Macroalgal protein hydrolysates from Palmaria palmata influence the 'incretin effect' in vitro via DPP-4 inhibition and upregulation of insulin, GLP-1 and GIP secretion.

PURPOSE: This study investigated metabolic benefits of protein hydrolysates from the macroalgae Palmaria palmata, previously shown to inhibit dipeptidylpeptidase-4 (DPP-4) activity in vitro. METHODS: Previously, Alcalase/Flavourzyme-produced P. palmata protein hydrolysate (PPPH) improved glycaemia and insulin production in streptozotocin-induced diabetic mice. Here the PPPH, was compared to alternative Alcalase, bromelain and Promod-derived hydrolysates and an unhydrolysed control. All PPPH's underwent simulated gastrointestinal digestion (SGID) to establish oral bioavailability. PPPH's and their SGID counterparts were tested in pancreatic, clonal BRIN-BD11 cells to assess their insulinotropic effect and associated intracellular mechanisms. PPPH actions on the incretin effect were assessed via measurement of DPP-4 activity, coupled with GLP-1 and GIP release from GLUTag and STC-1 cells, respectively. Acute in vivo effects of Alcalase/Flavourzyme PPPH administration on glucose tolerance and satiety were assessed in overnight-fasted mice. RESULTS: PPPH's (0.02-2.5 mg/ml) elicited varying insulinotropic effects (p < 0.05-0.001). SGID of the unhydrolysed protein control, bromelain and Promod PPPH's retained, or improved, bioactivity regarding insulin secretion, DPP-4 inhibition and GIP release. Insulinotropic effects were retained for all SGID-hydrolysates at higher PPPH concentrations. DPP-4 inhibitory effects were confirmed for all PPPH's and SGID counterparts (p < 0.05-0.001). PPPH's were shown to directly influence the incretin effect via upregulated GLP-1 and GIP (p < 0.01-0.001) secretion in vitro, largely retained after SGID. Alcalase/Flavourzyme PPPH produced the greatest elevation in cAMP (p < 0.001, 1.7-fold), which was fully retained post-SGID. This hydrolysate elicited elevations in intracellular calcium (p < 0.01) and membrane potential (p < 0.001). In acute in vivo settings, Alcalase/Flavourzyme PPPH improved glucose tolerance (p < 0.01-0.001) and satiety (p < 0.05-0.001). CONCLUSION: Bioavailable PPPH peptides may be useful for the management of T2DM and obesity.

Apelin-13 analogues show potent in vitro and in vivo insulinotropic and glucose lowering actions.

Nine structurally modified apelin-13 analogues were assessed for their in vitro and acute in vivo antidiabetic potential. Stability was assessed in mouse plasma and insulinotropic efficacy tested in cultured pancreatic BRIN-BD11 cells and isolated mouse pancreatic islets. Intracellular Ca2+ and cAMP production in BRIN-BD11 cells was determined, as was glucose uptake in 3T3-L1 adipocytes. Acute antihyperglycemic effects of apelin analogues were assessed following i.p. glucose tolerance tests (ipGGT, 18 mmol/kg) in normal and diet-induced-obese (DIO) mice and on food intake in normal mice. Apelin analogues all showed enhanced in vitro stability (up to 5.8-fold, t½â€¯= 12.8 h) in mouse plasma compared to native apelin-13 (t½â€¯= 2.1 h). Compared to glucose controls, stable analogues exhibited enhanced insulinotropic responses from BRIN-BD11 cells (up to 4.7-fold, p < 0.001) and isolated mouse islets (up to 5.3-fold) for 10-7 M apelin-13 amide (versus 7.6-fold for 10-7 M GLP-1). Activation of APJ receptors on BRIN-BD11 cells increased intracellular Ca2+ (up to 3.0-fold, p < 0.001) and cAMP (up to 1.7-fold, p < 0.01). Acute ipGTT showed improved insulinotropic and glucose disposal responses in normal and DIO mice (p < 0.05 and p < 0.01, respectively). Apelin-13 amide and (pGlu)apelin-13 amide were the most effective analogues exhibiting acute, dose-dependent and persistent biological actions. Both analogues stimulated insulin-independent glucose uptake by differentiated adipocytes (2.9-3.3-fold, p < 0.05) and inhibited food intake (26-33%, p < 0.001), up to 180 min in mice, versus saline. In contrast, (Ala13)apelin-13 and (Val13)apelin-13 inhibited insulin secretion, suppressed beta-cell signal transduction and stimulated food intake in mice. Thus, stable analogues of apelin-13 have potential for diabetes/obesity therapy.

