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  • br Materials and methods br Results br Discussion


    Materials and methods
    Discussion Ghrelin is required for maintenance of glucose homeostasis under fasting conditions, and GHSR-null mice have low fasting blood glucose levels [5], [9]. In this study, we found that restoration of GHSR in nodose ganglion neurons rescued low fasting blood glucose levels, suggesting that peripheral ghrelin signal transmission to the CNS via the vagus nerve is crucial for regulating blood glucose control under fasting conditions. The ghrelin receptor modulates the activity of the circadian system under normal conditions. Under constant light conditions, acrophases in GHSR-KO mice occur 2 h later than that in wild type-mice [10]. GHSR-null mice, but not GHSR/Phox2b mice, exhibit dysregulation of day and night feeding patterns, suggesting that GHSR restoration could modulate the system. Deletion of hypothalamic NPY and AgRP abolishes induction of food intake by intraperitoneal ghrelin administration [11], and direct microinjection of ghrelin into the dorsal vagal complex stimulates food intake in wild-type mice [12]. We found that in GHSR/Phox2b mice, the GHSR Disuflo Cy5 azide level in the nodose ganglion was restored to 60% of that in wild-type mice, but ghrelin administration did not induce feeding. Mice fed a high-fat diet for more than 4 weeks had lower levels of Ghsr mRNA in their nodose ganglion neurons and did not exhibit ghrelin-induced feeding [13]. These results suggest that the amount of GHSR protein and/or the number of GHSR-expressing neurons in the nodose ganglion are critical factors in peripheral ghrelin signaling. Few previous studies have analyzed ghrelin signaling using vagus nerve–specific transgenic animals. Indeed, the only prior study analyzed ghrelin signaling in the hindbrain, but not in the nodose ganglion [4]. In this study, we analyzed the role of GHSR-expressing neurons in the nodose ganglion and obtained results consistent with those of the prior study. However, because we used conditional transgenic animals, it is possible that compensatory mechanisms may have affected our results. Therefore, it will be necessary to analyze the role of ghrelin signaling via the vagus nerve in more detail using vagus nerve–specific and inducible transgenic animals. Cre recombinase–mediated DNA recombination efficiency is thought to be affected by the chromosomal location of loxP sites [14], the distance between loxP sites [15], and cell type–specific epigenetic context of floxed loci [16]. Fahs et al., used three types of Cre systems (Cdh5(Mila)-Cre, Cdh5(Spe)-Cre, and Tek-Cre) to generate tissue-specific KO mice, and found that the Cre-mediated recombination efficiencies differed among them (Cdh5(Mila)-Cre << Cdh5(Spe)-Cre < Tek-Cre) [17]. This result suggests that Cre-mediated recombination efficiency is also a function of promoter type and construction. Previous reports showed that in Phox2b-Cre mice, Cre is expressed in all nodose ganglion neurons [18], [19], [20]. One possible explanation of the low GHSR expression in GHSR/Phox2b mice is that the Cre expression level in nodose ganglion neurons is insufficient for Cre-mediated recombination in GHSR/Phox2b mice. Our results suggest that the abundance of GHSR-expressing neurons is critical for peripheral ghrelin signaling. Specific knockdown of the GLP-1 receptor in rat vagal afferent neurons caused attenuation of its anorectic effect when administered intraperitoneally [21]. Together, the gastrointestinal peptides and their receptors in the nodose ganglion may play key roles in feeding behavior. Further studies are required to elucidate the mechanisms regulating the expression of peptide receptors in the nodose ganglion.
    Acknowledgements The authors thank Itsuki Morinaga and Eiko Kurata for excellent technical assistance. We thank J.M. Zigman, M.M. Scott, and J.K. Elmquist for the GHSR-null and Phox2b-Cre mice. This research was supported by JSPS KAKENHI under Grant Number 16H05333 and AMED-CREST under Grant Number JP17gm0610016.