Distinct roles of the extracellular surface residues of glucagon-like peptide-1 receptor in ß-arrestin 1/2 signaling.

Glucagon-like peptide-1 receptor (GLP-1R) is a prime drug target for type 2 diabetes and obesity. The ligand initiated GLP-1R interaction with G protein has been well studied, but not with ß-arrestin 1/2. Therefore, bioluminescence resonance energy transfer (BRET), mutagenesis and an operational model were used to evaluate the roles of 85 extracellular surface residues on GLP-1R in ß-arrestin 1/2 recruitment triggered by three representative GLP-1R agonists (GLP-1, exendin-4 and oxyntomodulin). Residues selectively regulated ß-arrestin 1/2 recruitment for diverse ligands, and ß-arrestin isoforms were identified. Mutation of residues K130-S136, L142 and Y145 on the transmembrane helix 1 (TM1)-extracellular domain (ECD) linker decreased ß-arrestin 1 recruitment but increased ß-arrestin 2 recruitment. Other extracellular loop (ECL) mutations, including P137A, Q211A, D222A and M303A selectively affected ß-arrestin 1 recruitment while D215A, L217A, Q221A, S223A, Y289A, S301A, F381A and I382A involved more in ß-arrestin 2 recruitment for the ligands. Oxyntomodulin engaged more broadly with GLP-1R extracellular surface to drive ß-arrestin 1/2 recruitment than GLP-1 and exendin-4; I147, W214 and L218 involved in ß-arrestin 1 recruitment, while L141, D215, L218, D293 and F381 in ß-arrestin 2 recruitment for oxyntomodulin particularly. Additionally, the non-conserved residues on ß-arrestin 1/2 C-domains contributed to interaction with GLP-1R. Further proteomic profiling of GLP-1R stably expressed cell line upon ligand stimulation with or without ß-arrestin 1/2 overexpression demonstrated both commonly and biasedly regulated proteins and pathways associated with cognate ligands and ß-arrestins. Our study offers valuable information about ligand induced ß-arrestin recruitment mediated by GLP-1R and consequent intracellular signaling events.

OXM-104, a potential candidate for the treatment of obesity, NASH and type 2 diabetes.

OBJECTIVE: Dual glucagon-like peptide-1 (GLP-1) and glucagon receptor agonists are therapeutic agents with an interesting liver-specific mode of action suitable for metabolic complications. In this study, dual GLP-1 and glucagon receptor agonist OXM-104 is compared head-to-head with the once-daily dual GLP-1 and glucagon receptor agonist cotadutide and GLP-1 receptor agonist semaglutide to explore the metabolic efficacy of OXM-104. METHODS: The in vitro potencies of OXM-104, cotadutide and semaglutide were assessed using reporter assays. In addition, in vivo efficacy was investigated using mouse models of diet-induced obesity (DIO mice), diabetes (db/db mice) and diet-induced NASH mice (MS-NASH). RESULTS: OXM-104 was found to only activate the GLP-1 and glucagon with no cross-reactivity at the (GIP) receptor. Cotadutide was also found to activate the GLP-1 and glucagon receptors, whereas semaglutide only showed activity at the GLP-1 receptor. OXM-104, cotadutide, and semaglutide elicited marked reductions in body weight and improved glucose control. In contrast, hepatoprotective effects, i.e., reductions in steatosis and fibrosis, as well as liver fibrotic biomarkers, were more prominent with OXM-104 and cotadutide than those seen with semaglutide, demonstrated by an improved NAFLD activity score (NAS) by OXM-104 and cotadutide, underlining the importance of the glucagon receptor. CONCLUSION: These results show that dual GLP-1 and glucagon receptor agonism is superior to GLP-1 alone. OXM-104 was found to be a promising therapeutic candidate for the treatment of metabolic complications such as obesity, type 2 diabetes and NASH.

Self-assembled GLP-1/glucagon peptide nanofibrils prolong inhibition of food intake.

