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1.
Methods Enzymol ; 698: 195-219, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38886032

RESUMEN

Glucagon-like peptide-1, glucose-dependent insulinotropic polypeptide, and glucagon are three naturally occurring peptide hormones that mediate glucoregulation. Several agonists representing appropriately modified native ligands have been developed to maximize metabolic benefits with reduced side-effects and many have entered the clinic as type 2 diabetes and obesity therapeutics. In this work, we describe strategies for improving the stability of the peptide ligands by making them refractory to dipeptidyl peptidase-4 catalyzed hydrolysis and inactivation. We describe a series of alkylations with variations in size, shape, charge, polarity, and stereochemistry that are able to engender full activity at the receptor(s) while simultaneously resisting enzyme-mediated degradation. Utilizing this strategy, we offer a novel method of modulating receptor activity and fine-tuning pharmacology without a change in peptide sequence.


Asunto(s)
Péptido 1 Similar al Glucagón , Humanos , Péptido 1 Similar al Glucagón/química , Péptido 1 Similar al Glucagón/metabolismo , Diseño de Fármacos , Dipeptidil Peptidasa 4/química , Dipeptidil Peptidasa 4/metabolismo , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Péptidos/química , Polipéptido Inhibidor Gástrico/química , Polipéptido Inhibidor Gástrico/metabolismo , Alquilación , Glucagón/química , Glucagón/metabolismo , Animales , Ligandos , Hidrólisis , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo
2.
Bioconjug Chem ; 35(5): 693-702, 2024 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-38700695

RESUMEN

The development of oligomeric glucagon-like peptide-1 (GLP-1) and GLP-1-containing coagonists holds promise for enhancing the therapeutic potential of the GLP-1-based drugs for treating type 2 diabetes mellitus (T2DM). Here, we report a facile, efficient, and customizable strategy based on genetically encoded SpyCatcher-SpyTag chemistry and an inducible, cleavable self-aggregating tag (icSAT) scheme. icSAT-tagged SpyTag-fused GLP-1 and the dimeric or trimeric SpyCatcher scaffold were designed for dimeric or trimeric GLP-1, while icSAT-tagged SpyCatcher-fused GLP-1 and the icSAT-tagged SpyTag-fused GIP were designed for dual GLP-1/GIP (glucose-dependent insulinotropic polypeptide) receptor agonist. These SpyCatcher- and SpyTag-fused protein pairs were spontaneously ligated directly from the cell lysates. The subsequent icSAT scheme, coupled with a two-step standard column purification, resulted in target proteins with authentic N-termini, with yields ranging from 35 to 65 mg/L and purities exceeding 99%. In vitro assays revealed 3.0- to 4.1-fold increased activities for dimeric and trimeric GLP-1 compared to mono-GLP-1. The dual GLP-1/GIP receptor agonist exhibited balanced activity toward the GLP-1 receptor or the GIP receptor. All the proteins exhibited 1.8- to 3.0-fold prolonged half-lives in human serum compared to mono-GLP-1 or GIP. This study provides a generally applicable click biochemistry strategy for developing oligomeric or dual peptide/protein-based drug candidates.


Asunto(s)
Química Clic , Péptido 1 Similar al Glucagón , Péptido 1 Similar al Glucagón/química , Humanos , Receptores de la Hormona Gastrointestinal/agonistas , Receptores de la Hormona Gastrointestinal/química , Receptores de la Hormona Gastrointestinal/metabolismo , Diseño de Fármacos , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Polipéptido Inhibidor Gástrico/química , Polipéptido Inhibidor Gástrico/farmacología , Receptor del Péptido 1 Similar al Glucagón/agonistas
3.
Peptides ; 177: 171212, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38608836

RESUMEN

Surprisingly, agonists, as well as antagonists of the glucose-dependent insulinotropic polypeptide receptor (GIPR), are currently being used or investigated as treatment options for type 2 diabetes and obesity - and both, when combined with glucagon-like peptide 1 receptor (GLP-1R) agonism, enhance GLP-1-induced glycemia and weight loss further. This paradox raises several questions regarding not only the mechanisms of actions of GIP but also the processes engaged during the activation of both the GIP and GLP-1 receptors. Here, we provide an overview of studies of the properties and actions of peptide-derived GIPR antagonists, focusing on GIP(3-30)NH2, a naturally occurring N- and C-terminal truncation of GIP(1-42). GIP(3-30)NH2 was the first GIPR antagonist administered to humans. GIP(3-30)NH2 and a few additional antagonists, like Pro3-GIP, have been used in both in vitro and in vivo studies to elucidate the molecular and cellular consequences of GIPR inhibition, desensitization, and internalization and, at a larger scale, the role of the GIP system in health and disease. We provide an overview of these studies combined with recent knowledge regarding the effects of naturally occurring variants of the GIPR system and species differences within the GIP system to enhance our understanding of the GIPR as a drug target.


