Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle

PLOS ONE, Dec 2015

Cannabinoid CB1 receptors peripherally modulate energy metabolism. Here, we investigated the role of CB1 receptors in the expression of glucose/pyruvate/tricarboxylic acid (TCA) metabolism in rat abdominal muscle. Dihydrolipoamide dehydrogenase (DLD), a flavoprotein component (E3) of α-ketoacid dehydrogenase complexes with diaphorase activity in mitochondria, was specifically analyzed. After assessing the effectiveness of the CB1 receptor antagonist AM251 (3 mg kg-1, 14 days) on food intake and body weight, we could identified seven key enzymes from either glycolytic pathway or TCA cycle—regulated by both diet and CB1 receptor activity—through comprehensive proteomic approaches involving two-dimensional electrophoresis and MALDI-TOF/LC-ESI trap mass spectrometry. These enzymes were glucose 6-phosphate isomerase (GPI), triosephosphate isomerase (TPI), enolase (Eno3), lactate dehydrogenase (LDHa), glyoxalase-1 (Glo1) and the mitochondrial DLD, whose expressions were modified by AM251 in hypercaloric diet-induced obesity. Specifically, AM251 blocked high-carbohydrate diet (HCD)-induced expression of GPI, TPI, Eno3 and LDHa, suggesting a down-regulation of glucose/pyruvate/lactate pathways under glucose availability. AM251 reversed the HCD-inhibited expression of Glo1 and DLD in the muscle, and the DLD and CB1 receptor expression in the mitochondrial fraction. Interestingly, we identified the presence of CB1 receptors at the membrane of striate muscle mitochondria. DLD over-expression was confirmed in muscle of CB1-/- mice. AM251 increased the pyruvate dehydrogenase and glutathione reductase activity in C2C12 myotubes, and the diaphorase/oxidative activity in the mitochondria fraction. These results indicated an up-regulation of methylglyoxal and TCA cycle activity. Findings suggest that CB1 receptors in muscle modulate glucose/pyruvate/lactate pathways and mitochondrial oxidative activity by targeting DLD.

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Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle

