An update on galanin and spexin and their potential for the treatment of type 2 diabetes and related metabolic disorders.

Spexin (SPX) and galanin (GAL) are two neuropeptides widely expressed in the central nervous system as well as within peripheral tissues in humans and other species. SPX and GAL mediate their biological actions through binding and activation of galanin receptors (GALR), namely GALR1, GALR2 and GLAR3. GAL appears to trigger all three galanin receptors, whereas SPX interacts more specifically with GALR2 and GLAR3. Whilst the biological effects of GAL have been well-described over the years, in-depth knowledge of physiological action profile of SPX is still in its preliminary stages. However, it is recognised that both peptides play a significant role in modulating overall energy homeostasis, suggesting possible therapeutically exploitable benefits in diseases such as obesity and type 2 diabetes mellitus. Accordingly, although both peptides activate GALR's, it appears GAL may be more useful for the treatment of eating disorders such as anorexia and bulimia, whereas SPX may find therapeutic application for obesity and obesity-driven forms of diabetes. This short narrative review aims to provide an up-to-date account of SPX and GAL biology together with putative approaches on exploiting these peptides for the treatment of metabolic disorders.

Sustained glucagon receptor antagonism in insulin-deficient high-fat-fed mice.

Discerning modification to the amino acid sequence of native glucagon can generate specific glucagon receptor (GCGR) antagonists, that include desHis1Pro4Glu9-glucagon and the acylated form desHis1Pro4Glu9(Lys12PAL)-glucagon. In the current study, we have evaluated the metabolic benefits of once-daily injection of these peptide-based GCGR antagonists for 18 days in insulin-resistant high-fat-fed (HFF) mice with streptozotocin (STZ)-induced insulin deficiency, namely HFF-STZ mice. Administration of desHis1Pro4Glu9-glucagon moderately (P < 0.05) decreased STZ-induced elevations of food intake. Body weight was not different between groups of HFF-STZ mice and both treatment interventions delayed (P < 0.05) the onset of hyperglycaemia. The treatments reduced (P < 0.05-P < 0.001) circulating and pancreatic glucagon, whilst desHis1Pro4Glu9(Lys12PAL)-glucagon also substantially increased (P < 0.001) pancreatic insulin stores. Oral glucose tolerance was appreciably improved (P < 0.05) by both antagonists, despite the lack of augmentation of glucose-stimulated insulin release. Interestingly, positive effects on i.p. glucose tolerance were less obvious suggesting important beneficial effects on gut function. Metabolic benefits were accompanied by decreased (P < 0.05-P < 0.01) locomotor activity and increases (P < 0.001) in energy expenditure and respiratory exchange ratio in both treatment groups. In addition, desHis1Pro4Glu9-glucagon increased (P < 0.01-P < 0.001) O2 consumption and CO2 production. Together, these data provide further evidence that peptidic GCGR antagonists are effective treatment options for obesity-driven forms of diabetes, even when accompanied by insulin deficiency.

Metabolic effects of combined glucagon receptor antagonism and glucagon-like peptide-1 receptor agonism in high fat fed mice.

Ablation of glucagon receptor (GCGR) signalling is a potential treatment option for diabetes, whilst glucagon-like peptide-1 (GLP-1) receptor agonists are clinically approved for both obesity and diabetes. There is a suggestion that GCGR blockade enhances GLP-1 secretion and action, whilst GLP-1 receptor activation is known to inhibit glucagon release, implying potential for positive interactions between both therapeutic avenues. The present study has examined the ability of sustained GCGR antagonism, using desHis1Pro4Glu9-glucagon, to augment the established benefits of the GLP-1 mimetic, exendin-4, in high fat fed (HFF) mice. Twice-daily injection of desHis1Pro4Glu9-glucagon, exendin-4 or a combination of both peptides to groups of HFF mice for 10 days had no impact on body weight or energy intake. Circulating blood glucose and glucagon concentrations were significantly (P < 0.05-0.01) decreased by all treatment regimens, with plasma insulin levels elevated (P < 0.001) when compared to lean control mice. Intraperitoneal and oral glucose tolerance were improved (P < 0.05-0.01) by all treatments, despite lack of enhanced glucose-stimulated insulin secretion. Following exogenous glucagon administration, all HFF treatment groups displayed reduced (P < 0.05-0.001) glucose and insulin levels compared to HFF saline controls, although peripheral insulin sensitivity was largely unchanged across all animals. Interestingly, all treatments had tendency to increase pancreatic insulin content with pancreatic glucagon content significantly elevated (P < 0.05) by all interventions. These studies highlight the capacity of peptide-based GCGR inhibition, or GLP-1 receptor activation, to significantly improve metabolism in HFF mice but suggest no obvious additive benefits of combined therapy.

