The present study examined the glucose-lowering and insulinotropic properties of acylated GLP-1 (glucagon-like peptide-1) and GIP (glucose-dependent insulinotropic polypeptide) peptides in Type 2 diabetes and obesity. GLP-1, GIP, Liraglutide, N-AcGIP(Lys37Myr) (N-acetylGIP with myristic acid conjugated at Lys37), a simple combination of both peptides and a Lira–AcGIP preparation [overnight preparation of Liraglutide and N-AcGIP(Lys37Myr)] were incubated with DPP-IV (dipeptidyl peptidase-IV) to assess peptide stability, and BRIN–BD11 cells were used to evaluate cAMP production and insulin secretion. Acute glucose-lowering and insulinotropic actions were evaluated in Swiss TO mice. Subchronic studies on glucose homoeostasis, insulin secretion, food intake and bodyweight were evaluated in ob/ob mice. Liraglutide, N-AcGIP(Lys37Myr), a simple combination of both peptides and the Lira–AcGIP preparation demonstrated improved DPP-IV resistance (P<0.001), while stimulating cAMP production and insulin secretion (1.4–2-fold; P<0.001). The Lira–AcGIP preparation was more potent at lowering plasma glucose (20–51% reduction; P<0.05–P<0.001) and stimulating insulin secretion (1.5–1.8-fold; P<0.05–P<0.001) compared with Liraglutide and N-AcGIP(Lys37Myr) or a simple peptide combination. Daily administration of the Lira–AcGIP preparation to ob/ob mice lowered bodyweight (7–9%; P<0.05), food intake (23%; P<0.05) and plasma glucose (46% reduction; P<0.001), while increasing plasma insulin (1.5–1.6-fold; P<0.001). The Lira–AcGIP preparation enhanced glucose tolerance, insulin response to glucose and insulin content (P<0.05–P<0.001). These findings demonstrate that a combined preparation of the acylated GLP-1 and GIP peptides Liraglutide and N-AcGIP(Lys37Myr) markedly improved glucose-lowering and insulinotropic properties in diabetic obesity compared with either incretin mimetic given individually.

INTRODUCTION

Type 2 diabetes mellitus is a disorder with major socioeconomic implications and a rapidly escalating global incidence [1]. Recent estimates suggest that approximately $710 million (~7% of the U. K. National Health Service budget) was spent on glucose-lowering drugs and monitoring of blood glucose control in 2008 [2]. As current therapies do not prevent complications, there is a major unmet need for the development of more effective physiological drugs whose actions more tightly regulate the minute-to-minute variations of circulating blood glucose. The gut peptides GLP-1 (glucagon-like-peptide-1) and GIP (glucose-dependent insulinotropic polypeptide) are incretin hormones released by feeding, which specifically target pancreatic β-cells to enhance insulin secretion and aid reduction of postprandial hyperglycaemia [3]. Furthermore, incretin hormones only stimulate glucose-induced insulin release under hyperglycaemic conditions and, unlike other non-endogenous insulinotropic agents, are unlikely to result in hypoglycaemia [4]. It is principally this unique attribute that has led to much interest in exploiting the incretin hormones as potential therapeutic agents [5,6].

Despite their antidiabetic potential, both GLP-1 and GIP undergo enzymatic degradation by DPP-IV (dipeptidyl peptidase-IV) and rapid removal from the circulation [79]. Several strategies have now been successfully employed to circumvent these intrinsic limitations, including development of DPP-IV inhibitors and so-called incretin mimetics (for a review, see [10]). The first GLP-1 mimetic Exenatide (Byetta™) reached the market in 2005 closely followed by Liraglutide (Victoza®) [11]. Liraglutide shares a 97% sequence homology with human GLP-1, with structural variances restricted to amino acid replacement of Lys34 with an arginine residue and the addition of a C16 acyl moiety at position 26 via a γ-glutamyl linker [12]. As a full agonist of the GLP-1 receptor, Liraglutide stimulates glucose-induced insulin secretion [13], inhibits glucagon secretion [14], enhances β-cell mass [15], slows gastric emptying [16], restores β-cell sensitivity to glucose [17] and promotes satiety resulting in decreased energy intake and bw (body weight) loss [18]. Although GIP mimetics have not entered clinical development, preclinical studies clearly demonstrate that N-terminally modified and acylated GIP peptides exhibit improved DPP-IV resistance, and enhanced glucose-lowering and insulin-releasing actions in animal models of obesity diabetes [1929]. Furthermore, β-cell insensitivity to GIP observed in humans with Type 2 diabetes appears to be overcome by combination therapy using GIP with either sulfonylureas or insulin [30,31].

