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Gen Comp Endocrinol
2023 Jan 15;331:114167. doi: 10.1016/j.ygcen.2022.114167.
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Molecular cloning and analysis of the ghrelin/GHSR system in Xenopus tropicalis.
Wada R
,
Takemi S
,
Matsumoto M
,
Iijima M
,
Sakai T
,
Sakata I
.
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Ghrelin is a gut-derived peptide with several physiological functions, including feeding, gastrointestinal motility, and hormonal secretion. Recently, a host defense peptide, liver-expressed antimicrobial peptide-2 (LEAP2), was reported as an endogenous antagonist of growth hormone secretagogue receptor (GHS-R). The physiological relevance of the molecular LEAP2-GHS-R interaction in mammals has been explored; however, studies on non-mammals are limited. Here, we report the identification and functional characterization of ghrelin and its related molecules in Western clawed frog (Xenopus tropicalis), a known model organism. We first identified cDNA encoding X. tropicalis ghrelin and GHS-R. RT-qPCR revealed that ghrelin mRNA expression was most abundant in the stomach. GHS-R mRNA was widely distributed in the brain and peripheral tissues, and a relatively strong signal was observed in the stomach and intestine. In addition, LEAP2 was mainly expressed in intestinal tissues at higher levels than in the liver. In functional analysis, X. tropicalis ghrelin and human ghrelin induced intracellular Ca2+ mobilization with EC50 values in the low nanomolar range in CHO-K1 cells expressing X. tropicalis GHS-R. Furthermore, ghrelin-induced GHS-R activation was antagonized with IC50 values in the nanomolar range by heterologous human LEAP2. We also validated the expression of ghrelin and feeding-related factors under fasting conditions. After 2 days of fasting, no changes in ghrelin mRNA levels were observed in the stomach, but GHS-R mRNA levels were significantly increased, associated with significant downregulation of nucb2. In addition, LEAP2 upregulation was observed in the duodenum. These results provide the first evidence that LEAP2 functions as an antagonist of GHS-R in the anuran amphibian X. tropicalis. It has also been suggested that the ghrelin/GHS-R/LEAP2 system may be involved in energy homeostasis in X. tropicalis.
Fig. 1. Alignment of the deduced amino acid sequences of ghrelin in Xenopus tropicalis with sequences from the other vertebrates and phylogenetic analysis of ghrelin. (A) Multiple alignment of amino acid sequences of mature ghrelin in Xenopus tropicalis with ghrelin from the other amphibians. Identical amino acids among sequences are marked by asterisks. (B) Multiple alignment of amino acid sequences of prepro-ghrelin in Xenopus tropicalis with ghrelins from the other vertebrates. Identical amino acids among sequences are marked by asterisks. The mature ghrelin sequences are shaded, the proposed n-octanoic acid modified residues are in bold letters and typical dibasic processing amino acid sequences are boxed. The doted box indicates the putative obestatin sequences. (C) A phylogenetic tree showing the evolutionary relationships of the deduced amino acid sequences of Xenopus tropicalis ghrelin with those of other species was inferred by the neighbor-joining method using MEGA5. The numbers at tree nodes refer to percentage of trees in which the associated taxa clustered together in bootstrap test (1000 replicates). The evolutionary distances were computed using the p-distance method. The scale bar shows the genetic distance. Amino acid sequences are available from the GenBank databases (accession numbers BAA89371.1 (human; Homo sapiens), BAB19046.1 (mouse; Mus musculus), BAC24980.1 (chicken; Gallus gallus), NP_001118060.1 (rainbow trout; Oncorhynchus mykiss), NP_001077341.1 (zebrafish; Danio rerio), NP_001267573.1 (african clawed frog; Xenopus laevis), BAB71718.1 (american bullfrog; Rana catesbeianus), BAM29300.1 (japanese newt; Cynopus pyrrhogaster) and BAM65716.1 (red-eared slider; Trachemys scripta), respectively).
