RESUMEN
BACKGROUND/OBJECTIVES: The results of short-term studies in humans suggest that, compared with glucose, acute consumption of fructose leads to increased postprandial energy expenditure and carbohydrate oxidation and decreased postprandial fat oxidation. The objective of this study was to determine the potential effects of increased fructose consumption compared with isocaloric glucose consumption on substrate utilization and energy expenditure following sustained consumption and under energy-balanced conditions. SUBJECTS/METHODS: As part of a parallel arm study, overweight/obese male and female subjects, 40-72 years, consumed glucose- or fructose-sweetened beverages providing 25% of energy requirements for 10 weeks. Energy expenditure and substrate utilization were assessed using indirect calorimetry at baseline and during the 10th week of intervention. RESULTS: Consumption of fructose, but not glucose, led to significant decreases of net postprandial fat oxidation and significant increases of net postprandial carbohydrate oxidation (P<0.0001 for both). Resting energy expenditure (REE) decreased significantly from baseline values in subjects consuming fructose (P=0.031) but not in those consuming glucose. CONCLUSIONS: Increased consumption of fructose for 10 weeks leads to marked changes of postprandial substrate utilization including a significant reduction of net fat oxidation. In addition, we report that REE is reduced compared with baseline values in subjects consuming fructose-sweetened beverages for 10 weeks.
Asunto(s)
Metabolismo Basal/efectos de los fármacos , Metabolismo de los Hidratos de Carbono/efectos de los fármacos , Sacarosa en la Dieta/farmacología , Fructosa/farmacología , Glucosa/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Obesidad/metabolismo , Anciano , Bebidas , Ingestión de Energía , Metabolismo Energético/efectos de los fármacos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Oxidación-Reducción , Edulcorantes/farmacologíaAsunto(s)
Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/genética , Hiperglucemia/etiología , Niño , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 2/complicaciones , Diagnóstico Diferencial , Femenino , Proteínas de Homeodominio/genética , Humanos , Transactivadores/genéticaRESUMEN
Neurokinin A (NKA) is a tachykinin peptide that binds with high affinity to the tachykinin NK(2) receptor. Recent homologous binding studies, however, have shown that neurokinin A is also a high-affinity ligand for the tachykinin NK(1) receptor. In this report, we demonstrate that a photoreactive neurokinin A analogue specifically labels the NK(1) receptor in rat submandibular gland membranes and show via bioassay that neurokinin A is a potent stimulator of salivary secretion. Through the use of specific non-peptide antagonists in both photolabeling and functional assays, we unequivocally demonstrate that neurokinin A can specifically interact with the NK(1) receptor in vivo and elicit NK(1) receptor-mediated physiological responses.
Asunto(s)
Neuroquinina A/metabolismo , Receptores de Neuroquinina-1/metabolismo , Glándula Submandibular/metabolismo , Animales , Unión Competitiva , Indoles/farmacología , Radioisótopos de Yodo , Isoindoles , Membranas/efectos de los fármacos , Membranas/metabolismo , Neuroquinina A/farmacología , Antagonistas del Receptor de Neuroquinina-1 , Fenilalanina/análogos & derivados , Fenilalanina/metabolismo , Etiquetas de Fotoafinidad/metabolismo , Ratas , Ratas Sprague-Dawley , Salivación/efectos de los fármacos , Glándula Submandibular/efectos de los fármacos , Sustancia P/farmacologíaRESUMEN
Substance P (SP) belongs to the tachykinin family of bioactive peptides and exerts its many biological effects through functional interaction with its cell-surface, G protein-coupled neurokinin-1 receptor (NK-1R). Previous studies from our laboratory have shown that (125)I-Bolton-Hunter reagent-labeled p-benzoylphenylalanine(8)-SP (Bpa(8)SP) covalently attaches to Met(181), whereas (125)I-Bolton-Hunter reagent-labeled Bpa(4)SP covalently attaches to Met(174), both of which are located on the second extracellular loop (EC2) of the NK-1R. In this study, evidence has been obtained that at equilibrium, the photoreactive SP analogue (125)I-[D-Tyr(0)]Bpa(3)SP covalently labels residues in two distinct extracellular regions of the NK-1R. One site of (125)I-[D-Tyr(0)]Bpa(3)SP photoinsertion is located on EC2 within a segment of the receptor extending from residues 173 to 177; a second site of (125)I-[D-Tyr(0)]Bpa(3)SP photoinsertion is located on the extracellular N terminus within a segment of the receptor extending from residues 11 to 21, a sequence that contains both potential sites for N-linked glycosylation. Since competition binding data presented in this study do not suggest the existence of multiple peptide.NK-1R complexes, it is reasonable to assume that the receptor sequences within EC2 and N terminus identified by peptide mapping are in close proximity in the equilibrium complex.
