A Novel Glucagon-like Peptide-1 (GLP-1)/Glucagon Hybrid Peptide with Triple-acting Agonist Activity at Glucose-dependent Insulinotropic Polypeptide, GLP-1, and Glucagon Receptors and Therapeutic Potential in High Fat-fed Mice*

Background: Glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and glucagon have important gluco-regulatory actions. Results: Fusion of amino acid sequences of GLP-1, GIP, and glucagon produces hybrid peptides with triple-acting agonist activity. Conclusion: Hybrid peptides possess beneficial biological actions equivalent, or superior to, activation of single receptors. Significance: Multitargeting peptides offer a new class of therapeutics for obesity and diabetes. Glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and glucagon bind to related members of the same receptor superfamily and exert important effects on glucose homeostasis, insulin secretion, and energy regulation. The present study assessed the biological actions and therapeutic utility of novel GIP/glucagon/GLP-1 hybrid peptides. Nine novel peptides were synthesized and exhibited complete DPP-IV resistance and enhanced in vitro insulin secretion. The most promising peptide, [dA2]GLP-1/GcG, stimulated cAMP production in GIP, GLP-1, and glucagon receptor-transfected cells. Acute administration of [dA2]GLP-1/GcG in combination with glucose significantly lowered plasma glucose and increased plasma insulin in normal and obese diabetic (ob/ob) mice. Furthermore, [dA2]GLP-1/GcG elicited a protracted glucose-lowering and insulinotropic effect in high fat-fed mice. Twice daily administration of [dA2]GLP-1/GcG for 21 days decreased body weight and nonfasting plasma glucose and increased circulating plasma insulin concentrations in high fat-fed mice. Furthermore, [dA2]GLP-1/GcG significantly improved glucose tolerance and insulin sensitivity by day 21. Interestingly, locomotor activity was increased in [dA2]GLP-1/GcG mice, without appreciable changes in aspects of metabolic rate. Studies in knock-out mice confirmed the biological action of [dA2]GLP-1/GcG via multiple targets including GIP, GLP-1, and glucagon receptors. The data suggest significant promise for novel triple-acting hybrid peptides as therapeutic options for obesity and diabetes.

Peripheral signals that control glucose homeostasis and energy regulation are carefully balanced and encompass a number of factors, including a variety of peptide hormones (1). The major focus on gut hormone-based therapies over the past decades has concentrated on single molecules that target one specific pathway (2). Although specific glucagon-like peptide-1 (GLP-1) 2 mimetics are used clinically for type 2 diabetes, the glycemic control and weight reductions achieved with certain types of gastric bypass surgery is markedly superior (3). Recognition is growing that these beneficial effects reflect changes in circulating levels of multiple peptide hormones that trigger a broad spectrum of pathways involved in glucose regulation and energy balance (4). Therefore, combining the activity of two or more regulatory hormones, with complementary biological actions, offers a favorable approach for the treatment of obesity and diabetes. In this context, GLP-1, glucose-dependent insulinotropic polypeptide (GIP), and glucagon possesses a number of biological effects that would suggest significant combined therapeutic effectiveness (2).
Together GIP and GLP-1 account for almost all of the well established physiological incretin effect and have powerful insulin-releasing and gluco-regulatory properties (5). Moreover, both peptides appear to have important pancreatic betacell protective actions and additional extra-pancreatic glucose lowering effects that further promote therapeutic applicability for diabetes (6). On the other hand, glucagon is classically regarded as an important hormone in maintaining normal glucose concentrations through enhanced hepatic glucose production (7). However, recent evidence now suggests that glucagon can be exploited therapeutically as a satiety factor, which also increases energy expenditure and body weight loss (8). thermore, transgenic mice overexpressing the glucagon receptor (R) in pancreatic beta-cells demonstrate increased insulin secretion and pancreatic beta-cell mass, with protection against impaired glucose tolerance following high fat feeding (9). Thus, it follows that design of a single hybrid peptide, capable of simultaneous activation of GLP-1, GIP, and glucagon Rs, would have substantially enhanced therapeutic promise for obesity and diabetes.
To generate just such a compound, we have constructed nine novel GLP-1/GIP/glucagon hybrid peptides. These hybrid peptides have been created through fusion of the key amino acid sequences of GLP-1, GIP, and glucagon known to be important for biological activity (see Table 1). Importantly, because all three peptides are substrates for dipetidyl peptidase IV (10,11), hybrid peptides with GIP or GLP-1-like N termini have substitution of the naturally occurring alanine L-isomer residue for a D-isomer, whereas peptides with a glucagon-like N terminus have substitution of alanine for serine at position 2. These specific modifications are known to impart DPP IV resistance and improve biological activity of respective parent peptides (11)(12)(13). We initially examined DPP IV resistance, in vitro insulin secretion, and in vivo glucose-lowering and insulinotropic actions of all hybrid peptides. The acute antidiabetic effects of the most efficacious hybrids were then evaluated in obese diabetic (ob/ob) mice. The most effective peptide, [dAla 2 ]GLP-1glucagon-GLP-1 ([dA 2 ]GLP/GcG), was progressed to a twice daily injection regime in high fat-fed mice to examine effects of chronic treatment on body weight, food intake, energy expenditure, nonfasting glucose and insulin, glucose tolerance, insulin sensitivity, locomotor activity, and aspects of metabolic rate. Finally, to elucidate potential mechanism of action, cAMP production capabilities of [dA 2 ]GLP/GcG were examined in cells transfected with either the GLP-1, GIP, or glucagon R, and in vivo gluco-regulatory and insulin-releasing activity was assessed in GIP, GLP-1, and double incretin R knock-out mice.

