Abolishing ß-arrestin recruitment is necessary for the full metabolic benefits of G protein-biased glucagon-like peptide-1 receptor agonists.

AIM: Earlier studies have shown that peptide glucagon-like peptide-1 receptor (GLP-1R) agonists with reduced ß-arrestin recruitment show enhanced anti-hyperglycaemic efficacy through avoidance of GLP-1R desensitization. However, the ligand modifications needed to decrease ß-arrestin recruitment usually also reduces GLP-1R affinity, therefore higher doses are needed. Here we aimed to develop new, long-acting, G protein-biased GLP-1R agonists with acute signalling potency comparable with semaglutide, to provide insights into specific experimental and therapeutic scenarios. MATERIALS AND METHODS: New GLP-1R agonist peptides were assessed using a variety of in vitro and in vivo assays. RESULTS: First, we show that very substantial reductions in ß-arrestin recruitment efficacy are required to realize fully the benefits of GLP-1R agonism on blood glucose lowering in mice, with more moderate reductions being less effective. Secondly, our lead compound (SRB107) performs substantially better than semaglutide for effects on blood glucose and weight loss, which may be jointly attributable to its biased agonist action and protracted pharmacokinetics. Thirdly, we show that biased agonist-specific GLP-1R internalization profiles occur at clinically relevant pharmacological concentrations. Finally, we show that SRB107 cAMP signalling is differentially modulated by single and double GLP1R coding variants seen in human populations, with implications for GLP-1R agonist pharmacogenomics. CONCLUSIONS: Completely abolishing ß-arrestin recruitment improves the anti-hyperglycaemic effects of GLP-1R agonists in mice.

Adaptive infusion of a glucagon-like peptide-1/glucagon receptor co-agonist G3215, in adults with overweight or obesity: Results from a phase 1 randomized clinical trial.

AIMS: To determine whether a continuous infusion of a glucagon-like peptide receptor (GLP-1R)/glucagon receptor (GCGR) co-agonist, G3215 is safe and well tolerated in adults with overweight or obesity. METHODS: A phase 1 randomized, double blind, placebo-controlled trial of G3215 in overweight or obese participants, with or without type 2 diabetes. RESULTS: Twenty-six participants were recruited and randomized with 23 completing a 14-day subcutaneous infusion of G3215 or placebo. The most common adverse events were nausea or vomiting, which were mild in most cases and mitigated by real-time adjustment of drug infusion. There were no cardiovascular concerns with G3215 infusion. The pharmacokinetic characteristics were in keeping with a continuous infusion over 14 days. A least-squares mean body weight loss of 2.39 kg was achieved with a 14-day infusion of G3215, compared with 0.84 kg with placebo infusion (p < .05). A reduction in food consumption was also observed in participants receiving G3215 and there was no deterioration in glycaemia. An improved lipid profile was seen in G3215-treated participants and consistent with GCGR activation, a broad reduction in circulating amino acids was seen during the infusion period. CONCLUSION: An adaptive continuous infusion of the GLP-1/GCGR co-agonist, G3215, is safe and well tolerated offering a unique strategy to control drug exposure. By allowing rapid, response-directed titration, this strategy may allow for mitigation of adverse effects and afford significant weight loss within shorter time horizons than is presently possible with weekly GLP-1R and multi-agonists. These results support ongoing development of G3215 for the treatment of obesity and metabolic disease.

The postprandial secretion of peptide YY1-36 and 3-36 in obesity is differentially increased after gastric bypass versus sleeve gastrectomy.

