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Hypothalamic POMC neurons promote cannabinoid-induced feeding

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Marco Koch, Luis Varela, Jae Geun Kim, Jung Dae Kim, Francisco Hernández-Nuño, Stephanie E. Simonds, Carlos M. Castorena, Claudia R. Vianna, Joel K. Elmquist, Yury M. Morozov, Pasko Rakic, Ingo Bechmann, Michael A. Cowley, Klara Szigeti-Buck, Marcelo O. Dietrich, Xiao-Bing Gao, Sabrina Diano & Tamas L. Horvath

Affiliations

Nature (2015) doi:10.1038/nature14260
Received
Accepted
Published online
18 February 2015

Nature MedicineHypothalamic pro-opiomelanocortin (POMC) neurons promote satiety. Cannabinoid receptor 1 (CB1R) is critical for the central regulation of food intake. Here we test whether CB1R-controlled feeding in sated mice is paralleled by decreased activity of POMC neurons. We show that chemical promotion of CB1R activity increases feeding, and notably, CB1R activation also promotes neuronal activity of POMC cells. This paradoxical increase in POMC activity was crucial for CB1R-induced feeding, because designer-receptors-exclusively-activated-by-designer-drugs (DREADD)-mediated inhibition of POMC neurons diminishes, whereas DREADD-mediated activation of POMC neurons enhances CB1R-driven feeding. The Pomc gene encodes both the anorexigenic peptide α-melanocyte-stimulating hormone, and the opioid peptide β-endorphin. CB1R activation selectively increases β-endorphin but not α-melanocyte-stimulating hormone release in the hypothalamus, and systemic or hypothalamic administration of the opioid receptor antagonist naloxone blocks acute CB1R-induced feeding. These processes involve mitochondrial adaptations that, when blocked, abolish CB1R-induced cellular responses and feeding. Together, these results uncover a previously unsuspected role of POMC neurons in the promotion of feeding by cannabinoids.

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Author information

  1. Present address: Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon 406-772, South Korea.

    • Jae Geun Kim

Affiliations

  1. Program in Integrative Cell Signaling and Neurobiology of Metabolism, Section of Comparative Medicine, Yale University School of Medicine, New Haven, Connecticut 06520, USA

    • Marco Koch,
    • Luis Varela,
    • Jae Geun Kim,
    • Jung Dae Kim,
    • Francisco Hernández-Nuño,
    • Klara Szigeti-Buck,
    • Marcelo O. Dietrich,
    • Xiao-Bing Gao,
    • Sabrina Diano &
    • Tamas L. Horvath

  2. Institute of Anatomy, University of Leipzig, 04103 Leipzig, Germany

    • Marco Koch &
    • Ingo Bechmann

  3. Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, Connecticut 06520, USA

    • Jung Dae Kim,
    • Sabrina Diano &
    • Tamas L. Horvath

  4. Obesity & Diabetes Institute, Department of Physiology, Monash University, Clayton, Victoria 3800, Australia

    • Stephanie E. Simonds &
    • Michael A. Cowley

  5. Division of Endocrinology & Metabolism, Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

    • Carlos M. Castorena,
    • Claudia R. Vianna &
    • Joel K. Elmquist

  6. Department of Neurobiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA

    • Yury M. Morozov,
    • Pasko Rakic,
    • Marcelo O. Dietrich,
    • Sabrina Diano &
    • Tamas L. Horvath

