Benefits of Sustained Upregulated Unimolecular GLP-1 and CCK Receptor Signalling in Obesity-Diabetes

Combined activation of GLP-1 and CCK1 receptors has potential to synergistically augment the appetite-suppressive and glucose homeostatic actions of the individual parent peptides. In the current study, pancreatic beta-cell benefits of combined GLP-1 and CCK1 receptor upregulation were established, before characterising bioactivity and antidiabetic efficacy of an acylated dual-acting GLP-1/CCK hybrid peptide, namely [Lys12Pal]Ex-4/CCK. Both exendin-4 and CCK exhibited (p<0.001) proliferative and anti-apoptotic effects in BRIN BD11 beta-cells. Proliferative benefits were significantly (p<0.01) augmented by combined peptide treatment when compared to either parent peptide alone. These effects were linked to increases (p<0.001) in GLUT2 and glucokinase beta-cell gene expression, with decreased (p<0.05-p<0.001) expression of NFκB and BAX. [Lys12Pal]Ex-4/CCK exhibited prominent insulinotropic actions in vitro, coupled with beneficial (p<0.001) satiety and glucose homeostatic effects in the mice, with bioactivity evident 24 h after administration. Following twice daily injection of [Lys12Pal]Ex-4/CCK for 28 days in diabetic high fat fed (HFF) mice with streptozotocin (STZ)-induced compromised beta-cells, there were clear reductions (p<0.05-p<0.001) in energy intake and body weight. Circulating glucose was returned to lean control concentrations, with associated increases (p<0.001) in plasma and pancreatic insulin levels. Glucose tolerance and insulin secretory responsiveness were significantly (p<0.05-p<0.001) improved by hybrid peptide therapy. In keeping with this, evaluation of pancreatic histology revealed restoration of normal islet alpha- to beta-cell ratios and reduction (p<0.01) in centralised islet glucagon staining. Improvements in pancreatic islet morphology were associated with increased (p<0.05) proliferation and reduced (p<0.001) apoptosis of beta-cells. Together, these data highlight the effectiveness of sustained dual GLP-1 and CCK1 receptor activation by [Lys12Pal]Ex-4/CCK for the treatment of obesity-related diabetes.