Asunto(s)
Polipéptido Inhibidor Gástrico , Receptores de la Hormona Gastrointestinal , Receptores de la Hormona Gastrointestinal/antagonistas & inhibidores , Receptores de la Hormona Gastrointestinal/metabolismo , Humanos , Polipéptido Inhibidor Gástrico/farmacología , Polipéptido Inhibidor Gástrico/metabolismo , Polipéptido Inhibidor Gástrico/química , Animales , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/agonistas , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo
4.
J Med Chem ; 67(6): 4998-5010, 2024 Mar 28.
Artículo en Inglés | MEDLINE | ID: mdl-38458970

RESUMEN

Glucose-dependent insulinotropic peptide (GIP) is a 42-amino acid peptide hormone that regulates postprandial glucose levels. GIP binds to its cognate receptor, GIPR, and mediates metabolic physiology by improved insulin sensitivity, ß-cell proliferation, increased energy consumption, and stimulated glucagon secretion. Dipeptidyl peptidase-4 (DPP4) catalyzes the rapid inactivation of GIP within 6 min in vivo. Here, we report a molecular platform for the design of GIP analogues that are refractory to DPP4 action and exhibit differential activation of the receptor, thus offering potentially hundreds of GIP-based compounds to fine-tune pharmacology. The lead compound from our studies, which harbored a combination of N-terminal alkylation and side-chain lipidation, was equipotent and retained full efficacy at GIPR as the native peptide, while being completely refractory toward DPP4, and was resistant to trypsin. The GIP analogue identified from these studies was further evaluated in vivo and is one of the longest-acting GIPR agonists to date.


Asunto(s)
Polipéptido Inhibidor Gástrico , Receptores de la Hormona Gastrointestinal , Polipéptido Inhibidor Gástrico/farmacología , Polipéptido Inhibidor Gástrico/química , Polipéptido Inhibidor Gástrico/metabolismo , Insulina/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Péptido Hidrolasas , Péptidos , Endopeptidasas , Receptores de la Hormona Gastrointestinal/agonistas , Receptores de la Hormona Gastrointestinal/metabolismo
5.
Biochem Pharmacol ; 192: 114715, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34339714

RESUMEN

Glucose-dependent insulinotropic polypeptide (GIP) and its receptor (GIPR) are part of the incretin system that regulates glucose homeostasis. A series of GIPR residues putatively important for ligand binding and receptor activation were mutated and pharmacologically evaluated using GIPR selective agonists in cAMP accumulation, ERK1/2 phosphorylation (pERK1/2) and ß-arrestin 2 recruitment assays. The impact of mutation on ligand efficacy was determined by operational modelling of experimental data for each mutant, with results mapped onto the full-length, active-state GIPR structure. Two interaction networks, comprising transmembrane helix (TM) 7, TM1 and TM2, and extracellular loop (ECL) 2, TM5 and ECL3 were revealed, respectively. Both networks were critical for Gαs-mediated cAMP accumulation and the recruitment of ß-arrestin 2, however, cAMP response was more sensitive to alanine substitution, with most mutated residues displaying reduced signaling. Unlike the other two assays, activation of ERK1/2 was largely independent of the network involving ECL2, TM5 and ECL3, indicating that pERK1/2 is at least partially distinct from Gαs or ß-arrestin pathways and this network is also crucial for potential biased agonism at GIPR. Collectively, our work advances understanding of the structure-function relationship of GIPR and provides a framework for the design and/or interpretation of GIP analogues with unique signaling profiles.


Asunto(s)
Polipéptido Inhibidor Gástrico/genética , Polipéptido Inhibidor Gástrico/metabolismo , Receptores de la Hormona Gastrointestinal/genética , Receptores de la Hormona Gastrointestinal/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Células COS , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Polipéptido Inhibidor Gástrico/química , Polipéptido Inhibidor Gástrico/farmacología , Células HEK293 , Humanos , Mutación/fisiología , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Estructura Secundaria de Proteína , Receptores de la Hormona Gastrointestinal/química
6.
Sci Rep ; 11(1): 14470, 2021 07 14.
Artículo en Inglés | MEDLINE | ID: mdl-34262109

RESUMEN

We recently established a new plasma peptidomic technique and comprehensively identified a large number of low-molecular weight and low-abundance native peptides using a single drop of human plasma. To discover a novel polypeptide that potently modulates the cardiovascular system, we performed a bioinformatics analysis of the large-scale identification results, sequentially synthesized the selected peptide sequences, tested their biological activities, and identified a 30-amino-acid proatherogenic peptide, GIP_HUMAN[22-51], as a potent proatherosclerotic peptide hormone. GIP_HUMAN[22-51] has a common precursor with the glucose-dependent insulinotropic polypeptide (GIP) and is located immediately N-terminal to GIP. Chronic infusion of GIP_HUMAN[22-51] into ApoE-/- mice accelerated the development of aortic atherosclerotic lesions, which were inhibited by co-infusions with an anti-GIP_HUMAN[22-51] antibody. GIP_HUMAN[22-51] increased the serum concentrations of many inflammatory and proatherogenic proteins, whereas neutralising antibodies reduced their levels. GIP_HUMAN[22-51] induced IκB-α degradation and nuclear translocation of NF-κB in human vascular endothelial cells and macrophages. Immunoreactive GIP_HUMAN[22-51] was detected in human tissues but there was no colocalization with the GIP. The plasma GIP_HUMAN[22-51] concentration in healthy humans determined using a stable-isotope tagged peptide was approximately 0.6 nM. This study discovered a novel endogenous proatherogenic peptide by using a human plasma native peptidomic resource.