OBJECTIVES: Peptide tyrosine tyrosine (PYY) exists as two species, PYY1-36 and PYY3-36 , with distinct effects on insulin secretion and appetite regulation. The detailed effects of bariatric surgery on PYY1-36 and PYY3-36 secretion are not known as previous studies have used nonspecific immunoassays to measure total PYY. Our objective was to characterize the effect of sleeve gastrectomy (SG) and Roux-en-Y gastric bypass (RYGB) on fasting and postprandial PYY1-36 and PYY3-36 secretion using a newly developed liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. DESIGN AND SUBJECTS: Observational study in 10 healthy nonobese volunteers and 30 participants with obesity who underwent RYGB (n = 24) or SG (n = 6) at the Imperial Weight Centre [NCT01945840]. Participants were studied using a standardized mixed meal test (MMT) before and 1 year after surgery. The outcome measures were PYY1-36 and PYY3-36 concentrations. RESULTS: Presurgery, the fasting and postprandial levels of PYY1-36 and PYY3-36 were low, with minimal responses to the MMT, and these did not differ from healthy nonobese volunteers. The postprandial secretion of both PYY1-36 and PYY3-36 at 1 year was amplified after RYGB, but not SG, with the response being significantly higher in RYGB compared with SG. CONCLUSIONS: There appears to be no difference in PYY secretion between nonobese and obese volunteers at baseline. At 1 year after surgery, RYGB, but not SG, is associated with increased postprandial secretion of PYY1-36 and PYY3-36 , which may account for long-term differences in efficacy and adverse effects between the two types of surgery.

Acylation of the Incretin Peptide Exendin-4 Directly Impacts Glucagon-Like Peptide-1 Receptor Signaling and Trafficking.

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor and mainstay therapeutic target for the treatment of type 2 diabetes and obesity. Recent reports have highlighted how biased agonism at the GLP-1R affects sustained glucose-stimulated insulin secretion through avoidance of desensitization and downregulation. A number of GLP-1R agonists (GLP-1RAs) feature a fatty acid moiety to prolong their pharmacokinetics via increased albumin binding, but the potential for these chemical changes to influence GLP-1R function has rarely been investigated beyond potency assessments for cAMP. Here, we directly compare the prototypical GLP-1RA exendin-4 with its C-terminally acylated analog, exendin-4-C16. We examine relative propensities of each ligand to recruit and activate G proteins and ß-arrestins, endocytic and postendocytic trafficking profiles, and interactions with model and cellular membranes in HEK293 and HEK293T cells. Both ligands had similar cAMP potency, but exendin-4-C16 showed ∼2.5-fold bias toward G protein recruitment and a ∼60% reduction in ß-arrestin-2 recruitment efficacy compared with exendin-4, as well as reduced GLP-1R endocytosis and preferential targeting toward recycling pathways. These effects were associated with reduced movement of the GLP-1R extracellular domain measured using a conformational biosensor approach and a ∼70% increase in insulin secretion in INS-1 832/3 cells. Interactions with plasma membrane lipids were enhanced by the acyl chain. Exendin-4-C16 showed extensive albumin binding and was highly effective for lowering of blood glucose in mice over at least 72 hours. Our study highlights the importance of a broad approach to the evaluation of GLP-1RA pharmacology. SIGNIFICANCE STATEMENT: Acylation is a common strategy to enhance the pharmacokinetics of peptide-based drugs. This work shows how acylation can also affect various other pharmacological parameters, including biased agonism, receptor trafficking, and interactions with the plasma membrane, which may be therapeutically important.

Safety and efficacy of an extended-release peptide YY analogue for obesity: A randomized, placebo-controlled, phase 1 trial.

AIM: To report the results from a Phase 1 trial of an extended-release peptide YY analogue, Y14, developed for the treatment of obesity. METHODS: Y14 was evaluated in overweight/obese volunteers in a Phase 1 randomized placebo-controlled trial, conducted in a clinical trial unit in the United Kingdom. Part A was a blinded single-ascending-dose study evaluating doses up to 36 mg. Part B was double-blinded and tested multiple ascending doses between 9 and 36 mg, given at 7- to 14-day intervals, over the course of 28 days, with up to five doses given per participant. The primary outcome was safety and tolerability; the secondary outcome was assessment of pharmacokinetic (PK) characteristics. Exploratory outcomes included food intake, body weight change and glucose tolerance after multiple doses. RESULTS: Between April 11, 2017 and December 24, 2018, 53 participants were enrolled into Part A and 24 into Part B of the trial. The PK characteristics were compatible with administration every 7 to 14 days. The most common adverse events (AEs) were nausea, vomiting or administration site reactions, which were mild in most cases and settled with time. No serious AE occurred. Participants given multiple doses of Y14 lost between -2.87 and -3.58 kg body weight compared with placebo (P <0.0001) at 31 days from the first dose, with profound reductions in food intake of 38% to 55% (P <0.0001, compared to placebo) and there was no evidence of tachyphylaxis. CONCLUSIONS: Our results support the continued development of Y14 as a novel treatment for obesity.

