Kang Ling, Dunn-Meynell Ambrose A, Routh Vanessa H, Gaspers Larry D, Nagata Yasufumi, Nishimura Teruyuki, Eiki Junichi, Zhang Bei B, Levin Barry E
Department of Neurology and Neuroscience, New Jersey Medical School, Newark, USA.
Diabetes. 2006 Feb;55(2):412-20. doi: 10.2337/diabetes.55.02.06.db05-1229.
To test the hypothesis that glucokinase is a critical regulator of neuronal glucosensing, glucokinase activity was increased, using a glucokinase activator drug, or decreased, using RNA interference combined with calcium imaging in freshly dissociated ventromedial hypothalamic nucleus (VMN) neurons or primary ventromedial hypothalamus (VMH; VMN plus arcuate nucleus) cultures. To assess the validity of our approach, we first showed that glucose-induced (0.5-2.5 mmol/l) changes in intracellular Ca(2+) concentration (Ca(2+)) oscillations, using fura-2 and changes in membrane potential (using a membrane potential-sensitive dye), were highly correlated in both glucose-excited and -inhibited neurons. Also, glucose-excited neurons increased (half-maximal effective concentration [EC(50)] = 0.54 mmol/l) and glucose-inhibited neurons decreased (half-maximal inhibitory concentration [IC(50)] = 1.12 mmol/l) Ca(2+) oscillations to incremental changes in glucose from 0.3 to 5 mmol/l. In untreated primary VMH neuronal cultures, the expression of glucokinase mRNA and the number of demonstrable glucosensing neurons fell spontaneously by half over 12-96 h without loss of viable neurons. Transfection of neurons with small interfering glucokinase RNA did not affect survival but did reduce glucokinase mRNA by 90% in association with loss of all demonstrable glucose-excited neurons and a 99% reduction in glucose-inhibited neurons. A pharmacological glucokinase activator produced a dose-related increase in Ca(2+) oscillations in glucose-excited neurons (EC(50) = 0.98 mmol/l) and a decrease in glucose-inhibited neurons (IC(50) = 0.025 micromol/l) held at 0.5 mmol/l glucose. Together, these data support a critical role for glucokinase in neuronal glucosensing.
为了验证葡萄糖激酶是神经元葡萄糖感应的关键调节因子这一假设，我们在新鲜分离的腹内侧下丘脑核（VMN）神经元或原代腹内侧下丘脑（VMH；VMN加弓状核）培养物中，使用葡萄糖激酶激活药物提高葡萄糖激酶活性，或通过RNA干扰结合钙成像降低其活性。为评估我们方法的有效性，我们首先表明，使用fura - 2检测到的葡萄糖诱导的（0.5 - 2.5 mmol/L）细胞内Ca(2+)浓度（Ca(2+)）振荡变化与使用膜电位敏感染料检测到的膜电位变化在葡萄糖兴奋型和抑制型神经元中高度相关。此外，葡萄糖兴奋型神经元对葡萄糖从0.3到5 mmol/L的增量变化增加了（半数最大有效浓度[EC(50)] = 0.54 mmol/L）Ca(2+)振荡，而葡萄糖抑制型神经元则减少了（半数最大抑制浓度[IC(50)] = 1.12 mmol/L）。在未处理的原代VMH神经元培养物中，葡萄糖激酶mRNA的表达和可检测到的葡萄糖感应神经元数量在12 - 96小时内自发减少了一半，而存活神经元没有损失。用小干扰葡萄糖激酶RNA转染神经元不影响细胞存活，但确实使葡萄糖激酶mRNA减少了90%，同时所有可检测到的葡萄糖兴奋型神经元消失，葡萄糖抑制型神经元减少了99%。一种药理学葡萄糖激酶激活剂在葡萄糖兴奋型神经元中产生了与剂量相关的Ca(2+)振荡增加（EC(50) = 0.98 mmol/L），在葡萄糖抑制型神经元中产生了减少（IC(50) = 0.025 μmol/L），此时葡萄糖浓度维持在0.5 mmol/L。这些数据共同支持了葡萄糖激酶在神经元葡萄糖感应中起关键作用。