INTRODUCTION
The increasing use of GLP-1 mimetics to treat diabetes and obesity highlights the therapeutic importance of this class of drugs (1). Indeed, weight loss recently reported using the GLP-1 mimetic semaglutide in humans with obesity is highly impressive (2). Nevertheless, the pronounced metabolic benefits of bariatric surgery, often resulting in remission of diabetes, unquestionably exceed positive effects of GLP-1 mimetics, highlighting the important interplay between gut-derived hormones to induce distinct and sustained benefits in obesity-diabetes (3). In full agreement, administration of GLP-1 mimetics in combination with related gut-derived hormones imparts metabolic advantages beyond that seen with GLP-1 alone (4,5). Recent advances in peptide drug design have also witnessed generation of several unimolecular dual-or triple-acting hybrid peptides, that contain a GLP-1 backbone structure (6). For example, Tirzepatide, a synthetic dual-acting GIP and GLP-1 receptor agonist, demonstrates remarkable antidiabetic effectiveness in phase II clinical trials (7). With such successes in mind, a strong rational exists for generating unimolecular agents that mimic mealinduced gut hormone release by co-activating receptors for GLP-1 plus other hormones involved in conveying the metabolic and satiety signals initiated by feeding.
Cholecystokinin (CCK) is a hormone primarily secreted from gastrointestinal I-cells as well as neurons of the enteric and central nervous systems (8). The peptide circulates in various molecular forms, with the octapeptide CCK-8 being the smallest form to exhibit full biological activity (9). To impart its physiological actions, CCK binds and activates specific receptors, namely CCK1 and CCK2, that modulate various physiological processes including satiety, pancreatic endocrine, and exocrine function as well as gastric acid and bile secretion (8). In particular, CCK-based regulation of energy intake and pancreatic endocrine islet function make the hormone a potentially attractive therapeutic agent for obesity and diabetes (9). As such, CCK dose-dependently inhibits food intake consistently across species, from rodents (10) to non-human primates (11) and humans (12) mediated via CCK1 receptors (13). CCK also stimulates insulin secretion in rodents and humans (14,15), with sustained administration of a longacting CCK1 selective receptor agonist exerting notable metabolic benefits in rodent models of type 2 diabetes (16,17). In agreement, transgenic mice with genetically-induced CCK ablation (18) or overexpression (19) exhibit impaired or improved beta-cell function, respectively. Moreover, CCK has recently been shown to exert important beta-cell proliferative and anti-apoptotic effects (20), which might counter beta-cell loss in diabetes.
When taken together, the biological action profile of CCK has strong parallels with those of GLP-1 (9). Both hormones initiate separate, but complementary, cell signalling pathways via phospholipase C or adenylate cyclase, respectively (21). At the level of the pancreatic beta-cell an important GLP-1/CCK intraislet loop has also been described that helps protect against betacell apoptosis, with obvious benefits for diabetes (21). Thus, it is unsurprising that combined activation of CCK1 and GLP-1 receptors has been noted to induce numerous benefits on body weight reduction and blood glucose control (22)(23)(24). Preliminary studies also provide proof-of-principle evidence for this therapeutic approach, both in terms of combined administration of individual long-acting GLP-1 and CCK1 agonists (5,25), as well as dual-acting hybrid peptides (26,27). This is highly relevant for clinical translation since methods to augment the antidiabetic effectiveness of approved GLP-1 therapeutics are much sought after (28).
Two separate dual-acting GLP-1/CCK fusion peptides have been described to date, with both exerting remarkable benefits on body weight reduction and pancreatic islet morphology, resulting in improved metabolic control in obese high fat fed mice (26,27). Although uncharacterised in terms of in vivo islet effects, the fusion peptide described by Hornigold and colleagues (27) appeared to have the most attractive biological action profile, with confirmed balance between GLP-1 and CCK1 receptor activation. Based on this knowledge, and to progress attractiveness of the GLP-1/CCK hybrid peptide approach, we employed acylation of this molecule to create a compound capable of sustained, simultaneous activation of GLP-1 and CCK1 receptors, namely [Lys 12 Pal]Ex-4/CCK, with a pharmacodynamic profile more suitable for clinical development.
Thus, a C-16 fatty acid was conjugated to the free amino group of Lys 12 in the GLP-1/CCK hybrid peptide via a glutamyl linker ( Table 1).
Our primary objective was to examine the therapeutic efficacy and islet cell morphology after 28 days twice daily treatment with [Lys 12 Pal]Ex-4/CCK in high fat fed (HFF) mice with limited betacell compensation induced by low-dose STZ administration. Prior to this, bioactivity of [Lys 12 Pal]Ex-4/CCK was established in BRIN BD11 beta-cells, alongside assessment of acute and more prolonged in vivo effects on satiety and glucose homeostasis in mice. In addition, the positive interplay between GLP-1 and CCK receptor signalling on beta-cell growth and survival, with related impact on gene expression, was also studied. Ultimately, our data confirm that the profound beneficial pharmacological effects of combined activation GLP-1 and CCK receptor pathways in obesity-diabetes can be effectively harnessed within an appropriately designed single long-acting hybrid peptide.

Peptides
[Lys 12 Pal]Ex-4/CCK ( Table 1) and related peptides were synthesised by Synpeptide (Shanghai, China) at 95% purity and confirmed in-house by high-performance liquid

In Vitro Effects of GLP-1 and CCK1 Receptor Activation on Beta-Cell Gene Expression, Proliferation, and Apoptosis
Initial studies examined the impact of upregulated GLP-1 and CCK1 receptor signalling on the expression of key genes involved in insulin secretion and cell survival in BRIN BD11 beta-cells (29). Messenger RNA (mRNA) was isolated from cells (150,000 cells) following 18 h incubation with exendin-4, CCK-8, or a combination of both peptides (all at 10 -6 M) using the RNeasy mini kit (Qiagen, UK) following manufacturer's protocols. Utilising SuperScript ™ II Reverse Transcriptase (Invitrogen Life Technologies, Carlsbad, CA, USA), mRNA was converted to cDNA as described previously. Expression of genes associated with apoptosis (BAX and NFkB) and insulin secretion (Glut2 and Glucokinase) were determined by quantitative real-time PCR, normalised to internal house-keeping control GAPDH (30). Primer sequences for genes studied were as previously described from our laboratory (31 18-h fasted) and insulin sensitivity (25 U/kg bovine insulin; i.p.; non-fasted) tests were conducted. Terminal analyses involved dissection of pancreatic tissue, which was processed for quantification of hormone content or pancreatic islet morphology, following acid ethanol protein extraction or fixation in 4% PFA, respectively (33).

