Ghrelińs Orexigenic Effect Is Modulated via a Serotonin 2C Receptor Interaction

Understanding the intricate pathways that modulate appetite and subsequent food intake is of particular importance considering the rise in the incidence of obesity across the globe. The serotonergic system, specifically the 5-HT2C receptor, has been shown to be of critical importance in the regulation of appetite and satiety. The GHS-R1a receptor is another key receptor that is well-known for its role in the homeostatic control of food intake and energy balance. We recently showed compelling evidence for an interaction between the GHS-R1a receptor and the 5-HT2C receptor in an in vitro cell line system heterologously expressing both receptors. Here, we investigated this interaction further. First, we show that the GHS-R1a/5-HT2C dimer-induced attenuation of calcium signaling is not due to coupling to GαS, as no increase in cAMP signaling is observed. Next, flow cytometry fluorescence resonance energy transfer (fcFRET) is used to further demonstrate the direct interaction between the GHS-R1a receptor and 5-HT2C receptor. In addition, we demonstrate colocalized expression of the 5-HT2C and GHS-R1a receptor in cultured primary hypothalamic and hippocampal rat neurons, supporting the biological relevance of a physiological interaction. Furthermore, we demonstrate that when 5-HT2C receptor signaling is blocked ghreliņs orexigenic effect is potentiated in vivo. In contrast, the specific 5-HT2C receptor agonist lorcaserin, recently approved for the treatment of obesity, attenuates ghrelin-induced food intake. This underscores the biological significance of our in vitro findings of 5-HT2C receptor-mediated attenuation of GHS-R1a receptor activity. Together, this study demonstrates, for the first time, that the GHS-R1a/5-HT2C receptor interaction translates into a biologically significant modulation of ghreliņs orexigenic effect. This data highlights the potential development of a combined GHS-R1a and 5-HT2C receptor treatment strategy in weight management.Instituto Multidisciplinario de Biología Celula

Devil's Claw to suppress appetite--ghrelin receptor modulation potential of a Harpagophytum procumbens root extract.

Ghrelin is a stomach-derived peptide that has been identified as the only circulating hunger hormone that exerts a potent orexigenic effect via activation of its receptor, the growth hormone secretagogue receptor (GHS-R1a). Hence, the ghrelinergic system represents a promising target to treat obesity and obesity-related diseases. In this study we analysed the GHS-R1a receptor activating potential of Harpagophytum procumbens, popularly known as Devil's Claw, and its effect on food intake in vivo. H. procumbens is an important traditional medicinal plant from Southern Africa with potent anti-inflammatory and analgesic effects. This plant has been also used as an appetite modulator but most evidences are anecdotal and to our knowledge, no clear scientific studies relating to appetite modulation have been done to this date. The ghrelin receptor activation potential of an extract derived from the dried tuberous roots of H. procumbens was analysed by calcium mobilization and receptor internalization assays in human embryonic kidney cells (Hek) stably expressing the GHS-R1a receptor. Food intake was investigated in male C57BL/6 mice following intraperitoneal administration of H. procumbens root extract in ad libitum and food restricted conditions. Exposure to H. procumbens extract demonstrated a significant increased cellular calcium influx but did not induce subsequent GHS-R1a receptor internalization, which is a characteristic for full receptor activation. A significant anorexigenic effect was observed in male C57BL/6 mice following peripheral administration of H. procumbens extract. We conclude that H. procumbens root extract is a potential novel source for potent anti-obesity bioactives. These results reinforce the promising potential of natural bioactives to be developed into functional foods with weight-loss and weight maintenance benefits

Ghrelińs Orexigenic Effect Is Modulated via a Serotonin 2C Receptor Interaction

Understanding the intricate pathways that modulate appetite and subsequent food intake is of particular importance considering the rise in the incidence of obesity across the globe. The serotonergic system, specifically the 5-HT2C receptor, has been shown to be of critical importance in the regulation of appetite and satiety. The GHS-R1a receptor is another key receptor that is well-known for its role in the homeostatic control of food intake and energy balance. We recently showed compelling evidence for an interaction between the GHS-R1a receptor and the 5-HT2C receptor in an in vitro cell line system heterologously expressing both receptors. Here, we investigated this interaction further. First, we show that the GHS-R1a/5-HT2C dimer-induced attenuation of calcium signaling is not due to coupling to GαS, as no increase in cAMP signaling is observed. Next, flow cytometry fluorescence resonance energy transfer (fcFRET) is used to further demonstrate the direct interaction between the GHS-R1a receptor and 5-HT2C receptor. In addition, we demonstrate colocalized expression of the 5-HT2C and GHS-R1a receptor in cultured primary hypothalamic and hippocampal rat neurons, supporting the biological relevance of a physiological interaction. Furthermore, we demonstrate that when 5-HT2C receptor signaling is blocked ghreliņs orexigenic effect is potentiated in vivo. In contrast, the specific 5-HT2C receptor agonist lorcaserin, recently approved for the treatment of obesity, attenuates ghrelin-induced food intake. This underscores the biological significance of our in vitro findings of 5-HT2C receptor-mediated attenuation of GHS-R1a receptor activity. Together, this study demonstrates, for the first time, that the GHS-R1a/5-HT2C receptor interaction translates into a biologically significant modulation of ghreliņs orexigenic effect. This data highlights the potential development of a combined GHS-R1a and 5-HT2C receptor treatment strategy in weight management.Fil: Schellekens, Harriët. University College Cork; IrlandaFil: de Francesco, Pablo Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Kandil, Dalia. University College Cork; IrlandaFil: Theeuwes, Wessel F.. University College Cork; IrlandaFil: McCarthy, Triona. University College Cork; IrlandaFil: Van Oeffelen, Wesley E. P. A.. University College Cork; IrlandaFil: Perello, Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Giblin, Linda. Teagasc Food Research Centre; IrlandaFil: Dinan, Timothy G.. University College Cork; IrlandaFil: Cryan, John F.. University College Cork; Irland

<i>H. procumbens</i> root extract has no cytotoxicity on Hek cells (Hek293a).