Asunto(s)
Polipéptido Inhibidor Gástrico/química , Péptidos/sangre , Péptidos/farmacología , Animales , Aterosclerosis/inducido químicamente , Aterosclerosis/patología , Biomarcadores/sangre , Calcio/metabolismo , Células Cultivadas , Simulación por Computador , Polipéptido Inhibidor Gástrico/sangre , Humanos , Espectrometría de Masas , Ratones Noqueados para ApoE , FN-kappa B/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Péptidos/toxicidad
7.
Elife ; 102021 07 13.
Artículo en Inglés | MEDLINE | ID: mdl-34254582

RESUMEN

Glucose-dependent insulinotropic polypeptide (GIP) is a peptide hormone that exerts crucial metabolic functions by binding and activating its cognate receptor, GIPR. As an important therapeutic target, GIPR has been subjected to intensive structural studies without success. Here, we report the cryo-EM structure of the human GIPR in complex with GIP and a Gs heterotrimer at a global resolution of 2.9 Å. GIP adopts a single straight helix with its N terminus dipped into the receptor transmembrane domain (TMD), while the C terminus is closely associated with the extracellular domain and extracellular loop 1. GIPR employs conserved residues in the lower half of the TMD pocket to recognize the common segments shared by GIP homologous peptides, while uses non-conserved residues in the upper half of the TMD pocket to interact with residues specific for GIP. These results provide a structural framework of hormone recognition and GIPR activation.


Asunto(s)
Polipéptido Inhibidor Gástrico/química , Polipéptido Inhibidor Gástrico/metabolismo , Receptores de la Hormona Gastrointestinal/química , Receptores de la Hormona Gastrointestinal/metabolismo , Animales , Línea Celular , Microscopía por Crioelectrón , Humanos , Ligandos , Simulación de Dinámica Molecular , Conformación Proteica , Conformación Proteica en Hélice alfa , Dominios Proteicos
8.
PLoS One ; 16(3): e0249239, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33788878

RESUMEN

Combinatorial gut hormone therapy is one of the more promising strategies for identifying improved treatments for metabolic disease. Many approaches combine the established benefits of glucagon-like peptide-1 (GLP-1) agonism with one or more additional molecules with the aim of improving metabolic outcomes. Recent attention has been drawn to the glucose-dependent insulinotropic polypeptide (GIP) system due to compelling pre-clinical evidence describing the metabolic benefits of antagonising the GIP receptor (GIPR). We rationalised that benefit might be accrued from combining GIPR antagonism with GLP-1 agonism. Two GIPR peptide antagonists, GIPA-1 (mouse GIP(3-30)NH2) and GIPA-2 (NαAc-K10[γEγE-C16]-Arg18-hGIP(5-42)), were pharmacologically characterised and both exhibited potent antagonist properties. Acute in vivo administration of GIPA-1 during an oral glucose tolerance test (OGTT) had negligible effects on glucose tolerance and insulin in lean mice. In contrast, GIPA-2 impaired glucose tolerance and attenuated circulating insulin levels. A mouse model of diet-induced obesity (DIO) was used to investigate the potential metabolic benefits of chronic dosing of each antagonist, alone or in combination with liraglutide. Chronic administration studies showed expected effects of liraglutide, lowering food intake, body weight, fasting blood glucose and plasma insulin concentrations while improving glucose sensitivity, whereas delivery of either GIPR antagonist alone had negligible effects on these parameters. Interestingly, chronic dual therapy augmented insulin sensitizing effects and lowered plasma triglycerides and free-fatty acids, with more notable effects observed with GIPA-1 compared to GIPA-2. Thus, the co-administration of both a GIPR antagonist with a GLP1 agonist uncovers interesting beneficial effects on measures of insulin sensitivity, circulating lipids and certain adipose stores that seem influenced by the degree or nature of GIP receptor antagonism.


Asunto(s)
Polipéptido Inhibidor Gástrico/farmacología , Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/antagonistas & inhibidores , Glucosa/metabolismo , Secuencia de Aminoácidos , Animales , Glucemia/análisis , Peso Corporal/efectos de los fármacos , Dieta Alta en Grasa/veterinaria , Ácidos Grasos/sangre , Polipéptido Inhibidor Gástrico/química , Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Prueba de Tolerancia a la Glucosa , Secreción de Insulina , Liraglutida/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Curva ROC , Triglicéridos/sangre
9.
Cell Metab ; 33(4): 833-844.e5, 2021 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-33571454

RESUMEN

Uncertainty exists as to whether the glucose-dependent insulinotropic polypeptide receptor (GIPR) should be activated or inhibited for the treatment of obesity. Gipr was recently demonstrated in hypothalamic feeding centers, but the physiological relevance of CNS Gipr remains unknown. Here we show that HFD-fed CNS-Gipr KO mice and humanized (h)GIPR knockin mice with CNS-hGIPR deletion show decreased body weight and improved glucose metabolism. In DIO mice, acute central and peripheral administration of acyl-GIP increases cFos neuronal activity in hypothalamic feeding centers, and this coincides with decreased body weight and food intake and improved glucose handling. Chronic central and peripheral administration of acyl-GIP lowers body weight and food intake in wild-type mice, but shows blunted/absent efficacy in CNS-Gipr KO mice. Also, the superior metabolic effect of GLP-1/GIP co-agonism relative to GLP-1 is extinguished in CNS-Gipr KO mice. Our data hence establish a key role of CNS Gipr for control of energy metabolism.


