Regulation of metabolism and food intake by enteropancreatic hormones

Enteropancreatic hormones such as pancreatic polypeptide (PP), peptide YY (PYY) and glucagon-like peptide-1 (GLP-1) are secreted post-prandially by the gut and the pancreas. They act to regulate metabolism and appetite. An understanding of the physiology of these hormones and how they can be delivered in a practical manner is required to allow their translation into clinical treatments for obesity and diabetes. Work in this thesis investigated the effect of subcutaneously injected PP in healthy human volunteers, and demonstrated a significant reduction in food intake. A novel peptidase resistant analogue of PP, PP 1420, was administered in combination with metformin to rodents. This combination reduced food intake and body weight additively, suggesting that the combination of PP 1420 and metformin may well be beneficial in patients with obesity and diabetes. A subsequent study of PP 1420 in human volunteers, in a first-in-man Phase 1 trial, confirmed that PP 1420 was safe, well tolerated and possessed an extended terminal elimination half-life compared to native PP. In this thesis, I also explored the physiological effects of gut hormone combinations. The administration of single gut hormones such as PP, PYY or GLP-1 can reduce food intake, but may cause side effects such as nausea. The combination of gut hormones offers the possibility of increased efficacy with fewer side effects, for example, PYY+GLP-1 in combination have previously been shown to possess additive effects on food intake. The effects of a PYY+GLP-1 combination on carbohydrate metabolism have not yet been investigated. Work in this thesis examined the effects of a PYY+GLP-1 combination intravenous infusion on insulin secretion and sensitivity in healthy volunteers. Administration of PYY alone did not significantly affect insulin secretion. PYY+GLP-1 in combination stimulated insulin secretion to a similar extent to GLP-1 alone. There were no significant acute effects of PYY, GLP-1 or PYY+GLP-1 on insulin sensitivity. These findings suggest that gut hormone analogues may represent safe, effective and practical treatments for obesity. Combination PYY+GLP-1 treatment may provide the metabolic benefits of bariatric surgery without the surgery itself.Open Acces

Identification of plasma protease derived metabolites of glucagon and their formation under typical laboratory sample handling conditions

Copyright © 2014 John Wiley & Sons, Ltd. RATIONALE Glucagon modulates glucose production, and it is also a biomarker for several pathologies. It is known to be unstable in human plasma, and consequently stabilisers are often added to samples, although these are not particularly effective. Despite this, there have not been any studies to identify in vitro plasma protease derived metabolites; such a study is described here. Knowledge of metabolism should allow the development of more effective sample stabilisation strategies. METHODS Several novel metabolites resulting from the incubation of glucagon in human plasma were identified using high-resolution mass spectrometry with positive electrospray ionisation. Tandem mass spectrometric (MS/MS) scans were acquired for additional confirmation using a QTRAP. Separation was performed using reversed-phase ultra-high-performance liquid chromatography. The formation of these metabolites was investigated during a time-course experiment and under specific stress conditions representative of typical laboratory handling conditions. Clinical samples were also screened for metabolites. RESULTS Glucagon 3-29 and [pGlu] 3 glucagon 3-29 were the major metabolites detected, both of which were also present in clinical samples. We also identified two oxidised forms of [pGlu] 3 glucagon 3-29 as well as glucagon 19-29 , or 'miniglucagon', along with the novel metabolites glucagon 20-29 and glucagon 21-29 . The relative levels of these metabolites varied throughout the time-course experiment, and under the application of the different sample handling conditions. Aprotinin stabilisation of samples had negligible effect on metabolite formation. CONCLUSIONS Novel plasma protease metabolites of glucagon have been confirmed, and their formation characterised over a time-course experiment and under typical laboratory handling conditions. These metabolites could be monitored to assess the effectiveness of new sample stabilisation strategies, and further investigations into their formation could suggest specific enzyme inhibitors to use to increase sample stability. In addition the potential of the metabolites to affect immunochemistry-based assays as a result of cross-reactivity could be investigated

Development of a high-throughput UHPLC-MS/MS (SRM) method for the quantitation of endogenous glucagon from human plasma