Biochemical Analyses
Blood samples were obtained from conscious mice via the cut tip on the tail vein and blood glucose immediately measured using an Ascencia Contour blood glucose meter (Bayer Healthcare Newbury, UK). Blood was collected in chilled heparin/fluoride coated microcentrifuge tubes (Sarstedt, Numbrecht, Germany) and centrifuged for 15 minutes at 12,000 rpm using a Beckman microcentrifuge (Beckman Instruments, Galway, Ireland) to separate plasma. Insulin and glucagon were then measured by radioimmunoassay (32) or commercially available ELISA (EZGLU-30K, Merck Millipore, Burlington, Massachusetts), respectively.

Immunohistochemistry
Immunohistochemistry was used to examine islet morphology by staining for insulin (1:400; ab6995, AbCam) or glucagon (1:1000; ab92517, AbCam). Image analysis was carried out using Cell F image analysis software (Olympus Soft Imaging Solutions, GmbH, Münster, Germany). To assess islet morphology, areas of insulin and glucagon positive staining were quantified using a "closed polygon" and expressed as islet/beta-/alpha-cell areas in µm 2 , as described previously (34). To assess beta-cell proliferation and apoptosis, co-staining of insulin with Ki-67 (1:400; ab15580, AbCam) or TUNEL (In situ cell death kit, Fluorescein; Roche Diagnostics) was conducted. For quantification, the number of insulin-positive cells coexpressing Ki-67 or TUNEL respectively were counted using ImageJ software, with >80 islets analysed per treatment group. In all cases, following incubation with primary antibodies, the following secondary antibodies Alexa Fluor594 goat antimouse IgG and Alexa Fluor488 goat anti-rabbit, were used as appropriate (1:400; ThermoFisher Scientific). Slides underwent a final incubation with DAPI before being mounted for imaging using a fluorescence microscope (Olympus system microscope, model BX51) fitted with DAPI (350 nm), FITC (488 nm) and TRITC (594 nm) filters and an Olympus XM10 camera system.

Statistical Analyses
Statistical tests were conducted using GraphPad PRISM software (Version 5.0). Values are expressed as mean ± SEM. Comparative analyses between groups were performed using a one-way ANOVA with Bonferroni's post hoc test, a two-way ANOVA with Bonferroni's post hoc test or Student's unpaired t-test, as appropriate. Differences were deemed significant if p<0.05.

Effects of [Lys 12 Pal]Ex-4/CCK on Body
Weight, Food Intake as well as Circulating Glucose, Insulin, and Glucagon Twice daily treatment with [Lys 12 Pal]Ex-4/CCK in HFF mice with STZ-induced compromised beta-cells, namely HFF-STZ mice, resulted in significant (p<0.05) weight loss on day 7, that persisted until the end of the study ( Figure 5A). Interestingly, there was a small decline of body weight in all mice on day 3, likely as a result of treatment regimen acclimatisation, with body weights of saline treated mice then stabilising compared with a continued gradual reduction of weight in mice receiving [Lys 12 Pal]Ex-4/CCK ( Figure 5A). Indeed, when body weight change over the 28 days was analysed, only [Lys 12 Pal]Ex-4/CCK treated HFF-STZ mice presented with a reduction (p<0.05) in body weight gain ( Figure 5B). [Lys 12 Pal]Ex-4/CCK induced body weight reductions were associated with consistent decreases (p<0.05-p<0.001) in energy intake ( Figure 5C). On day 28, circulating glucose in [Lys 12 Pal]Ex-4/CCK HFF-STZ mice was similar to lean controls, and significantly (p<0.001) reduced when compared to HFF-STZ control mice ( Figure 5D). Corresponding plasma insulin levels were decreased in HFF-STZ control mice, and elevated (p<0.01) to levels above lean controls by [Lys 12 Pal]Ex-4/CCK intervention ( Figure 5E). Interestingly, plasma glucagon was not different between lean and diabetic control mice on day 28, but [Lys 12 Pal]Ex-4/CCK decreased (p<0.05) circulating glucagon when compared to HFF-STZ control mice ( Figure 5F).