EXPERIMENTAL PROCEDURES
Peptides- Table 1 displays the amino acid sequence of the nine hybrid peptides used in this study, which were based on the structures of GLP-1, GIP, and glucagon. In addition, native GLP-1, GIP, and glucagon, along with [dAla 2 ]GIP, [dSer 2 ]glucagon, and [dAla 2 ]GLP-1 were used as control peptides. All peptides were purchased from GL Biochem Ltd. (Shanghai, China; greater than 90% purity). In addition to quality control data supplied with peptide purchased, all pep-tides were characterized in-house using MALDI-TOF MS, as described previously (14).
DPP-IV Degradation Assay-Peptides were incubated at 37°C in 50 mmol/liter TEA-HCl (pH 7.8; Sigma-Aldrich) with purified porcine DPP-IV (5 milliunits; Sigma-Aldrich) for 0, 2, 4, and 8 h. Degradation profiles were obtained using RP-HPLC analysis as described previously (14), and the HPLC peak area data were used to calculate the percentage of intact peptide remaining at time points during the incubation.
In Vitro Insulin Secretion-Effects of peptides on in vitro insulin secretion were examined using BRIN-BD11 cells whose characteristics have been reported previously (15). Briefly, BRIN-BD11 cells were seeded (150,000 cells/well) into 24-well plates (Nunc, Roskilde, Denmark) and allowed to attach overnight at 37°C. Following 40 min of preincubation (1.1 mmol/ liter glucose; 37°C), cells were incubated (20 min; 37°C) in the presence of 5.6 and 16.7 mmol/liter glucose with a range of peptide concentrations (10 Ϫ12 -10 Ϫ6 mol/liter). After 20 min of incubation, buffer was removed from each well, and aliquots (200 l) were stored at Ϫ20°C prior to determination of insulin by radioimmunoassay (16).
In Vitro cAMP Production-Effects of [DA 2 ]GLP-1/GcG, GLP-1, GIP, and glucagon on cAMP production were assessed in Chinese hamster lung cells transfected with either the human GIP-or GLP-1-R, as well as human embryonic kidney (HEK293) cells transfected with the human glucagon R (17). Cells were seeded (200,000 cells/well) into 96-well plates (Nunc) and washed with Hanks' balanced salt solution buffer before incubation with test peptides (10 Ϫ6 -10 Ϫ12 mol/liter) in the presence of 200 mol/liter 3-isobutyl-1-methylxanthine for 20 min at 37°C. After incubation, medium was removed, and the cells were lysed before measurement of cAMP using Parameter cAMP assay (R&D Systems, Abingdon, UK) according to the manufacturer's instructions.
Animals-Acute animal studies were carried out in male National Institutes of Health Swiss mice (Harlan Ltd., Blackthorne, UK; 12-14 weeks old), obese (ob/ob) mice (derived from the colony originally maintained at Aston University (18); 14 -16 weeks old), and also C57BL/6 mice with genetic deletion of either the GIP-or GLP-1 R and both incretin Rs (the background and generation of GIP, GLP-1, and double incretin R knock-out mice has been previously described (19)). Longer term experiments were performed in National Institutes of Health Swiss mice previously fed a high fat diet for 140 days    2 ]GLP/GcG, exendin-4 (both at 25 nmol/kg of body weight; intraperitoneal), or saline vehicle (0.9% (w/v) NaCl) were administered at 09:00 and 16:00 h over 21 days to high fat-fed mice. Food intake, body weight, nonfasting plasma glucose, and insulin concentrations were monitored at 2-4-day intervals. Oral and intraperitoneal glucose tolerance (both 18 mmol/kg of body weight) and insulin sensitivity (15 units/kg of body weight; intraperitoneal) tests were performed after 21 days of treatment. At the end of the treatment period, locomotor activity and aspects of metabolic rate were assessed over a 22-h period using Complete Laboratory Animal Monitoring System metabolic chambers (Columbus Instruments, Columbus, OH), as described previously (14).
Biochemical Analyses-Blood samples were collected from the cut tip on the tail vein of conscious mice into chilled fluoride/heparin glucose microcentrifuge tubes (Sarstedt, Numbrecht, Germany) at the time points indicated in the figures. Samples were immediately centrifuged using a Beckman microcentrifuge (Beckman Instruments, Galway, Ireland) for 1 min at 13,000 ϫ g. Plasma glucose was assayed by an automated glucose oxidase procedure using a Beckman glucose analyzer II (Beckman Instruments). Plasma insulin was assayed by a modified dextran-coated charcoal RIA (16).
Statistical Analysis-The results are expressed as means Ϯ S.E., and the data were compared using the unpaired Student's t test. Where appropriate, data were compared using repeated measures or one-way analysis of variance, followed by the Student-Newman-Keuls post hoc test. Incremental area under the curve (AUC) analyses for plasma glucose and insulin were calculated using GraphPad Prism version 5.0. Groups of data were considered to be significantly different if p Ͻ 0.05.  Table 2). In addition, all novel hybrid peptides were completely stable to the actions of DPP-IV up to and including 8-h incubations ( Table 2).