© 2014 Future Science Ltd. Background: Published LC-MS/MS methods are not sensitive enough to quantify endogenous levels of glucagon. Results: An ultra high performance liquid chromatography-MS/MS (SRM) method for the quantitation of endogenous levels glucagon was successfully developed and qualified. A novel 2D extraction procedure was used to reduce matrix suppression, background noise and interferences. Glucagon levels in samples from healthy volunteers were found to agree with radioimmunoassay (RIA) derived literature values. Bland-Altman analysis showed a concentration-dependent positive bias of the LC/MS-MS assay versus an RIA. Both assays produced similar pharmacokinetic profiles, both of which were feasible considering the nature of the study. Conclusion: Our method is the first peer reviewed LC-MS/MS method for the quantitation of endogenous levels of glucagon, and offers a viable alternative to RIA-based approaches

Development of a UHPLC-MS/MS (SRM) method for the quantitation of endogenous glucagon and dosed GLP-1 from human plasma

© 2017 Future Science Ltd. Aim: The performance of glucagon and GLP-1 immunoassays is often poor, but few sensitive LC-MS/MS methods exist as alternatives. Experimental: A multiplexed LC-MS/MS method using a 2D extraction technique was developed. Results: The method was established for the quantitation of endogenous glucagon (LLOQ: 15 pg/ml) and dosed GLP-1 (LLOQ: 25 pg/ml) in human plasma, and is the first such method avoiding immunoenrichment. Specificity of endogenous glucagon quantitation was assured using a novel approach with a supercharging mobile phase additive to access a sensitive qualifier SRM. Endogenous glucagon concentrations were within the expected range, and showed good reproducibility after extended sample storage. A cross-validation against established immunoassays using physiological study samples demonstrated some similarities between methods. Conclusion: The LC-MS/MS method offers a viable alternative to immunoassays for quantitation of endogenous glucagon, dosed glucagon and/or dosed GLP-1

Early childhood respiratory morbidity and antibiotic use in ex-preterm infants: A primary care population-based cohort study

Background Globally, bronchopulmonary dysplasia (BPD) continues to increase in preterm infants. Recent studies exploring subsequent early childhood respiratory morbidity have been small or focused on hospital admissions.Primary aim Examine early childhood rates of primary care consultations for respiratory tract infections (RTI), lower respiratory tract infections (LRTI), wheeze and antibiotic prescriptions (Abx Px) in ex-preterm and term children. Secondary aim: examine differences between preterm infants discharged home with or without oxygen.Methods Retrospective cohort study using linked electronic primary care and hospital databases of children born between 1997 and 2014. We included 253 677 eligible children, with 1666 born preterm [less than] 32 weeks' gestation, followed up from primary care registration to age 5 years. Adjusted incidence rate ratios (aIRR) were calculated.Results Ex-preterm infants had higher rates of morbidity across all respiratory outcomes. After adjusting for confounders, aIRRs for RTI (1.37, 95% CI 1.33–1.42), LRTI (2.79, 95% CI 2.59–3.01), wheeze (3.05, 95% CI 2.64–3.52) and Abx Px (1.49, 95% CI 1.44–1.55) were higher for ex-preterm infants. Ex-preterm infants discharged home on oxygen had significantly greater morbidity across all respiratory diagnoses and Abx Px compared to those without home oxygen. The highest rates of respiratory morbidity were observed in children from the most deprived socioeconomic groups.Conclusion Ex-preterm infants, particularly those with BPD requiring home oxygen, have significant respiratory morbidity and antibiotic prescriptions in early childhood. With the increasing prevalence of BPD, further research should focus on strategies to reduce the burden of respiratory morbidity in these high-risk infants after hospital discharge

Determining the role of novel metabolic pathways in driving intracranial pressure reduction after weight loss

