Department of Physiology, The University of Melbourne, Melbourne, Victoria, Australia.
Department of Medicine, St. Vincent's Campus, The University of Melbourne, Melbourne, Victoria, Australia.
PLoS One. 2014 May 23;9(5):e97570. doi: 10.1371/journal.pone.0097570. eCollection 2014.
Ischemic stroke is the combinatorial effect of many pathological processes including the loss of energy supplies, excessive intracellular calcium accumulation, oxidative stress, and inflammatory responses. The brain's ability to maintain energy demand through this process involves metabolism of glycogen, which is critical for release of stored glucose. However, regulation of glycogen metabolism in ischemic stroke remains unknown. In the present study, we investigate the role and regulation of glycogen metabolizing enzymes and their effects on the fate of glycogen during ischemic stroke.
Ischemic stroke was induced in rats by peri-vascular application of the vasoconstrictor endothelin-1 and forebrains were collected at 1, 3, 6 and 24 hours post-stroke. Glycogen levels and the expression and activity of enzymes involved in glycogen metabolism were analyzed. We found elevated glycogen levels in the ipsilateral hemispheres compared with contralateral hemispheres at 6 and 24 hours (25% and 39% increase respectively; P<0.05). Glycogen synthase activity and glycogen branching enzyme expression were found to be similar between the ipsilateral, contralateral, and sham control hemispheres. In contrast, the rate-limiting enzyme for glycogen breakdown, glycogen phosphorylase, had 58% lower activity (P<0.01) in the ipsilateral hemisphere (24 hours post-stroke), which corresponded with a 48% reduction in cAMP-dependent protein kinase A (PKA) activity (P<0.01). In addition, glycogen debranching enzyme expression 24 hours post-stroke was 77% (P<0.01) and 72% lower (P<0.01) at the protein and mRNA level, respectively. In cultured rat primary cerebellar astrocytes, hypoxia and inhibition of PKA activity significantly reduced glycogen phosphorylase activity and increased glycogen accumulation but did not alter glycogen synthase activity. Furthermore, elevated glycogen levels provided metabolic support to astrocytes during hypoxia.
Our study has identified that glycogen breakdown is impaired during ischemic stroke, the molecular basis of which includes reduced glycogen debranching enzyme expression level together with reduced glycogen phosphorylase and PKA activity.
缺血性中风是多种病理过程的综合效应，包括能量供应的丧失、细胞内钙的过度积累、氧化应激和炎症反应。大脑通过代谢糖原维持能量需求的能力对于释放储存的葡萄糖至关重要。然而，缺血性中风中糖原代谢的调节仍不清楚。在本研究中，我们研究了糖原代谢酶的作用和调节及其在缺血性中风期间对糖原命运的影响。
通过血管收缩剂内皮素-1 血管周围给药诱导大鼠缺血性中风，并在中风后 1、3、6 和 24 小时采集大脑前额叶。分析了糖原水平以及参与糖原代谢的酶的表达和活性。我们发现，与对侧半球相比，同侧半球在 6 和 24 小时时糖原水平升高（分别增加 25%和 39%；P<0.05）。在同侧、对侧和假对照半球之间，糖原合酶活性和糖原分支酶表达相似。相比之下，糖原分解限速酶糖原磷酸化酶在同侧半球的活性降低了 58%（P<0.01）（中风后 24 小时），相应的 cAMP 依赖性蛋白激酶 A（PKA）活性降低了 48%（P<0.01）。此外，中风后 24 小时糖原去分支酶表达水平在蛋白和 mRNA 水平上分别降低了 77%（P<0.01）和 72%（P<0.01）。在培养的大鼠原代小脑星形胶质细胞中，缺氧和 PKA 活性抑制显著降低糖原磷酸化酶活性并增加糖原积累，但不改变糖原合酶活性。此外，升高的糖原水平为星形胶质细胞在缺氧期间提供代谢支持。
我们的研究表明，在缺血性中风期间，糖原分解受损，其分子基础包括糖原去分支酶表达水平降低以及糖原磷酸化酶和 PKA 活性降低。