Asunto(s)
Peso Corporal/efectos de los fármacos , Ingestión de Alimentos/efectos de los fármacos , Polipéptido Inhibidor Gástrico/farmacología , Receptores de la Hormona Gastrointestinal/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Sistema Nervioso Central/metabolismo , Dieta Alta en Grasa , Polipéptido Inhibidor Gástrico/química , Péptido 1 Similar al Glucagón/farmacología , Humanos , Hipotálamo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Obesidad/metabolismo , Obesidad/patología , Obesidad/prevención & control , Proteínas Proto-Oncogénicas c-fos/metabolismo , Receptores de la Hormona Gastrointestinal/deficiencia , Receptores de la Hormona Gastrointestinal/genética
10.
Peptides ; 125: 170224, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-31809770

RESUMEN

Enzymatic cleavage of endogenous peptides is a commonly used principle to initiate, modulate and terminate action for instance among cytokines and peptide hormones. The incretin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), and the related hormone glucagon-like peptide-2 (GLP-2) are all rapidly N-terminally truncated with severe loss of intrinsic activity. The most abundant circulating form of full length GIP(1-42) is GIP(3-42) (a dipeptidyl peptidase-4 (DPP-4) product). GIP(1-30)NH2 is another active form resulting from prohormone convertase 2 (PC2) cleavage of proGIP. Like GIP(1-42), GIP(1-30)NH2 is a substrate for DPP-4 generating GIP(3-30)NH2 which, compared to GIP(3-42), binds with higher affinity and very efficiently inhibits GIP receptor (GIPR) activity with no intrinsic activity. Here, we review the action of these four and multiple other N- and C-terminally truncated forms of GIP with an emphasis on molecular pharmacology, i.e. ligand binding, subsequent receptor activation and desensitization. Our overall conclusion is that the N-terminus is essential for receptor activation as GIP N-terminal truncation leads to decreased/lost intrinsic activity and antagonism (similar to GLP-1 and GLP-2), whereas the C-terminal extension of GIP(1-42), as compared to GLP-1, GLP-2 and glucagon (29-33 amino acids), has no apparent impact on the GIPR in vitro, but may play a role for other properties such as stability and tissue distribution. A deeper understanding of the molecular interaction of naturally occurring and designed GIP-based peptides, and their impact in vivo, may contribute to a future therapeutic targeting of the GIP system - either with agonists or with antagonists, or both.


Asunto(s)
Polipéptido Inhibidor Gástrico/metabolismo , Fragmentos de Péptidos/metabolismo , Receptores de la Hormona Gastrointestinal/metabolismo , Animales , Polipéptido Inhibidor Gástrico/química , Humanos , Fragmentos de Péptidos/química , Receptores de la Hormona Gastrointestinal/química , Relación Estructura-Actividad
11.
Basic Clin Pharmacol Toxicol ; 126 Suppl 6: 122-132, 2020 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-31299132

RESUMEN

In patients with type 2 diabetes mellitus (T2DM), the insulinotropic action of the GIP system is desensitized, whereas this is not the case for the GLP-1 system. This has raised an interesting discussion of whether GIP agonists or antagonists are most suitable for future treatment of T2DM together with GLP-1-based therapies. Homozygous carriers of the GIP receptor (GIPR) variant, [E354Q], display lower bone mineral density, increased bone fracture risk and slightly increased blood glucose. Here, we present an in-depth molecular pharmacological phenotyping of GIPR-[E354Q]. In silico modelling suggested similar interaction of the endogenous agonist GIP(1-42) to [E354Q] as to GIPR wt. This was supported by homologous competition binding in COS-7 cells revealing GIPR wt-like affinities of GIP(1-42) with Kd values of ~2 nmol/L and wt-like agonist association rates (Kon ). In contrast, the dissociation rates (Koff ) were slower, resulting in 25% higher agonist residence time for GIPR-[E354Q]. Moreover, in Gαs signalling (cAMP production) GIP(1-42) was ~2-fold more potent and more efficacious on GIPR-[E354Q] compared to wt with 17.5% higher basal activity. No difference from GIPR wt was found in the recruitment of ß-arrestin 2, whereas the agonist-induced internalization rate was 2.1- to 2.3-fold faster for [E354Q]. Together with the previously described impaired recycling of [E354Q], our findings with enhanced signalling and internalization rate possibly explained by an altered ligand-binding kinetics will lead to receptor desensitization and down-regulation. This could explain the long-term functional impairment of the GIP system in bone metabolism and blood sugar maintenance for [E354Q] carriers and may shed light on the desensitization of the insulinotropic action of GIP in patients with T2DM.


