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.


Introduction
It has been well established that abnormal elevation in circulating glucagon leads to an increase in hepatic glucose production and glycogen metabolism that contribute to hyperglycaemia in diabetes (Unger 1978). For this reason, blockade of glucagon receptor (GCGR) signalling has been widely regarded as a potential therapeutic option to help control blood glucose levels for the treatment of diabetes (Patil et al. 2020, Lafferty et al. 2021). In addition, some recent observations (Wang et al. 2021), coupled with earlier R A Lafferty et al. 255:2 et al. , Pettus et al. 2020, monoclonal antibodies (Kim et al. 2012, Okamoto et al. 2015 or antisense oligonucleotides (Liang et al. 2004, Morgan et al. 2019. Although all approaches possess robust glucose-lowering actions, the adverse side effect profile of each has been questioned (Patil et al. 2020, Lafferty et al. 2021. To date, it appears that peptide-based GCGR antagonists offer the best efficacy vs side effect profile , 2022, McShane et al. 2014. Whether this relates to the composition of the compounds in question, or overall potency and degree of GCGR blockade, remains to be determined. However, a wealth of data suggests that organic peptides, such as desHis 1 Pro 4 Glu 9 -glucagon, represent highly effective GCGR antagonists , Franklin et al. 2022. Indeed, other truncated glucagon-based peptides have recently been shown to yield selective, high potency, GCGR antagonists (Yang et al. 2021), supporting this as an effective approach to decrease GCGR activity. Moreover, an acylated, longer-acting, version of desHis 1 Pro 4 Glu 9glucagon has been described, namely desHis 1 Pro 4 Glu 9glucagon(Lys 12 PAL), that also effectively antagonises the GCGR . This analogue has a palmitic acid covalently attached to the Lys 12 residue of desHis 1 Pro 4 Glu 9 -glucagon via a γ-glutamyl spacer molecule, delivering a significantly extended pharmacodynamic profile . Notably, our previous work fully characterises the in vitro and acute in vivo biological action profile of both desHis 1 Pro 4 Glu 9 -glucagon and desHis 1 Pro 4 Glu 9 -glucagon(Lys 12 PAL), including effects on cAMP accumulation, insulin secretion, inhibition of glucagon action, glucose disposal and islet hormone secretion . Moreover, we have also previously shown that sustained administration of desHis 1 Pro 4 Glu 9 -glucagon, or its Lys 12 acylated counterpart, can reverse aspects of genetically induced and dietary-induced obesity-related diabetes in obese-diabetic (ob/ob) and high-fat-fed (HFF) mice, respectively . However, both these murine models of diabetes are associated with adaptive beta-cell expansion prior to the development of overt diabetes. In this regard, administration of the beta-cell toxin, streptozotocin (STZ), can counter betacell compensation and prevent such innate adaptations (Furman 2015). Thus, HFF mice with STZ-induced compromised beta-cells are characterised by obstruction of the classical beta-cell hypertrophy in response to prolonged high-fat feeding (Tanday et al. 2021). Therefore, this HFF-STZ murine model represents an ideal tool to fully explore the positive effects of peptide-based GCGR antagonists in obesity-driven forms of diabetes, where restoration of functional beta-cell mass would be highly advantageous. Notably, the benefits of GCGR blockade are believed to require at least some residual beta-cell function (Damond et al. 2016), which would be the case for HFF-STZ mice (Tanday et al. 2021).

Journal of Endocrinology
Consequently, in the current study, we have investigated the impact of once-daily treatment with desHis 1 Pro 4 Glu 9 -glucagon or desHis 1 Pro 4 Glu 9glucagon(Lys 12 PAL) in HFF-STZ mice for 18 days. Effects on food and fluid intake as well as body weight and circulating glucose were assessed at regular intervals. The metabolic status of the mice was then examined at the end of the treatment period through glucose and insulin tolerance tests. Finally, aspects of indirect calorimetry and pancreatic hormone content were also investigated. Taken together, we reveal that peptidic GCGR antagonists possess metabolic benefits following STZ-induced betacell insult in insulin-resistant HFF mice, which merits further investigation in terms of translation to the clinical setting.

