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ABSTRACT Hypothalamic 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. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS

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institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS ADRENERGIC MODULATION OF MELANOCORTIN PATHWAY BY HUNGER SIGNALS Article Open

access 19 October 2023 GLUTAMATERGIC PROJECTIONS FROM HOMEOSTATIC TO HEDONIC BRAIN NUCLEI REGULATE INTAKE OF HIGHLY PALATABLE FOOD Article Open access 16 December 2020 _CANNABIS SATIVA_

TARGETS MEDIOBASAL HYPOTHALAMIC NEURONS TO STIMULATE APPETITE Article Open access 27 December 2023 CHANGE HISTORY * _ 04 MARCH 2015 Minor changes were made to citations in the Methods. _

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(2011) Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS The authors thank M. Shanabrough and J. Bober for technical support and R. Jakab for assisting with the

illustrations. This work was supported by the US National Institutes of Health (DP1 DK098058, R01 DK097566, R01 AG040236 and P01 NS062686), the American Diabetes Association, The Klarmann

Family Foundation, the Helmholtz Society (ICEMED) and the Deutsche Forschungsgemeinschaft SFB 1052/1 (Obesity Mechanisms). AUTHOR INFORMATION Author notes * Jae Geun Kim Present address:

Present address: Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon 406-772, South Korea., AUTHORS AND AFFILIATIONS * Program in

Integrative Cell Signaling and Neurobiology of Metabolism, Section of Comparative Medicine, Yale University School of Medicine, New Haven, 06520, Connecticut, 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 * Institute of Anatomy, University of

Leipzig, 04103 Leipzig, Germany, Marco Koch & Ingo Bechmann * Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, 06520,

Connecticut, USA Jung Dae Kim, Sabrina Diano & Tamas L. Horvath * Department of Physiology, Obesity & Diabetes Institute, Monash University, Clayton, 3800, Victoria, Australia

Stephanie E. Simonds & Michael A. Cowley * Division of Endocrinology & Metabolism, Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas,

75390, Texas, USA Carlos M. Castorena, Claudia R. Vianna & Joel K. Elmquist * Department of Neurobiology, Yale University School of Medicine, New Haven, 06520, Connecticut, USA Yury M.

Morozov, Pasko Rakic, Marcelo O. Dietrich, Sabrina Diano & Tamas L. Horvath * Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, 06520, Connecticut, USA

Pasko Rakic & Tamas L. Horvath Authors * Marco Koch View author publications You can also search for this author inPubMed Google Scholar * Luis Varela View author publications You can

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author inPubMed Google Scholar 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. CORRESPONDING AUTHOR Correspondence to Tamas L. Horvath.

ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. EXTENDED DATA FIGURES AND TABLES EXTENDED DATA FIGURE 1 CHARACTERIZATION OF CB1R-DEPENDENT FOOD

INTAKE. 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, _P_ values (unpaired comparisons) by two-tailed Student’s _t_-test. Scale bars, 25 μm. Source data EXTENDED DATA

FIGURE 2 DREADD-MEDIATED REGULATION OF POMC NEURONS. 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). Source data EXTENDED DATA FIGURE 3 HYPERPHAGIC CB1R ACTIVATION SELECTIVELY INCREASED PVN Β-ENDORPHIN. 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.

Source data EXTENDED DATA FIGURE 4 BIMODAL CHARACTER OF ARC CB1R-DRIVEN Β-ENDORPHIN INCREASE. 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. Source data 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. 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. Source data EXTENDED DATA FIGURE 6 BIMODAL CB1R-DEPENDENT REGULATION OF MITOCHONDRIAL RESPIRATION AND UCP2-DEPENDENT CONTROL OF POMC. A, B, Bimodal

CB1R-controlled mitochondrial respiration in hippocampus. A, Hyperphagic (1 mg kg−1 body weight ACEA, intraperitoneal) CB1R activation increased _ex vivo_ mitochondrial 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−1body 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 (pre-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. Source

data SUPPLEMENTARY INFORMATION SUPPLEMENTARY TABLE This file contains Supplementary Table 1. (PDF 103 kb) POWERPOINT SLIDES POWERPOINT SLIDE FOR FIG. 1 POWERPOINT SLIDE FOR FIG. 2 POWERPOINT

SLIDE FOR FIG. 3 POWERPOINT SLIDE FOR FIG. 4 POWERPOINT SLIDE FOR FIG. 5 SOURCE DATA SOURCE DATA TO FIG. 1 SOURCE DATA TO FIG. 2 SOURCE DATA TO FIG. 3 SOURCE DATA TO FIG. 4 SOURCE DATA TO

FIG. 5 SOURCE DATA TO EXTENDED DATA FIG. 6 SOURCE DATA TO EXTENDED DATA FIG. 7 SOURCE DATA TO EXTENDED DATA FIG. 8 SOURCE DATA TO EXTENDED DATA FIG. 9 SOURCE DATA TO EXTENDED DATA FIG. 10

SOURCE DATA TO EXTENDED DATA FIG. 11 RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Koch, M., Varela, L., Kim, J. _et al._ Hypothalamic POMC neurons

promote cannabinoid-induced feeding. _Nature_ 519, 45–50 (2015). https://doi.org/10.1038/nature14260 Download citation * Received: 22 March 2014 * Accepted: 23 January 2015 * Published: 18

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