  7. Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06520, USA

    • Pasko Rakic &
    • Tamas L. Horvath

Contributions

M.K., S.D. and T.L.H. developed the conceptual framework of this study. M.K., M.O.D., X.-B.G., S.D. and T.L.H. interpreted results. M.K. performed experiments and analysed results. Experimental contributions: L.V. contributed to Figs 4h–j, 5d and Extended Data Figs 1b, 5e and 6a, b; J.G.K. contributed to Figs 2e, f, 3i, 5a, b and Extended Data Fig. 2g; J.D.K. contributed to Figs 3b–d, 5e–g and Extended Data Figs 5c and 6c; F.H. contributed to Figs 4a, 5c and Extended Data Fig. 5a, b, d; S.E.S. contributed to Fig. 3a; C.M.C., C.R.V. and J.K.E. provided key animal models; Y.M.M. and P.R. contributed to Fig. 3b and Extended Data Fig. 1c; P.R., I.B. and M.A.C. provided materials, animals and equipment; K.S.-B. contributed to Figs 3f and 4d–g; X.-B.G. contributed to Figs 1C, Da–c and 3j. M.K. and T.L.H. wrote the paper.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Characterization of CB1R-dependent food intake. (449 KB)
    a, Bimodal effects of different ACEA doses on food intake in fed mice (vehicle, n = 23 mice, 100 ± 16.3%; ACEA (in mg kg−1 body weight, intraperitoneal): 0.1, n = 8, 104.5 ± 46.6%; 0.5, n = 3, 190.8 ± 40.4%; 1.0, n = 19, 196.7 ± 30%; 2.5, n = 16, 87.1 ± 18%; 5.0, n = 11, 59.2 ± 15.5%; P < 0.01 versus vehicle, one-way ANOVA, followed by Dunnett’s multiple comparisons test; six independent experiments with litters from different parents). b, Neutral dose of ACEA on feeding (5 mg kg−1 body weight, intraperitoneal) did not alter locomotor activity of fed mice (n = 3 mice/group; P > 0.05). c, Impaired feeding response to ACEA (1 mg kg−1 body weight, intraperitoneal) in CB1R-heterozygote mice (Cnr1+/−, n = 6 mice, 1 h: 0.04 ± 0.01 g, 2 h: 0.07 ± 0.01 g) and CB1R-deficient mice (Cnr1−/−, 1 h: n = 6, 0.02 ± 0.01 g, 2 h: n = 4, 0.03 ± 0.01 g) mice, when compared to CB1R wild-type mice (Cnr1+/+, 1 h: n = 12, 0.13 ± 0.01 g, 2 h: n = 4, 0.18 ± 0.04 g; P < 0.01, P < 0.001 versus wild-type; two independent experiments). d, Central, local ACEA injection into the ARC induced food intake (vehicle, n = 4 mice, 1 h: 0.05 ± 0.03 g, 2 h: 0.12 ± 0.01 g; ACEA, n = 4, 1 h: 0.25 ± 0.03 g; 2 h: 0.43 ± 0.05 g; P < 0.01, P < 0.001). e, Verification of correct ARC cannula placement by HOECHST (blue) injection (representative image (two different magnifications) of four independent experiments). f, Hyperphagic CB1R activation (1 mg kg−1 body weight ACEA, intraperitoneal) was abolished by central, local ARC RIMO-mediated CB1R blockade (vehicle plus vehicle, n = 8 mice, 0.05 ± 0.01 g; vehicle plus ACEA, n = 8, 0.15 ± 0.02 g; RIMO plus vehicle, n = 8, 0.09 ± 0.02 g; RIMO plus ACEA, n = 8, 0.09 ± 0.02 g; P < 0.05, #P < 0.05 for interaction between RIMO and ACEA, two-way ANOVA, followed by Šidák’s multiple comparisons test; two independent experiments). g, Hyperphagic CB1R activation (1 mg kg−1 body weight WIN, intraperitoneal) was reduced by local ARC RIMO-mediated CB1R blockade (vehicle plus WIN, n = 8 mice, 0.21 ± 0.03 g; RIMO+WIN, n = 8, 0.1 ± 0.02 g; P < 0.01). h, RIMO-induced hypophagic blockade of CB1R in fasted mice (vehicle, n = 10 mice, 1 h: 0.76 ± 0.07 g, 2 h: 1.18 ± 0.07 g; RIMO, n = 11 mice, 1 h: 0.42 ± 0.05 g, 2 h: 0.75 ± 0.08 g; P < 0.01, P < 0.001; two independent experiments). Values (biological replicates) denote mean ± s.e.m. If not otherwise stated, Pvalues (unpaired comparisons) by two-tailed Student’s t-test. Scale bars, 25 μm.
