Causes and consequences of AMPK activation in the heart

描述（由申请人提供）：AMP活化蛋白激酶（AMPK）作为细胞低燃料警告系统。AMP浓度的增加（[AMP]）激活AMPK，而高浓度的ATP（[ATP]）拮抗AMPK激活。在该警告系统被激活后，它启动保护措施以保存ATP或促进ATP生成的替代方法。 我们使用代谢抑制剂增加[AMP]，并在灌流的大鼠心脏中测量AMPK活性和[AMP]之间的关系。AMPK的半数最大激活浓度为1.8 +/- 0.3 μ M，10至30 μ M胞质[AMP]导致AMPK活性最大增加两倍。在这项研究中，[ATP]大于或等于7 mM。这些结果表明，在心脏ATP不拮抗AMP激活，因为它在试管研究。在缺血大鼠心脏中，我们发现10至30 fM胞质[AMP]导致AMPK活性增加8倍。在缺血中AMPK激活增加了四倍，表明AMP以外的因素引起AMPK激活。这些观察结果导致我们的第一个假设，即AMPK激活的因果因素是代谢活性的胞质[AMP]（而不是总AMP），减少的[ATP]和细胞内H+（[H+]i）。 我们提出了两个具体的目标来检验这一假设。首先，在离体灌流的小鼠心脏中确定AMPK活性与[AMP]之间的关系。[AMP]、[ATP]和[H+]i将使用能量代谢、代谢抑制剂、缺氧和缺血的三种操作来改变。第二，确定在离体灌注小鼠心脏中恢复正常[AMP]后AMPK保持激活的时间。 我们的第二个假设是AMPK激活的主要后果是底物代谢的转变，特别是AMPK导致乙酰辅酶A羧化酶磷酸化导致的脂肪酸氧化加速。第三个具体目标将定义在底物利用率的改变，导致在分离的灌注小鼠心脏中AMPK活性增加。 我们的长期目标是确定AMPK活性的原因和AMPK活性对美国两种最危险的心血管疾病（缺血和心力衰竭）心肌底物代谢的影响。

DESCRIPTION (provided by applicant): AMP-activated protein kinase (AMPK) acts as a cellular low fuel warning system. Increases in AMP concentration ([AMP]) activate AMPK, while high concentrations of ATP ([ATP]) antagonize AMPK activation. After this warning system is activated it initiates protective measures to either conserve ATP or promote alternative methods of ATP generation. We increased [AMP] using metabolic inhibitors and measured the relationship between AMPK activity and [AMP] in the perfused rat heart. AMPK's half maximal activating concentration of AMP was 1.8 +/- 0.3 muM and 10 to 30 muM cytosolic [AMP] resulted in a two-fold maximal increase in AMPK activity. In this study [ATP] was greater than or equal too 7 mM. These results suggest that in the heart ATP does not antagonize AMP activation as it does in test tube studies. In the ischemic rat heart we found that 10 to 30 fM cytosolic [AMP] resulted in an eight-fold increase in AMPK activity. This four times greater AMPK activation in ischemia indicates that factors other than AMP cause AMPK activation. These observations lead to our first hypothesis that the causal factors for AMPK activation are the metabolically active cytosolic [AMP] (not total AMP), reduced [ATP], and intracellular H+ ([H+]i). We propose two specific aims to test this hypothesis. First, to define the relationship between AMPK activity and the [AMP] in the isolated perfused mouse heart. The [AMP], [ATP], and [H+]i, will be altered using three manipulations of energy metabolism, metabolic inhibitors, hypoxia, and ischemia. Second, to define how long AMPK remains activated after restoration of normal [AMP] in the isolated perfused mouse heart. Our second hypothesis that the predominant consequence of AMPK activation is a shift in substrate metabolism, in particular, an acceleration of fatty acid oxidation resulting from acetyl-CoA carboxylase phosporylation by AMPK. The third specific aim will define the alteration in substrate utilization resulting from increased AMPK activity in the isolated perfused mouse heart. Our long-term objective is to determine the causes of AMPK activity and the consequences of AMPK activity for myocardial substrate metabolism during the two most dangerous cardiovascular diseases, ischemia and heart failure, in the United States.