Asunto(s)
Polipéptido Inhibidor Gástrico/metabolismo , Animales , Células COS , Chlorocebus aethiops , Polipéptido Inhibidor Gástrico/agonistas , Polipéptido Inhibidor Gástrico/química , Células HEK293 , Humanos , Estructura Molecular , Transducción de Señal , beta-Arrestinas
12.
PLoS One ; 14(1): e0208892, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30650080

RESUMEN

A disturbance of glucose homeostasis leading to type 2 diabetes mellitus (T2DM) is one of the severe side effects that may occur during a prolonged use of many drugs currently available on the market. In this manuscript we describe the most common cases of drug-induced T2DM, discuss available pharmacotherapies and propose new ones. Among various pharmacotherapies of T2DM, incretin therapies have recently focused attention due to the newly determined crystal structure of incretin hormone receptor GLP1R. Incretin hormone receptors: GLP1R and GIPR together with the glucagon receptor GCGR regulate food intake and insulin and glucose secretion. Our study showed that incretin hormone receptors, named also gut hormone receptors as they are expressed in the gastrointestinal tract, could potentially act as unintended targets (off-targets) for orally administrated drugs. Such off-target interactions, depending on their effect on the receptor (stimulation or inhibition), could be beneficial, like in the case of incretin mimetics, or unwanted if they cause, e.g., decreased insulin secretion. In this in silico study we examined which well-known pharmaceuticals could potentially interact with gut hormone receptors in the off-target way. We observed that drugs with the strongest binding affinity for gut hormone receptors were also reported in the medical information resources as the least disturbing the glucose homeostasis among all drugs in their class. We suggested that those strongly binding molecules could potentially stimulate GIPR and GLP1R and/or inhibit GCGR which could lead to increased insulin secretion and decreased hepatic glucose production. Such positive effect on the glucose homeostasis could compensate for other, adverse effects of pharmacotherapy which lead to drug-induced T2DM. In addition, we also described several top hits as potential substitutes of peptidic incretin mimetics which were discovered in the drug repositioning screen using gut hormone receptors structures against the ZINC15 compounds subset.


Asunto(s)
Diabetes Mellitus Tipo 2/inducido químicamente , Diabetes Mellitus Tipo 2/metabolismo , Animales , Polipéptido Inhibidor Gástrico/química , Polipéptido Inhibidor Gástrico/metabolismo , Receptor del Péptido 1 Similar al Glucagón/química , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Humanos , Estructura Secundaria de Proteína , Receptores de la Hormona Gastrointestinal/química , Receptores de la Hormona Gastrointestinal/metabolismo , Receptores de Glucagón/química , Receptores de Glucagón/metabolismo
13.
Eur J Pharmacol ; 828: 31-41, 2018 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-29577894

RESUMEN

Huntington's disease (HD) is an inherited complex progressive neurodegenerative disorder with an established etiopathology linked to neuronal oxidative stress and corticostriatal excitotoxicity. Present study explores the effects of glucose-dependent insulinotropic polypeptide (GIP) receptor agonist on the neurobehavioral sequelae of quinolinic acid-induced phenotype of Huntington's disease in rats. Bilateral administration of quinolinic acid (300 nmol/4 µl) to the rat striatum led to characteristic deficits in, locomotor activity, motor coordination, neuromuscular coordination and short-term episodic memory. Therapeutic treatment for 14 days with a stable and brain penetrating GIP receptor agonist, D-Ala2GIP (100 nmol/kg, i.p.), attenuated the neurobehavioral deficits due to quinolinic acid (QA) administration. Protective actions of D-Ala2GIP were sensitive to blockade with a GIP receptor antagonist, (Pro3)GIP (50 nmol/kg, i.p.), indicating specific involvement of GIP receptor signaling pathway. Stimulation of GIP receptor with D-Ala2GIP attenuated lipid peroxidation, evidenced by reduced levels of brain malondialdehyde (MDA), and restoration of reduced glutathione (GSH) levels in brain. Quinolinic acid administration led to significant loss of striatal monoamines, e.g., norepinephrine, epinephrine, serotonin, dopamine, and metabolites, 3,4-Dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-Hydroxyindoleacetic acid (5-HIAA). D-Ala2GIP attenuated the QA-induced depletion of striatal monoamines, without affecting the monoamine degradation pathways. Thus, observed effects with D-Ala2GIP in the QA-induced Huntington's disease model could be attributable to reduction in lipid peroxidation, restoration of endogenous antioxidants and decreased striatal monoamine levels. These findings together suggest that stimulation of GIP receptor signaling pathway in brain could be a potential therapeutic strategy in the symptomatic management of Huntington's disease.


Asunto(s)
Conducta Animal/efectos de los fármacos , Monoaminas Biogénicas/metabolismo , Polipéptido Inhibidor Gástrico/farmacología , Enfermedad de Huntington/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Neostriado/metabolismo , Receptores de la Hormona Gastrointestinal/agonistas , Animales , Peso Corporal/efectos de los fármacos , Modelos Animales de Enfermedad , Interacciones Farmacológicas , Marcha/efectos de los fármacos , Polipéptido Inhibidor Gástrico/química , Glutatión/metabolismo , Fuerza de la Mano , Enfermedad de Huntington/inducido químicamente , Enfermedad de Huntington/fisiopatología , Locomoción/efectos de los fármacos , Masculino , Malondialdehído/metabolismo , Memoria Episódica , Neostriado/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Ácido Quinolínico/efectos adversos , Ratas , Ratas Wistar
14.
Sci Rep ; 8(1): 2948, 2018 02 13.
Artículo en Inglés | MEDLINE | ID: mdl-29440684

