Jaimini Cegla

Coinfusion of Low-Dose GLP-1 and Glucagon in Man Results in a Reduction in Food Intake

Obesity is a growing epidemic, and current medical therapies have proven inadequate. Endogenous satiety hormones provide an attractive target for the development of drugs that aim to cause effective weight loss with minimal side effects. Both glucagon and GLP-1 reduce appetite and cause weight loss. Additionally, glucagon increases energy expenditure. We hypothesized that the combination of both peptides, administered at doses that are individually subanorectic, would reduce appetite, while GLP-1 would protect against the hyperglycemic effect of glucagon. In this double-blind crossover study, subanorectic doses of each peptide alone, both peptides in combination, or placebo was infused into 13 human volunteers for 120 min. An ad libitum meal was provided after 90 min, and calorie intake determined. Resting energy expenditure was measured by indirect calorimetry at baseline and during infusion. Glucagon or GLP-1, given individually at subanorectic doses, did not significantly reduce food intake. Coinfusion at the same doses led to a significant reduction in food intake of 13%. Furthermore, the addition of GLP-1 protected against glucagon-induced hyperglycemia, and an increase in energy expenditure of 53 kcal/day was seen on coinfusion. These observations support the concept of GLP-1 and glucagon dual agonism as a possible treatment for obesity and diabetes.

Gut-brain cross-talk in appetite regulation.

Purpose of reviewTo review the role of gut hormones involved in appetite regulation and their potential in the treatment of obesity. Recent findingsThe medical treatment of obesity has been fraught with challenges. With two centrally acting agents having been recently withdrawn from the market, new therapies are required. The gut hormones are especially promising therapeutic targets. This article looks at the interplay between gut and brain which mediates the regulation of appetite and energy expenditure. Some recent developments that point the way towards the future development of gut hormone analogues as treatments for obesity are reviewed. SummaryOur understanding of the interaction between gut hormones and the neuronal circuits controlling appetite regulation has made great advances over recent years. This knowledge is essential if these peptides are to become potential obesity therapies, both from an efficacy and safety perspective.

Obesity: Lifestyle management, bariatric surgery, drugs, and the therapeutic exploitation of gut hormones

Abstract Obesity is on the rise and the pursuit of efficient and safe treatment is ongoing. Available anti-obesity medical therapies have so far proved to be disappointing, whereas bariatric surgery is leading the way and offers long-term health benefits. Part of the success of bariatric surgery is thought to be mediated by gut hormones. A better understanding of the role of gut hormones within the gut-brain signaling pathway in the control of hunger, satiety, and energy homeostasis, has led to their therapeutic exploitation as possible anti-obesity drugs. In this review, we provide a summary of currently available treatment options for obesity from simple lifestyle modifications and bariatric surgery to traditional and novel medical therapies.

Comparison of the overnight metyrapone and glucagon stimulation tests in the assessment of secondary hypoadrenalism

The insulin tolerance test (ITT) is contraindicated in a proportion of patients with suspected ACTH deficiency. The aim of this study was to investigate the diagnostic accuracy of the glucagon stress test (GST) compared with the overnight metyrapone test (OMT) in patients with contraindications to ITT.

RAMP2 influences glucagon receptor pharmacology via trafficking and signaling

Endogenous satiety hormones provide an attractive target for obesity drugs. Glucagon causes weight loss by reducing food intake and increasing energy expenditure. To further understand the cellular mechanisms by which glucagon and related ligands activate the glucagon receptor (GCGR), we investigated the interaction of the GCGR with receptor activity modifying protein (RAMP)2, a member of the family of receptor activity modifying proteins. We used a combination of competition binding experiments, cell surface enzyme-linked immunosorbent assay, functional assays assessing the Gαs and Gαq pathways and β-arrestin recruitment, and small interfering RNA knockdown to examine the effect of RAMP2 on the GCGR. Ligands tested were glucagon; glucagonlike peptide-1 (GLP-1); oxyntomodulin; and analog G(X), a GLP-1/glucagon coagonist developed in-house. Confocal microscopy was used to assess whether RAMP2 affects the subcellular distribution of GCGR. Here we demonstrate that coexpression of RAMP2 and the GCGR results in reduced cell surface expression of the GCGR. This was confirmed by confocal microscopy, which demonstrated that RAMP2 colocalizes with the GCGR and causes significant GCGR cellular redistribution. Furthermore, the presence of RAMP2 influences signaling through the Gαs and Gαq pathways, as well as recruitment of β-arrestin. This work suggests that RAMP2 may modify the agonist activity and trafficking of the GCGR, with potential relevance to production of new peptide analogs with selective agonist activities.

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

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.

Pharmacokinetics and pharmacodynamics of subcutaneously administered PYY3–36 and its analogues in vivo

