Protective Effects of Hexarelin and JMV2894 in a Human Neuroblastoma Cell Line Expressing the SOD1-G93A Mutated Protein.

Amyotrophic lateral sclerosis (ALS) is an incurable motor neuron disease whose etiology remains unresolved; nonetheless, mutations of superoxide dismutase 1 (SOD1) have been associated with several variants of ALS. Currently available pharmacologic interventions are only symptomatic and palliative in effect; therefore, there is a pressing demand for more effective drugs. This study examined potential therapeutic effects of growth hormone secretagogues (GHSs), a large family of synthetic compounds, as possible candidates for the treatment of ALS. Human neuroblastoma cells expressing the SOD1-G93A mutated protein (SH-SY5Y SOD1G93A cells) were incubated for 24 h with H2O2 (150 µM) in the absence, or presence, of GHS (1 µM), in order to study the protective effect of GHS against increased oxidative stress. The two GHSs examined in this study, hexarelin and JMV2894, protected cells from H2O2-induced cytotoxicity by activating molecules that regulate apoptosis and promote cell survival processes. These findings suggest the possibility of developing new GHS-based anti-oxidant and neuroprotective drugs with improved therapeutic potential. Further investigations are required for the following: (i) to clarify GHS molecular mechanisms of action, and (ii) to envisage the development of new GHSs that may be useful in ALS therapy.

miRNA Expression Profiling in Subcutaneous Adipose Tissue of Monozygotic Twins Discordant for HIV Infection: Validation of Differentially Expressed miRNA and Bioinformatic Analysis.

Combined AntiRetroviral Treatments (cARTs) used for HIV infection may result in varied metabolic complications, which in some cases, may be related to patient genetic factors, particularly microRNAs. The use of monozygotic twins, differing only for HIV infection, presents a unique and powerful model for the controlled analysis of potential alterations of miRNAs regulation consequent to cART treatment. Profiling of 2578 mature miRNA in the subcutaneous (SC) adipose tissue and plasma of monozygotic twins was investigated by the GeneChip® miRNA 4.1 array. Real-time PCR and ddPCR experiments were performed in order to validate differentially expressed miRNAs. Target genes of deregulated miRNAs were predicted by the miRDB database (prediction score > 70) and enrichment analysis was carried out with g:Profiler. Processes in SC adipose tissue most greatly affected by miRNA up-regulation included (i) macromolecular metabolic processes, (ii) regulation of neurogenesis, and (iii) protein phosphorylation. Furthermore, KEGG analysis revealed miRNA up-regulation involvement in (i) insulin signaling pathways, (ii) neurotrophin signaling pathways, and (iii) pancreatic cancer. By contrast, miRNA up-regulation in plasma was involved in (i) melanoma, (ii) p53 signaling pathways, and (iii) focal adhesion. Our findings suggest a mechanism that may increase the predisposition of HIV+ patients to insulin resistance and cancer.

Growth Hormone Secretagogues and the Regulation of Calcium Signaling in Muscle.

Growth hormone secretagogues (GHS) are a family of synthetic molecules, first discovered in the late 1970s for their ability to stimulate growth hormone (GH) release. Many effects of GHS are mediated by binding to GHS-R1a, the receptor for the endogenous hormone ghrelin, a 28-amino acid peptide isolated from the stomach. Besides endocrine functions, both ghrelin and GHS are endowed with some relevant extraendocrine properties, including stimulation of food intake, anticonvulsant and anti-inflammatory effects, and protection of muscle tissue in different pathological conditions. In particular, ghrelin and GHS inhibit cardiomyocyte and endothelial cell apoptosis and improve cardiac left ventricular function during ischemia-reperfusion injury. Moreover, in a model of cisplatin-induced cachexia, GHS protect skeletal muscle from mitochondrial damage and improve lean mass recovery. Most of these effects are mediated by GHS ability to preserve intracellular Ca2+ homeostasis. In this review, we address the muscle-specific protective effects of GHS mediated by Ca2+ regulation, but also highlight recent findings of their therapeutic potential in pathological conditions characterized by skeletal or cardiac muscle impairment.

