CCB-Biocenter, Institute of Neurobiochemistry, Medical University of Innsbruck, 6020 Innsbruck, Austria.
Department of Biochemistry, Institute of Bioanalytic & Intermediary Metabolism, University of Innsbruck, 6020 Innsbruck, Austria.
Mol Metab. 2024 Oct;88:102018. doi: 10.1016/j.molmet.2024.102018. Epub 2024 Aug 24.
Glucose-1,6-bisphosphate (G-1,6-BP), a byproduct of glycolysis that is synthesized by phosphoglucomutase 2 like 1 (PGM2L1), is particularly abundant in neurons. G-1,6-BP is sensitive to the glycolytic flux, due to its dependence on 1,3-bisphosphoglycerate as phosphate donor, and the energy state, due to its degradation by inosine monophosphate-activated phosphomannomutase 1. Since the exact role of this metabolite remains unclear, our aim was to elucidate the specific function of G-1,6-BP in the brain.
The effect of PGM2L1 on neuronal post-ischemic viability was assessed by siRNA-mediated knockdown of PGM2L1 in primary mouse neurons. Acute mouse brain slices were used to correlate the reduction in G-1,6-BP upon ischemia to changes in carbon metabolism by C-glucose tracing. A drug affinity responsive target stability assay was used to test if G-1,6-BP interacts with the mitochondrial pyruvate carrier (MPC) subunits in mouse brain protein extracts. Human embryonic kidney cells expressing a MPC bioluminescence resonance energy transfer sensor were used to analyze how PGM2L1 overexpression affects MPC activity. The effect of G-1,6-BP on mitochondrial pyruvate uptake and oxygen consumption rates was analyzed in isolated mouse brain mitochondria. PGM2L1 and a predicted upstream kinase were overexpressed in a human neuroblastoma cell line and G-1,6-BP levels were measured.
We found that G-1,6-BP in mouse brain slices was quickly degraded upon ischemia and reperfusion. Knockdown of PGM2L1 in mouse neurons reduced post-ischemic viability, indicating that PGM2L1 plays a neuroprotective role. The reduction in G-1,6-BP upon ischemia was not accompanied by alterations in glycolytic rates but we did see a reduced C-glucose incorporation into citrate, suggesting a potential role in mitochondrial pyruvate uptake or metabolism. Indeed, G-1,6-BP interacted with both MPC subunits and overexpression of PGM2L1 increased MPC activity. G-1,6-BP, at concentrations found in the brain, enhanced mitochondrial pyruvate uptake and pyruvate-induced oxygen consumption rates. Overexpression of a predicted upstream kinase inhibited PGM2L1 activity, showing that besides metabolism, also signaling pathways can regulate G-1,6-BP levels.
We provide evidence that G-1,6-BP positively regulates mitochondrial pyruvate uptake and post-ischemic neuronal viability. These compelling data reveal a novel mechanism by which neurons can couple glycolysis-derived pyruvate to the tricarboxylic acid cycle. This process is sensitive to the glycolytic flux, the cell's energetic state, and upstream signaling cascades, offering many regulatory means to fine-tune this critical metabolic step.
葡萄糖-1,6-二磷酸（G-1,6-BP）是糖酵解的副产物，由磷酸葡糖变位酶 2 样 1（PGM2L1）合成，在神经元中特别丰富。G-1,6-BP 对糖酵解通量敏感，因为它依赖于 1,3-双磷酸甘油酸作为磷酸供体，并且由于其被肌苷单磷酸激活的磷酸甘露糖变位酶 1 降解而对能量状态敏感。由于这种代谢物的确切作用尚不清楚，我们的目的是阐明 G-1,6-BP 在大脑中的特定功能。
通过 siRNA 介导的 PGM2L1 在原代小鼠神经元中的敲低，评估 PGM2L1 对神经元缺血后存活的影响。使用急性小鼠脑切片来关联缺血时 G-1,6-BP 的减少与 C-葡萄糖示踪法测定的碳代谢变化。药物亲和反应靶标稳定性测定用于测试 G-1,6-BP 是否与小鼠脑蛋白提取物中的线粒体丙酮酸载体（MPC）亚基相互作用。表达 MPC 生物发光共振能量转移传感器的人胚肾细胞用于分析 PGM2L1 过表达如何影响 MPC 活性。在分离的小鼠脑线粒体中分析 G-1,6-BP 对线粒体丙酮酸摄取和耗氧量的影响。在人神经母细胞瘤细胞系中过表达 PGM2L1 和预测的上游激酶，并测量 G-1,6-BP 水平。
我们发现，在缺血再灌注期间，小鼠脑切片中的 G-1,6-BP 迅速降解。PGM2L1 在小鼠神经元中的敲低降低了缺血后的存活能力，表明 PGM2L1 发挥了神经保护作用。缺血时 G-1,6-BP 的减少并未伴随着糖酵解速率的改变，但我们确实看到 C-葡萄糖掺入柠檬酸的减少，这表明其可能在线粒体丙酮酸摄取或代谢中发挥作用。事实上，G-1,6-BP 与 MPC 亚基相互作用，PGM2L1 的过表达增加了 MPC 活性。G-1,6-BP，以大脑中发现的浓度，增强了线粒体丙酮酸摄取和丙酮酸诱导的耗氧量。预测的上游激酶的过表达抑制了 PGM2L1 的活性，表明除了代谢之外，信号通路也可以调节 G-1,6-BP 水平。
我们提供的证据表明，G-1,6-BP 正向调节线粒体丙酮酸摄取和缺血后神经元存活。这些令人信服的数据揭示了神经元可以将糖酵解衍生的丙酮酸与三羧酸循环偶联的新机制。该过程对糖酵解通量、细胞能量状态和上游信号级联敏感，为精细调节这一关键代谢步骤提供了许多调节手段。
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