟基红花黄色素A刺激组均对3T3-L1前脂肪细胞增殖起显著抑制作用,且作用无明显剂量依赖关系,而100 mg/L组仅在24小时对增殖有显著抑制作用,其后的48、72及96小时测定与对照无显著差别。至120小时时,羟基红花黄色素A对细胞增殖均呈促进作用,且促进随浓度增加而增强。
2.羟基红花黄色素A对3T3-L1前脂肪细胞分化为脂肪细胞有抑制和增强两种作用。0.01、1 mg/L羟基红花黄色素A组呈现先抑制后促进的现象,在分化的第4、8、12天,HSYA组细胞内脂质含量显著降低,且降低具剂量依赖关系;而在分化第16天时,细胞内脂质含量较对照组升高,但无统计学差异。而100 mg/L羟基红花黄色素A组细胞内脂质含量则在分化第4天及16天时较对照组显著增多。
3.羟基红花黄色素A对于小鼠3T3-L1前脂肪细胞分化过程中脂代谢调节酶脂肪酸合成酶和激素敏感性脂肪酶主要起促进作用。
结论
1.羟基红花黄色素A对于3T3—L1前脂肪细胞增殖的影响随不同浓度、不同作用时间有所不同。主要趋势为在一定浓度范围内的羟基红花黄色素A刺激作用可在一定时间内抑制前脂肪细胞的增殖,但随着细胞培养时间的延长、细胞密度增加,逐渐表现为促进细胞增殖。
2.羟基红花黄色素A对于3T3—L1前脂肪细胞分化的影响随不同浓度、不同作用时间有所不同。即0.01及1 mg/L羟基红花黄色素A组在3T3—L1前脂肪细胞诱导分化过程中主要起抑制分化作用。而100 mg/L羟基红花黄色素A组则主要表现为促进分化。
3.羟基红花黄色素A对于小鼠3T3-L1前脂肪细胞分化过程中脂代谢调节酶脂肪酸合成酶和激素敏感性脂肪酶都主要起促进作用,但对于激素敏感性脂肪酶的促进作用大于脂肪酸合成酶,可能是导致分化过程中脂质合成少于对照组的原因。
Objective:
Obesity has already been a worldwide epidemic disease and it can lead patients to lots of serious diseases,such as coronary heart disease,stroke,hypertension,type 2 diabetes,hyperlipidemia,sleep apnea,and et al.Excess energy intake and stored in the white adipocytes,as well as the proliferation and differentiation of preadipocytes are the two core factors in the development of obesity.Our previous study showed that the main component of Carthamus tinctorius L.,hydroxysafflor yellow A could modulate the activity of PPARγ,a key transcription factor during preadipocyte differentiation.And the result showed the possible roles of hydroxysafflor yellow A in adipocytes.The aim of this study is to learn the effect of hydroxysafflor yellow A on the proliferation and differentiation of 3T3-L1 preadipocyte as well as the activity of fatty acid synthase and hormone sensitive lipase.
Methods:
1.To study the effect of hydroxysafflor yellow A on the proliferation of 3T3-L1 preadipocytes by MTT detection.
2.To study the effect of hydroxysafflor yellow A on the differentiation of 3T3-L1 preadipocytes by the means of Oil Red O staining and Triglyceride GPO-POD enzymatic detection.
3.To study the effects of hydroxysafflor yellow A on the activity of fatty acid synthase and hormone sensitive lipase by real time PCR and Dual luciferase Reporter Assay system.
Results:
1.Hydroxysafflor yellow A could enhance or inhibit the proliferation of 3T3-L1 preadipocytes.During 24 to 96 hours,0.01、0.1、1、10mg/L hydroxysafflor yellow A could significantly inhibit the proliferation of 3T3-L1 preadipocytes.And 100 mg/L hydroxysafflor yellow A could only inhibit the proliferation in the 24-hour action time significantly.After 120 hours,hydroxysafflor yellow A could stimulate the growth of 3T3-L1 preadipocytes in a dose-dependent manner.
2.Hydroxysafflor yellow A could stimulate or repress the differentiation of 3T3-L1 preadipocytes.0.01、1 mg/L hydroxysafflor yellow A decreased the lipid content in the differentiation day 4,8 and 12,while increased it in day 16.100 mg/L hydroxysafflor yellow A mainly stimulated the lipid formation in the day 4 and 16 during the differentiation of 3T3-L1 preadipocytes.
3.Hydroxysafflor yellow A enhanced the activity of fatty acid synthase and hormone sensitive lipase during the differentiation of 3T3-L1 preadipocytes.
Conclusions
1.Hydroxysafflor yellow A could enhance or inhibit the proliferation of 3T3-L1 preadipocytes in specific proliferation stages with specific concentration。
2.Hydroxysafflor yellow A could stimulate or repress the differentiation of 3T3-L1 preadipocytes in specific stages with specific concentration.
3.Hydroxysafflor yellow A enhanced the activity of fatty acid synthase and hormone sensitive lipase during the differentiation of 3T3-L1 preadipocytes.The stronger stimulation effect of hormone sensitive lipase than fatty acid synthase may the main cause lead to the reduction of lipid formation during differentiation of 3T3-L1 preadipocytes.
