Leptin receptor-expressing nucleus tractus solitarius neurons suppress food intake independently of GLP1 in mice

Abstract

Leptin receptor-expressing (LepRb-expressing) neurons of the nucleus tractus solitarius (NTS; LepRbNTS neurons) receive gut signals that synergize with leptin action to suppress food intake. NTS neurons that express preproglucagon (Ppg) (and that produce the food intake-suppressing PPG cleavage product glucagon-like peptide-1 [GLP1]) represent a subpopulation of mouse LepRbNTS cells. Using Leprcre, Ppgcre, and Ppgfl mouse lines, along with Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), we examined roles for Ppg in GLP1NTS and LepRbNTS cells for the control of food intake and energy balance. We found that the cre-dependent ablation of NTS Ppgfl early in development or in adult mice failed to alter energy balance, suggesting the importance of pathways independent of NTS GLP1 for the long-term control of food intake. Consistently, while activating GLP1NTS cells decreased food intake, LepRbNTS cells elicited larger and more durable effects. Furthermore, while the ablation of NTS Ppgfl blunted the ability of GLP1NTS neurons to suppress food intake during activation, it did not impact the suppression of food intake by LepRbNTS cells. While Ppg/GLP1-mediated neurotransmission plays a central role in the modest appetite-suppressing effects of GLP1NTS cells, additional pathways engaged by LepRbNTS cells dominate for the suppression of food intake.

Keywords: Endocrinology; Leptin; Metabolism; Obesity; Peptides.

Figure 1. Colocalization of neuronal markers with LepRb^NTS neurons.

(A–D) Representative images showing LepRb^NTS neurons (using leptin-induced pSTAT3-IR [A, purple] or GFP-IR in LepRb^eGFP mice [B–D, green]) and CCK (GFP-IR in CCK^eGFP mice; A, green), PRLH (B, purple), TH (C, purple), and choline acetyltransferase (ChAT, D, purple). (E) Representative image showing colocalization of NTS GLP1-IR (purple) with LepRb (mCherry-IR in AAV^Flex-mCherry transduced Lepr^cre mice, green). All panels are representative of n ≥ 3 similar images. (F–H) LepRb^eGFP mice were fasted overnight (F) or fasted overnight and then re-fed for 2 hours (G) before perfusion for the detection of LepRb (GFP, green) and FOS (purple). F and G show representative images (from n = 3 cases). (H) Colocalization of LepRb and FOS is shown. Data are shown as mean ± SEM; P value by unpaired 2-tailed t test is shown. AP, area postrema; cc, central canal. Scale bars: 150 μm.

Figure 2. Ablation of Ppg in LepRb^NTS and GLP1^NTS neurons.

(A) Schematic diagram showing the cross of Ppg^fl with Lepr^cre and Ppg^cre mice to generate Ppg^LepRbKO and Ppg^GLP1-NTSKO (Ppg^ppgKO) mice. (B–D) Representative images showing GLP1-IR (purple) in WT (B), Ppg^LepRbKO (C), and Ppg^ppgKO mice (D). All panels are representative of n ≥ 3 similar images. (E–J) Body weight (measurements for each animal are normalized to its own baseline weight) (E and H), food intake (FI) from the 12th week of age (F and I), and body composition at approximately 16 weeks of age (G and J) are shown for Ppg^LepRbKO and Ppg^ppgKO mice (data are from both sexes; data for each sex separately are shown in Supplemental Figure 1, A–H). Data are shown as mean ± SEM; n = 11–19 (E), n = 6 (G and F), n = 7 (H–J). All comparisons not significant using 2-way ANOVA with Sidak’s multiple comparisons test (E and H) or unpaired 2-tailed t test (F, G, I, and J). AP, area postrema; cc, central canal. Scale bars: 150 μm.

Figure 3. Viral-mediated Ppg KO in the NTS.

(A) Schematic diagram showing deletion of NTS Ppg by delivering mCherry-tagged AAV^cre (AAV^cre-mCherry) into the NTS of Ppg^fl mice. (B and C) Representative images of GFP-IR in mice injected with AAV^GFP (top panel, green) or mCherry-IR in AAV^cre-mCherry (bottom panel, red) (B); GLP1-IR (purple) for similar mice is shown in C. All panels are representative of n ≥ 10 similar images. (D) Weekly body weight change on chow and HFD (measurements for each animal were normalized to its baseline weight). (E and F) Food intake is shown for the 8th week after surgery (E), and body composition is shown for 2 months after surgery (F). Data are from male animals; female body weight data are shown in Supplemental Figure 1I. Data are shown as mean ± SEM; D (n = 11–13), E (n = 6) and F (n = 5). All comparisons, P > 0.05 using repeated measures 2-way ANOVA with Sidak’s multiple comparisons test (D) and 2-tailed unpaired t test (E and F). AP, area postrema; cc, central canal. Scale bar: 150 μm.

Figure 4. Activation of LepRb^NTS or GLP1^NTS neurons suppressed food intake and body weight.