Novel dual agonist peptide analogues derived from dogfish glucagon show promising in vitro insulin releasing actions and antihyperglycaemic activity in mice.

The antidiabetic potential of thirteen novel dogfish glucagon derived analogues were assessed in vitro and in acute in vivo studies. Stable peptide analogues enhanced insulin secretion from BRIN-BD11 ß-cells (p < 0.001) and reduced acute glycaemic responses following intraperitoneal glucose (25 nmol/kg) in healthy NIH Swiss mice (p < 0.05-p<0.001). The in vitro insulinotropic actions of [S2a]dogfish glucagon, [S2a]dogfish glucagon-exendin-4(31-39) and [S2a]dogfish glucagon-Lys(30)-γ-glutamyl-PAL, were blocked (p < 0.05-p<0.001) by the specific GLP-1 and glucagon receptor antagonists, exendin-4(9-39) and (desHis(1)Pro(4)Glu(9))glucagon amide but not by (Pro(3))GIP, indicating lack of GIP receptor involvement. These analogues dose-dependently stimulated cAMP production in GLP-1 and glucagon (p < 0.05-p<0.001) but not GIP-receptor transfected cells. They improved acute glycaemic and insulinotropic responses in high-fat fed diabetic mice and in wild-type C57BL/6J and GIPR-KO mice (p < 0.05-p<0.001), but not GLP-1R-KO mice, confirming action on GLP-1 but not GIP receptors. Overall, dogfish glucagon analogues have potential for diabetes therapy, exerting beneficial metabolic effects via GLP-1 and glucagon receptors.

Glucagon receptor antagonist and GIP agonist combination for diet-induced obese mice.

Ablation of glucagon receptor signaling represents a potential treatment option for type 2 diabetes (T2DM). Additionally, activation of glucose-dependent insulinotropic polypeptide (GIP) receptor signaling also holds therapeutic promise for T2DM. Therefore, this study examined both independent and combined metabolic actions of desHis(1)Pro(4)Glu(9)(Lys(12)PAL)-glucagon (glucagon receptor antagonist) and d-Ala(2)GIP (GIP receptor agonist) in diet-induced obese mice. Glucagon receptor binding has been linked to alpha-helical structure and desHis(1)Pro(4)Glu(9)(Lys(12)PAL)-glucagon displayed enhanced alpha-helical content compared with native glucagon. In clonal pancreatic BRIN-BD11 beta-cells, desHis(1)Pro(4)Glu(9)(Lys(12)PAL)-glucagon was devoid of any insulinotropic or cAMP-generating actions, and did not impede d-Ala(2)GIP-mediated (P<0.01 to P<0.001) effects on insulin and cAMP production. Twice-daily injection of desHis(1)Pro(4)Glu(9)(Lys(12)PAL)-glucagon or d-Ala(2)GIP alone, and in combination, in high-fat-fed mice failed to affect body weight or energy intake. Circulating blood glucose levels were significantly (P<0.05 to P<0.01) decreased by all treatments regimens, with plasma and pancreatic insulin elevated (P<0.05 to P<0.001) in all mice receiving d-Ala(2)GIP. Interestingly, plasma glucagon concentrations were decreased (P<0.05) by sustained glucagon inhibition (day 28), but increased (P<0.05) by d-Ala(2)GIP therapy, with a combined treatment resulting in glucagon concentration similar to saline controls. All treatments improved (P<0.01) intraperitoneal and oral glucose tolerance, and peripheral insulin sensitivity. d-Ala(2)GIP-treated mice showed increased glucose-induced insulin secretion in response to intraperitoneal and oral glucose. Metabolic rate and ambulatory locomotor activity were increased (P<0.05 to P<0.001) in all desHis(1)Pro(4)Glu(9)(Lys(12)PAL)-glucagon-treated mice. These studies highlight the potential of glucagon receptor inhibition alone, and in combination with GIP receptor activation, for T2DM treatment.