引文
1.Bray GA,Bellanger T.Epidemiology,trends,and morbidities of obesity and the metabolic syndrome.Endocrine.2006,29(1):109-18.
2.Wyatt SB,Winters KP,Dubbert PM.Overweight and obesity:prevalence,consequences,and causes of a growing public health problem.Am J Med Sci.2006,331(4):166-174.
3.武阳丰,马冠生,胡永华,等.中国居民的超重和肥胖流行现状。中华预防医学杂志 2005,39(5):316-320.
4.董秋婷,顼志敏,代谢综合征防治进展,中华老年心脑血管病杂志,2008,10(1):66-68.
5.Halford JC.Obesity drugs in clinical development.Curr Opin Investig Drugs.2006,7(4):312-318.
6.施峰,刘焱文,红花的化学成分及药理研究进展,时珍国医国药,2006,17(9):1666-1667.
7.祝美珍,胡国恒,肖健,羟基红花黄色素A的应用研究进展,中西医结合心脑血管病杂志,2007,(11):1108-1110.
8.He,H.,X.Yang,et al.Protective effects of hydroxysafflor yellow A on acute and chronic congestive cardiac failure mediated by reducing ET-1,NOS and oxidative stress in rats.J Pharm Pharmacol,2008,60(1):115-23.
9.Liu,Y.N.,Z.M.Zhou,et al.Evidence that hydroxysafflor yellow A protects the heart against ischaemia-reperfusion injury by inhibiting mitochondrial permeability transition pore opening.Clin Exp Pharmacol Physiol,2008,35(2):211-6.
10.Tian,J.W.,F.H.Fu,et al,Protective effect of hydroxysafflor yellow A against rat cortex mitochondrial injuries induced by cerebral ischemia,Yao Xue Xue Bao,2004,39(10):774-7.
11.Wei,X.,H.Liu,et al.Hydroxysafflor yellow A protects rat brains against ischemia-reperfusion injury by antioxidant action,Neurosci Lett,2005,386(1):58-62.
12.Zhu,H.B.,Z.H.Wang,et al.Protective effect of hydroxysafflor yellow A on experimental cerebral ischemia in rats,Yao Xue Xue Bao,2005 40(12):1144-6.
13.Zhu,H.B.,L.Zhang,et al.Therapeutic effects of hydroxysafflor yellow A on focal cerebral ischemic injury in rats and its primary mechanisms.J Asian Nat Prod Res,2005,7(4):607-13.
14.徐剑.以过氧化物酶体增殖物受体γ为靶点的天然减肥药物的初步筛选[D].中 国协和医科大学:,2006。
15.Rosen,E.D.and O.A.MacDougald.Adipocyte differentiation from the inside out,Nat Rev Mol Cell Biol 2006,7(12):885-96.
16.Guan HP,Li Y,Jensen M,et al.A futile metabolic cycle activated in adipocytes by antidiabetic agents.Nat Med 2002,8:1122-1128
17.Barroso I,Gumell M,Crowley VEF,et al.Dominant negative mutations in human PPARγ associated with severe insulin resistance,diabetes mellitus,and hypertension.Nature 1999;402:880-883
18.Wolf G.Insulin resistance and obesity:resistin,a hormone secreted by adipose tissue.Nutr Rev.2004;62(10):389-94
19.Lin Y,Rajala M,Berger J,et al.Hyperglycemia-induced production of acute phase reactants.J Biol Chem 2001;276:42077-42083
20.Smith S,Witkowski A,Joshi AK.Structural and functional organization of the animal fatty acid synthase.Prog Lipid Res.2003,42(4):289-317;
21.Loftus,T.M.,D.E.Jaworsky,et al..Reduced food intake and body weight in mice treated with fatty acid synthase inhibitors.Science,2000,288(5475):2379-81;
22.Large V,Reynisdottir S,Langin D,et al.Decreased expression and function of adipocyte hormone-sensitive lipase in subcutaneous fat cells of obese subjects.J Lipid Res,1999,40:2059-2065
23.Roduit R,Masiello P,Wang SP,et al.A role for hormone-sensitive lipase in glucose stimulated insulin secretion.Diabetes.2001,50:1970-1975
24.Mular H,Holst LS,Svensson H,et al.Hormone-sensitive lipase,the rate-limiting enzyme in triglyceride hydrolysis,is expressed and actived in β-cells.Diabetes,1999,48:228-232.
25.Green H,Kehinde O.Sublines of mouse 3T3 cells that accumulate lipid.Cell 1974;1(1):113-6.
26.Green H,Meuth M.An established pre-adipose cell line and its differentiation in culture.Cell 1974;3(2):127-33.
27.Green H,Kehinde O.An established preadipose cell line and its differentiation in culture.Ⅱ.Factors affecting the adipose conversion.Cell 1975;5(1):19-27.
28.Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.Methods(San Diego,Calif 2001;25(4):402-8.
29.张前,牛欣,闫妍等.羟基红花黄色素A对体外培养人脐静脉内皮细胞增殖的抑制作用.中国医药学报,2004,19(6):379-381.