TLQP-21, A VGF-Derived Peptide Endowed of Endocrine and Extraendocrine Properties: Focus on In Vitro Calcium Signaling.

VGF gene encodes for a neuropeptide precursor of 68 kDa composed by 615 (human) and 617 (rat, mice) residues, expressed prevalently in the central nervous system (CNS), but also in the peripheral nervous system (PNS) and in various endocrine cells. This precursor undergoes proteolytic cleavage, generating a family of peptides different in length and biological activity. Among them, TLQP-21, a peptide of 21 amino acids, has been widely investigated for its relevant endocrine and extraendocrine activities. The complement complement C3a receptor-1 (C3aR1) has been suggested as the TLQP-21 receptor and, in different cell lines, its activation by TLQP-21 induces an increase of intracellular Ca2+. This effect relies both on Ca2+ release from the endoplasmic reticulum (ER) and extracellular Ca2+ entry. The latter depends on stromal interaction molecules (STIM)-Orai1 interaction or transient receptor potential channel (TRPC) involvement. After Ca2+ entry, the activation of outward K+-Ca2+-dependent currents, mainly the KCa3.1 currents, provides a membrane polarizing influence which offset the depolarizing action of Ca2+ elevation and indirectly maintains the driving force for optimal Ca2+ increase in the cytosol. In this review, we address the main endocrine and extraendocrine actions displayed by TLQP-21, highlighting recent findings on its mechanism of action and its potential in different pathological conditions.

Intra-Articular Cytokine Levels in Adolescent Patients after Anterior Cruciate Ligament Tear.

The treatment of anterior cruciate ligament (ACL) injuries in children and adolescents is challenging. Preclinical and clinical studies investigated ACL repairing techniques in skeletally immature subjects. However, intra-articular bioenvironment following ACL tear has not yet been defined in skeletally immature patients. The aim of this study was to measure cytokine concentrations in the synovial fluid in adolescent population. Synovial levels of IL-1ß, IL-1ra, IL-6, IL-8, IL-10, and TNF-α were measured in 17 adolescent patients (15 boys) with ACL tears who underwent ACL reconstruction including acute (5), subacute (7), and chronic (5) phases. Femoral growth plates were classified as "open" in three patients, "closing" in eight, and "closed" in six. Eleven patients presented an ACL tear associated with a meniscal tear. The mean Tegner and Lysholm scores (mean ± SD) of all patients were 8 ± 1 and 50.76 ± 26, respectively. IL-8, TNF-α, and IL-1ß levels were significantly greater in patients with "open" physes. IL-1ra and IL-1ß levels were significantly higher in patients with ACL tear associated with a meniscal tear. Poor Lysholm scores were associated with elevated IL-6 and IL-10 levels. IL-10 levels positively correlated with IL-6 and IL-8 levels, whereas TNF-α concentration negatively correlated with IL-6 levels. Skeletally immature patients with meniscal tears and open growth plates have a characteristic cytokine profile with particularly elevated levels of proinflammatory cytokines including IL-8, TNF-α, and IL-1ß. This picture suggests that the ACL tear could promote an intra-articular catabolic response in adolescent patients greater than that generally reported for adult subjects. The study lacks the comparison with synovial samples from healthy skeletally immature knees due to ethical reasons. Overall, these data contribute to a better knowledge of adolescent intra-articular bioenvironment following ACL injuries.

Neurotrophic and Neuroregenerative Effects of GH/IGF1.