Peptides
All peptides were synthesised by Synpeptide (Shanghai, China) at 95% purity, which was confirmed in-house by high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry (MS), as previously described (Lafferty et al. 2020).

Animals
Young male NIH Swiss mice (10-week-old; n = 8) were maintained on high-fat diet (45% fat, 20% protein and 25% carbohydrates; percent of total energy of 26.15 kJ/g; Special Diets Services, Witham, Essex, UK) for 12 weeks, by which stage obesity was clearly manifested. After this period, mice were administered with a single large i.p. dose of STZ (4-h fast, 125 mg/kg bw, dissolved in sodium citrate buffer, pH 4.5). A separate group of HFF mice that did not receive STZ injection were employed as an additional control group. Appropriate numbers of non-diabetic control mice were not available for inclusion in the current study, but the basic phenotypes of HFF mice such as obesity, impaired, glucose R A Lafferty et al.

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Journal of Endocrinology tolerance, hyperinsulinaemia and insulin resistance were confirmed.

Chronic in vivo experiments
Groups (n = 8) of HFF-STZ mice received once-daily i.p. injections (10:00 h) of saline vehicle (0.9% (w/v) NaCl), desHis 1 Pro 4 Glu 9 -glucagon or desHis 1 Pro 4 Glu 9glucagon(Lys 12 PAL) (both at 25 nmol/kg bw) for 18 days, starting on the same day that STZ was administered. To acclimatise mice to the injection regimen, all mice received once-daily i.p. injections of saline over a 6-day run in period.

Biochemical analyses
Blood samples were obtained from conscious mice via the cut tip on the tail vein, and blood glucose was immediately measured using an Ascencia Contour blood glucose meter (Bayer Healthcare, Newbury, UK). Pancreatic or plasma insulin and glucagon, as appropriate, were measured by in-house RIA (Flatt & Bailey 1981) or commercially available ELISA (EZGLU-30K, Merck Millipore), respectively.

Statistical analyses
Statistical tests were conducted using GraphPad PRISM software (Version 5.0). Values are expressed as mean ± s.e.m. Comparative analyses between groups were performed using a one-way or two-way ANOVA with Bonferroni's post hoc test, as appropriate. Differences were deemed significant if P < 0.05.

Results
Effects of desHis 1 Pro 4 Glu 9 -glucagon or desHis 1 Pro 4 Glu 9 -glucagon(Lys 12 PAL) on food and fluid intake, body weight, circulating glucose and glucagon in HFF-STZ mice Food intake was significantly (P < 0.05-P < 0.001) increased in all HFF mice that received STZ injection (Fig. 1A). Only treatment with desHis 1 Pro 4 Glu 9 -glucagon led to reductions (P < 0.05) of STZ-induced elevations of food intake that was evident on days 10 and 12