Fig. 2. Structure based alignment of the deduced amino acid sequences of GHS-R in Xenopus tropicalis with sequences from the other vertebrates and phylogenetic analysis of GHS-R. (A)Multiple alignment of the deduced amino acid sequence of Xenopus tropicalis GHS-R with the other amphibians and human GHS-R. Identical amino acids among sequences are shaded across all species. The amino acid sequences of seven transmembrane domains (TM1-TM7) predicted by the crystallization construct of human GHS-R (Shiimura et al., 2020) are boxed. Intra- and extracellular loops are marked by ICL and ECL, respectively. (B) A phylogenetic tree showing the evolutionary relationships of the deduced amino acid sequences of Xenopus tropicalis GHS-R with those of other species was inferred by the neighbor-joining method using MEGA5. The numbers at tree nodes refer to percentage of trees in which the associated taxa clustered together in bootstrap test (1000 replicates). The evolutionary distances were computed using the p-distance method. The scale bar shows the genetic distance. Amino acid sequences are available from the GenBank databases (accession numbers NP_940799.1 (human; Homo sapiens), NP_796304.1 (mouse; Mus musculus), NP_989725.1 (chicken; Gallus gallus), NP_001118066.1 (rainbow trout; Oncorhynchus mykiss), NP_001139744.1 (zebrafish; Danio rerio), XP_018121186.1 (african clawed frog; Xenopus laevis), BAB11343.1 (american bullfrog; Rana catesbeianus), BAS66832.1 (Japanese newt; Cynopus pyrrhogaster) and XP_034637794.1 (Red-eared Slider; Trachemys scripta), respectively).
Fig. 3. Functional analysis of Xenopus tropicalis GHS-R. Dose-response relationships of ghrelin-mediated intracellular Ca2+ mobilization in Chinese hamster ovary (CHO) -K1 cells transiently expressing Xenopus tropicalis GHS-R. (A) Dose-response curves showing GHS-R activation induced by Xenopus tropicalis ghrelin (black circle, EC50 = 2.74 nM) or human ghrelin (black triangle, EC50 = 4.84 nM). (B) Dose-response curves showing human LEAP2 antagonizing activation of GHS-R induced by EC80 (30 nM) of Xenopus tropicalis ghrelin (black circle, IC50 = 4.25 nM) and human ghrelin (black triangle, IC50 = 5.28 nM). (C) Dose-response curve of Xenopus tropicalis ghrelin-induced GHS-R activation. In the presence of 10 nM human LEAP2 (black triangle), the maximal activation efficacy of Xenopus tropicalis ghrelin was drastically decreased compared to without LEAP2 treatment (black circle). All experiments were performed independently-three times in duplicate. Representative results are expressed as mean ± SEM (n = 3). The EC50 and IC50 values were calculated by nonlinear regression using GraphPad Prism 5 software.
Fig. 4. Tissue distribution of ghrelin, LEAP2, GOAT and GHS-R mRNAs in Xenopus tropicalis. mRNA expression analysis of Xenopus tropicalis ghrelin (A), GHS-R (ghsr) (B), liver-enriched antimicrobial peptide 2 (leap2) (C), ghrelin O-acyltransferase (mboat4) (D), and nucleobindin 2 (nucb2) (D) in various tissues. RT-qPCR was performed using equal amounts of total RNA isolated from male Xenopus tropicalis tissues. The number of transcript copies is shown on a logarithmic scale, with the exception of mboat4. Data are expressed as the mean ± SEM (n = 4). For the different genes, the following tissues had the highest expression: ghrelin, stomach; ghsr, stomach; leap2, intestine-1 (upper intestine); mboat4, stomach; and nucb2, testis.
Fig. 5. Effects of short-term fasting on the mRNA expression of ghrelin and the other ghrelin related genes in Xenopus tropicalis. RT-qPCR was performed on equal amounts of total RNA isolated from the brain (A), liver (B), stomach (C), and duodenum (D) of male Xenopus tropicalis at the metabolic status for fed (Fed) or fasted for 2 days (Fasted). mRNA expression levels of GHS-R (ghsr) (A, C, and D), nucleobindin 2 (nucb2) (A, B, C, and D), liver-enriched antimicrobial peptide 2 (leap2) (B and D), ghrelin (C), and ghrelin O-acyltransferase (mboat4) (C). Relative expression values were calculated using Ct values normalized to the ribosomal protein L8 (rpl8). Data are expressed as the mean ± SEM (n = 3). Asterisks indicate significant differences compared with the control (Fed) value by unpaired Student’s t-test (p < 0.05).