Nesfatin-1, identified as an anorexigenic peptide, regulates the energy metabolism by suppressing food intake. The majority of nesfatin-1-synthesizing neurons are concentrated in various hypothalamic nuclei, especially in the supraoptic (SON), arcuate (ARC) and paraventricular nuclei (PVN). We tested the hypothesis that the glutamatergic system regulates nesfatin-1 neurons through glutamate receptors. Therefore, the first aim of the proposed studies was to examine effects of different glutamate agonists in the activation of nesfatin-1 neurons using c-Fos double immunohistochemical labeling. Experimental groups were formed containing male and female rats which received intraperitoneal injections of glutamate agonists kainic acid, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) while the control rats received vehicle. The significant increase in the number of c-Fos-expressing nesfatin-1 neurons after agonist injections were observed both in female and male subjects and some of these effects were found to be sexually dimorphic. In addition, treatment with specific glutamate antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or dizocilpine (MK-801) before each of the three agonist injections caused a statistically significant reduction in the number of activated nesfatin-1 neurons in the hypothalamic nuclei including supraoptic, paraventricular and arcuate nuclei. The second aim of the study was to determine the expression of glutamate receptor subunit proteins in the nesfatin-1 neurons by using a double immunofluorescence technique. The results showed that the glutamate receptor subunits, which may form homomeric or heteromeric functional receptor channels, were expressed in the nesfatin-1 neurons. In conclusion, the results of this study suggest that nesfatin-1 neurons respond to glutamatergic signals in the form of neuronal activation and that the glutamate receptors that are synthesized by nesfatin-1 neurons may participate in the glutamatergic regulation of these neurons.

Neuronostatin, a newly identified anorexigenic peptide, is present in the central nervous system. We tested the hypothesis that neuronostatin neurons are activated by feeding as a peripheral factor and that the glutamatergic system has regulatory influences on neuronostatin neurons. The first set of experiments analyzed the activation of neuronostatin neurons by refeeding as a physiological stimulus and the effectiveness of the glutamatergic system on this physiological stimulation. The subjects were randomly divided into three groups: the fasting group, refeeding group, and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)+refeeding group. We found that refeeding increased the phosphorylated signal transducers and transcription activator-5 (pSTAT5) expression in neuronostatin-positive neurons and that the CNQX injection significantly suppressed the number of pSTAT5-expressing neuronostatin neurons. The second set of experiments analyzed the activation pathways of neuronostatin neurons and the regulating effects of the glutamatergic system on neuronostatin neurons. The animals received intraperitoneal injections of glutamate receptor agonists (kainic acid, α-amino-3-hydroxy-5methyl-4-isoazepropionic acid (AMPA), and N-methyl-D-aspartate (NMDA)) or 0.9% NaCl. The number of c-Fos-expressing neuronostatin neurons significantly increased following the AMPA and NMDA injections. In conclusion, we found that the neuronostatin neurons were activated by peripheral or central signals, including food intake and/or glutamatergic innervation, and that the glutamate receptors played an important role in this activation.

The R-spondin protein family is a group of proteins that enhance Wnt/b-catenin signaling and have pleiotropic functions in stem cell growth and development. In the literature reviews, there is no histomorphological study showing the localization and distribution of R-spondins in different hypothalamic nuclei. For this reason, the purpose of this study was to determine the localization, distribution characteristics, and densities in the hypothalamic nuclei of neurons expressing Rspo1 and Rspo3 proteins. The free-floating brain sections of the male rats who were not exposed to any treatment were stained with the indirect immunoperoxidase method using the relevant antibodies. As a result of the immunohistochemical studies, it was determined that neurons expressing the Rspo1 protein were found in large numbers in the supraoptic nucleus (SON), the suprachiasmatic nucleus (SCh), anterior paraventricular nucleus, periventricular hypothalamic nucleus (PeV), anterior hypothalamic area, magnocellular preoptic nucleus (MCPO) and the lateral hypothalamic area (LH) from the hypothalamic nuclei, while they were localized in fewer numbers in the arcuate nucleus (ARC). Rspo3 protein expression was found in neurons localized in the hypothalamic nuclei SON, paraventricular nucleus (PVN), PeV, ARC, ventromedial nucleus (VMH), LH, anterior parvicellular nucleus, and zona inserta (ZI). In addition, neurons synthesizing both peptides were found in the cortex and hippocampus regions (H). Rspo1 and 3 proteins are expressed in hypothalamic energy homeostatic areas, thus these proteins may be involved in the regulation of food intake.

