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N-Oleoylglycine-induced hyperphagia was associated with the activation of AgRP neuron by CB1R Junguo Wu, Canjun Zhu, Liusong Yang, Zhonggang Wang, Lina Wang, Songbo Wang, Ping Gao, Yongliang Zhang, Qingyan Jiang, Gang Shu, and Xiaotong Zhu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05281 • Publication Date (Web): 19 Jan 2017 Downloaded from http://pubs.acs.org on January 23, 2017

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Journal of Agricultural and Food Chemistry

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N-Oleoylglycine-induced hyperphagia was associated with the

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activation of AgRP neuron by CB1R

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Junguo Wu†,‡, Canjun Zhu†,‡, Liusong Yang†,‡, Zhonggang Wang#, Lina Wang†,‡,

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Songbo Wang†,‡, Ping Gao†,‡, Yongliang Zhang†,‡, Qingyan Jiang†,‡, Xiaotong Zhu*,†,‡,

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Gang Shu*,†,‡

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†College

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Swine Industry, South China Agricultural University, Guangzhou 510640, P. R. China.

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‡ALLTECH-SCAU

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Agricultural University, Guangzhou 510642, P. R. China

of Animal Science & National Engineering Research Center for Breeding

Animal Nutrition Control Research Alliance, South China

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#Huahong

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Guangdong, 430051, P. R. China

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*

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[email protected]. Phone: +86 20 85284901. Fax: +86 20 85284901

Engineering and Research Center of Agricultural and Livestock. Zhaoqing,

Correspondence

should

be

addressed

to

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ACS Paragon Plus Environment

[email protected]

and


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Abstract

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N-acyl amino acids (NAAAs) are conjugate products of fatty acids and amino

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acids, which are available in animal-derived food. We compared the effects of N-

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arachidonoyl glycine (NAGly), N-arachidonoyl serine (NASer), and N-Oleoylglycine

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(OLGly) on in vivo food intake and in vitro [Ca2+]i of AgRP neurons to identify the role

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of these compounds in energy homeostasis. The hypothalamic neuropeptide expression

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and anxiety behavior in response to OLGly were also tested. To further identify the

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underlying mechanism of OLGly on food intake, we first detected the expression level

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of potential OLGly receptors. The cannabinoid receptor type 1 (CB1R) antagonist was

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co-treated with OLGly to analyze the activation of AgRP neuron, including [Ca2+]i,

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expression level of PKA, CREB, c-Fos, and neuropeptide secretion. Results

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demonstrated that only OLGly (intrapertioneal injection 6 mg/kg) can induce

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hyperphagia without changing the expression of hypothalamic neuropeptides and

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anxiety-like behavior. Moreover, 20 μM OLGly robustly enhances [Ca2+]i, c-Fos

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protein expression in AgRP neuron, and AgRP content in the culture medium. OLGly-

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induced activation of AgRP neuron was completely abolished by CB1R specific

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antagonist, AM251. In summary, this study is the first to demonstrate the association of

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OLGly-induced hyperphagia with the activation of AgRP neuron by CB1R. These

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findings open avenues for investigation and application of OLGly to modulate energy

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homeostasis.

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Keywords: N-acyl amino acids; Food intake; Anxiety behavior; CB1R; AgRP

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secretion


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Introduction

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Lipoamino acids or N-acyl amino acids (NAAAs) are a group of nutritional

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metabolites produced from the fatty acid conjugation of amino acids. NAAAs exist as

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endogenous substances1, found in skeletal muscle, lung, kidney, and brain of human

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and domestic animals2, 3. At present, over 50 NAAAs had been identified in different

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tissues4. For example, OLGly (300–700 pmol/g) is abundant in the skin, spinal cord,

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and lung3, whereas N-arachidonoyl amino acids (50–140 pmol/g) are ubiquitous in

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intestinal, kidney, and central nerve system5, 6. Several studies have revealed the diverse

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biological activities of these compounds, such as vasodilatory and neuroprotective

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effects7, 8, bone turnover9, inflammation10, cell proliferation11, and calcium ion

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mobilization12. However, the effects of NAAAs on food intake and energy homeostasis

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are still unknown.