(A and B) Representative images of DREADD-hM3Dq-mCherry (purple) and FOS-IR (green) in CNO-treated (1 mg/kg i.p., 2 hours) Ppg^cre (A) and Lepr^cre (B) mice subjected to the injection of AAV^hM3Dq into the GLP1^NTS-Dq (Ppg^Dq) and LepRb^NTS-Dq (LepRb^Dq) mice, respectively. All panels are representative of n ≥ 3 similar images. AP, area postrema; cc, central canal. Scale bar: 150 μm. (C–E) Food intake following vehicle (Veh) or CNO (1 mg/kg i.p.) treatment of Ppg^Dq and LepRb^Dq mice during refeeding following a fast (C; n = 5 and 8 in Ppg^Dq and LepRb^Dq groups, respectively) or at the onset of the dark cycle (DC) on normal chow (D, n = 13 and 20 in Ppg^Dq and LepRb^Dq groups, respectively) or HFD (E, n = 6 and 5 Ppg^Dq and LepRb^Dq groups, respectively). (F and G) Daily food intake (F) and body weight relative to baseline (G) (n = 5–6 [F], 11–13 [G] in Ppg^Dq and LepRb^Dq groups, respectively) during multiday treatment with CNO (1 mg/kg, i.p., bid). Data are from both sexes; for data separated by sex, see Supplemental Figure 1, J–N. Data are shown as mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons test was performed for each time point in each panel; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 for CNO groups compared with vehicle. ^#P < 0.05, ^####P < 0.0001 for comparisons between CNO groups. Scale bar: 150 μm.

Figure 5. GLP1^NTS neurons require Ppg to mediate the suppression of food intake.

Representative images of DREADD-hM3Dq-mCherry (purple) and FOS-IR (green) in CNO-treated (1 mg/kg i.p., 2 hours) Ppg^cre (A) and Ppg^GLP1-NTSKO (Ppg^PpgKO) (B) mice subjected to the injection of AAV^hM3Dq into the NTS (Ppg-Dq and Ppg^PpgKO-Dq mice, respectively). All panels are representative of n ≥ 3 similar images. AP, area postrema; cc, central canal. Scale bars: 150 μm. (C) Food intake in Ppg-Dq and Ppg^PpgKO-Dq mice during the first 4 hours of the dark cycle with chow or HFD, as indicated; n = 19 (chow) or 9 (HFD) in Ppg-Dq and n = 8 (both chow and HFD) in Ppg^PpgKO-Dq groups. (D) Food intake in vehicle-treated (Veh-treated) or CNO-treated (1 mg/kg i.p.) Ppg-Dq and Ppg^PpgKO-Dq mice following an overnight fast; n = 13, 5, and 8 in Veh, Ppg-Dq, and Ppg^PpgKO-Dq groups, respectively. (E and F) Daily food intake (E) and body weight (F) relative to baseline during multiday treatment with CNO (1 mg/kg, i.p., bid); n = 10, 8, and 8 in Veh, Ppg-Dq, and Ppg^PpgKO-Dq groups, respectively. Data are from both sexes; for data separated by sex, see Supplemental Figure 2, A–D. Data are shown as mean ± SEM. Two-way ANOVA with Sidak’s multiple comparisons test was performed for chow and HCD conditions (separately) in C. Two-way ANOVA with Tukey’s multiple comparisons test was performed for D–F. Different letters indicate difference (P < 0.05) in C. (D–F) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus Veh. ^##P < 0.01, ^####P < 0.0001 for Ppg-Dq versus Ppg^PpgKO-Dq.

Figure 6. NTS Ppg is not required for the suppression of food intake by LepRb^NTS neurons.

Representative images of DREADD-hM3Dq-mCherry (purple) and FOS-IR (green) in CNO-treated (1 mg/kg i.p., 2 hours) Lepr^cre (A) and Ppg^LepRbKO (B) mice subjected to the injection of AAV^hM3Dq into the NTS (LepRb^NTS-Dq and Ppg^LepRbKO-Dq mice, respectively). All panels are representative of n ≥ 3 similar images. AP, area postrema; cc, central canal. Scale bars: 150 μm. (C) Food intake in LepRb^NTS-Dq and Ppg^LepRbKO-Dq mice during the first 4 hours of the dark cycle with Chow or HFD, as indicated; n = 14 (chow) or 8 (HFD) in Ppg-Dq groups, n = 8 (both chow and HFD) in Ppg^LepRbKO-Dq groups. (D) Food intake in vehicle-treated (Veh-treated) or CNO-treated (1 mg/kg i.p.) LepRb^NTS-Dq and Ppg^LepRbKO-Dq mice following an overnight fast; n = 7, 11, and 8 in Veh, LepRb^NTS-Dq, and Ppg^LepRbKO-Dq groups, respectively. (E and F) Daily food intake (E) and body weight relative to baseline (F) during multiday treatment with CNO (1 mg/kg, i.p., bid); n = 12, 13, and 9 in Veh, LepRb^NTS-Dq, and Ppg^LepRbKO-Dq groups, respectively. Data are from both sexes; for data separated by sex, see Supplemental Figure 2, E–H. Data are shown as mean ± SEM. Two-way ANOVA with Sidak’s multiple comparisons was performed for chow and HCD conditions (separately) in C. Two-way ANOVA analysis with Tukey’s multiple comparisons test was performed for D–F. Different letters indicate difference (P < 0.05) in C. (D–F) **P < 0.01, ***P < 0.001, ****P < 0.0001 versus Veh.