Epinephrine responses to hypoglycemia are probably

Epinephrine responses to hypoglycemia are probably triggered centrally, perhaps with input from peripheral hypoglycemia sensing [3], [4]. We observed increased epinephrine responses in GCK-MODY and I366F diabetic mice. Consistent with a role for Triflusal mg GCK in detecting a low blood glucose, we also saw increased epinephrine responses to hypoglycemia in brain GCKKO mice. It is unclear why there were no observed differences in epinephrine responses to hypoglycemia in I366F mice given the findings in the other 3 models studied. The clamp protocol in I366F mice was different from that used for I366F diabetic and brain GCKKO mice. However, we would anticipate that the double catheter technique with mice being free moving and unhandled would carry less potential for the confounding effects of stress from study conditions. In these murine studies, we were limited by sampling volumes to a single measure of counter-regulation at the end of studies and it is possible that a more complete time series might have unmasked differences. Brain GCK expression is restricted to key brain glucose-sensing areas including areas in the basomedial hypothalamus such as the ARC and VMN [16], [17], [18], [19]. A recent study examined electromagnetic inhibition of VMN GCK-neurons, finding blunted hyperglycemic response to 2-deoxyglucose induced glucopenia (although individual counter-regulatory hormone responses were not reported) [38]. A previous study used short hairpin RNA-knockdown of VMN GCK in rats, reporting increased epinephrine responses to non-clamped (insulin-bolus induced) hypoglycemia [39]. It is possible that VMN neurons involved in GCK-mediated counter-regulation will be non-SF1 cells given that genetic inactivation of GCK specifically within VMN SF1 cells does not increase epinephrine or glucagon release in response to hypoglycemia [40]. We did not see significantly different responses between GCK-MODY and T2D groups for the other measured counter-regulatory hormones in human studies. This suggests either that GCK is less important in these responses and/or that adaptation occurs. Transcriptional profiling of hypothalamic GCK cells in a model revealed a population of hypoglycemia-activated growth hormone releasing hormone cells, suggesting a role for brain GCK in generating growth hormone responses to hypoglycemia [20]. It is unclear if this reflects species differences. Given the volumes required for sampling, it was not possible to measure additional hormones in our mice models. Two studies in humans examined brain GCK and hypoglycemia indirectly, using fructose infusion and hypothesizing that this acted via increased brain fructose-6 phosphate to activate GCK regulatory protein (GKRP), leading in turn to inhibition of brain GCK. Systemic fructose infusion amplified glucagon and epinephrine responses to hypoglycemia in healthy subjects and increased epinephrine responses in patients with type 1 diabetes [41], [42]. However, it is unclear whether brain GCK inhibition is indeed the mechanism of action of fructose which may have other actions such as hypothalamic AMPK activation [43]. The strengths of our experimental approach are that we studied defined molecular perturbations in GCK and used insulin clamp techniques across all models to create carefully controlled and matched hypoglycemic challenges (as opposed to insulin bolus-induced hypoglycemia or glucoprivation). Of note, each of our three murine models (I366F, β-cell ablated I366F and brain GCKKO) were studied on different genetic backgrounds and/or gender. Previous data have shown quantitative differences between strains in the magnitude and threshold of counter-regulatory responses to hypoglycemia [32]. We used female mice in our streptozotocin studies to maximize efficient use of the I366F breeding colony. Gender differences in counter-regulation have been reported in human and murine studies [40], [44]. It is thus possible that background strain and/or gender might have altered our findings.