Uncovering the role of Gpr45 in obesity regulation

Abstract

Objectives: G protein-coupled receptors (GPCRs) are the most druggable targets in biology due to their cell-type specificity, ligand binding, and cell surface accessibility. Underscoring this, agonists for GPCRs have recently revolutionized the treatment of diabetes and obesity. The rampant success of these compounds has invigorated interest in identifying additional GPCRs that modulate appetite and body weight homeostasis. One such potential therapeutic target is G-protein couped receptor 45 (Gpr45), an orphan GPCR expressed both centrally and peripherally. We aimed to explore the role of Gpr45 as well as neurons expressing Gpr45 in energy balance.

Methods: Three novel transgenic mouse models were engineered to investigate the functional contribution of Gpr45 to body weight and appetite regulation: 1) a global Gpr45 knockout, 2) a conditional floxed Gpr45 allele, and 3) a Gpr45-CreERT2 knock-in. Metabolic profiling was performed in global Gpr45 knockout animals including body weight, food intake, body mass, energy expenditure, and body temperature measurements. Animals harboring a conditional floxed Gpr45 allele were bred to mice expressing Cre-recombinase in excitatory neurons labeled via Vesicular glutamate transporter 2 (Vglut2), inhibitory cells expressing Vesicular GABA transporter (Vgat), or neurons marked by the transcription factor Single-minded 1 (Sim1) and monitored for body weight and food consumption. Additionally, floxed Gpr45 mice were bilaterally injected with AAV-Cre targeting the paraventricular nucleus of the hypothalamus (PVH) and body weight and food intake were evaluated. The Gpr45-CreERT2 knock-in model was used to express chronic and acute actuators to the PVH to assess the role of PVH^Gpr45 neurons in energy homeostasis.

Results: Global Gpr45 disruption caused marked weight gain, increased food intake and fat mass, but no detectable alterations in core temperature or energy output. Selective deletion of Gpr45 from Sim1+ or excitatory Vglut2+ but not inhibitory Vgat+, neurons produced obesity and hyperphagia. Targeted deletion of Gpr45 from the PVH phenocopies these metabolic changes suggesting a major site of action of Gpr45 signaling is glutamatergic neurons residing in the PVH. Tetanus toxin light chain (TeNT) was used to permanently silence PVH^Gpr45 neuronal activity in Gpr45-CreER mice leading to rapid weight accumulation and escalated food intake. These experiments highlight the critical role of both Gpr45 signaling and neural network activity in the regulation of body weight and appetite. A mutated version of the bacterial sodium channel, NaChBac, was used to constitutively activate PVH^Gpr45 neuronal activity in Gpr45-CreER mice with limited to no effect on body weight and food consumption, implicating redundant circuitry acting in concert to bias weight loss protection. Acute chemogenetic stimulation of PVH^Gpr45 neurons durably suppressed food intake regardless of caloric need state or food palatability demonstrating the capacity of these cells to curb appetite.

Conclusions: Gpr45 is a putative therapeutic candidate that could be targeted to combat obesity and overeating.

Keywords: G protein-coupled receptor 45; behavioral neuroscience; body weight regulation; food intake.

Figure 1

Metabolic phenotyping of global Gpr45 knockout mouse model. (A) Gpr45 knockout (KO) mice gain significantly more weight at room temperature on standard chow diet than age/sex-matched wildtype (WT) controls (n = 23 Gpr45 KO, n = 29 WT, mixed sex; repeated measures two-way ANOVA followed by Šídák's multiple comparison test). (B) Gpr45 knockout (KO) mice consume significantly more standard chow diet than age/sex-matched wildtype (WT) controls (n = 6 Gpr45 KO, n = 8 WT; repeated measures two-way ANOVA followed by Šídák's multiple comparison test). (C) Gpr45 knockout (KO) mice accrue significantly more fat and lean mass on standard chow diet than age/sex-matched wildtype (WT) controls (n = 10 Gpr45 KO, n = 12 WT; unpaired t test, two-tailed). (D) Gpr45 knockout (KO) mice gain significantly more weight at 27 °C on standard chow diet than age/sex-matched wildtype (WT) controls (n = 10 Gpr45 KO, n = 12 WT, mixed sex; repeated measures two-way ANOVA followed by Šídák's multiple comparison test). (E) Gpr45 knockout (KO) mice and age/sex-matched wildtype (WT) controls exhibit comparable core body temperatures (n = 8 Gpr45 KO, n = 6 WT; unpaired t test, two-tailed). (F) Gpr45 knockout (KO) mice and age/sex-matched wildtype (WT) controls exhibit comparable total energy expenditure values considering body weight differences (n = 6 Gpr45 KO, n = 8 WT; simple linear regression). All error bars represent s.e.m. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Figure 2