Dual-agonist incretin peptides from fish with potential for obesity-related Type 2 diabetes therapy - A review.

The long-acting glucagon-like peptide-1 receptor (GLP1R) agonist, semaglutide and the unimolecular glucose-dependent insulinotropic polypeptide receptor (GIPR)/GLP1R dual-agonist, tirzepatide have been successfully introduced as therapeutic options for patients with Type-2 diabetes (T2DM) and obesity. Proglucagon-derived peptides from phylogenetically ancient fish act as naturally occurring dual agonists at the GLP1R and the glucagon receptor (GCGR) with lamprey GLP-1 and paddlefish glucagon being the most potent and effective in stimulating insulin release from BRIN-BD11 clonal ß-cells. These peptides were also the most effective in lowering blood glucose and elevating plasma insulin concentrations when administered intraperitoneally to overnight-fasted mice together with a glucose load. Zebrafish GIP acts as a dual agonist at the GIPR and GLP1R receptors. Studies with the high fat-fed mouse, an animal model with obesity, impaired glucose-tolerance and insulin-resistance, have shown that twice-daily administration of the long-acting analogs [D-Ala2]palmitoyl-lamprey GLP-1 and [D-Ser2]palmitoyl-paddlefish glucagon over 21 days improves glucose tolerance and insulin sensitivity. This was associated with ß-cell proliferation, protection of ß-cells against apoptosis, decreased pancreatic glucagon content, improved lipid profile, reduced food intake and selective alteration in the expression of genes involved in ß-cell stimulus-secretion coupling. In insulin-deficient GluCreERT2;ROSA26-eYFP transgenic mice, the peptides promoted an increase in ß-cell mass with positive effects on transdifferentiation of glucagon-producing to insulin-producing cells. Naturally occurring fish dual agonist peptides, particularly lamprey GLP-1 and paddlefish glucagon, provide templates for development into therapeutic agents for obesity-related T2DM.

Proglucagon-Derived Peptides as Therapeutics.

Initially discovered as an impurity in insulin preparations, our understanding of the hyperglycaemic hormone glucagon has evolved markedly over subsequent decades. With description of the precursor proglucagon, we now appreciate that glucagon was just the first proglucagon-derived peptide (PGDP) to be characterised. Other bioactive members of the PGDP family include glucagon-like peptides -1 and -2 (GLP-1 and GLP-2), oxyntomodulin (OXM), glicentin and glicentin-related pancreatic peptide (GRPP), with these being produced via tissue-specific processing of proglucagon by the prohormone convertase (PC) enzymes, PC1/3 and PC2. PGDP peptides exert unique physiological effects that influence metabolism and energy regulation, which has witnessed several of them exploited in the form of long-acting, enzymatically resistant analogues for treatment of various pathologies. As such, intramuscular glucagon is well established in rescue of hypoglycaemia, while GLP-2 analogues are indicated in the management of short bowel syndrome. Furthermore, since approval of the first GLP-1 mimetic for the management of Type 2 diabetes mellitus (T2DM) in 2005, GLP-1 therapeutics have become a mainstay of T2DM management due to multifaceted and sustainable improvements in glycaemia, appetite control and weight loss. More recently, longer-acting PGDP therapeutics have been developed, while newfound benefits on cardioprotection, bone health, renal and liver function and cognition have been uncovered. In the present article, we discuss the physiology of PGDP peptides and their therapeutic applications, with a focus on successful design of analogues including dual and triple PGDP receptor agonists currently in clinical development.

A Narrative Review of the Anti-Hyperglycemic and Satiating Effects of Fish Protein Hydrolysates and Their Bioactive Peptides.

Prevalence of type 2 diabetes and overweight/obesity are increasing globally. Food supplementation as a preventative option has become an attractive option in comparison to increased pharmacotherapy dependency. Hydrolysates of fish processing waste and by-products have become particularly interesting in a climate of increased food wastage awareness and are rapidly gaining traction in food research. This review summarizes the available research so far on the potential effect of these hydrolysates on diabetes and appetite suppression. Scopus and Web of Science are searched using eight keywords (fish, hydrolysate, peptides, satiating, insulinotropic, incretin, anti-obesity, DPP-4 [dipeptidylpeptidase-4/IV]) returning a total of 2549 results. Following exclusion criteria (repeated appearances, non-fish marine sources [e.g., macroalgae], and irrelevant bioactivities [e.g., immunomodulatory, anti-thrombotic]), 44 relevant publications are included in this review. Stimulation of hormone secretion, regulation of glucose uptake, anorexigenic potential, identified mechanisms of action, and research conducted on the most potent bioactive peptides identified within these hydrolysates are all specifically addressed. Results of this review conclude that despite wide methodological variation between studies, there is significant potential for the application of fish protein hydrolysates in the management of bodyweight and hyperglycemia.