INTRODUCTION: Human neurodegenerative diseases increase progressively with age and present a high social and economic burden. Growth hormone (GH) and insulin-like growth factor-1 (IGF-1) are both growth factors exerting trophic effects on neuronal regeneration in the central nervous system (CNS) and peripheral nervous system (PNS). GH and IGF-1 stimulate protein synthesis in neurons, glia, oligodendrocytes, and Schwann cells, and favor neuronal survival, inhibiting apoptosis. This study aims to evaluate the effect of GH and IGF-1 on neurons, and their possible therapeutic clinical applications on neuron regeneration in human subjects. METHODS: In the literature, we searched the clinical trials and followed up studies in humans, which have evaluated the effect of GH/IGF-1 on CNS and PNS. The following keywords have been used: "GH/IGF-1" associated with "neuroregeneration", "amyotrophic lateral sclerosis", "Alzheimer disease", "Parkinson's disease", "brain", and "neuron". RESULTS: Of the retrieved articles, we found nine articles about the effect of GH in healthy patients who suffered from traumatic brain injury (TBI), and six studies (four using IGF-1 and two GH therapy) in patients with amyotrophic lateral sclerosis (ALS). The administration of GH in patients after TBI showed a significantly positive recovery of brain and mental function. Treatment with GH and IGF-1 therapy in ALS produced contradictory results. CONCLUSIONS: Although strong findings have shown the positive effects of GH/IGF-1 administration on neuroregeneration in animal models, a very limited number of clinical studies have been conducted in humans. GH/IGF-1 therapy had different effects in patients with TBI, evidencing a high recovery of neurons and clinical outcome, while in ALS patients, the results are contradictory. More complex clinical protocols are necessary to evaluate the effect of GH/IGF-1 efficacy in neurodegenerative diseases. It seems evident that GH and IGF-1 therapy favors the optimal recovery of neurons when a consistent residual activity is still present. Furthermore, the effect of GH/IGF-1 could be mediated by, or be overlapped with that of other hormones, such as estradiol and testosterone.

Effects of ACL Reconstructive Surgery on Temporal Variations of Cytokine Levels in Synovial Fluid.

Anterior cruciate ligament (ACL) reconstruction restores knee stability but does not reduce the incidence of posttraumatic osteoarthritis induced by inflammatory cytokines. The aim of this research was to longitudinally measure IL-1ß, IL-6, IL-8, IL-10, and TNF-α levels in patients subjected to ACL reconstruction using bone-patellar tendon-bone graft. Synovial fluid was collected within 24-72 hours of ACL rupture (acute), 1 month after injury immediately prior to surgery (presurgery), and 1 month thereafter (postsurgery). For comparison, a "control" group consisted of individuals presenting chronic ACL tears. Our results indicate that levels of IL-6, IL-8, and IL-10 vary significantly over time in reconstruction patients. In the acute phase, the levels of these cytokines in reconstruction patients were significantly greater than those in controls. In the presurgery phase, cytokine levels in reconstruction patients were reduced and comparable with those in controls. Finally, cytokine levels increased again with respect to control group in the postsurgery phase. The levels of IL-1ß and TNF-α showed no temporal variation. Our data show that the history of an ACL injury, including trauma and reconstruction, has a significant impact on levels of IL-6, IL-8, and IL-10 in synovial fluid but does not affect levels of TNF-α and IL-1ß.

Changes in subcutaneous adipose tissue microRNA expression in HIV-infected patients.

OBJECTIVES: We evaluated the possibility that a pattern of abnormal microRNA (miRNA) expression could be fuelling the mechanisms causing HIV-associated lipodystrophy (HAL). METHODS: In this case-control study, samples of subcutaneous adipose tissue from eight consecutive HIV-infected patients on combination antiretroviral therapy with HAL (cases) were compared with those of eight HIV-negative subjects (controls). Human miRNA microarrays were used to probe the transcriptomes of the samples. Analysis of differentially expressed miRNAs was performed using DataAssist v2.0 software, applying a paired Student's t-test. RESULTS: Data showed that 21 miRNAs out of 754 were overexpressed in the patient group. Ten of these (i.e. miR-186, miR-199a-3p, miR-214, miR-374a, miR-487b, miR-532-5p, miR-628-5p, miR-874, miR-125-b-1* and miR-374b*) were up-regulated to a significant degree (fold change >2.5; P < 0.01). Eleven other miRNAs (i.e. miR-let-7d, miR-24, miR-30c, miR-125a-3p, miR-149, miR-191, miR-196-b, miR-218, miR-342-3p, miR-452 and miR-454*) were 2- to 2.5-fold more expressed in HIV+ samples than in controls. Levels of mRNA for lipin 1, the target of miR-218, were significantly lower in subcutaneous adipose tissue from HIV patients. CONCLUSIONS: In adipocytes of HIV-infected patients, the up-regulation of specific miRNAs could lead to an increased 'activation' that might contribute to the pathogenesis of HAL by increasing cell turnover and/or promotion of apoptosis.