Introduction: Oxyntomodulin (Oxm) hormone peptide has a number of beneficial effects on nutrition and metabolism including increased energy expenditure and reduced body weight gain. Despite its many advantages as a potential therapeutic agent, Oxm is subjected to rapid renal clearance and protease degradation limiting its clinical application. Previously, we have shown that subcutaneous administration of a fibrillar Oxm formulation can significantly prolong its bioactivity in vivo from a few hours to a few days. Methods: We used a protease resistant analogue of Oxm, Aib2-Oxm, to form nanfibrils depot and improve serum stability of released peptide. The nanofibrils and monomeric peptide in solution were characterized by spectroscopic, microscopic techniques, potency assay, QCM-D and in vivo studies. Results: We show that in comparison to Oxm, Aib2-Oxm fibrils display a slower elongation rate requiring higher ionic strength solutions, and a higher propensity to dissociate. Upon subcutaneous administration of fibrillar Aib2-Oxm in rodents, a 5-fold increase in bioactivity relative to fibrillar Oxm and a significantly longer bioactivity than free Aib2-Oxm were characterized. Importantly, a decrease in food intake was observed up to 72-hour post-administration, which was not seen for free Aib2-Oxm. Conclusion: Our findings provides compelling evidence for the development of long-lasting peptide fibrillar formulations that yield extended plasma exposure and enhanced in vivo pharmacological response.

Emerging roles of oxyntomodulin-based glucagon-like peptide-1/glucagon co-agonist analogs in diabetes and obesity.

Oxyntomodulin (OXM) is an endogenous peptide hormone secreted from the intestines following nutrient ingestion that activates both glucagon-like peptide-1 (GLP-1) and glucagon receptors. OXM is known to exert various effects, including improvement in glucose tolerance, promotion of energy expenditure, acceleration of liver lipolysis, inhibition of food intake, delay of gastric emptying, neuroprotection, and pain relief. The antidiabetic and antiobesity properties have led to the development of biologically active and enzymatically stable OXM-based analogs with proposed therapeutic promise for metabolic diseases. Structural modification of OXM was ongoing to enhance its potency and prolong half-life, and several GLP-1/glucagon dual receptor agonist-based therapies are being explored in clinical trials for the treatment of type 2 diabetes mellitus and its complications. In the present article, we provide a brief overview of the physiology of OXM, focusing on its structural-activity relationship and ongoing clinical development.

Discovery of novel OXM-based glucagon-like peptide 1 (GLP-1)/glucagon receptor dual agonists.

Novel glucagon receptor (GCGR) and glucagon-like peptide 1 receptor (GLP-1R) dual agonists are reported to have improved efficacy over GLP-1R mono-agonists in treating type 2 diabetes (T2DM) and obesity. Here, we describe the discovery of a novel oxyntomodulin (OXM) based GLP-1R/GCGR dual agonist with potent and balanced potency toward GLP-1R and GCGR. The lead peptide OXM-7 was obtained via stepwise rational design and long-acting modification. In ICR and db/db mice, OXM-7 exhibited prominent acute and long-acting hypoglycemic effects. In diet-induced obesity (DIO) mice, twice-daily administration of OXM-7 produced significant weight loss, normalized lipid metabolism, and improved glucose control. In DIO-nonalcoholic steatohepatitis (NASH) mice, OXM-7 treatment significantly reversed hepatic steatosis, and reduced serum and hepatic lipid levels. These preclinical data suggest the therapeutic potential of OXM-7 as a novel anti-diabetic, anti-steatotic and/or anti-obesity agent.

BI 456906: Discovery and preclinical pharmacology of a novel GCGR/GLP-1R dual agonist with robust anti-obesity efficacy.