Selective deletion of Gpr45 from Vglut2+ excitatory hypothalamic neurons results in obesity and hyperphagia. (A) Expression pattern of anatomical brain regions marked by Vesicular glutamate transporter 2 (Vglut2) where Gpr45 is deleted including the cerebral cortex (CTX), piriform cortex (PIR), paraventricular nucleus of the hypothalamus (PVN), lateral olfactory tract (LOT), thalamus (TH), basal lateral amygdala (BLA), posterior hypothalamus (PH), periaqueductal grey (PAG), dorsal raphe (DR), lateral parabrachial nucleus (LPB), and nucleus of the solitary tract (NTS). (B–C) Mice with specific deletion of Gpr45 from excitatory Vglut2+ neurons (B) gain significantly more weight and (C) consume significantly more standard chow diet than age/sex-matched heterozygous controls (n = 8 Vglut2-ires-Cre; Gpr45 flox/flox, n = 7 Vglut2-ires-Cre, n = 6 Gpr45 flox/flox, mixed sex; repeated measures two-way ANOVA followed by Tukey's multiple comparison test). (D) Expression pattern of anatomical brain regions marked by Vesicular GABA transporter (Vgat) where Gpr45 is deleted, including the caudate putamen (CP), nucleus accumbens (ACB), olfactory tubercle (TU), lateral septum (LS), bed nucleus of the stria terminalis (BST), suprachiasmatic nucleus (SCh), arcuate nucleus of the hypothalamus (ARC), central amygdala (CeA), substantia nigra pars reticulata (SNr), superior colliculus (SC), and dorsal cochlear nucleus (DC). (E–F) Mice with specific deletion of Gpr45 from inhibitory Vgat + neurons exhibit comparable (E) body weight and (F) standard chow diet intake as age/sex-matched heterozygous controls (n = 8 Vgat-ires-Cre; Gpr45 flox/flox, n = 6 Vgat-ires-Cre, n = 6 Gpr45 flox/flox, mixed sex; repeated measures two-way ANOVA followed by Tukey's multiple comparison test). (G) Expression pattern of anatomical brain regions marked by Single-minded 1 (Sim1) where Gpr45 is deleted including the paraventricular nucleus of the hypothalamus (PVH), nucleus of lateral olfactory tract (NLOT), medial amygdala (MeA), and dorsal motor vagus (DMV). (H–I) Mice with specific deletion of Gpr45 from excitatory Sim1+ neurons (H) gain significantly more weight and (I) consume significantly more standard chow diet than age/sex-matched heterozygous controls (n = 10 Vglut2-ires-Cre; Gpr45 flox/flox, n = 7 Sim1-ires-Cre, n = 5 Gpr45 flox/flox, mixed sex; repeated measures two-way ANOVA followed by Tukey's multiple comparison test). Panels (A) and (D) adapted from [15]. Panel (G) adapted from [22]. All error bars represent s.e.m. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Figure 3

Selective deletion of Gpr45 from PVH neurons results in obesity and hyperphagia. (A) Brain schematic representing bilateral injection of AAV-hSyn-mCherry-P2A-Cre-WPRE into the PVH of Gpr45 flox/flox or age/sex-matched WT controls. (B) Representative epifluorescent image of a brain slice after bilateral injection of AAV-hSyn-mCherry-P2A-Cre-WPRE into the PVH of a Gpr45 flox/flox mouse. (C–D) Mice with specific deletion of Gpr45 from the PVH (C) gain significantly more weight and (D) consume significantly more standard chow diet than age/sex-matched WT controls (n = 15 Gpr45 flox/flox, n = 9 WT, mixed sex; repeated measures two-way ANOVA followed by Šídák's multiple comparison test). All error bars represent s.e.m. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Figure 4

