Impaired Glutamatergic Neurotransmission in the VMH May Contribute to Defective Counterregulation in Recurrently Hypoglycemic Rats.

: The objectives of this study were to understand the role of ventromedial hypothalamic (VMH) glutamatergic neurotransmission in response to hypoglycemia and to elucidate the effects of recurrent hypoglycemia (RH) on this neurotransmitter. We measured 1) changes in interstitial VMH glutamate levels using microdialysis and biosensors, 2) identified the receptors that mediate glutamate’s stimulatory effects on the counterregulatory responses, 3) quantified glutamate metabolic enzyme levels in the VMH, 4) examined astrocytic glutamate reuptake mechanisms and 5) used 1 H-[ 13 C]-NMR spectroscopy to evaluate the effects of RH on neuronal glutamate metabolism. We demonstrated that glutamate acts through kainic acid receptors in the VMH to augment the counterregulatory responses. Biosensors showed the normal transient rise in glutamate levels in response to hypoglycemia is absent in RH animals. More importantly, RH reduced extracellular glutamate concentrations and this was due partly to decreased glutaminase expression. Decreased glutamate was also associated with reduced astrocytic glutamate transport in the VMH. NMR analysis revealed a decrease in 13 C 4 -glutamate but unaltered 13 C 4 -glutamine concentrations in the VMH of RH animals. Our data suggests glutamate release is important for proper activation of the counterregulatory response to hypoglycemia and impairment of glutamate metabolic and re-synthetic pathways with RH may contribute to counterregulatory failure. To assess the effect of deprivation on astrocytic primary rat (Invitrogen) were cultured in DMEM containing high glucose, and were until they reached 90% confluence. Subsequently, 1x10 6 cells were transferred to 6-well plates with fresh medium containing 2.5mM glucose. Controls were incubated in 2.5mM glucose medium. Additional wells of cells were initially incubated in 2.5mM glucose medium before undergoing the recurrent glucose deprivation protocol: the was to 0.2mM glucose for 3hrs/day before being changed back to fresh DMEM containing 2.5mM glucose. This procedure was repeated for 3 consecutive days. On day 4, glutamate uptake was evaluated in the astrocyte cultures under baseline conditions, where the astrocytes were incubated in 2.5mM glucose medium or following a low glucose challenge by switching to medium containing 0.2mM glucose for 90mins. To assess glutamate uptake, the astrocytes were washed twice with PBS buffer that was warmed to 37 o C for 10min. The PBS buffer was removed and the cells incubated for 5min in 0.5mL fresh PBS buffer containing 100uM unlabeled glutamate and 0. 2µCi/mL 14 C-labeled glutamate for 5min. After 5min, the reaction was stopped by washing the cells twice in ice cold Subsequently, the cells were solubilized in 0.5mL 1% SDS/0.1N NaOH solution and counted (Tri-Carb 2800TR LSC, Perkin Elmer). Radioactivity count was standardized to total protein content. A second subset of cells were treated as described above (without undergoing the uptake assay) on day 4 for of GLT-1 mRNA qRT-PCR. lower in the VMH of RH animals at baseline and their metabolic response to hypoglycemia is markedly blunted to nil. The relative maintenance of labeled glutamine in astrocytes suggests that glial metabolism is less affected (or possibly augmented) by RH. However, RH VMH glutamatergic neurotransmission by reducing the capacity for glutamate re-synthesis from glutamine in neurons (PAG reduction) which ultimately reduces neurotransmitter glutamate release. In addition to the changes in neurons, RH led to significant reductions in the astroglial transporter, GLT-1, which may be an adaptation to reduced interstitial glutamate levels. Taken together, our findings indicate that reduced VMH glutamatergic neurotransmission contributes to the development of defective counterregulation in RH animals. Treatments which normalize interstitial glutamate levels by augmenting neuronal PAG or enhancing glutamatergic function could be of potential therapeutic value. While our studies identified defects in pre-synaptic glutamatergic neurons, we have not identified the origins of these neurons nor the post-synaptic targets of VMH kainic acid receptors which will be the focus of future studies.