Roles for Prlhr/GPR10 and Npffr2/GPR74 in feeding responses to PrRP

Abstract

Objective: Several groups of neurons in the NTS suppress food intake, including Prlh-expressing neurons (NTS^Prlh cells). Not only does the artificial activation of NTS^Prlh cells decrease feeding, but also the expression of Prlh (which encodes the neuropeptide PrRP) and neurotransmission by NTS^Prlh neurons contributes to the restraint of food intake and body weight, especially in animals fed a high fat diet (HFD). We set out to determine roles for putative PrRP receptors in the response to NTS PrRP and exogenous PrRP-related peptides.

Methods: We used animals lacking PrRP receptors GPR10 and/or GPR74 (encoded by Prlhr and Npffr2, respectively) to determine roles for each in the restraint of food intake and body weight by the increased expression of Prlh in NTS^Prlh neurons (NTS^PrlhOX mice) and in response to the anorectic PrRP analog, p52.

Results: Although Prlhr played a crucial role in the restraint of food intake and body weight in HFD-fed control animals, the combined absence of Prlhr and Npffr2 was required to abrogate the restraint of food intake in NTS^PrlhOX mice. p52 suppressed feeding independently of both receptors, however.

Conclusions: Hence, each receptor can participate in the NTS^Prlh-mediated suppression of food intake and body weight gain, while PrRP analog treatment can mediate its effects via distinct systems. While Prlhr plays a crucial role in the physiologic restraint of weight gain, the action of either receptor is capable of ameliorating obesity in response to enhanced NTS^Prlh signaling.

Keywords: Food intake; NTS; Npffr2; Obesity; Prlh; Prlhr.

Figure 1

Ablation of Prlh from the NTS promotes increased body weight in HFD-fed mice. A. Representative image of PrRP-IR in NTS injection sites for AAV-GFP (left) and AAV-CRE (right) injected Prlh^Flox/Flox mice. B. Quantification of PrRP-IR cell bodies per section from AAV-GFP (n = 6) and AAV-CRE (n = 6) injected animals, as shown in (A). C. Body weight from AAV-GFP (n = 11; black line) and AAV-CRE (n = 8; red line) injected mice over 6 post-surgical weeks on chow and an additional 6 weeks on HFD. Body weight is normalized to the first week's measurement. All graphs: Shown is mean ± SEM. Two-way ANOVA, Sidak's multiple comparisons test and unpaired t-test were used; p values are shown for significant comparisons. ∗: 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).

Figure 2

Increased Prlh expression in NTS^Prlhneurons restrains food intake and body weight gain in HFD-fed mice. A. Representative image of PrRP-IR (magenta) and GFP (green) in the NTS of control (Ctrl-WT, left) or NTS^PrlhOX mice (PrlhOX-WT, right) on the Cre-dependent GFP-Sun1 reporter background. B. Quantification of PrRP-IR normalized per unit area of the NTS section from CTRL-WT (n = 11) and PrlhOX-WT (n = 10), as shown in (A). C, D Cumulative food intake (C) and normalized body weight (D) for HFD-fed control (Ctrl-WT, n = 16) and NTS^PrlhOX (PrlhOX-WT, n = 15) mice. Body weight is shown normalized to the first week's measurement. All graphs: Shown is mean ± SEM. Two-way ANOVA, Sidak's multiple comparisons test and unpaired t-test were used; All images were taken at same magnification; scale bar equals 100 μm. P values are shown for significant comparisons. ∗: 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).

Figure 3

Generation and baseline metabolic parameters in Prlhr^KO(10KO) and Npffr2KO (74KO) mice. A, B Exon 1 of the Prlhr gene (A, encoding GPR10) and exon 2 of the Npffr2 gene (B, encoding GPR74) were deleted in wild-type mice, respectively, using CRISPR technology. C, D Relative expression of Prlhr (C) and Npffr2 (D) in the hypothalamus of wild-type mice and mice with individual and combined 10KO and 74KO genetic backgrounds. ND-not detected. E–H The body weight (E), lean mass percentage (F), fat percentage (G) and serum leptin (H) in 12-week-old chow fed mice on the individual or combined 10KO and 74 KO genetic backgrounds. All graphs: Shown is mean ± SEM. One-way ANOVA, Tukey's multiple comparisons test was used; p values are shown for significant comparisons. ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗: p < 0.0001.

Figure 4

Combined absence of GPR10 and GPR74 abrogates the suppression of food intake and body weight by NTS^PrlhOXin HFD-fed mice. (A–B) Cumulative food intake (A) and body weight (B) following surgery for Ctrl-CRE (n = 16), Ctrl-10KO (n = 6), Ctrl-74KO (n = 6), and Ctrl-10&74KO (n = 11) mice. (C–D) Cumulative food intake (C) and body weight (D) following surgery for control (Ctrl-10KO, n = 6; solid red line) and NTS^PrlhOX (PrlhOX-10KO, n = 6; dashed red line) mice on the GPR10 KO background. (E–F) Cumulative food intake (E) and body weight (F) following surgery for control (Ctrl-74KO, n = 6; solid blue line) and NTS^PrlhOX (PrlhOX-74KO, n = 6; dashed blue line) mice on the GPR74 KO background. (G–H) Cumulative food intake (left) and body weight (right) following surgery for control (Ctrl-10/74KO, n = 11; solid green line) and NTS^PrlhOX (PrlhOX-10/74KO, n = 12; dashed green line) mice on the combined 10KO/74KO background. Body weight is shown normalized to the first week's measurement for each strain. All graphs: Shown is mean ± SEM. Two-way ANOVA, Sidak's multiple comparisons test was used; p values are shown for significant comparisons. ∗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).

Figure 5

p52 suppress food intake independently of GPR10 and GPR74 expression in mice. A. Wild-type mice were treated with vehicle, liraglutide (dose) or the indicated concentration of p52 at the onset of the dark cycle and food intake was monitored over the subsequent 4 h (n = 8 per group). B Wild-type (WT) (n = 7), 10KO (n = 9), 74KO (n = 5), and 10/74KO (n = 5) mice were treated with vehicle or p52 (5 mg/kg) at the onset of the dark cycle and food intake was monitored over the subsequent 4 h. All graphs: Shown is mean ± SEM. One-way ANOVA, Dunnett's multiple comparisons test and unpaired t-test were used; p values are shown for significant comparisons. ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗: p < 0.0001.

Figure 6

p52, but not NTS^PrlhOXor the activation of NTS Prlh neurons, suppresses locomotor activity in mice. A. Locomotor activity in wild-type mice treated with vehicle and p52 (n = 12, 5 mg/kg IP), respectively. B. Locomotor activity in Prlh^Cre (n = 6; Ctrl-WT) and NTS^PrlhOX (n = 7, PrlhOX-WT) mice. C. Locomotor activity in control mice (Ctrl; n = 9) and NTS^Prlh−Dq mice (Dq; n = 3) treated with either saline or CNO (1 mg/kg IP) twice daily. All graphs: Shown is mean beam breaks per hour ± SEM for the light cycle, the dark cycle, and averaged over the entire 24-hour day. Multiple paired t-tests, Holm-Šídák method and Šídák-Bonferroni method were used; p values are shown for significant comparisons. ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗: p < 0.0001.