Characterization of murine melanocortin receptors mediating adipocyte lipolysis and examination of signalling pathways involved

International audienceThe melanocortin receptors (MCRs) belong to the G-protein coupled receptors (family A). So far, 5 different subtypes have been described (MC1R- MC5R) and of these MC2R and MC5R have been proposed to act directly in adipocytes and regulate lipolysis in rodents. Using ACTH and a-melanocyte stimulating hormone (a-MSH) generated from proopiomelanocortin (POMC), as well as synthetic MSHanalogues to stimulate lipolysis in murine 3T3-L1 adipocytes it is shown that MC2R and MC5R are lipolytic mediators in differentiated 3T3-L1 adipocytes. Involvement of cAMP, phosphorylated extracellular signal-regulated kinase (ERK) 1/2, protein kinase B (PKB), adenosine 5' monophosphateactivated protein kinase (AMPK) and Jun-amino-terminal kinase (JNK) in MCR mediated lipolysis were studied. Interestingly, results obtained in 3T3-L1 cells suggest that lipolysis stimulated by a-MSH, NDPa- MSH, MT-II, SHU9119 and PG-901 is mediated through MC5R in a cAMP independent manner. Finally, we identify essential differences in MCR mediated lipolysis when using 3T3-L1 cells compared to primary adipocytes

<新刊紹介>パリ法科大學教授ノガロー原著小樽高等商業學校教授手塚嘉郎著「國際貿易に於ける貨幣の職分と貨幣數量説」

Glucagon-like peptide-1 (GLP-1) activates the GLP-1 receptor (GLP-1R), which belongs to family B of the G-protein-coupled receptors. We previously identified a selective small molecule ligand, compound 2, that acted as a full agonist and allosteric modulator of GLP-1R. In this study, the structurally related small molecule, compound 3, stimulated cAMP production from GLP-1R, but not from the homologous glucagon receptor (GluR). The receptor selectivity encouraged a chimeric receptor approach to identify domains important for compound 3-mediated activation of GLP-1R. A subsegment of the GLP-1R transmembrane domain containing TM2 to TM5 was sufficient to transfer compound 3 responsiveness to GluR. Therefore, divergent residues in this subsegment of GLP-1R and GluR are responsible for the receptor selectivity of compound 3. Functional analyses of other chimeric receptors suggested that the existence of a helix-helix interface between TM1 and TM7 is important for the compound 3 response. Furthermore, site-directed mutagenesis revealed that a Phe195-Leu substitution in TM2 and a Thr391-Ala substitution in TM7 increased and decreased the efficacy of compound 3 without disturbing the potency or efficacy of GLP-1. Collectively, differential effects of receptor mutations suggest that TM2 and/or TM7 are important for compound 3-mediated activation of GLP-1R.</jats:p

α-MSH stimulates glucose uptake in mouse muscle and phosphorylates Rab-GTPase-activating protein TBC1D1 independently of AMPK

The melanocortin system includes five G-protein coupled receptors (family A) defined as MC1R-MC5R, which are stimulated by endogenous agonists derived from proopiomelanocortin (POMC). The melanocortin system has been intensely studied for its central actions in body weight and energy expenditure regulation, which are mainly mediated by MC4R. The pituitary gland is the source of various POMC-derived hormones released to the circulation, which raises the possibility that there may be actions of the melanocortins on peripheral energy homeostasis. In this study, we examined the molecular signaling pathway involved in α-MSH-stimulated glucose uptake in differentiated L6 myotubes and mouse muscle explants. In order to examine the involvement of AMPK, we investigate -MSH stimulation in both wild type and AMPK deficient mice. We found that -MSH significantly induces phosphorylation of TBC1 domain (TBC1D) family member 1 (S237 and T596), which is independent of upstream PKA and AMPK. We find no evidence to support that -MSH-stimulated glucose uptake involves TBC1D4 phosphorylation (T642 and S704) or GLUT4 translocation

α-MSH (100 nM) stimulated TBC1D1 S237 and T596 phosphorylation in WT and AMPK KD mice.

<p>TBC1D1 S237 and T596 phosphorylation sites were measured in soleus muscle using WB as described (n indicated in the individual bars). Phosphorylation of TBC1D1 S237 and T596 is normalized to total TBC1D1. Findings are shown as representative immunoblots and pooled data is quantified in bar graphs as arbitrary units. 2-way RM ANOVA was used to calculate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle). ^# indicates a significant effect of genotype (^#p < 0.05, ^##p < 0.01, ^###p < 0.001).</p

α-MSH (100 nM) stimulated 2-Deoxy Glucose uptake and TBC1D1 S237, T596 and S700 phosphorylation +/- H89 in dissected soleus explants from WT mice.

<p>A: Soleus muscle was dissected, stimulated and 2-DG was measured as described (n indicated in the individual bars). B: Phosphorylation of TBC1D1 was measured in soleus muscle using WB as described. TBC1D1 S237, T596 and S700 phosphorylation is normalized to total TBC1D1. Findings are shown as a representative immunoblot and pooled data quantified in bar graphs as arbitrary units. 2-way RM ANOVA was used to calculate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle). ^# indicates a significant effect of genotype (^#p < 0.05, ^##p < 0.01, ^###p < 0.001). Data generated in the experiment are only obtained from experiment day 4.</p

α-MSH stimulated 2-Deoxy Glucose uptake in soleus and extensor digitorum longus (EDL) muscle explants.

<p>3.A: soleus explants from WT and AMPK KD mice were stimulated with α-MSH (100 nM). Data is presented as the mean ± SEM of pooled data from a series of experiments (see individual bars). 3.B: EDL explants from WT and AMPK KD mice were stimulated with α-MSH (100 nM). 3.C: Phosphorylation of Akt was measured in soleus explants after α-MSH-stimulation (100 nM). Data is presented as the mean ± SEM. 2-way RM ANOVA was used to calculate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle). ^# indicates a significant effect of genotype (^#p < 0.05, ^##p < 0.01, ^###p < 0.001).</p

α-MSH induced phosphorylation of AMPK in differentiated L6 myotubes and in soleus explants dissected from lean and diet-induced obese (DIO) mice.

<p>A: Phosphorylation of AMPK in lean and DIO mice in basal conditions in soleus muscle explants (n=4). 2.B: α-MSH-phosphorylation of AMPK in lean and DIO soleus muscle explants (n=6). 2.C: Phosphorylation of AMPK after α-MSH-stimulation (100 nM) in differentiated L6 myotubes (n=4). AMPK phosphorylation is normalized to total AMPK. Unpaired one-tailed t-test was used to calculate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle)</p

Overview of cellular signaling induced by α-MSH in skeletal muscle.

<p>α-MSH stimulates glucose uptake by an unknown mechanism, which acts independently of AMPK, TBC1D1 and GLUT4 translocation. α-MSH stimulates phosphorylation of TBC1D1 independently of PKA and AMPK. α-MSH stimulates phosphorylation of AMPK independently of PKA.</p