Abstract
Hypoglycemia, a complication of insulin or sulfonylurea therapy in diabetic patients, leads to brain damage. Furthermore, glucose replenishment following hypoglycemic coma induces neuronal cell death. In this study, we investigated the molecular mechanism underlying glucose deficiency-induced cytotoxicity and the protective effect of d-β-hydroxybutyrate (D-BHB) using SH-SY5Y cells. The cytotoxic mechanism of metformin under glucose deficiency was also examined. Cell viability under 1 mM glucose (glucose deficiency) was significantly decreased which was accompanied by increased production of reactive oxygen species (ROS) and decreased phosphorylation of extracellular signal-regulated kinase (ERK) and glycogen synthase 3 (GSK3β). ROS inhibitor reversed the glucose deficiency-induced cytotoxicity and restored the reduced phosphorylation of ERK and GSK3β. While metformin did not alter cell viability in normal glucose media, it further increased cell death and ROS production under glucose deficiency. However, D-BHB reversed cytotoxicity, ROS production, and the decrease in phosphorylation of ERK and GSK3β induced by the glucose deficiency. ERK inhibitor reversed the D-BHB-induced increase in cell viability under glucose deficiency, whereas GSK3β inhibitor did not restore glucose deficiency-induced cytotoxicity. Finally, the protective effect of D-BHB against glucose deficiency was confirmed in primary neuronal cells. We demonstrate that glucose deficiency-induced cytotoxicity is mediated by ERK inhibition through ROS production, which is attenuated by D-BHB and intensified by metformin.
Highlights
Glucose is the key source of energy necessary for brain functioning, and hypoglycemia has damaging effects on various organs, including the brain [1]
We demonstrate that glucose deficiency-induced cell death is mediated by reactive oxygen species (ROS) production, which is attenuated by treatment with ketone body D-BHB
Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay showed that as time passed, cell viability was decreased from 2 h onwards, and it was significantly decreased at 8 h, and was further decreased at 24 h (Figure 1A)
Summary
Glucose is the key source of energy necessary for brain functioning, and hypoglycemia has damaging effects on various organs, including the brain [1]. D-BHB administration decreased ROS levels and reversed neuronal cell death in the cortex of hypoglycemic rats [12]. It was reported that lowering the glucose level potentiated metformin-induced cytotoxicity in breast cancer cells [25]. In this regard, the glucose level might account for this discrepancy between the anti-cancer effect and neuroprotection in SH-SY5Y cells. We examined the effects of metformin and D-BHB in a glucose deficient condition and elucidated their underlying molecular mechanisms. We demonstrate that glucose deficiency-induced cell death is mediated by ROS production, which is attenuated by treatment with ketone body D-BHB. Metformin has a cytotoxic effect only in a glucose deprivation condition, which is mediated by ROS production
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