Characterization of a novel peripheral pro-lipolytic mechanism in mice: role of VGF-derived peptide TLQP-21.

The peptides encoded by the VGF gene are gaining biomedical interest and are increasingly being scrutinized as biomarkers for human disease. An endocrine/neuromodulatory role for VGF peptides has been suggested but never demonstrated. Furthermore, no study has demonstrated so far the existence of a receptor-mediated mechanism for any VGF peptide. In the present study, we provide a comprehensive in vitro, ex vivo and in vivo identification of a novel pro-lipolytic pathway mediated by the TLQP-21 peptide. We show for the first time that VGF-immunoreactivity is present within sympathetic fibres in the WAT (white adipose tissue) but not in the adipocytes. Furthermore, we identified a saturable receptor-binding activity for the TLQP-21 peptide. The maximum binding capacity for TLQP-21 was higher in the WAT as compared with other tissues, and selectively up-regulated in the adipose tissue of obese mice. TLQP-21 increases lipolysis in murine adipocytes via a mechanism encompassing the activation of noradrenaline/ß-adrenergic receptors pathways and dose-dependently decreases adipocytes diameters in two models of obesity. In conclusion, we demonstrated a novel and previously uncharacterized peripheral lipolytic pathway encompassing the VGF peptide TLQP-21. Targeting the sympathetic nerve-adipocytes interaction might prove to be a novel approach for the treatment of obesity-associated metabolic complications.

Potential Applications for Growth Hormone Secretagogues Treatment of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) arises from neuronal death due to complex interactions of genetic, molecular, and environmental factors. Currently, only two drugs, riluzole and edaravone, have been approved to slow the progression of this disease. However, ghrelin and other ligands of the GHS-R1a receptor have demonstrated interesting neuroprotective activities that could be exploited in this pathology. Ghrelin, a 28-amino acid hormone, primarily synthesized and secreted by oxyntic cells in the stomach wall, binds to the pituitary GHS-R1a and stimulates GH secretion; in addition, ghrelin is endowed with multiple extra endocrine bioactivities. Native ghrelin requires esterification with octanoic acid for binding to the GHS-R1a receptor; however, this esterified form is very labile and represents less than 10% of circulating ghrelin. A large number of synthetic compounds, the growth hormone secretagogues (GHS) encompassing short peptides, peptoids, and non-peptidic moieties, are capable of mimicking several biological activities of ghrelin, including stimulation of GH release, appetite, and elevation of blood IGF-I levels. GHS have demonstrated neuroprotective and anticonvulsant effects in experimental models of pathologies both in vitro and in vivo. To illustrate, some GHS, currently under evaluation by regulatory agencies for the treatment of human cachexia, have a good safety profile and are safe for human use. Collectively, evidence suggests that ghrelin and cognate GHS may constitute potential therapies for ALS.

Novel domain-selective ACE-inhibiting activity of synthetic growth hormone secretagogues.

The mechanisms of cardiovascular protective effects of ghrelin and its synthetic analogs are still largely unknown. Our first aim was to ascertain whether or not natural and synthetic ligands of GHS-R1a are capable of interfering with the activity of the renin-angiotensin system. Second, since polymorphisms in the ACE gene have been associated with Alzheimer's dementia (AD) and ACE is potentially involved in brain ß-amyloid degradation, we also investigated the state of ghrelin axis and inflammatory markers in patients with AD and vascular dementia (VaD). Desacyl ghrelin, hexarelin, EP80317, and GHRP-6 all significantly inhibited ACE activity in vitro; by comparison, the efficacies of ghrelin and MK-0677 were significantly lower, suggesting that ACE-inhibiting activity is unrelated to ligand affinity to GHS-R1a. ACE was capable of cleaving Aßin vitro, reducing its ability to aggregate in fibrillar Aß. Interestingly, this protective effect of ACE was blunted by enalapril but not hexarelin or EP80317. Desacyl ghrelin levels were lower in VaD subjects compared with AD and control subjects, whereas ghrelin and TNF-α levels were similar in all groups. VaD subjects demonstrated greater levels of mRNA for GHS-R1a, PPAR-γ and CD36 in peripheral blood lymphocytes compared with other groups. In conclusion, some GHSs are effective ACE-inhibitors, and this activity may contribute to their cardiovascular effects. Hexarelin or EP80317 do not inhibit the N-domain of ACE, which is also involved in the metabolism of ß-amyloid, suggesting the possibility of developing new antihypertensive drugs with improved therapeutic potential.