30.张前,赵言群,解华,牛欣,.中药对肿瘤血管生成的影响.中华中医药杂志,2006,21(4):242-245.
31.张前.羟基红花黄色素A对血管内皮细胞调控机理的研究[D].北京中医药大学:,2005.
32.张前,牛欣,闰妍,金鸣,杨向竹,李金荣.羟基红花黄色素A抑制新生血管形成的机制研究.北京中医药大学学报,2004,27(3):25-29.
33.SONG Yan,ZHANG Ling,QU Kai,et al,Hydroxysafflor Yellow A Promotes Vascular Endothelial Cell Proliferation via VEGF/VEGF Receptor,Journal of Chinese Pharmaceutical Sciences 2005,14(3):181-185.
34.吉登波,李长龄,朱美财等,从红花中提取的单体化合物-羟基红花黄色素A通过上调HIF-1α-VEGF信号通路和调控Bcl-2/Bax表达比率促进低氧状态下人血管内皮细胞存活的研究,第九届全国心血管药理学术会议论文集,14-15.
35.陈粉粉.EGCG对猪前体脂肪细胞增殖分化和脂解的影响[D].西北农林科技大学:,2007.
36.李宗孝,温普红,袁美娟,肉桂中查耳酮的类似胰岛素作用,中医药学报,2004,32(5):29-31.
37.莫尚武,陈恒留,刘宁,金建南,张淑渊,李文学,陈治恒,刘开明.用～(45)Ca研究红花对大鼠胸主动脉Ca-(2+)内流的影响,中草药,1995,(10):532-534.
38.王玢,郭锡熔,陈荣华,钙-肥胖的重要调节者,医学综述,2005,11(4):300-303.
39.Hsu,C.L.and G.C.Yen,Effects of flavonoids and phenolic acids on the inhibition of adipogenesis in 3T3-L1 adipocytes.J Agric Food Chem,2007,55(21):8404-10.
40.刘新迎,周联,梁瑞燕,柚皮苷对前脂肪细胞3T3-L1增殖和诱导分化的影响,中药新药与临床药理,2007,18(3):176-179.
41.金鸣,李金荣,吴伟,羟基红花黄色素A抗氧化作用的研究,中草药,2004,35(6):665-666.
1.Peter Arner,Adipose Tissue as an Endocrine Organ,Best Practice & Research Clinical Endocrinology & Metabolism,2005,19,471-482;
2.Diraison F,Beylot M.Role of human liver lipogenesis and reesterification in triglycerides secretion and in FFA reesterification.Am J Physiol,1998,274,E321-7;
3.Aarsland A,Chinkes D,Wolfe R.Hepatic and whole body fat synthesis in humans during carbohydrate overfeeding.Am J Clin Nutr,1997,65,1174-82;
4.Letexier D,Pinteur C,Large V,Fr(?)ring V and Beylot M.Comparison of the expression and activity of the lipogenic pathway in human and rat adipose tissue.J Lipid Res,2003,44,2127-34;
5.Diraison F.,V.Yankah,D.Letexier,E.Dusserre,P.Jones,and M.Beylot.Differences in the regulation of adipose tissue and liver lipogenesis by carbohydrates in humans.J.Lipid Res.44:846-853;
6.Kersten S.Mechanisms of nutritional and hormonal regulation of lipogenesis.EMBO reports 2001,2(4),282-6;
7. Mead JR, Irvine SA, Ramji D. Lipoprotein lipase: structure, function, regulation and role in disease. J Mol Med, 2002, 80, 753-69;
8. Tacken PJ, Hofker MH, Havekes L, et al. Living up to a name: the role of the VLDL receptor in lipid metabolism. Cur Opin Lipidol, 2001,12,275-9.
9. Goudriaan JR, Tacken PJ, Dahlmans VE, et al. Protection from obesity in mice lacking the VLDL receptor. Arterioscler Thromb Vasc Biol, 2001 21,1488-93.
10. Jianqiang M, Francesco JD, Huiguang L, et al. Liver-specific deletion of acetyl-CoA carboxylase 1 reduces hepatic triglyceride accumulation without affecting glucose homeostasis, Proc Natl Acad Sci ,103(22): 8552-8557.
