Nesfatin-1通过脑桥外侧臂旁核（LPBN）参与1型糖尿病小鼠低血糖危象的调节及其机制的研究

Patients with type 1 diabetes mellitus (T1DM) are characterized by a lower sensitivity and impairment of counter-regulatory responses (CRR) to hypoglycemia, and are thus susceptible to a life-threatening hypoglycemic crisis. For now, the therapia to alleviate CRR impairment in T1DM patients have not been well established. We first revealed improved CRR after microinjection of Nesfatin-1 into LPBN and a significantly reduced occurrence of seizure and mortality under insulin induced hypoglycemia. Molecular and electrophysiological techniques (patch clamps and in vivo electrophysiological technique), primary cell culture, and Tag-lite technique will be used in the following experiments: 1. To determine whether Nesfatin-1 could alleviate CRR impairment through LPBN glucose inhibited (GI) neurons. 2. To investigate the participance of Nesfatin-1 in neurohumoral (sympathetic nerve-adrenal medulla) regulation of CRR. 3. To investigate the role of lipid metabolism in regulation of hypoglycemic crisis by Nesfatin-1. 4. To prove the relative specificity in combination of nesfatin-1 and growth hormone secretagogue receptor (GHSR), and further validate the molecular pathway after that combination. This project will provide a new view for the prevention and treatment of hypoglycemia in T1DM patients.

1型糖尿病（T1DM）患者对于低血糖的敏感性下降，低血糖反向调节（CRR）明显受损，易发生低血糖危象；但如何改善T1DM患者CRR受损的方法尚未明确。本课题组首次发现LPBN注射Nesfatin-1可增强正常鼠CRR，并显著降低正常鼠在外周胰岛素诱导的低血糖状态下的惊厥及死亡率。本课题组拟采用分子生物学、电生理（膜片钳和在体电生理）、细胞培养以及Tag-lite等技术探究：1. Nesfatin-1能否通过LPBN核团中GI神经元调节T1DM小鼠CRR受损；2. Nesfatin-1参与T1DM小鼠CRR的神经-体液（交感神经-肾上腺髓质）调节机制；3. Nesfatin-1调节脂代谢在改善T1DM小鼠低血糖危象中的作用；4. 证实Nesfatin-1与GHSR结合的相对特异性，进一步明确Nesfatin-1与 GHSR作用后的分子信号通路。将为T1DM患者低血糖的预防和治疗提供新的思路。

1型糖尿病（T1DM）患者更容易发生低血糖现象，低血糖反向调节（CRR）明显受损，易发生低血糖危象。Nesfatin-1是厌食肽，LPBN是参与能量平衡调节的重要核团。本课题组首次发现LPBN注射Nesfatin-1可显著降低健康大鼠在外周胰岛素诱导的低血糖状态下的惊厥及死亡率。然而，目前尚不清楚LPBN中nesfatin-1是否参与了T1DM血糖的调节以及是否介导了胰岛素降低血糖的作用？T1DM大鼠LPBN中的葡萄糖敏感神经元是否受到nesfatin-1的调节而影响外周糖脂代谢？本课题研究发现：1.低血糖可以激活LPBN核团中nesfatin-1阳性神经元。2.LPBN微量注射nesfatin-1可明显降低wistar大鼠和T1DM大鼠夜间摄食。3.在正常wistar大鼠腹腔注射10IU/Kg胰岛素时，LPBN注射nesfatin-1可以起到改善低血糖危象，缓解血糖过低作用（2.62±0.10 vs. 2.33±0.05）；在T1DM大鼠腹腔注射30IU/Kg胰岛素时，LPBN 注射nesfatin-1可以起到协助胰岛素降血糖的作用（7.63±0.44 vs. 12.26±0.95）。4.T1DM大鼠血浆中nesfatin-1水平低于正常wistar大鼠 (259.44±6.01 vs. 283.63±6.5)。5.细胞外在体电生理结果显示，正常wistar大鼠LPBN中GS神经元的GI居多（50.0% vs. 38.8%），T1DM大鼠LPBN中GS神经元的GE居多（48.9% vs. 36.4%）。 Wistar大鼠的LPBN微量注射nesfatin-1主要对GI神经元起兴奋作用，对GE神经元起抑制作用；T1DM大鼠LPBN微量注射nesfatin-1主要对GE神经元起兴奋作用，对GI神经元起抑制作用。结论，低血糖可激活LPBN中的Nesfatin-1免疫阳性神经元，且LPBN中的Nesfatin-1可与胰岛素协同降低T1DM大鼠的血糖。T1DM大鼠LPBN中葡萄糖兴奋敏感神经元(GE)比例高于健康大鼠，可被Nesfatin-1刺激。我们的研究为治疗和预防T1DM中CRR受损提供了新的思路。