荧光素酶报告基因检测系统原理

1、The Central Melanocortin System Can Directly Regulate Serum Insulin LevelsReporter：Ziqi Zhang Author: Roger D. Cone et al.From: Endocrinology (2000)2015/6/22BackgroundMethodprocessConclusionContents BackgroundObesity HyperinsulinemiaInsulin resistanceType 2 diabetesThe mechanistic relationship betwe

2、en obesity, hyperinsulinemia, insulin resistance, and diabetes is not entirely clear. hyperphagia, hyperinsulinemic, hypoactivity, and obesity hyperphagia, severe obesity, hyperinsulinemia, late-onset hyperglycemia, hyperphagia and obesity and insulin resistance obesity, insulin resistance, and dysl

3、ipidemia.Avy Mice ectopically expressing agoutiMice overexpressing the hypothalamic AGRPMice lacking POMC derived peptideMice lacking MC4RBackgroundMouse modelsHyperinsulinemia is one of common features among all the animal models of obesity, and also one of the earlist metabolic alterations observe

4、d in models.The development is similar to the process of type 2 diabetesThe Central Melanocortin system Energy intake Energy expenditureGlucose homeostasisBackgroundMC4RMC3RHypothesis: the central melanocortin system may be involved in the regulation of glucose homeostasisGlucose homeostasisBackgrou

5、nd外周组织对胰岛素的敏感性中央黑皮质素信号传中央黑皮质素信号传导缺陷（未知导缺陷（未知）肥胖组织增生干扰胰岛素作用（有报道）Background其他未知瘦素缺陷瘦素缺陷（有报道）胰岛素敏感性下降血糖升高血糖升高胰岛补偿性分泌胰岛素增加血糖失衡血液胰岛素增加高胰岛素血症高胰岛素血症胰岛素抗性胰岛素抗性血糖下降暂时性血糖平衡糖尿病糖尿病Objective 1. Central melanocortin receptor signaling may play a role in the control of glucose homeostasis.2.A hypothesis: the melan

6、ocortin agonist lowers serum insulin by stimulating the sympathetic drive to the pancreas known to inhibit insulin release.3. Whether the hyperinsulinemia in the melanocortin obesity syndromes is primary to MC4R blockade, a development consequence of MC4R blockade, or secondary to te hyperphagia and

7、 obesity.Method Surgical procedureRIA assay for serum or plasma insulinBlood glucose and glucose tolerance testNonesterified fatty acid assayInsulin tolerance testProcess 1 Ob/ob mouse Icv injection of MT (0.3、1、3nmol) 、ACSF、LeptinAfter 30-60min, RIA assay for serum insulin At 60 min after 1nmol MT,

8、 the blood glucose testThe effect of melanocortin adminstration on serum insulinMTII 是MC4R/MC3R 的激动剂，是-MSH的类似物。ACSF人工脑脊液Ob/ob 老鼠是瘦素缺陷老鼠，具有高胰岛素血症。Results Fig 1. 在ob/ob老鼠中，注射黑皮质素激动剂对血清胰岛素的影响。 72.8%30-60minConclusion 1.A中与两个对照相比，MTII 降低血清中的胰岛素水平，并具有剂量反应2.B中注射MTII后一小时检测到血糖浓度有明显增加。a.中央黑皮质素受体可能参与在血糖调节过程。b

9、.MTII对胰岛素分泌的抑制不是由于降低血糖浓度而间接起作用的。Process 2 Ob/ob mouse Icv injection of MT、ACSF、Leptin RIA assay for serum insulin to test the hypothesis Ip injection with phentolamine or salinePhentolamine :芬妥胺，非特异性-肾上腺素受体的反向激动剂，对交感神经对胰岛素分泌的抑制有阻断作用。Conclusion Fig 2. MTII注射后降低血清胰岛素水平是通过刺激交感神经而起作用的。S: salineP: phento

