The Effects of Glucose Deprivation and Ketone Supplementation on the Growth and Function of Neural Stem and Neuronal Precursor Cells

Abstract

The cellular and phenotypic effects that carbohydrate restriction have on brain development are unclear. Ketone bodies become a primary energy source in periods of prolonged glucose deprivation, due to dieting, strenuous exercise, starvation and fasting. Moreover, recent studies have highlighted how the ketone bodies β-hydroxybutyrate (βOHB) and Acetoacetate (AcAc) are vital metabolic and signalling mediators in the mature and developing brain even when glucose is in abundance. The goals of this study are to elucidate the effects of glucose deprivation and ketone body supplementation on the growth, development, and health of rapidly dividing neural stem and neural precursor cells. Neuroblastoma (NB) derived SH-SY5Y cells and neuroepithelial cell derived NE-4C cells are characterised by a high proliferative capacity and glucose demand as a hallmark of their growth and metabolism. However, the impact of glucose deprivation and ketone body supplementation on SH-SY5Y and NE-4C cell growth, lineage and metabolic health are unclear due to conflicting reports in the literature and complexities underlying the Warburg effect. Firstly, a systematic review of literature researching the impact of ketone bodies, glucose and other media formulations in vitro was undertaken to determine if they exert a positive or negative effect on cell growth. The species of cell line, type of cell line, experimental duration, concentration, and media formulations used in each study were assessed. It was determined that over half of the studies that used ketone bodies on cell growth in vitro produced a positive result. The most used ketone body was βOHB and the experimental concentration ranges were between 5 and 10 mM. The systematic review also showed that human derived neuronal cells were the most frequently utilized species and type of cell line. There were inconsistent data gathered with respect to media formulations used in each study. Most studies did not outline whether glucose, pyruvate or glutamine were included in their culturing media. Based on data generated from the systematic review and the inconsistencies in reported data in outlining the specific culture formulations, we investigated the impact of ketone bodies and glucose deprivation, and pyruvate and glutamine deprivation on neural stem and neural precursor cell growth. Ketone supplementation did not reveal an effect on cell growth and health in culture formulations deprived of glucose but containing glutamine and pyruvate. Given the potentially confounding effects of other substrates in media formulations, the next set of experiments aimed to clarify the metabolic roles of culture substrates on the growth and health of NE-4C neuroepithelial cells and SH-SY5Y human NB cells in the presence and absence of βOHB. SH-SY5Y and NE-4C cells survived and appeared morphologically healthy in media deprived of all anaplerotic inputs and supplemented with ketone bodies; however, their growth patterns and metabolic activities were reduced. Pyruvate appeared to mask the effects of ketone supplementation, most likely due to competition at the monocarboxylate transporters, which shuttles βOHB into cells, but is also involved in lactate, an end-product of glycolysis, efflux. Experimental analysis enabled the development of standard media control culture conditions for use in future experiments, in which βOHB appeared most available for metabolism. This standard media culture condition consisted of low glucose, supplemented with βOHB in the absence of pyruvate and glutamine. This study next aimed to reveal the metabolic roles of glucose, pyruvate and glutamine on the lineage potentials and metabolic health of differentiating NE-4C and SH-SY5Y cells in the presence and absence of βOHB as their metabolic profile shifts from aerobic glycolysis to oxidative phosphorylation (OXPHOS). Glucose and substrate deprivation is detrimental to NE-4C and SH-SY5Y cell metabolic heath and phenotype during differentiation. Supplementation with βOHB over 5 and 10 days in vitro (DIV) failed to rescue cell growth in both lines; however, cells were partially rescued at 15DIV, indicating a shift from glycolytic metabolism to OXPHOS. From the data gathered in the systematic review and experiments undertaken in the present study, the role of culture metabolic substrates in experimental paradigms appears to be critical when understanding how cells tolerate and grow in media supplemented with βOHB and deprived of glucose. Pyruvate appears to play a key role in brain cell metabolism in the absence of glucose in culture, which impacts their capacity to metabolise ketone bodies. These data contribute to research underlying the role of ketone body metabolism on neural stem and neural progenitor growth in vitro and may have implications for our understanding of the roles of ketone body metabolism on brain growth and function