OBJECTIVE: Obesity and its associated comorbidities represent a global health challenge with a need for well-tolerated, effective, and mechanistically diverse pharmaceutical interventions. Oxyntomodulin is a gut peptide that activates the glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R) and reduces bodyweight by increasing energy expenditure and reducing energy intake in humans. Here we describe the pharmacological profile of the novel glucagon receptor (GCGR)/GLP-1 receptor (GLP-1R) dual agonist BI 456906. METHODS: BI 456906 was characterized using cell-based in vitro assays to determine functional agonism. In vivo pharmacological studies were performed using acute and subchronic dosing regimens to demonstrate target engagement for the GCGR and GLP-1R, and weight lowering efficacy. RESULTS: BI 456906 is a potent, acylated peptide containing a C18 fatty acid as a half-life extending principle to support once-weekly dosing in humans. Pharmacological doses of BI 456906 provided greater bodyweight reductions in mice compared with maximally effective doses of the GLP-1R agonist semaglutide. BI 456906's superior efficacy is the consequence of increased energy expenditure and reduced food intake. Engagement of both receptors in vivo was demonstrated via glucose tolerance, food intake, and gastric emptying tests for the GLP-1R, and liver nicotinamide N-methyltransferase mRNA expression and circulating biomarkers (amino acids, fibroblast growth factor-21) for the GCGR. The dual activity of BI 456906 at the GLP-1R and GCGR was supported using GLP-1R knockout and transgenic reporter mice, and an ex vivo bioactivity assay. CONCLUSIONS: BI 456906 is a potent GCGR/GLP-1R dual agonist with robust anti-obesity efficacy achieved by increasing energy expenditure and decreasing food intake.

S3-2, a novel long-lasting oxyntomodulin derivative, exerts improvement on diabesity and renal injury through activating GLP-1 and glucagon receptors.

AIMS: To prolong the short lifespan of oxyntomodulin (OXM) for treating obesity and diabetes, we designed a novel fused OXM analog, containing an albumin-binding sequence, a protease cleavable tetrapeptide, and a mutated OXM. MAIN METHODS: We screened two albumin-binding sequences (S3 and S6) to construct OXM derivatives, termed S3-2 (with two cysteines) and S6-0 (without cysteine). After peptides were synthesized, isothermal titration calorimetry (ITC) was applied to assess binding-affinity for HSA. Further in vivo acute efficacies evaluation and candidate selection were performed in diabetic db/db mice via oral glucose tolerance test (OGTT) and glucose-lowering duration test. Chronic efficacy test of selected candidate was also performed in diabetic mice. RESULTS: Firstly, S3-2 and S6-0 with purity over 99% were prepared. ITC measurements demonstrated that S3-2 and S6-0 associate with HSA with high-affinity (Kd = 12.81 ± 1.11 nM and 26.98 ± 2.39 nM, respectively). Then hypoglycemic efficacies showed deoxidation S3-2 (S3-2re) showed longer hypoglycemic duration than the oxidation one (S3-2ox), and better blood glucose level (BGL) control effect than S6-0. OGTTs in diabetic mice revealed the glucose-lowering efficacies of S3-2re were similar to Liraglutide. The protracted antidiabetic effects of S3-2re were further confirmed by multiple OGTTs in db/db mice. Furthermore, twice weekly injection of S3-2re to db/db mice achieved beneficial effects on body weight gain, glucose tolerance, postprandial BGL and obesity. Moreover, S3-2 produces significantly protective effects on the impaired renal functions of the diabetic mice. CONCLUSION: S3-2re exhibits outstanding therapeutical potential as a candidate drug for treating the obesity and diabetes.

Novel peptidic dual GLP-1/glucagon receptor agonist alleviates diabetes and diabetic complications in combination with low-intensity ultrasound.