Figure 1
Effects of once-daily administration of desHis 1 Pro 4 Glu 9 -glucagon or desHis 1 Pro 4 Glu 9glucagon (Lys 12 PAL) (each at 25 nmol/kg bw) for 18 days on cumulative food intake (A), cumulative fluid intake (B), body weight (C) and blood glucose (D) in HFF-STZ mice. Measurements were taken 6 days prior to and throughout the treatment period, at regular intervals. The treatment period is highlighted by the horizontal black bar parallel to x-axis. Values are mean ± s.e.m. (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001 compared to HFF-STZ saline controls. Ω P < 0.05 compared to HFF saline controls. R A Lafferty et al.
Effects of desHis 1 Pro 4 Glu 9 -glucagon or desHis 1 Pro 4 Glu 9 -glucagon(Lys 12 PAL) on glucose tolerance and insulin sensitivity in HFF-STZ mice Following an i.p. glucose challenge, glucose levels were significantly lower (P < 0.05) 15 min post injection in both desHis 1 Pro 4 Glu 9 -glucagon and desHis 1 Pro 4 Glu 9glucagon(Lys 12 PAL)-treated HFF-STZ mice when compared to saline controls ( Fig. 2A). However, this reduction was not sustained at 30 and 60 min ( Fig. 2A), and there was no difference in 0-60 min glucose AUC values between all HFF-STZ groups of mice (Fig. 2B). Glucose-induced insulin secretory responses were almost absent in all HFF-STZ mice, with only control HFF mice displaying any real glucose-induced elevations of insulin concentrations ( Fig. 2C and D). The benefits of desHis 1 Pro 4 Glu 9 -glucagon and desHis 1 Pro 4 Glu 9 -glucagon(Lys 12 PAL) treatment were more prominent following an oral glucose tolerance challenge ( Fig. 3A and B). Thus, although individual glucose levels were still elevated in the treatment groups compared to HFF controls (Fig. 3A), 0-60 min AUC values were decreased (P < 0.05) by desHis 1 Pro 4 Glu 9 -glucagon and desHis 1 Pro 4 Glu 9 -glucagon(Lys 12 PAL) when compared to STZ-diabetic control mice, and not significantly different from HFF controls (Fig. 3B). However, glucose-induced insulin concentrations were not noticeably amplified by either treatment (Fig. 3C and D). In some agreement with this, the hypoglycaemic action of exogenous insulin was significantly (P < 0.05-P < 0.001) augmented by desHis 1 Pro 4 Glu 9 -glucagon and desHis 1 Pro 4 Glu 9glucagon(Lys 12 PAL), when compared to HFF-STZ or HFF control mice ( Fig. 4A and B). Interestingly, STZ administration alone also appeared to enhance (P < 0.05) peripheral insulin action in HFF mice ( Fig. 4A and B). As anticipated, administration of STZ significantly (P < 0.001) depressed pancreatic insulin content, but 18 days therapy with desHis 1 Pro 4 Glu 9 -glucagon(Lys 12 PAL) was able to partially reverse (P < 0.001) this detrimental effect (Fig. 4C). STZ also increased (P < 0.001) pancreatic glucagon content of HFF mice, but this effect was fully reversed by both desHis 1 Pro 4 Glu 9 -glucagon and desHis 1 Pro 4 Glu 9glucagon(Lys 12 PAL) treatment (Fig. 4D).

Figure 3
Effects of once-daily administration of desHis 1 Pro 4 Glu 9 -glucagon or desHis 1 Pro 4 Glu 9glucagon(Lys 12 PAL) (each at 25 nmol/kg bw) for 18 days on oral glucose tolerance in HFF-STZ mice (18 mmol/kg bw). Blood glucose (A) and associated plasma insulin responses (C) with respective areas under the curves (B and D) are provided. Values are mean ± s.e.m. (n = 8). *P < 0.05 compared to HFF-STZ saline controls. Ω P < 0.05 compared to HFF saline controls.

Figure 4
Effects of once-daily administration of desHis 1 Pro 4 Glu 9 -glucagon or desHis 1 Pro 4 Glu 9glucagon(Lys 12 PAL) (each at 25 nmol/kg bw) for 18 days on insulin sensitivity (A) in HFF-STZ mice, with the related area above the curve (B) presented. Insulin was administered by i.p. injection at 5 IU/kg/bw in non-fasted mice. Additionally, effects on pancreatic hormone content were assessed on day 18. Pancreatic glucagon (C) and insulin (D) levels were assessed in excised pancreatic tissue via a commercially available ELISA or in-house RIA, respectively.

255:2
Journal of Endocrinology decreased (P < 0.05-P < 0.01) X beam ambulatory breaks vs HFF-STZ controls during both the light and dark phases (Fig. 6A, B and C). A similar effect of desHis 1 Pro 4 Glu 9glucagon was also noted in terms of Z-beam breaks during the light phase (P < 0.05), which represent vertical activity levels such as mouse rearing events (Fig. 6D, E and F). Both peptide treatments had significantly (P < 0.05-P < 0.001) reduced X and Z beam breaks when compared to salinetreated HFF controls (Fig. 6A, B, C, D, E and F).

