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 3 h at room temperature or on ice, treated with various enzyme inhibitors, after up to three thawing–refreezing cycles, and after storage at −20 and −80 °C for up to 1 year. Recoveries were unaffected by freezing cycles or if plasma was stored on ice for up to 3 h, but were impaired when samples stood at RT for more than 1 h. 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.
Nicolai J Wewer Albrechtsen, Monika J Bak, Bolette Hartmann, Louise Wulff Christensen, Rune E Kuhre, Carolyn F Deacon, and Jens J Holst
Justyna Modrzynska, Christine F Klein, Kasper Iversen, Henning Bundgaard, Bolette Hartmann, Maike Moss, Nikolaj Rittig, Niels Moeller, Jens J Holst, and Nicolai J Wewer Albrechtsen
Objective: Glucagon and glucagon-like peptide-1 (GLP-1) originate from the common precursor, proglucagon, and their plasma concentrations have been reported to be increased during inflammatory conditions. Increased blood glucose levels are frequently observed in septic patients, and therefore we hypothesized that glucagon, but not GLP-1, is increased in individuals with inflammation.
Design: Prospective longitudinal cohort study.
Materials and Methods: We measured glucagon and GLP-1 in plasma sampled consecutively in three cohorts consisting of patients with infective endocarditis (n=16), urosepsis (n=28) and post-operative inflammation following percutaneous aortic valve implantation or thoracic endovascular aortic repair (n=5). Correlations between C-Reactive Protein (CRP), a marker of systemic inflammation, and glucagon and GLP-1 concentrations were investigated. Additionally, glucagon and GLP-1 concentrations were measured after a bolus infusion of lipopolysaccharide (LPS, 1ng/kg) in nine healthy young males.
Results: Glucagon and CRP were positively and significantly correlated (r=0.27; P=0.0003), whereas no significant association between GLP-1 and CRP was found (r=0.08, P=0.30). LPS infusion resulted in acute systemic inflammation reflected by increased temperature, pulse, tumor necrosis factor-α (TNFα), interleukin-6 (IL-6) and concomitantly increased concentrations of glucagon (P<0.05) but not GLP-1.
Conclusions :Systemic inflammation caused by bacterial infections or developed as a non-infected condition is associated with increased plasma concentration of glucagon, but not GLP-1. Hyperglucagonemia may contribute to the impaired glucose control in patients with systemic inflammatory diseases.
Marie Reeberg Sass, Nicolai Jacob Wewer Albrechtsen, Jens Pedersen, Kristine Juul Hare, Nis Borbye-Lorenzen, Katalin Kiss, Tina Vilsbøll, Filip Krag Knop, Steen Seier Poulsen, Niklas Rye Jørgensen, Jens Juul Holst, Cathrine Ørskov, and Bolette Hartmann
Parathyroid hormone (PTH) is a key hormone in regulation of calcium homeostasis and its secretion is regulated by calcium. Secretion of PTH is attenuated during intake of nutrients, but the underlying mechanism(s) are unknown. We hypothesized that insulin acts as an acute regulator of PTH secretion.
Intact PTH was measured in plasma from patients with T1D and matched healthy individuals during 4-h oral glucose tolerance tests (OGTT) and isoglycemic i.v. glucose infusions on 2 separate days. In addition, expression of insulin receptors on surgical specimens of parathyroid glands was assessed by immunochemistry (IHC) and quantitative PCR (qPCR).
The inhibition of PTH secretion was more pronounced in healthy individuals compared to patients with T1D during an OGTT (decrementalAUC0–240min: −5256 ± 3954 min × ng/L and −2408 ± 1435 min × ng/L, P = 0.030). Insulin levels correlated significantly and inversely with PTH levels, also after adjusting for levels of several gut hormones and BMI (P = 0.002). Expression of insulin receptors in human parathyroid glands was detected by both IHC and qPCR.
Our study suggests that insulin may act as an acute regulator of PTH secretion in humans.
Amalie R Lanng, Lærke S Gasbjerg, Natasha C Bergmann, Sigrid Bergmann, Mads M Helsted, Matthew P Gillum, Bolette Hartmann, Jens J Holst, Tina Vilsbøll, and Filip K Knop
Ingestion of the calorically dense compound alcohol may cause metabolic disturbances including hypoglycaemia, hepatic steatosis and insulin resistance, but the underlying mechanisms are uncertain. The gastrointestinal tract is well recognised as a major influencer on glucose, protein and lipid metabolism, but its role in alcohol metabolism remains unclear.
To examine the effects of oral and intravenous alcohol, respectively, on plasma concentrations of several gluco-regulatory hormones including serum/plasma insulin, C-peptide, glucagon, glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide 1 (GLP-1) and fibroblast growth factor 21 (FGF21).
Design and methods
In a double-blinded, randomised, crossover design, we subjected 12 healthy men to intragastric ethanol infusion (IGEI) and an isoethanolaemic intravenous ethanol infusion (IVEI) (0.7 g alcohol per kg body weight), respectively, on two separate experimental days.
Isoethanolaemia during the two alcohol administration forms was obtained (P = 0.38). During both interventions, plasma glucose peaked after ~30 min and thereafter fell below baseline concentrations. GIP and GLP-1 concentrations were unaffected by the two interventions. Insulin concentrations were unaffected by IGEI but decreased during IVEI. C-peptide, insulin secretion rate and glucagon concentrations were lowered similarly during IGEI and IVEI. FGF21 concentrations increased dramatically (nine-fold) and similarly during IGEI and IVEI.
Alcohol does not seem to affect the secretion of incretin hormones but decreased insulin and glucagon secretion independently of gut-derived factors. IGEI as well as IVEI potently stimulate FGF21 secretion indicating a gut-independent effect of alcohol on FGF21 secretion in humans.