Physiological glucose is critical for optimized neuronal viability and AMPK responsiveness in vitro

Abstract

Understanding the mechanisms that govern neuronal responses to oxidative and metabolic stress is essential for therapeutic intervention. In vitro modeling is an important approach for these studies, as the metabolic environment influences neuronal responses. Surprisingly, most neuronal culture methods employ conditions that are non-physiological, especially with regards to glucose concentrations, which often exceed 20 mM. This concentration is a significant departure from physiological glucose levels, and even several-fold greater than that seen during severe hyperglycemia. The goal of this study was to establish a physiological neuronal culture system that will facilitate the study of neuronal energy metabolism and responses to metabolic stress. We demonstrate that the metabolic environment during preparation, plating, and maintenance of cultures affects neuronal viability and the response of neuronal pathways to changes in energy balance.

Modeling Ischemic Stroke In Vitro: Status Quo and Future Perspectives

“…However, glucose levels found in cell cultures (designed for rapid cell growth) far exceed physiological levels, often containing >20mM glucose. In severe hyperglycemia plasma levels may reach 15.2mM, and in hypoglycemia, may drop to 2.8mM, while brain levels only reach 4.5mM, or fall as low as 0.16mM 41 . As such, neurons in culture are consistently exposed to >10 times the levels found in vivo .…”

“…In vivo , plasma glucose can range from 5.5 to 7.8mM, while brain glucose levels have been reported ranging from 0.82 to 2.4mM 41 , 42 . However, glucose levels found in cell cultures (designed for rapid cell growth) far exceed physiological levels, often containing >20mM glucose.…”

“…As such, neurons in culture are consistently exposed to >10 times the levels found in vivo . High levels of glucose in standard media have been shown to negatively affect long term neuronal viability and influence AMPK signaling 41 . Levels of other energy sources including lactate, pyruvate, fatty acids, and ketone bodies have been demonstrated to influence neuronal metabolism both in vivo and in vitro 41 and should be considered when investigating neuronal energy crisis.…”

Role of 5′-Adenosine Monophosphate-Activated Protein Kinase in Cell Survival and Death Responses in Neurons

“…Moreover, the pharmacological inhibition or gene deletion of AMPK can be neuroprotective in stroke (70,78). The discrepancies between in vivo and in vitro studies elucidating the role of AMPK in neuronal survival (28,78) could be due to differences in glucose concentrations, which has been previously shown to be critical for determining neuronal survival and the levels of AMPK activation (62,91). Therefore, by using the similar metabolic conditions our laboratory has further explored the protective versus cell death-inducing effects of AMPK activation in neurons exposed to metabolic stress, as exemplified in a model of excitotoxic Ca 2þ overloading.…”

Selenoprotein S Reduces Endoplasmic Reticulum Stress-Induced Phosphorylation of Tau: Potential Role in Selenate Mitigation of Tau Pathology

“…We found that hyperglycemic conditions (>10 mM glucose) for neuronal cells in culture suppressed SelS mRNA and protein levels. SelS expression was greatest at a glucose concentration near the normal range in brain (5 mM) [25] and decreased as glucose increased (Fig. 1C, above) [10, 11, 21].…”