Hypothalamic and brainstem glucose-dependent insulinotropic polypeptide receptor neurons employ distinct mechanisms to affect feeding

Abstract

Central glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) signaling is critical in GIP-based therapeutics' ability to lower body weight, but pathways leveraged by GIPR pharmacology in the brain remain incompletely understood. We explored the role of Gipr neurons in the hypothalamus and dorsal vagal complex (DVC) - brain regions critical to the control of energy balance. Hypothalamic Gipr expression was not necessary for the synergistic effect of GIPR/GLP-1R coagonism on body weight. While chemogenetic stimulation of both hypothalamic and DVC Gipr neurons suppressed food intake, activation of DVC Gipr neurons reduced ambulatory activity and induced conditioned taste avoidance, while there was no effect of a short-acting GIPR agonist (GIPRA). Within the DVC, Gipr neurons of the nucleus tractus solitarius (NTS), but not the area postrema (AP), projected to distal brain regions and were transcriptomically distinct. Peripherally dosed fluorescent GIPRAs revealed that access was restricted to circumventricular organs in the CNS. These data demonstrate that Gipr neurons in the hypothalamus, AP, and NTS differ in their connectivity, transcriptomic profile, peripheral accessibility, and appetite-controlling mechanisms. These results highlight the heterogeneity of the central GIPR signaling axis and suggest that studies into the effects of GIP pharmacology on feeding behavior should consider the interplay of multiple regulatory pathways.

Keywords: Diabetes; Metabolism; Neuroendocrine regulation; Neuroscience; Obesity.

Figure 1. Hypothalamic Gipr expression is not necessary for GIPR/GLP-1R dual agonism–mediated weight loss.

(A) DIO Gipr^ΔHyp and Gipr^{WT Hyp} mice were dosed with vehicle, GLP-140 (30 nmol/kg, s.c.), or GLP-140 (30 nmol/kg, s.c.) + GIP-085 (300 nmol/kg s.c.) for 12 days. (B) Gipr^ΔSst and Gipr^WT mice were dosed with GLP-140 (30 nmol/kg, s.c.) + GIP-085 (300 nmol/kg s.c.) for 12 days. Daily body weight and food intake were measured throughout the study. Changes in body weight were calculated as a percentage of the body weight of the same animal prior to the first injection. Statistical comparisons made using a repeated measures 2-way ANOVA with a Sidak’s post hoc test. *P <0.05 GLP-140 versus GLP-140 + GIP-085 same genotype, ^$P <0.05 GLP-140 + GIP-085 in Gipr^ΔHyp versus Gipr^{WT Hyp}, ^#P <0.05 GLP-140 in Gipr^ΔHyp versus Gipr^{WT Hyp}; n = 5–11.

Figure 2. Gipr expression in the DVC and vagal afferents.

(A) Coronal sections from Gipr^GCaMP3 mice were stained for GFP (green). Nuclei were counterstained with DAPI (blue). Photomicrograph is representative of experiments conducted in tissue from 5 separate mice. Original magnification: ×20 (A and B). (B) Coronal sections of mouse brain stem tissue from C57BL/6 mice were probed for Gipr expression using FISH (green). Nuclei are counterstained with DAPI (blue). Photomicrograph is representative of experiments conducted in tissue from 3 separate mice. (C) qPCR was performed in pooled samples of nodose ganglia (n = 5–6 mice per replicate) for Gipr and Glp1r expression. Expression levels were calculated relative to Actb. Data presented as 2^ΔCT ± SEM. (D) Sections of nodose ganglia isolated from C57BL/6 mice were probed for Gipr (magenta), Oxtr (yellow), and Glp1r (cyan) expression using FISH. Neurons were stained for HuC/D (gray). Photomicrograph is representative of experiments conducted in tissue from 4 separate mice. Scale bars: 100 μm and 10 μm (insets). Arrows indicate Gipr postitive cells.

