Activation of temperature-sensitive TRPV1-like receptors in ARC POMC neurons reduces food intake

Abstract

Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARC) respond to numerous hormonal and neural signals, resulting in changes in food intake. Here, we demonstrate that ARC POMC neurons express capsaicin-sensitive transient receptor potential vanilloid 1 receptor (TRPV1)-like receptors. To show expression of TRPV1-like receptors in ARC POMC neurons, we use single-cell reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, electrophysiology, TRPV1 knock-out (KO), and TRPV1-Cre knock-in mice. A small elevation of temperature in the physiological range is enough to depolarize ARC POMC neurons. This depolarization is blocked by the TRPV1 receptor antagonist and by Trpv1 gene knockdown. Capsaicin-induced activation reduces food intake that is abolished by a melanocortin receptor antagonist. To selectively stimulate TRPV1-like receptor-expressing ARC POMC neurons in the ARC, we generate an adeno-associated virus serotype 5 (AAV5) carrying a Cre-dependent channelrhodopsin-2 (ChR2)-enhanced yellow fluorescent protein (eYFP) expression cassette under the control of the two neuronal POMC enhancers (nPEs). Optogenetic stimulation of TRPV1-like receptor-expressing POMC neurons decreases food intake. Hypothalamic temperature is rapidly elevated and reaches to approximately 39 °C during treadmill running. This elevation is associated with a reduction in food intake. Knockdown of the Trpv1 gene exclusively in ARC POMC neurons blocks the feeding inhibition produced by increased hypothalamic temperature. Taken together, our findings identify a melanocortinergic circuit that links acute elevations in hypothalamic temperature with acute reductions in food intake.

Fig 1. ARC POMC neurons express temperature-sensitive TRPV1 receptors.

(A) Single-cell RT-PCR analysis of POMC neurons showing Trpv1 mRNA expression. Images showing POMC-eGFP neurons in hypothalamic slices (left). Right: Transcripts were collected from 20 different POMC neurons (lane, 1–20). L: ladder (Trpv1, 120 bp; Pomc, 170 bp; 18S rRNA, 121 bp). P: positive control. (B) Images of fluorescence confocal microscopy showing TRPV1 receptor expression in the ARC of WT and TRPV1 KO mice. Scale bar: 100 μm. (C) TRPV1 receptor expression in the ARC of WT and TRPV1 KO mice. (D) Images of fluorescence confocal microscopy showing TRPV1 receptor (red) expression in the ARC of POMC-eGFP mice. Arrowheads represent TRPV1-positive ARC POMC neurons. Scale bar: 20 μm. (E) Images of fluorescence confocal microscopy showing TRPV1 receptor-positive POMC neurons (red) in the ARC of TRPV1-Cre mice injected with AAV5-DIO-GFP (Arrowheads). Approximately 90% of GFP-positive cells contained POMC (n = 357 out of 401 GFP-positive cells, n = 3 mice). Scale bar: 20 μm. (F) Representative traces showing depolarization of POMC neurons in response to increased temperature. This depolarization was abolished by the TRPV1 receptor antagonist and by knockdown of TRPV1 receptors with TRPV1 receptor shRNA. Scale bar: 25 mV, 1 min. (G) Pooled data of mean membrane potential in response to elevated bath temperature from 34 °C to 38 °C (Control, n = 10 neurons, AMG9810 [10 μM], n = 11 neurons, and Trpv1 shRNA, n = 11 neurons, *p < 0.05, **p < 0.01, ***p < 0.001 versus 34 °C). (H) Pooled data of mean membrane potential at 34 °C and 38 °C (38 °C, Control, ΔVm, 11.6 ± 2.1 mV, n = 10 neurons, AMG9810, ΔVm, 2.5 ± 0.5 mV, n = 11 neurons, TRPV1 shRNA, ΔVm, 3.6 ± 1.0 mV, n = 11 neurons, **p < 0.01, ***p < 0.001 versus 34 °C). Data are shown as mean ± SEM. AAV5, adeno-associated virus serotype 5; ARC, arcuate nucleus of the hypothalamus; DIO, double-floxed inversed open reading frame; GFP, green fluorescent protein; KO, knock-out; POMC, proopiomelanocortin; RT-PCR, reverse transcription-polymerase chain reaction; shRNA, short hairpin RNA; TRPV1, transient receptor potential vanilloid 1 receptor; VMH, ventromedial nucleus of the hypothalamus; WT, wild-type; 18S, 18S ribosomal RNA.