OBJECTIVE: To design and evaluate a novel oxyntomodulin (OXM) derivative with albumin-binding helix domain and dual GLP-1 receptor (GLP-1R) and glucagon receptor (GcgR) activation activity to achieve metabolize improvement on the diabetes-related complication. MATERIALS AND METHODS: Mutation (D-Ser2) on OXM was performed and then different helix albumin-binding domains were fused to the mutated OXM via a thrombin-cleavable linker to generate seven fusion peptides, named LM01-LM07. Seven LM peptides were synthesized and screened via in vitro receptor activation test, albumin binding measurement and protease cleavage assay to select potent candidate peptide for further in vivo study. Moreover, acute and chronic efficacy studies were conducted to evaluate the efficacy of selected candidate using db/db mice. RESULTS: LM06, as selected OXM derivative, exhibited higher albumin-binding affinity, sustained-release efficiency and balanced activation activities on both GLP-1R and GcgR compared with other ones. Moreover, LM06 was demonstrated with improved hypoglycemic and insulinotropic abilities in receptor-deficient mice via activating GLP-1R. In addition, prolonged anti-diabetic efficacies of LM06 were demonstrated via hypoglycemic duration assay and OGTT in db/db mice. Further pharmacokinetic test of LM06 in both rats and monkeys identified improved half-life and other metabolic characteristics. Nevertheless, 8-week subcutaneously dosed LM06 in db/db mice achieved prominent efficacies on glucostasis, weight-lowering, pancreatic function and adipogenesis via activating GLP-1R and GcgR. Moreover, LM06 also could accelerate diabetic skin wound closure in combination with low-intensity ultrasound. CONCLUSIONS: LM06, as a long-acting dual GLP-1R/GcgR agonist, exerts potential as a once-weekly therapeutic candidate against diabetes-related complication in combination with low-intensity ultrasound.

Conjugation of a peptide to an antibody engineered with free cysteines dramatically improves half-life and activity.

The long circulating half-life and inherently bivalent architecture of IgGs provide an ideal vehicle for presenting otherwise short-lived G-protein-coupled receptor agonists in a format that enables avidity-driven enhancement of potency. Here, we describe the site-specific conjugation of a dual agonist peptide (an oxyntomodulin variant engineered for potency and in vivo stability) to the complementarity-determining regions (CDRs) of an immunologically silent IgG4. A cysteine-containing heavy chain CDR3 variant was identified that provided clean conjugation to a bromoacetylated peptide without interference from any of the endogenous mAb cysteine residues. The resulting mAb-peptide homodimer has high potency at both target receptors (glucagon receptor, GCGR, and glucagon-like peptide 1 receptor, GLP-1R) driven by an increase in receptor avidity provided by the spatially defined presentation of the peptides. Interestingly, the avidity effects are different at the two target receptors. A single dose of the long-acting peptide conjugate robustly inhibited food intake and decreased body weight in insulin resistant diet-induced obese mice, in addition to ameliorating glucose intolerance. Inhibition of food intake and decrease in body weight was also seen in overweight cynomolgus monkeys. The weight loss resulting from dosing with the bivalently conjugated dual agonist was significantly greater than for the monomeric analog, clearly demonstrating translation of the measured in vitro avidity to in vivo pharmacology.

A novel long-acting oxyntomodulin analogue eliminates diabetes and obesity in mice.

Oxyntomodulin (OXM) was identified as a glucagon (GCG) receptor (GCGR) and glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) dual agonist to suppress appetite, increase energy expenditure, and induce body weight loss in obese humans. However, the activities of native OXM to activate GCGR and GLP-1R in vitro were much weaker than the natural ligands. To address this gap, structural modifications were adopted and novel OXM analogues were obtained through chimeric peptide sequence design. One specific analogue with enhanced and balanced GCGR/GLP-1R activations was chemically conjugated with polyethylene glycol (PEG) to achieve sustained release in vivo. This PEGylated analogue was further explored pharmacologically in db/db and diet-induced obese (DIO) mice models. Chronic weekly administration significantly induced hypoglycemic effects and body weight loss with dose dependency, along with normalized adiposity, lipid metabolism, and liver steatosis. Based on its profiles in vitro and in vivo, the analogue has the great potential to develop as a novel anti-diabetic and/or anti-obese candidate. As observed more insulin stimulation and improved insulin resistance, it may be also explored for the treatment of nonalcoholic steatohepatitis (NASH) in the future.

Evaluation of biased agonism mediated by dual agonists of the GLP-1 and glucagon receptors.