10、lamineA: ACSFM: MTL: LeptinConclusion 1.芬妥胺对胰岛素分泌有促进作用，与文献报道效果相同芬妥胺能阻断交感神经对胰岛素分泌的抑制作用。2.芬妥胺对MTII在胰岛素分泌上的抑制作用具有完全阻断效果。3.芬妥胺对瘦素在胰岛素分泌上的抑制作用具有部分阻断效果。a.MTII对胰岛素分泌的抑制效果可能只通过一种方式起作用，通过刺激交感神经驱使胰岛抑制胰岛素的分泌。b.瘦素除了通过交感神经抑制胰岛分泌外，可能还有其他方式。Process 3 C57BL/6J mouse Icv injection of MT(1nmol、3nmol)、ACSF RIA assay f

11、or plasma insulin At 60 min after 3nmol MT, the blood glucose testThe effect of melanocortin adminstration on plasma insulin and glucose tolerancePVH injection of MT（0.45nmol/0.3ul ）,ip injection glucose,test blood glucose levelResults Fig 3.在瘦的C57BL/6j小鼠中MTII对胰岛素的分泌和葡萄糖耐性的影响。Conclusion 1.MTII对正常小鼠的

12、基础胰岛素水平也有剂量依赖的抑制作用2.在注射MTII一小时后血糖浓度同样有显著的升高3. 在PVH注射MTII后进行葡萄糖耐受测试，结果表明与对照相比，注射MTII血糖浓度波动较大，葡萄糖耐性降低a.进一步表明中央黑皮质素受体受激动剂刺激后，可能参与血糖平衡调节，降低胰岛素水平，葡萄糖耐性降低。Process 4 Lean MC4R KO mouseThe basal insulin level and insulin tolerance test, measure body weightTo determine the change on plasma insulin, body weig

13、ht, insulin tolerance when MC4R defectResults Fig 4 在瘦小的MC4R敲除小鼠中血浆胰岛素水平增加，外周胰岛素抗性降低。Age: 4 weeksmaleAge: 6-7 weeksmaleResults Fig 4femalemale6-7 weeksResults Fig 48-9 weeks malemaleConclusion a. 在MC4R敲除小鼠生长过程中，首先发现的是胰岛素水平上升，而不是体重增加。b. MC4R缺陷小鼠对胰岛素的耐性下降，出现胰岛素抗性，在体重开始发生差异时，胰岛素抗性更强。2. C、D组结果表明，无论雌雄，皮下

14、注射胰岛素后，6-7周大的MC4R缺陷小鼠与野生型相比，血糖降低幅度较小。3. 而8-9周大的MC4R敲除小鼠与野生型相比，在注射胰岛素后，血糖下降幅度更缓和。而且此时MC4R敲除小鼠体重开始表现出略大于野生型小鼠。1. MC4R KO小鼠四周大时测量其血浆胰岛素水平，与野生型相比，其胰岛素水平显著增加。而在6-7周测其体重变化，发现基本与野生型一样重。Process 5 6-7weeks MC4R KO mouseNonesterified fatty acid assayTo determine if the MC4R KO mice has a defect in trigyceride

15、 metabolismglucose tolerance testFig 5Results 6-7 weeks female9-10 weeks male6-7 weeks female6-7 weeks maleConclusion 1. 无论6-7周大雌性还是8-9周大的雄性MC4R敲除小鼠与野生型在葡萄糖耐性上没有明显区别。2.无论雌雄，6-7周大的MC4R敲除小鼠与对照相比在血清NEFA游离脂肪酸水平上没有明显区别。a.此时，MC4R KO 小鼠甘油三酯代谢正常b.MC4R KO小鼠的胰岛素抗性并非是由于非酯化脂肪酸的增加诱导产生的。c.葡萄糖耐性正常可能是因为胰岛素补偿性增加弥补了胰岛素抗性作用。In general 1.中央黑皮质素受体信号传导可能对胰岛素的分泌有强的抑制作用，而且该系统可能独立参与血糖平衡控制过程。2. 中央黑皮质素肽可能作用于黑皮质素受体去增加交感神经兴奋，作用于胰岛，以发挥对胰岛素分泌的抑制作用