The Orphan G Protein-Coupled Receptor Gene GPR178 Is Evolutionary Conserved and Altered in Response to Acute Changes in Food Intake

Abstract

G protein-coupled receptors (GPCRs) are a class of integral membrane proteins mediating physiological functions fundamental for survival, including energy homeostasis. A few years ago, an amino acid sequence of a novel GPCR gene was identified and named GPR178. In this study, we provide new insights regarding the biological significance of Gpr178 protein, investigating its evolutionary history and tissue distribution as well as examining the relationship between its expression level and feeding status. Our phylogenetic analysis indicated that GPR178 is highly conserved among all animal species investigated, and that GPR178 is not a member of a protein family. Real-time PCR and in situ hybridization revealed wide expression of Gpr178 mRNA in both the brain and periphery, with high expression density in the hypothalamus and brainstem, areas involved in the regulation of food intake. Hence, changes in receptor expression were assessed following several feeding paradigms including starvation and overfeeding. Short-term starvation (12-48h) or food restriction resulted in upregulation of Gpr178 mRNA expression in the brainstem, hypothalamus and prefrontal cortex. Conversely, short-term (48h) exposure to sucrose or Intralipid solutions downregulated Gpr178 mRNA in the brainstem; long-term exposure (10 days) to a palatable high-fat and high-sugar diet resulted in a downregulation of Gpr178 in the amygdala but not in the hypothalamus. Our results indicate that hypothalamic Gpr178 gene expression is altered during acute exposure to starvation or acute exposure to palatable food. Changes in gene expression following palatable diet consumption suggest a possible involvement of Gpr178 in the complex mechanisms of feeding reward.

Fig 1. Consensus phylogenetic tree of GPR178.

The consensus phylogenetic tree of GPR178 amino acid sequences from 16 different species. The numbers in percentage indicates the amino acid identity to human GPR178. The sequence alignment used for phylogenetic calculations was based on sequences starting from TM1 and constructed with MAFFT-GINSI. The consensus tree is based on 100 Maximum Parsimony trees and the branch lengths of the tree are calculated with Tree-Puzzle 5.2 using JTT model of substitution on the topology obtained from parsimony. Alignment and phylogenetic analysis of GPR178 amino acid sequences: GPR178 protein sequences were retrieved from GenBank using tblastn (http://blast.ncbi.nlm.nih.gov/Blast.cgi): human (Homo sapiens) NP 065874, house mouse (Mus musculus) NP 001028350, Norway rat (Rattus norvegicus) NP 001124411, domestic cow (Bos taurus) XP 870842, dog (Canis lupus familiaris) XP 855028, gray short-tailed opossum (Monodelphis domestica) XP 001381390, chicken (Gallus gallus) XP 419698, western clawed frog (Xenopus tropicalis) NP 001039119, zebrafish (Danio rerio) NP 001038305, purple sea urchin (Strongylocentrotus purpuratus) XP 001183797, fruit fly (Drosophila melanogaster) NP 651004, malaria mosquito (Anopheles gambiae) XP 311316, honey bee (Apis mellifera) XP 624574, red flour beetle (Tribolium castaneum) XP 969527, starlet sea anemone (Nematostella vectensis) XP 001618510 and Trichoplax adhaerens XP 002118380.

Fig 2. Gpr178 mRNA expression in rat and mouse.

mRNA expression levels of Gpr178 in tissues from rat (A) and mouse (B). Results are shown as relative expression to minimum (fold increase). In rat panel (A), abbreviations II–VIII indicate brain cross sections adapted from [13]. Intestine was divided into four parts of equal size during dissection and the most proximal 25% denoted I and the most distal 25% denoted IV.

Fig 3. Gpr178 mRNA expression during food intake experiments.

(A) Gpr178 mRNA expression after short-term exposure to starvation in mice; control group, had unlimited access to chow diet; n = 8 each group. (B) Gpr178 mRNA expression after long-term exposure (48 hours) in rat. Control group had unlimited access to chow diet, whereas restricted group was provided with 45% of the total daily caloric intake of the control group. Restricted group had free access to food until 48h before the end point of the experiment. N = 8 each group. (C) Gpr178 mRNA expression in mice after short-term (48 hours) full access to palatable drinking solutions (4.1% Intralipid or 10% sucrose); n = 8 each group. (D) Gpr178 mRNA expression in mice after long-term (10days) exposure to high-fat or high-carbohydrate diet; n = 8. BS, Brainstem; HYP, Hypothalamus; PFC, Prefrontal cortex; AMY, Amygdala. Data were analysed by two-way ANOVA followed by post hoc LSD (A, 12h starvation/24h starvation; B, restricted/48h starvation; C, Intralipid/10% sucrose; D, high-sugar/high-fat or high-carbohydrate diet). * p<0.05 ** p<0.01 ***p<0.001.

Fig 4. Floating in situ hybridization experiment in mouse brain.

Floating in situ hybridization using 300 ng of digoxigenin labeled mouse Gpr178 antisense probe (A-N). Abbreviations and organization of the brain are depicted using Franklin and Paxinos [16].