RESUMEN

Somatostatin receptor targeting is considered the standard nuclear medicine technique for visualization of neuroendocrine tumors (NET). Since not all NETs over-express somatostatin receptors, the search for novel targets, visualizing these NETs, is ongoing. Many NETs, expressing low somatostatin receptor levels, express glucose-dependent insulinotropic polypeptide (GIP) receptors (GIPR). Here, we evaluated the performance of [Lys37(DTPA)]N-acetyl-GIP1-42, a newly synthesized GIP analogue to investigate whether NET imaging via GIPR targeting is feasible. Therefore, [Lys37(DTPA)]N-acetyl-GIP1-42 was radiolabeled with 111In with specific activity up to 1.2 TBq/µmol and both in vitro and in vivo receptor targeting properties were examined. In vitro, [Lys37(111In-DTPA)]N-acetyl-GIP1-42 showed receptor-mediated binding to BHK-GIPR positive cells, NES2Y cells and isolated islets. In vivo, both NES2Y and GIPR-transfected BHK tumors were visualized on SPECT/CT. Furthermore, co-administration of an excess unlabeled GIP1-42 lowered tracer uptake from 0.7 ± 0.2%ID/g to 0.6 ± 0.01%ID/g (p = 0.78) in NES2Y tumors and significantly lowered tracer uptake from 3.3 ± 0.8 to 0.8 ± 0.2%ID/g (p = 0.0001) in GIPR-transfected BHK tumors. In conclusion, [Lys37(111In-DTPA)]N-acetyl-GIP1-42 shows receptor-mediated binding in various models. Furthermore, both GIPR-transfected BHK tumors and NES2Y tumors were visible on SPECT/CT using this tracer. Therefore, [Lys37(111In-DTPA)]N-acetyl-GIP1-42 SPECT seems promising for visualization of somatostatin receptor negative NETs.


Asunto(s)
Polipéptido Inhibidor Gástrico/química , Radioisótopos de Indio/química , Tumores Neuroendocrinos/diagnóstico por imagen , Animales , Línea Celular Tumoral , Estabilidad de Medicamentos , Femenino , Polipéptido Inhibidor Gástrico/metabolismo , Polipéptido Inhibidor Gástrico/farmacocinética , Humanos , Marcaje Isotópico , Cinética , Ratones , Tumores Neuroendocrinos/metabolismo , Tumores Neuroendocrinos/patología , Transporte de Proteínas , Trazadores Radiactivos , Tomografía Computarizada por Tomografía Computarizada de Emisión de Fotón Único , Distribución Tisular
15.
Peptides ; 100: 202-211, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29412820

RESUMEN

Combined modulation of peptide hormone receptors including, glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and xenin, have established benefits for the treatment of diabetes. The present study has assessed the biological actions and therapeutic efficacy of a novel exendin-4/xenin-8-Gln hybrid peptide, both alone and in combination with the GIP receptor agonist (DAla2)GIP. Exendin-4/xenin-8-Gln was enzymatically stable and exhibited enhanced insulin secretory actions when compared to its parent peptides. Exendin-4/xenin-8-Gln also possessed ability to potentiate the in vitro actions of GIP. Acute administration of exendin-4/xenin-8-Gln in mice induced appetite suppressive effects, as well as significant and protracted glucose-lowering and insulin secretory actions. Twice daily administration of exendin-4/xenin-8-Gln, alone or in combination with (DAla2)GIP, for 21-days significantly reduced non-fasting glucose and increased circulating insulin levels in high fat fed mice. In addition, all exendin-4/xenin-8-Gln treated mice displayed improved glucose tolerance, insulin sensitivity and metabolic responses to GIP. Combination therapy with (DAla2)GIP did not result in any obvious further benefits. Metabolic improvements in all treatment groups were accompanied by reduced pancreatic beta-cell area and insulin content, suggesting reduced insulin demand. Interestingly, body weight, food intake, circulating glucagon, metabolic rate and amylase activity were unaltered by the treatment regimens. However, all treatment groups, barring (DAla2)GIP alone, exhibited marked reductions in total- and LDL-cholesterol. Furthermore, exendin-4 therapy also reduced circulating triacylglycerol. This study highlights the positive antidiabetic effects of exendin-4/xenin-8-Gln, and suggests that combined modulation of GLP-1 and xenin related signalling pathways represents an exciting treatment option for type 2 diabetes.


Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Polipéptido Inhibidor Gástrico/administración & dosificación , Péptido 1 Similar al Glucagón/administración & dosificación , Hipoglucemiantes/administración & dosificación , Animales , Glucemia/efectos de los fármacos , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Combinación de Medicamentos , Exenatida/administración & dosificación , Exenatida/química , Polipéptido Inhibidor Gástrico/química , Glucagón/química , Glucagón/metabolismo , Péptido 1 Similar al Glucagón/química , Glucosa/metabolismo , Humanos , Hipoglucemiantes/química , Insulina/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Ratones , Neurotensina/administración & dosificación , Neurotensina/química
16.
Peptides ; 100: 219-228, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29412822