Beneficial actions of a long-acting apelin analogue in diabetes are related to positive effects on islet cell turnover and transdifferentiation.

AIM: The current study has tested the hypothesis that the positive effects of apelin receptor activation in diabetes are linked to benefits on islet cell apoptosis, proliferation and transdifferentiation using Ins1Cre/+ ;Rosa26-eYFP transgenic mice and induction of diabetes-like syndromes by streptozotocin (STZ) or high-fat feeding. MATERIALS AND METHODS: Groups (n = 6-8) of streptozotocin (STZ)-induced diabetic and high-fat diet (HFD)-fed mice received once-daily injection (25 nmol/kg) of the long-acting acylated apelin-13 analogue, pGlu(Lys8 Glu-PAL)apelin-13 amide, for 10 or 12 days, respectively. RESULTS: pGlu(Lys8 Glu-PAL)apelin-13 amide treatment partly reversed body weight loss induced by STZ and normalized circulating insulin. There was no effect of pGlu(Lys8 Glu-PAL)apelin-13 amide on these variables in HFD-fed mice, but an increase in pancreatic insulin content was observed. pGlu(Lys8 Glu-PAL)apelin-13 amide also fully, or partially, reversed the detrimental effects of STZ and HFD on plasma and pancreatic glucagon concentrations. In HFD-fed mice, the apelin analogue decreased dietary-induced elevations of islet, ß- and α-cell areas, whilst reducing α-cell area in STZ-induced diabetic mice. In terms of islet cell lineage, pGlu(Lys8 Glu-PAL)apelin-13 amide effectively reduced ß- to α-cell transdifferentiation and helped maintain ß-cell identity, which was linked to elevated Pdx-1 expression. These islet effects were coupled with decreased ß-cell apoptosis and α-cell proliferation in both models, and there was an accompanying increase of ß-cell proliferation in STZ-induced diabetic mice. CONCLUSION: Taken together these data demonstrate, for the first time, that pancreatic islet benefits of sustained APJ receptor activation in diabetes are linked to favourable islet cell transition events, leading to maintenance of ß-cell mass.

Identification and characterisation of peptides from a boarfish (Capros aper) protein hydrolysate displaying in vitro dipeptidyl peptidase-IV (DPP-IV) inhibitory and insulinotropic activity.

Twenty-two novel dipeptidyl peptidase-IV (DPP-IV) inhibitory peptides (with IC50 values <200 µM) and fifteen novel insulinotropic peptides were identified in a boarfish protein hydrolysate generated at semi-pilot scale using Alcalase 2.4L and Flavourzyme 500L. This was achieved by bioassay-driven semi-preparative reverse phase-high performance liquid chromatography fractionation, liquid chromatography-mass spectrometry and confirmatory studies with synthetic peptides. The most potent DPP-IV inhibitory peptide (IPVDM) had a DPP-IV half maximal inhibitory concentration (IC50) value of 21.72 ±â€¯1.08 µM in a conventional in vitro and 44.26 ±â€¯0.65 µM in an in situ cell-based (Caco-2) DPP-IV inhibition assay. Furthermore, this peptide stimulated potent insulin secretory activity (1.6-fold increase compared to control) from pancreatic BRIN-BD11 cells grown in culture. The tripeptide IPV exhibited potent DPP-IV inhibitory activity (IC50: 5.61 ±â€¯0.20 µM) comparable to that reported for the known DPP-IV inhibitor IPI (IC50: 3.20 µM). Boarfish proteins contain peptide sequences with potential to play a role in glycaemic management in vivo.

Chronic apelin analogue administration is more effective than established incretin therapies for alleviating metabolic dysfunction in diabetic db/db mice.

