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. 2019 Jan;68(1):21-33.
doi: 10.2337/db18-0729. Epub 2018 Oct 2.

Preproglucagon Neurons in the Nucleus of the Solitary Tract Are the Main Source of Brain GLP-1, Mediate Stress-Induced Hypophagia, and Limit Unusually Large Intakes of Food

Marie K Holt  1 , James E Richards  1 , Daniel R Cook  1 , Daniel I Brierley  1 , Diana L Williams  2 , Frank Reimann  3 , Fiona M Gribble  3 , Stefan Trapp  4
Affiliations

Preproglucagon Neurons in the Nucleus of the Solitary Tract Are the Main Source of Brain GLP-1, Mediate Stress-Induced Hypophagia, and Limit Unusually Large Intakes of Food

Marie K Holt et al. Diabetes. 2019 Jan.

Abstract

Centrally administered glucagon-like peptide 1 (GLP-1) supresses food intake. Here we demonstrate that GLP-1-producing (PPG) neurons in the nucleus tractus solitarii (NTS) are the predominant source of endogenous GLP-1 within the brain. Selective ablation of NTS PPG neurons by viral expression of diphtheria toxin subunit A substantially reduced active GLP-1 concentrations in brain and spinal cord. Contrary to expectations, this loss of central GLP-1 had no significant effect on the ad libitum feeding of mice, affecting neither daily chow intake nor body weight or glucose tolerance. Only after bigger challenges to homeostasis were PPG neurons necessary for food intake control. PPG-ablated mice increased food intake after a prolonged fast and after a liquid diet preload. Consistent with our ablation data, acute inhibition of hM4Di-expressing PPG neurons did not affect ad libitum feeding; however, it increased refeeding intake after fast and blocked stress-induced hypophagia. Additionally, chemogenetic PPG neuron activation through hM3Dq caused a strong acute anorectic effect. We conclude that PPG neurons are not involved in primary intake regulation but form part of a secondary satiation/satiety circuit, which is activated by both psychogenic stress and large meals. Given their hypophagic capacity, PPG neurons might be an attractive drug target in obesity treatment.