DPP-IV Stability-As
In Vitro Insulin Secretion- Table 2 displays the effects of all test peptides on insulin secretion at 5.6 and 16.7 mmol/liter glucose in BRIN-BD11 cells. At both glucose concentrations, all peptides (10 Ϫ6 mol/liter), with the exception of native glucagon at 16.7 mmol/liter glucose, significantly (p Ͻ 0.05 to p Ͻ 0.001) increased insulin secretion compared with respective glucose controls. All hybrid peptides displayed similar insulin secretory potencies (Table 2). Thus, none of the hybrid peptides displayed absolute superior insulinotropic actions when compared with the native or positive control peptides ( Table 2).
Acute Glucose-lowering and Insulinotropic Actions of Hybrid Peptides in Normal and ob/ob Mice-Native GIP and glucagon failed to elicit any significant insulin-releasing or glucose-modulating actions in normal mice at the dose employed when compared with glucose alone controls ( Table 2). In contrast, GLP-1 significantly reduced (p Ͻ 0.05) overall 0 -60 min AUC plasma glucose values and increased (p Ͻ 0.01) the overall insulin secretory response when compared with controls (  (Table 2). Consequently, the insulin-releasing and glucose-lowering capabilities of these three novel hybrid peptides were examined in ob/ob mice (  (Table 2). In ob/ob mice, [DS 2 ]GcG/ GIP exhibited only a mildly enhanced insulin secretory action when compared with glucose alone control (Fig. 1, a and b), whereas [DA 2 ]GIP/GLP-1 had no obvious beneficial effects (Fig. 1, e and f). However, [DA 2 ]GLP-1/GcG retained substantial and significant (p Ͻ 0.05 to p Ͻ 0.01) glucose-lowering and insulin-releasing actions in ob/ob mice (Fig. 1, c and d).
Persistent Glucose-lowering and Insulinotropic Actions of [DA 2 ]GLP-1/GcG in High Fat-fed Mice-When administered 4 ( Fig. 2, a and b) or 8 (Fig. 2, c and d) h prior to a glucose load [DA 2 ]GLP-1/GcG significantly reduced individual post-injection and overall 0 -60 min AUC glucose values in high fat-fed mice when compared with injection of native GLP-1 (Fig. 2, a  and c). In agreement, post-injection and overall glucose-induced insulin concentrations were markedly (p Ͻ 0.05 to p Ͻ 0.001) elevated by [DA 2 ]GLP-1/GcG administration 4 or 8 h prior to a glucose challenge when compared with native GLP-1 (Fig. 2, b and d).