RESUMEN

Nine structurally modified apelin-13 analogues were assessed for their in vitro and acute in vivo antidiabetic potential. Stability was assessed in mouse plasma and insulinotropic efficacy tested in cultured pancreatic BRIN-BD11 cells and isolated mouse pancreatic islets. Intracellular Ca2+ and cAMP production in BRIN-BD11 cells was determined, as was glucose uptake in 3T3-L1 adipocytes. Acute antihyperglycemic effects of apelin analogues were assessed following i.p. glucose tolerance tests (ipGGT, 18 mmol/kg) in normal and diet-induced-obese (DIO) mice and on food intake in normal mice. Apelin analogues all showed enhanced in vitro stability (up to 5.8-fold, t½â€¯= 12.8 h) in mouse plasma compared to native apelin-13 (t½â€¯= 2.1 h). Compared to glucose controls, stable analogues exhibited enhanced insulinotropic responses from BRIN-BD11 cells (up to 4.7-fold, p < 0.001) and isolated mouse islets (up to 5.3-fold) for 10-7 M apelin-13 amide (versus 7.6-fold for 10-7 M GLP-1). Activation of APJ receptors on BRIN-BD11 cells increased intracellular Ca2+ (up to 3.0-fold, p < 0.001) and cAMP (up to 1.7-fold, p < 0.01). Acute ipGTT showed improved insulinotropic and glucose disposal responses in normal and DIO mice (p < 0.05 and p < 0.01, respectively). Apelin-13 amide and (pGlu)apelin-13 amide were the most effective analogues exhibiting acute, dose-dependent and persistent biological actions. Both analogues stimulated insulin-independent glucose uptake by differentiated adipocytes (2.9-3.3-fold, p < 0.05) and inhibited food intake (26-33%, p < 0.001), up to 180 min in mice, versus saline. In contrast, (Ala13)apelin-13 and (Val13)apelin-13 inhibited insulin secretion, suppressed beta-cell signal transduction and stimulated food intake in mice. Thus, stable analogues of apelin-13 have potential for diabetes/obesity therapy.


Asunto(s)
Hipoglucemiantes/administración & dosificación , Insulina/metabolismo , Péptidos y Proteínas de Señalización Intercelular/administración & dosificación , Obesidad/tratamiento farmacológico , Células 3T3-L1 , Animales , Glucemia/efectos de los fármacos , AMP Cíclico/metabolismo , Polipéptido Inhibidor Gástrico/química , Glucagón/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Glucosa/metabolismo , Prueba de Tolerancia a la Glucosa , Humanos , Hipoglucemiantes/química , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Péptidos y Proteínas de Señalización Intercelular/química , Ratones , Ratones Obesos , Obesidad/metabolismo , Obesidad/patología , Ratas , Receptores de Glucagón/metabolismo
17.
Curr Pharm Biotechnol ; 18(10): 840-848, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29205117

RESUMEN

BACKGROUND: Glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP) play a similar but complementary role in the regulation of glucose levels in islet ß-cells. This study was aimed to develop a fusion peptide, which combines 4 tandem repeated GLP-1 and 4 tandem repeated GIP (4rolGG), and to investigate its therapeutic effect on type 2 diabetes using a diabetic mice model. METHODS: A 4rolGG expression plasmid was constructed and expressed in BL21 (DE3). By inducting with IPTG, 4rolGG was expressed at a high level, which was confirmed by SDS-PAGE electrophoresis and Western Blotting. Subsequently, 4rolGG was purified by Ni-NTA affinity chromatography and the purity of 4rolGG was up to 90%. RESULT AND CONCLUSION: After oral administration of 4rolGG for 4 weeks, streptozotocin-induced diabetic mice showed a dramatic reduction in the levels of plasma glucose, GHbA1C, TC and TG, while the insulin levels were increased significantly.


Asunto(s)
Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Polipéptido Inhibidor Gástrico/química , Péptido 1 Similar al Glucagón/química , Fragmentos de Péptidos/uso terapéutico , Administración Oral , Secuencia de Aminoácidos , Animales , Glucemia/análisis , Polipéptido Inhibidor Gástrico/genética , Péptido 1 Similar al Glucagón/genética , Humanos , Insulina/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Plásmidos , Ingeniería de Proteínas
18.
J Am Chem Soc ; 139(46): 16688-16695, 2017 11 22.
Artículo en Inglés | MEDLINE | ID: mdl-29130686

RESUMEN

Peptide hormones are attractive as injectable therapeutics and imaging agents, but they often require extensive modification by mutagenesis and/or chemical synthesis to prevent rapid in vivo degradation. Alternatively, the single-atom, O-to-S modification of peptide backbone thioamidation has the potential to selectively perturb interactions with proteases while preserving interactions with other proteins, such as target receptors. Here, we use the validated diabetes therapeutic, glucagon-like peptide-1 (GLP-1), and the target of clinical investigation, gastric inhibitory polypeptide (GIP), as proof-of-principle peptides to demonstrate the value of thioamide substitution. In GLP-1 and GIP, a single thioamide near the scissile bond renders these peptides up to 750-fold more stable than the corresponding oxopeptides toward cleavage by dipeptidyl peptidase 4, the principal regulator of their in vivo stability. These stabilized analogues are nearly equipotent with their parent peptide in cyclic AMP activation assays, but the GLP-1 thiopeptides have much lower ß-arrestin potency, making them novel agonists with altered signaling bias. Initial tests show that a thioamide GLP-1 analogue is biologically active in rats, with an in vivo potency for glycemic control surpassing that of native GLP-1. Taken together, these experiments demonstrate the potential for thioamides to modulate specific protein interactions to increase proteolytic stability or tune activation of different signaling pathways.


