RESUMEN
BACKGROUND: Proglucagon-derived peptides (PGDPs), which include glucagon-like peptide (GLP)-1, glucagon, and oxyntomodulin, are key regulators of glucose homeostasis and satiety. These peptide hormones are typically measured with immuno-based assays (e.g., ELISA, RIA), which often suffer from issues of selectivity. METHODS: We developed a multiplexed assay for measuring PGDPs including GLP-1 (7-36) amide, GLP-1 (9-36) amide, glucagon, and oxyntomodulin by mass spectrometry and used this assay to examine the effect of a meal tolerance test on circulating concentrations of these hormones. Participants fasted overnight and were either given a meal (n = 8) or continued to fast (n = 4), with multiple blood collections over the course of 3 h. Plasma samples were analyzed by microflow immunoaffinity (IA)-LC-MS/MS with an isotope dilution strategy. RESULTS: Assay performance characteristics were examined and established during analytical validation for all peptides. Intra- and interassay imprecision were found to be 2.2%-10.7% and 6.8%-22.5%, respectively. Spike recovery was >76%, and dilution linearity was established up to a 16-fold dilution. Immediately after the meal tolerance test, GLP-1 and oxyntomodulin concentrations increased and had an almost identical temporal relationship, and glucagon concentrations increased with a slight delay. CONCLUSIONS: IA-LC-MS/MS was used for the simultaneous and selective measurement of PGDPs. This work includes the first indication of the physiological concentrations and modulation of oxyntomodulin after a meal.
Asunto(s)
Ayuno , Péptido 1 Similar al Glucagón/sangre , Glucagón/sangre , Inmunoensayo , Oxintomodulina/sangre , Espectrometría de Masas en Tándem , Animales , Cromatografía Líquida de Alta Presión , Glucagón/inmunología , Péptido 1 Similar al Glucagón/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Oxintomodulina/inmunologíaRESUMEN
Dietary advanced glycation end products (AGE) formed during heating of food have gained interest as potential nutritional toxins with adverse effects on inflammation and glucose metabolism. In the present study, we investigated the short-term effects of high and low molecular weight (HMW and LMW) dietary AGE on insulin sensitivity, expression of the receptor for AGE (RAGE), the AGE receptor 1 (AGER1) and TNF-α, F2-isoprostaglandins, body composition and food intake. For 2 weeks, thirty-six Sprague-Dawley rats were fed a diet containing 20% milk powder with different proportions of this being given as heated milk powder (0, 40 or 100%), either native (HMW) or hydrolysed (LMW). Gene expression of RAGE and AGER1 in whole blood increased in the group receiving a high AGE LMW diet, which also had the highest urinary excretion of the AGE, methylglyoxal-derived hydroimidazolone 1 (MG-H1). Urinary excretion of N ε-carboxymethyl-lysine increased with increasing proportion of heat-treated milk powder in the HMW and LMW diets but was unrelated to gene expression. There was no difference in insulin sensitivity, F2-isoprostaglandins, food intake, water intake, body weight or body composition between the groups. In conclusion, RAGE and AGER1 expression can be influenced by a high AGE diet after only 2 weeks in proportion to MG-H1 excretion. No other short-term effects were observed.
Asunto(s)
Dieta/efectos adversos , Productos Finales de Glicación Avanzada/efectos adversos , Hexosiltransferasas/metabolismo , Receptor para Productos Finales de Glicación Avanzada/agonistas , Regulación hacia Arriba , Animales , Biomarcadores/sangre , Biomarcadores/orina , Ingestión de Energía , Productos Finales de Glicación Avanzada/administración & dosificación , Productos Finales de Glicación Avanzada/química , Productos Finales de Glicación Avanzada/orina , Hexosiltransferasas/sangre , Hexosiltransferasas/química , Hexosiltransferasas/genética , Calor/efectos adversos , Imidazoles/orina , Imidazolinas/orina , Lisina/análogos & derivados , Lisina/orina , Masculino , Proteínas de la Leche/administración & dosificación , Proteínas de la Leche/efectos adversos , Proteínas de la Leche/química , Peso Molecular , Proteolisis , Distribución Aleatoria , Ratas Sprague-Dawley , Receptor para Productos Finales de Glicación Avanzada/sangre , Receptor para Productos Finales de Glicación Avanzada/genética , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Eliminación Renal , Pruebas de Toxicidad Subaguda , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
PURPOSE: Advanced glycation end products (AGEs) formed in food during high-heat cooking may induce overeating and inflammation. We investigated whether AGE contents in a single meal affect postprandial appetite and markers of inflammation, endothelial activation, and oxidative stress. METHODS: In total, 19 healthy overweight individuals completed a crossover meal test with two meals of identical ingredients prepared by roasting (H-AGE) or steaming (L-AGE), respectively. Postprandial blood samples were analysed for N(ε)-carboxymethyl-lysine (CML), appetite-regulating gut hormones, glucose, insulin, triacylglycerol, and markers of inflammation and endothelial activation. Subjective appetite ratings and subsequent food intake were also assessed, and urine was analysed for CML, methylglyoxal-derived hydroimidazolone (MG-H1), and F2-isoprostanes. RESULTS: CML content of the H- and L-AGE meals was 5.0 and 2.8 mg, respectively. Plasma CML and urinary CML and MG-H1 tended to be higher after the H-AGE meal. There was no change in subsequent food intake, appetite sensations, or appetite hormone responses between meals, except for the overall ghrelin response, which was higher after the H-AGE meal compared with the L-AGE meal (p = 0.016). There was an increased glycaemic response to the H-AGE meal (p = 0.027) compared with the L-AGE meal. Inflammatory and endothelial activation markers did not differ between meals, but there was an overall effect on endothelial activation (p = 0.021) and on the oxidative marker, F2-isoprostanes, in urine (p = 0.013). CONCLUSION: The present study did not show any pronounced effects of AGEs on appetite and markers of inflammation, but did indicate that AGEs may affect postprandial ghrelin, oxidative stress, and glucose responses.