Effects of [Lys 12 Pal]Ex-4/CCK on Glucose Tolerance, Insulin Sensitivity, and Pancreatic Insulin and Glucagon Content
Following a glucose challenge on day 28, [Lys 12 Pal]Ex-4/CCK mice presented with substantially (p<0.001) improved glucose homeostasis ( Figures 6A, B), that was associated with a significant (p<0.05) augmentation of glucose-induced insulin release ( Figures 6C, D), when compared to HFF control mice Values are mean ± SEM (n=4). lll p < 0.001 compared to untreated controls. FFF p < 0.001 compared to cytokine mix. *p < 0.05, **p < 0.01, ***p < 0.001 compared to exendin-4 alone. D p < 0.05 and DDD p < 0.001 compared to CCK-8 alone.  Figures 7A, B). However, elevations (p<0.001) of alpha-cell area in HFF-STZ mice were fully reversed by [Lys 12 Pal]Ex-4/CCK treatment ( Figure 7C), which was associated with a trend towards normalisation of alpha:beta ratio in these mice ( Figure 7D). Moreover, the characteristic appearance of increased glucagon staining within the centre of islets in HFF-STZ mice was reversed by [Lys 12 Pal]Ex-4/CCK treatment ( Figure 7E). In addition, beta-cell proliferation was increased (p<0.05) and beta-cell apoptosis decreased (p<0.001), respectively, by [Lys 12 Pal]Ex-4/CCK treatment compared to HFF/STZ mice ( Figures 7F, G), with beta-cell proliferation also increased (p<0.001) when compared to lean control mice ( Figure 7F).  receptor activation have been documented previously (23,36). In the current study, we have exploited this in terms of realising a new avenue for the treatment of T2DM. First, we examined the potential benefits of upregulated GLP-1 and CCK1 receptor activation on pancreatic beta-cells, given the recent observations that CCK may be less influential in terms of augmenting GLP-1 induced beta-cell insulin secretion (37). This is an important aspect to understand because GLP-1 and CCK are not only synthesised in the intestine but also released locally by islet cells to exert autocrine or paracrine actions (19,20). Independent benefits on beta-cell growth and survival following upregulation of GLP-1 and CCK receptor activation were verified (18,34). Although additive effects were not apparent in terms of protection against apoptosis, there were clear benefits of combined receptor upregulation on beta-cell proliferation. Interestingly, interactions between islet-derived GLP-1 and CCK have been suggested to be important for betacell survival, rather than growth (21). Our contrasting observations may be linked to the experimental system employed, as well as differences in duration and extent of receptor activation. However, proliferative benefits of GLP-1 and CCK in the current setting are fully supported by changes in gene expression. As such, beta-cell regeneration is believed to be controlled by glucose metabolism and metabolic demand (38), with combination GLP-1 and CCK-8 treatment resulting in distinct increases of glucose sensor glucokinase gene expression (39). Combined treatment also resulted in a reduction in proapoptotic NFkB and BAX gene expression in line with known downstream actions of CCK (40). When viewed in conjunction with previous observations (5,25), these data provide a compelling case to further explore the therapeutic potential of GLP-1/CCK dual-acting hybrid peptides (26,27).
To this end, [Lys 12 Pal]Ex-4/CCK was engineered as a longacting GLP-1 and CCK1 receptor agonist (Table 1), based on earlier positive observations of pronounced bioactivity of a GLP-1/CCK-8 fusion peptide and the pharmacodynamic benefits of peptide acylation (27,41). As such, [Lys 12 Pal]Ex-4/CCK combines the key complementary glucose-lowering and satiety actions of exendin-4 and CCK-8, possessing an exendin-4 like Nterminus fused to a C-terminal CCK1 receptor agonist through use of a polyether linking compound ( Table 1). Consistent with previous studies, activation of GLP-1 and CCK1 receptors by [Lys 12 Pal]Ex-4/CCK stimulated insulin secretion from BRIN   (26,27). Encouraged by these observations, we embarked on a longerterm in vivo study in mice that were administered low dose injections of STZ daily over 14 days to compromise beta-cell function in association with ad libitum access to high fat diet to induce insulin resistance and beta-cell stress (33). The resulting phenotype was characterised by failure of classical islet hypertrophy and beta-cell compensation in HFF-STZ mice, culminating in severe hyperglycaemia with blood glucose >12 mmol/l. Twice daily injection of these mice with [Lys 12 Pal]Ex-4/CCK for 28 days led to significant reductions in energy intake, body weight and blood glucose levels, in keeping with upregulated GLP-1 and CCK1 receptor activity (9). Importantly, these beneficial effects were apparent early in the treatment regimen and persisted throughout, with no indication of desensitisation (42). This highlights the long-acting nature and efficacy of [Lys 12 Pal]Ex-4/ CCK. Development of a specific assay to measure [Lys 12 Pal]Ex-4/ CCK in plasma, as well as tissue distribution, would be useful to provide more precise details of