Idiopathic intracranial hypertension, a disease classically occurring in women with obesity, is characterised by raised intracranial pressure. Weight loss leads to reduction in intracranial pressure. Additionally, pharmacological glucagon-like peptide-1 agonism reduces cerebrospinal fluid secretion and intracranial pressure. The potential mechanisms by which weight loss reduces intracranial pressure are unknown and was the focus for this study.Meal stimulation tests (fasted plasma sample, then samples at 15, 30, 60, 90 and 120 minutes following a standardised meal) were conducted pre- and post-bariatric surgery (early (2 weeks) and late (12 months)) in patients with active idiopathic intracranial hypertension. Dynamic changes in gut neuropeptides (glucagon-like peptide-1, gastric inhibitory polypeptide, and ghrelin) and metabolites (untargeted ultra-high performance liquid chromatography-mass spectrometry) were evaluated. We determined the relationship between gut neuropeptides, metabolites, and intracranial pressure.18 idiopathic intracranial hypertension patients were included (Roux-En-Y gastric bypass n=7, gastric banding n=6, or sleeve gastrectomy n=5). At 2 weeks post-bariatric surgery, despite similar weight loss, Roux-En-Y gastric bypass had a two-fold (50%) greater reduction in intracranial pressure compared to sleeve. Increased meal stimulated glucagon-like peptide-1 secretion was observed after Roux-En-Y gastric bypass (+600 %) compared to sleeve (+319 %). There was no change in gastric inhibitory polypeptide and ghrelin. Dynamic changes in meal stimulated metabolites after bariatric surgery consistently identified changes in lipid metabolites, predominantly ceramides, glycerophospholipids and lysoglycerophospholipids, which correlated with intracranial pressure. A greater number of differential lipid metabolites were observed in the Roux-En-Y gastric bypass cohort at 2 weeks, and these also correlated with intracranial pressure.In idiopathic intracranial hypertension, we identified novel changes in lipid metabolites and meal stimulated glucagon-like peptide-1 levels following bariatric surgery which were associated with changes in intracranial pressure. Roux-En-Y gastric bypass was most effective at reducing intracranial pressure despite analogous weight loss to gastric sleeve at 2 weeks post-surgery and was associated with more pronounced changes in these metabolite pathways. We suggest that these novel perturbations in lipid metabolism and glucagon-like peptide-1 secretion are mechanistically important in driving reduction in intracranial pressure following weight loss in patients with idiopathic intracranial hypertension. Therapeutic targeting of these pathways, for example with glucagon-like peptide-1 agonist infusion, could represent a therapeutic strategy

Tripeptide gut hormone infusion does not alter food preferences or sweet taste function in volunteers with obesity and prediabetes/diabetes but promotes restraint eating: A secondary analysis of a randomized single‐blind placebo‐controlled study

Aims To investigate whether the elevation in postprandial concentrations of the gut hormones glucagon-like peptide-1 (GLP-1), oxyntomodulin (OXM) and peptide YY (PYY) accounts for the beneficial changes in food preferences, sweet taste function and eating behaviour after Roux-en-Y gastric bypass (RYGB). Materials and methods This was a secondary analysis of a randomized single-blind study in which we infused GLP-1, OXM, PYY (GOP) or 0.9% saline subcutaneously for 4 weeks in 24 subjects with obesity and prediabetes/diabetes, to replicate their peak postprandial concentrations, as measured at 1 month in a matched RYGB cohort (ClinicalTrials.gov NCT01945840). A 4-day food diary and validated eating behaviour questionnaires were completed. Sweet taste detection was measured using the method of constant stimuli. Correct sucrose identification (corrected hit rates) was recorded, and sweet taste detection thresholds (EC50s: half maximum effective concencration values) were derived from concentration curves. The intensity and consummatory reward value of sweet taste were assessed using the generalized Labelled Magnitude Scale. Results Mean daily energy intake was reduced by 27% with GOP but no significant changes in food preferences were observed, whereas a reduction in fat and increase in protein intake were seen post-RYGB. There was no change in corrected hit rates or detection thresholds for sucrose detection following GOP infusion. Additionally, GOP did not alter the intensity or consummatory reward value of sweet taste. A significant reduction in restraint eating, comparable to the RYGB group was observed with GOP. Conclusion The elevation in plasma GOP concentrations after RYGB is unlikely to mediate changes in food preferences and sweet taste function after surgery but may promote restraint eating