Asunto(s)
Fenilalanina/análogos & derivados , Fenilalanina/metabolismo , Receptores de Neuroquinina-1/metabolismo , Sustancia P/metabolismo , Animales , Células CHO , Cricetinae , Hidrólisis , Mapeo Peptídico , Etiquetas de Fotoafinidad , Unión Proteica , Ratas , Receptores de Neuroquinina-1/química , Tripsina/metabolismoRESUMEN
Molecular models for the interaction of substance P (SP) with its G protein-coupled receptor, the neurokinin-1 receptor (NK-1R), have been developed. The ligand.receptor complex is based on experimental data from a series of photoaffinity labeling experiments and spectroscopic structural studies of extracellular domains of the NK-1R. Using the ligand/receptor contact points derived from incorporation of photolabile probes (p-benzoylphenylalanine (Bpa)) into SP at positions 3, 4, and 8 and molecular dynamics simulations, the topological arrangement of SP within the NK-1R is explored. The model incorporates the structural features, determined by high resolution NMR studies, of the second extracellular loop (EC2), containing contact points Met(174) and Met(181), providing important experimentally based conformational preferences for the simulations. Extensive molecular dynamics simulations were carried out to probe the nature of the two contact points identified for the Bpa(3)SP analogue (Bremer, A. A., Leeman, S. E., and Boyd, N. D. (2001) J. Biol. Chem. 276, 22857-22861), examining modes of ligand binding in which the contact points are fulfilled sequentially or simultaneously. The resulting ligand.receptor complex has the N terminus of SP projecting toward transmembrane helix (TM) 1 and TM2, exposed to the solvent. The C terminus of SP is located in proximity to TM5 and TM6, deeper into the central core of the receptor. The central portion of the ligand, adopting a helical loop conformation, is found to align with the helices of the central regions EC2 and EC3, forming important interactions with both of these extracellular domains. The model developed here allows for atomic insight into the biochemical data currently available and guides targeting of future experiments to probe specific ligand/receptor interactions and thereby furthers our understanding of the functioning of this important neuropeptide system.
Asunto(s)
Modelos Moleculares , Receptores de Neuroquinina-1/metabolismo , Sustancia P/metabolismo , Humanos , Etiquetas de Fotoafinidad , Conformación Proteica , Receptores de Neuroquinina-1/química , Sustancia P/químicaRESUMEN
Although neurokinin A (NKA), a tachykinin peptide with sequence homology to substance P (SP), is a weak competitor of radiolabeled SP binding to the NK-1 receptor (NK-1R), more recent direct binding studies using radiolabeled NKA have demonstrated an unexpected high-affinity interaction with this receptor. To document the site of interaction between NKA and the NK-1R, we have used a photoreactive analogue of NKA containing p-benzoyl-L-phenylalanine (Bpa) substituted in position 7 of the peptide. Peptide mapping studies of the receptor photolabeled by (125)I-iodohistidyl(1)-Bpa(7)NKA have established that the site of photoinsertion is located within a segment of the receptor extending from residues 178 to 190 (VVCMIEWPEHPNR). We have previously shown that (125)I-BH-Bpa(8)SP, a photoreactive analogue of SP, covalently attaches to M(181) within this same receptor sequence. Importantly, both of these peptides ((125)I-iodohistidyl(1)-Bpa(7)NKA and (125)I-BH-Bpa(8)SP) have the photoreactive amino acid in an equivalent position within the conserved tachykinin carboxyl-terminal tail. In this report, we also show that site-directed mutagenesis of M(181) to A(181) in the NK-1R results in a complete loss of photolabeling of both peptides to this receptor site, indicating that the equivalent position of SP and NKA, when bound to the NK-1R, contact the same residue.