Ghrelin acylation by ghrelin- O-acyltransferase can occur in healthy part of oncological liver in humans.

Activation of ghrelin is controlled by the enzyme ghrelin- O-acyl transferase (GOAT). In humans, localization of this acylation is poorly understood. The aim of this study is to explore GOAT localization and activation in the human liver by evaluating both bioactive and non-bioactive ghrelin in the bloodstream entering and leaving the liver and to simultaneously evaluate GOAT mRNA expression in the liver. A healthy part of oncologic hepatic tissue collected from nine patients undergoing hepatectomy was used to evaluate GOAT mRNA expression by quantitative real-time polymerase chain reaction (RT-qPCR). Simultaneously, blood from the portal vein, the suprahepatic vein, the subclavicular vein, and the radial artery was also sampled to assay total and acylated ghrelin. Acylated ghrelin level was significantly increased in the suprahepatic vein compared with the portal vein level (385 ± 42 ng/ml vs. 268 ± 24 ng/ml, P = 0.04). Suprahepatic-to-portal vein ratio for acylated ghrelin (acylation ratio) is 1.4 ± 0.1. Mean expression of GOAT mRNA in the liver, expressed as 2-∆Ct·µg total RNA-1·1 µl of liver tissue-1 was at 0.042 ± 0.021 arbitrary units. GOAT mRNA expression in the liver was correlated with acylated-to-total ghrelin ratio in the suprahepatic vein ( P = 0.016, R = 0.75) and with the acylation liver ratio ( P = 0.05, R = 0.61). Blood concentration of acylated ghrelin was found significantly increased after its passage through the liver, suggesting that acylation can occur in the liver. RT-qPCR data confirmed the presence of GOAT in the liver, with a positive correlation between GOAT expression and acylated ghrelin liver ratio. This study strongly suggests that the liver is a site of ghrelin acylation in humans. NEW & NOTEWORTHY Although the activation of ghrelin by the enzyme ghrelin- O-acyl transferase (GOAT) is yet well demonstrated, its localization, especially in humans, remains poorly understood. We explored GOAT localization and activation in the human liver by simultaneously evaluating both bioactive and non-bioactive ghrelin in the bloodstream entering and leaving the liver and also GOAT mRNA expression in the liver. We therefore showed for the first time, to our knowledge, that GOAT localized in the liver is active and takes part in ghrelin activation.

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 Dunnett's 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.

Learning curve of vessel cannulation in rats using cumulative sum analysis.