Asunto(s)
Polipéptido Inhibidor Gástrico/química , Péptido 1 Similar al Glucagón/química , Tioamidas/química , Polipéptido Inhibidor Gástrico/uso terapéutico , Péptido 1 Similar al Glucagón/uso terapéutico , Estabilidad Proteica , Proteolisis
19.
Eur J Pharmacol ; 804: 38-45, 2017 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-28366809

RESUMEN

The aim of the present study was to evaluate the ability of D-Ala2GIP, a gastric inhibitory polypeptide (GIP) receptor agonist, to attenuate the behavioral phenotype of Parkinson's disease caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration in mice. In the behavioral studies, MPTP administration led to spontaneous locomotor activity deficits, impaired rotarod performance, akinesia, muscular rigidity and increased tremor amplitude, which was attenuated by pretreatment with D-Ala2GIP (50-100 nmol/kg, i.p.). This acute neuroprotective response by D-Ala2GIP was found to be blocked by a selective GIP receptor antagonist, (Pro3)GIP (50 nmol/kg, i.p.), indicating that the observed effects are mediated through GIP receptor mediated signaling pathway. Biochemical studies revealed that D-Ala2GIP reduced the brain malondialdehyde levels and enhanced the brain glutathione levels, thereby mitigating the MPTP-induced oxidative stress. MPTP administration resulted in reduction of the striatal concentration of dopamine and its metabolites, homovanillic acid (HVA) and 3, 4-Dihydroxyphenylacetic acid (DOPAC). Pretreatment with D-Ala2GIP attenuated the loss of striatal dopamine levels without affecting the normal dopamine catabolism. Thus, the observed effects in the MPTP-induced Parkinsonism model could be in part attributable to the antioxidant properties of D-Ala2GIP and enhanced turnover of dopamine in the nigrostriatal pathways in mouse brain. These findings together suggest that GIP receptor could be a therapeutic target in the management of symptoms of Parkinson's disease.


Asunto(s)
1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Polipéptido Inhibidor Gástrico/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Receptores de la Hormona Gastrointestinal/agonistas , Ácido 3,4-Dihidroxifenilacético/metabolismo , Animales , Dopamina/metabolismo , Polipéptido Inhibidor Gástrico/química , Polipéptido Inhibidor Gástrico/uso terapéutico , Glutatión/metabolismo , Ácido Homovanílico/metabolismo , Locomoción/efectos de los fármacos , Masculino , Malondialdehído/metabolismo , Ratones , Ratones Endogámicos C57BL , Neostriado/efectos de los fármacos , Neostriado/metabolismo , Neostriado/patología , Neuronas/metabolismo , Fármacos Neuroprotectores/uso terapéutico , Temblor/tratamiento farmacológico
20.
Biochem Pharmacol ; 131: 78-88, 2017 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-28237651

RESUMEN

Alternative processing of the precursor protein pro-GIP results in endogenously produced GIP(1-30)NH2, that by DPP-4 cleavage in vivo results in the metabolite GIP(3-30)NH2. We showed previously that GIP(3-30)NH2 is a high affinity antagonist of the human GIPR in vitro. Here we determine whether it is suitable for studies of GIP physiology in rats since effects of GIP agonists and antagonists are strictly species-dependent. Transiently transfected COS-7 cells were assessed for cAMP accumulation upon ligand stimulation or assayed in competition binding using human 125I-GIP(1-42) as radioligand. In isolated perfused rat pancreata, insulin, glucagon, and somatostatin-releasing properties were evaluated. Competition binding demonstrated that on the rat GIP receptor (GIPR), rat GIP(3-30)NH2 bound with high affinity (Ki of 17nM), in contrast to human GIP(3-30)NH2 (Ki of 250nM). In cAMP studies, rat GIP(3-30)NH2 inhibited GIP(1-42)-induced rat GIPR activation and schild-plot analysis showed competitive antagonism with a pA2 of 13nM and a slope of 0.9±0.09. Alone, rat GIP(3-30)NH2 displayed weak, low-potent partial agonistic properties (EC50>1µM) with an efficacy of 9.4% at 0.32µM compared to GIP(1-42). In perfused rat pancreata, rat GIP(3-30)NH2 efficiently antagonized rat GIP(1-42)-induced insulin, somatostatin, and glucagon secretion. In summary, rat GIP(3-30)NH2 is a high affinity competitive GIPR antagonist and effectively antagonizes GIP-mediated G protein-signaling as well as pancreatic hormone release, while human GIP(3-30)NH2, despite a difference of only one amino acid between the two (arginine in position 18 in rat GIP(3-30)NH2; histidine in human), is unsuitable in the rat system. This underlines the importance of species differences in the GIP system, and the limitations of testing human peptides in rodent systems.


Asunto(s)
Polipéptido Inhibidor Gástrico/fisiología , Glucagón/metabolismo , Insulina/metabolismo , Fragmentos de Péptidos/farmacología , Receptores de la Hormona Gastrointestinal/antagonistas & inhibidores , Somatostatina/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Polipéptido Inhibidor Gástrico/química , Polipéptido Inhibidor Gástrico/farmacología , Humanos , Secreción de Insulina , Masculino , Fragmentos de Péptidos/química , Fragmentos de Péptidos/fisiología , Ratas , Ratas Wistar , Homología de Secuencia de Aminoácido
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