Characterization of microRNA Levels in Synovial Fluid from Knee Osteoarthritis and Anterior Cruciate Ligament Tears.

This study investigated modifications of microRNA expression profiles in knee synovial fluid of patients with osteoarthritis (OA) and rupture of the anterior cruciate ligament (ACL). Twelve microRNAs (26a-5p, 27a-3p, let7a-5p, 140-5p, 146-5p, 155-5p, 16-5p,186-5p, 199a-3p, 210-3p, 205-5p, and 30b-5p) were measured by real-time quantitative polymerase chain reaction (RT-qPCR) in synovial fluids obtained from 30 patients with ACL tear and 18 patients with knee OA. These 12 miRNAs were chosen on the basis of their involvement in pathological processes of bone and cartilage. Our results show that miR-26a-5p, miR-186-5p, and miR-30b-5p were expressed in the majority of OA and ACL tear samples, whereas miR-199a-3p, miR-210-3p, and miR-205-5p were detectable only in a few samples. Interestingly, miR-140-5p was expressed in only one sample of thirty in the ACL tear group. miR-140-5p has been proposed to modulate two genes (BGN and COL5A1100) that are involved in ligamentous homeostasis; their altered expression could be linked with ACL rupture susceptibility. The expression of miR-30b-5p was higher in OA and chronic ACL groups compared to acute ACL samples. We provide evidence that specific miRNAs could be detected not only in synovial fluid of patients with OA, but also in post-traumatic ACL tears.

Hexarelin Modulation of MAPK and PI3K/Akt Pathways in Neuro-2A Cells Inhibits Hydrogen Peroxide-Induced Apoptotic Toxicity.

Hexarelin, a synthetic hexapeptide, exerts cyto-protective effects at the mitochondrial level in cardiac and skeletal muscles, both in vitro and in vivo, may also have important neuroprotective bioactivities. This study examined the inhibitory effects of hexarelin on hydrogen peroxide (H2O2)-induced apoptosis in Neuro-2A cells. Neuro-2A cells were treated for 24 h with various concentrations of H2O2 or with the combination of H2O2 and hexarelin following which cell viability and nitrite (NO2-) release were measured. Cell morphology was also documented throughout and changes arising were quantified using Image J skeleton and fractal analysis procedures. Apoptotic responses were evaluated by Real-Time PCR (caspase-3, caspase-7, Bax, and Bcl-2 mRNA levels) and Western Blot (cleaved caspase-3, cleaved caspase-7, MAPK, and Akt). Our results indicate that hexarelin effectively antagonized H2O2-induced damage to Neuro-2A cells thereby (i) improving cell viability, (ii) reducing NO2- release and (iii) restoring normal morphologies. Hexarelin treatment also reduced mRNA levels of caspase-3 and its activation, and modulated mRNA levels of the BCL-2 family. Moreover, hexarelin inhibited MAPKs phosphorylation and increased p-Akt protein expression. In conclusion, our results demonstrate neuroprotective and anti-apoptotic effects of hexarelin, suggesting that new analogues could be developed for their neuroprotective effects.

Desacyl-ghrelin and synthetic GH-secretagogues modulate the production of inflammatory cytokines in mouse microglia cells stimulated by beta-amyloid fibrils.