The protracted duration of action of Liraglutide is understood to arise primarily from two mechanisms, namely: (i) non-covalent reversible binding to serum albumin; and (ii) the ability of the peptide to self-associate in solution, thus slowing absorption and facilitating resistance to degradation by DPP-IV [32]. Given this, it seems likely that acylated GIP peptides would behave in a similar manner to Liraglutide with regards to both of these mechanisms. Indeed, from a therapeutic perspective, it would be attractive to develop an incretin-based preparation that would activate both arms of the enteroinsular axis normally triggered by feeding. We hypothesized that combining Liraglutide with the acylated GIP peptide N-AcGIP(Lys37Myr) (N-acetylGIP with myristic acid conjugated at Lys37) into a single preparation would facilitate peptide self-association, thereby providing a particularly effective means of exploiting both incretin pathways for the treatment of diabetes. Therefore the present study examined the effects of a combined preparation of the acylated GLP-1 and GIP peptides Liraglutide and N-AcGIP(Lys37Myr) on in vitro DPP-IV resistance and biological activity, as well as in vivo actions on glucose-lowering and insulin secretion. The effects were compared with the actions of either peptide alone.

MATERIALS AND METHODS

Peptide synthesis and characterization

GLP-1, GIP and Liraglutide were purchased from GL Biochem. N-AcGIP(Lys37Myr) was synthesized on an Applied BioSystems ABI 432A Peptide Synthesiser with an Fmoc (fluoren-9-ylmethoxycarbonyl)-Gln(Trt)-Wang resin (Bachem) using Fmoc chemistry [29]. Briefly, the ϵ-amino group at Lys37 of GIP was conjugated with C14 (myristic acid) and an acetyl adduct incorporated at Tyr1. Peptides were purified using rp-HPLC (reverse-phase HPLC) on a Waters Millennium 2010 Chromatography System [29]. The identity of peptides was confirmed using Voyager-DE BioSpectrometry MALDI–TOF-MS (matrix-assisted laser-desorption ionization–time-of-flight MS) (PerSeptive BioSystems), as described previously [29].

Preparation of peptide solutions

Prior to all experimentation (unless otherwise specified), GLP-1, GIP, Liraglutide and N-AcGIP(Lys37Myr) and a Lira–AcGIP preparation [overnight preparation of equal concentrations of both Liraglutide and N-AcGIP(Lys37Myr)] were aliquoted into Sterlin tubes containing experimental buffer [50 mM triethanolamine/HCl/Krebs–Ringer bicarbonate buffer or saline vehicle (0.9% NaCl)]. Peptide solutions were then placed on an orbital shaker and agitated overnight at 4°C for 12 h. A similar protocol has been used by others for self-association of acylated peptides, including GLP-1 [33]. HPLC analysis following overnight incubations revealed minimal loss of peptide (98.3±0.8% intact peptide remaining) with no significant differences between Liraglutide, N-AcGIP(Lys37Myr) or the Lira–AcGIP preparation. For the simple combination of Liraglutide plus N-AcGIP(Lys37Myr), the same protocol was followed with the exception that peptides were incubated separately overnight, then combined prior to experimentation.

Degradation by DPP-IV

GLP-1, GIP, Liraglutide, N-AcGIP(Lys37Myr), a simple combination of Liraglutide plus N-AcGIP(Lys37Myr) and the Lira–AcGIP preparation were incubated at 37°C in 50 mM triethanolamine/HCl (pH 7.8) with DPP-IV (5 m-units; Sigma–Aldrich) for 0, 2, 4, 8 and 24 h. Enzymatic reactions were terminated by the addition of 15μl of 10% (v/v) TFA (trifluoroacetic acid)/water. Reaction products were separated on a Vydac C4 column (4.6 mm × 250 mm; The Separations Group), and intact peptide were separated from associated major degradation products GIP-(3–42) and GLP-1-(9–36)amide. Absorption was monitored at 206 nm using a SpectraSystem UV2000 detector (Thermoquest). HPLC peak area data were used to calculate the percentage of intact peptide and peptide purity.

In vitro cAMP production and insulin secretion

The effects of GLP-1, GIP, Liraglutide, N-AcGIP(Lys37Myr), a simple combination of Liraglutide plus N-AcGIP(Lys37Myr) and the Lira–AcGIP preparation on stimulation of cAMP production and insulin secretion were measured in BRIN–BD11 cells. For cAMP studies, cells (100000 cells/well) were seeded on to 96-well plates (Nunc) and washed with HBS (Hepes-buffered saline) prior to incubation with peptides (20 min at 37°C) in the presence of 200 mM IBMX (isobutylmethylxanthine). After incubation, the medium was removed and cells were lysed prior to measurement of cAMP using an HTS Immunoassay Kit (Millipore). For insulin-release studies, BRIN–BD11 cells (150000 cells/well) were seeded on to 24-well plates and allowed to attach overnight at 37°C. Following a 40 min pre-incubation with 1.1 mM glucose, cells were incubated (20 min; 37°C) in the presence of 5.6 mM glucose with a range of peptide concentrations (10−12–10−6 M). After incubation for 20 min, the buffer was removed from each well and insulin concentrations were determined using an RIA.