Stable apelin-13 peptide analogues have shown promising acute antidiabetic effects in mice with diet-induced obesity diabetes. Here the efficacy of (pGlu)apelin-13 amide (apelin amide) and the acylated analogue (pGlu)(Lys8GluPAL)apelin-13 amide (apelin FA), were examined following chronic administration in db/db mice, a genetic model of degenerative diabetes. Groups of 9-week old male db/db mice (n = 8) received twice daily injections (09:00 and 17:00 h; i.p.) or saline vehicle, apelin amide, apelin FA, or the established incretin therapies, exendin-4(1-39) or liraglutide, all at 25 nmol/kg body weight for 21 days. Control C57BL/6J mice were given saline twice daily. No changes in body weight or food intake were observed with either apelin or liraglutide treatments, but exendin-4 showed a reduction in cumulative food intake (p < 0.01) compared with saline-treated db/db mice. Apelin analogues and incretin mimetics induced sustained improvements of glycaemia (p < 0.05 to p < 0.001, from day 9-21), lowered HbA1c at 21 days (p < 0.05) and raised plasma insulin concentrations. The treatments also improved OGTT and ipGTT with enhanced insulin responses compared with saline-treated control db/db mice (p < 0.05 to p < 0.001). Apelin amide was superior to incretin mimetics in lowering plasma triglycerides by 34% (p < 0.05). Apelin analogues unlike both incretin mimetics reduced pancreatic α-cell area (p < 0.05 to p < 0.01) and all peptide treatments enhanced pancreatic insulin content (p < 0.05 to p < 0.01). In conclusion, longer-term administration of apelin-13 analogues, induced similar and in some respects more effective metabolic improvements than incretin mimetics in db/db mice, providing a viable alternative approach for counteracting metabolic dysfunction for mild and more degenerative forms of the disease.

Blockade of gastric inhibitory polypeptide (GIP) action as a novel means of countering insulin resistance in the treatment of obesity-diabetes.

Gastric inhibitory polypeptide (GIP) is a 42 amino acid hormone secreted from intestinal K-cells in response to nutrient ingestion. Despite a recognised physiological role for GIP as an insulin secretagogue to control postprandial blood glucose levels, growing evidence reveals important actions of GIP on adipocytes and promotion of fat deposition in tissues. As such, blockade of GIP receptor (GIPR) action has been proposed as a means to counter insulin resistance, and improve metabolic status in obesity and related diabetes. In agreement with this, numerous independent observations in animal models support important therapeutic applications of GIPR antagonists in obesity-diabetes. Sustained administration of peptide-based GIPR inhibitors, low molecular weight GIPR antagonists, GIPR neutralising antibodies as well as genetic knockout of GIPR's or vaccination against GIP all demonstrate amelioration of insulin resistance and reduced body weight gain in response to high fat feeding. These observations were consistently associated with decreased accumulation of lipids in peripheral tissues, thereby alleviating insulin resistance. Although the impact of prolonged GIPR inhibition on bone turnover still needs to be determined, evidence to date indicates that GIPR antagonists represent an exciting novel treatment option for obesity-diabetes.

Long-term treatment with acylated analogues of apelin-13 amide ameliorates diabetes and improves lipid profile of high-fat fed mice.

Previous studies have shown that modified apelin analogues exhibited enzyme resistance in plasma and improved circulating half-life compared to apelin-13. This study investigated the antidiabetic effects of chronic administration of stable long acting fatty acid modified apelin analogues, namely, (Lys8GluPAL)apelin-13 amide and pGlu(Lys8GluPAL)apelin-13 amide, in high-fat fed obese-diabetic mice. Male NIH Swiss mice (groups n = 8) were maintained either on a high-fat diet (45% fat) from 8 to 28 weeks old, or control mice were fed a normal diet (10% fat). When diet induced obesity-diabetes was established after high-fat feeding, mice were injected i.p. once daily with apelin analogues, liraglutide (25 nmol/kg) or saline (controls). Administration of (Lys8GluPAL)apelin-13 amide and pGlu(Lys8GluPAL)apelin-13 amide for 28 days significantly reduced food intake and decreased body weight. Non-fasting glucose was reduced (p<0.01 to p<0.001) and plasma insulin concentrations increased (p<0.01 to p<0.001). This was accompanied by enhanced insulin responses (p<0.01 to p<0.001) and significant reductions in glucose excursion after oral (p<0.01) or i.p. (p<0.01) glucose challenges and feeding. Apelin analogues also significantly improved HbA1c (p<0.01), enhanced insulin sensitivity (p<0.01), reduced triglycerides (p<0.001), increased HDL-cholesterol (p<0.01) and decreased LDL-cholesterol (p<0.01), compared to high-fat fed saline treated control mice. Cholesterol levels were decreased (p<0.01) by pGlu(Lys8GluPAL)apelin-13 amide and both apelin treated groups showed improved bone mineral content, reduced fat deposits and increased plasma GLP-1. Daily treatment with liraglutide mirrored many of these changes (not on bone or adipose tissue), but unlike apelin analogues increased plasma amylase. Consumption of O2, production of CO2, respiratory exchange ratio and energy expenditure were improved by apelin analogues. These results indicate that long-term treatment with acylated analogues (Lys8GluPAL)apelin-13 amide and particularly pGlu(Lys8GluPAL)apelin-13 amide resulted in similar or enhanced therapeutic responses to liraglutide in high-fat fed mice. Fatty acid derived apelin analogues represent a new and exciting development in the treatment of obesity-diabetes.

