Abstract
Glucagon-like peptide-1 (GLP-1), a metabolic signal molecule, regulates reproduction, although, the involved molecular mechanisms have not been elucidated, yet. Therefore, responsiveness of gonadotropin-releasing hormone (GnRH) neurons to the GLP-1 analog Exendin-4 and elucidation of molecular pathways acting downstream to the GLP-1 receptor (GLP-1R) have been challenged. Loose patch-clamp recordings revealed that Exendin-4 (100 nM–5 μM) elevated firing rate in hypothalamic GnRH-GFP neurons of male mice via activation of GLP-1R. Whole-cell patch-clamp measurements demonstrated increased excitatory GABAergic miniature postsynaptic currents (mPSCs) frequency after Exendin-4 administration, which was eliminated by the GLP-1R antagonist Exendin-3(9–39) (1 μM). Intracellular application of the G-protein inhibitor GDP-β-S (2 mM) impeded action of Exendin-4 on mPSCs, suggesting direct excitatory action of GLP-1 on GnRH neurons. Blockade of nitric-oxide (NO) synthesis by Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME; 100 μM) or N5-[Imino(propylamino)methyl]-L-ornithine hydrochloride (NPLA; 1 μM) or intracellular scavenging of NO by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO; 1 mM) partially attenuated the excitatory effect of Exendin-4. Similar partial inhibition was achieved by hindering endocannabinoid pathway using cannabinoid receptor type-1 (CB1) inverse-agonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidyl) pyrazole-3-carboxamide (AM251; 1 μM). Simultaneous blockade of NO and endocannabinoid signaling mechanisms eliminated action of Exendin-4 suggesting involvement of both retrograde machineries. Intracellular application of the transient receptor potential vanilloid 1 (TRPV1)-antagonist 2E-N-(2, 3-Dihydro-1,4-benzodioxin-6-yl)-3-[4-(1, 1-dimethylethyl)phenyl]-2-Propenamide (AMG9810; 10 μM) or the fatty acid amide hydrolase (FAAH)-inhibitor PF3845 (5 μM) impeded the GLP-1-triggered endocannabinoid pathway indicating an anandamide-TRPV1-sensitive control of 2-arachidonoylglycerol (2-AG) production. Furthermore, GLP-1 immunoreactive (IR) axons innervated GnRH neurons in the hypothalamus suggesting that GLP-1 of both peripheral and neuronal sources can modulate GnRH neurons. RT-qPCR study confirmed the expression of GLP-1R and neuronal NO synthase (nNOS) mRNAs in GnRH-GFP neurons. Immuno-electron microscopic analysis revealed the presence of nNOS protein in GnRH neurons. These results indicate that GLP-1 exerts direct facilitatory actions via GLP-1R on GnRH neurons and modulates NO and 2-AG retrograde signaling mechanisms that control the presynaptic excitatory GABAergic inputs to GnRH neurons.
Highlights
Glucagon-like peptide-1 (GLP-1) is one of the metabolic signal molecules, discovered in humans in 1987 (Kreymann et al, 1987)
: (1) GLP-1 excites firing and increases frequency of GABAergic miniature postsynaptic currents (mPSCs) in gonadotropinreleasing hormone (GnRH) neurons via GLP-1 receptor (GLP-1R); (2) downstream events of GLP-1R involve two retrograde signaling pathways: activation of nitric oxide (NO)- and suppression of 2-AG signaling mechanisms; (3) suppression of 2-AG pathway is mediated via anandamide-transient receptor potential vanilloid 1 (TRPV1) signaling
Our results demonstrated that GLP-1 agonist exerts excitatory effect in GnRH neurons
Summary
Glucagon-like peptide-1 (GLP-1) is one of the metabolic signal molecules, discovered in humans in 1987 (Kreymann et al, 1987) It is secreted by the intestinal L-cells as a gut hormone (Mojsov et al, 1990; Baggio and Drucker, 2007). GLP-1R is widely expressed in the human and rodent brains such as in neurons of the circumventricular organs, amygdala, hypothalamic nuclei, ventrolateral medulla, NST, thalamic paraventricular nucleus, hippocampus and cortex, in various loci for hypothalamic regulation of glucose homeostasis, and parabrachial nucleus a regulatory center of feeding behavior (Goke et al, 1995; Wei and Mojsov, 1995; Scrocchi et al, 1996; Li et al, 2003; Sandoval et al, 2008; Richard et al, 2014; Richards et al, 2014; Cork et al, 2015; Sandoval and Sisley, 2015)
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