Asunto(s)
Apetito/efectos de los fármacos , Dieta , Endotelio/fisiología , Productos Finales de Glicación Avanzada/administración & dosificación , Inflamación , Sobrepeso/fisiopatología , Adulto , Glucemia/análisis , Índice de Masa Corporal , Estudios Cruzados , Endotelio/efectos de los fármacos , Ingestión de Energía , F2-Isoprostanos/orina , Femenino , Ghrelina/sangre , Péptido 1 Similar al Glucagón/sangre , Calor , Humanos , Insulina/sangre , Lisina/análogos & derivados , Lisina/sangre , Masculino , Persona de Mediana Edad , Estrés Oxidativo/efectos de los fármacos , Péptido YY/sangre , Periodo Posprandial , Vapor , Triglicéridos/sangreRESUMEN
To investigate the stability of glucagon-like peptide 1 (GLP-1) and glucagon in plasma under short- and long-term storage conditions. Pooled human plasma (n=20), to which a dipeptidyl peptidase 4 (DPP4) inhibitor and aprotinin were added, was spiked with synthetic GLP-1 (intact, 7-36NH2 as well as the primary metabolite, GLP-1 9-36NH2) or glucagon. Peptide recoveries were measured in samples kept for 1 and 3âh at room temperature or on ice, treated with various enzyme inhibitors, after up to three thawing-refreezing cycles, and after storage at -20 and -80â°C for up to 1 year. Recoveries were unaffected by freezing cycles or if plasma was stored on ice for up to 3âh, but were impaired when samples stood at RT for more than 1âh. Recovery of intact GLP-1 increased by addition of a DPP4 inhibitor (no ice), but was not further improved by neutral endopeptidase 24.11 inhibitor or an inhibitor cocktail. GLP-1, but not glucagon, was stable for at least 1 year. Surprisingly, the recovery of glucagon was reduced by almost 50% by freezing compared with immediate analysis, regardless of storage time. Plasma handling procedures can significantly influence results of subsequent hormone analysis. Our data support addition of DPP4 inhibitor for GLP-1 measurement as well as cooling on ice of both GLP-1 and glucagon. Freeze-thaw cycles did not significantly affect stability of GLP-1 or glucagon. Long-term storage may affect glucagon levels regardless of storage temperature and results should be interpreted with caution.
RESUMEN
AIM: To determine the specificity and sensitivity of assays carried out using commercially available kits for glucagon and/or oxyntomodulin measurements. METHODS: Ten different assay kits used for the measurement of either glucagon or oxyntomodulin concentrations were obtained. Solutions of synthetic glucagon (proglucagon (PG) residues 3361), oxyntomodulin (PG residues 3369) and glicentin (PG residues 169) were prepared and peptide concentrations were verified by quantitative amino acid analysis and a processing-independent in-house RIA. Peptides were added to the matrix (assay buffer) supplied with the kits (concentration range: 1.25-300âpmol/l) and to human plasma and recoveries were determined. Assays yielding meaningful results were analysed for precision and sensitivity by repeated analysis and ability to discriminate low concentrations. RESULTS AND CONCLUSION: Three assays were specific for glucagon (carried out using the Millipore (Billerica, MA, USA), Bio-Rad (Sundbyberg, Sweden), and ALPCO (Salem, NH, USA) and Yanaihara Institute (Shizuoka, Japan) kits), but none was specific for oxyntomodulin. The assay carried out using the Phoenix (Burlingame, CA, USA) glucagon kit measured the concentrations of all three peptides (total glucagon) equally. Sensitivity and precision were generally poor; the assay carried out using the Millipore RIA kit performed best with a sensitivity around 10âpmol/l. Assays carried out using the BlueGene (Shanghai, China), USCN LIFE (Wuhan, China) (oxyntomodulin and glucagon), MyBioSource (San Diego, CA, USA) and Phoenix oxyntomodulin kits yielded inconsistent results.