LEPTIN RECEPTOR AND THE OBESITY/DIABETES SYNDROME

本项目旨在确定瘦素受体在神经元和脂肪细胞中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。对人类和啮齿类动物瘦素的研究表明，瘦素的主要体重调节作用发生在下丘脑。由神经肽含量定义的特定神经元群，如NPY/AGRP和POMC/CART，是瘦素作用的重要介质。然而，瘦素对脂肪细胞有作用，如调节脂肪分解，这可能导致肥胖表型。本提案的目标是：A)评估仅在神经元、仅在脂肪细胞和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、仅在脂肪细胞和仅在神经元和脂肪细胞中消除瘦素受体表达的肥胖产生效应；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对肥胖和糖尿病的降低作用。对于A，带有细胞类型特异性启动子/增强子的转基因将用于驱动LeprB表达。这些转基因将与db-3J突变结合，db-3J突变是编码外显子12的17bp缺失，可阻止所有膜结合LEPR同种异构体的合成。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。胚胎干细胞的同源重组将用于引入编码外显子17的两个loxP位点。含有麻风基因柔化等位基因的被操纵的胚胎干细胞将被用于产生携带麻风基因柔化的小鼠。以组织特异性方式表达cre重组酶的转基因，仅在神经元或仅在脂肪细胞中表达，将与Lepr flox小鼠杂交，获得组织特异性缺失Lepr表达的动物。一种基因敲入方法(C)将被用于识别特定的神经元群，这些神经元群介导瘦素对肥胖和糖尿病的影响。通过在Pomc和Agrp基因的3'非翻译区插入IRES- LeprB/IRES- β -半乳糖苷酶cDNA盒（内部核糖体进入位点），产生三反子mRNA，可以在化学定义的神经元中表达LeprB。因此，可以在db-3J/db-3J小鼠中评估LeprB在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。这些实验将为AGRP/NPY神经元和POMC/CART神经元中瘦素受体信号在肥胖/糖尿病综合征中的作用提供数据。

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.