Metabolic diseases such as obesity, diabetes, and their comorbidities have converged as one of the most serious health concerns on a global scale. Selective glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonists are one of the major therapeutics for type 2 diabetes and obesity. Polypharmacological approaches that enable modulation of multiple metabolic targets in a single drug have emerged as a potential avenue to improve therapeutic outcomes. Among numerous peptides under development are those targeting the GLP-1R and either the glucagon receptor (GCGR), glucose-dependent insulinotropic peptide receptor (GIPR) or all 3 receptors, as dual- or tri- peptide agonists. Despite many of them entering into clinical trials, current development has been based on only a limited understanding of the spectrum of potential pharmacological properties of these ligands beyond binding selectivity. In the present study, we examined the potential for agonists that target both GLP-1R and GCGR to exhibit biased agonism, comparing activity across proximal activation of Gs protein, cAMP accumulation, pERK1/2 and ß-arrestin recruitment. Three distinct dual agonists that have different relative cAMP production potency for GLP-1R versus GCGR, "peptide 15", MEDI0382 and SAR425899, and one triagonist of the GLP-1R, GCGR and GIPR were examined. We demonstrated that all novel peptides have distinct biased agonism profiles relative to either of the cognate agonists of the receptors, and to each other. This is an important feature of the pharmacology of this drug class that needs to be considered alongside selectivity, bioavailability and pharmacokinetics for rational optimization of new therapeutics.

Oxyntomodulin induces satiety and activates the arcuate nucleus of the hypothalamus in Japanese quail.

Oxyntomodulin (OXM) is a proglucagon-derived peptide that suppresses hunger in humans. There are some differences in its food intake-inhibitory effects among species. The central mechanisms are unclear and it is unknown if OXM is more efficacious in a gallinaceous species that has not undergone as much selection for growth as the chicken. The objective was thus to determine the effects of OXM on food and water intake and hypothalamic physiology in Japanese quail. At 7 days post-hatch, 6-h-fasted quail were injected intracerebroventricularly (ICV) or intraperitoneally (IP) with 0.32, 0.65, or 1.3 nmol of OXM. All doses decreased food intake for 180 min post-ICV injection. On a cumulative basis, water intake was not affected until 120 min, with the lowest and highest doses decreasing water intake after ICV injection. The two highest doses were anorexigenic when administered via the IP route, whereas all doses were anti-dipsogenic starting at 30 min post-injection. In hypothalamic samples collected at 1-h post-ICV injection, there was an increase in c-Fos immunoreactivity, an indicator of recent neuronal activation, in the arcuate nucleus (ARC) and dorsomedial nucleus (DMN) of the hypothalamus in OXM-injected individuals. Results suggest that quail are more sensitive than chickens to the satiety-inducing effects of OXM. The central mechanism is likely mediated through a pathway in the ARC that is conserved among species, and through activation of the DMN, an effect that is unique to quail. Such knowledge is critical for facilitating the development of novel, side effect-free anti-eating strategies to promote weight-loss in obesity.

New Generation Oxyntomodulin Peptides with Improved Pharmacokinetic Profiles Exhibit Weight Reducing and Anti-Steatotic Properties in Mice.

Oxyntomodulin (OXM) is an intestinal peptide hormone that activates both glucagon-like peptide-1 (GLP-1) and glucagon (GCG) receptors. The natural peptide reduces body weight in obese subjects and exhibits direct acute glucoregulatory effects in patients with type II diabetes. However, the clinical utility of OXM is limited due to its lower in vitro potency and short in vivo half-life. To overcome these issues, we developed stapled, long-acting, and highly potent OXM analogs with balanced activities at both GLP-1 and GCG receptors. The lead molecule O14 exhibits potent and long-lasting effects on glucose control, body weight loss, and reduction of hepatic fat reduction in DIO mice. Importantly, O14 significantly reversed hepatic steatosis; reduced liver weight, total cholesterol, and hepatic triglycerides; and improved markers of liver function in a nonalcoholic steatohepatitis (NASH) mouse model. A symmetrical version of the peptide was also shown to be more efficacious and long-lasting in controlling glucose than semaglutide and the clinical candidate cotadutide in wild-type mice, highlighting the utility of our designs of the dual agonist as a potential new therapy for diabetes and liver diseases.

A dual GLP-1 and Gcg receptor agonist rescues spatial memory and synaptic plasticity in APP/PS1 transgenic mice.