Graphical Polycystic ovary syndrome (PCOS) is a complex reproductive–endocrine disorder characterized by impaired folliculogenesis, anovulation, and hormonal imbalance. While PNX-14 is known as a hypothalamic peptide that modulates GnRH signaling, emerging evidence indicates its local ovarian expression, particularly in granulosa and luteal cells, suggesting potential peripheral roles in follicular development. This study aimed to evaluate the therapeutic potential of PNX-14 in a dehydroepiandrosterone (DHEA)-induced rat model of PCOS, with a focus on its potential peripheral effects on ovarian tissue. Female rats were divided into control, PCOS, and PCOS + PNX-14 (2.5, 5, or 30 nmol) groups. PCOS was induced by DHEA administration (60 mg/kg/day, s.c.) for 21 days. Vaginal cytology, hormone assays (FSH, LH, E2, P4, and testosterone), histological analysis, PCNA immunohistochemistry, and western blot were performed. DHEA-induced PCOS rats exhibited disrupted estrous cycles, suppressed gonadotropins and estradiol, and polycystic ovarian morphology with extensive follicular atresia and reduced PCNA expression. PNX-14 treatment, particularly at 5 nmol, significantly improved estrous cyclicity, restored primordial and antral follicle populations, reduced atretic and cystic follicles, and normalized PCNA levels in granulosa cells. Serum hormones showed trends toward recovery without consistent statistical significance after 3 days of treatment. This study provides the first in vivo evidence that peripheral administration of this peptide ameliorates key morphological and cellular features of PCOS in rats, primarily by enhancing granulosa cell proliferation and early folliculogenesis. These findings support further investigation into PNX-14 as a novel therapeutic candidate as a local ovarian modulator for PCOS, potentially offering disease-modifying effects beyond conventional symptom-targeted therapies. Lay summary: PCOS is a leading cause of infertility and affects millions of women worldwide. It disrupts normal egg development, causing hormonal imbalances and cyst formation in the ovaries. Current treatments mainly control symptoms but do not repair the ovary itself. We investigate PNX-14, a natural peptide present in the brain and ovary that supports early egg growth. We tested this peptide found in the brain and ovary that supports early egg growth, in a well-established rat model of PCOS. After three days of treatment, it improved ovarian structure and restored regular reproductive cycling. Importantly, it significantly reduced degenerating follicles, helping to preserve healthy early-stage follicles. These benefits occurred even though major blood hormone levels were largely unchanged, suggesting that this peptide acts locally within the ovary. This is the first in vivo evidence that it can directly counter key cellular and structural damage caused by PCOS. The findings highlight PNX-14 as a potential new therapy to protect ovarian health and improve fertility in people with PCOS.

R-spondin 1 (RSPO1) and R-spondin 3 (RSPO3) contribute to reproductive development and feeding regulation, but their hypothalamic glutamatergic regulation remains unclear. We hypothesized that NMDA and non-NMDA receptor mechanisms contribute to hypothalamic RSPO neuronal activation. Given steroid-sensitive hypothalamic glutamatergic signaling, we compared activation and antagonist-mediated attenuation between sexes. Adult male and metestrous female Sprague–Dawley rats received kainic acid or AMPA with or without CNQX pretreatment, or NMDA with or without MK-801 pretreatment. RSPO/c-Fos labeling assessed activation in the supraoptic (SON), paraventricular (PVN), and arcuate (ARC) nuclei; ELISA assessed accompanying changes in serum RSPO concentrations. All agonists activated both populations in all nuclei. Only kainate-induced PVN RSPO3 activation differed between sexes (females, 89.3%; males, 72.8%; unadjusted p = 0.039), an isolated finding among 18 agonist comparisons. Antagonists attenuated all responses, although activation remained above control in 17 of 36 conditions. Females showed greater attenuation in selected kainate and NMDA conditions in the SON and PVN. Serum changes did not parallel neuronal activation. These findings support NMDA and non-NMDA receptor involvement and sex-dependent attenuation of RSPO neuronal activation. Given the central feeding effects of RSPOs, their sex-dependent glutamatergic regulation within neuroendocrine nuclei may contribute to sex differences in metabolic control.

Merkezi sinir sisteminde (MSS) nöron fonksiyonlarının düzenlenmesinde çok çeşitli nörotransmitter madde rol oynar. Glutamat, hipotalamusta en önemli eksitatör amino asit nörotransmitterlerden biri olarak yer alır ve nöronlar üzerindeki düzenleyici işlevini iyonotropik ve metabotropik olarak iki alt ailede sınıflandırılan reseptörlerine bağlanarak gerçekleştirir. Glutamatın bazı nöroendokrin nöronlar üzerinde etkili olduğu yapılan elektrofizyolojik ve farmakolojik çalışmalarla belirlenmiştir. Çalışmamız kapsamında, glutamaterjik innervasyonun histomorfolojik olarak desteklendiği çalışmaların bulunmaması sebebiyle hipotalamik glutamat reseptör alt birimlerine odaklanılmıştır. İyonotropik glutamat reseptör alt birimlerinden AMPA (GluA1, GluA2, GluA3, GluA4), Kainik asit (GluK5, GluK1, GluK2, GluK3) ve NMDA’nın (GluN1, GluN2A) hipotalamustaki dağılımları immünohistokimya yöntemi kullanılarak araştırılmıştır. Dişi sıçanların beyin dokularından vibratom aracılığıyla alınan kesitler, hipotalamik nöronlardaki glutamat reseptör alt birimlerinin varlığı ve yoğunluğu açısından değerlendirilmiştir. Çalışmada, AMPA reseptörlerinden GluA1 ve GluA2’nin yaygın olarak hipotalamusta lokalize olduğu, GluA3 ve GluA4 immünoreaksiyonunun ise çok sınırlı olduğu; kainat reseptör alt birimlerinden GluK5’in çok yaygın olduğu ve GluK1/2/3 içeren nöronların daha az sayıda olduğu, NMDA reseptör alt birimlerinden GluN1’in ise hipotalamusta yoğun olarak bulunduğu saptanmıştır. Sonuç olarak, bu çalışma ile hipotalamik birçok nöronun iyon spesifik iGluR kanallarını farklı miktar ve yoğunlukta eksprese edebildiği, bunun da hedef nöronların glutamat tarafından farklı şekilde düzenlenmesine izin verebileceği gösterilmiştir.