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Figures

Figure 1
Figure 1
Pharmacogenetic activation of PPG neurons robustly reduces food intake. A: Expression of hM3Dq:mCherry (magenta) in PPG neurons (detected with an anti-GFP antibody; green) 3 weeks after stereotaxic injection of AAV2-hM3Dq:mCherry into the NTS of Glu-Cre/GCaMP3 mice. Scale bar, 100 µm. B: Increase in [Ca2+]i in GCaMP3-expressing PPG neurons in response to superfusion of ex vivo brainstem slices with 1 μmol/L CNO. Data are displayed as traces (top left panel) and a heat map (bottom left panel) representing the fractional change in fluorescence from baseline. Right panel: Representative pseudocolored cell responding to 1 μmol/L CNO with an increase in [Ca2+]i. Scale bar, 10 μm; n = 11 cells. C: Expression of the immediate early gene cFOS (green) in PPG neurons expressing hM3Dq:mCherry (magenta) after i.p. injection of 2 mg/kg CNO (top panels) or saline (bottom panels). White arrows, representative cFOS-positive hM3Dq-expressing cells; white stars, representative cFOS-negative hM3Dq-positive cells. Scale bars: middle panel, 100 μm; right panel, 20 μm. Noncumulative (D) and cumulative (E) food intake in the first 4 h of dark phase after injection of CNO (2 mg/kg i.p.) or saline. CNO was delivered 30 min prior to dark onset. Data are given as the mean ± SEM; n = 9 mice. D: No significant time × drug interaction (F(2,16) = 2.897, P = 0.0844), but a significant main effect of drug treatment (F(1,8) = 17.31, P = 0.0032). E: Significant time × drug interaction (F(2,16) = 5.626, P = 0.0141). One-hour P = 0.0005; 2-h P < 0.0001; 4-h P < 0.0001 (Sidak multiple-comparisons test). Inset: P = 0.20 (paired t test). F: Daily chow intake in hM3Dq-expressing (n = 7) and control (n = 6) Glu-Cre mice in response to twice-daily i.p. injection of 2 mg/kg CNO (indicated with black arrows). Significant time × virus interaction (F(11,121) = 2.06, P = 0.0283); day 6 P = 0.0119 (Sidak multiple-comparisons test). Data are given as the mean ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001.
Figure 2
Figure 2
NTS PPG neurons are the main source of brain GLP-1. A: Expression of mCherry and GCaMP3 (as a marker for PPG neurons) 4, 7, and 14 days after unilateral stereotaxic injection of AAV8-mCherry-FLEX-DTA into the NTS of a Glu-Cre/GCaMP3 mouse (schematic on left). White arrows indicate the remaining GCaMP3-positive PPG neurons. Scale bars: top panels, 100 μm; inset, 20 μm. B: Protein levels of active GLP-1 (normalized to total protein) detected in several brain regions after bilateral stereotaxic injection of AAV8-mCherry-FLEX-DTA or a control virus (AAV1/2-FLEX-Perceval). Brainstem P = 0.0317; hypothalamus P = 0.0079 (Mann-Whitney U test); spinal cord P = 0.0004 (unpaired t test). Data are given as the mean ± SEM; n = 5 in each group. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 3
Figure 3
Ablation of NTS PPG neurons has no impact on body weight, food intake, or glucose tolerance. Body weight change (A) and daily chow intake (B) after stereotaxic injection of AAV8-mCherry-FLEX-DTA or control virus. Body weight was measured every 2–3 days over 2 months. Data are given as the mean ± SEM; n = 7 (control), n = 6 (DTA). A: No significant time × virus interaction (F(1,12) = 0.08578, P = 0.7746) and no significant main effect of virus (F(1,12) = 0.08578, P = 0.7746). B: P = 0.45 (unpaired t test). Blood glucose in response to an i.p. injection of glucose (1 g/kg) at t = 0 7 weeks after stereotaxic injection of DTA or control virus in six male (C) and six female (D) Glu-Cre mice. Area under the curve (AUC) of the i.p. glucose tolerance test for each group is given on the right of each graph. Data are given as the mean ± SEM. Glucose concentrations: there was a significant time × virus interaction for males (F(5,20) = 3.83, P = 0.014), but no significant difference between DTA and control mice at any timepoint. There was no significant time × virus interaction for females (F(5,20) = 0.19, P = 0.96) and no significant main effect of virus (F(1,4) = 1.08, P = 0.36). AUC: no effect of virus for males (P > 0.99, Mann-Whitney U test) or females (P = 0.34, unpaired t test). a.u., arbitrary units; n.s., not significant.
Figure 4
Figure 4