Animals

Acute animal studies were carried out in normal male NIH (National Institutes of Health) Swiss TO mice (Harlan; 50–55-weeks old), whereas subchronic experiments were performed in obese diabetic (ob/ob) mice (20–22-weeks old) derived from the Aston University colony [34]. Mice were age-matched, divided into groups (n=8 or 9) and housed individually in an air-conditioned room (22±2°C) with a 12-h light/12-h dark cycle. Animals had free access to drinking water and normal laboratory chow (Trouw Nutrition). All animal experiments were conducted according to U.K. Home Office Regulations (U.K. Animals Scientific Procedures Act 1986) and the ‘Principles of Laboratory Animal Care’ (NIH Publication no. 86–23, revised 1985). No adverse effects were observed following administration of any of the peptides.

Acute glucose-lowering and insulinreleasing properties of GLP-1 and GIP peptides in Swiss TO mice

Plasma glucose and insulin concentrations were measured prior to and after intraperitoneal administration of glucose (18 mmol/kg of bw) in animals injected 4 h previously with saline vehicle (0.9% NaCl), Liraglutide, N-AcGIP(Lys37Myr) (each at 50 nmol/kg of bw), a simple combination of Liraglutide plus N-AcGIP(Lys37Myr) (each at 50 nmol/kg of bw) or the Lira–AcGIP preparation (100 nmol/kg of bw). Blood samples were collected at the times shown in the Figures and glucose and insulin concentrations measured as indicated below. All peptides were administered by i.p. (intraperitoneal) injection.

Effects of once-daily administration of Liraglutide, N-AcGIP(Lys37Myr) or the Lira–AcGIP preparation in ob/ob mice

Once-daily injections of saline vehicle (0.9% NaCl), Liraglutide, N-AcGIP(Lys37Myr) (each at 50 nmol/kg of bw) or the Lira–AcGIP preparation (100 nmol/kg of bw) were administered intraperitoneally at 16:00 h over 21 days to ob/ob mice. Food intake, bw, plasma glucose and insulin concentrations were monitored at 2–4-day intervals. Glucose tolerance (18 mmol/kg of bw; i.p.) and insulin sensitivity (50 units/kg of bw; i.p.) tests were performed following 21 days of treatment. At termination, blood for circulating triacylglycerols (triglycerides) was taken, and pancreatic tissues were excised and processed for measurement of insulin following extraction with ice-cold ethanol (5 ml/g of tissue) as described previously [29].

Biochemical analysis

Blood samples were collected from the cut tip on the tail vein of conscious mice into chilled fluoride/heparin glucose-treated microcentrifuge tubes (Sarstedt). Samples were immediately centrifuged using a microcentrifuge (Beckman Instruments) for 30 s at 13000 g. Plasma glucose was measured from whole blood using the new ‘plasma calibrated’ Ascensia Contour® Blood Glucose Meter (Bayer AG). Plasma and pancreatic insulin were assayed by a modified dextran-coated charcoal RIA [35]. Plasma triacylglycerol levels were measured using a Hitachi Automatic Analyser 912 (Boehringer Mannheim).

Statistical analysis

Results are expressed as means±S.E.M. and were compared using the unpaired Student's t test with a χ2 analysis for correlations. Where appropriate, data were compared using repeated measures ANOVA or one-way ANOVA, followed by Student–Newman–Keuls post hoc test. Incremental AUC (area under the curve) analyses for plasma glucose and insulin were calculated using GraphPad Prism version 3.02. Groups of data were considered to be significantly different if P<0.05.

RESULTS

Structural characteristics of peptides

The retention times and observed molecular masses for GLP-1, GIP, Liraglutide and N-AcGIP(Lys37Myr) using rp-HPLC and MALDI–TOF-MS respectively are displayed (Table 1). Peptide purity was calculated as being greater than 95% from HPLC AUC analysis using appropriate standards. The molecular mass for each peptide corresponded closely to theoretical values, thereby confirming correct peptide identity.

Table 1
Structural characteristics of GLP-1 and GIP peptides using rp-HPLC and MALDI–TOF-MS

Peptides were purified by rp-HPLC [29] and retention times were recorded. Purified samples were subsequently mixed with α-cyano-4-hydroxycinnamic acid, applied to sample plate of a Voyager-DE BioSpectrometry Workstation and the m/z ratio was compared with the relative peak intensity recorded. The theoretical mass was included for comparative purposes.

rp-HPLCMALDI–TOF-MS
retentionTheoreticalObserved
Peptidetime (min)mass (Da)mass (Da)
GLP-1 19.8 3297.0 3298.4 
GIP 18.6 4982.4 4982.9 
Liraglutide 26.2 3751.0 3752.2 
N-AcGIP(Lys37Myr) 46.8 5235.8 5236.4 
rp-HPLCMALDI–TOF-MS
retentionTheoreticalObserved
Peptidetime (min)mass (Da)mass (Da)
GLP-1 19.8 3297.0 3298.4 
GIP 18.6 4982.4 4982.9 
Liraglutide 26.2 3751.0 3752.2 
N-AcGIP(Lys37Myr) 46.8 5235.8 5236.4 