RESEARCH ARTICLE Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle Sergio Arrabal1,2, Miguel Angel Lucena1, Miren Josune Canduela3, Almudena RamosUriarte3, Patricia Rivera1,2, Antonia Serrano1,2, Francisco Javier Pavón1,2, Juan Decara1,2, Antonio Vargas1,2, Elena Baixeras1,2, Mercedes Martín-Rufián4, Javier Márquez5, Pedro Fernández-Llébrez6, Baukje De Roos7, Pedro Grandes3, Fernando Rodríguez de Fonseca1,2*, Juan Suárez1,2* OPEN ACCESS Citation: Arrabal S, Lucena MA, Canduela MJ, Ramos-Uriarte A, Rivera P, Serrano A, et al. (2015) Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle. PLoS ONE 10(12): e0145244. doi:10.1371/ journal.pone.0145244 Editor: Vasu D. Appanna, Laurentian University, CANADA Received: September 9, 2015 Accepted: November 30, 2015 Published: December 15, 2015 Copyright: © 2015 Arrabal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information file. Funding: This work was supported by CIBERobn (CB06/03/1008), Ministerio de Economía y Competitividad (MINECO) (PG: BFU2012-33334), Instituto de Salud Carlos III (ISCIII), MINECO, cofunded by UE-ERDF program (JS: CP12/03109), Red de Trastornos Adictivos (FRF: RD12/0028/0001, PG: RD12/0028/0004, JM: RD12/0028/0013), The Basque Country Government (PG: BCG IT764-13), Consejería de Economía, Innovación y Ciencia, Junta 1 UGC Salud Mental, Instituto de Investigación Biomédica de Málaga (IBIMA), Universidad de MálagaHospital Universitario Regional de Málaga, Málaga, Spain, 2 CIBER OBN, Instituto de Salud Carlos III, Madrid, Spain, 3 Department of Neurosciences, University of the Basque Country UPV/EHU, Leioa, Spain, 4 ECAI de Proteómica, Instituto de Investigación Biomédica de Málaga (IBIMA), Universidad de Málaga, Málaga, Spain, 5 Departamento de Biología Molecular y Bioquímica, Instituto de Investigación Biomédica de Málaga (IBIMA), Universidad de Málaga, Málaga, Spain, 6 Departamento de Biología Celular, Genética y Fisiología, Instituto de Investigación Biomédica de Málaga (IBIMA), Universidad de Málaga, Málaga, Spain, 7 University of Aberdeen, Rowett Institute of Nutrition & Health, Aberdeen, United Kingdom * (JS); (FRF) Abstract Cannabinoid CB1 receptors peripherally modulate energy metabolism. Here, we investigated the role of CB1 receptors in the expression of glucose/pyruvate/tricarboxylic acid (TCA) metabolism in rat abdominal muscle. Dihydrolipoamide dehydrogenase (DLD), a flavoprotein component (E3) of α-ketoacid dehydrogenase complexes with diaphorase activity in mitochondria, was specifically analyzed. After assessing the effectiveness of the CB1 receptor antagonist AM251 (3 mg kg-1, 14 days) on food intake and body weight, we could identified seven key enzymes from either glycolytic pathway or TCA cycle—regulated by both diet and CB1 receptor activity—through comprehensive proteomic approaches involving two-dimensional electrophoresis and MALDI-TOF/LC-ESI trap mass spectrometry. These enzymes were glucose 6-phosphate isomerase (GPI), triosephosphate isomerase (TPI), enolase (Eno3), lactate dehydrogenase (LDHa), glyoxalase-1 (Glo1) and the mitochondrial DLD, whose expressions were modified by AM251 in hypercaloric diet-induced obesity. Specifically, AM251 blocked high-carbohydrate diet (HCD)-induced expression of GPI, TPI, Eno3 and LDHa, suggesting a down-regulation of glucose/pyruvate/lactate pathways under glucose availability. AM251 reversed the HCD-inhibited expression of Glo1 and DLD in the muscle, and the DLD and CB1 receptor expression in the mitochondrial fraction. Interestingly, we identified the presence of CB1 receptors at the membrane of striate muscle mitochondria. DLD over-expression was confirmed in muscle of CB1-/- mice. AM251 increased the pyruvate dehydrogenase and glutathione reductase activity in C2C12 myotubes, and the diaphorase/oxidative activity in the mitochondria fraction. These results PLOS ONE | DOI:10.1371/journal.pone.0145244 December 15, 2015 1 / 23 Anti-Obesity Role of CB1 Receptor in Muscle Metabolism de Andalucía, UE-ERDF (FRF: CTS-8221, JM: CVI6656), Consejería de Salud, Junta de Andalucía, UEERDF (FRF: SAS111224), and University of the Basque Country UPV/EHU (PG: UFI11/41). JS, FJP and AS hold “Miguel Servet” research contracts from the National System of Health, ISCIII, UE-ERDF (CP12/03109, CP14/00212, and CP14/00173 respectively). Competing Interests: The authors have declared that no competing interests exist. Abbreviations: ACEA, arachidonyl-2’chloroethylamide; Actb, β-actin; CB1, cannabinoid receptor type 1; Cox4i1, cytochrome c oxidase subunit 4 isoform 1, mitochondrial; DLD, dihydrolipoamide dehydrogenase; ECS, endocannabinoid system; Eno3, β-enolase; GADPH, glyceraldehyde 3-phosphate dehydrogenase; Glo1, glyoxilase 1; GPI, glucose-6-phosphate isomerase; GSH, reduced glutathione; GSSG, oxidized glutathione; Gusb, β-glucuronidase; HCD, high carbohydrate diet; HFD, high fat diet; HPLC-ESI MS, high-performance liquid chromatography-electrospray ionization mass spectrometry; LDHa, lactate dehydrogenase; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; NADPH, nicotinamide adenine dinucleotide phosphate; NBT, nitroblue tetrazolium; PKM, pyruvate kinase; STD, standard diet; TPI, triosephosphate isomerase 1. indicated an up-regulation of methylglyoxal and TCA cycle activity. Findings suggest that CB1 receptors in muscle modulate glucose/pyruvate/lactate pathways and mitochondrial oxidative activity by targeting DLD. Introduction The endocannabinoid signaling system (ECS) plays a fundamental role in the onset of obesity and metabolic disorders, implicating central and peripheral mechanisms predominantly via the activation of the cannabinoid CB1 receptors [1–4]. Obesity is associated with an increase in circulating endocannabinoid levels as a consequence of an altered expression of the endocannabinoid-metabolizing enzymes [5,6]. CB1 receptors are mainly expressed in the brain, but also in many peripheral organs involved in energy metabolism, including striate muscle and adipose tissue [7–9]. We now know that cannabinoids facilitates energy intake and, perhaps even more important, enhance energy storage into adipose tissue and reduce energy expenditure in muscle via lipid and glucose metabolism [5,10]. The endocannabinoid anandamide can modify β-oxidation pathways in the striate muscle, suggesting that CB1 receptor antagonism could be an important strategy in the regulation of energy expenditure needed to fight obesity [11]. Importantly, metabolic alte (...truncated)


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Sergio Arrabal, Miguel Angel Lucena, Miren Josune Canduela, Almudena Ramos-Uriarte, Patricia Rivera, Antonia Serrano, Francisco Javier Pavón, Juan Decara, Antonio Vargas, Elena Baixeras, Mercedes Martín-Rufián, Javier Márquez, Pedro Fernández-Llébrez, Baukje De Roos, Pedro Grandes, Fernando Rodríguez de Fonseca, Juan Suárez. Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle, PLOS ONE, 2015, Volume 10, Issue 12, DOI: 10.1371/journal.pone.0145244