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

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, co-funded 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 de Andalucía, UE-ERDF (FRF: CTS-8221, JM: CVI-6656), Consejería de Salud, Junta de Andalucía, UE-ERDF (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)Peer reviewedPublisher PD

Protein identification and characterization in muscle of DIO rats treated with AM251.

<p>Seven proteins in the abdominal muscle of STD, HFD and HCD-fed rats treated with AM251 were identified by using 2-dimensional (2D) electrophoresis and MALDI-TOF/LC-ESI trap mass spectrometry. <b>A)</b> A representative 2D polyacrylamide gel showing the selected spots. <b>B-D</b>) Scheme of the glucose and pyruvate pathways and Krebs cycle showing the step reaction of the seven enzymes identified. <b>E</b>) Fatty acid metabolism. Dihydrolipoamide dehydrogenase (DLD) is an E3 component with dehydrogenase/diaphorase activity of the mitochondrial pyruvate¹/α-ketoglutarate²/branched-chain α-ketoacid³ dehydrogenase complexes.</p

AM251 regulates the protein expressions of glucose/pyruvate-metabolizing enzymes.

<p>AM251 effects on the protein expressions of GPI, TPI, Eno3, PKM1, LDHa and Glo1 in the abdominal muscle of STD, HFD and HCD-fed rats (<b>A-F</b>). Representative 2D polyacrylamide gels showing the intensity of the identified spots (<b>G</b>). Bonferroni’s test (<i>n</i> = 6): *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 <i>vs</i>. STD-vehicle; ^#<i>P</i><0.05, ^##<i>P</i><0.01, ^###<i>P</i><0.001 <i>vs</i>. HFD-vehicle; ^$<i>P</i><0.05, ^{$ $$}<i>P</i><0.001 <i>vs</i>. HCD-vehicle.</p

AM251 effectiveness on food intake and body weight in DIO rats.

<p>Dose-response of AM251 (0.3, 1, 3, 10 mg kg^-1) in cumulative food intake (<b>A</b>) and experimental design (<b>B</b>) for cumulative food/caloric intake and body weight gain (<b>C</b>-<b>H</b>) during STD, HFD and HCD feeding for 84 days and repeated AM251 administration (3 mg kg^-1) for 14 days. Bonferroni’s test (<i>n</i> = 8): *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 <i>vs</i>. STD; ^#<i>P</i><0.05, ^###<i>P</i><0.001 <i>vs</i>. HFD; ^$<i>P</i><0.05, ^{$ $$}<i>P</i><0.001 <i>vs</i>. HFD-vehicle; ^&&<i>P</i><0.01, ^&&&<i>P</i><0.001 <i>vs</i>. HCD-vehicle. Haematoxylin-eosin staining in the abdominal muscle of the rats fed with STD, HFD and HCD for 84 days and repeatedly treated with AM251 (3 mg kg^-1) for 14 days (<b>I</b>-<b>N</b>).</p

Scheme showing the enzymatic reactions that catalyze the pyruvate dehydrogenase complex, including pyruvate dehydrogenase activity and diaphorase activity of DLD (A) as well as the glutathione reductase/peroxidase activity (B).

<p>NBT staining is proportional to O^- production from redox activity.</p

AM251 regulates the expression of DLD and Cox4i1.

<p>AM251 effects on the protein and gene expressions of DLD (<b>A</b>,<b>B</b>) and the gene expression of <i>Cox4i1</i> (<b>C</b>) in the abdominal muscle of STD, HFD and HCD-fed rats. Representative 2D polyacrylamide gels showing the intensity of the identified spots for DLD (<b>D</b>). Bonferroni’s test (<i>n</i> = 6–8): *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 <i>vs</i>. STD-vehicle; ^#<i>P</i><0.05 <i>vs</i>. HFD-vehicle; ^$<i>P</i><0.05 <i>vs</i>. HCD-vehicle. Gene (<b>D</b>) and protein (<b>E</b>) expressions of DLD in the abdominal muscle of wild-type and <i>CB</i>_<i>1</i>^<i>-/-</i> mice. Representative immunoblots (<b>F</b>). Student’s <i>t</i> test (<i>n</i> = 6): *<i>P</i><0.05, ***<i>P</i><0.001 <i>vs</i>. WT.</p

Expression of the metabolic enzymes in the abdominal muscle of <i>CB</i>_<i>1</i>^-/- mice.

<p>Gene (<b>A</b>) and protein (<b>B</b>) expressions of GPI, TPI, Eno3, PKM1, LDHa and Glo1 in the abdominal muscle of wild-type and <i>CB</i>_<i>1</i>^-/- mice. <b>C)</b> Representative immunoblots. Student’s <i>t</i> test (<i>n</i> = 6): *<i>P</i><0.05 <i>vs</i>. WT.</p

AM251 regulates the gene expressions of glucose/pyruvate-metabolizing enzymes.

<p>AM251 effects on the gene expressions of <i>Gpi</i>, <i>Tpi1</i>, <i>Eno3</i>, <i>Pkm</i>, <i>Ldha</i> and <i>Glo1</i> in the abdominal muscle of STD, HFD and HCD-fed rats. Bonferroni’s test (<i>n</i> = 8): *<i>P</i><0.05, ***<i>P</i><0.001 <i>vs</i>. STD-vehicle; ^$<i>P</i><0.05 <i>vs</i>. HCD-vehicle.</p