DPP-IV stability, in vitro cAMP production and insulin secretion

Native GLP-1 and GIP were progressively degraded over 24 h with estimated half-lives of 4.5 and 2.2 h, respectively (Figure 1A). In contrast, Liraglutide, N-AcGIP(Lys37Myr), a simple combination of Liraglutide plus N-AcGIP(Lys37Myr) and the Lira–AcGIP preparation maintained a significantly higher percentage of intact peptide over the 24 h (P<0.05–P<0.001). All test agents exhibited equipotent stimulation of cAMP production with EC50 values ranging from 2.1 to 4.0 nM (P>0.05; Figure 1B). Similarly, all peptides and combinations significantly stimulated insulin secretion from BRIN–BD11 cells in a concentration-dependent manner compared with control (1.4–2-fold; P<0.01–P<0.001; Figure 1C).

DPP-IV resistance, cAMP production and insulin secretion of GLP-1 and GIP peptides

Figure 1
DPP-IV resistance, cAMP production and insulin secretion of GLP-1 and GIP peptides

(A) Resistance of GIP, GLP-1, Liraglutide, N-AcGIP(Lys37Myr), a simple combination of Liraglutide plus N-AcGIP(Lys37Myr) and the Lira–AcGIP preparation to degradation by DPP-IV (5 m-units) was measured (n=3) following incubation for 0, 2, 4, 8 and 24 h. Reaction products were subsequently separated by rp-HPLC and degradation expressed as a percentage of intact peptide. ***P<0.001 compared with native GIP; ΔP<0.05 and ΔΔΔP<0.001 compared with native GLP-1. (B) BRIN–BD11 cells were exposed to various peptide concentrations for 20 min (n=4) and cAMP production assayed using ELISA. (C) BRIN–BD11 cells were incubated with a range of peptide concentrations for 20 min (n=8) in the presence of 5.6 mM glucose and insulin release was measured using RIA. Values represent means±S.E.M. **P<0.01 and ***P<0.001 compared with 5.6 mM glucose control.

Figure 1
DPP-IV resistance, cAMP production and insulin secretion of GLP-1 and GIP peptides

(A) Resistance of GIP, GLP-1, Liraglutide, N-AcGIP(Lys37Myr), a simple combination of Liraglutide plus N-AcGIP(Lys37Myr) and the Lira–AcGIP preparation to degradation by DPP-IV (5 m-units) was measured (n=3) following incubation for 0, 2, 4, 8 and 24 h. Reaction products were subsequently separated by rp-HPLC and degradation expressed as a percentage of intact peptide. ***P<0.001 compared with native GIP; ΔP<0.05 and ΔΔΔP<0.001 compared with native GLP-1. (B) BRIN–BD11 cells were exposed to various peptide concentrations for 20 min (n=4) and cAMP production assayed using ELISA. (C) BRIN–BD11 cells were incubated with a range of peptide concentrations for 20 min (n=8) in the presence of 5.6 mM glucose and insulin release was measured using RIA. Values represent means±S.E.M. **P<0.01 and ***P<0.001 compared with 5.6 mM glucose control.

Acute glucose-lowering and insulinotropic actions of GLP-1 and GIP peptides in Swiss TO mice

Liraglutide, N-AcGIP(Lys37Myr) and a simple combination of Liraglutide plus N-AcGIP(Lys37Myr) significantly reduced the glycaemic response following intraperitoneal glucose compared with saline (25–35% reduction; P<0.05–P<0.001) (Figure 2A). However, administration of the Lira–AcGIP preparation 4 h previously resulted in a significant improvement in glycaemic control relative to all groups of mice (17–45% reduction; P<0.05–P<0.001). This trend was confirmed by AUC analysis for Liraglutide, N-AcGIP(Lys37Myr), the simple combination of Liraglutide plus N-AcGIP(Lys37Myr) and the Lira–AcGIP preparation, which revealed decreases of 26, 25, 30 and 47% respectively compared with saline-treated mice (P<0.05–P<0.001; Figure 2A). Importantly, the Lira–AcGIP preparation significantly lowered plasma glucose (P<0.05) compared with a simple combination of Liraglutide plus N-AcGIP(Lys37Myr) (Figure 2A). A simple combination of Liraglutide plus N-AcGIP(Lys37Myr) significantly enhanced plasma insulin concentrations (1.6-fold; P<0.05) (Figure 2B). The Lira–AcGIP preparation was significantly more potent at stimulating insulin release (1.5- to 1.8-fold; P<0.05–P<0.001) compared with saline-treated animals. This was confirmed by a 1.6-fold increase in plasma insulin AUC (P<0.01; Figure 2B). Administration of native GLP-1 and GIP 4 h previously did not result in significant modification of glycaemic or insulin responses compared with saline-treated controls (results not shown).