Data from Alzheimer's disease (AD) patients and AD animal models demonstrate the accumulation of inflammatory microglia at sites of insoluble fibrillar beta-amyloid protein (fAbeta) deposition. It is known that fAbeta binds to CD36, a type B scavenger receptor also involved in internalization of oxidized low-density lipoprotein (LDL), and initiate a signaling cascade that regulates microglial recruitment, activation, and secretion of inflammatory mediators leading to neuronal dysfunction and death. The recent demonstration of a binding site for the growth hormone secretagogues (GHS) on CD36 prompted us to ascertain whether ghrelin and synthetic GHS could modulate the synthesis of inflammatory cytokines in fAbeta-activated microglia cells. We demonstrate that N9 microglia cells express the CD36 and are a suitable model to study the activation of inflammatory cytokines synthesis. In fact, in N9 cells exposed to fAbeta(25-35) for 24 hr, the expression of interleukin (IL)-1beta and IL-6 mRNA significantly increased. Interestingly, 10(-7) M desacyl-ghrelin, hexarelin, and EP80317 in the nanomolar range effectively counteracted fAbeta(25-35) stimulation of IL-6 mRNA levels, whereas ghrelin was ineffective. Similarly, the effects of fAbeta(25-35) on IL-1beta mRNA levels were attenuated by desacyl-ghrelin, hexarelin, and EP80317, but not ghrelin. Because we have observed that the specific GHS receptor GHS-R1a is not expressed in N9 cells, the actions of GHS should be mediated by different receptors. Reportedly, hexarelin and EP80317 are capable of binding the CD36 in mouse macrophages and reducing atherosclerotic plaque deposition in mice. We conclude that desacyl-ghrelin, hexarelin, and EP80317 might interfere with fAbeta activation of CD36 in microglia cells.

miRNA-218 Targets Lipin-1 and Glucose Transporter Type 4 Genes in 3T3-L1 Cells Treated With Lopinavir/Ritonavir.

Background: Metabolic complications represent a common and serious problem associated with HIV infection and combined Antiretroviral Therapy (cART). Alterations in body fat distribution are associated with significantly increased risks of (i) metabolic derangements, (ii) cardiovascular pathologies, and (iii) insulin resistance. A case control study showed that in subcutaneous adipose tissue from HIV-infected patients on cART presenting lipodystrophy (LS), the levels of miRNA-218 were upregulated and those of lipin-1, a putative target gene of miRNA-218, were downregulated compared with HIV-negative subjects. Lipin-1 is one of the most important factors linked to development of LS. Lipin-1, by controlling PPARγ2, regulates the expression of specific genes, such as that of glucose transporter type 4 (GLUT-4), required for maturation and maintenance of adipocytes. Objectives: To determine whether lopinavir/ritonavir (LPV/RTV) can modulate lipogenesis in adipocytes affecting miRNA-218 and lipin-1 mRNA expression, and to investigate the functional link between miRNA-218 and GLUT-4 mRNA expression. Methods: Differentiated 3T3-L1 cells were treated with various combinations of LPV/RTV, followed by measurements of cell viability, lipid accumulation, lipin-1 and GLUT-4 mRNA and miRNA-218 levels. Transfection of anti-miR-218 or a miRNA-218 mimic were used to investigate the role of miRNA-218 in lipogenesis. Results: LPV/RTV treatment of 3T3-L1 cells did not affect the viability of differentiated 3T3-L1 cells, but caused (i) a significant decrease of lipid accumulation, (ii) an overexpression of miRNA-218, and (iii) a reduction of lipin-1 and GLUT-4 mRNA levels. The anti-miR-218 transfection of 3T3-L1 cells significantly ameliorated the adipogenic dysfunction and restored mRNA levels of lipin-1 and GLUT-4 consequent to LPV/RTV treatment. By contrast, 3T3-L1 cells transfected with a specific miRNA-218 mimic showed (i) an overexpression of miRNA-218, (ii) a reduced cellular lipid fraction, and (iii) decreased levels of mRNA for lipin-1 and GLUT-4. Conclusion: 3T3-L1 cells, treated with LPV/RTV, show altered lipid content due to increased miRNA-218 levels, which affects lipin-1 mRNA. Moreover, increased miRNA-218 levels were inversely correlated with changes in GLUT-4 expression, which suggests a role for miRNA-218 in mediating the insulin resistance consequent to cART.

New trisubstituted 1,2,4-triazole derivatives as potent ghrelin receptor antagonists. 3. Synthesis and pharmacological in vitro and in vivo evaluations.

