Estrogen (E2) has been demonstrated to possess protective effects from hypoglycemic toxicity, particularly in the pancreas. In the central nervous system, several brain regions, such as the hypothalamus, are highly vulnerable to hypoglycemic injuries that may lead to seizures, coma, and mortality. The present study performed a novel in vitro assay of hypoglycemic injury to hypothalamic cells, and is the first study, to the best of our knowledge, to demonstrate that E2 protects hypothalamic cells from hypoglycemic toxicity. The toxic effects of hypoglycemia on hypothalamic cells in vitro was determined by performing cell counts, together with MTT and lactate dehydrogenase assays, using the N38 murine hypothalamic cell line. Following 24 and 48 h in hypoglycemic conditions, a 60 and 75% reduction in cell number and mitochondrial function was observed, which reached 80 and ~100% by 72 and 96 h, respectively. E2 treatment prevented the hypoglycemia‑induced loss in cell number and mitochondrial toxicity at 24 and 48 h. However at 72 and 96 h of hypoglycemic conditions, the neuroprotective effects of E2 on cell number or mitochondrial function was not significant or not present at all. In order to determine whether E2 exerted its effects through the AKT signaling pathway, the expression of proline‑rich AKT substrate of 40 kDa (PRAS40) was analyzed. No alterations in PRAS40 expression were observed when N38 cells were exposed to hypoglycemic shock. From the biochemical and molecular data obtained, the authors speculated that E2 exhibits neuroprotective effects against hypoglycemic shock in hypothalamic cells, which dissipates with time. Despite demonstrating no significant effect on total AKT/PRS40 activity, it is possible that E2 may mediate these neuroprotective effects by upregulating the phosphorylated‑AKT/pPRAS40 signaling pathway. The present study presented, to the best of our knowledge, the first in vitro model for hypoglycemic toxicity to hypothalamic cells, and provided evidence to suggest that E2 may protect hypothalamic cells from the damaging effects of hypoglycemia.
Glucose is the main metabolic fuel of the brain. and its availability is directly linked to neuronal activity. In the absence of glucose i.e. hypoglycemia, there is loss of neurons, impaired IQ and can ultimately lead to morbidity. In the present study we have reported the neuroprotection of estrogen on hypoglycemic injury in hypothalamic cell line. There was a marked decrease in the cell count and mitochondrial function when exposed to hypoglycemia which increased in presence of estrogen (42.8 vs 98.5, 24 hours, p<0.0001). The attenuation of the death pathways was only seen within the first 24 hours and gradually decreased with time. DNA microarray studies showed AKT, a promoter of cell survival, was down‐regulation in the absence of glucose and in the presence of estrogen an increase in gene expression. RTPCR showed decrease in AKT expression with a slight increase in presence of estrogen. PRAS 40, proline rich substrate for AKT was measured. Results show low expression of PRAS40 in hypoglycemic conditions in 24 hours and 72 hours with a trend to increase in presence of estrogen. Therefore, it seems that one of the mechanism by which estrogen shows its neuroprotection is through the Akt‐GSK3b pathway activation.
Glucose is the main metabolic fuel of the brain. Recurrent episodes of hypoglycemia can lead to seizures, coma, and even death. The hypothalamus; a region involved in feeding and energy balance is most vulnerable to this injury. In the present study we report neuroprotection of estrogen on hypoglycemic injury in a hypothalamic cell line. The cell viability, toxicity and proliferation were determined using MTT, lactate dehydrogenase (LDH) assay, and cell count. There was a significant increase in the cell count and increase in mitochondrial function in the presence of estrogen (p<0.0001) when exposed to hypoglycemia within 24 hours. The attenuation of the death pathways was only seen within the first 24 hours and gradually decreased with time. Percent cytotoxicity also increased in the absence of glucose and decreased in the presence of estrogen within 24 hours. Two pathways seemed viable in mediating estrogen action, the phospho‐AKT pathway, and a pathway involving caveolin‐1 (CAV‐1). In order to determine estrogen mode of action, PRAS 40 was analyzed via ELISA assay, which showed no change when exposed to hypoglycemic shock. This suggested that the phospho‐AKT pathway was not involved in estrogen mode of action. Our current efforts are focused on determining whether estrogen neuroprotection is mediated through CAV‐1 and re‐testing the phospho‐AKT pathway via western blot analysis.
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