Alzheimer's disease (AD) is a neurodegenerative disease that severely affects the health and lifespan of the elderly worldwide. Recently, the correlation between AD and type 2 diabetes mellitus (T2DM) has received intensive attention, and a promising new anti-AD strategy is the use of anti-diabetic drugs. Oxyntomodulin (Oxm) is a peptide hormone and growth factor that acts on neurons in the hypothalamus. OXM activates glucagon-like peptide 1 (GLP-1) and glucagon (Gcg) receptors, facilitates insulin signaling and has neuroprotective effects against Aß1-42-induced cytotoxicity in primary hippocampal neurons. Here, we tested the effects of the protease-resistant analogue (D-Ser2)Oxm on spatial memory and synaptic plasticity and the underlying molecular mechanisms in the APP/PS1 transgenic mouse model of AD. The results showed that (D-Ser2)Oxm not only alleviated the impairments of working memory and long-term spatial memory, but also reduced the number of Aß plaques in the hippocampus, and reversed the suppression of hippocampal synaptic long-term potentiation (LTP). Moreover, (D-Ser2)Oxm administration significantly increased p-PI3K/p-AKT1 expression and decreased p-GSK3ß levels in the hippocampus. These results are the first to show an in vivo neuroprotective role of (D-Ser2)Oxm in APP/PS1 mice, and this role involves the improvement of synaptic plasticity, clearance of Aß and normalization of PI3K/AKT/GSK3ß cell signaling in the hippocampus. This study suggests that (D-Ser2)Oxm holds promise for the prevention and treatment of AD.

Effects of long-acting GIP, xenin and oxyntomodulin peptide analogues on alpha-cell transdifferentiation in insulin-deficient diabetic GluCreERT2;ROSA26-eYFP mice.

Enzyme-resistant long-acting forms of the gut-derived peptide hormones, glucose-dependent insulinotropic polypeptide (GIP), xenin and oxyntomodulin (Oxm) have been generated, and exert beneficial effects on diabetes control and pancreatic islet architecture. The current study has employed alpha-cell lineage tracing in GluCreERT2;ROSA26-eYFP transgenic mice to investigate the extent to which these positive pancreatic effects are associated with alpha- to beta-cell transdifferentiation. Twice-daily administration of (D-Ala2)GIP, xenin-25[Lys13PAL] or (D-Ser2)-Oxm[Lys38PAL] for 10 days to streptozotocin (STZ)-induced diabetic mice did not affect body weight, food intake or blood glucose levels, but (D-Ser2)-Oxm[Lys38PAL] reduced (P < 0.05 to P < 0.001) fluid intake and circulating glucagon. (D-Ala2)GIP and (D-Ser2)-Oxm[Lys38PAL] also augmented (P < 0.05 and P < 0.01, respectively) pancreatic insulin content. Detrimental changes of pancreatic morphology induced by STZ in GluCreERT2;ROSA26-eYFP mice were partially reversed by all treatment interventions. This was associated with reduced (P < 0.05) apoptosis and increased (P < 0.05 to P < 0.01) proliferation of beta-cells, alongside opposing effects on alpha-cells, with (D-Ala2)GIP and (D-Ser2)-Oxm[Lys38PAL] being particularly effective in this regard. Alpha-cell lineage tracing revealed that induction of diabetes was accompanied by increased (P < 0.01) transdifferentiation of glucagon positive alpha-cells to insulin positive beta-cells. This islet cell transitioning process was augmented (P < 0.01 and P < 0.001, respectively) by (D-Ala2)GIP and (D-Ser2)-Oxm[Lys38PAL]. (D-Ser2)-Oxm[Lys38PAL] also significantly (P < 0.05) promoted loss of alpha-cell identity in favour of other endocrine islet cells. These data highlight intra-islet benefits of (D-Ala2)GIP, xenin-25[Lys13PAL] and (D-Ser2)-Oxm[Lys38PAL] in diabetes with beta-cell loss induced by STZ. The effects appear to be independent of glycaemic change, and associated with alpha- to beta-cell transdifferentiation for the GIP and Oxm analogues.