11.Abu-Elheiga L, Jayakumar A, Baldini A, et al. Human acetyl-CoA carboxylase: characterization, molecular cloning, and evidence for two isoforms. Proc Natl Acad Sci. 1995,92(9):4011-5;
12. Abu EL, Almarza DB, Baldini A, et al, Human acetyl-CoA carboxylase 2. Molecular cloning, characterization, chromosomal mapping, and evidence for two isoforms. J Biol Chem, 272(16): 10669-77;
13. Barber MC, Price NT, Travers MT. Structure and regulation of acetyl-CoA carboxylase genes of metazoa. Biochim Biophys Acta. 2005,1733(1):1-28;
14. Smith S, Witkowski A, Joshi AK. Structural and functional organization of the animal fatty acid synthase. Prog Lipid Res. 2003,42(4):289-317;
15. Loftus, T. M., D. E. Jaworsky, et al.. Reduced food intake and body weight in mice treated with fatty acid synthase inhibitors. Science, 2000, 288(5475): 2379-81;
16. Abumrad NA, El-Maghrabi MR, Amri EZ, et al. Cloning of a rat adipocyte membrane protein implicated in binding or transport of long-chain fatty acids that is induced during preadipocyte differentiation. J Biol Chem, 1993,268,17665-8;
17. Schaffer JE, Lodish HF. Expression cloning and characterization of a novel adipocytes long chain fatty acid transport protein. Cell, 1994, 79,427-36;
18. Isola LM, Zhou SL, Kiang CL, et al. 3T3 fibroblasts transfected with a cDNA for mitochondrial aspartate aminotransferase express plasma membrane fatty acid-binding protein and saturable fatty acid uptake. Proc Natl Acad Sci, 1995, 92, 9866-70;
19. Hermann T, Buchkremer F, Gosch I, et al. Mouse fatty acid transpoter protein 4 (FATP4): characterization of the gene and functional assessment as a very long chain acyl-CoA synthetase. Gene, 2001, 270, 31-40;
20. Bonen A, Luiken JJFP, Arumugam Y, et al. Acute regulation of fatty acid uptake involves the cellular redistribution of fatty acid translocase. J Biol Chem, 2000, 275, 14501-8;
21. Dyck DJ, Steinberg G, Bonen A. Insulin increase FFA uptake and esterification but reduces lipid utilization in isolated contracting muscle. Am J Physiol Endocrinol Metab, 2001,281, E600-7;
22. Weisiger RA. Cytosolic fatty acid binding proteins catalyze two distinct steps in intracellular transport of their ligands. Mol Cel Biochem, 2002,239, 35-42;
23. Franckhauser S, Munoz S, Pujol A, et al. Increased fatty acid re-esterification by PEPCK overexpression in adipose tissue leads to obesity without insulin resistance. Diabetes, 2002, 51, 624-30;
24. Kragelund BB, Knudsen J, Poulsen FM. Acyl-coenzymeA binding protein (ACBP). Biochim Biophys Acta, 1999,1441,150-61;
25. Agarwal AK, Garg A. Congenital generalized lipodystrophy: significance of triglyceride biosynthetic pathways. Trends in Endocrinology and Metabolism, 2003, 14, 214-21;
26. Hammond LE, Gallagher PA, Wang S, et al. Mitochondrial glycerol-3-phosphate acyltransferase-deficient mice have reduced weight and liver triacylglycerol content and altered glycerolipid fatty acid composition. Mol Cell Biol, 2002,22, 8204-14;
27. James G, Hsiao-Ping H. Moore, Location, location: protein trafficking and lipolysis in adipocytes, TRENDS in Endocrinology and Metabolism, 2007,19(1), 3-9;
28 Dominique Langin, Adipose tissue lipolysis as a metabolic pathway to define pharmacological strategies against obesity and the metabolic syndrome, Pharmacological Research, 2006, 53: 482-491;
29.C.Holm, Molecular mechanisms regulating hormone-sensitive lipase and lipolysis, Biochemical Society Transactions, 2003, 31( 6), 1120— 1124;
30. Lafontan M, Berlan M. Fat cell adrenergic receptors and the control of white and brown fat cell function. J Lip Res, 1993, 34, 1057-91.
31 . Degerman E, Belfrage P, Manganiello VC. Structure and regulation of cGMP-inhibited phosphodiesterase (PDE3). J Biol Chem, 1997, 272, 6823-6.
32. Kishida K, Shimomura I, Kondo H, et al. Genomic structure and insulin-mediated repression of the aquaporin adipose (Aqap), adiposespecific glycerol channel. J Biol Chem, 2001, 276(39), 36251-60;