BACKGROUND: Intravascular access routes are widely used for administering agents or taking blood samples in rodents. Vessel cannulation in rats is a technically challenging procedure with a risk for significant complications. The use of cumulative sum (CUSUM) analysis allows continuous monitoring of the performer's outcomes to evaluate the learning curve for a particular procedure. The aim of the present study was to assess a researcher's learning curve in the cannulation of the jugular and femoral vein in rats using CUSUM analysis. MATERIALS AND METHODS: A single researcher performed two hundred microsurgical operations between September 2012 and September 2013. The animals (male Wistar rats) were anesthetized with isoflurane whereas the right jugular vein and the left femoral vein were catheterized. Prospective data were collected and analyzed using CUSUM analysis. For the purposes of the study, the rat population was divided in four groups based on the order of studies; group 1 represents the first 50 animals cannulated, group 2 the next batch of 50 animals, and so forth. RESULTS: The operating times required for cannulation of the jugular vein for groups 1, 2, 3, and 4 were 24.6 ± 4.8, 15.9 ± 2.5, 15.2 ± 3.2, and 15.7 ± 3.3 min, respectively. Group 1's operating time was significantly longer than all the other groups (P < 0.001 compared with all other groups). The operating times for groups 2, 3, and 4 did not differ significantly (P > 0.05). The cannulation of the femoral vein required a mean of 32 ± 5.3 min for group 1, 24.9 ± 5.7 min for group 2, 18.4 ± 4 min for group 3, and 17.2 ± 3.4 min for group 4. The operating time of group 1 was significantly longer when compared with all groups (P < 0.001 for all groups). Group 2 also had a longer operating time than groups 3 and 4 (P < 0.001 compared with both groups). Groups 3 and 4 did not show any statistical significant difference when their operating time was compared (P > 0.05). CUSUM analysis suggested that the number of cases required to achieve the required experience to most effectively cannulate the jugular and femoral vein is approximately 50 and 100 cases, respectively. The adverse effects of the procedure included two unexpected deaths, both of which occurred in group 1 (0.5% in total). CONCLUSIONS: The authors' experience regarding the learning curve of the cannulation of the femoral and jugular vein in rats from 200 animals operated over a period of 1 y for the evaluation of the pharmacokinetic properties of drug candidates suggests significant experience is required to optimize the operating time required for the procedure.

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

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.

Investigating the Glucagon Receptor and Glucagon-Like Peptide 1 Receptor Activity of Oxyntomodulin-Like Analogues in Male Wistar Rats.

AIMS: To investigate the effect of Glu-3 OXM-like analogues on food intake and bodyweight in male rats. BACKGROUND: Oxyntomodulin (OXM) is a natural agonist at both the glucagon receptor (GCGr) and the glucagon-like peptide 1 receptor (GLP-1r), and peripheral administration reduces food intake and increases energy expenditure in rodents and humans. Substituting the native glutamine (Gln) at amino acid position 3 of OXM for glutamate (Glu) has previously been shown to diminish GCGr activity without affecting GLP-1r activity. The effects of Glu-3 OXM analogues have not been investigated in rats. METHODS: The effect of 2 Glu-3-substituted OXM-like analogues (eg, OXM14E3 and OXM15E3) on food intake and body weight was investigated in male Wistar rats during 6 days of daily subcutaneous (SC) administration. The effects of Glu-3 substitution on analogue binding and activity at the rat GCGr and rat GLP-1 receptor were investigated in vitro using Chinese hamster ovary or Chinese hamster lung cells. RESULTS: We report the novel finding that 2 5-nmol/kg Glu-3 OXM-like analogues (OXM14E3 and OXM15E3) significantly increased rat body weight by up to 4% compared with the equivalent non-Glu-3 analogues (OXM14 and OXM15), without affecting food intake. The effect of OXM15E3 on body weight was dose-dependent. Glu-3 analogues, including Glu-3 OXM, decreased glucagon-mediated cyclic adenosine monophosphate accumulation in Chinese hamster ovary cells expressing the rat GCGr, suggesting they may be acting as antagonists. CONCLUSIONS: The results indicate Glu-3 OXM-like analogues might not be suitable tools to investigate the mechanism of OXM analogue action in a rat model because they significantly increase body weight independent of food intake. Glu-3 OXM analogues are partial agonists at the rat GCGr and may also act as antagonists, possibly resulting in the observed increase in body weight.

Chronic treatment with glucagon-like peptide-1 and glucagon receptor co-agonist causes weight loss-independent improvements in hepatic steatosis in mice with diet-induced obesity.

