LEPTIN RECEPTOR AND THE OBESITY/DIABETES SYNDROME

本项目旨在确定神经元和脂肪细胞中瘦素受体的表达对能量平衡的控制作用。瘦素受体(Lepr)的缺乏会导致肥胖和糖尿病综合征。对人类和啮齿动物的瘦素研究表明，瘦素的主要体重调节作用发生在下丘脑。特定的神经细胞群，如NPY/AGRP和POMC/CART的神经肽含量，是瘦素作用的重要中介。然而，瘦素对脂肪细胞有作用，如调节脂肪分解，这可能与肥胖表型有关。这项建议的目标是：A)评估仅在神经元、仅在脂肪细胞、在神经元和脂肪细胞中表达LeprB的减肥效果；B)评估仅在神经元、仅在脂肪细胞、在神经元和脂肪细胞中取消瘦素受体表达的肥胖产生效果；C)确定在POMC/CART神经元、AGRP/NPY神经元、POMC/CART神经元和AGRP/NPY神经元中表达LeprB的减肥和糖尿病效果。对于A，带有细胞类型特异性启动子/增强子的转基因将被用来驱动LeprB的表达。转基因将与db-3J突变结合在一起，db-3J突变是编码外显子12的17bp缺失，阻止了所有膜结合的LEPR亚型的合成。对于B，将使用cre-lox系统获得LepR编码外显子17的组织特异性批次缺失。胚胎干细胞中的同源重组将被用来在编码外显子17的两侧引入两个loxP位点。含有Lepr等位基因的操纵ES细胞将被用来产生携带Lepr-Flox的小鼠。以组织特异性方式表达cre重组酶的转基因，仅限神经元或仅脂肪细胞，将与Lepr Flox小鼠繁殖，以获得组织特异性缺失Lepr表达的动物。基因敲入方法(C)将被用来识别介导瘦素对肥胖和糖尿病影响的特定神经元群体。LeprB在化学定义的神经元中的表达将通过在POMC和AgRP基因的3‘非翻译区插入IRES-LeprB/IRES-β-Galactosidase cDNA盒(内部核糖体进入位点)，产生一个三顺反子mRNA来实现。因此，可以评价LeprB在db-3J/db-3J小鼠AGRP/NPY神经元和/或POMC/CART神经元中的表达对肥胖/糖尿病表型的影响。这些实验将提供有关瘦素受体信号在AGRP/NPY神经元和POMC/CART神经元在肥胖/糖尿病综合征中的作用的数据。

This project is designed to determine the role of leptin receptor expression in neurons and adipocytes to the control of energy balance. The lack of leptin receptor (Lepr) results in a syndrome of obesity and diabetes. Investigations with leptin in humans and rodents suggest that the major weight-regulating action of leptin occurs in the hypothalamus. Specific neuronal populations, defined by their neuropeptide content such as NPY/AGRP and POMC/CART, are important mediators of leptin action. However, leptin has actions on adipocytes, such as the regulation of lipolysis, that may contribute to the obese phenotype. The goals of this proposal are: A) Evaluate the obesity-reducing effects of the expressing LeprB in neurons only, in adipocytes only, and in neurons and adipocytes; B) Evaluate the obesity-producing effects of abrogating leptin receptor expression in neurons only, in adipocytes only, and in neurons and adipocytes; C) Determine the obesity- and diabetes-reducing effects of expressing LeprB in POMC/CART neurons, in AGRP/NPY neurons, and in POMC/CART neurons and AGRP/NPY neurons. For A, transgenes with cell type-specific promoter/enhancers will be used to drive LeprB expression. The transgenes will be combined with the db-3J mutation, a 17bp deletion of coding exon 12 that prevents the synthesis of all membrane-bound LEPR isoforms. For B, the cre-lox system will be used to obtain tissue-specific lot deletion of coding exon 17 of Lepr. Homologous recombination in embryonal stem cells will be used to introduce two loxP sites that flank coding exon 17. The manipulated ES cells containing the floxed Lepr allele will be used to generate mice that carry Lepr-flox. Transgenes that express cre recombinase in a tissue- specific manner, neurons only or adipocytes only, will be bred with Lepr flox mice to obtain animals with tissue-specific deletion of Lepr expression. A gene knock-in method (C) will be used to identify specific neuronal populations that mediate the effects of leptin on obesity and diabetes. Expression of LeprB in chemically defined neurons will be achieved by the insertion of an IRES- LeprB/IRES-beta-Galactosidase cDNA cassette (internal ribosomal entry site) in the 3' untranslated regions of the Pomc and Agrp genes, generating a tricistronic mRNA. Thus, the effect of expression of LeprB in AGRP/NPY neurons and/or POMC/CART neurons on the obese/diabetic phenotype can be evaluated in db-3J/db-3J mice. These experiments will provide data regarding the role of leptin receptor signaling in AGRP/NPY neurons and POMC/CART neurons to the obesity/diabetes syndrome.