Sheriff S, Chance W T, Fischer J E, Balasubramaniam A
Department of Surgery, University of Cincinnati, Ohio 45267-0558, USA.
Mol Pharmacol. 1997 Apr;51(4):597-604. doi: 10.1124/mol.51.4.597.
Intrahypothalamic (IHT) administration of neuropeptide Y (NPY) induces a robust feeding response in rats. We have shown previously that NPY-induced feeding is mediated by a pertussis-toxin-sensitive G protein in rats. NPY receptors are coupled to cAMP and Ca2+. Because these second messengers are known to activate cAMP response element binding proteins, (CREB), cAMP response element modulators, or activating transcription factor 1, we investigated the involvement of these transcription factors in NPY-induced feeding in rats. Compared with control injections of cerebrospinal fluid (1 microl), IHT administration of NPY increased cAMP response element (CRE) binding to rat hypothalamic nuclear extracts in a time-dependent manner, as detected by an electrophoretic mobility shift assay. In contrast, IHT administration of the anorectic neuropeptide, pituitary adenylate cyclase activating polypeptide, strongly inhibited the CRE binding. Food deprivation for 48 hr also increased CRE binding, whereas 8 hr of refeeding normalized CRE activity. Preincubation of the hypothalamic nuclear extracts of NPY-treated and unfed rats with antibody specific to CREB blocked CRE binding, whereas preincubation with phosphoCREB antibody retarded the migration of CRE-protein complex, indicating that phosphoCREB is involved in this process. Consistently, immunohistochemical studies with food-deprived rats showed an intense phosphoCREB signal in the paraventricular nuclei and ventromedial hypothalamus in comparison to rats fed ad libitum. Hypothalamic calcium/calmodulin-dependent protein kinase II activity was also increased by IHT-NPY. These results suggest that calcium/calmodulin-dependent protein kinase II induced phosphorylation of CREB may be involved in regulating feeding behavior induced by NPY.
下丘脑内注射神经肽Y（NPY）可在大鼠中引发强烈的进食反应。我们之前已经表明，NPY诱导的进食是由大鼠体内一种对百日咳毒素敏感的G蛋白介导的。NPY受体与cAMP和Ca2+偶联。由于已知这些第二信使可激活cAMP反应元件结合蛋白（CREB）、cAMP反应元件调节剂或激活转录因子1，因此我们研究了这些转录因子在NPY诱导的大鼠进食中的作用。与注射脑脊液（1微升）的对照相比，下丘脑内注射NPY以时间依赖性方式增加了cAMP反应元件（CRE）与大鼠下丘脑核提取物的结合，这通过电泳迁移率变动分析检测到。相比之下，下丘脑内注射厌食神经肽垂体腺苷酸环化酶激活多肽则强烈抑制了CRE结合。禁食48小时也增加了CRE结合，而重新进食8小时使CRE活性恢复正常。用特异性针对CREB的抗体对NPY处理的大鼠和未进食大鼠的下丘脑核提取物进行预孵育可阻断CRE结合，而用磷酸化CREB抗体预孵育则使CRE - 蛋白复合物的迁移减慢，表明磷酸化CREB参与了这一过程。同样，对禁食大鼠的免疫组织化学研究表明，与随意进食的大鼠相比，室旁核和腹内侧下丘脑有强烈的磷酸化CREB信号。下丘脑内注射NPY也增加了钙/钙调蛋白依赖性蛋白激酶II的活性。这些结果表明，钙/钙调蛋白依赖性蛋白激酶II诱导的CREB磷酸化可能参与调节NPY诱导的进食行为。