OBJECTIVES: Co-agonists at the glucagon-like peptide-1 and glucagon receptors (GLP1R/GCGR) show promise as treatments for metabolic dysfunction-associated steatotic liver disease (MASLD). Although most co-agonists to date have been heavily GLP1R-biased, glucagon directly acts on the liver to reduce fat content. The aims of this study were to investigate a GCGR-biased co-agonist as treatment for hepatic steatosis in mice. METHODS: Mice with diet-induced obesity (DIO) were treated with Dicretin, a GLP1/GCGR co-agonist with high potency at the GCGR, Semaglutide (GLP1R monoagonist) or food restriction over 24 days, such that their weight loss was matched. Hepatic steatosis, glucose tolerance, hepatic transcriptomics, metabolomics and lipidomics at the end of the study were compared with Vehicle-treated mice. RESULTS: Dicretin lead to superior reduction of hepatic lipid content when compared to Semaglutide or equivalent weight loss by calorie restriction. Markers of glucose tolerance and insulin resistance improved in all treatment groups. Hepatic transcriptomic and metabolomic profiling demonstrated many changes that were unique to Dicretin-treated mice. These include some known targets of glucagon signaling and others with as yet unclear physiological significance. CONCLUSIONS: Our study supports the development of GCGR-biased GLP1/GCGR co-agonists for treatment of MASLD and related conditions.

Pharmacokinetics, adverse effects and tolerability of a novel analogue of human pancreatic polypeptide, PP 1420.

AIMS: The objectives of this phase 1 study were to confirm the tolerability of single ascending subcutaneous doses of PP 1420 in healthy subjects, to assess its adverse effects and to investigate the drug's pharmacokinetics and dose proportionality. METHODS: This was a double-blind, placebo-controlled, randomized study. There were three dosing periods. Each subject (n= 12) was randomized to receive one dose of placebo and two ascending doses of PP 1420, given as a subcutaneous injection. Blood samples were taken over 24 h to assess pharmacokinetics. Standard safety and laboratory data were collected. The primary endpoint was the tolerability of PP 1420. The secondary endpoint was exposure to PP 1420 as assessed by C(max) and AUC(0,∞). RESULTS: PP 1420 was well tolerated by all subjects with no serious adverse effects. Following single subcutaneous doses of PP 1420 at 2, 4 and 8 mg to male subjects, C(max) was reached at a median t(max) of approximately 1 h post dose (range 0.32-2.00 h). Thereafter, plasma concentrations of PP 1420 declined with geometric mean apparent terminal elimination t(1/2) ranging from 2.42-2.61 h (range 1.64-3.95 h) across all dose levels. CONCLUSIONS: Subcutaneous PP 1420 was well tolerated in healthy human subjects at single doses between 2-8 mg, with no tolerability issues arising. Where observed, adverse events were not serious, and there was no evidence of a dose-relationship to frequency of adverse events. The results therefore support the conduct of clinical trials to investigate efficacy, tolerability and pharmacokinetics during repeated dosing.

The Metabolomic Effects of Tripeptide Gut Hormone Infusion Compared to Roux-en-Y Gastric Bypass and Caloric Restriction.

CONTEXT: The gut-derived peptide hormones glucagon-like peptide-1 (GLP-1), oxyntomodulin (OXM), and peptide YY (PYY) are regulators of energy intake and glucose homeostasis and are thought to contribute to the glucose-lowering effects of bariatric surgery. OBJECTIVE: To establish the metabolomic effects of a combined infusion of GLP-1, OXM, and PYY (tripeptide GOP) in comparison to a placebo infusion, Roux-en-Y gastric bypass (RYGB) surgery, and a very low-calorie diet (VLCD). DESIGN AND SETTING: Subanalysis of a single-blind, randomized, placebo-controlled study of GOP infusion (ClinicalTrials.gov NCT01945840), including VLCD and RYGB comparator groups. PATIENTS AND INTERVENTIONS: Twenty-five obese patients with type 2 diabetes or prediabetes were randomly allocated to receive a 4-week subcutaneous infusion of GOP (n = 14) or 0.9% saline control (n = 11). An additional 22 patients followed a VLCD, and 21 underwent RYGB surgery. MAIN OUTCOME MEASURES: Plasma and urine samples collected at baseline and 4 weeks into each intervention were subjected to cross-platform metabolomic analysis, followed by unsupervised and supervised modeling approaches to identify similarities and differences between the effects of each intervention. RESULTS: Aside from glucose, very few metabolites were affected by GOP, contrasting with major metabolomic changes seen with VLCD and RYGB. CONCLUSIONS: Treatment with GOP provides a powerful glucose-lowering effect but does not replicate the broader metabolomic changes seen with VLCD and RYGB. The contribution of these metabolomic changes to the clinical benefits of RYGB remains to be elucidated.

