Examination of the Influence of Leptin and Acute Metabolic Challenge on RFRP-3 Neurons of Mice in Development and Adulthood

Abstract

Background: The neuropeptide RFamide-related peptide-3 (RFRP-3; mammalian ortholog to gonadotropin-inhibiting hormone) can inhibit luteinizing hormone (LH) release and increases feeding, but the regulation and development of RFRP-3 neurons remains poorly characterized, especially in mice. Methods and Results: We first confirmed that peripheral injections of murine RFRP-3 peptide could markedly suppress LH secretion in adult mice, as in other species. Second, given RFRP-3's reported orexigenic properties, we performed double-label in situ hybridization for metabolic genes in Rfrp neurons of mice. While Rfrp neurons did not readily coexpress neuropeptide Y, thyrotropin-releasing hormone, or MC4R, a small subset of Rfrp neurons did express the leptin receptor in both sexes. Surprisingly, we identified no changes in Rfrp expression or neuronal activation in adult mice after acute fasting. However, we determined that Rfrp mRNA levels in the dorsal-medial nucleus were significantly reduced in adult obese (Ob) mice of both sexes. Given the lower Rfrp levels observed in adult Ob mice, we asked whether leptin might also regulate RFRP-3 neuron development. Rfrp gene expression changed markedly over juvenile development, correlating with the timing of the juvenile ‘leptin surge' known to govern hypothalamic feeding circuit development. However, the dramatic developmental changes in juvenile Rfrp expression did not appear to be leptin driven, as the pattern and timing of Rfrp neuron development were unaltered in Ob juveniles. Conclusion: Leptin status modulates RFRP-3 expression in adulthood, but is not required for normal development of the RFRP-3 system. Leptin's regulation of adult RFRP-3 neurons likely occurs primarily via indirect signaling, and may be secondary to obesity, as only a small subset of RFRP-3 neurons express the long form of the leptin receptor (LepRb).

The involvement of gonadotropin inhibitory hormone and kisspeptin in the metabolic regulation of reproduction

“…These neurons, containing KP and GnIH, are also expressing receptors of the metabolic hormones (Smith et al 2006, Poling et al 2014. Importantly, the activities of these neurons, as will be discussed below, are altered during negative energy balance conditions (Castellano & Tena-Sempere 2013, Wahab et al 2013a, Roa & Tena-Sempere 2014.…”

“…Important candidates in this respect are peripheral metabolic cues and their receptive hypothalamic neuronal circuits. Important metabolic cues are adiponectin, glucose, ghrelin, cortisol, insulin, leptin, or other metabolic molecules (Kinoshita et al 2003, Budak et al 2006, Wahab et al 2010, 2011c, 2013a, Poling et al 2014, Roa & Tena-Sempere 2014. Glucose is a ubiquitous cue for all organisms.…”

“…Although recently it has been noted that GnIH neurons are not responsive to leptin, glucocorticoid receptor expression has been observed on GnIHcontaining neurons which are upregulated by both acute and chronic stress (Gojska & Belsham 2014, Poling et al 2014, Rizwan et al 2014. As fasting is a type of metabolic stress and an increase in glucocorticoid has been reported in this condition (Wahab et al 2008), it is possible that glucocorticoid may alter the GnIH system, which may communicate inhibitory information to the reproductive axis (Smith & Grove 2002).…”

“…As a number of studies have reported that leptin infusions also rescue reproductive quiescence in Ob/Ob mice (Mounzih et al 1997, Cleary et al 2001, we cautiously propose that the impaired KPKiss1r signaling contributes at least partly to reproductive impairments caused by leptin deficiency. More recently, analyses of the function of leptin on the GnIH-GnIHR system have been carried out in these mice (Poling et al 2014, Rizwan et al 2014. Only a small subset of GnIH neurons expresses the LepR (Poling et al 2014).…”

Neuroendocrine integration of nutritional signals on reproduction

“…Among these, for example, are RFamide-related peptide (RFRP), melanin-concentrating hormone (MCH) and orexin-expressing neurons. A small subpopulation of RFRP neurons, which are the mammalian orthologs to avian gonadotropin-inhibiting hormone neurons, were recently shown to co-express LepR (Poling et al 2014), for example. However, leptin's regulation of RFRP neurons appears to occur via indirect signaling (Rizwan et al 2014).…”