Atlantic salmon (Salmo salar) co-product-derived protein hydrolysates: A source of antidiabetic peptides.

Large quantities of low-value protein rich co-products, such as salmon skin and trimmings, are generated annually. These co-products can be upgraded to high-value functional ingredients. The aim of this study was to assess the antidiabetic potential of salmon skin gelatin and trimmings-derived protein hydrolysates in vitro. The gelatin hydrolysate generated with Alcalase 2.4L and Flavourzyme 500L exhibited significantly higher (p < 0.001) insulin and GLP-1 secretory activity from pancreatic BRIN-BD11 and enteroendocrine GLUTag cells, respectively, when tested at 2.5 mg/mL compared to hydrolysates generated with Alcalase 2.4L or Promod 144MG. The gelatin hydrolysate generated with Alcalase 2.4L and Flavourzyme 500L showed significantly more potent (p < 0.01) DPP-IV inhibitory activity than those generated with Alcalase 2.4L or Promod 144MG. No significant difference was observed in the insulinotropic activity mediated by any of the trimmings-derived hydrolysates when tested at 2.5 mg/mL. However, the trimmings hydrolysate generated with Alcalase 2.4L and Flavourzyme 500L exhibited significantly higher DPP-IV inhibitory (p < 0.05:Alcalase 2.4L and p < 0.01:Promod 144MG) and GLP-1 (p < 0.001, 2.5 mg/mL) secretory activity than those generated with Alcalase 2.4L or Promod 144MG. The salmon trimmings hydrolysate generated with Alcalase 2.4L and Flavourzyme 500L when subjected to simulated gastrointestinal digestion (SGID) was shown to retain its GLP-1 secretory and DPP-IV inhibitory activities, in addition to improving its insulin secretory activity. However, the gelatin hydrolysate generated with Alcalase 2.4L and Flavourzyme 500L was shown to lose GLP-1 secretory activity following SGID. A significant increase in membrane potential (p < 0.001) and intracellular calcium (p < 0.001) by both co-product hydrolysates generated with Alcalase 2.4L and Flavourzyme 500L suggest that both hydrolysates mediate their insulinotropic activity through the KATP channel-dependent pathway. Additionally, by stimulating a significant increase in intracellular cAMP release (p < 0.05) it is likely that the trimmings-derived hydrolysate may also mediate insulin secretion through the protein kinase A pathway. The results presented herein demonstrate that salmon co-product hydrolysates exhibit promising in vitro antidiabetic activity.

Beneficial long-term antidiabetic actions of N- and C-terminally modified analogues of apelin-13 in diet-induced obese diabetic mice.

AIMS: To investigate the chronic effects of twice-daily administration of stable apelin analogues, apelin-13 amide and pyroglutamyl (pGlu) apelin-13 amide, on metabolic variables in glucose-intolerant and insulin-resistant diet-induced obese mice fed a high-fat diet for 150 days. METHODS: Groups of mice received twice-daily (9 am and 5 pm) injections of saline vehicle, apelin-13 amide, (pGlu)apelin-13 amide or exendin-4(1-39) for 28 days (all at 25 nmol/kg). Energy intake, body weight, non-fasting blood glucose, plasma insulin, glucose tolerance, metabolic response to feeding and insulin sensitivity, together with pancreatic hormone content and biochemical variables such as lipids and total GLP-1 were monitored. Dual-energy X-ray absorptiometry analysis and indirect calorimetry were also performed. RESULTS: Administration of apelin-13 amide, (pGlu)apelin-13 amide or exendin-4 significantly decreased body weight, food intake and blood glucose and increased plasma insulin compared with high-fat-fed saline-treated controls (P < .05 and P < .001), Additionally, all peptide-treated groups exhibited improved glucose tolerance (oral and intraperitoneal), metabolic responses to feeding and associated insulin secretion. (pGlu)apelin-13 amide also significantly improved glycated haemoglobin and insulin sensitivity after 28 days. Both (pGlu)apelin-13 amide and exendin-4 increased bone mineral content and decreased respiratory exchange ratio, whereas only (pGlu)apelin-13 amide increased energy expenditure. All treatment groups displayed reduced circulating triglycerides and increased glucagon-like peptide-1 concentrations, although only (pGlu)apelin-13 amide significantly reduced LDL cholesterol and total body fat, and increased pancreatic insulin content. CONCLUSION: These data indicate the therapeutic potential of stable apelin-13 analogues, with effects equivalent to or better than those of exendin-4.

Acylated apelin-13 amide analogues exhibit enzyme resistance and prolonged insulin releasing, glucose lowering and anorexic properties.

