The Effect And Mechanism Of Glucose Decrease On [Ca²⁺]_i In Hypothalamic ARC-NPY Neuron Of Rat

Aim: As a center of energy metabolism, hypothalamus can sense the concentration changesof blood glucose and feedback regulation.therefore, we investigated the response of arcuatenucleus （ARC） NPY neurons to glucose decrease when they were perfused with low glucoseperfusion fluid.Methods: The immunocytochemical technique was used to identify NPY neurons, and the [Ca²⁺]ichanges of hypothalamic ARC-NPY neurons induced by glucose decrease was evaluatedusing Fura-2based calcium imaging techniques.Results: The decrease in extracellular glucose concentration from10mM to1mM inducedthe elevation of F340nm/F380nm ratio from0.55±0.04to0.73±0.11in ARC NPYneurons by28.7±8.5%.Conclusion: Glucose decrease can induce an [Ca²⁺]ielevation in ARC NPY neurons. Aim: The changes of extracellular glucose concentration induced an hypothalamic specificincrease in [Ca²⁺]iin hypothalamic NPY neurons, which will subsequently phosphorylationof downstream kinases, like CaMKK II etc, to play its physiological effect. But, little isknown about the pathway that mediated glucose decrease-induced elevation of [Ca²⁺]i.Furthermore, there is still no report concerning if this elevtion induced by an influx ofcalcium mediated by calcium channel or cytosolic calcium release Thus, we investigatedthe source of [Ca²⁺]ielevation in the hypothalamic ARC-NPY neurons treated with lowglucose perfusion fluid.Methods: We evaluated the glucose decrease induced [Ca²⁺]ielevation of hypothalamicARC-NPY neurons using Fura-2based calcium imaging techniques. In calcium imaginginvestigations, in order to explore the effects of glucose decrease induced [Ca²⁺]ielevation,calcium free perfusion fluid and tool agents were used to identify the source of Ca²⁺.Results:（1） Glucose decrease did not induce [Ca²⁺]iin hippocampal and cortical neurons,but induced an increase in ARC-NPY neurons.（2） Calcium-free perfusion fluid couldabolish the increase in [Ca²⁺]iin ARC-NPY neurons induced by glucose decrease.（3）AMPA receptor blocker CNQX, NMDA receptor blocker D-APV and MK-801, specific Ltype calcium channel blocker nifedipine, specific N-type calcium channel blockerω-conotoxin GVIA, can not abolish the [Ca²⁺]ielevation of hypothalamic ARC-NPYneurons, except specific P/Q-type calcium channel blocker ω-agatoxin TK.Conclusion: The glucose decrease-induced [Ca²⁺]ielevation is NPY neurons specific. Thiselevation is extracellular calcium depedent and mediated by P/Q type calcium channel. Aim: AMPK is reported to be the crucial molecule in food intake and energy expenditureregulation. AMPK will be activated when energy absence induced a ratio change ofAMP/ATP. As the downstream of AMPK, GSK3β can influence P/Q type calcium channel.Here, we investigated if AMPK and GSK3β were involved in glucose decrease-induced[Ca²⁺]ielevation in ARC-NPY neurons.Methods:（1） Western blot analysis was used to investigate the influence of glucosedecrease on AMPK and GSK3β phosphorylation.（2） Fura-2based calcium imagingtechnique was used to examinate if AMPK and GSK3βwere involved in glucose decreaseinduced [Ca²⁺]ielevation in ARC-NPY neurons.Results:（1） The phosphorylation of AMPK and GSK3β increased in ARC-NPY neurons withperfused with low glucose solution.（2） AMPK activator can enhance the amplification ofglucose decrease-induced [Ca²⁺]ielevation. This effect can be mimicked by GSK3βinhibitorConclusion: Glucose decrease activates AMPK, and then inhibits GSK3β in ARC-NPYneurons, which underlying the mechanism of glucose decrease-induced [Ca²⁺]ielevation inNPY neurons.