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NAAAs are structurally similar to endocannabinoids, whose main receptors have

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been identified as either cannabinoid receptor type 1 or 2 (CB1R or CB2R)13 or

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transient receptor potential channels (TRPVs)14-16. Likewise, these receptors mediate

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the biological function of different NAAAs in a structure-dependent manner12, 17. Most

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importantly, both CB receptors and TRPVs are widely expressed in different tissues,

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including central nerve system, peripheral tissues, immune system and specialized

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sensory neurons18-20. Activation of these receptors suppresses transmitter release21, 22,

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mediates anxiety-like behavior23,

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evidence lead to the hypothesis that CB1R, CB2R or TRPV1 may mediate the effect of

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NAAAs on food intake.

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, and modulates appetite25-27. These pieces of

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To test the hypothesis, we compared the effects of three common NAAAs (NAGly,

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NASer, and OLGly) on food intake and found that only OLGly can effectively induce

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hyperphagia. In addition, we further identified the role of OLGly in the activation and



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neuropeptide release of the hypothalamic AgRP neurons, which are mediated by CB1R.

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The results of this paper are the first to demonstrate the novel role of OLGly in appetite

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regulation, which open avenues for investigating and applying this compound in the

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modulation of energy homeostasis.

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Materials and methods

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Chemicals: N-Oleoyl glycine (OLGly) and AM251 (CB1R antagonist) were purchased

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from Sigma Aldrich (St. Louis, MO, USA). N-arachidonoyl glycine (NAGly) and N-

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arachidonoyl serine (NASer) were purchased from Santa (Santa Cruz, CA). AgRP-

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related protein (AGRP) (82–131) amid (Mouse) ELISA kit was purchased from bioleaf

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(Shanghai, China). Fluorescent-8® calcium indicators were purchased from Tianjin

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Biolit Biotech Co., Ltd. (Tianjin, China).

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Antibodies: Phospho-PKA (Ser339) and PKA were purchased from Bioss (Beijing,

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China). Phosphor-CREB, CREB, and c-Fos were purchased from Cell Signaling

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Technology Inc. (Danvers, MA, USA). β-tubulin was purchased from Bioworld

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Technology, Inc. (St. Louis Park, MN, USA). Neuropeptide Y (NPY) and Agouti-

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related protein (AgRP) were purchased from Santa (Santa Cruz, CA). CB1, TRPV1,

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and proopiomelanocortin (POMC) were purchased from Abcam Biotechnology, Inc.

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(Shanghai, China). Hipure gel pure DNA kits were purchased from Magen (Guangzhou,

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Guangdong, China). Go Taq® qPCR Master Mix was purchased from PROMEGA

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(Beijing, China).

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Animal and anxiety behavior test

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All animals used in these experiments were reared and sacrificed with the approval

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of the College of Animal Science, South China Agricultural University. All experiments

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were conducted in accordance with “the instructive notions with respect to caring for

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laboratory animals” issued by the Ministry of Science and Technology of the People’s

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Republic of China.

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C57/BL6J mice were purchased from the Medical Experimental Animal Center of

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Guangdong Province (Guangzhou, Guangdong, China). All animals were individually

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housed for one week before intraperitoneal injection and maintained on a 12 h light/dark



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cycle with temperature of 23 ± 3 °C and relative humidity of 70 ± 10% throughout the

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experimental period. These mice had ad libitum access to standard pellets (crude protein

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18%, crude fat 4%, and crude ash 8%). In acute experiment, 8–10-week-old male mice,

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weighing 22–25 g, were divided into two groups and intraperitoneally injected with

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vehicle (5% Tween-80) and 6 mg/kg of NAAAs respectively. After 30 min, the mice

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were given standard pellets to measure their food consumption at different time points.

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Elevated-Plus Maze (EPM) and open field test (OFT) were used for anxiety

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behavior test of rodents. Following 30 min of rest after intraperitoneal injection,

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animals from both groups were exposed to EPM test with a duration of 5 min according

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to previously reported protocols28, 29. For OFT, animals were placed in the center of an

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open-field area and movement for 20 min based on a previously has been described

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protocol30. Both tests were video-recorded and analyzed by ANY-Maze software.