The adipokine, apelin has many biological functions but its activity is curtailed by rapid plasma degradation. Fatty acid derived apelin analogues represent a new and exciting avenue for the treatment of obesity-diabetes. This study explores four novel fatty acid modified apelin-13 analogues, namely, (Lys8GluPAL)apelin-13 amide, pGlu(Lys8GluPAL)apelin-13 amide, Lys8GluPAL(Tyr13)apelin-13 and Lys8GluPAL(Val13)apelin-13. Fatty acid modification extended the half-life of native apelin-13 to >24 h in vitro. pGlu(Lys8GluPAL)apelin-13 amide was the most potent insulinotropic analogue in BRIN-BD11 cells and isolated islets with maximal stimulatory effects of up to 2.7-fold (p < .001). (Lys8GluPAL)apelin-13 amide (1.9-fold) and Lys8GluPAL(Tyr13)apelin-13 (1.7-fold) were less effective, whereas Lys8GluPAL(Val13)apelin-13 had an inhibitory effect on insulin secretion. Similarly, pGlu(Lys8GluPAL)apelin-13 amide was most potent in increasing beta-cell intracellular Ca2+ concentrations (1.8-fold, p < .001) and increasing glucose uptake in 3T3-L1 adipocytes (2.3-fold, p < .01). Persistent biological action was observed with both pGlu(Lys8GluPAL)apelin-13 amide and (Lys8GluPAL)apelin-13 amide significantly reducing blood glucose (39-43%, p < .01) and enhancing insulin secretion (43-56%, p < .001) during glucose tolerance tests in diet-induced obese mice. pGlu(Lys8GluPAL)apelin-13 amide and (Lys8GluPAL)apelin-13 amide also inhibited feeding (28-40%, p < .001), whereas Lys8GluPAL(Val13)apelin-13 increased food intake (8%, p < .05) in mice. These data indicate that novel enzymatically stable analogues of apelin-13 may be suitable for future development as therapeutic agents for obesity-diabetes.

Comparison of stability, cellular, glucose-lowering and appetite supressing effects of oxyntomodulin analogues modified at the N-terminus.

Oxyntomodulin (Oxm) possesses beneficial biological actions for the potential treatment of obesity-diabetes. However, rapid inactivation by dipeptidyl peptidase-4 (DPP-4) results in a short half-life, hindering therapeutic applicability. In the present study, six Oxm analogues namely, (Thr(2))Oxm, (Asp(3))Oxm, (Aib(2))Oxm, (d-Ser(2))Oxm, (N-acetyl)Oxm and (d-Ser(2))Oxm-Lys-γ-glutamyl-PAL were synthesised and tested for DPP-4 stability and biological activity. Native Oxm, (Thr(2))Oxm and (Asp(3))Oxm were rapidly degraded by DPP-4, while (Aib(2))Oxm, (d-Ser(2))Oxm, (N-acetyl)Oxm and (d-Ser(2))Oxm-Lys-γ-glutamyl-PAL were resistant to degradation. All peptides stimulated cAMP production (P<0.01 to P<0.001) in GLP-1-R, but not in GIP-R, transfected cells. In glucagon-R transfected cells, all peptides except (N-acetyl)Oxm and (Thr(2))Oxm evoked significant cAMP generation. Similarly, all analogues, except (N-acetyl)Oxm, exhibited prominent (P<0.05 to P<0.001) insulinotropic activity in BRIN BD11 cells. When administered in conjunction with glucose to normal mice only native Oxm, (Aib(2))Oxm and (d-Ser(2))Oxm significantly (P<0.05 to P<0.01) increased overall plasma insulin levels. The corresponding glycaemic excursion was significantly (P<0.05 to P<0.001) lowered by all Oxm peptides, barring (N-acetyl)Oxm. Further investigations revealed persistent glucose-lowering and insulin-releasing actions of (d-Ser(2))Oxm-Lys-γ-glutamyl-PAL. Studies in GIP- and GLP-1-receptor KO mice with (Aib(2))Oxm, (d-Ser(2))Oxm, and (d-Ser(2))Oxm-Lys-γ-glutamyl-PAL highlighted the importance of GLP-1 receptor signalling for the beneficial glucose homoeostatic actions of these analogues. All peptides, except (N-acetyl)Oxm, possessed significant appetite suppressive effects in mice. These data highlight the significant therapeutic promise of enzymatically stable Oxm-based peptides, particularly with position 2 modifications, for the treatment of obesity-diabetes.

Ablation of glucagon receptor signaling by peptide-based glucagon antagonists improves glucose tolerance in high fat fed mice.