Ad libitum food intake is unaffected by ablation or acute inhibition of NTS PPG neurons. Cumulative (A) and noncumulative (B) food intake of control and PPG-ablated (DTA) mice in the first 4 h of the dark phase. Data are given as the mean ± SEM; n = 7 (control), n = 6 (DTA). n.s., not significant. A: No significant time × virus interaction (F(2,22) = 0.5406, P = 0.5900) and no significant main effect of virus (F(1,11) = 0.012, P = 0.91). B: No significant time × virus interaction (F(2,22) = 0.834, P = 0.4476) and no significant main effect of virus (F(1,11) = 0.1472, P = 0.7085). C: Cre-dependent expression of hM4Di and EGFP as control in the NTS of Glu-Cre/GCaMP3 mice. Scale bar, 100 μm. Bottom left: Schematic of bilateral injections. Bottom right: Representative voltage-clamp recording (top) and summary data (bottom) of hM4Di-expressing PPG neurons in ex vivo slice preparation superfused with CNO (1 μmol/L). Data are given as the mean ± SEM; n = 5. *P < 0.05 (paired t test). Cumulative (D) and noncumulative (E) food intake of hM4Di-expressing mice injected with saline or CNO (2 mg/kg i.p.; 30 min prior to dark onset) in the first 4 h of the dark phase. Data are given as the mean ± SEM; n = 12. D: No significant main effect of virus (F(1,21) = 1.19, P = 0.29) or drug (F(1,21) = 0.002, P = 0.96). E: No significant main effect of virus (F(1,20) = 0.11, P = 0.75) or drug (F(1,20) = 0.045, P = 0.83). Data from mice expressing control virus (Supplementary Fig. 4) are included in the analysis. n.s., not significant.
Figure 5
Figure 5
Ablation or acute inhibition of PPG neurons increases food intake only after a large meal. Cumulative (A and B) and noncumulative (C) food intake of control and PPG-ablated mice in the first 4 h (A and C) and 21 h (B) after the onset of the dark phase after 18 h of food deprivation prior to the onset of dark. Data are given as the mean ± SEM; n = 7 (control), n = 6 (DTA). A: No significant virus × time interaction (F(2,22) = 1.81, P = 0.19), but a significant main effect of virus (F(1,11) = 8.0, P = 0.016). B: P = 0.056 (unpaired t test). C: No significant virus × time interaction (F(2,22) = 0.75, P = 0.49), but a significant main effect of virus (F(1,11) = 6.1, P = 0.031). Cumulative (D and E) and noncumulative (F) food intake of hM4Di-expressing mice injected with saline or CNO (2 mg/kg i.p.; 30 min prior to dark onset) in the first 4 h of the dark phase after 18 h of food deprivation prior to the onset of dark. Data are given as the mean ± SEM; n = 12. D: There was a significant virus × drug interaction at hour 1 (F(1,21) = 4.733, P = 0.0411), P = 0.038 (CNO vs. saline, Sidak multiple comparisons test). E: No significant virus × drug interaction (F(1,21) = 1.245, P = 0.28) and no significant main effect of drug (F(1,21) = 1.67, P = 0.21) or virus (F(1,21) = 0.084, P = 0.77). F: There was a significant virus × drug interaction at hour 1 (F(1,21) = 4.733, P = 0.0411), P = 0.038 (CNO vs. saline; Sidak multiple-comparisons test). Data from mice expressing control virus are included in analysis. *P < 0.05. n.s., not significant.
Figure 6
Figure 6
Intake of large volumes of highly palatable diet activates PPG neurons. A: Expression of the immediate early gene cFOS (green) in PPG neurons after 30 min access to Vanilla Ensure or no access to food (control). White arrows: representative cFOS-positive PPG neurons. Scale bar, 100 μm. B: Percentage of PPG neurons expressing cFOS 90 min after 30-min access to Vanilla Ensure or no access to food. Data are given as the mean ± SEM; n = 3 (control), n = 3 (Ensure). P = 0.0079 (Mann-Whitney U test). C: Ensure intake during 15-min access at dark onset over several days of habituation in control and PPG-ablated (DTA) mice. Data are given as the mean ± SEM; n = 7 (control), n = 7 (DTA). No interaction of virus × time (F(3,36) = 0.592, P = 0.62) and no significant main effect of virus (F(1,12) = 1.135, P = 0.31) or time (F(3,36) = 0.19, P = 0.90).). D: Ensure intake during the 15-min access period on the test day in control and PPG-ablated (DTA) mice. Data are given as the mean ± SEM; n = 7 (control), n = 7 (DTA). P = 0.70 (unpaired t test). E: Chow intake of control and PPG-ablated (DTA) mice for 1 h after 15-min access to Vanilla Ensure. Data given as the mean ± SEM; n = 7 (control), n = 7 (DTA). P = 0.0052 (unpaired t test). **P < 0.01. n.s., not significant.
Figure 7
Figure 7
Stress-induced hypophagia requires PPG neurons. Cumulative (A) and noncumulative (B) chow intake of mice expressing EGFP or hM4Di injected with 2 mg/kg CNO i.p. 60 min prior to dark onset and left undisturbed or exposed to 30 min of restraint stress 30 min prior to dark onset. Also included in A are data from the same EGFP- and hM4Di-expressing mice injected with saline and left undisturbed. Data are given as the mean ± SEM; n = 9 (control), n = 12 (hM4Di). A: Significant main effect of stress for control (F(1,8) = 14.14, P = 0.0055), but not hM4Di (F(1,11) = 0.8684, P = 0.37). No effect of CNO on food intake in both control (P = 0.5) and PPG-inhibited mice (P = 0.98) compared with saline vehicle in the absence of stress. B: Hour 0–1: Significant effect of stress in the EGFP group (P = 0.0008), but not in the hM4Di group (P = 0.25) (Sidak multiple-comparisons test). Hour 1–2: No significant virus × stress interaction (F(1,19) = 0.091, P = 0.77) and no main effect of virus (F(1,19) = 0.49, P = 0.49) or stress (F(1,19) = 1.16, P = 0.29). **P < 0.01; ***P < 0.001. n.s., not significant.
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