Hypothalamic glucokinase in obesity and diabetes

下丘脑神经元可以感受葡萄糖浓度的变化并作出反应，但功能性神经元的功能性变化不明显。 这种葡萄糖感测特性的重要性仍有待确定。在胰腺β细胞中， 葡萄糖激酶（GK）构成下丘脑葡萄糖敏感机制的关键组分。纯合 GK基因的全身消融产生致命的新生儿糖尿病，杂合子全身消融 GK基因的突变导致葡萄糖稳态受损和肥胖表型，包括POMC减少和 下丘脑神经元AgRP基因表达增加。有趣的是，纯合子全身消融 胰岛素受体也会产生致命的新生儿糖尿病， 受体部分挽救了这种表型。我们现在已经证明了杂合子和纯合子 特异性地在神经元中消除GK基因重现了相应的全身GK敲除 表型另一方面，其他研究表明，下丘脑伸长细胞（一种类型的 神经胶质细胞）也在代谢稳态中起作用。因此，我们建议，代谢的不同方面， 稳态依赖于POMC神经元、AgRP神经元或伸长细胞中GK的表达。解决 基于这一假设，在具体目标1中，我们提出通过交叉 在POMC启动子控制下表达cre-重组酶的转基因小鼠（POMC-cre）， GK基因的侧翼是lox位点。为了补充这些研究，我们建议恢复GK 特别是在（纯合或杂合）全身GK敲除小鼠的POMC神经元中，使用 Okamoto等人通过恢复胰岛素受体挽救致命的新生儿糖尿病的“敲入”策略 特别是对全身胰岛素受体敲除小鼠的神经元。小鼠将维持在低脂肪或高脂肪的环境中。 高脂肪饮食。代谢表型，包括瘦素敏感性、葡萄糖和胰岛素耐量试验、食物摄入， 将评估体重、肥胖、代谢率和体温。小鼠将被处死，基因 将在下丘脑和其它组织中测定表达。我们建议在《特定目标》中进行类似的研究 图2和图3中所示的方法，使用AgRP或GFAP cre重组酶（在伸长细胞中表达）特异性消融或恢复GK， 在AgRP神经元中，或在tanyctes中消融GK。我们预期，在特定的下丘脑细胞中， 类型将部分重演GK全身消融产生的特定代谢损伤， 将GK恢复为POMC或AgRP神经元将逆转由整体- 体GK缺乏症

Hypothalamic neurons can sense and respond to changes in glucose concentration, but the functional significance of this glucose-sensing property remains to be determined. As in pancreatic beta cells, glucokinase (GK) constitutes a key component of the hypothalamic glucose-sensing mechanism. Homozygous whole-body ablation of the GK gene produces lethal neonatal diabetes, and heterozygous whole-body ablation of the GK gene causes impaired glucose homeostasis and obese phenotypes, including reduced POMC and increased AgRP gene expression in hypothalamic neurons. Interestingly, homozygous whole-body ablation of the insulin receptor also produces lethal neonatal diabetes, and neuron-specific expression of the insulin receptor partially rescues this phenotype. We have now demonstrated that heterozygous and homozygous ablation of the GK gene specifically in neurons recapitulates the respective whole-body GK knockout phenotypes. On the other hand, other studies have suggested that GK in hypothalamic tanycytes (a type of glial cell) also plays a role in metabolic homeostasis. We therefore propose that different aspects of metabolic homeostasis are dependent on GK expression in POMC neurons, AgRP neurons, or tanycytes. To address this hypothesis, in Specific Aim 1 we propose to ablate GK specifically from POMC neurons by crossing transgenic mice expressing cre-recombinase under control of the POMC promoter (POMC-cre) with mice in which the GK gene is flanked by lox sites. Complementing these studies, we propose to restore GK specifically in POMC neurons in (homozygous or heterozygous) whole-body GK knockout mice, using the "knock-in" strategy by which Okamoto et al. rescued lethal neonatal diabetes by restoring insulin receptors specifically to neurons in whole-body insulin receptor knockout mice. Mice will be maintained on a low-fat or a high-fat diet. Metabolic phenotypes, including leptin sensitivity, glucose and insulin tolerance tests, food intake, body weight, adiposity, metabolic rate, and temperature will be assessed. Mice will be sacrificed, and gene expression will be determined in hypothalamus and other tissues. We propose similar studies in Specific Aims 2 and 3, using AgRP, or GFAP cre-recombinase (expressed in tanycytes) to ablate or restore GK specifically in in AgRP neurons, or to ablate GK in tanyctes. We anticipate that ablation of GK in specific hypothalamic cell types will partially recapitulate specific metabolic impairments produced by whole-body ablation of GK, and that restoration of GK to POMC or AgRP neurons will reverse specific metabolic impairments caused by whole- body GK deficiency