Modification to the structure of glucagon has provided a number of glucagon receptor antagonists with possible therapeutic application for diabetes. These novel peptide analogs include desHis(1)Pro(4)Glu(9)-glucagon and desHis(1)Pro(4)Glu(9)(Lys(30)PAL)-glucagon. This study has evaluated the metabolic benefits of once daily administration of desHis(1)Pro(4)Glu(9)-glucagon and desHis(1)Pro(4)Glu(9)(Lys(30)PAL)-glucagon in high fat (45%) fed mice for 15 days. Administration of desHis(1)Pro(4)Glu(9)-glucagon and desHis(1)Pro(4)Glu(9)(Lys(30)PAL)-glucagon had no significant effect on body weight, food intake or circulating glucose concentrations during the treatment period. However, both peptides significantly (P<0.05 to P<0.01) reduced circulating plasma insulin concentrations from day 6 onwards. Oral glucose tolerance and insulin sensitivity, as assessed by exogenous insulin administration, were significantly (P<0.01 to P<0.001) improved by both desHis(1)Pro(4)Glu(9)-glucagon and desHis(1)Pro(4)Glu(9)(Lys(30)PAL)-glucagon. These metabolic benefits were accompanied by significantly (P<0.01) increased pancreatic insulin stores. No significant differences in blood triacylglycerol or cholesterol levels were noted with desHis(1)Pro(4)Glu(9)-glucagon, however desHis(1)Pro(4)Glu(9)(Lys(30)PAL)-glucagon treatment significantly (P<0.01) increased HDL-cholesterol levels. Glucagon-mediated elevations of glucose and insulin were effectively (P<0.01 to P<0.001) annulled in both treatment groups on day 15. Interestingly, glucose levels during an intraperitoneal glucose tolerance test were not altered by either desHis(1)Pro(4)Glu(9)-glucagon or desHis(1)Pro(4)Glu(9)(Lys(30)PAL)-glucagon treatment. These data provide further evidence that glucagon antagonism could provide an effective means of treating T2DM.

A novel DPP IV-resistant C-terminally extended glucagon analogue exhibits weight-lowering and diabetes-protective effects in high-fat-fed mice mediated through glucagon and GLP-1 receptor activation.

AIMS/HYPOTHESIS: Modification of the structure of glucagon could provide useful compounds for the potential treatment of obesity-related diabetes. METHODS: This study evaluated N-acetyl-glucagon, (D-Ser(2))glucagon and an analogue of (D-Ser(2))glucagon with the addition of nine amino acids from the C-terminal of exendin(1-39), namely (D-Ser(2))glucagon-exe. RESULTS: All analogues were resistant to dipeptidyl peptidase IV degradation. N-Acetyl-glucagon lacked acute insulinotropic effects in BRIN BD11 cells, whereas (D-Ser(2))glucagon and (D-Ser(2))glucagon-exe evoked significant (p < 0.001) insulin release. (D-Ser(2))glucagon-exe stimulated cAMP production (p < 0.001) in glucagon- and GLP-1-receptor (GLP-1R)-transfected cells but not in glucose-dependent insulinotropic polypeptide-receptor-transfected cells. In normal mice, N-acetyl-glucagon and (D-Ser(2))glucagon retained glucagon-like effects of increasing (p < 0.001) plasma glucose and insulin levels. (D-Ser(2))glucagon-exe was devoid of hyperglycaemic actions but substantially (p < 0.001) increased plasma insulin levels. (D-Ser(2))glucagon-exe reduced the glycaemic excursion (p < 0.01) and increased the insulin secretory (p < 0.01) response following a glucose challenge 12 h after administration. Studies in GLP-1R knockout mice confirmed involvement of the GLP-1R pathway in the biological actions of (D-Ser(2))glucagon-exe. Twice-daily administration of (D-Ser(2))glucagon-exe to high-fat-fed mice for 28 days significantly (p < 0.05 to p < 0.001) reduced body weight, energy intake and non-fasting glucose levels, as well as increasing insulin concentrations. Glucose tolerance and insulin sensitivity were significantly (p < 0.01) improved and energy expenditure, O2 consumption and locomotor activity were (p < 0.05 to p < 0.001) augmented. The metabolic benefits were accompanied by increases in pancreatic islet number (p < 0.001) and area (p < 0.05), as well as beta cell area (p < 0.05). Beneficial effects were largely retained for 14 days following cessation of treatment. CONCLUSIONS/INTERPRETATION: This study emphasises the potential of (D-Ser(2))glucagon-exe for the treatment of obesity-related diabetes.

Glucagon induces translocation of glucokinase from the cytoplasm to the nucleus of hepatocytes by transfer between 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase-2 and the glucokinase regulatory protein.

