SAT-407 Evidence for Impaired Kisspeptin Signaling During Metabolic Stress in Female Mice

Stress impairs reproduction, at least in part, by suppressing gonadotropin secretion. One common metabolic stressor is hypoglycemia, which occurs in a variety of pathologies and in healthy people, particularly athletes. Insulin-induced hypoglycemia (IIH) is a repeatable, reliable, and quantifiable acute model of metabolic stress that inhibits pulsatile luteinizing hormone (LH) secretion, though little is known about the underlying mechanism. We have observed that IIH robustly suppresses LH pulses in female mice, which could be mediated by either a pituitary or hypothalamic effect. In the present study, we performed a kisspeptin challenge to determine whether downstream cells involved in LH pulsatility are impaired during IIH. Adult mice (C57/BL6, n = 8/group) were ovariectomized and acclimated to handling for tail-tip blood collection and i.p. injection. On the day of each challenge, mice were fasted for 5 hr and then received an i.p. injection of either saline or insulin (0.75mU/kg). All animals then received an injection of kisspeptin (2μg/g), 45 min later. This time point represents a consistent period of LH pulse suppression following IIH. Frequent blood samples were collected for 30 min before and after kisspeptin injection. Two-weeks later, the experiment was repeated with each mouse receiving the alternate treatment (i.e. insulin or saline) in a cross-over design. Mean LH concentrations were significantly increased after kisspeptin injection in animals treated with saline (saline, pre: 3.8 ± 0.6 vs. post: 7.8 ± 0.5 ng/ml). Interestingly, the LH response to kisspeptin in animals treated with insulin was also markedly increased (insulin, pre: 3.2 ± 0.6 vs. post: 6.4 ± 0.3 ng/ml), but not significantly different from animals treated with saline. We conclude from this study that impaired GnRH or gonadotrope cell function is not the primary cause of reduced LH secretion in this model of metabolic stress. Since kisspeptin neurons in the arcuate nucleus likely form the GnRH/LH pulse generator, we next determined whether arcuate kisspeptin cells were inhibited during IIH. For this purpose, we used a mouse model expressing GFP in kisspeptin cells (Kiss1-Cre-GFP). Mice were fasted and injected with insulin or saline, as above, and fixed neural tissue was collected for assessment of c-Fos immunoreactivity in arcuate Kiss1 cells. Two hours following saline injection ~20% of Kiss1 neurons contained c-Fos. In contrast, only 6.6 ± 0.5% of Kiss1 neurons contained c-Fos in animals treated with insulin. Thus, reduced gonadotropin secretion in IIH may be mediated by an inhibition of the ARC kisspeptin cells. Together these data support the hypothesis that inhibition of LH pulses by metabolic stress is mediated by neural factors including arcuate kisspeptin cells. Future work will investigate kisspeptin cell afferents that convey the inhibitory signal initiated by metabolic stressors.