Characterisation of structurally modified analogues of glucagon as potential glucagon receptor antagonists.

Acute in vitro and in vivo biological activities of four novel structural analogues of glucagon were tested. desHis(1)Pro(4)-glucagon, desHis(1)Pro(4)Glu(9)-glucagon, desHis(1)Pro(4)Glu(9)Lys(12)FA-glucagon and desHis(1)Pro(4)Glu(9)Lys(30)FA-glucagon were stable to DPP-4 degradation and dose-dependently inhibited glucagon-mediated cAMP production (p<0.05 to p<0.001). None stimulated insulin secretion in vitro above basal levels, but all inhibited glucagon-induced insulin secretion (p<0.01 to p<0.001). In normal mice all analogues antagonised acute glucagon-mediated elevations of blood glucose (p<0.05 to p<0.001) and blocked corresponding insulinotropic responses. In high-fat fed mice, glucagon-induced increases in plasma insulin (p<0.05 to p<0.001) and glucagon-induced hyperglycaemia were blocked (p<0.05 to p<0.01) by three analogues. In obese diabetic (ob/ob) mice only desHis(1)Pro(4)Glu(9)-glucagon effectively (p<0.05 to p<0.01) inhibited both glucagon-mediated glycaemic and insulinotropic responses. desHis(1)Pro(4)-glucagon and desHis(1)Pro(4)Glu(9)-glucagon were biologically ineffective when administered 8h prior to glucagon, whereas desHis(1)Pro(4)Glu(9)Lys(12)FA-glucagon retained efficacy (p<0.01) for up to 24h. Such peptide-derived glucagon receptor antagonists have potential for type 2 diabetes therapy.

Comparison of the independent and combined metabolic effects of subchronic modulation of CCK and GIP receptor action in obesity-related diabetes.

OBJECTIVE: Compromise of gastric inhibitory polypeptide (GIP) receptor action and activation of cholecystokinin (CCK) receptors represent mechanistically different approaches to the possible treatment of obesity-related diabetes. In the present study, we have compared the individual and combined effects of (Pro(3))GIP[mPEG] and (pGlu-Gln)-CCK-8 as an enzymatically stable GIP receptor antagonist and CCK receptor agonist molecule, respectively. RESULTS: Twice-daily injections of (pGlu-Gln)-CCK-8 alone and in combination with (Pro(3))GIP[mPEG] in high-fat-fed mice for 34 days significantly decreased the energy intake throughout the entire study (P<0.05 to P<0.01). Body weights were significantly depressed (P<0.05 to P<0.01) in all treatment groups from day 18 onwards. Administration of (pGlu-Gln)-CCK-8, (Pro(3))GIP[mPEG] or a combination of both peptides significantly (P<0.01 to P<0.001) decreased the overall glycaemic excursion in response to both oral and intraperitoneal glucose challenge when compared with the controls. Furthermore, oral glucose tolerance returned to lean control levels in all treatment groups. The beneficial effects on glucose homeostasis were not associated with altered insulin levels in any of the treatment groups. In keeping with this, the estimated insulin sensitivity was restored to control levels by twice-daily treatment with (pGlu-Gln)-CCK-8, (Pro(3))GIP[mPEG] or a combination of both peptides. The blood lipid profile on day 34 was not significantly different between the high-fat controls and all treated mice. CONCLUSION: These studies highlight the potential of (pGlu-Gln)-CCK-8 and (Pro(3))GIP[mPEG] in the treatment of obesity-related diabetes, but there was no evidence of a synergistic effect of the combined treatment.