PVH^Gpr45 neurons bidirectionally modulate appetite. (A) Brain schematic representing bilateral injection of AAV-CMV-DIO-eGFP-2A-TeNT into the PVH of Gpr45-CreER or age/sex-matched WT controls. (B) Representative epifluorescent image of a brain slice after bilateral injection of AAV-CMV-DIO-eGFP-2A-TeNT into the PVH of a Gpr45-CreER mouse. (C–D) Synaptic silencing of PVH^Gpr45 neurons in Gpr45-CreER mice injected with AAV-CMV-DIO-eGFP-2A-TeNT into the PVH (C) gain significantly more weight and (D) consume significantly more standard chow diet than age/sex-matched WT controls (n = 9 Gpr45-CreER, n = 9 WT, mixed sex; repeated measures two-way ANOVA followed by Šídák's multiple comparison test). (E) Brain schematic representing bilateral injection of AAV-hSyn-DIO-hM3dq-mCherry into the PVH of Gpr45-CreER. (F) Representative epifluorescent image of a brain slice after bilateral injection of AAV-hSyn-DIO-hM3dq-mCherry into the PVH of a Gpr45-CreER mouse. (G–I) Acute, CNO-induced chemogenetic activation of PVH^Gpr45 neurons in Gpr45-CreER mice injected with AAV-hSyn-DIO-hM3dq-mCherry into the PVH significantly attenuates (G) standard chow diet food intake in a fast-refeed paradigm (n = 7 Gpr45-CreER, mixed sex; repeated measures two-way ANOVA followed by Šídák's multiple comparison test), (H) standard chow diet food intake in an ad libitum fed paradigm (n = 7 Gpr45-CreER; repeated measures two-way ANOVA followed by Šídák's multiple comparison test), and (I) high fat diet (HFD) food intake in a fast-refeed paradigm (n = 6 Gpr45-CreER; repeated measures two-way ANOVA followed by Šídák's multiple comparison test), compared to the same animals following a vehicle saline injection. Shaded blue area in (C–D) and (I) represent HFD homecage availability. All error bars represent s.e.m. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. S1

Expression pattern of Gpr45, Vglut2, and Vgat transcripts in the hypothalamus and hindbrain. (A) DAPI (blue) expression highlighting hypothalamic structures including the paraventricular nucleus of the hypothalamus (PVH), anterior hypothalamic area (AHA), suprachiasmatic nucleus (SCN), and zona incerta (ZI). Vglut2 (red), Vgat (magenta), and Gpr45 (green) expression in these regions. (B) DAPI (blue) expression highlighting hypothalamic structures including the dorsal medial hypothalamus (DMH), ventral medial hypothalamus (VMH), and arcuate nucleus (ARC). Vglut2 (red), Vgat (magenta), and Gpr45 (green) expression in these regions. (C) DAPI (blue) expression highlighting hindbrain structures including the are postrema. (AP) and nucleus of the solitary tract (NTS). Vglut2 (red), Vgat (magenta), and Gpr45 (green) expression in these regions.

Fig. S2

Generation and validation of conditional Gpr45 floxed mouse model. (A) Schematic of Gpr45 flox/flox allele generated using CRISPR/Cas9 technology. LoxP sites were inserted in frame with the mouse Gpr45 locus 254 bp upstream of exon 2 and 35 bp downstream of the stop codon. (B) Relative expression levels in the mediobasal hypothalamus detected via RT-PCR of Gpr45, but not Brs3, transcript levels are significantly lower in Vglut2-ires-Cre; Gpr45 flox/flox animals compared to Gpr45 flox/flox controls (n = 3 Vglut2-ires-Cre; Gpr45 flox/flox, n = 3 Gpr45 flox/flox; unpaired t test, two-tailed). (C) Relative expression levels in the mediobasal hypothalamus detected via RT-PCR of Gpr45, but not Mc4r, transcript levels are significantly lower in Vgat-ires-Cre; Gpr45 flox/flox animals compared to Gpr45 flox/flox controls (n = 3 Vgat-ires-Cre; Gpr45 flox/flox, n = 3 Gpr45 flox/flox; unpaired t test, two-tailed). (D) Body weights at week 18 of Vglut2-ires-Cre; Gpr45 flox/flox mice are significantly higher than Sim1-Cre; Gpr45 flox/flox subjects, but both are significantly higher than Vgat-ires-Cre; Gpr45 flox/flox mice (n = 8 Vglut2-ires-Cre; Gpr45 flox/flox, n = 8 Vgat-ires-Cre; Gpr45 flox/flox, n = 10 Sim1-Cre; Gpr45 flox/flox, mixed sex; repeated measures one-way ANOVA followed by Tukey's multiple comparison test). All error bars represent s.e.m. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Fig. S3