Effects of Twice Daily Administration of [DA 2 ]GLP-1/GcG or Exenatide on Body Weight, Energy Intake, Nonfasting Plasma Glucose, and Insulin Concentrations in High Fat-fed Mice-
Twice daily administration of exenatide or [DA 2 ]GLP-1/GcG had no significant effect on accumulated energy intake over the course of the 21 days (Fig. 3a). There was an obvious trend for reduced body weight gain with [DA 2 ]GLP-1/GcG treatment, and this was significant on days 10 and 16 when compared with the saline control group (Fig. 3b). Similarly, nonfasting plasma glucose levels were not significantly different between groups at the individual observation points, but the overall glucose exposure during the 21-day period was significantly (p Ͻ 0.05) reduced by [DA 2 ]GLP-1/GcG treatment (Fig. 3c). Circulating insulin concentrations were significantly (p Ͻ 0.01) increased on day 21 in exenatide and [DA 2 ]GLP-1/GcG mice, with accompanying elevations (p Ͻ 0.05 and p Ͻ 0.01; respectively) of overall insulin levels during the entire 21-day treatment period (Fig. 3d).

Effects of Twice Daily Administration of [DA 2 ]GLP-1/GcG or Exenatide on Glucose Tolerance, Plasma Insulin Response to
Glucose, and Insulin Sensitivity in High Fat-fed Mice-Following intraperitoneal (Fig. 4, a and b) or oral (Fig. 4, c and d) glucose challenge on day 21, plasma glucose levels had a strong tendency to be reduced in exenatide and [DA 2 ]GLP-1/GcGtreated mice, but this failed to reach significance (Fig. 4, a and  c). Furthermore, there was a significant (p Ͻ 0.05 to p Ͻ 0.01) elevation of both post-injection and 0 -60 min overall AUC glucose-stimulated plasma insulin concentrations in exenatide and [DA 2 ]GLP-1/GcG mice following either intraperitoneal or oral glucose administration on day 21 (Fig. 4, b and d). The effects of [DA 2 ]GLP-1/GcG and exenatide were broadly similar (Fig. 4, a-d). In addition, the hypoglycemic action of exogenous insulin was substantially and similarly (p Ͻ 0.001) augmented in exenatide and [DA 2 ]GLP-1/GcG mice 30 and 60 min postinsulin injection on day 21 (Fig. 4e). This was corroborated from overall 0 -60 min glucose values, where both treatment groups significantly (p Ͻ 0.05) improved insulin action compared with saline controls (Fig. 4f).

Effects of Twice Daily Administration of Exenatide or [DA 2 ]GLP-1/GcG on Locomotor Activity and Metabolic Rate in
High Fat-fed Mice-There were no differences in O 2 consumption, CO 2 production, respiratory exchange ratio, and energy expenditure in any of the groups of mice on day 21 (data not shown). However, although there were also no significant differences in ambulatory activity between groups (Fig. 5, a and b), treatment with exenatide and [DA 2 ]GLP-1/GcG significantly (p Ͻ 0.05 and p Ͻ 0.01; respectively) increased rearing and jumping episodes during the light phase, as assessed by Z beam breaks (Fig. 5c). In addition, [DA 2 ]GLP-1/GcG administration significantly (p Ͻ 0.01) increased Z beam breaks during the dark phase (Fig. 5d).

