A comparative transcriptomic analysis of Glucagon-like peptide-1 receptor- and Glucose-dependent insulinotropic polypeptide receptor-expressing cells in the hypothalamus

OBJECTIVE: The hypothalamus is a key region of the brain implicated in homeostatic regulation, and is an integral centre for the control of feeding behaviour. Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are incretin hormones with potent glucoregulatory function through engagement of their respective cognate receptors, GLP-1R and GIPR. Recent evidence indicates that there is a synergistic effect of combining GIP- and GLP-1-based pharmacology on appetite and body weight. The mechanisms underlying the enhanced weight loss exhibited by GIPR/GLP-1R co-agonism are unknown. Gipr and Glp1r are expressed in the hypothalamus in both rodents and human. To better understand incretin receptor-expressing cell populations, we compared the cell types and expression profiles of Gipr- and Glp1r- expressing hypothalamic cells using single-cell RNA sequencing. METHODS: Using Glp1r-Cre or Gipr-Cre transgenic mouse lines, fluorescent reporters were introduced into either Glp1r- or Gipr-expressing cells, respectively, upon crossing with a ROSA26-EYFP reporter strain. From the hypothalami of these mice, fluorescent Glp1r(EYFP+) or Gipr(EYFP+) cells were FACS purified and sequenced using single-cell RNA sequencing. Transcriptomic analysis provided a survey of both non-neuronal and neuronal cells, and comparisons between Glp1r(EYFP+) and Gipr(EYFP+) populations were made. RESULTS: A total of 14,091 Glp1r(EYFP+) and Gipr(EYFP+) cells were isolated, sequenced and taken forward for bioinformatic analysis. Both Glp1r(EYFP+) and Gipr(EYFP+) hypothalamic populations were transcriptomically highly heterogeneous, representing vascular cell types, oligodendrocytes, astrocytes, microglia, and neurons. The majority of Gipr(EYFP+) cells were non-neuronal, whereas the Glp1r(EYFP+) population was evenly split between neuronal and non-neuronal cell types. Both Glp1r(EYFP+) and Gipr(EYFP+) oligodendrocytes express markers for mature, myelin-forming oligodendrocytes. While mural cells are represented in both Glp1r(EYFP+) and Gipr(EYFP+) populations, Glp1r(EYFP+) mural cells are largely smooth muscle cells, while the majority of Gipr(EYFP+) mural cells are pericytes. The co-expression of regional markers indicate that clusters of Glp1r(EYFP+) and Gipr(EYFP+) neurons have been isolated from the arcuate, ventromedial, lateral, tuberal, suprachiasmatic, and premammillary nuclei of the hypothalamus. CONCLUSIONS: We have provided a detailed comparison of Glp1r and Gipr cells of the hypothalamus with single-cell resolution. This resource will provide mechanistic insight into how engaging Gipr and Glp1r cells of the hypothalamus may result in changes in feeding behaviour and energy balance.