Early administration of the glucose-dependent insulinotropic polypeptide receptor antagonist (Pro3)GIP prevents the development of diabetes and related metabolic abnormalities associated with genetically inherited obesity in ob/ob mice.

AIMS/HYPOTHESIS: Ablation of gastric inhibitory polypeptide (GIP) receptor action is reported to protect against obesity and associated metabolic abnormalities. The aim of this study was to use prediabetic ob/ob mice to examine whether 60 days of chemical GIP receptor ablation with (Pro(3))GIP is able to counter the development of genetic obesity-related diabetes. MATERIALS AND METHODS: Young (5-7 weeks) ob/ob mice received once daily i.p. injections of either saline vehicle or (Pro(3))GIP (25 nmol kg(-1) day(-1)) over a 60 day period. Food intake, body weight and circulating glucose and insulin were measured at frequent intervals. At 60 days, glucose tolerance, response to native GIP, postprandial responses, insulin sensitivity, HbA(1c), circulating hormones and plasma lipids were assessed. RESULTS: Body weight and food intake in (Pro(3))GIP-treated mice did not differ from ob/ob controls. GIP receptor blockade significantly improved non-fasting glucose (p < 0.001), HbA(1c) (p < 0.05), glucose tolerance (p < 0.001), meal tolerance (p < 0.001) and insulin sensitivity (p < 0.05). Remarkably, (Pro(3))GIP treatment prevented the age-related development of diabetes, as none of these parameters differed significantly between treated ob/ob mice and normal age-matched lean controls. Circulating levels of glucagon, corticosterone, adiponectin and total cholesterol were unchanged by (Pro(3))GIP, while levels of triacylglycerol, LDL-cholesterol and resistin were decreased (p < 0.05) compared with those in control ob/ob mice. Plasma and pancreatic insulin concentrations were generally lower after (Pro(3))GIP treatment than in control ob/ob mice (p < 0.01), but plasma insulin levels remained substantially raised (p < 0.001) compared with those observed in lean controls. CONCLUSIONS/INTERPRETATION: These data indicate that sustained GIP receptor antagonism provides an effective means of preventing the development of many of the metabolic abnormalities of obesity-driven diabetes.

Effects of sub-chronic exposure to naturally occurring N-terminally truncated metabolites of glucose-dependent insulinotrophic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), GIP(3-42) and GLP-1(9-36)amide, on insulin secretion and glucose homeostasis in ob/ob mice.

Glucose-dependent insulinotrophic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are important enteroendocrine hormones that are rapidly degraded by an ubiquitous enzyme dipeptidyl peptidase IV to yield truncated metabolites GIP(3-42) and GLP-1(9-36)amide. In this study, we investigated the effects of sub-chronic exposure to these major circulating forms of GIP and GLP-1 on blood glucose control and endocrine pancreatic function in obese diabetic (ob/ob) mice. A once daily injection of either peptide for 14 days had no effect on body weight, food intake or pancreatic insulin content or islet morphology. GLP-1(9-36)amide also had no effect on plasma glucose homeostasis or insulin secretion. Mice receiving GIP(3-42) exhibited small but significant improvements in non-fasting plasma glucose, glucose tolerance and glycaemic response to feeding. Accordingly, plasma insulin responses were unchanged suggesting that the observed enhancement of insulin sensitivity was responsible for the improvement in glycaemic control. These data indicate that sub-chronic exposure to GIP and GLP-1 metabolites does not result in physiological impairment of insulin secretion or blood glucose control. GIP(3-42) might exert an overall beneficial effect by improving insulin sensitivity through extrapancreatic action.

Function of a long-term, GLP-1-treated, insulin-secreting cell line is improved by preventing DPP IV-mediated degradation of GLP-1.