Fig 2. Activation of TRPV1 receptors in the ARC reduces food intake.

(A) Schematic drawing of our experimental configuration (left panel). Saline and drug were injected into the ARC of POMC-GFP mice (right panel). (B) Scheme depicting food intake measurement. (C) Pooled data from 17 mice of food intake following local microinjection of capsaicin into the ARC. *p < 0.05, ***p < 0.001. (D) Pooled data from 7 mice showing blockade of the effect of capsaicin with AMG9810 (3 μg/μL). ***p < 0.001, *p < 0.05. (E) Schematic drawing of our experimental configuration. shRNAs were bilaterally injected into the ARC (left panel). Images of fluorescence microscopy showing transfection of control shRNA in the ARC (right panel). White oval circle shows shRNA injection site. (F) Trpv1 mRNA expression in mice injected with control and Trpv1 shRNA. mRNAs were extracted from the ARC of mice injected with control (n = 6 animals) and Trpv1 shRNA (n = 6 animals). Tprv1 mRNA expression was significantly reduced by injection of TRPV1 shRNA (**p < 0.01). (G and H) Pooled data showing that capsaicin no longer reduces food intake in mice injected with Trpv1 shRNA into the ARC (control sgRNA, n = 9 mice; Trpv1 sgRNA, n = 9 mice). **p < 0.01, ***p < 0.001. Data are shown as mean ± SEM. ARC, arcuate nucleus of the hypothalamus; cap, capsaicin; GFP, green fluorescent protein; POMC, proopiomelanocortin; sal, saline; sgRNA, single guide RNA; shRNA, short hairpin RNA; TRPV1, transient receptor potential vanilloid 1 receptor.

Fig 3. Activation of TRPV1 receptors in ARC POMC neurons reduces food intake.

(A) Schematic drawing of our experimental configuration showing that Cre-inducible AAV-G_i/o-DREADD was injected into the ARC of POMC-Cre mice. (B) Expression of G_i/o-DREADD in POMC neurons in the ARC (top left panel, scale bar: 20 μm). Arrowheads represent POMC neurons that co-expressed mCherry (n = 493 out of 529 mCherry-positive neurons, n = 3 mice). Treatment with CNO (10 μM) hyperpolarized POMC neurons (top right panel; Control, −40.7 ± 1.5 mV; CNO, −47.1 ± 2.3 mV, n = 6 neurons, **p < 0.01). Scale bar: 25 mV, 2 min. Bottom panel: In the presence of CNO, treatment of the TRPV1 agonist was not able to depolarize POMC neurons (n = 3 neurons). Scale bar: 20 mV, 100 s. (C and D) Pooled data from 8 mice showing blockade of the effect of capsaicin by activation of G_i/o-DREADD in ARC POMC neurons. **p < 0.01, ***p < 0.001. (E) Pooled data from 11 mice showing blockade of the effect of capsaicin by the MC3/4 receptor antagonist SHU9119 (5 μM). p > 0.05. Data are shown as mean ± SEM. AAV, adeno-associated virus; ARC, arcuate nucleus of the hypothalamus; cap, capsaicin; CNO, clozapine-N-oxide; Cre, Cre recombinase; DREADD, designer receptor exclusively activated by designer drugs; hSyn, human synapsin I promoter; i.p., Intraperitoneal; mCherry, monomeric cherry fluorescent protein; MC3/4, melanocortin 3/4; POMC, proopiomelanocortin; RTX, resiniferatoxin; sal, saline; TRPV1, transient receptor potential vanilloid 1 receptor.

Fig 4. Knockdown of the Trpv1 gene in ARC POMC neurons blocks the anorexigenic effect of capsaicin.