Discussion
The interplay between pancreatic alpha-and beta-cell signalling is intriguing, with secretions from beta-cells directly inhibiting alpha-cell function, whilst alpha-cells release factors that are stimulatory for beta-cells (English & Irwin 2019, Moede et al. 2020. Coupled with recent awareness that mature beta-cells de-differentiate into alpha-cells (Weir et al. 2013) and that alpha-cells can act as progenitors for functional beta-cells (Habener & Stanojevic 2012), modulation of alpha-cell activity could hold promise for the treatment of diabetes. Indeed, alpha-tobeta-cell lineage conversion is enhanced in GCGR KO mice (Damond et al. 2016), and more recently, human alphacells were shown to be capable of reprogramming into glucose-sensitive insulin-secreting cells to help ameliorate diabetes in mice (Furuyama et al. 2019). In this regard, inhibition of GCGR signalling has long been considered as a potential means of effectively controlling blood glucose levels (Unger 1978).
In the current study, we employed a high single dose of STZ as an established method to induce beta-cell cytotoxicity and perturb insulin secretory function in HFF mice (Deeds et al. 2011, Millar et al. 2017. Thus, HFF mice are classically characterised by the manifestation of insulin resistance leading to subsequent compensatory beta-cell expansion and hyperinsulinaemia (Ahrén et al. 2010). Notably, saline-treated control HFF mice did not present with overt hyperglycaemia, but as expected, glucose intolerance was evident following a glucose challenge likely as a result of dietary-induced insulin resistance (Ahrén et al. 2010). However, hyperglycaemia was clearly apparent in all GCGR antagonist-treated HFF-STZ mice by day 18, treatment intervention appeared to delay onset. Indeed, the acylated GCGR analogue partially restored pancreatic insulin concentrations, which may be linked to the more protracted bioactive profile of

Figure 5
Effects of once-daily administration of desHis 1 Pro 4 Glu 9 -glucagon or desHis 1 Pro 4 Glu 9 -glucagon(Lys 12 PAL) (each at 25 nmol/kg bw) for 18 days on O 2 consumption (A and B), CO 2 production (C and D), respiratory exchange ratio (RER) (E and F) and energy expenditure (G and H) in HFF-STZ mice. Mice were placed in CLAMS metabolic chambers for 18 h to acclimatise, and measurements were obtained over a further 24 h period (12 h dark period as shown by black bar parallel to x-axis) at the end of the treatment period. O 2 consumption and CO 2 production were measured for 30 s at 25 min intervals (A and C). RER was calculated by dividing VCO 2 by VO 2 (E and F). Energy expenditure was calculated using RER with the following equation: (3.815 + 1.232 × RER) × VO 2 (G). Average energy expenditure is also provided (H), separated into the light (LP) and dark phases (DP). Overall incremental data are presented in panels B, D, F and G, where each data point represents information collected at individual time-points over the 24 h period. Values are mean ± s.e.m. (n = 6). **P < 0.01, ***P < 0.001 compared to HFF-STZ saline controls. Ω P < 0.05, ΩΩ P < 0.01, ΩΩΩ P < 0.001 compared to HFF saline controls. R A Lafferty et al.