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Cell culture and treatment

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Embryonic mouse hypothalamic AgRP cell line N38 (mHypoE-38, Cellutions,

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CLU118) (Burlington, Ontario, Canada) was cultured in low-glucose DMEM (Gibco

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BRL, Carlsbad, CA, USA), containing 10% FBS (Gibco BRL, Carlsbad, CA, USA),

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1×105 units/L of penicillin sodium, and 100 mg/L of streptomycin sulfate (Gibco BRL,

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Carlsbad, CA, USA) solution with (0, 2, 20 μM) OLGly for 12 h to investigate the the

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dose effects of OLGly at 37 °C in a humidified atmosphere that contained 5% CO2. In

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addition, the cells were cultured with 20 μM OLGly for 0.5 and 1 h to explore the

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expression of c-Fos, p-PKA, and p-CREB at different time points. The cells were

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washed twice with phosphate-buffered saline (PBS) before protein extraction. The

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samples were then applied for western blot or qPCR.

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Assay of [Ca2+]i


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[Ca2+]i was measured by calcium fluorometry using fluo-8 AM. The cells were

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seeded in a 24-well plate and cultured for 24 h until they reached 50% confluence. The

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cells were washed twice with Hank’s Balanced Salt Solution (HBSS, pH=7.2–7.4)

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containing 8 g/L NaCl, 0.4 g/L KCl, 0.1 g/L MgSO4.7H2O, 0.1 g/L MgCl2.6H2O, 0.06

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g/L Na2HPO4.2H2O, 0.06 g/L KH2PO4, 1 g/L glucose, 0.14 g/L CaCl2, and 0.35 g/L

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NaHCO3 and incubated with 10 μM fluo-8-AM at 37 °C. After 1 h incubation, the cells

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were washed twice with HBSS, and the calcium response assay was initiated by manual

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addition of NAAAs (NAGly, NASer, and OLGly) equipped with Nikon Eclipse Ti-s

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microscopy. Fluorometric data were acquired at excitation and emission wavelengths

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of 490 and intensity at 525 nm (490/525 nm) for every 5 s interval over a 180 s period.

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Immunocytochemistry

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N38 cells were cultured in LG-DMEM with 20 μM OLGly and AM251 for 30 min

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in six-well plates, and used for immunocytochemistry of c-Fos, based on procedures

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we previously described

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gently rinsed with PBS thrice at room temperature. The cells were then fixed by

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incubation in 4% (v/v) paraformaldehyde in PBS for 20 min at room temperature. The

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cells were rinsed thrice with PBS, permeabilized with 0.4% Triton X-100, and blocked

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with PBS containing 1% goat serum for 1 h at room temperature. The cells were

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immunostained with c-Fos antibody at 4 °C overnight. Cells were incubated in FITC

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second antibody (1:1000; bioss) for 1 h at room temperature. Cells were observed using

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Nikon Eclipse Ti-s microscopy, and images were captured using Nis-Elements BR

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software (Nikon Instruments, Tokyo, Japan). The number of cells used for the statistics

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was 30–60 in different fields of vision31, 32.

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. The culture medium was aspirated from each well and



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Western blot analysis

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The protein expression levels of AgRP (1:500), c-Fos (1:5000), phosphor-CREB

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(1:1000), CREB (1:3000), phosphor-PKA (1:1000), PKA (1:2000), CB1 (1:3000),

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TRPV1 (1:3000), and β-tubulin (1:5000) in N38 cells were detected by western blot

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analysis as we previously described33. In addition, the protein expression levels of

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hypothalamic AgRP, POMC (1:2000), CB1, TRPV1, and β-tubulin were also detected

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by western blot analysis.

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Real-time quantitative PCR

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The extraction and reverse transcription of RNA were conducted as previously

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described32. Template DNA was amplified, excised from the agarose gel, and purified

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with Hipure Gel Pure DNA kits. The concentration of DNA was detected and diluted

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containing 101 to 1012 copies as standards. Gene expression levels were examined by

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real-time quantitative PCR as we previously described17. We calculated the gene copies

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according to standard curves. The primers for amplification are shown in Table 1.

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Statistical analysis

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All data are presented as means ± standard error of the mean (SEM). Statistical

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analysis was performed using GraphPad Prism 6.0. Differences between various groups

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in the dose-effect experiment were determined by one-way ANOVA. Differences

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between the control and the treated group were analyzed by Student’s t-test. A

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confidence level of P < 0.05 was considered statistically significant.

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Results

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Effects of NAAAs on food intake and hypothalamic neuropeptide expression.

