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

Abstract

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 humans. 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-expressing cells of the hypothalamus may result in changes in feeding behaviour and energy balance.

Keywords: Appetite; Feeding; Glucagon-like peptide-1; Glucose-dependent insulinotropic polypeptide; Gut-brain axis; Hypothalamus; Transcriptomics.