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Journal of Endocrinology desHis 1 Pro 4 Glu 9 -glucagon(Lys 12 PAL) over desHis 1 Pro 4 Glu 9glucagon . In agreement, GCGR blockade has recently been demonstrated to promote recovery of functional beta-cell mass in diabetic mice (Wang et al. 2021). Regrettably, a technical issue during tissue processing thwarted our efforts to investigate aspects of pancreatic islet morphology, including beta-cell mass and turnover, that would help to validate our observations. Interestingly, the augmented pancreatic insulin content, which was particularly apparent in desHis 1 Pro 4 Glu 9glucagon(Lys 12 PAL)-treated HFF-STZ mice, was not matched by prominent improvements in insulin secretory responses or glucose levels. Thus, further investigation of beta-cell secretory function and responsiveness would be required to uncover the relationship between increased insulin stores and translation to more obvious improvements of metabolism in these mice, although improvements in insulin action might also be important in this regard. Circulating and pancreatic glucagon levels were reduced in all GCGR antagonist-treated HFF-STZ, which contrasts with observations using small molecule GCGR inhibitors (Mu et al. 2011(Mu et al. , 2012, but complements previous work with peptidic GCGR antagonists , McShane et al. 2014. This also highlights the improved adverse effect profile of peptidic GCGR antagonists over other methods employed to inhibit GCGR signalling. As such, rebound hyperglycaemia has been observed on treatment termination with some small molecule GCGR antagonists (Sloop et al. 2004), likely because of their actions to elevate circulating glucagon. Benefits on glucose tolerance were more apparent following an oral as opposed to i.p. glucose challenge in desHis 1 Pro 4 Glu 9 -glucagon and desHis 1 Pro 4 Glu 9glucagon(Lys 12 PAL)-treated HFF-STZ mice. In accordance with this, GCGR antagonism-mediated improvements in glycaemic control have been suggested to be dependent on functional GLP-1 receptors (Gu et al. 2010). Indeed, more recent studies have demonstrated that GCGR blockade can promote intestinal L-cell proliferation (Lang et al. 2020a) and inhibit L-cell apoptosis (Lang et al. 2020b), leading to elevated GLP-1 synthesis and secretion. In agreement, inhibition of the incretin hormone degrading enzyme, dipeptidyl peptidase-4, improves the effectiveness of GCGR inhibition in diabetic mice (Mu et al. 2011).