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Mice were fasted overnight and intraperitoneally injected with 6 mg/kg NAGly, NASer,

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and OLGly, respectively, to investigate the effects of NAAAs on food intake. As shown

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in Figs. 1A–C, only OLGly increased at 24 h food intake, whereas the two NAAAs had

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no effect on appetite during the first 6 h post-injection. Similarly, OLGly also enhanced

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the food intake of mice in dark cycle (18:00–06:00) without fasting (Fig. 1D). However,

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the protein expression of hypothalamic agouti-related protein (AgRP), neuropeptide Y

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(NPY), and proopiomelanocortin (POMC) in OLGly group were comparable with those

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the control group (Fig.1E and F), which means OLGly-induced hyperphagia may be

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independent of neuropeptide expression.

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Effect of OLGly on the anxiety behavior of C57BL6/J mice. To further test

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whether anxiety behavior is responsible for OLGly-induced hyperphagia, elevated-plus

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maze (EPM) and OFT were adopted to analyze the behavior change in response to

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intraperitoneal injection of OLGly. However, OLGly unaltered the open-time ratio (Fig.

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2A), open-distance traveled (Fig. 2B), and head dip times (Fig. 2C) during EPM

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analysis. Similarly, no significant differences were observed in the total distance (Fig.

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2D), center distance (Fig. 2E), and center time ratio (Fig. 2F) during OFT. These data

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indicated that anxiety might not have any effect on OLGly-induced hyperphagia.

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OLGly evoked [Ca2+]i response and AgRP secretion in N38 cells.

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Neurotransmitter release is the direct evidence of neuron activation. We further

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investigated the effects of NAAAs on the activation of AgRP cells (hypothalamic N38

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cell line). The results were consistent with our previous in vivo food intake data, where

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only OLGly significantly elevates the [Ca2+]i levels (Fig. 3A). In addition, 20 μM

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OLGly increased AgRP secretion (Fig. 3B; 3C) in N38 cell. Accordingly, c-Fos



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expression was enhanced by OLGly administration for 30 min (Figs. 3G–J). However,

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cAMP cascade (PKA and CREB) remained constant in response to OLGly (Figs. 3D–

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F). Together, these data supported that OLGly-induced AgRP secretion by evoking

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[Ca2+]i cascade.

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Involvement of CB1R in OLGly-induced hypothalamic AgRP secretion. To

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identify the receptor of NAAAs, we first examined the expression of CB1R, CB2R, and

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TRPV1 in N38 cell and hypothalamus. The results indicated that CB1R were highly

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expressed in N38 cells and hypothalamus (Fig. 4A; 4B). However, OLGly did not affect

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CB1R expression level (Figs. 4C–D). The CB1 antagonist, AM251, completely

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abolished the effects of OLGly on [Ca2+]i, c-Fos expression, and AgRP secretion in N38

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cells (Figs. 4E–I). Hence, our present data supported that CB1R is the dominant

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receptor mediating the role of OLGly in AgRP secretion and appetite regulation.

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Discussion

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N-Arachidonoyl serine is involved in angiogenesis34, TNF-α formation35, and cell

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migration36. Meanwhile, N-arachidonoyl glycine reduces pain, inflammation3, 37, and

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apoptosis38. Furthermore, our previous investigation revealed the positive role of N-

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Oleoyl glycine in adipogenesis17. NAAAs are structurally similar to endocannabinoids,

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2-arachidonoylglycerol (2-AG), or arachidonyl-ethanolamide (AEA). Both 2-AG and

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AEA are important in food intake and energy homeostasis39. Based on this, we first

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tested the effect of three common NAAAs (NAGly, NASer, and OLGly) on the food

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intake of mice. Among the three NAAAs, only OLGly can induce hyperphagia.

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Previous evidence also demonstrated that the distinct role of NAAAs may be related to

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their structures. Burstein et al. revealed that NAGly and LinGly (N-linoleoyl glycine)

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suppress the proliferation of murine macrophage cell line, RAW264.7, whereas OLGly

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and PalGly (N-palmitoyl glycine) have no effect40. However, the structure activity

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relationship of NAAAs needs further investigation. Arachidonic acids are prone to

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oxidation and form oxidative decomposition products. Hence, the stability of NAGly

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and NASer might influence the effect of these compounds on food intake.