<p>Cellular viability was around 100% after exposure to different concentrations of <i>H. procumbens</i> root extract up to 10 mg/mL (A) or purified Harpagoside up to 1 mg/mL (B). Results are expressed as percentage of viability with respect to the control (cells in 1x HBSS containing 20 mM HEPES). Graph represents the mean ± SEM of triplicate samples from one representative assay.</p

<i>H. procumbens</i> root extract induces GHS-R1a-mediated calcium influx.

<p>Calcium influx in Hek293a (wild type) cells versus Hek-GHS-R1a-EGFP cells (<b>A</b>) and dose response curves of <i>H. procumbens</i> root extract (<b>B</b>) and ghrelin (<b>C</b>) are depicted. Exposure to ghrelin, the endogenous ghrelin receptor ligand, and <i>H. procumbens</i> root extract potently increase intracellular calcium through activation of the GHS-R1a receptor in a dose dependent manner. Calcium increase was depicted as a percentage of maximal calcium increase as elicited by control in each separate experiment (3.33% FBS). The data represents the mean ± SEM of a representative experiment out of three independent experiments with each concentration point performed in triplicate.</p

Anorexigenic effect of <i>H. procumbens</i> root extract in <i>ad libitum</i> conditions.

<p>Cumulative food intake (<b>A</b>,<b>B</b>) and food intake per time bin (<b>C,D</b>) in <i>ad libitum</i> fed C57BL/6 mice following intraperitoneal administration of <i>H. procumbens</i> root extract (200 and 500 mg/kg; 2.5% DMSO) and vehicle (saline; 2.5% DMSO). Results are depicted in line graphs ± SEM. Statistical significant differences compared to vehicle are depicted as * <i>p</i><0.05, ** <i>p</i><0.01 or *** <i>p</i><0.001, n = 10 per group.</p

<i>H. procumbens</i> root extract does not internalize the GHS-R1a receptor.

<p>Hek cells stably expressing the GHS-R1a receptor as a C-terminal EGFP fusion protein were visualized using the IN Cell Analyser 1000 (GE Healthcare) following different treatments: untreated (<b>A</b>), ghrelin (<b>B</b>,<b>C</b>), [D-Arg1, D-Phe5, D-Trp7,9, Leu11]-substance P (SP-analogue) (<b>D</b>), (Dlys³)-GHRP-6 (<b>E</b>) and <i>H. procumbens</i> root extract (<b>F</b>) at the indicated concentrations for 1 h at 37°C. Ligand-mediated GHS-R1a-EGFP translocation is quantified following the EGFP fluorescent trafficking away from membrane into vesicles within the cytosol. Graph represents the mean ± SEM of the fluorescence intensity of perinuclear receptor expression versus plasma membrane receptor expression from a representative experiment out of two independent experiments with each treatment performed in triplicate (<b>G</b>). Significant increased internalization is depicted as *** <i>p</i><0.001, and significant decreased internalization is depicted as ##<i>p</i><0.01, #<i>p</i><0.05 with respect to internalization obtained from assay buffer (blanc).</p

<i>H. procumbens</i> root extract specifically activates GHS-R1a receptor independent of harpagoside.

<p>Calcium mobilization upon exposure to <i>H. procumbens</i> root extract and harpagoside in Hek cells stably expressing the GHS-R1a receptor as an EGFP fusion protein. <i>H. procumbens</i> root extract induced GHS-R1a receptor activation was enhancedfollowing attenuation of constitutive receptor activity by pre-treatment with the GHS-R1a receptor inverse agonist, [D-Arg1, D-Phe5, D-Trp7,9, Leu11]-substance P (500 nM, SP-analogue) (<b>A</b>). The iridoid glycoside harpagoside, one of the main compounds present in <i>H. procumbens</i>, did not show an enhanced GHS-R1a receptor-mediated calcium influx, suggesting that the activity observed in this extract is due to others compounds present (<b>B</b>). Graph represents the mean ± SEM of a representative experiment from three (<b>A</b>) or two (<b>B</b>) independents experiments with each concentration point performed in triplicate. Intracellular calcium increase was depicted as a percentage of maximal calcium increase as elicited by control (3.3% FBS). ***<i>p</i><0.001, **<i>p</i><0.01 compared with no [D-Arg1, D-Phe5, D-Trp7,9, Leu11]-substance P pre-treatment.</p

Anorexigenic effect of <i>H. procumbens</i> root extract in food restricted condition.

<p>Cumulative food intake (<b>A</b>) and food intake per time bin (<b>B</b>) in 16 h food restricted C57BL/6 mice following intraperitoneal administration of <i>H. procumbens</i> extract (500 mg/kg; 2.5% DMSO) and vehicle (saline; 2.5% DMSO). Results are depicted in line graphs ± SEM. Statistical significant differences compared to vehicle are depicted as * <i>p</i><0.05, ** <i>p</i><0.01 or *** <i>p</i><0.001, n = 10 per group.</p

Chemical composition of the dried <i>H. procumbens</i> root powder.

<p><b>*</b>calculated by difference.</p