Glucagon-like peptide-1 (GLP-1) is an important insulinotropic hormone with potential in the treatment of type 2 diabetes. However, the short biological half-life of the peptide after cleavage by dipeptidylpeptidase IV (DPP IV) is a major limitation. Inhibition of DPP IV activity and the development of resistant GLP-1 analogues is the subject of ongoing research. In this study, we determined cell growth, insulin content, insulin accumulation and insulin secretory function of a insulin-secreting cell line cultured for 3 days with either GLP-1, GLP-1 plus the DPP IV inhibitor diprotin A (DPA) or stable N-acetyl-GLP-1. Native GLP-1 was rapidly degraded by DPP IV during culture with accumulation of the inactive metabolite GLP-1(9-36)amide. Inclusion of DPA or use of the DPP IV-resistant analogue, N-acetyl-GLP-1, improved cellular function compared to exposure to GLP-1 alone. Most notably, basal and accumulated insulin secretion was enhanced, and glucose responsiveness was improved. However, prolonged GLP-1 treatment resulted in GLP-1 receptor desensitization regardless of DPP IV status. The results indicate that prevention of DPP IV action is necessary for beneficial effects of GLP-1 on pancreatic beta cells and that prolonged exposure to GLP-1(9-36)amide may be detrimental to insulin secretory function. These observations also support the ongoing development of DPP-IV-resistant forms of GLP-1, such as N-acetyl-GLP-1.

A comparison of the cellular and biological properties of DPP-IV-resistant N-glucitol analogues of glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide.

AIM: The two major incretin hormones--glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP)--are being actively researched by the pharmaceutical industry because of their glucose-lowering and potential anti-diabetic properties. Unfortunately, the inactivation of GLP-1 and GIP in the circulation brought about by dipeptidyl-peptidase-IV (DPP-IV) degradation makes their biological actions short-lived. This study directly compares the cellular and biological properties of GLP-1, GIP and their N-terminally modified counterparts, with glucitol extension at positions His7 and Tyr1 respectively, to confer DPP-IV resistance. METHODS: Using both the glucose-responsive pancreatic beta-cell line, BRIN BD11, and the obese diabetic (ob/ob) mouse, we assessed adenosine 3',5'-cyclic monophosphate (cAMP) production and insulinotropic action in vitro as well as in vivo glucose-lowering and insulin-releasing actions. RESULTS: The results reveal that glycation of the N-terminus of GLP-1 or GIP stabilized both peptides against DPP-IV degradation. However, N-glucitol-GLP-1 displayed reduced cAMP production, insulinotropic activity and glucose-lowering potency, compared to native GLP-1. By contrast, N-glucitol-GIP exhibited substantially improved biological activities, compared to native GIP, and possessed similar or enhanced in vivo potency to GLP-1. N-terminal extension by means of glucitol addition is more beneficial to bioactivity of GIP than it is to GLP-1. CONCLUSIONS: N-terminal glycation generates a super GIP agonist, which possesses acute in vivo glucose-lowering and insulinotropic actions superior to native GLP-1. Therefore, N-glucitol-GIP is a particularly attractive potential candidate molecule for drug therapy of type 2 diabetes.

Inhibition of dipeptidyl peptidase IV activity by oral metformin in Type 2 diabetes.

AIMS: Glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP) are important insulinotropic hormones that enhance the insulin secretory response to feeding. Their potential for treating Type 2 diabetes is limited by short biological half-life owing to degradation by dipeptidyl peptidase IV (DPP IV). We investigated the acute effects of metformin on DPP IV activity in Type 2 diabetes to elucidate inhibition of DPP IV as a possible mechanism of action. METHODS: Eight fasting subjects with Type 2 diabetes (5M/3F, age 53.1+/-4.2 years, BMI 36.8+/-1.8 kg/m2, glucose 8.9+/-1.2 mmol/l, HbA1c 7.8+/-0.6%) received placebo or metformin 1 g orally 1 week apart in a random, crossover design. RESULTS: Following metformin, DPP IV activity was suppressed compared with placebo (AUC0-6 h 3230+/-373 vs. 5764+/-504 nmol ml/l, respectively, P=0.001). Circulating glucose, insulin and total GLP-1 were unchanged. Metformin also concentration-dependently inhibited endogenous DPP IV activity in vitro in plasma from Type 2 diabetic subjects. CONCLUSION: Oral metformin effectively inhibits DPP IV activity in Type 2 diabetic patients, suggesting that the drug may have potential for future combination therapy with incretin hormones.