Hypoaminoacidemia underpins glucagon-mediated energy expenditure and weight loss.

Glucagon analogs show promise as components of next-generation, multi-target, anti-obesity therapeutics. The biology of chronic glucagon treatment, in particular, its ability to induce energy expenditure and weight loss, remains poorly understood. Using a long-acting glucagon analog, G108, we demonstrate that glucagon-mediated body weight loss is intrinsically linked to the hypoaminoacidemia associated with its known amino acid catabolic action. Mechanistic studies reveal an energy-consuming response to low plasma amino acids in G108-treated mice, prevented by dietary amino acid supplementation and mimicked by a rationally designed low amino acid diet. Therefore, low plasma amino acids are a pre-requisite for G108-mediated energy expenditure and weight loss. However, preventing hypoaminoacidemia with additional dietary protein does not affect the ability of G108 to improve glycemia or hepatic steatosis in obese mice. These studies provide a mechanism for glucagon-mediated weight loss and confirm the hepatic glucagon receptor as an attractive molecular target for metabolic disease therapeutics.

A glucagon analogue decreases body weight in mice via signalling in the liver.

Glucagon receptor agonists show promise as components of next generation metabolic syndrome pharmacotherapies. However, the biology of glucagon action is complex, controversial, and likely context dependent. As such, a better understanding of chronic glucagon receptor (GCGR) agonism is essential to identify and mitigate potential clinical side-effects. Herein we present a novel, long-acting glucagon analogue (GCG104) with high receptor-specificity and potent in vivo action. It has allowed us to make two important observations about the biology of sustained GCGR agonism. First, it causes weight loss in mice by direct receptor signalling at the level of the liver. Second, subtle changes in GCG104-sensitivity, possibly due to interindividual variation, may be sufficient to alter its effects on metabolic parameters. Together, these findings confirm the liver as a principal target for glucagon-mediated weight loss and provide new insights into the biology of glucagon analogues.

Partial agonism improves the anti-hyperglycaemic efficacy of an oxyntomodulin-derived GLP-1R/GCGR co-agonist.

OBJECTIVE: Glucagon-like peptide-1 and glucagon receptor (GLP-1R/GCGR) co-agonism can maximise weight loss and improve glycaemic control in type 2 diabetes and obesity. In this study, we investigated the cellular and metabolic effects of modulating the balance between G protein and ß-arrestin-2 recruitment at GLP-1R and GCGR using oxyntomodulin (OXM)-derived co-agonists. This strategy has been previously shown to improve the duration of action of GLP-1R mono-agonists by reducing target desensitisation and downregulation. METHODS: Dipeptidyl dipeptidase-4 (DPP-4)-resistant OXM analogues were generated and assessed for a variety of cellular readouts. Molecular dynamic simulations were used to gain insights into the molecular interactions involved. In vivo studies were performed in mice to identify the effects on glucose homeostasis and weight loss. RESULTS: Ligand-specific reductions in ß-arrestin-2 recruitment were associated with slower GLP-1R internalisation and prolonged glucose-lowering action in vivo. The putative benefits of GCGR agonism were retained, with equivalent weight loss compared to the GLP-1R mono-agonist liraglutide despite a lesser degree of food intake suppression. The compounds tested showed only a minor degree of biased agonism between G protein and ß-arrestin-2 recruitment at both receptors and were best classified as partial agonists for the two pathways measured. CONCLUSIONS: Diminishing ß-arrestin-2 recruitment may be an effective way to increase the therapeutic efficacy of GLP-1R/GCGR co-agonists. These benefits can be achieved by partial rather than biased agonism.

The role of gut hormones and the hypothalamus in appetite regulation.

