Hypothalamic RFamide-related peptide-3 neurons do not play a major role in mediating the actions of leptin or insulin in mice

Leptin, an adipose-derived anorectic hormone, modulates a number of hypothalamic functions including a permissive regulation of fertility. Leptin ‘sends’ information to the hypothalamic gonadotrophin-releasing hormone (GnRH) neuronal system regarding the body’s metabolic status. Our laboratoy has shown that leptin does not directly act on GnRH neurons, and the indirect mechanisms by which leptin modulates GnRH release remains unclear. When the hypothalamic leptin receptor is stimulated, signal transducer and activator of transcription-3 (STAT3) is phosphorylated mainly in the regions of the arcuate nucleus, ventral premamillary nucleus, medial preoptic area, and to a lesser extent the dorsomedial hypothalamus (DMH). Insulin on the other hand is a hormone produced from the pancreas, and its main role is the regulation of fat and carbohydrate metabolism by insulin-sensitive tissues such as the liver, skeletal muscle and adipose tissue. It has also been shown to play a role in fertility. Similar to leptin, insulin responsive cells have been located in various regions of the hypothalamus and to a lesser extent in the DMH, but insulin probably does not directly regulate GnRH neurons. RFRP-3 neurons are located in the DMH, and have been shown to directly act on GnRH neurons to inhibit reproduction. We tested whether leptin or insulin could indirectly act via RFRP-3 neurons to regulate GnRH activity. To examine this, I used three approaches. First, the presence of leptin-induced STAT3 phosphorylation (pSTAT3), and insulin-induced Akt phosphorylation (pAkt) in RFRP-3 neurons were examined in female and male wild type C57BL/6J mice. Mice were given an acute leptin (1 mg/kg) and insulin (1IU/kg) challenge followed by brain fixation by perfusion and dual label immunohistochemistry for RFRP-3 and pSTAT3 or RFRP-3 and pAkt. Results revealed that although being present in the DMH and in close proximity to RFRP-3 neurons, there was no colocalization between RFRP-3 neurons and pSTAT3 or pAkt. Secondly, we investigated the effects of leptin deficiency on numbers of RFRP-3 neurons. In both wt and leptin-deficient ob/ob male and female mice, no significant effect of leptin deficiency in the number of immunoreactive RFRP-3 cell bodies was observed. However, males expressed a significantly higher number of RFRP-3 immunoreactive cells compared to females, regardless of their genotype. Finally, qPCR was used to compare RFRP mRNA levels between leptin-deficient ob/ob mice and wild type mice of both sexes. There was no significant difference between the levels of RFRP mRNA in wild type versus ob/ob males and females. Collectively, these results indicate that although it is able to act in the same region, leptin does not act on RFRP-3 neurons to modulate fertility. As for insulin, the preliminary data suggests that RFRP-3 does not mediate insulin’s actions on fertility. However, due to the cytoplasmic nature of pAkt, the current tools available are insufficient to study this conclusively. New tools, such as genetically modified cre-lox GFP mice for insulin receptors and RFRP-3 are currently being developed and will be used to investigate this matter in the near future