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Agouti-related protein (AgRP), neuropeptide Y (NPY), and proopiomelanocortin

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(POMC) are three neuropeptides that regulate food intake and energy homeostasis in

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mammals41. To investigate the underlying mechanism of OLGly-induced hyperphagia,

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we first test the expression levels of hypothalamic neuropeptides. Nevertheless, we only

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found a slightly increase of the AgRP protein in hypothalamus after OLGly treatment.

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Furthermore, the protein expression levels of NPY and POMC remained constant.

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These data indicated that the expression of these neuropeptides are possibly not

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involved in OLGly-induced hyperorexia. Although anxiety behavior can mediate the

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effect of endocannabinoids on feeding42, 43, EPM and OFT results indicated that anxiety



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behavior is independent of response to OLGly administration.

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We further examined the effect of OLGly on neurotransmitter release by AgRP

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neurons, which has an indispensable role in appetite41 and has full access to plasma

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nutrients33, 44. Calcium is a universal intracellular messenger, which initiates many

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neuronal responses, including secretion of neurotransmitters. [Ca2+]i activation induces

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neuronal excitability and promotes the secretion of neurotransmitters45. Our results

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indicated that OLGly has the highest potential of elevating [Ca2+]i in N38 cells, an

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immobility cell model for AgRP neurons, and increases the AgRP release of cells to the

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culture medium. We emphasized that several calcium ion channels12 or G protein-

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coupled receptors (GPCRs)46 mediate the effect of NAAAs. We then detected

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PKA/CREB cascade activation. However, the protein levels of p-PKA/PKA and p-

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CREB/CREB in N38 cells remained constant, even though the c-Fos protein

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dramatically increased from OLGly treatment. Depending on the data of [Ca2+]i and c-

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Fos, we reasonably speculate that either calcium ion channel or Gq subfamily GPCRs

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are involved in OLGly-induced AgRP release of N38 cells.

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Subsequently, we compared the expression levels of CB1R, CB2R, and TRPV1 in

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N38 cells and hypothalamus. We found that CB1R is abundantly expressed in the cells

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and tissue. In addition, CB1R-specific antagonist, AM251, abolished OLGly-induced

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activation of AgRP neuron, which means that CB1R mediates the effect of OLGly.

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CB1R is a member of the heptahelical GPCR superfamily47. Coupling of Gα to CB1R

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is dependent on its ligand. Endogenous cannabinoid binding of CB1R recruited by Gi/o

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stimulates mitogen-activated kinases and decreases the generation of [Ca2+]i48,

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However, one cannabinoid agonist, WIN55212-2, binding CB1R could couple Gq and

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increase the concentration of [Ca2+]i 50, 51. The expression of c-Fos, an indirect marker

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of neuronal activity, needs induction from the increase of [Ca2+]i52, 53. Therefore, our


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.

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[Ca2+]i and c-Fos data support the idea that OLGly may induce Gq activation through

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CB1R binding and eventually increase AgRP release in N38 cells.

250 251

In short, OLGly induces hyperphagia and AgRP release of hypothalamic AgRP cells by CB1R.

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Abbreviations Used

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NAAAs, N-acyl amino acids; CB1R, cannabinoid receptor type 1; CB2R,

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cannabinoid receptor type 2; NAGly, N-arachidonoyl glycine; NASer, N-arachidonoyl

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serine; OLGly, N-Oleoyl glycine; TRPVs, transient receptor potential channels;

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TRPV1, transient receptor potential channel 1; AgRP, AgRP-related protein; NPY,

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neuropeptide Y; POMC, proopiomelanocortin; FBS, fetal bovine serum; PKA, protein

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kinase A; CREB, cAMP-response element binding protein; EPM, elevated-plus maze;

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OFT, open field test. LinGly, N-linoleoyl glycine; AEA, arachidonyl-ethanolamide;

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PalGly, N-Palmitoyl Glycine; GPCR, G protein-coupled receptor.

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Funding This study was supported by National Basic Research Program of China (2013CB127306) and National Natural Science Foundation of China (31572480).