Comparative effects of acylated GLP-1 and GIP peptides plus peptide combinations on glucose tolerance and insulin response to glucose in Swiss TO mice

Figure 2
Comparative effects of acylated GLP-1 and GIP peptides plus peptide combinations on glucose tolerance and insulin response to glucose in Swiss TO mice

(A) Plasma glucose and (B) insulin concentrations were measured prior to and after administration of glucose (18 mmol/kg of bw) in animals injected previously (−240 min) with saline vehicle (0.9% NaCl), Liraglutide, N-AcGIP(Lys37Myr), a simple combination of Liraglutide plus N-AcGIP(Lys37Myr) or the Lira–AcGIP preparation (100 nmol/kg of bw). All peptides were administered i.p. at a dose of 50 nmol/kg of bw unless otherwise stated. Time of injection is indicated by the arrow. Plasma glucose and insulin AUC values for 0–60 min post-injection are included. Values represent means±S.E.M. for eight mice. *P<0.05, **P<0.01 and ***P<0.001 compared with saline control; ΔP<0.05 compared with N-AcGIP(Lys37Myr)-treated mice; +P<0.05 compared with Liraglutide-treated mice; and. oP>0.05 compared with a simple combination of Liraglutide plus N-AcGIP(Lys37Myr)-treated mice.

Figure 2
Comparative effects of acylated GLP-1 and GIP peptides plus peptide combinations on glucose tolerance and insulin response to glucose in Swiss TO mice

(A) Plasma glucose and (B) insulin concentrations were measured prior to and after administration of glucose (18 mmol/kg of bw) in animals injected previously (−240 min) with saline vehicle (0.9% NaCl), Liraglutide, N-AcGIP(Lys37Myr), a simple combination of Liraglutide plus N-AcGIP(Lys37Myr) or the Lira–AcGIP preparation (100 nmol/kg of bw). All peptides were administered i.p. at a dose of 50 nmol/kg of bw unless otherwise stated. Time of injection is indicated by the arrow. Plasma glucose and insulin AUC values for 0–60 min post-injection are included. Values represent means±S.E.M. for eight mice. *P<0.05, **P<0.01 and ***P<0.001 compared with saline control; ΔP<0.05 compared with N-AcGIP(Lys37Myr)-treated mice; +P<0.05 compared with Liraglutide-treated mice; and. oP>0.05 compared with a simple combination of Liraglutide plus N-AcGIP(Lys37Myr)-treated mice.

Effects of once-daily Liraglutide, N-AcGIP(Lys37Myr) or the Lira–AcGIP preparation on bw, food intake, plasma glucose and insulin in ob/ob mice

Daily administration of Liraglutide and N-AcGIP(Lys37Myr) did not alter bw or food intake in ob/ob mice (Figures 3A and 3B). In contrast, the Lira–AcGIP preparation significantly reduced bw by day 18–21 (7–9% decrease; P<0.05; Figure 3A) and decreased food intake by day 21 (23% decrease; P<0.05; Figure 3B). Non-fasting plasma glucose concentrations were significantly improved following Liraglutide (29% reduction; P<0.01), N-AcGIP(Lys37Myr) (23% reduction; P<0.05) and the Lira–AcGIP preparation (46% reduction; P<0.001) (Figure 3C). In addition, the Lira–AcGIP preparation significantly reduced plasma glucose concentrations relative to N-AcGIP(Lys37Myr)-treated animals (30% reduction; P<0.05) by day 21. Improvements in glucose-lowering were accompanied by significantly increased non-fasting plasma insulin concentrations following administration of Liraglutide (1.3–1.4-fold increase; P<0.05), N-AcGIP(Lys37Myr) (1.2–1.3-fold increase; P<0.05) and the Lira–AcGIP preparation (1.5–1.6-fold increase; P<0.001; Figure 3D).

Effects of daily administration of Liraglutide, N-AcGIP(Lys37Myr) and the Lira–AcGIP preparation on (A) bw, (B) food intake, (C) plasma glucose and (D) plasma insulin in ob/ob mice

Figure 3
Effects of daily administration of Liraglutide, N-AcGIP(Lys37Myr) and the Lira–AcGIP preparation on (A) bw, (B) food intake, (C) plasma glucose and (D) plasma insulin in ob/ob mice

Parameters were measured prior to and 21 days during treatment with saline vehicle (0.9% NaCl, i.p.), Liraglutide, N-AcGIP(Lys37Myr) (each at 50 nmol/kg of bw, i.p.) or the Lira–AcGIP preparation (100 nmol/kg of bw, i.p.). Values represent means±S.E.M. for nine mice. *P<0.05, **P<0.01 and ***P<0.001 compared with saline-treated group; ΔP<0.05 compared with N-AcGIP(Lys37Myr)-treated mice.