Oxyntomodulin attenuates TNF­α induced neuropathic pain by inhibiting the activation of the NF­κB pathway.

Neuropathic pain is rarely diagnosed. Oxyntomodulin is peripherally and centrally distributed; however, the potential mechanisms underlying the effects of oxyntomodulin in attenuating nociception remain unclear; thus, we aimed to explore them in the present study. A neuropathic pain model in male C57BL/6 mice was induced by intrathecal injection of tumor necrosis factor­α (TNF­α), and the duration of nociceptive behavioral responses was measured with a stop­watch timer within 30 min. Western blotting was used to explore the protein levels of ionized calcium binding adaptor molecule­1 (IBA1), nuclear factor­κB (NF­κB) phosphorylated­p65, interleukin (IL)­6 and IL­1ß. We performed reverse transcription­quantitative polymerase chain reaction and ELISA were performed to determine the mRNA and protein expression levels of IL­6 and IL­1ß, respectively. An MTT assay was conducted to detect BV2 cell viability. Oxyntomodulin was observed to attenuate TNF­α­induced pain hypersensitivity in mice, as well as the expression of IBA1, NF­κB p­p65, IL­6 and IL­1ß in the spinal cord. Oxyntomodulin exhibited no cytotoxicity on BV2 cells, and attenuated TNF­α­induced IL­6 and IL­1ß production and release in BV2 cells and culture medium, respectively. Collectively, we proposed oxyntomodulin to attenuate TNF­α induced neuropathic pain associated with the release of glial cytokines IL­6 and IL­1ß via inhibiting the activation of the NF­κB pathway.

Design, screening and biological evaluation of novel fatty acid chain-modified oxyntomodulin-based derivatives with prolonged glucose-lowering ability and potent anti-obesity effects.

Recently, oxyntomodulin (OXM) has emerged as a treatment option for type 2 diabetes mellitus and obesity. In order to develop more promising novel OXM derivatives combining glycemic effects of glucagon-like peptide-1 (GLP-1) and lipolytic properties of glucagon, six 12-mer GLP-1 receptor agonists (PP01-PP06) were screened using a phage display method and then fused to OXM (3-37) to generate hybrid OXM derivatives (PP07-PP12). PP11, as a selected starting point, was further site-specifically modified with three lengths of fatty acid chains to provide long-acting conjugates PP13-PP24, among which PP18 was found not only to retain almost the entire balanced activation potency of PP11 in GLP-1/glucagon receptors but also to enhance plasma stability and prolong hypoglycemic activity. PP18 was further confirmed as an insulin secretagogue and glycemic agent in gene knockout mice. The protracted antidiabetic effects and in vivo half-life of PP18 were further proved by hypoglycemic efficacies in diet-induced obesity (DIO) mice and pharmacokinetics tests in Sprague Dawley (SD) rats, respectively. Nevertheless, administration of PP18 once per day normalized food intake, body weight, blood biochemical indexes, insulin resistance and islet function of DIO mice. These preclinical results suggested that PP18, as a novel OXM-based dual GLP-1 and glucagon receptor agonist, may serve as a novel therapeutic approach to treat T2DM and obesity.

Glucagon-related peptides from phylogenetically ancient fish reveal new approaches to the development of dual GCGR and GLP1R agonists for type 2 diabetes therapy.