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431



432

Figure captions

433

Figure 1. Effects of NAAAs on mice food intake hypothalamic neuropeptide

434

expression. The control group mice were intraperitoneally administered vehicle (5%

435

Tween-80), whereas the other group was administered with 6 mg/kg NASer, NAGly,

436

or OLGly, respectively. (A–C) Food intake of mice, fasted overnight, after NAAAs

437

injection (n=6–10). (D) Food intake of mice in dark cycle after OLGly administration

438

(n=13). (E and F) Protein expression of hypothalamic AgRP (n=3), NPY (n=3), and

439

POMC (n=3). Data were expressed as the mean±SEM. A confidence level of P < 0.05

440

was considered statistically significant.

441 442

Figure 2. Effect of OLGly on the anxiety behavior of C57BL6/J mice. EPM and OFT

443

were used to reflect the anxiety-like behavior of mice. Mice were intraperitoneally

444

administered vehicle and OLGly for 30 min before tests. (A–C) Open time ratio, open

445

distance traveled, and head dip times in EPM for 5 min (n=10). (D–F) Total distance,

446

center distance, and center time ratio in OFT for 15 min (n=11). Data were expressed

447

as the mean±SEM.

448 449

Figure 3. Activation of N38 cells and AgRP secretion increase by OLGly. (A) The

450

change of [Ca2+]i after NASer, NAGly, and OLGly were treated on N38 cells (n=30).

451

(B) AgRP content in N38 culture medium detected by western blot (n=4). (C) AgRP

452

content in N38 culture medium detected by ELISA Kit (n=8). (D–F) Western blot

453

analysis of p-CREB and p-PKA at 15, 30 min (n=6). (G–H) Western blot analysis of c-

454

Fos in N38 cells 15 and 30 min treatment of OLGly (n=6). (I–J) Immunocytochemistry

455

of c-Fos after NAAAs (OLGly, NAGly, and NASer) treatment in N38 cells at 0.5 h


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456

(n=3). Data were expressed as the mean±SEM. A confidence level of P < 0.05 was

457

considered statistically significant.

458 459

Figure 4. Involvement of CB1R in OLGly-induced hypothalamic AgRP secretion. (A)

460

The protein expression of CB1R, CB2R, and TRPV1 in the hypothalamus and N38

461

cells (n=6). The numbers in parentheses were exposure time of proteins. (B) Copies of

462

CB1R, CB2R, and TRPV1 genes in hypothalamus and N38 cells (n=6). (C–D) The

463

protein expression of TRPV1 and CB1 in hypothalamus after OLGly treatment (n=6).

464

(E) The change of calcium in N38 cells after treatment with CB1R antagonist AM251

465

(n=30). (F–G) AgRP content in N38 culture medium detected by western blot (n=6).

466

(H) AgRP content in N38 culture medium detected by ELISA kit (n=8). (I)

467

Immunocytochemistry of c-Fos after OLGly and AM251 treated for 0.5 h (n=3). Data

468

were expressed as the mean±SEM. A confidence level of P < 0.05 was considered

469

statistically significant.

470



471

Tables

472

Table 1 PCR primer sequences and amplification parameters Gene β-actin

Primer sequence (5'-3') S: 5'- GGTCATCACTATTGGCAACGAG -3'

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Product size (bp)

Tm (°C)

142

57

123

59

147

59

142

59

A: 5'- GAGGTCTTTACGGATGTCAACG -3' CB1

S: 5'- AAGTCGATCTTAGACGGCCTT -3' A: 5'- TCCTAATTTGGATGCCATGTCTC -3'

CB2

S: 5'- ACGGTGGCTTGGAGTTCAAC -3' A: 5'- GCCGGGAGGACAGGATAAT -3'

TRPV1

S: 5'- CCGGCTTTTTGGGAAGGGT -3' A: 5'- GAGACAGGTAGGTCCATCCAC -3'

473


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474 475


Figure graphics Figure 1

476 477



478

Figure 2

479 480


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Figure 3

482 483



484

Figure 4

485 486


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487


Graphic for table of contents

488 489

OLGly increased mice food intake, which is associated with the activation of

490

AgRP neuron and neuropeptide release. Briefly, OLGly activated the CB1R in AgRP

491

neuron and induced the peak of intracellular calcium and c-Fos expression. The

492

accumulation of [Ca2+]i promoted the neurotransmitter vesicle to merge with plasma

493

membrane and, therefore, release AgRP. However, PKA/CREB signaling pathway was

494

not involved in OLGly-induced AgRP release.

495


(AgRP) Neuron by Cannabinoid Receptor Type 1 ... - ACS Publications

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