  2. Extended Data Figure 2: DREADD-mediated regulation of POMC neurons. (622 KB)
    a, Selective DREADD expression specified by local ARC mCherry fluorescence. b, POMC neurons (green) contain mCherry-labelled DREADD (red, arrowheads). c, CNO-activated inhibitory DREADD reduced ARC cFOS immunolabelled neurons in fed mice (arrowheads). Representative images of four independent experiments (a–c). d, e, CNO-activated inhibitory DREADD blocked ACEA-induced POMC activation (cFOS; vehicle plus ACEA, n= 6 mice, 60.4 ± 3.6%; CNO plus ACEA, n = 5, 32.3 ± 2.5%; P < 0.001). f, CNO-activated POMC-specific inhibitory DREADD did not acutely affect feeding but enhanced it after 8 h (vehicle, n = 17 mice, 0.42 ± 0.04 g; CNO, n = 16, 0.58 ± 0.04 g; 24 h after injection: vehicle, n = 5 mice, 2.57 ± 0.07 g; CNO, n = 5, 3.37 ± 0.18 g; P < 0.01 versus vehicle; three independent experiments). g, CNO-activated POMC-specific stimulating DREADD did not acutely affect feeding but reduced it after 8 h (vehicle, n = 6 mice, 0.58 ± 0.05 g; CNO, n = 6, 0.34 ± 0.05 g; P < 0.01 versus vehicle; 24 h after injection: vehicle, 3.96 ± 0.15 g; CNO, 3.65 ± 0.21 g; P > 0.05 versus vehicle). Values (biological replicates) denote mean ± s.e.m. If not otherwise stated, P values (unpaired comparisons) by two-tailed Student’s t-test. Scale bars, 100 μm (a), 25 μm (b) and 50 μm (c, d).
  3. Extended Data Figure 3: Hyperphagic CB1R activation selectively increased PVN β-endorphin. (735 KB)
    a–d, i, PVN α-MSH remained unchanged after hyperphagic CB1R activation (PVN unilateral analysis; vehicle, n = 6 values (technical replicates)/6 sections/3 mice (biological replicates); 60 min ACEA, n = 10/10/5; 90 min ACEA, n = 6/6/3; values, see Extended Data Table 1a). e–h, j, In contrast, hyperphagic ACEA increased PVN β-endorphin 60 and 90 min after application (PVN unilateral analysis; vehicle, n = 13 values/13 sections/6 mice; 60 min ACEA, n = 4/4/4; 90 min ACEA, n = 14/14/7; values, see Extended Data Table 1b. P < 0.001, P < 0.05 versus vehicle, one-way ANOVA, followed by Dunnett’s multiple comparisons test, two independent experiments using litters from different parents). Error bars indicate mean ± s.e.m. Scale bars, 25 μm.
  4. Extended Data Figure 4: Bimodal character of ARC CB1R-driven β-endorphin increase. (239 KB)
    a, Compared to vehicle (bilateral PVN analysis; n = 22 values (technical replicates)/11 sections/4 mice (biological replicates), hyperphagic doses (1 mg kg−1 body weight, respectively) of WIN (n = 24/12/4) or ACEA (n = 18/9/3) induced PVN β-endorphin immunoreactivity. Neutral dose (5 mg kg−1 BW) of ACEA (n = 18/9/3) on feeding showed no effects (see Extended Data Table 2 for all values). P < 0.05, P < 0.01, P < 0.001 versus vehicle, one-way ANOVA, followed by Dunnett’s multiple comparisons test. b, Representative binary images of four independent experiments showing β-endorphin immunoreactivity after thresholding (image segmentation) using ImageJ software (see Methods). c, Compared to vehicle (unilateral PVN analysis; n = 4 mice (biological replicates), 2–3 sections (technical replicates) per mouse), central, hyperphagic local ARC injection of ACEA (n = 5 mice, 3 sections per mouse) increased PVN β-endorphin immunoreactivity (see Extended Data Table 3 for all values; P < 0.05, P < 0.01). Error bars indicate mean ± s.e.m. If not otherwise stated, P values (unpaired comparisons) by two-tailed Student’s t-test. Scale bars, 100 μm.