33. Kondo H, Shimomura I, Kishida K, et al. Human aquaporin adipose (AQPap) gene. Eur J Biochem, 2002, 269, 1814-26.
34. Abumrad N, Harmon C, Ibrahimi A. Membrane transport of longchain fatty acids: evidence for a falicitated process. J lipid Res, 1998, 39, 2309-18;
35. Kraemer FB , Shen WJ, Hormone sensitive lipase : control of intracellular tri (di) acylglycerol and cholesteryl ester hydrolysis, J Lipid Res, 2002 ,43:1585—1594;
36. Shen WJ , Liang Y, Hong R , et al, Characterization of the functional interaction of adipocyte lipid binding protein with hormone sensitive lipase, J Biol Chem , 2001, 276:49443-49448
37. Anthonsen MW, Ronnstrand L, Wernstedt C ,et al, Indentification of Novel Phosphorylation Sites in Hormone sensitive Lipase That Are Phosphorylated in Response to Isoproterenol and Govern Activation Properties in Vitro, J Biol Chem ,1998 ,273:215-211;
38. Greenberg AS , Shen WJ , Muliro K, et all Stimulation of lipolysis and hormone sensitive lipase via t he extracellular signal regulated kinase pathwayl J Biol Chem , 2001,276:45456-45461;
39. Osuga J, Ishibashi S, Oka T, et al. Targeted disruption of hormone sensitive lipase results in male sterility and adipocyte hypertrophy but not in obesity. Proc Natl Acad Sci, 2000, 97,787-92;
40. John.T, Carole. S,, Erica. H, et al, The Central Role of Perilipin A in Lipid Metabolism and Adipocyte Lipolysis , IUBMB Life, 2004, 56(7), 379 - 385;
41. Brasaemle DL, Rubin B, Harten IA, et al. Perilipin A increases triacylglycerol storage by decreasing the rate of triacylglycerol hydrolysis. J Biol Chem, 2000, 275, 38486-93;
42.Hideaki M, Sandra S, Hui-Hong Z, et al, Perilipin Promotes Hormone-sensitive Lipase-mediated Adipocyte Lipolysis via Phosphorylation-dependent and independent Mechanisms, J Biol Chem, 2006, 281(23),15837-15844;
43. Marcus C, Ehreen H, Bolme P, et al. Regulation of lipolysis during the neonatal period. J Clin Invest, 1988, 82,1793-7;
44. Carlson MG, Snead WL, Campbell PJ. Regulation of free fatty acid metabolism by glucagon. J Clin Endocrinol Metab, 1993,77,11-5;
45. Hellstrom L, Wahrenberg H, Reynisdottir S, et al. catecholamines induced lipolysis in human hyperthyroidism. J Clin Endocrinol Metab, 1997, 82,159-66;
46. Marcus C, Bolme P, Micha-Johansson G, et al. Growth hormone increases the lipolytic sensitivity for cathecholamines in adipocytes from healthy adults. Life sciences, 1994,54, 1335-41;
47. V Large, O Peroni, D Letexier, et al, Metabolism of lipids in human white adipocyte, Diabetes Metab 2004, 30, 294-309;
48. Botion LM, Green A. Long-term regulation of lipolysis and hormone sensitive lipase by insulin and glucose. Diabetes, 1999, 48, 1691-7;
49. Engfeldt P, Hellmer J, Wahrenberg H, et al. Effects of insulin on adrenoceptor binding and the role of catecholamine-induced lipolysis in isolated human fat cells. J Biol Chem, 1988,263,15553-60;
50. Boulware SD, Tamborlane WV, Matthews LS, et al. Diverse effects of insulin-like growth factor I on glucose, lipid, and amino acid metabolism. Am J Physiol, 1992, 262, E130-3;
51. Lonnroth P, Jansson PA, Fredholm BB, et al. Microdialysis of intracellular adenosine concentration in subcutaneous tissue in humans. Am J Physiol, 1989,256, E250-5.
52. Strouch MB, Jackson EK, Mi Z, et al. Extracellular cyclic AMP-adenosine pathway in isolated adipocytes and adipose Tissue, Obes Res. 2005 ,13 (6): 974-81
2.Wyatt SB,Winters KP,Dubbert PM.Overweight and obesity:prevalence,consequences,and causes of a growing public health problem.Am J Med Sci.2006,331(4):166-174.
3.武阳丰,马冠生,胡永华,等.中国居民的超重和肥胖流行现状。中华预防医学杂志 2005,39(5):316-320.
4.董秋婷,顼志敏,代谢综合征防治进展,中华老年心脑血管病杂志,2008,10(1):66-68.
5.Halford JC.Obesity drugs in clinical development.Curr Opin Investig Drugs.2006,7(4):312-318.
6.施峰,刘焱文,红花的化学成分及药理研究进展,时珍国医国药,2006,17(9):1666-1667.
7.祝美珍,胡国恒,肖健,羟基红花黄色素A的应用研究进展,中西医结合心脑血管病杂志,2007,(11):1108-1110.
8.He,H.,X.Yang,et al.Protective effects of hydroxysafflor yellow A on acute and chronic congestive cardiac failure mediated by reducing ET-1,NOS and oxidative stress in rats.J Pharm Pharmacol,2008,60(1):115-23.
9.Liu,Y.N.,Z.M.Zhou,et al.Evidence that hydroxysafflor yellow A protects the heart against ischaemia-reperfusion injury by inhibiting mitochondrial permeability transition pore opening.Clin Exp Pharmacol Physiol,2008,35(2):211-6.
10.Tian,J.W.,F.H.Fu,et al,Protective effect of hydroxysafflor yellow A against rat cortex mitochondrial injuries induced by cerebral ischemia,Yao Xue Xue Bao,2004,39(10):774-7.
11.Wei,X.,H.Liu,et al.Hydroxysafflor yellow A protects rat brains against ischemia-reperfusion injury by antioxidant action,Neurosci Lett,2005,386(1):58-62.
12.Zhu,H.B.,Z.H.Wang,et al.Protective effect of hydroxysafflor yellow A on experimental cerebral ischemia in rats,Yao Xue Xue Bao,2005 40(12):1144-6.
13.Zhu,H.B.,L.Zhang,et al.Therapeutic effects of hydroxysafflor yellow A on focal cerebral ischemic injury in rats and its primary mechanisms.J Asian Nat Prod Res,2005,7(4):607-13.
14.徐剑.以过氧化物酶体增殖物受体γ为靶点的天然减肥药物的初步筛选[D].中 国协和医科大学:,2006。
15.Rosen,E.D.and O.A.MacDougald.Adipocyte differentiation from the inside out,Nat Rev Mol Cell Biol 2006,7(12):885-96.
