A comparative transcriptomic analysis of glucagon-like peptide-1 receptor- and glucose-dependent insulinotropic polypeptide-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 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 Glp1rEYFP+ 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.

Automated summary

This study compared the cell types and expression profiles of Glp1r and Gipr cells in the hypothalamus using single-cell RNA sequencing. The results showed that Glp1r and Gipr cells in the hypothalamus exhibit high heterogeneity in cell types and functions. This study provides mechanistic insights into the roles of Glp1r and Gipr in feeding behavior and energy balance.