  5. Extended Data Figure 5: Post-transcriptional regulation of hypothalamic pro-protein convertases, normal Cnr1 expression in Ucp2−/− mice and presence of CB1R in POMC neurons. (289 KB)
    a, b, ACEA did not affect transcripts of pro-protein convertases 1 (Pcsk1) and 2 (Pcsk2) (in fold change; Pcsk1: vehicle, n = 11 mice, 1.00 ± 0.07; ACEA, n = 10 mice, 1.17 ± 0.09; Pcsk2: vehicle, n = 11 mice, 1.00 ± 0.13; ACEA, n = 11 mice, 1.14 ± 0.19; P > 0.05; two independent experiments). c, Representative western blot membranes for PC-1 (~80 kilodaltons (kDa)) and PC-2 (~72 kDa) immunolabelling. d, Equal Cnr1 expression in wild-type and Ucp2−/− mice (in fold change: all groups n = 6 mice; wild type, 1.00 ± 0.1; Ucp2−/−, 0.98 ± 0.12; P > 0.05). e, We have previously shown that antibodies raised against CB1R also recognized the mitochondrial protein, stomatin-like protein 2 (ref. 21). In line with this, mitochondrial labelling of CB1R was found substantially diminished but not completely eliminated in CB1R-KO (Cnr1−/−) mice23, 24, 25. We observed that in contrast to wild-type animals (Cnr1+/+ mice), which showed ~80% (77 out of 97, 79.5 ± 3.9%) of POMC neurons (red fluorescence) to contain labelling with the CB1R antisera (green fluorescence), in CB1R knockout (KO; Cnr1−/−) mice, less than 30% (37 out of 128, 29.2 ± 3.3%) of POMC neurons retained immunolabelling. Thus, we concluded that a large population of POMC neurons contains CB1R (P < 0.001). All values (biological replicates: a–c, d; biological replicates including technical replicates: e) denote mean ± s.e.m. If not otherwise stated, P values (unpaired comparisons) by two-tailed Student’s t-test. Scale bar, 25 μm.
  6. Extended Data Figure 6: Bimodal CB1R-dependent regulation of mitochondrial respiration and UCP2-dependent control of POMC. (511 KB)
    a, b, Bimodal CB1R-controlled mitochondrial respiration in hippocampus. a, Hyperphagic (1 mg kg−1 body weight ACEA, intraperitoneal) CB1R activation increased ex vivomitochondrial respiration (in nmol O2 min−1 mg−1 protein; state 3: vehicle, n = 6 mice, 170.7 ± 12; ACEA, n = 8, 252.7 ± 17.2; state 4: vehicle, 92.7 ± 5.4; ACEA, 139.7 ± 6; P < 0.01, P < 0.001). b, Neutral dose of ACEA on feeding (5 mg kg−1 body weight, intraperitoneal) reduced mitochondrial respiration (state 3: vehicle, n = 7 mice, 178.2 ± 12.2; ACEA, n = 5, 118.9 ± 9.4; state 4: vehicle, 100 ± 5.1; ACEA, 64.3 ± 6.3; two independent experiments). c, Representative western blot membranes for POMC (pro-POMC, ~31 kDa; POMC, ~27 kDa). d, The 24-h food intake did not differ between wild-type (n = 28 mice, 100 ± 3.2%) and Ucp2−/− (n = 29, 98.9 ± 4.7%; P > 0.05) mice after ACEA (1 mg kg−1 body weight, intraperitoneal) treatment (six independent experiments using litters from different parents). All values (biological replicates) denote ± s.e.m. If not otherwise stated, P values (unpaired comparisons) by two-tailed Student’s t-test.

Extended Data Tables

  1. Extended Data Table 1: Semi-quantitative measurements of α-MSH and β-endorphin immunoreactivity (144 KB)
  2. Extended Data Table 2: Semi-quantitative measurements of β-endorphin immunoreactivity (111 KB)
  3. Extended Data Table 3: Semi-quantitative measurements of β-endorphin immunoreactivity (58 KB)
  4. Extended Data Table 4: Semi-quantitative measurements of β-endorphin immunoreactivity (101 KB)

Supplementary information

PDF files

  1. Supplementary Table (103 KB)
    This file contains Supplementary Table 1.twin memes II