Figure 3
Effects of daily administration of Liraglutide, N-AcGIP(Lys37Myr) and the Lira–AcGIP preparation on (A) bw, (B) food intake, (C) plasma glucose and (D) plasma insulin in ob/ob mice

Parameters were measured prior to and 21 days during treatment with saline vehicle (0.9% NaCl, i.p.), Liraglutide, N-AcGIP(Lys37Myr) (each at 50 nmol/kg of bw, i.p.) or the Lira–AcGIP preparation (100 nmol/kg of bw, i.p.). Values represent means±S.E.M. for nine mice. *P<0.05, **P<0.01 and ***P<0.001 compared with saline-treated group; ΔP<0.05 compared with N-AcGIP(Lys37Myr)-treated mice.

Effects of once-daily Liraglutide, N-AcGIP(Lys37Myr) or the Lira–AcGIP preparation on glucose tolerance and insulin response to glucose in ob/ob mice

Liraglutide, N-AcGIP(Lys37Myr) and the Lira–AcGIP preparation significantly reduced plasma glucose levels at 15, 30 and 60 min post-glucose injection (17–51% decrease; P<0.05–P<0.001) (Figure 4A), which was corroborated by plasma glucose AUC analysis (21–40% reduction; P<0.01–P<0.001). Importantly, the Lira–AcGIP preparation exhibited a significantly improved glycaemic excursion compared with either Liraglutide or N-AcGIP(Lys37Myr) alone (20–36% reduction; P<0.05–P<0.01; Figure 4A). Glucose-lowering actions were mirrored by significantly increased plasma insulin response to glucose for Liraglutide and N-AcGIP(Lys37Myr) (1.3–1.6-fold increase; P<0.05) and the Lira–AcGIP preparation (1.9-fold increase; P<0.05–P<0.001) relative to saline-treated animals (Figure 4B).

Effects of daily administration of Liraglutide, N-AcGIP(Lys37Myr) and the Lira–AcGIP preparation on (A) glucose tolerance and (B) plasma insulin response to glucose in ob/ob mice

Figure 4
Effects of daily administration of Liraglutide, N-AcGIP(Lys37Myr) and the Lira–AcGIP preparation on (A) glucose tolerance and (B) plasma insulin response to glucose in ob/ob mice

Parameters were measured prior to and after administration of glucose (18 mmol/kg of bw, i.p.) in animals receiving 21 days daily treatment with saline vehicle (0.9% NaCl, i.p.), Liraglutide, N-AcGIP(Lys37Myr) (each at 50 nmol/kg of bw, i.p.) or the Lira–AcGIP preparation (100 nmol/kg of bw, i.p.). The time of injection is indicated by the arrow. Values represent means±S.E.M. for nine mice. *P<0.05, **P<0.01 and ***P<0.001 compared with saline-treated mice'; ΔP<0.05 and ΔΔP<0.01 compared with N-AcGIP(Lys37Myr)-treated mice; and +P<0.05 compared with Liraglutide-treated mice.

Figure 4
Effects of daily administration of Liraglutide, N-AcGIP(Lys37Myr) and the Lira–AcGIP preparation on (A) glucose tolerance and (B) plasma insulin response to glucose in ob/ob mice

Parameters were measured prior to and after administration of glucose (18 mmol/kg of bw, i.p.) in animals receiving 21 days daily treatment with saline vehicle (0.9% NaCl, i.p.), Liraglutide, N-AcGIP(Lys37Myr) (each at 50 nmol/kg of bw, i.p.) or the Lira–AcGIP preparation (100 nmol/kg of bw, i.p.). The time of injection is indicated by the arrow. Values represent means±S.E.M. for nine mice. *P<0.05, **P<0.01 and ***P<0.001 compared with saline-treated mice'; ΔP<0.05 and ΔΔP<0.01 compared with N-AcGIP(Lys37Myr)-treated mice; and +P<0.05 compared with Liraglutide-treated mice.

Effects of once-daily Liraglutide, N-AcGIP(Lys37Myr) or the Lira–AcGIP preparation on insulin sensitivity, pancreatic insulin and circulating triacylglycerols in ob/ob mice

No improvements on insulin sensitivity (Figure 5A) or circulating triacylglycerols (Figure 5B) were observed following daily treatment with Liraglutide, N-AcGIP(Lys37Myr) or the Lira–AcGIP preparation. All treatment groups exhibited significantly enhanced pancreatic insulin content (1.3–1.7-fold increase; P<0.05–P<0.01; Figure 5C).

