Influence of altered life conditions on the regulation of AMP-activated kinase (AMPK) in inflammatory pain

AMP激活的激酶(AMPK)是一种细胞能量感受器，在消耗ATP的过程中，细胞内AMP浓度的增加会激活AMP。AMPK调节多种细胞功能，包括糖脂代谢、氧化还原信号和消炎作用。此外，我们在以前的工作中可以证明，激活AMPK可以产生抗伤害效应。这种激活主要是通过药物实现的，分子机制只在分离的细胞中研究过，或者在我的工作组中，在中枢神经系统中。根据目前的研究结果，必须假设AMPK介导的抗伤害感觉在很大程度上是基于炎症周围组织中的分子机制。因此，基于我们之前的结果，计划项目的目标之一是在炎症组织中确定AMPK激活的分子效应的细胞类型特异性。不同的细胞类型将通过细胞特异性标记的免疫组织化学染色在组织冰冻切片中进行研究，或者将通过MACS/FACS分析进行分离和分类。一方面，这使得检测与潜在的AMPK靶基因的共定位成为可能，另一方面，利用qRT-PCR和Western Blot可以评估不同分选的细胞中AMPK的激活和调控。由于生活条件的调节也可以调节AMPK的激活，我们将进一步评估运动训练、减量饮食或年龄是否会影响不同组织中的AMPK活性，特别是神经元组织中的AMPK活性，从而影响伤害性反应。我们将研究AMPK基因的表观遗传调节以及已知的AMPK介导的信号转导机制的调节。在实验中，小鼠将通过在跑步机上跑步几周来进行训练，或者将接受减量饮食。为了研究年龄的影响，我们将使用12到24个月大的小鼠，并将它们与更年轻的小鼠(6-8周)进行比较。之后，我们将确定AMPK启动子的甲基化状态作为表观遗传调控的参数，并测量AMPK活性和AMPK靶标的调控。伤害性行为将在已建立的炎性疼痛模型中进行分析。在项目结束时，我们想发表一项声明，即AMPK是否可以在不使用药物的情况下被刺激和表观遗传调节，以及这种激活是否足以抑制伤害性感受。此外，我们将在炎症组织中AMPK的调节方面获得新的见解，这也可能为疼痛提供新的治疗选择。

The AMP-activated kinase (AMPK) is a cellular energy sensor which is activated by an increase in intracellular AMP-concentration occurring after ATP-consuming processes. AMPK regulates a number of cellular functions including glucose and lipid metabolism, redox signaling and resolution of inflammation. Furthermore, we could show in our previous work that activation of AMPK leads to antinociceptive effects. This activation has been mostly achieved by drugs and the molecular mechanisms have only been investigated in isolated cells or, in my working group, in the central nervous system. Based on current results, it has to be assumed that AMPK-mediated antinociception is largely based on molecular mechanisms in the inflamed peripheral tissue. Therefore, one of the aims of the planned project is, based on our previous results, cell-type specific determination of molecular effects of AMPK-activation in the inflamed tissues. The different cell types will be investigated in tissue cryoslides by immunohistochemical staining for cell-specific markers or will be separated and sorted by MACS/FACS-analyses. On the one hand, this makes it possible to detect co-localization with potential AMPK target genes and on the other hand different sorted cells can be assessed for AMPK activation and regulation of AMPK target genes using qRT-PCR und Western Blot.Since modulation of life conditions can also modulate AMPK activation, we will further assess if exercise training, reduction diet or age can affect the AMPK activity in different tissues, particularly in neuronal tissue, and thereby the nociceptive response. We will investigate epigenetic modulations of the AMPK gene as well as regulations of already known AMPK-mediated signal transduction mechanisms. In the experiments, mice will be trained by running on a treadmill for several weeks or will be subjected to a reduction diet. To investigate effects of age we will use mice between 12 and 24 months of age and compare them with younger mice (6-8 weeks). Afterwards, we will determine the methylation status of the AMPK promoter as a parameter of epigenetic regulations and measure AMPK activity and regulation of AMPK targets. The nociceptive behavior will be analyzed in well established models of inflammatory pain.At the end of the project we want to make a statement if AMPK can be stimulated and epigenetically regulated without using drugs and if this activation is sufficient to inhibit nociception. Moreover, we will gain new insight in the regulation of AMPK in the inflamed tissue, which might also provide novel treatment options for pain.