in vivo bioavailability. Improvements in glucose tolerance in HFF mice with STZinduced compromised beta-cells, although likely to be partially dependent on weight loss (43), were associated with significantly increased glucose-induced insulin concentrations. Indeed, circulating and pancreatic insulin were substantially increased in [Lys 12 Pal]Ex-4/CCK mice at the end of the 28-day study. It would also have been useful to examine metabolic effects following an oral nutrient challenge, but unfortunately that was not possible in the current study. Interestingly, the characteristic insulin resistance evoked by sustained high fat feeding (44) did not appear to be appreciably improved by [Lys 12 Pal]Ex-4/CCK intervention. Evidence for a small but significant enhancement of AAC glucose was noted, but this modest effect could be related to the fact that blood glucose levels in [Lys 12 Pal]Ex-4/CCK treated mice were well below 5 mmol/l during the experiment. This presumably resulted in activation of adaptive responses to counter the hypoglycaemia induced by bolus injection of exogenous insulin. In contrast to the small magnitude of this effect, [Lys 12 Pal]Ex-4/CCK evoked particularly prominent improvements in islet architecture in the HFF-STZ mice (19). Such morphological benefits which were closely linked to elevations in beta-cell proliferation (20,34), in direct agreement with our in vitro observations. We also observed reductions in beta-cell apoptosis with sustained [Lys 12 Pal]Ex-4/CCK therapy that would also contribute to improved islet architecture (21). Furthermore, observed reductions in alpha-cell area and overall alpha:beta cell ratios were paralleled by decreases in pancreatic and plasma glucagon in the peptide treated mice. This observation fits well with reported glucagonostatic effects of GLP-1 receptor activation (45). However, it does contrast somewhat with the reported direct glucagonotropic actions of CCK (8), and detailed study of the overall impact of activation of GLP-1 and CCK1 receptors by [Lys 12 Pal]Ex-4/CCK in this regard is required.
Despite the obvious therapeutic attractiveness of [Lys 12 Pal]Ex-4/CCK, there are a number of significant matters that need to be considered with regards possible translation towards clinical development. Although initial fears of increased pancreatitis risk with GLP-1 mimetic therapy in humans has now been fully allayed (46), extremely high doses of CCK are employed to create experimental rodent models of pancreatic inflammation (47). However, it should be noted that the CCK1 receptor is expressed at much higher levels in pancreatic acinar cells of rodents than humans (48). Moreover, 28 days treatment with [Lys 12 Pal]Ex-4/CCK did not lead to any obvious detrimental pancreatic histopathological findings in our study. As noted above, alterations in endocrine islet morphology induced by [Lys 12 Pal]Ex-4/CCK were consistently linked to correction of the diabetes phenotype. In addition, previous studies employing sustained co-activation of GLP-1 and CCK1 receptors in rodent models reported no effect, or even reductions, in amylase and lipase secretion (25,26). The possible impact of combined GLP-1 and CCK1 receptor activation on gastric emptying would also need to be considered (49,50). However, we observed no obvious behavioural changes in [Lys 12 Pal]Ex-4/CCK treated mice. Finally, there is a suggestion that GLP-1 mimetics suppress CCK secretion in humans, leading to adverse gallbladder events (51). Thus, dual activation of both receptor pathways by [Lys 12 Pal]Ex-4/CCK could help to alleviate this potential problem.
In conclusion, the present study has reaffirmed clear benefits of combined GLP-1 and CCK1 receptor signalling. [Lys 12 Pal]Ex-4/CCK, created as a long-acting, dual GLP-1 and CCK1 receptor activating hybrid peptide, displayed prominent antidiabetic actions in HFF-STZ mice that were directly linked to improved islet morphology and beta-cell function. Such benefits were also coupled to induction of satiety and sustained reductions in body weight. These exciting preclinical observations necessitate the further consideration of combined GLP-1 and CCK1 receptor activation as a potential treatment option for the increasing numbers of people living with obesity and related diabetes.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by the Ulster University Animal Welfare and Ethical Review Body (AWERB).

AUTHOR CONTRIBUTIONS
NI and PF contributed to the overall concept and experimental design, and reviewed the manuscript. AE, NT, and RL researched data, contributed to data interpretation, and edited the manuscript. NI, NT, and PF wrote the manuscript. All authors contributed to the article and approved the submitted version. show (H) insulin (red) and glucagon (green), (I) insulin (red) and Ki-67 (green) or (J) insulin (red) and TUNEL (green) from each treatment group. Values are mean ± SEM for 8 mice. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with HFF-STZ diabetic control. DD p < 0.01 and DDD p < 0.001 compared with lean control.