Effects of daily administration of Liraglutide, N-AcGIP(Lys37Myr) and the Lira–AcGIP preparation on (A) insulin sensitivity, (B) circulating triacylglycerols and (C) pancreatic insulin content in ob/ob mice

Figure 5
Effects of daily administration of Liraglutide, N-AcGIP(Lys37Myr) and the Lira–AcGIP preparation on (A) insulin sensitivity, (B) circulating triacylglycerols and (C) pancreatic insulin content in ob/ob mice

Parameters were measured after 21 days daily treatment with saline vehicle (0.9% NaCl, i.p.), Liraglutide, N-AcGIP(Lys37Myr) (each at 50 nmol/kg of bw, i.p.) or the Lira–AcGIP preparation (100 nmol/kg of bw, i.p.). Insulin (50 units/kg of bw, i.p.) was injected at time indicated by the arrow. Values represent means±S.E.M. for nine mice. *P<0.05 and **P<0.01 compared with saline-treated mice.

Figure 5
Effects of daily administration of Liraglutide, N-AcGIP(Lys37Myr) and the Lira–AcGIP preparation on (A) insulin sensitivity, (B) circulating triacylglycerols and (C) pancreatic insulin content in ob/ob mice

Parameters were measured after 21 days daily treatment with saline vehicle (0.9% NaCl, i.p.), Liraglutide, N-AcGIP(Lys37Myr) (each at 50 nmol/kg of bw, i.p.) or the Lira–AcGIP preparation (100 nmol/kg of bw, i.p.). Insulin (50 units/kg of bw, i.p.) was injected at time indicated by the arrow. Values represent means±S.E.M. for nine mice. *P<0.05 and **P<0.01 compared with saline-treated mice.

DISCUSSION

One successful strategy which has been employed to overcome the therapeutic barrier posed by the very short half-life of native GLP-1 is the use of peptide acylation, which promotes both stability to enzymatic degradation by DPP-IV and non-covalent binding to serum proteins, thereby significantly prolonging biological half-life [6,32]. Similarly, acylation of GIP has been shown to greatly improve bioactivity and therapeutic effects in diabetic animal models, especially when combined with N-terminal acetylation [2326,29]. Perhaps much less well recognized is the concept that acylation of peptides, including insulin and GLP-1, facilitates the process of self-association [32,36]. Indeed, Liraglutide forms strongly self-associated heptamers in solution, which appears as a key for protracted absorption after injection and the pharmacokinetic profile suitable for once-daily administration [37,38]. As such, it is possible that a preparation of combined acylated GLP-1 and GIP peptides could provide additional non-covalent binding of peptide fatty acid chains thus promoting stability, potency, biological effectiveness and at the same time enabling activation of both important physiological arms of the enteroinsular axis. In this study, we have examined the effects of a Liraglutide and N-AcGIP(Lys37Myr) preparation (Lira–AcGIP), formed by simple self-association, on DPP-IV stability, in vitro cAMP production and insulin secretion, and in vivo glucose-lowering effects in normal and obese diabetic (ob/ob) mice. The actions of the Lira–AcGIP preparation were compared with either peptide given alone.

As observed in previous studies, both native GLP-1 and GIP were rapidly degraded by DPP-IV in vitro [29,39]. Liraglutide, N-AcGIP(Lys37Myr), a simple combination of both peptides and the Lira–AcGIP preparation remained intact throughout the entire 24 h with significantly more intact peptide remaining at all time points compared with native GLP-1 and GIP. This enhanced DPP-IV resistance probably results from masking the enzyme cleavage site [40], alterations to peptide charge or is possibly related to hydroaffinity [24,40]. Importantly, the Lira–AcGIP preparation was completely stable over the incubation period, highlighting that combining Liraglutide with N-AcGIP(Lys37Myr) did not have any detrimental effects on susceptibility to DPP-IV degradation.

Native GLP-1, GIP, Liraglutide, N-AcGIP(Lys37Myr), a simple combination of both Liraglutide plus N-AcGIP(Lys37Myr) and the Lira–AcGIP preparation concentration-dependently stimulated cAMP production in an equipotent manner in BRIN–BD11 cells. Estimated EC50 values correlated well with previous observations and were not significantly different for any of the test groups [29,39]. These observations demonstrate that peptide modifications, and especially formation of the Lira–AcGIP preparation, did not adversely affect cAMP second messenger production. Similarly, all test agents, including the Lira–AcGIP preparation, stimulated insulin secretion equally, again with no apparent loss of potency relative to native ligands.

To assess the acute glucose-lowering and insulinotropic actions of the GLP-1 and GIP peptides, normal Swiss TO mice were employed. The native incretins were inactive when administered 4 h prior to an intraperitoneal glucose load, whereas Liraglutide and N-AcGIP(Lys37Myr) both displayed notable effects on the glycaemic control, as described previously [29,39]. Simple combination of Liraglutide plus N-AcGIP(Lys37Myr) resulted in more prominent responses than saline controls, but did not reveal significant differences compared with single doses of Liraglutide or N-AcGIP(Lys37Myr) given alone. In contrast, the Lira–AcGIP preparation exhibited a significantly improved glycaemic profile compared with Liraglutide, N-AcGIP(Lys37Myr) and a simple combination of both Liraglutide plus N-AcGIP(Lys37Myr). In line with effects on glucose-lowering, the Lira–AcGIP preparation resulted in significantly greater stimulation of insulin release. The greater potency of the pharmacological preparations in vivo presumably reflects differences in the bioavailability and pharmacokinetics of injected preparations. These results demonstrate the beneficial effects of the Lira–AcGIP preparation over simple combination and use of either constituent incretin mimetic given alone.