16.Guan HP,Li Y,Jensen M,et al.A futile metabolic cycle activated in adipocytes by antidiabetic agents.Nat Med 2002,8:1122-1128
17.Barroso I,Gumell M,Crowley VEF,et al.Dominant negative mutations in human PPARγ associated with severe insulin resistance,diabetes mellitus,and hypertension.Nature 1999;402:880-883
18.Wolf G.Insulin resistance and obesity:resistin,a hormone secreted by adipose tissue.Nutr Rev.2004;62(10):389-94
19.Lin Y,Rajala M,Berger J,et al.Hyperglycemia-induced production of acute phase reactants.J Biol Chem 2001;276:42077-42083
20.Smith S,Witkowski A,Joshi AK.Structural and functional organization of the animal fatty acid synthase.Prog Lipid Res.2003,42(4):289-317;
21.Loftus,T.M.,D.E.Jaworsky,et al..Reduced food intake and body weight in mice treated with fatty acid synthase inhibitors.Science,2000,288(5475):2379-81;
22.Large V,Reynisdottir S,Langin D,et al.Decreased expression and function of adipocyte hormone-sensitive lipase in subcutaneous fat cells of obese subjects.J Lipid Res,1999,40:2059-2065
23.Roduit R,Masiello P,Wang SP,et al.A role for hormone-sensitive lipase in glucose stimulated insulin secretion.Diabetes.2001,50:1970-1975
24.Mular H,Holst LS,Svensson H,et al.Hormone-sensitive lipase,the rate-limiting enzyme in triglyceride hydrolysis,is expressed and actived in β-cells.Diabetes,1999,48:228-232.
25.Green H,Kehinde O.Sublines of mouse 3T3 cells that accumulate lipid.Cell 1974;1(1):113-6.
26.Green H,Meuth M.An established pre-adipose cell line and its differentiation in culture.Cell 1974;3(2):127-33.
27.Green H,Kehinde O.An established preadipose cell line and its differentiation in culture.Ⅱ.Factors affecting the adipose conversion.Cell 1975;5(1):19-27.
28.Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.Methods(San Diego,Calif 2001;25(4):402-8.
29.张前,牛欣,闫妍等.羟基红花黄色素A对体外培养人脐静脉内皮细胞增殖的抑制作用.中国医药学报,2004,19(6):379-381.
30.张前,赵言群,解华,牛欣,.中药对肿瘤血管生成的影响.中华中医药杂志,2006,21(4):242-245.
31.张前.羟基红花黄色素A对血管内皮细胞调控机理的研究[D].北京中医药大学:,2005.
32.张前,牛欣,闰妍,金鸣,杨向竹,李金荣.羟基红花黄色素A抑制新生血管形成的机制研究.北京中医药大学学报,2004,27(3):25-29.
33.SONG Yan,ZHANG Ling,QU Kai,et al,Hydroxysafflor Yellow A Promotes Vascular Endothelial Cell Proliferation via VEGF/VEGF Receptor,Journal of Chinese Pharmaceutical Sciences 2005,14(3):181-185.
34.吉登波,李长龄,朱美财等,从红花中提取的单体化合物-羟基红花黄色素A通过上调HIF-1α-VEGF信号通路和调控Bcl-2/Bax表达比率促进低氧状态下人血管内皮细胞存活的研究,第九届全国心血管药理学术会议论文集,14-15.
35.陈粉粉.EGCG对猪前体脂肪细胞增殖分化和脂解的影响[D].西北农林科技大学:,2007.
36.李宗孝,温普红,袁美娟,肉桂中查耳酮的类似胰岛素作用,中医药学报,2004,32(5):29-31.
37.莫尚武,陈恒留,刘宁,金建南,张淑渊,李文学,陈治恒,刘开明.用～(45)Ca研究红花对大鼠胸主动脉Ca-(2+)内流的影响,中草药,1995,(10):532-534.
38.王玢,郭锡熔,陈荣华,钙-肥胖的重要调节者,医学综述,2005,11(4):300-303.
39.Hsu,C.L.and G.C.Yen,Effects of flavonoids and phenolic acids on the inhibition of adipogenesis in 3T3-L1 adipocytes.J Agric Food Chem,2007,55(21):8404-10.
40.刘新迎,周联,梁瑞燕,柚皮苷对前脂肪细胞3T3-L1增殖和诱导分化的影响,中药新药与临床药理,2007,18(3):176-179.
41.金鸣,李金荣,吴伟,羟基红花黄色素A抗氧化作用的研究,中草药,2004,35(6):665-666.