(A) Images of fluorescence confocal microscopy showing GFP-positive POMC neurons in the ARC of POMC-Cre::CRISPR-Cas-9-eGFP mice. Scale bar: 50 μm. (B) Images of fluorescence confocal microscopy showing that local injection of capsaicin induced expression of c-fos protein in POMC neurons in the ARC. (C) Images of fluorescence confocal microscopy showing that local injection of capsaicin did not induce c-fos protein expression in mice lacking TRPV1 receptors in ARC POMC neurons. Scale bar: 50 μm. (D) Pooled data show the percentage of POMC neurons expressing c-fos protein in control mice and mice lacking TRPV1 receptors in ARC POMC neurons following treatment with saline and capsaicin (n = 5, 5, and 3 animals, respectively). (E and F) Schematic drawing of our experimental configuration. Trpv1 sgRNA was bilaterally injected into the ARC of POMC-Cre and POMC-Cre::CRISPR-Cas-9-eGFP mice (E). Direct injection of capsaicin into ARC did not reduce food intake in mice silencing the Trpv1 gene in ARC POMC neurons (F) (n = 6 and 7 mice, respectively). Mice were fasted for 6 h (1 PM to 7 PM) ***p < 0.001. Data are shown as mean ± SEM. ARC, arcuate nucleus of the hypothalamus; cap, capsaicin; Cre, Cre recombinase; CRISPR-Cas-9, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9; eGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; POMC, proopiomelanocortin; sal, saline; sgRNA, single guide RNA; TRPV1, transient receptor potential vanilloid 1 receptor.

Fig 5. Activation of TRPV1-like receptor-expressing POMC neurons reduces feeding.

(A) Schematic drawing of the double-floxed Cre-dependent AAV vector expressing ChR2–eYFP under the control of the nPE-globin. (B) Images of confocal microscopy showing co-expression of POMC and YFP in the ARC of TRPV1 Cre mice injected with AAV5-nPE-globin-DIO-ChR2-eYFP in the ARC (arrowheads; top panel). Bottom panel: Co-expression of TRPV1 and YFP (arrowheads). Scale bar: 20 μm. (C) Schematic drawing of our experimental configuration. AAV5-nPE-ChR2 viruses were bilaterally injected into the ARC of TRPV1-Cre mice. An optic cannula was implanted to the ARC to optogenetically stimulate TRPV1-like receptor-expressing POMC neurons. (D) Pooled data showing effect of optogenetic stimulation of TRPV1-like receptor-expressing POMC in the ARC. Ten-hertz stimulation significantly reduced food intake for 1 h (without stimulation, 2.1 ± 0.04 mL; with stimulation, 0.6 ± 0.04 mL, n = 7 mice, ***p < 0.001). Data are shown as mean ± SEM. AAV, adeno-associated virus; ChR2, channelrhodopsin-2; Cre, Cre recombinase; DIO, double-floxed inversed open reading frame; eYFP, enhanced YFP; ITR, inverted terminal repeat; LoxP, locus of X(cross)-over in P1; nPE, neuronal POMC enhancer; POMC, proopiomelanocortin; TRPV1, transient receptor potential vanilloid 1 receptor; TTX, tetrodotoxin; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; YFP, yellow fluorescent protein.

Fig 6. Exercise increases body and ARC temperatures.

(A) Scheme depicting the mouse treadmill protocol. Mice were always warmed up for 5 min at 7 m/min and the speed was slowly ramped up to 18 m/min. (B and C) Pooled data showing changes in body and ARC temperature during treadmill running (gray line, warming up for 5 min and black line, running for 35 min [B]). Plot showing mean temperature at 0, 5, and 40 min exercise (at 40 min, mean body temperature, 38.9 ± 0.1 °C; mean ARC temperature, 38.6 ± 0.2 °C). (D) Pooled data from 9 mice showing the effect of exercise on liquid food intake for 1 h (*p < 0.05, ***p < 0.001). (E) Pooled data from 9 mice showing the effect of exercise on food intake for 1 h with an MC3/4R antagonist. Exercise-induced anorexigenic effect was abolished by local injection of SHU9001. (F) Pooled data from 9 mice showing the effect of exercise on food intake for 1 h in mice injected with control sgRNA. Exercise-induced anorexigenic effect is absent in mice silencing the Trpv1 gene in ARC POMC neurons. *p < 0.05, **p < 0.01, ***p < 0.001. (G) Pooled data from 9 mice showing the effect of exercise on food intake for 1 h in mice silencing the Trpv1 gene in POMC neurons. Exercise no longer reduced food intake in mice lacking the Trpv1 gene in POMC neurons. Data are shown as mean ± SEM. ARC, arcuate nucleus of the hypothalamus; Ex, exercise; MC3/4R, melanocortin 3 and 4 receptor; POMC, proopiomelanocortin; sgRNA, single guide RNA; Trpv1, transient receptor potential vanilloid 1 receptor; Veh, vehicle.