The World Health Organisation has estimated that by 2015 approximately 2.3 billion adults will be overweight and more than 700 million obese. Obesity is associated with an increased risk of diabetes, cardiovascular events, stroke and cancer. The hypothalamus is a crucial region for integrating signals from central and peripheral pathways and plays a major role in appetite regulation. In addition, there are reciprocal connections with the brainstem and higher cortical centres. In the arcuate nucleus of the hypothalamus, there are two major neuronal populations which stimulate or inhibit food intake and influence energy homeostasis. Within the brainstem, the dorsal vagal complex plays a role in the interpretation and relaying of peripheral signals. Gut hormones act peripherally to modulate digestion and absorption of nutrients. However, they also act as neurotransmitters within the central nervous system to control food intake. Peptide YY, pancreatic polypeptide, glucagon-like peptide-1 and oxyntomodulin suppress appetite, whilst ghrelin increases appetite through afferent vagal fibres to the caudal brainstem or directly to the hypothalamus. A better understanding of the role of these gut hormones may offer the opportunity to develop successful treatments for obesity. Here we review the current understanding of the role of gut hormones and the hypothalamus on food intake and body weight control.

Signalling, trafficking and glucoregulatory properties of glucagon-like peptide-1 receptor agonists exendin-4 and lixisenatide.

BACKGROUND AND PURPOSE: Amino acid substitutions at the N-termini of glucagon-like peptide-1 (GLP-1) receptor agonist peptides result in distinct patterns of intracellular signalling, sub-cellular trafficking and efficacy in vivo. Here, we to determine whether sequence differences at the ligand C-termini of clinically approved GLP-1 receptor agonists exendin-4 and lixisenatide lead to similar phenomena. EXPERIMENTAL APPROACH: Exendin-4, lixisenatide and N-terminally substituted analogues with biased signalling characteristics were compared across a range of in vitro trafficking and signalling assays in different cell types. Fluorescent ligands and new time-resolved FRET approaches were developed to study agonist behaviours at the cellular and sub-cellular level. Anti-hyperglycaemic and anorectic effects of each parent ligand and their biased derivatives were assessed in mice. KEY RESULTS: Lixisenatide and exendin-4 showed equal binding affinity, but lixisenatide was fivefold less potent for cAMP signalling. Both peptides induced extensive GLP-1 receptor clustering in the plasma membrane and were rapidly endocytosed, but the GLP-1 receptor recycled more slowly to the cell surface after lixisenatide treatment. These combined deficits resulted in reduced maximal sustained insulin secretion and reduced anti-hyperglycaemic and anorectic effects in mice with lixisenatide. N-terminal substitution of His1 by Phe1 to both ligands had favourable effects on their pharmacology, resulting in improved insulin release and lowering of blood glucose. CONCLUSION AND IMPLICATIONS: Changes to the C-terminus of exendin-4 affect signalling potency and GLP-1 receptor trafficking via mechanisms unrelated to GLP-1 receptor occupancy. These differences were associated with changes in their ability to control blood glucose and therefore may be therapeutically relevant.

Disconnect between signalling potency and in vivo efficacy of pharmacokinetically optimised biased glucagon-like peptide-1 receptor agonists.

OBJECTIVE: The objective of this study was to determine how pharmacokinetically advantageous acylation impacts on glucagon-like peptide-1 receptor (GLP-1R) signal bias, trafficking, anti-hyperglycaemic efficacy, and appetite suppression. METHODS: In vitro signalling responses were measured using biochemical and biosensor assays. GLP-1R trafficking was determined by confocal microscopy and diffusion-enhanced resonance energy transfer. Pharmacokinetics, glucoregulatory effects, and appetite suppression were measured in acute, sub-chronic, and chronic settings in mice. RESULTS: A C-terminally acylated ligand, [F1,G40,K41.C16 diacid]exendin-4, was identified that showed undetectable ß-arrestin recruitment and GLP-1R internalisation. Depending on the cellular system used, this molecule was up to 1000-fold less potent than the comparator [D3,G40,K41.C16 diacid]exendin-4 for cyclic AMP signalling, yet was considerably more effective in vivo, particularly for glucose regulation. CONCLUSIONS: C-terminal acylation of biased GLP-1R agonists increases their degree of signal bias in favour of cAMP production and improves their therapeutic potential.