AbstractsBiology & Animal Science

Central insulin signaling plays a key role in regulating the neuroendocrine reproductive axis. Mice exhibiting brain-specific insulin receptor gene (Insr) deletion (NIRKO mice) or diabetic insulin insufficiency display hypothalamic hypogonadism in association with absent or decreased insulin signaling. However, the specific neuronal target cells mediating insulin’s central effects on the reproductive axis remain largely unidentified. Using a combination of techniques, including immunohistochemistry to identify insulin responsive neurons as well as cell type-specific insulin receptor (InsR) knockout (KO) experiments, I investigated whether insulin’s central effects on the neuroendocrine reproductive axis are mediated directly via gonadotropin-releasing hormone (GnRH neurons), which are the final central drivers of reproductive function, or indirectly via putative insulin-sensitive neuronal populations implicated in the metabolic control of fertility. My first aim was to determine whether GnRH neurons are direct targets of insulin signaling. Encouragingly, I detected Insr mRNA and InsR protein in GnRH-immunolabeled neurons. However, I found no evidence of insulin-induced phosphorylated AKT (pAKT) or phosphorylated ERK1/2 (pERK1/2) in GnRH neurons. These results demonstrate GnRH neurons can directly respond to insulin; however, there was no clear evidence of insulin-induced signaling in GnRH neurons. This suggests insulin’s central effects on the reproductive axis are likely mediated by GnRH afferent neurons. Therefore, I next investigated whether insulin’s central effects on fertility are mediated via kisspeptin neurons, which are the most potent known activators of GnRH neurons. I used the Cre-lox system to generate kisspeptin-specific InsR knockout (KIRKO) mice and then assessed multiple reproductive parameters. Disappointingly, no significant differences in puberty onset, estrous cyclicity, or fertility were observed in the female or male KIRKO mice in comparison to their control littermates. The results from this experiment demonstrate InsR signaling via kisspeptin cells is not critically involved in mediating insulin’s central effects on the neuroendocrine reproductive axis. I then broadened my approach and next investigated whether insulin signaling via either gamma-aminobutyric acid (GABA) or glutamate-expressing neurons plays a critical role in the metabolic regulation of reproduction by generating GABA-specific and glutamate-specific InsR knockout mice, again using the Cre-lox system. Surprisingly, given the widespread expression of GABAergic and glutamatergic neurons, and hence the significant reduction in hypothalamic Insr mRNA expression in both GABA-specific and glutamate-specific InsR knockout mice, no reproductive abnormalities were found. However, female GABA-specific InsR knockout mice exhibited a significant metabolic phenotype, demonstrating insulin signaling via GABAergic neurons plays a critical role in the regulation of energy homeostasis. Nevertheless, the neuronal mediators of insulin’s…