Results from acute in vivo studies provided a strong basis for the subsequent 21-day subchronic study using genetically obese diabetic (ob/ob) mice [34]. Furthermore, we chose to administer the Lira–AcGIP preparation once daily and to compare this with either Liraglutide or N-AcGIP(Lys37Myr) given alone. As expected, N-AcGIP(Lys37Myr) treatment did not affect bw or food intake corroborating other findings that GIP does not affect feeding activity [2339,41]. Although both Liraglutide and the Lira–AcGIP preparation significantly reduced food intake towards the end of the study period, only the Lira–AcGIP preparation led to a significant reduction in bw by day 18 onwards. This is most likely due to CNS (central nervous system) actions as Liraglutide and other GLP-1 analogues have been shown to cross the blood–brain barrier [4245], and peripheral administration of Liraglutide activates several CNS nuclei [45,46]. However, further studies examining the effects of Lira–AcGIP on weight reduction, including gene and protein expression, is necessary. Importantly, during routine physical examination of the mice over the 21-day study period, we did not observe any visible negative or toxic effects of drug treatment. Liraglutide, N-AcGIP(Lys37Myr) and the Lira–AcGIP preparation significantly decreased non-fasting plasma glucose levels, accompanied by significantly elevated plasma insulin concentrations. These effects were particularly prominent in the Lira–AcGIP preparation-treated group, with significantly improved glucose-lowering compared with N-AcGIP(Lys37Myr). Consistent with these actions, the Lira–AcGIP preparation significantly improved glucose tolerance compared with either incretin mimetic given alone. This was accompanied by a significant enhancement of the plasma insulin response to glucose, demonstrating its ability to overcome the β-cell defect in this animal model [47]. Thus a large part of the glucose-lowering actions of these pharmacological preparations stems from their potent insulinotropic actions. However, other receptor-mediated biochemical effects, such as inhibition of glucagon secretion, may be involved in improving metabolic control. Although all of the treatments improved pancreatic insulin content, changes in glucose homoeostasis were not associated with any change of insulin sensitivity or circulating triacylglycerols highlighting a primary mode of action on the pancreatic β-cell.

Defects in GLP-1 secretion and GIP action contribute to the pathogenesis of diabetes, and the introduction of stable, long-acting GLP-1 mimetics represents one of the most significant therapeutic developments over recent years [1,6,48]. Reconstitution of normal physiological incretin action in diabetes using a combination of GLP-1 and GIP may enable further improvements in glycaemic control. Additional therapeutic strategies leading to improvement of β-cell responsiveness to GIP are needed. However, the results of the present study demonstrate that an acylated GLP-1 and GIP preparation offers beneficial long-acting glucose-lowering and insulinotropic actions compared with single-component peptide injections. These findings point towards the possibility of developing novel formulations of acylated incretin peptides that will be effective in exploiting both arms of the enteroinsular axis for the treatment of diabetes.

FUNDING

This work was supported by The SAAD Contracting and Trading Company and University of Ulster Strategic Research Funding.

We thank Professor Clifford Bailey (School of Life and Health Science, Aston University, Birmingham, U.K.) for providing the breeding pairs of ob/+ mice.

Abbreviations

     
  • AUC

    area under the curve

  •  
  • bw

    body weight

  •  
  • CNS

    central nervous system

  •  
  • DPP-IV

    dipeptidyl peptidase-IV

  •  
  • Fmoc

    fluoren-9-ylmethoxycarbonyl

  •  
  • GIP

    glucose-dependent insulinotropic polypeptide

  •  
  • GLP-1

    glucagon-like peptide-1

  •  
  • i.p.

    intraperitoneal(ly)

  •  
  • Lira–AcGIP preparation

    overnight preparation of equal concentrations of Liraglutide and N-AcGIP(Lys37Myr) (N-acetylGIP with myristic acid conjugated at Lys37)

  •  
  • MALDI–TOF-MS

    matrix-assisted laser-desorption ionization–time-of-flight MS

  •  
  • rp-HPLC

    reverse-phase HPLC

AUTHOR CONTRIBUTION

Victor Gault and Peter Flatt conceived the experiments, co-wrote and checked the manuscript prior to submission. Barry Kerr performed all of the experiments, analysed the data and co-wrote the manuscript. Patrick Harriott synthesized the N-AcGIP(Lys37Myr) peptide.

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