CNS Counterregulatory Mechanisms

描述(由申请人提供)：低血糖、意识不清和反调节失败是使用强化胰岛素治疗控制1型糖尿病的严重和危及生命的缺点。肾上腺髓质肾上腺素的分泌是对抗低血糖最迅速有效和最容易保存的防御措施之一。阐明逆调节反应，特别是肾上腺素调节的神经通路，将有助于设计治疗方法来对抗低血糖无意识。我们最近发现，大脑组胺能活动减少与逆向调节受损相关，抑制组胺合成会降低肾上腺素对低血糖的反应。作为确定无意识低血糖的神经机制的总体目标的一部分，我们假设大脑组胺神经元参与低血糖诱导的肾上腺素的分泌，可能还有其他反调节激素。我们进一步假设，中枢组胺水平的操纵将有助于识别控制这些反应的其他神经通路。为了解决我们的假设，我们将：(1)使用特定的组胺拮抗剂和测量反调节激素来测试低血糖诱导的肾上腺素分泌是否会选择性地(A)被脑穿透性组胺H1或H2受体拮抗剂抑制，(B)不受非渗透性拮抗剂影响，以及(C)通过H3自身受体拮抗剂硫代巴比妥钠增强中枢组胺。我们还将(2)在无拘无束的、长期空腹的小鼠身上整合降糖钳技术，以测试(A)链脲佐菌素(STZ)-糖尿病对反调节失败进展的影响，以及(B)假设硫代巴比妥胺诱导的中枢组胺增加将逆转STZ小鼠反复低血糖引起的反调节缺陷。最后，使用来自AIMS 1和AIMS 2的小鼠大脑，我们将结合神经元c-fos基因表达的原位杂交分析与组氨酸脱羧酶mRNA的组织化学分析或组胺拮抗剂的应用，来检验以下假设：(A)明确的神经通路，包括下丘脑组胺神经元，在反复低血糖后表现出改变的活性，以及(B)中枢组胺能张力的实验变化将揭示与反调节相关的额外神经元群体。这些研究将为血糖控制的神经药理学提供新的信息，可能揭示预防低血糖无意识的治疗策略。

DESCRIPTION (provided by applicant): Hypoglycemia unawareness and counterregulatory failure are serious and life-threatening drawbacks to the use of intensive insulin therapy to control type 1 diabetes. Adrenomedullary epinephrine secretion is one of the most rapidly effective and readily preserved defenses against hypoglycemia. Elucidating neural pathways underlying the regulation of counterregulatory responses, and of epinephrine in particular, will aid in designing therapies to combat hypoglycemia unawareness. We have recently found that reduced brain histaminergic activity correlates with impaired counterregulation, and that inhibiting histamine synthesis decreases epinephrine responses to hypoglycemia. As part of an overall goal to identify neural mechanisms for hypoglycemia unawareness, we hypothesize that brain histamine neurons are involved in hypoglycemia-induced secretion of epinephrine, and possibly other counterregulatory hormones. We further hypothesize that manipulation of central histamine levels will aid in identifying additional neural pathways controlling these responses. To address our hypotheses, we will: (1) use specific histamine antagonists and measure counterregulatory hormones to test if hypoglycemia-induced epinephrine secretion will be selectively (a) inhibited by brain-penetrant histamine H1- or H2- receptor antagonists, (b) unaffected by nonpenetrant antagonists, and (c) enhanced by increasing central histamine with the H3 autoreceptor antagonist, thioperamide. We will also (2) integrate hypoglycemic clamp techniques in unrestrained, chronically cannulated mice to test (a) the impact of streptozotocin (STZ)-diabetes on the progression of counterregulatory failure and (b) the hypothesis that thioperamide-evoked increases in central histamine will reverse counterregulatory deficits induced by recurrent hypoglycemia in STZ mice. Finally (3), using brains of mice from Aims 1 and 2, we will combine in situ hybridization analysis of neuronal c-fos gene expression with histochemical analysis (histidine decarboxylase mRNA) or histamine antagonist administration, to test the hypotheses that (a) defined neural pathways, including hypothalamic histamine neurons, exhibit altered activity after recurrent hypoglycemia, and (b) experimental alterations in central histaminergic tone will reveal additional neuron populations relevant to counterregulation. These studies will provide novel information on the neuropharmacology of glucose control that may reveal therapeutic strategies to prevent hypoglycemia unawareness.