Figure 3. Acute activation of Gipr neurons in the DVC and hypothalamus reduce food intake with differing effects on energy expenditure and locomotion.

(A–D) Gipr-Cre mice were injected with AAV-hSyn-DIO-hM3D(Gq)-mCherry into the DVC (A–C) or the hypothalamus (D) to produce Gipr^DVC–Dq or Gipr^Hyp–Dq mice, respectively. Mice were housed in indirect calorimetry cages equipped with continuous monitoring. CNO (1 mg/kg) or vehicle was injected i.p. at the onset of the dark phase. Mice were given standard chow and drinking water (A and B), a choice of standard chow or 45% HFD and drinking water (C), or standard chow with a choice of drinking water or 10% sucrose (D). Data are plotted as mean ± SEM. Statistical comparisons made using a repeated measures 2-way ANOVA with a Sidak’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n = 8–16.

Figure 4. Acute activation of Gipr neurons in the DVC induces conditioned taste avoidance.

(A) Schematic illustrating CTA protocol. Taste avoidance conditioned by CNO (1 mg/kg) or vehicle administration in Gipr^DVC–Dq and Gipr^Hyp–Dq mice. Data are plotted as mean ± SEM. Statistical analysis performed using a 1-way ANOVA with a Dunnett’s post hoc test. ***P < 0.001; n = 4–11. (B) c-Fos activation (green) in the DVC from Gipr^DVC–Dq mice following administration of CNO or vehicle. Image of hM3Dq-mCherry staining in DVC from Gipr^DVC–Dq mouse representative of n = 13 experiments. Images of c-Fos staining are representative of n = 5–8 experiments. (C) Quantification of c-Fos in indicated brain regions. Data are plotted as mean ± SEM. Statistical comparisons made using a repeated measures 2-way ANOVA with a Sidak’s post hoc test. **P < 0.01, ***P < 0.001; n = 5–8. PVH, paraventricular nucleus of the hypothalamus; SO, supraoptic nucleus; PSTh, parasubthalamic nucleus; LPBN, lateral parabrachial nucleus; MPBN, medial parabrachial nucleus; scp, superior cerebellar peduncle; AP, area postrema; NTS, nucleus tractus solitarius.

Figure 5. Gipr neurons in the NTS project to the PBN and PVH.

(A and B) Gipr-Cre mice were injected with AAV-hSyn-DIO-hM3D(Gq)-mCherry into the DVC. mCherry⁺ fibers were visualized by IHC in serial brain sections localising in the PVH (A) and LPBN (B) Scale bars: 50 μm (A), 100 μm (B). (C and D) AAVs optimized for retrograde transport packaging hSyn-DIO-hM3D(Gq)-mCherry (AAVret-DIO hM3Dq-mCherry) were injected into the PVH (C) and the LPBN (D). mCherry⁺ cell bodies were visualized in the DVC by IHC. Photomicrographs are representative of experiments conducted in tissue from 3 separate mice. PVH, paraventricular nucleus of the hypothalamus; LPBN, lateral parabrachial nucleus. Scale bars: 100 μm.

Figure 6. Transcriptomic characterization of Gipr-expressing cells in the hindbrain.

Gipr cells were isolated from single-cell digests of hindbrain sections from Gipr^EYFP mice via FACS, and their transcriptomes were characterized via scRNA-Seq followed by clustering analysis. (A) Uniform Manifold Approximation and Projection (UMAP) visualization of Gipr^EYFP+ cells. Cell types were assigned according to expression of marker genes (Peri.1, Peri.2 = pericytes; EC.1, EC.2 = endothelial cells; OD.ma = mature ODs; OD.my = myelinating ODs; OD.3 = ODs, SMC.1, SMC.2 = smooth muscle cells; VLMC = vascular leptomenigeal cells; Neuron = neurons; Ast/Ep = astroependymal cells; MG = microglia) (B). (C) Dual-label FISH showing colocalization of Gipr (green) and Syt1 (red) transcript in coronal sections of mouse brain stem tissue from C57BL/6 mice. Nuclei are counterstained with DAPI (blue). Gipr/Syt1 coexpression was quantified in sections from 3 mice. Scale bar: 20 μm. Arrows indicate cells expressing both Gipr and Syt1. (D) Principal component analysis of Gipr^EYFP+ versus Glp1r^EYFP+ neurons (left). Dot plot of selected differentially expressed genes in Gipr^EYFP+ versus Glp1r^EYFP+ neurons (right).