Gpr45 is ubiquitously expressed in the ARC but selective deletion of Gpr45 from AgRP neurons has no effect on body weight regulation. (A–D) FeaturePlots showing ARC neurons and clusters in UMAP space with (A) Slc17a6 (Vglut2), (B) Slc32a1 (Vgat), (C)Agrp, and (D)Gpr45 expression. (E). Mice with specific deletion of Gpr45 from AgRP + neurons exhibit comparable body weight as age/sex-matched heterozygous controls (n = 5 AgRP-ires-Cre; Gpr45 flox/flox, n = 5 AgRP-ires-Cre, n = 5 Gpr45 flox/flox, mixed sex; repeated measures two-way ANOVA followed by Tukey's multiple comparison test). All error bars represent s.e.m. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Fig. S4

Generation and body weight characterization of knock-in Gpr45-CreER mice. (A) Schematic of Gpr45-CreER knock-in model generated using CRISPR/Cas9 technology. The CreERT2 cassette was inserted in frame with the mouse Gpr45 locus and after the coding sequence. (B) Brain schematic representing bilateral injection of AAV-CMV-DIO-GFP into the PVH of Gpr45-CreER or WT littermate mice. (C) Gpr45-CreER mice injected with AAV-CMV-DIO-GFP into the PVH exhibit comparable bodyweight as littermate WT controls injected with AAV-CMV-DIO-GFP into the PVH. (n = 7 Gpr45-CreER::GFP, n = 7 WT::GFP, mixed sex; repeated measures two-way ANOVA followed by Šídák's multiple comparison test). Shaded blue area in (C) represent HFD homecage availability. All error bars represent s.e.m. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Fig. S5

Chronic PVH^Gpr45 neuronal hyperexcitability fails to alter body weight despite pronounced alterations in electrical activity. (A) Brain schematic representing bilateral injection of AAV-EF1a-FLEX-eGFP-P2A-mNaChBac into the PVH of Gpr45-CreER or age/sex-matched WT controls. (B) Representative epifluorescent image of a brain slice after bilateral injection of AAV-EF1a-FLEX-eGFP-P2A-mNaChBac into the PVH of a Gpr45-CreER mouse. (C) Mice with constitutive PVH^Gpr45 neural activity in Gpr45-CreER mice injected with AAV-EF1a-FLEX-eGFP-P2A-mNaChBac into the PVH show comparable body weights to age/sex-matched WT controls injected with AAV-EF1a-FLEX-eGFP-P2A-mNaChBac into the PVH on both a standard chow diet and 60% HFD (n = 7 Gpr45-CreER, n = 7 WT, mixed sex; repeated measures two-way ANOVA followed by Šídák's multiple comparison test). (D) Representative traces showing resting membrane potential (RMP) and action potentials in control (top) and NaChBach-expressing cells (bottom). (E) Quantification of RMP (n = 16 in 3 mice for control, n = 15 in 5 mice for NaChBac, unpaired two-tailed t-test, p = 0.023) and (F) Input resistance (n = 12 in 3 mice for control, n = 16 in 5 mice for NaChBac, unpaired two-tailed t-test, p = 0.026). (G) Representative traces of elicited action potentials by injecting step-wise depolarizing currents in control cells (top) and NaChBac-expressing cells (bottom). First steps that elicited AP highlighted in red. (H) Quantification of action potential threshold (n = 11 in 3 mice for control, n = 11 in 3 mice for NaChBac, unpaired two-tailed t-test, p < 0.0001) and (I) Rheobase (n = 11 in 3 mice for control, n = 11 in 3 mice for NaChBac, unpaired two-tailed t-test, p = 0.007). Shaded blue area in (C) represent HFD homecage availability. All error bars represent s.e.m. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.