Research articleCAPS2 deficiency induces proopiomelanocortin accumulation in pituitary and affects food intake behavior in mice
Introduction
Proopiomelanocortin (POMC) is a precursor polypeptide (241 amino acids) that undergoes cell type-specific proteolytic processing for the generation of ten different neuropeptides [1,2]. The predominant POMC synthesis sites in the central nervous system are the anterior and intermediate pituitary lobes, a small group of neurons in the solitary tract nucleus (NTS) of the brainstem, and the arcuate nucleus (ARC) of the hypothalamus. The tridecapeptide alpha-melanocyte-stimulating hormone (α-MSH) is the N-acetylated form of adrenocorticotropic hormone (ACTH)(1-13)NH2 (deacetylated α-MSH) derived from POMC. POMC neurons in the ARC project to the paraventricular nucleus (PVN) of the hypothalamus and regulate feeding, sexual behavior, and energy homeostasis via the activation of the α-MSH receptor, MC4R [3]. α-MSH produced in the pituitary is released into the blood and plays important roles in peripheral tissues, including the promotion of prolactin secretion [4,5]. Recently, blood α-MSH was reported to affect food intake by inducing peptide YY (PYY) and glucagon-like peptide-1 (GLP-1) secretion via activation of MC4R in enteroendocrine L cells, which predominate in the distal ilium and colon [6]. In addition, Mc3r or Mc4r knockout (KO) mice presented with obesity and hyperphagia [[7], [8], [9]]. Thus, the POMC–α-MSH pathway is considered to be important for energy homeostasis. However, the molecular mechanism underlying POMC-derived peptide secretion remains to be elucidated.
Ca2+-dependent activator protein for secretion 1 (CAPS1) regulates the release of catecholamines from neuroendocrine cells by promoting the priming step of dense-core vesicle (DCV) exocytosis triggered by an increase in intracellular Ca2+ [[10], [11], [12]]. We previously showed that CAPS2, a CAPS1 paralogue, enhanced the release of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) from neurons [[13], [14], [15]], indicating the involvement of CAPS proteins in exocytosis of neuropeptide-containing DCVs. Intriguingly, compared with control mice, Caps2-KO mice exhibited smaller body size, and lower body weight throughout the postnatal age [16,17]. Moreover, CAPS2 is expressed in mouse hypothalamus and pituitary, where several neuropeptides involved in regulation of appetite and energy expenditure are produced and/or released [18]. However, the involvement of CAPS2 in DCV-mediated release of these appetite/energy expenditure-related peptides has not yet been reported.
In this study, we analyzed CAPS2 expression in POMC-producing neurons of the hypothalamic ARC and the intermediate pituitary to investigate the involvement of CAPS2 in the release of POMC-derived peptides including α-MSH. We also assessed the effect of MC4R activation in Caps2-KO mice in regard to food intake and body weight. Our results suggest that CAPS2 regulates the secretion of POMC-derived α-MSH in the mouse hypothalamic ARC and intermediate pituitary and is thereby involved in feeding and energy metabolism.
Section snippets
Animals
We used male Caps2-KO mice [17] and their wild-type (WT) littermate controls (background: C57BL/6J). All mice (1–2-month-old) were group-housed and maintained under a 12:12 light-dark cycle. The experiments were approved by the Institutional Animal Care and Use Committee of Tokyo University of Science. All experiments were conducted in accordance with the Regulations for Animal Research of the Tokyo University of Science.
Antibodies
The following primary antibodies were used: guinea pig anti-CAPS2 (1:100) [
Distribution of CAPS2 in POMC-positive neurons in the hypothalamic ARC and pituitary
To assess the involvement of CAPS2 in POMC-derived peptide secretion, we analyzed the distribution of CAPS2 in the ARC neurons of hypothalamus using IHC. POMC expression was detected primarily in a punctate pattern in the ARC, whereas CAPS2 was expressed in the soma and also in probably synaptic areas in a punctate pattern (Fig. 1A). These CAPS2-immunopositive puncta were largely colocalized to POMC-immunopositive puncta (Fig. 1A-a, b, a + b). In addition to anorexigenic POMC neurons,
Discussion
In this study we showed the expression of CAPS2, a DCV exocytosis regulator, in the POMC-producing cells of the hypothalamic ARC and the intermediate pituitary lobe in mice. We suggest that CAPS2 is involved in the DCV-mediated exocytosis of POMC-derived peptides, including α-MSH, which modulate food intake and energy metabolism. We previously reported considerably smaller body size in mice lacking CAPS2-dependent facilitation of DCV exocytosis throughout their lifespan [16,17]. Collectively,
Declaration of competing interest
The authors declare no competing financial interests.
Funding
This work was supported by a Grant-in-Aid for KAKENHI from the Japan Society for the Promotion of Science (JSPS) and the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan [grant numbers 17H03563, 17K19638, 26290026, 15K14356, 15K21013].
CRediT authorship contribution statement
Shuhei Fujima: Conceptualization, Methodology, Investigation, Formal analysis, Writing - original draft. Natsuki Amemiya: Methodology, Investigation, Formal analysis. Tomoki Arima: Investigation, Data curation. Yoshitake Sano: Funding acquisition, Project administration. Teiichi Furuichi: Conceptualization, Funding acquisition, Supervision, Writing - review & editing.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgment
We thank Ms. Natsumi Shibano for technical assistance.
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Present address: Kobe University, Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuoh-ku, Kobe 650-0017, Japan.