Figure 7. Transcriptomic analysis of Gipr Neurons in the hindbrain.

(A) Uniform Manifold Approximation and Projection (UMAP) showing clusters of isolated Gipr neurons following dimensionality reduction and unsupervised clustering. (B) The top 15 markers for each cluster were cross-referenced with published brain region–specific transcriptional markers and the Allen Brain Atlas for region specific cluster assignments. (C) Violin plots of neurotransmitters, secreted products, and cell surface receptors or ion channels enriched in each cluster. Data are plotted in CPM. (D) Dual-label FISH showing colocalization of Gipr with either Slc17a6, Cartpt, or Th transcript in the NTS of brain stem tissue from C57BL/6 mice. Nuclei are counterstained with DAPI (blue). (E) Dual-label FISH showing colocalization of Gipr with either Penk, Npy2r, or Oxtr transcript in the AP of brain stem tissue from C57BL/6 mice. Nuclei are counterstained with DAPI (blue). Gipr/Syt1, Gipr/Npy2r, or Gipr/Oxtr coexpression was quantified in sections from 3 mice. Scale bars: 20 μm. Arrows represent colocalization of probes as indicated.

Figure 8. Stabilized, fluorescently labeled GIP peptides are specific and effective GIPR agonists that access circumventricular organs in the CNS.

(A) Taste avoidance conditioned by vehicle or GIP-532 (10 nmol/kg, s.c.) or LiCl (0.4M, i.p.) in WT mice. Data are plotted as mean ± SEM. Statistical analysis performed using a 1-way ANOVA with a Dunnett’s post hoc test. n = 4–5. ***P < 0.001. (B) Schematic showing nature and binding of the stabilized red (sGIP549) and far red (sGIP648) GIPR probes (GIPR pdb: 7ra3). (C) sGIP549, sGIP648, and native GIP(1-42) cAMP signaling responses in T-REx-SNAP-GIPR cells, n = 3. (D and E) sGIP648 (D) and sGIP549 (E) label GIPR^GCaMP3 reporter islets, showing colocalization with GCaMP3⁺ cells (n = 11-12 islets, 2 animals). (F) sGIP648 labels β cells, identified using LUX551, in Gipr^fl/fl control but not Gipr^{–/–βcell} islets. Arrows show GIPR labeling only in LUX551^– cells, presumed to be α cells (n = 63 islets, 10 animals). (G) sGIP549 labels β cells, identified using LUX645, in Gipr^fl/fl control but not Gipr^{–/–βcell} islets. Arrows show GIPR labeling only in LUX645^– cells, presumed to be α cells (n = 56 islets, 10 animals). (H) sGIP648 labels the DVC and MBH following i.v. administration in mice. (Veh: n = 4, sGIP648: n = 7). (I) c-Fos activation in the DVC following i.v. injection of vehicle or sGIP648 into Gipr^+/+ or Gipr^–/– mice (n = 3 mice per genotype per treatment). (J and K) Maximum intensity projection of the average signal computed from individual brains (n = 4) overlaid onto the Common Coordinate Framework V3 template from AIBS for mice treated with vehicle (J) or D-alaGIP/IR800 (K). CP, choroid plexus; DVC, dorsal vagal complex; MBH mediobasal hypothalamus; ARH, arcuate nucleus of the hypothalamus; ME, median eminence; NTS, nucleus tractus solitarius; AP, area postrema; Scale bar: 53 μm (D–G).