A Dairy-Derived Ghrelinergic Hydrolysate Modulates Food Intake In Vivo.

Recent times have seen an increasing move towards harnessing the health-promoting benefits of food and dietary constituents while providing scientific evidence to substantiate their claims. In particular, the potential for bioactive protein hydrolysates and peptides to enhance health in conjunction with conventional pharmaceutical therapy is being investigated. Dairy-derived proteins have been shown to contain bioactive peptide sequences with various purported health benefits, with effects ranging from the digestive system to cardiovascular circulation, the immune system and the central nervous system. Interestingly, the ability of dairy proteins to modulate metabolism and appetite has recently been reported. The ghrelin receptor (GHSR-1a) is a G-protein coupled receptor which plays a key role in the regulation of food intake. Pharmacological manipulation of the growth hormone secretagogue receptor-type 1a (GHSR-1a) receptor has therefore received a lot of attention as a strategy to combat disorders of appetite and body weight, including age-related malnutrition and the progressive muscle wasting syndrome known as cachexia. In this study, a milk protein-derivative is shown to increase GHSR-1a-mediated intracellular calcium signalling in a concentration-dependent manner in vitro. Significant increases in calcium mobilisation were also observed in a cultured neuronal cell line heterologously expressing the GHS-R1a. In addition, both additive and synergistic effects were observed following co-exposure of GHSR-1a to both the hydrolysate and ghrelin. Subsequent in vivo studies monitored standard chow intake in healthy male and female Sprague-Dawley rats after dosing with the casein hydrolysate (CasHyd). Furthermore, the provision of gastro-protected oral delivery to the bioactive in vivo may aid in the progression of in vitro efficacy to in vivo functionality. In summary, this study reports a ghrelin-stimulating bioactive peptide mixture (CasHyd) with potent effects in vitro. It also provides novel and valuable translational data supporting the potential role of CasHyd as an appetite-enhancing bioactive. Further mechanistic studies are required in order to confirm efficacy as a ghrelinergic bioactive in susceptible population groups.

Irish Cheddar cheese increases glucagon-like peptide-1 secretion in vitro but bioactivity is lost during gut transit.

Appetite can be effectively reduced by targeting the production, secretion, circulation time or receptor of the enteric satiety hormone glucagon-like peptide-1 (GLP-1). The objective of this study was to investigate the potency of Irish Cheddar cheeses to modulate GLP-1 levels. Nine out of ten water-soluble extracts (WSEs) of representative Irish Cheddar cheeses, post 6 months ripening, significantly (p < 0.05) stimulated active GLP-1 secretion from the mouse enteroendocrine cell line STC-1. This secretion was associated with protein content and ripening time of cheese. C-57BL/6 mice (n = 9/10), who received the most potent sample, C2-WSE-8 M, had a significantly reduced cumulative food intake at 6 h compared to control (p < 0.05), but not overall treatment × time effect over a 7 h period. Simulated in vitro gastrointestinal digestion, that models the upper human gut, indicated loss of GLP-1 stimulating activity once C2-WSE-8M entered the intestinal phase, suggesting efficacy of C2-WSE-8M will depend on protection during gut transit.

Quinolones Modulate Ghrelin Receptor Signaling: Potential for a Novel Small Molecule Scaffold in the Treatment of Cachexia.

Cachexia is a metabolic wasting disorder characterized by progressive weight loss, muscle atrophy, fatigue, weakness, and appetite loss. Cachexia is associated with almost all major chronic illnesses including cancer, heart failure, obstructive pulmonary disease, and kidney disease and significantly impedes treatment outcome and therapy tolerance, reducing physical function and increasing mortality. Current cachexia treatments are limited and new pharmacological strategies are needed. Agonists for the growth hormone secretagogue (GHS-R1a), or ghrelin receptor, prospectively regulate the central regulation of appetite and growth hormone secretion, and therefore have tremendous potential as cachexia therapeutics. Non-peptide GHS-R1a agonists are of particular interest, especially given the high gastrointestinal degradation of peptide-based structures, including that of the endogenous ligand, ghrelin, which has a half-life of only 30 min. However, few compounds have been reported in the literature as non-peptide GHS-R1a agonists. In this paper, we investigate the in vitro potential of quinolone compounds to modulate the GHS-R1a in both transfected human cells and mouse hypothalamic cells. These chemically synthesized compounds demonstrate a promising potential as GHS-R1a agonists, shown by an increased intracellular calcium influx. Further studies are now warranted to substantiate and exploit the potential of these novel quinolone-based compounds as orexigenic therapeutics in conditions of cachexia and other metabolic and eating disorders.

Sustained-release multiparticulates for oral delivery of a novel peptidic ghrelin agonist: Formulation design and in vitro characterization.

There is an impetus to provide appropriate sustained release oral delivery vehicles to protect biofunctional peptide loads from gastric degradation in vivo. This study describes the generation of a high load capacity pellet formulation for sustained release of a freely water-soluble dairy-derived hydrolysate, FHI-2571. The activity of this novel peptidic ghrelin receptor agonist is reported using in vitro calcium mobilization assays. Conventional extrusion spheronization was then used to prepare peptide-loaded pellets which were subsequently coated with ethylcellulose (EC) film coats using a fluid bed coating system in bottom spray (Wurster) mode. Aqueous-based EC coating dispersions produced mechanically brittle coats which fractured due to osmotic pressure build-up within pellets in simulated media. In contrast, an ethanolic-based EC coating solution provided robust, near zero-order release in both USP Type 1 and Type 4 dissolution studies. Interestingly, the functionality of aqueous-based EC film coats was restored by first layering pellets with a methacrylic acid copolymer (MA) subcoat, thereby hindering pellet core swelling in acidic media. Broadband Acoustic Resonance Dissolution Spectroscopy (BARDS) was utilised as a complementary technique to confirm the results seen in USP dissolution studies. Retention of activity of the ghrelinergic peptide hydrolysate in the final encapsulated product was confirmed as being greater than 80%. The described pellet formulation is amenable to oral dosing in small animal studies in order to assess in vivo efficacy of the whey-derived ghrelinergic hydrolysate. In more general terms, it is also suitable as a delivery vehicle for peptide-based bioactives to special population groups e.g paediatric and geriatric.

A Novel Non-Peptidic Agonist of the Ghrelin Receptor with Orexigenic Activity In vivo.

Loss of appetite in the medically ill and ageing populations is a major health problem and a significant symptom in cachexia syndromes, which is the loss of muscle and fat mass. Ghrelin is a gut-derived hormone which can stimulate appetite. Herein we describe a novel, simple, non-peptidic, 2-pyridone which acts as a selective agonist for the ghrelin receptor (GHS-R1a). The small 2-pyridone demonstrated clear agonistic activity in both transfected human cells and mouse hypothalamic cells with endogenous GHS-R1a receptor expression. In vivo tests with the hit compound showed significant increased food intake following peripheral administration, which highlights the potent orexigenic effect of this novel GHS-R1a receptor ligand.

Ghrelin's 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 ghrelin's 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 ghrelin's orexigenic effect. This data highlights the potential development of a combined GHS-R1a and 5-HT2C receptor treatment strategy in weight management.