The insulinotropic and antihyperglycaemic properties of glucagons from the sea lamprey (Petromyzontiformes), paddlefish (Acipenseriformes) and trout (Teleostei) and oxyntomodulin from dogfish (Elasmobranchii) and ratfish (Holocephali) were compared with those of human glucagon and GLP-1 in mammalian test systems. All fish peptides produced concentration-dependent stimulation of insulin release from BRIN-BD11 rat and 1.1 B4 human clonal ß-cells and isolated mouse islets. Paddlefish glucagon was the most potent and effective peptide. The insulinotropic activity of paddlefish glucagon was significantly (P < 0.01) decreased after incubating BRIN-BD11 cells with the GLP1R antagonist, exendin-4(9-39) and the GCGR antagonist [des-His1,Pro4, Glu9] glucagon amide but GIPR antagonist, GIP(6-30)Cex-K40[palmitate] was without effect. Paddlefish and lamprey glucagons and dogfish oxyntomodulin (10 nmol L-1) produced significant (P < 0.01) increases in cAMP concentration in Chinese hamster lung (CHL) cells transfected with GLP1R and human embryonic kidney (HEK293) cells transfected with GCGR. The insulinotropic activity of paddlefish glucagon was attenuated in CRISPR/Cas9-engineered GLP1R knock-out INS-1 cells but not in GIPR knock-out cells. Intraperitoneal administration of all fish peptides, except ratfish oxyntomodulin, to mice together with a glucose load produced significant (P < 0.05) decreases in plasma glucose concentrations and paddlefish glucagon produced a greater release of insulin compared with GLP-1. Paddlefish glucagon shares the sequences Glu15-Glu16 and Glu24-Trp25-Leu26-Lys27-Asn28-Gly29 with the potent GLP1R agonist, exendin-4 so may be regarded as a naturally occurring, dual-agonist hybrid peptide that may serve as a template design of new drugs for type 2 diabetes therapy.

Glucagon-like peptide-1 receptor internalisation controls spatiotemporal signalling mediated by biased agonists.

The glucagon-like peptide-1 receptor (GLP-1R) is a major therapeutic target in the treatment of type 2 diabetes due to its roles in regulating blood glucose and in promoting weight loss. Like many GPCRs, it is pleiotropically coupled, can be activated by multiple ligands and is subject to biased agonism. The GLP-1R undergoes agonist mediated receptor internalisation that may be associated with spatiotemporal control of signalling and biased agonism, although to date, this has not been extensively explored. Here, we investigate GLP-1R trafficking and its importance with regard to signalling, including the localisation of key signalling molecules, mediated by biased peptide agonists that are either endogenous GLP-1R ligands or are used clinically. Each of the agonists promoted receptor internalisation through a dynamin and caveolae dependent mechanism and traffic the receptor to both degradative and recycling pathways. This internalisation is important for signalling, with cAMP and ERK1/2 phoshorylation (pERK1/2) generated by both plasma membrane localised and internalised receptors. Further assessment of pERK1/2 revealed that all peptides induced nuclear ERK activity, but ligands, liraglutide and oxyntomodulin that are biased towards pERK1/2 relative to cAMP (when compared to GLP-1 and exendin-4), also stimulated pERK1/2 activity in the cytosol. This compartmentalisation of ERK1/2 signalling was reliant on receptor internalisation, with restriction of receptor localisation to the plasma membrane limiting ERK1/2 signalling to the cytosol. Thus, this study implicates a role of receptor internalisation in spatiotemporal control of ERK1/2 signalling that may contribute to GLP-1R biased agonism.

Oxyntomodulin analogue increases energy expenditure via the glucagon receptor.

The gut hormone oxyntomodulin (OXM) causes weight loss by reducing appetite and increasing energy expenditure. Several analogues are being developed to treat obesity. Exactly how oxyntomodulin works, however, remains controversial. OXM can activate both glucagon and GLP-1 receptors but no specific receptor has been identified. It is thought that the anorectic effect occurs predominantly through GLP-1 receptor activation but, to date, it has not been formally confirmed which receptor is responsible for the increased energy expenditure. We developed OX-SR, a sustained-release OXM analogue. It produces a significant and sustained increase in energy expenditure in rats as measured by indirect calorimetry. We now show that this increase in energy expenditure occurs via activation of the glucagon receptor. Blockade of the GLP-1 receptor with Exendin 9-39 does not block the increase in oxygen consumption caused by OX-SR. However, when activity at the glucagon receptor is lost, there is no increase in energy expenditure. Glucagon receptor activity therefore appears to be essential for OX-SR's effects on energy expenditure. The development of future 'dual agonist' analogues will require careful balancing of GLP-1 and glucagon receptor activities to obtain optimal effects.