Glucokinase activity is a major determinant of hepatic glucose metabolism and blood glucose homeostasis. Liver glucokinase activity is regulated acutely by adaptive translocation between the nucleus and the cytoplasm through binding and dissociation from its regulatory protein (GKRP) in the nucleus. Whilst the effect of glucose on this mechanism is well established, the role of hormones in regulating glucokinase location and its interaction with binding proteins remains unsettled. Here we show that treatment of rat hepatocytes with 25mM glucose caused decreased binding of glucokinase to GKRP, translocation from the nucleus and increased binding to 6-phosphofructo 2-kinase/fructose 2,6 bisphosphatase-2 (PFK2/FBPase2) in the cytoplasm. Glucagon caused dissociation of glucokinase from PFK2/FBPase2, concomitant with phosphorylation of PFK2/FBPase2 on Ser-32, uptake of glucokinase into the nucleus and increased interaction with GKRP. Two novel glucagon receptor antagonists attenuated the action of glucagon. This establishes an unequivocal role for hormonal control of glucokinase translocation. Given that glucagon excess contributes to the pathogenesis of diabetes, glucagon may play a role in the defect in glucokinase translocation and activity evident in animal models and human diabetes.

Effects of short-term chemical ablation of glucagon signalling by peptide-based glucagon receptor antagonists on insulin secretion and glucose homeostasis in mice.

Glucagon is a hormone with important effects on blood glucose regulation. This study has utilized the stable glucagon receptor antagonists, desHis¹Pro4Glu9-glucagon and desHis¹Pro4Glu9(Lys¹²PAL)-glucagon, to evaluate the effects of sustained inhibition of glucagon receptor signalling in normal mice. Twice-daily injection of either analogue for 10 days had no effect on food intake, body weight and non-fasting plasma glucose concentrations. However, insulin levels were significantly raised (p<0.05 to p<0.01) from day 3 onwards in desHis¹Pro4Glu9-glucagon mice. After 10 days, glucose tolerance was improved (p<0.05) in desHis¹Pro4Glu9-glucagon treated mice. Glucose-mediated insulin secretion and circulating cholesterol levels were significantly (p<0.05 to p<0.01) decreased in both treatment groups. Importantly, the effects of glucagon to increase blood glucose and insulin concentrations were still annulled on day 10. Insulin sensitivity was almost identical in all groups of mice at the end of the study. In addition, no changes in pancreatic insulin and glucagon content or islet morphology were observed in either treatment group. Finally, acute injection of desHis¹Pro4Glu9-glucagon followed by a 24-h fast in treatment naïve mice was not associated with any hypoglycaemic episodes. These data indicate that peptide-based glucagon receptor antagonists represent safe and effective treatment options for type 2 diabetes.

desHis¹Glu9-glucagon-[mPEG] and desHis¹Glu9(Lys³°PAL)-glucagon: long-acting peptide-based PEGylated and acylated glucagon receptor antagonists with potential antidiabetic activity.

Glucagon is hormone secreted from the pancreatic alpha-cells that is involved in blood glucose regulation. As such, antagonism of glucagon receptor signalling represents an exciting approach for treating diabetes. To harness these beneficial metabolic effects, two novel glucagon analogues, desHis¹Glu9-glucagon-[mPEG] and desHis¹Glu9(Lys³°PAL)-glucagon, has been evaluated for potential glucagon receptor antagonistic properties. Both novel peptides were completely resistant to enzymatic breakdown and significantly (P<0.05 to P<0.001) inhibited glucagon-mediated elevations of cAMP production in glucagon receptor transfected cells. Similarly, desHis¹Glu9-glucagon-[mPEG] and desHis¹Glu9(Lys³°PAL)-glucagon effectively antagonised glucagon-induced increases of insulin secretion from BRIN BD11 cells. When administered acutely to normal, high fat fed or ob/ob mice, both analogues had no significant effects on overall blood glucose or plasma insulin levels when compared to saline treated controls. However, desHis¹Glu9-glucagon-[mPEG] significantly (P<0.05) annulled glucagon-induced increases in blood glucose and plasma insulin levels in normal mice and had similar non-significant tendencies in high fat and ob/ob mice. In addition, desHis¹Glu9(Lys³°PAL)-glucagon effectively (P<0.05 to P<0.001) antagonised glucagon-mediated elevations of blood glucose levels in high fat fed and ob/ob mice, but was less efficacious in normal mice. Further studies confirmed the significant persistent glucagon receptor antagonistic properties of both novel enzyme-resistant analogues 4h post administration in normal mice. These studies emphasise the potential of longer-acting peptide-based glucagon receptor antagonists, and particularly acylated versions, for the treatment of diabetes.