BACKGROUND Obesity is an emergent epidemic associated with morbidity, mortality, and psychosocial effects. One of the key gut hormones that controls appetite is peptide tyrosine-tyrosine 3-36 (PYY3-36) whose circulating half-life is only 8 min. A long-acting analogue of PYY3-36 would therefore have great potential as an antiobesity agent. The aims of this study were to investigate the effect of various aminoacid modifications of PYY3-36 on pharmacokinetics and their ability to suppress food intake. METHODS To investigate the pharmacokinetics of PYY3-36 and three modified analogues, serial sampling of plasma peptide levels via cannulation of the jugular vein was performed after subcutaneous injection of the peptide in rats (n=4 per peptide, 80 nmol/kg). To investigate the effect of these peptides on food intake, mice were injected subcutaneously (1000 nmol/kg) and food intake was assessed at timed intervals over 24 h (n=8 per peptide). FINDINGS One-way ANOVA with post-hoc Dunnetts test was used in which each comparison was with the PYY3-36 or saline group. Plasma concentrations of the modified analogue, PYY-AP3H, were significantly higher than PYY3-36 up to 24 h post injection (p=0·0008 at 4 h, p=0·0028 at 24 h). The results confirm that modification of the native peptide, by addition of an α-helix stabilising sequence and histidine residues, lengthens the pharmacokinetic profile. Furthermore, PYY-AP3H significantly reduced food intake for up to 24 h compared with saline (p<0·0001) and native PYY3-36 (p<0·0001). INTERPRETATION The rationally designed analogue, PYY-AP3H, has potential as a once-a-day subcutaneously administered preparation for the treatment of obesity. FUNDING UK Medical Research Council, Biotechnology and Biological Sciences Research Council, National Institute for Health Research (NIHR), an Integrative Mammalian Biology (IMB) Capacity Building award, an FP7-HEALTH-2009-241592 EuroCHIP grant, NIHR Imperial Biomedical Research Centre Funding Scheme.

Clinical roles in clinical biochemistry: a national survey of practice in the UK

Background Using an online survey, we collected data to present a picture of how clinical authorization is performed in the UK. Methods A 21-question survey was uploaded to www.surveymonkey.com, and responses were invited via the mail base of the Association for Clinical Biochemistry and Laboratory Medicine. The questionnaire examined the intensity and function of the duty biochemist role and how different types of authorization are used to handle and release results. Results Of 70 responses received, 60 were suitable for analysis. Responses were received from every region of the UK. A typical duty biochemist shift started on average at 8:50, and finished at 17:25. The mean duration was 8 h 58 min. Clinical scientists are the most abundantly represented group on duty biochemist rotas. Higher banded clinical scientists and chemical pathologists covered out-of-hours shifts. Results were handled differently depending on the level of abnormality and the requesting area. Normal results tended to be released either directly from the analyser or after technical then autoauthorization (90%). A greater preference for clinical authorization was seen for abnormal and critical results originating from outpatients (49% and 69%, respectively) or general practice (51% and 71%) than for inpatients (33% and 53%) or A&E (25% and 37%). Conclusions The handling and authorization of biochemistry results varies greatly between laboratories. The role is clearly heterogeneous in the UK. Guidance from the Association for Clinical Biochemistry and Royal College of Pathologists may help to clarify the essential roles of the duty biochemist.

The preanalytical stability of glucagon as measured by liquid chromatography tandem mass spectrometry and two commercially available immunoassays

Background One of the main challenges in the measurement of glucagon is the premise that it is unstable in human plasma. Traditionally, protease inhibitors have been used to prevent its degradation; however, their use is controversial. Here, we investigated the optimal method of sample collection for glucagon, with measurement by liquid chromatography tandem mass spectrometry (LC-MS/MS) and two commercially available immunoassays. Methods Blood from healthy fasting volunteers (n = 10) was processed under a variety of preanalytical conditions including collection in EDTA vs. lithium heparin tubes and the addition of aprotinin and/or a dipeptidyl-peptidase IV (DPPIV) inhibitor. Additionally, the effect of freeze thaw was assessed. Plasma glucagon concentrations were measured by LC-MS/MS and two commercially available immunoassays (HTRF® sandwich immunoassay, Cisbio and Milliplex MAP Human Metabolic Hormone Panel, Merck Millipore). Results A systematic bias of Milliplex > LC-MS/MS > HTRF was noted and plasma glucagon concentrations were significantly different between methods (Milliplex vs. LC-MS/MS P < 0.01; Milliplex vs. HTRF P < 0.0001; LC-MS/MS vs. HTRF P < 0.001). The addition of aprotinin, DPPIV inhibitor or a combination of aprotinin and DPPIV inhibitor had no effect on plasma glucagon concentrations when compared to ‘non-stabilized’ samples or each other. Whether samples were taken in EDTA tubes or lithium heparin tubes made no difference to plasma glucagon concentrations. These findings were consistent for all three methods. Plasma glucagon concentrations were not significantly different after two freeze–thaw cycles (performed on samples in EDTA tubes containing aprotinin and DPPIV inhibitor). Conclusions This study demonstrates that glucagon is stable in both EDTA and lithium heparin tubes when stored at −80℃. Furthermore, the addition of aprotinin and DPPIV inhibitors is unnecessary.

Liraglutide safety and efficacy in patients with non-alcoholic steatohepatitis (LEAN): a multicentre, double-blind, randomized, placebo-controlled phase 2 study.

The March edition of the JCEM included a series of four notable articles highlighting the presentation, diagnosis, management and epidemiology of hypoparathyroidism. Following the recently published European Society of Endocrinology clinical guidelines, these constitute the consensus guidelines from the first International Conference on the Management of Hypoparathyroidism. Of the four articles, the Epidemiology and Diagnosis statement paper may be of particular interest to the clinical biochemistry laboratory and discusses the use of particular PTH assays. It advocates the use of secondand third-generation PTH assays (specific for the PTH carboxyand amino-terminals, respectively) for the diagnosis of hypoparathyroidism. This point has, to my knowledge, not been mentioned in previous guidelines. Interestingly, this paper suggests that the third-generation assays, while theoretically better, have not proven superior in clinical practice. The paper mentions a recent report (J Bone Miner Res 2015; 30: 1803–1813) describing the influence of genetic mutations on the diagnostic performance of the secondand third-generation assays. In summary, this series of papers addresses key aspects of diagnosis, evaluation and management of hypoparathyroidism.