Ghrelin receptor ligands based on trisubstituted 1,2,4-triazole structure were synthesized and evaluated for their in vitro binding and biological activity. In this study, we explored the replacement of the alpha-aminoisobutyryl moiety by aromatic or heteroaromatic groups. Compounds 5 and 34 acted as potent in vivo antagonists of hexarelin-stimulated food intake. These two compounds did not stimulate growth hormone secretion in rodents and did not antagonize growth hormone secretion induced by hexarelin.

STIM Proteins and Orai Ca2+ Channels Are Involved in the Intracellular Pathways Activated by TLQP-21 in RAW264.7 Macrophages.

TLQP-21 is a neuropeptide which has been implicated in regulation of nociception and other relevant physiologic functions. Although recent studies identified C3a and gC1q receptors as targets for TLQP-21, its intracellular molecular mechanisms of action remain largely unidentified. Our aim was (i) to explore the intracellular signaling pathway(s) activated by JMV5656, a novel derivative of TLQP-21, in RAW264.7 macrophages, and (ii) to assess linkages of these pathways with its purported receptors. JMV5656 stimulated, in a dose-dependent fashion, a rapid and transient increase in intracellular Ca2+ concentrations in RAW264.7 cells; repeated exposure to the peptide resulted in a lower response, suggesting a possible desensitization mechanism of the receptor. In particular, JMV5656 increased cytoplasmic Ca2+ levels by a PLC-dependent release of Ca2+ from the endoplasmic reticulum. STIM proteins and Orai Ca2+ channels were activated and played a crucial role. In fact, treatment of the cells with U73122 and thapsigargin modulated the increase of intracellular Ca2+ levels stimulated by JMV5656. Moreover, in RAW264.7 cells intracellular Ca2+ increases did not occur through the binding of JMV5656 to the C3a receptor, since the increase of intracellular Ca2+ levels induced by JMV5656 was not affected by specific siRNA against C3aR. In summary, our study provides new indications for the downstream effects of JMV5656 in macrophages, suggesting that it could activate receptors different from the C3aR.

Toward potent ghrelin receptor ligands based on trisubstituted 1,2,4-triazole structure. 2. Synthesis and pharmacological in vitro and in vivo evaluations.

A series of ghrelin receptor ligands based on the trisubstituted 1,2,4-triazole structure were synthesized and evaluated for their in vitro binding and biological activity. In this study, we explored the significance of the aminoisobutyryl (Aib) moiety, a common feature in numerous growth hormone secretagogues described in the literature. Potent agonist and antagonist ligands of the growth hormone secretagogue receptor type 1a (GHS-R1a) were obtained, i.e., compounds 41 (JMV2894) and 17 (JMV3031). The best compounds were evaluated for their in vivo activity on food intake, after sc injection in rodents. Among the tested compounds, few of them were able to stimulate food intake and some others, i.e., compounds 4 (JMV2959), 17, and 52 (JMV3021), acted as potent in vivo antagonist of hexarelin-stimulated food intake. These compounds did not stimulate growth hormone secretion in rats and furthermore did not antagonize growth hormone secretion induced by hexarelin, revealing that it is possible to modulate food intake without altering growth hormone secretion.

Synthesis and pharmacological in vitro and in vivo evaluations of novel triazole derivatives as ligands of the ghrelin receptor. 1.

A new series of growth hormone secretagogue (GHS) analogues based on the 1,2,4-triazole structure were synthesized and evaluated for their in vitro binding and their ability to stimulate intracellular calcium release to the cloned hGHS-1a ghrelin receptor expressed in LLC PK-1 cells. We have synthesized potent ligands of this receptor, some of them behaving as agonists, partial agonists, or antagonists. Some compounds among the most potent, i.e., agonist 29c (JMV2873), partial agonists including 21b (JMV2810), antagonists 19b (JMV2866) and 19c (JMV2844), were evaluated for their in vivo activity on food intake, after sc injection in rodents. Some compounds were found to stimulate food intake like hexarelin; some others were identified as potent hexarelin antagonists in this assay. Among the tested compounds, 21b was identified as an in vitro ghrelin receptor partial agonist, as well as a potent in vivo antagonist of hexarelin-stimulated food intake in rodents. Compound 21b was without effect on GH release from rat. However, in this series of compounds, it was not possible to find a clear correlation between in vitro and in vivo results.