1.Peter Arner,Adipose Tissue as an Endocrine Organ,Best Practice & Research Clinical Endocrinology & Metabolism,2005,19,471-482;
2.Diraison F,Beylot M.Role of human liver lipogenesis and reesterification in triglycerides secretion and in FFA reesterification.Am J Physiol,1998,274,E321-7;
3.Aarsland A,Chinkes D,Wolfe R.Hepatic and whole body fat synthesis in humans during carbohydrate overfeeding.Am J Clin Nutr,1997,65,1174-82;
4.Letexier D,Pinteur C,Large V,Fr(?)ring V and Beylot M.Comparison of the expression and activity of the lipogenic pathway in human and rat adipose tissue.J Lipid Res,2003,44,2127-34;
5.Diraison F.,V.Yankah,D.Letexier,E.Dusserre,P.Jones,and M.Beylot.Differences in the regulation of adipose tissue and liver lipogenesis by carbohydrates in humans.J.Lipid Res.44:846-853;
6.Kersten S.Mechanisms of nutritional and hormonal regulation of lipogenesis.EMBO reports 2001,2(4),282-6;
7. Mead JR, Irvine SA, Ramji D. Lipoprotein lipase: structure, function, regulation and role in disease. J Mol Med, 2002, 80, 753-69;
8. Tacken PJ, Hofker MH, Havekes L, et al. Living up to a name: the role of the VLDL receptor in lipid metabolism. Cur Opin Lipidol, 2001,12,275-9.
9. Goudriaan JR, Tacken PJ, Dahlmans VE, et al. Protection from obesity in mice lacking the VLDL receptor. Arterioscler Thromb Vasc Biol, 2001 21,1488-93.
10. Jianqiang M, Francesco JD, Huiguang L, et al. Liver-specific deletion of acetyl-CoA carboxylase 1 reduces hepatic triglyceride accumulation without affecting glucose homeostasis, Proc Natl Acad Sci ,103(22): 8552-8557.
11.Abu-Elheiga L, Jayakumar A, Baldini A, et al. Human acetyl-CoA carboxylase: characterization, molecular cloning, and evidence for two isoforms. Proc Natl Acad Sci. 1995,92(9):4011-5;
12. Abu EL, Almarza DB, Baldini A, et al, Human acetyl-CoA carboxylase 2. Molecular cloning, characterization, chromosomal mapping, and evidence for two isoforms. J Biol Chem, 272(16): 10669-77;
13. Barber MC, Price NT, Travers MT. Structure and regulation of acetyl-CoA carboxylase genes of metazoa. Biochim Biophys Acta. 2005,1733(1):1-28;
14. Smith S, Witkowski A, Joshi AK. Structural and functional organization of the animal fatty acid synthase. Prog Lipid Res. 2003,42(4):289-317;
15. Loftus, T. M., D. E. Jaworsky, et al.. Reduced food intake and body weight in mice treated with fatty acid synthase inhibitors. Science, 2000, 288(5475): 2379-81;
16. Abumrad NA, El-Maghrabi MR, Amri EZ, et al. Cloning of a rat adipocyte membrane protein implicated in binding or transport of long-chain fatty acids that is induced during preadipocyte differentiation. J Biol Chem, 1993,268,17665-8;
17. Schaffer JE, Lodish HF. Expression cloning and characterization of a novel adipocytes long chain fatty acid transport protein. Cell, 1994, 79,427-36;
18. Isola LM, Zhou SL, Kiang CL, et al. 3T3 fibroblasts transfected with a cDNA for mitochondrial aspartate aminotransferase express plasma membrane fatty acid-binding protein and saturable fatty acid uptake. Proc Natl Acad Sci, 1995, 92, 9866-70;
19. Hermann T, Buchkremer F, Gosch I, et al. Mouse fatty acid transpoter protein 4 (FATP4): characterization of the gene and functional assessment as a very long chain acyl-CoA synthetase. Gene, 2001, 270, 31-40;
20. Bonen A, Luiken JJFP, Arumugam Y, et al. Acute regulation of fatty acid uptake involves the cellular redistribution of fatty acid translocase. J Biol Chem, 2000, 275, 14501-8;
21. Dyck DJ, Steinberg G, Bonen A. Insulin increase FFA uptake and esterification but reduces lipid utilization in isolated contracting muscle. Am J Physiol Endocrinol Metab, 2001,281, E600-7;
22. Weisiger RA. Cytosolic fatty acid binding proteins catalyze two distinct steps in intracellular transport of their ligands. Mol Cel Biochem, 2002,239, 35-42;
23. Franckhauser S, Munoz S, Pujol A, et al. Increased fatty acid re-esterification by PEPCK overexpression in adipose tissue leads to obesity without insulin resistance. Diabetes, 2002, 51, 624-30;
24. Kragelund BB, Knudsen J, Poulsen FM. Acyl-coenzymeA binding protein (ACBP). Biochim Biophys Acta, 1999,1441,150-61;
25. Agarwal AK, Garg A. Congenital generalized lipodystrophy: significance of triglyceride biosynthetic pathways. Trends in Endocrinology and Metabolism, 2003, 14, 214-21;
26. Hammond LE, Gallagher PA, Wang S, et al. Mitochondrial glycerol-3-phosphate acyltransferase-deficient mice have reduced weight and liver triacylglycerol content and altered glycerolipid fatty acid composition. Mol Cell Biol, 2002,22, 8204-14;
27. James G, Hsiao-Ping H. Moore, Location, location: protein trafficking and lipolysis in adipocytes, TRENDS in Endocrinology and Metabolism, 2007,19(1), 3-9;
28 Dominique Langin, Adipose tissue lipolysis as a metabolic pathway to define pharmacological strategies against obesity and the metabolic syndrome, Pharmacological Research, 2006, 53: 482-491;
29.C.Holm, Molecular mechanisms regulating hormone-sensitive lipase and lipolysis, Biochemical Society Transactions, 2003, 31( 6), 1120— 1124;
30. Lafontan M, Berlan M. Fat cell adrenergic receptors and the control of white and brown fat cell function. J Lip Res, 1993, 34, 1057-91.
