Causes and consequences of AMPK activation in the heart

描述(由申请人提供)：AMP激活的蛋白激酶(AMPK)作为细胞低燃料预警系统。增加AMP浓度([AMP])激活AMPK，而高浓度的ATP([ATP])拮抗AMPK激活。在这个预警系统被激活后，它会启动保护措施，以保存ATP或促进替代的ATP生成方法。 我们使用代谢抑制剂增加了[AMP]，并在灌流的大鼠心脏中测量了AMPK活性与[AMP]的关系。AMPK对AMP的半数最大激活浓度为1.8+/-0.3um，10~30um的胞浆[AMP]可使AMPK活性增加2倍。在本研究中，[ATP]大于或等于7 mm。这些结果表明，在心脏中，ATP并不像在试管研究中那样拮抗AMP的激活。在缺血大鼠心脏中，我们发现10到30个FM胞浆[AMP]导致AMPK活性增加8倍。AMPK在缺血时的4倍以上激活表明，AMP以外的其他因素导致AMPK激活。这些观察结果导致了我们的第一个假设，即AMPK激活的原因是代谢活跃的胞浆[AMP](而不是总AMP)、降低的[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，明确AMPK活性与[AMP]在离体小鼠心脏中的关系。[AMP]、[ATP]和[H+]i将使用能量代谢、代谢抑制剂、缺氧和缺血三种操作来改变。第二，确定AMPK在离体灌流小鼠心脏恢复正常后多长时间保持激活。 我们的第二个假设是AMPK激活的主要结果是底物代谢的变化，特别是AMPK导致的乙酰辅酶A羧基酶磷酸化导致的脂肪酸氧化加速。第三个特定目标将定义由于AMPK活性增加而导致的底物利用的变化。 我们的长期目标是确定AMPK活性的原因以及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.