146-OR: Causative Role of Hypothalamic Ventromedial Glucose-Inhibited Neurons in Impaired Counterregulatory Responses to Hypoglycemia

The development of impaired counterregulatory responses (CRR) after recurrent hypoglycemia (RH) is one of the most life-threatening and debilitating aspects of Type 1 Diabetes (T1D). Numerous scientific evidence points to the ventromedial nucleus of the hypothalamus (VMH) glucose-inhibited (GI) neurons as major players in the development of impaired CRR. However, there is no current evidence of a causative role of VMH GI neurons in impaired CRR. In this study, we used a protocol in which mice were injected with insulin (2.5 U/kg, i.p.) for 4 consecutive days. These mice displayed impaired CRR after 2-deoxy-D-glucose (2-DG) administration (200 mg/kg, i.p. injected on the 5^th day) with blunted levels of circulating glucose, corticosterone, and epinephrine in comparison to mice with intact CRR after 2-DG administration. Using a TRAP2:tdTomato mouse model, we labeled (tdTomato) GI neurons by injecting 4-hydroxytamoxifen after 2-DG administration. Four weeks later, the mice were injected with either insulin or vehicle for 4 days followed by 2-DG administration, of which the brains were then immunostained and analyzed for cFos in the VMH. We observed a significant decrease in double-labeled tdTomato/cFos cells in mice with impaired CRR compared to the controls, thus suggesting an altered VMH GI neuronal activation following insulin-induced RH. In support of this, chemogenetic activation of VMH GI neurons in mice with impaired CRR significantly increased blood glucose levels following 2-DG administration compared to mice with impaired CRR. Overall, our data provide evidence for a direct and causal involvement of VMH GI neurons in the development of impaired CRR following RH.