Comparative effects of GLP-1 and GIP on cAMP production, insulin secretion, and in vivo antidiabetic actions following substitution of Ala8/Ala2 with 2-aminobutyric acid.

The two major incretin hormones, glucagon-like peptide-1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP), are currently being considered as prospective drug candidates for treatment of type 2 diabetes. Interest in these gut hormones was initially spurred by their potent insulinotropic activities, but a number of other antihyperglycaemic actions are now established. One of the foremost barriers in progressing GLP-1 and GIP to the clinic concerns their rapid degradation and inactivation by the ubiquitous enzyme, dipeptidyl peptidase IV (DPP IV). Here, we compare the DPP IV resistance and biological properties of Abu8/Abu2 (2-aminobutyric acid) substituted analogues of GLP-1 and GIP engineered to impart DPP IV resistance. Whereas (Abu8)GLP-1 was completely stable to human plasma (half-life >12 h), GLP-1, GIP, and (Abu2)GIP were rapidly degraded (half-lives: 6.2, 6.0, and 7.1 h, respectively). Native GIP, GLP-1, and particularly (Abu8)GLP-1 elicited significant adenylate cyclase and insulinotropic activity, while (Abu2)GIP was less effective. Similarly, in obese diabetic (ob/ob) mice, GIP, GLP-1, and (Abu8)GLP-1 displayed substantial glucose-lowering and insulin-releasing activities, whereas (Abu2)GIP was only weakly active. These studies illustrate divergent effects of penultimate amino acid Ala8/Ala2 substitution with Abu on the biological properties of GLP-1 and GIP, suggesting that (Abu8)GLP-1 represents a potential candidate for future therapeutic development.

N-terminal His(7)-modification of glucagon-like peptide-1(7-36) amide generates dipeptidyl peptidase IV-stable analogues with potent antihyperglycaemic activity.

Glucagon-like peptide-1(7-36)amide (GLP-1) possesses several unique and beneficial effects for the potential treatment of type 2 diabetes. However, the rapid inactivation of GLP-1 by dipeptidyl peptidase IV (DPP IV) results in a short half-life in vivo (less than 2 min) hindering therapeutic development. In the present study, a novel His(7)-modified analogue of GLP-1, N-pyroglutamyl-GLP-1, as well as N-acetyl-GLP-1 were synthesised and tested for DPP IV stability and biological activity. Incubation of GLP-1 with either DPP IV or human plasma resulted in rapid degradation of native GLP-1 to GLP-1(9-36)amide, while N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 were completely resistant to degradation. N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 bound to the GLP-1 receptor but had reduced affinities (IC(50) values 32.9 and 6.7 nM, respectively) compared with native GLP-1 (IC(50) 0.37 nM). Similarly, both analogues stimulated cAMP production with EC(50) values of 16.3 and 27 nM respectively compared with GLP-1 (EC(50) 4.7 nM). However, N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 exhibited potent insulinotropic activity in vitro at 5.6 mM glucose (P<0.05 to P<0.001) similar to native GLP-1. Both analogues (25 nM/kg body weight) lowered plasma glucose and increased plasma insulin levels when administered in conjunction with glucose (18 nM/kg body weight) to adult obese diabetic (ob/ob) mice. N-pyroglutamyl-GLP-1 was substantially better at lowering plasma glucose compared with the native peptide, while N-acetyl-GLP-1 was significantly more potent at stimulating insulin secretion. These studies indicate that N-terminal modification of GLP-1 results in DPP IV-resistant and biologically potent forms of GLP-1. The particularly powerful antihyperglycaemic action of N-pyroglutamyl-GLP-1 shows potential for the treatment of type 2 diabetes.