31 . Degerman E, Belfrage P, Manganiello VC. Structure and regulation of cGMP-inhibited phosphodiesterase (PDE3). J Biol Chem, 1997, 272, 6823-6.
32. Kishida K, Shimomura I, Kondo H, et al. Genomic structure and insulin-mediated repression of the aquaporin adipose (Aqap), adiposespecific glycerol channel. J Biol Chem, 2001, 276(39), 36251-60;
33. Kondo H, Shimomura I, Kishida K, et al. Human aquaporin adipose (AQPap) gene. Eur J Biochem, 2002, 269, 1814-26.
34. Abumrad N, Harmon C, Ibrahimi A. Membrane transport of longchain fatty acids: evidence for a falicitated process. J lipid Res, 1998, 39, 2309-18;
35. Kraemer FB , Shen WJ, Hormone sensitive lipase : control of intracellular tri (di) acylglycerol and cholesteryl ester hydrolysis, J Lipid Res, 2002 ,43:1585—1594;
36. Shen WJ , Liang Y, Hong R , et al, Characterization of the functional interaction of adipocyte lipid binding protein with hormone sensitive lipase, J Biol Chem , 2001, 276:49443-49448
37. Anthonsen MW, Ronnstrand L, Wernstedt C ,et al, Indentification of Novel Phosphorylation Sites in Hormone sensitive Lipase That Are Phosphorylated in Response to Isoproterenol and Govern Activation Properties in Vitro, J Biol Chem ,1998 ,273:215-211;
38. Greenberg AS , Shen WJ , Muliro K, et all Stimulation of lipolysis and hormone sensitive lipase via t he extracellular signal regulated kinase pathwayl J Biol Chem , 2001,276:45456-45461;
39. Osuga J, Ishibashi S, Oka T, et al. Targeted disruption of hormone sensitive lipase results in male sterility and adipocyte hypertrophy but not in obesity. Proc Natl Acad Sci, 2000, 97,787-92;
40. John.T, Carole. S,, Erica. H, et al, The Central Role of Perilipin A in Lipid Metabolism and Adipocyte Lipolysis , IUBMB Life, 2004, 56(7), 379 - 385;
41. Brasaemle DL, Rubin B, Harten IA, et al. Perilipin A increases triacylglycerol storage by decreasing the rate of triacylglycerol hydrolysis. J Biol Chem, 2000, 275, 38486-93;
42.Hideaki M, Sandra S, Hui-Hong Z, et al, Perilipin Promotes Hormone-sensitive Lipase-mediated Adipocyte Lipolysis via Phosphorylation-dependent and independent Mechanisms, J Biol Chem, 2006, 281(23),15837-15844;
43. Marcus C, Ehreen H, Bolme P, et al. Regulation of lipolysis during the neonatal period. J Clin Invest, 1988, 82,1793-7;
44. Carlson MG, Snead WL, Campbell PJ. Regulation of free fatty acid metabolism by glucagon. J Clin Endocrinol Metab, 1993,77,11-5;
45. Hellstrom L, Wahrenberg H, Reynisdottir S, et al. catecholamines induced lipolysis in human hyperthyroidism. J Clin Endocrinol Metab, 1997, 82,159-66;
46. Marcus C, Bolme P, Micha-Johansson G, et al. Growth hormone increases the lipolytic sensitivity for cathecholamines in adipocytes from healthy adults. Life sciences, 1994,54, 1335-41;
47. V Large, O Peroni, D Letexier, et al, Metabolism of lipids in human white adipocyte, Diabetes Metab 2004, 30, 294-309;
48. Botion LM, Green A. Long-term regulation of lipolysis and hormone sensitive lipase by insulin and glucose. Diabetes, 1999, 48, 1691-7;
49. Engfeldt P, Hellmer J, Wahrenberg H, et al. Effects of insulin on adrenoceptor binding and the role of catecholamine-induced lipolysis in isolated human fat cells. J Biol Chem, 1988,263,15553-60;
50. Boulware SD, Tamborlane WV, Matthews LS, et al. Diverse effects of insulin-like growth factor I on glucose, lipid, and amino acid metabolism. Am J Physiol, 1992, 262, E130-3;
51. Lonnroth P, Jansson PA, Fredholm BB, et al. Microdialysis of intracellular adenosine concentration in subcutaneous tissue in humans. Am J Physiol, 1989,256, E250-5.
52. Strouch MB, Jackson EK, Mi Z, et al. Extracellular cyclic AMP-adenosine pathway in isolated adipocytes and adipose Tissue, Obes Res. 2005 ,13 (6): 974-81