Swain Sarpras, Roberts David M, Chowdhry Saad, Durbin Ryan, Boyd Reece, Petereit Juli, Renden Robert
Department of Physiology and Cell Biology, Reno School of Medicine, University of Nevada, Reno, Nevada, USA.
Nevada Bioinformatics Center (RRID:SCR_017802), University of Nevada, Reno, Reno, Nevada, USA.
J Neurochem. 2025 Jun;169(6):e70125. doi: 10.1111/jnc.70125.
Neurons are almost exclusively cultured in media containing glucose at much higher concentrations than found in the brain. To test whether these "standard" hyperglycemic culture conditions affect neuronal respiration relative to near-euglycemic conditions, we compared neuronal cultures grown with minimal glial contamination from the hippocampus and cortex of neonatal C57BL/6NCrl mice in standard commercially available media (25 mM Glucose) and in identical media with 5 mM glucose. Neuronal growth in both glucose concentrations proceeded until at least 14 days in vitro, with similar morphology and synaptogenesis. Neurons grown in high glucose were highly dependent on glycolysis as their primary source of ATP, measured using ATP luminescence and cellular respirometry assays. In contrast, neurons grown in 5 mM glucose showed a more balanced dependence on glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), greater reserve mitochondrial respiration capacity, and increased mitochondrial population relative to standard media. Our results show that neurons cultured in artificially high glucose-containing media preferentially use glycolysis, opposite to what is known for neurons in vivo as the primary pathway for ATP maintenance. Changes in gene and protein expression levels corroborate these changes in function and additionally suggest that high glucose culture media increases neuronal inflammation. We suggest using neuronal culture systems in 5 mM glucose to better represent physiologically relevant neuronal respiration.
神经元几乎完全是在含有葡萄糖的培养基中培养的，其葡萄糖浓度比大脑中的浓度高得多。为了测试这些“标准”高血糖培养条件相对于近正常血糖条件是否会影响神经元呼吸，我们比较了在市售标准培养基（25 mM葡萄糖）和含5 mM葡萄糖的相同培养基中培养的、来自新生C57BL/6NCrl小鼠海马体和皮质且胶质细胞污染最小的神经元培养物。两种葡萄糖浓度下的神经元生长均持续至体外至少14天，形态和突触形成相似。使用ATP发光和细胞呼吸测定法测量，在高葡萄糖中生长的神经元高度依赖糖酵解作为其ATP的主要来源。相比之下，在5 mM葡萄糖中生长的神经元对糖酵解和线粒体氧化磷酸化（OXPHOS）的依赖性更为平衡，线粒体呼吸储备能力更强，且相对于标准培养基，线粒体数量增加。我们的结果表明，在人工高糖培养基中培养的神经元优先使用糖酵解，这与体内神经元维持ATP的主要途径相反。基因和蛋白质表达水平的变化证实了这些功能变化，此外还表明高糖培养基会增加神经元炎症。我们建议使用含5 mM葡萄糖的神经元培养系统，以更好地体现生理相关的神经元呼吸。