Glucose-lowering and Insulinotropic Actions of [DA 2 ]GLP-1/ GcG in GLP-1, GIP, and Double Incretin R Knock-out Mice-As
would be expected, native GIP and GLP-1 were without biological effects in GIP and GLP-1 R knock-out mice, respectively (Fig. 6, a-d). In addition, administration of GIP or GLP-1 had no consequence in double incretin R knock-out mice (Fig. 6, e and f). In contrast, [DA 2 ]GLP-1/GcG significantly (p Ͻ 0.05 to p Ͻ 0.001) increased glucose-stimulated insulin secretion in GLP-1, GIP, and double incretin R knock-out mice (Fig. 6, b, d,  and f). The glucose-lowering actions of [DA 2 ]GLP-1/GcG were particularly evident in GIP R knock-out mice, with significant

DPP-IV stability, in vitro insulin secretory activity, and in vivo glucose lowering and insulin releasing actions of native, control, and novel hybrid peptides
Resistance of peptides to degradation by DPP-IV (5 milliunits) was measured (n ϭ 3) following 0, 2, 4, and 8 h of incubation. Reaction products were subsequently analyzed by HPLC. For in vitro insulin secretory studies, peptides (10 Ϫ6 M) were incubated with BRIN-BD11 cells in the presence of 5.6 or 16.7 mM glucose (20 min; n ϭ 8), and insulin release was measured by radioimmunoassay and presented as a percentage of respective control. For in vivo studies, plasma glucose and insulin concentrations were measured immediately prior to and 15, 30, and 60 min after intraperitoneal administration of glucose alone (18 mmol/kg of body weight; n ϭ 8) or in combination with respective peptides (each at 25 nmol/kg of body weight) in 18-h fasted NIH Swiss normal mice. The data are expressed as means Ϯ S.E.

GLP-1/Glucagon Hybrid Peptide
reductions in 30 and 60 min post-injection values (p Ͻ 0.001), as well as overall 0 -60 min AUC (p Ͻ 0.05) values, when compared with glucose controls (Fig. 6a). In addition, there was also a strong trend for decreased glucose levels with [DA 2 ]GLP-1/ GcG treatment in GLP-1R knock-out mice (Fig. 6c), and increased sample size may have improved the statistical power of this experiment. Interestingly, glucagon induced significant (p Ͻ 0.05 to p Ͻ 0.01) elevations of the overall glycemic excursion in GIP and double incretin R knock-out mice, but not in GLP-1 R knock-out mice (Fig. 6).

DISCUSSION
In the present study, we have evaluated the biological actions and therapeutic applicability of a series of novel GLP-1/GIP/ glucagon hybrid peptides. These peptides were engineered to combine the energy liberating action of glucagon (8), with the robust insulin-releasing actions of GIP and GLP-1 (6), in a single compound. Unlike native GIP, GLP-1, or glucagon, all novel peptides were completely stable to enzymatic breakdown by DPP-IV and exhibited significantly enhanced insulinotropic actions in clonal beta-cells. These observations are in harmony with previous studies that clearly reveal that the N-terminal modifications employed in the current study mask the DPP-IVbinding site and increase intrinsic biological activity of GIP, GLP-1, and glucagon (11)(12)(13). Thus, importantly our data show that the modified hybrid peptides still retain the ability to activate important corresponding pathways that lead to insulin secretion.
To screen acute in vivo properties of positive control and novel hybrid peptides, they were co-administered with glucose to normal mice. Only

GLP-1/Glucagon Hybrid Peptide
nificant reductions in body weight gain. Thus, it follows that combining the activity of two or more regulatory hormones, to concomitantly activate related biological pathways, offers a more favorable approach for the treatment of obesity and diabetes than activation of lone pathways (2). The slightly greater weight loss with [DA 2 ]GLP-1/GcG, as opposed to exenatide treatment, could also be an important factor. The observed effects of [DA 2 ]GLP-1/GcG were independent of changes in energy intake, despite related satiating effects of GLP-1 and glucagon (21,22). The lack of effect of exenatide on energy intake and body weight likely reflects up-regulation of inherent adaptive mechanisms to normalize energy balance and body weight regulation when only one signaling pathway is activated, with similar observations reported previously in our laboratory and others (23,24). However, this does contrast with similar studies in genetically obese or diabetic animals showing that longer term administration of exenatide significantly reduced food intake, causing weight loss (24 -26). The most plausible explanation for the lack of such chronic effects of exenatide in the current study therefore lies with the dose, possible GLP-1 receptor desensitization, or background genetics. It seems unlikely to us that the dose is an issue, because other studies with exenatide in animal models have employed doses of 1 nmol/kg (27), 24 nmol/kg (25), or 50 nmol/kg (24). The alter-  DECEMBER 6, 2013 • VOLUME 288 • NUMBER 49 native that GLP-1 receptor desensitization occurs is perhaps plausible from in vitro studies (28), although this phenomenon has not been observed to any appreciable extent in vivo (29,30). Thus, the observation of lack of effect of exenatide on energy intake and body weight regulation would appear to be speciesand animal model-specific.