Figure 6
Effects of once-daily administration of desHis 1 Pro 4 Glu 9 -glucagon or desHis 1 Pro 4 Glu 9 -glucagon(Lys 12 PAL) (each at 25 nmol/kg bw) for 18 days on locomotor activity in HFF-STZ mice. Mice were placed in CLAMS metabolic chambers for 18 h to acclimatise, and measurements were obtained over a further 24 h period (12 h dark period as shown by black bar parallel to x-axis) at the end of the treatment period. Activity counts on x-axis (A, B and C) and z-axis (D, E and F) were recorded at 60-s intervals. Overall incremental data are presented in panels B, C, E and F, where each data point represents information collected at individual time-points over the 24 h period. Values are mean ± s.e.m. (n = 6). *P < 0.05, **P < 0.01 compared to HFF-STZ saline controls. Ω P < 0.05, ΩΩ P < 0.01, ΩΩΩ P < 0.001 compared to HFF saline controls.
It has also been suggested that combined GLP-1 receptor activation and GCGR inhibition possesses beneficial actions (Claus et al. 2007). Unfortunately, we were unable to measure circulating GLP-1 concentrations in the current study due to the limited volume of blood that can be withdrawn from mice. However, we have recently shown that combined administration of a peptidic GCGR antagonist, with the well-characterised GLP-1 receptor mimetic exendin-4, exerts limited additive metabolic benefits (Franklin et al. 2022). Thus, activation of receptors for the sister incretin hormone of GLP-1, namely glucosedependent insulinotropic polypeptide, may offer a more attractive paradigm in terms of combination therapy with GCGR antagonism (McShane et al. 2016). However, intestinal L-cell number has also been demonstrated to be reduced by STZ administration (Vasu et al. 2015), which could represent another confounding factor in our current observations. Thus, both pancreatic beta-cells and enteroendocrine L-cells appear to have limited antioxidant defence mechanisms (Lenzen 2008, Vasu et al. 2015. Although, in this respect, it should be noted that by their very nature, intestinal mucosal cell turnover is rapid, with efficient cellular replacement by differentiating stem cells that arise from intestinal crypts (Roth & Gordon 1990, Schonhoff et al. 2004. Of note is the improvement of glucose handling in the absence of any real augmentation of insulin concentrations, this being despite elevated pancreatic insulin content in desHis 1 Pro 4 Glu 9 -glucagon(Lys 12 PAL)treated HFF-STZ mice. It follows that insulin action must be enhanced in these mice, which was indeed apparent following exogenous insulin injection. Similar observations have been made previously following STZ treatment in GCGR KO mice (Lee et al. 2011). In the absence of GCGR signalling, hepatic glucose output and the positive effects of GCGR signalling on basal metabolic rate are also likely to be much reduced (Breton et al. 1983). In good accord with this, in the current study, the peptidic GCGR antagonists both decreased physical activity in HFF-STZ mice. However, the ability of desHis 1 Pro 4 Glu 9glucagon(Lys 12 PAL), and particularly desHis 1 Pro 4 Glu 9glucagon, to increase energy expenditure does contrast with this notion, but this may simply highlight the plasticity of signalling pathways involved in energy homeostasis (Smith et al. 2018). The slight difference in efficacy between desHis 1 Pro 4 Glu 9 -glucagon and desHis 1 Pro 4 Glu 9glucagon(Lys 12 PAL) in terms of indirect calorimetry data could be related to free vs albumin-bound drug, where it is often considered that albumin binding reduces bioactivity of peptides (Miyakawa et al. 2013). However, more detailed pharmacokinetic studies, that are outside the scope of the current investigation, would be required to confirm this. In a similar fashion, there were also slight differences between the effect of both peptides on food and fluid intake. Thus, desHis 1 Pro 4 Glu 9 -glucagon had a mild and transient impact on moderating STZ-induced elevations of feeding, whereas desHis 1 Pro 4 Glu 9 -glucagon(Lys 12 PAL) exerted more enduring effects to counter increased fluid intake in HFF-STZ mice. Although peptide pharmacodynamic profiles may also be important in this observation, we are unable to discount alterations in the passage of either peptide through the blood-brain barrier and the subsequent impact on hypothalamic circuits that regulate energy intake and thirst (Woods 2013).
It is established that glucagon plays an important role in lipid oxidation and metabolism (Galsgaard et al. 2019), and our observed increases in RER evoked by sustained GCGR antagonism likely partly reflects this. Thus, carbohydrate oxidation drives RER to a value closer to 1.0, with fatty acid oxidation reducing this to 0.7 (Rosenkilde et al. 2010, Purdom et al. 2018. Hyperaminoacidaemia has also been reported following inhibition of GCGR signalling and assessment of plasma amino acid levels would have been interesting in this regard (Richter et al. 2022). The impact of the high-fat background diet (45%), enduring insulin deficiency and small GCGR antagonist-induced changes in food intake and body weight need to be considered in terms of overall effects on carbohydrate metabolism. In that respect, GCGR KO mice are reported to be resistant to highfat feeding-induced obesity (Conarello et al. 2007), but the possibility for life-long adaptations in these animals should not be overlooked. However, differences in the magnitude of GCGR signalling annulment between genetic and chemical receptor blockade could also be a factor. Thus, similar to the current setting, prolonged treatment with a small molecule GCGR antagonist did not affect body weight in HFF mice (Mu et al. 2011), this being despite increased energy expenditure with desHis 1 Pro 4 Glu 9 -glucagon and desHis 1 Pro 4 Glu 9 -glucagon(Lys 12 PAL) therapy. The current treatment regimen entailed once-daily peptide treatment for 18 days and whether extended dosing periods would lead to more discernible benefits on metabolism in HFF-STZ mice still needs to be established.
In summary, the current study establishes that peptide-based GCGR antagonism exerts notable benefits in obesity-driven forms of diabetes, even in the presence of insulin deficiency. As well as delaying the onset of hyperglycaemia, desHis 1 Pro 4 Glu 9 -glucagon, and particularly desHis 1 Pro 4 Glu 9 -glucagon(Lys 12 PAL), improved glucose handling and insulin action in addition to augmenting pancreatic insulin stores. Our observations further support the promise of peptidic GCGR antagonists as a new class of drugs for the management of various forms of diabetes.

Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding
This work was supported by an Invest Northern Ireland Proof-of-Concept grant (PoC106), a Department for the Economy, Northern Ireland PhD studentship and Ulster University Selective Research Funding

Data availability statement
The authors declare that the data supporting the findings of this study are available within the article. Any additional raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
Author contribution statement N I and F O H conceived/designed and supervised the study. N I, R A L and P R F drafted the manuscript. L M McS and Z J F and participated in the conduct/data collection and analysis and interpretation of data. All authors revised the manuscript critically for intellectual content and approved the final version of the manuscript.