GLP-1/Glucagon Hybrid Peptide
Intraperitoneal and oral glucose tolerance were marginally improved to a similar extent by 21-day twice daily treatment with [DA 2 ]GLP-1/GcG and exenatide. This was associated with significantly increased insulin levels following nutrient challenge. Thus, as would be expected, the metabolic benefits of [DA 2 ]GLP-1/GcG and exenatide are likely mediated predominantly by direct insulin secretory actions (21). Interestingly, substantial similar insulin-induced reductions of blood glucose levels were observed in [DA 2 ]GLP-1/GcG and exenatide mice, highlighting beneficial effects independent of pancreatic beta-cell function. This facet of biological action does not appear to be a direct consequence of reduced adipose tissue mass and thus most likely reflects actions of GLP-1 to improve insulin resistance (31). Thus, [DA 2 ]GLP-1/GcG appears to have both beneficial effects of pancreatic beta-cell function and also a direct or indirect augmentation of peripheral insulin action. We were unable to perform pharmacokinetic analysis of [DA 2 ]GLP-1/GcG because of the requirement for generation of a specific antibody. Thus, the possibility that [DA 2 ]GLP-1/ GcG has altered binding kinetics or an extended half-life as compared with exenatide cannot be discounted.
To further clarify the mechanism behind the observed effects of [DA 2 ]GLP-1/GcG or exenatide, we assessed aspects of locomotor activity and metabolic rate following 21-day treatment. Locomotor activity was unchanged, but explorative episodes (Z-beam breaks) were elevated during the light phase in both treatment groups, but only by [DA 2 ]GLP-1/GcG, and not exenatide, during the dark phase. This is interesting because the activity of mice is normally much less during the light phase and merits further investigation. Given the prominent effects of glucagon and GLP-1 on energy balance (32), the elevations of energy expenditure may have been predicted in the current study. However, this was not the case, because neither treatment regimen altered energy expenditure or the respiratory exchange ratio. In contrast, the beneficial metabolic actions of other co-agonists reported to date generally appear to center around effects on thermogenesis in brown adipose tissue and increased energy expenditure (2). However, it is unclear whether this effect would be fully translated to the obese insulin-resistant human form of type 2 diabetes. Thus, [DA 2 ]GLP-1/GcG may possess a distinct advantage over other similar coagonists and represent a particularly attractive candidate for further development.
Finally, in an attempt to delineate the receptors involved in the biological actions of [DA 2 ]GLP-1/GcG, we conducted studies in genetically transfected cells and knock-out mice. Interestingly, [DA 2 ]GLP-1/GcG stimulated cAMP production in GLP-1, GIP, and glucagon R-transfected cells with comparable, or even increased, efficacy when compared with the native peptide. Thus, our data clearly illustrate that [DA 2 ]GLP-1/GcG is a potent triple agonist, with cross-talk between GLP-1, GIP, and glucagon Rs. The rationale as to why a GLP-1/glucagon hybrid would efficiently activate GIP Rs is unclear, but it does demonstrate the marked similarities and sequence overlap between peptides and receptors of the same glucagon superfamily (33).
Moreover, recent data relating to a modified glucagon/GIP peptide hybrid clearly show that this molecule was capable of activating GLP-1 Rs (17). Further studies in GIP, GLP-1, and double incretin R knock-out mice confirmed our initial in vitro findings, with [DA 2 ]GLP-1/GcG displaying prominent insulin secretory actions in all three models, corroborating triple agonist properties. However, the glucose-lowering action of [DA 2 ]GLP-1/GcG was different in GIP and GLP-1 R knock-out mice, despite comparable insulin-releasing actions, indicating possible differences in insulin action between knock-out models.
In conclusion, the present study has demonstrated the hybrid peptide analog, [DA 2 ]GLP-1/GcG, is a DPP-IV resistant, potent, triple acting GIP, GLP-1, and glucagon R agonist.
GcG are due to concurrent activation of receptors on the same or distinct cell types, with subsequent stimulation of complementary signaling pathways, still needs to be determined. However, it is clear that multitargeting peptides are an attractive new class of therapeutics for the treatment of type 2 diabetes.