Lys9 for Glu9 substitution in glucagon-like peptide-1(7-36)amide confers dipeptidylpeptidase IV resistance with cellular and metabolic actions similar to those of established antagonists glucagon-like peptide-1(9-36)amide and exendin (9-39).

The incretin hormone glucagon-like peptide-1(7-36)amide (GLP-1) has been deemed of considerable importance in the regulation of blood glucose. Its effects, mediated through the regulation of insulin, glucagon, and somatostatin, are glucose-dependent and contribute to the tight control of glucose levels. Much enthusiasm has been assigned to a possible role of GLP-1 in the treatment of type 2 diabetes. GLP-1's action unfortunately is limited through enzymatic inactivation caused by dipeptidylpeptidase IV (DPP IV). It is now well established that modifying GLP-1 at the N-terminal amino acids, His(7) and Ala(8), can greatly improve resistance to this enzyme. Little research has assessed what effect Glu(9)-substitution has on GLP-1 activity and its degradation by DPP IV. Here, we report that the replacement of Glu(9) of GLP-1 with Lys dramatically increased resistance to DPP IV. This analogue, (Lys(9))GLP-1, exhibited a preserved GLP-1 receptor affinity, but the usual stimulatory effects of GLP-1 were completely eliminated, a trait duplicated by the other established GLP-1-antagonists, exendin (9-39) and GLP-1(9-36)amide. We investigated the in vivo antagonistic actions of (Lys(9))GLP-1 in comparison with GLP-1(9-36)amide and exendin (9-39) and revealed that this novel analogue may serve as a functional antagonist of the GLP-1 receptor.

Novel dipeptidyl peptidase IV resistant analogues of glucagon-like peptide-1(7-36)amide have preserved biological activities in vitro conferring improved glucose-lowering action in vivo.

Although the incretin hormone glucagon-like peptide-1 (GLP-1) is a potent stimulator of insulin release, its rapid degradation in vivo by the enzyme dipeptidyl peptidase IV (DPP IV) greatly limits its potential for treatment of type 2 diabetes. Here, we report two novel Ala(8)-substituted analogues of GLP-1, (Abu(8))GLP-1 and (Val(8))GLP-1 which were completely resistant to inactivation by DPP IV or human plasma. (Abu(8))GLP-1 and (Val(8))GLP-1 exhibited moderate affinities (IC(50): 4.76 and 81.1 nM, respectively) for the human GLP-1 receptor compared with native GLP-1 (IC(50): 0.37 nM). (Abu(8))GLP-1 and (Val(8))GLP-1 dose-dependently stimulated cAMP in insulin-secreting BRIN BD11 cells with reduced potency compared with native GLP-1 (1.5- and 3.5-fold, respectively). Consistent with other mechanisms of action, the analogues showed similar, or in the case of (Val(8))GLP-1 slightly impaired insulin releasing activity in BRIN BD11 cells. Using adult obese (ob/ob) mice, (Abu(8))GLP-1 had similar glucose-lowering potency to native GLP-1 whereas the action of (Val(8))GLP-1 was enhanced by 37%. The in vivo insulin-releasing activities were similar. These data indicate that substitution of Ala(8) in GLP-1 with Abu or Val confers resistance to DPP IV inactivation and that (Val(8))GLP-1 is a particularly potent N-terminally modified GLP-1 analogue of possible use in type 2 diabetes.

DPP IV resistance and insulin releasing activity of a novel di-substituted analogue of glucose-dependent insulinotropic polypeptide, (Ser2-Asp13)GIP.

Structure-function studies suggest that preservation of the N-terminus and secondary structure of glucose-dependent insulinotropic polypeptide (GIP) is important for biological activity. Therefore, a novel di-substituted analogue of GIP, (Ser(2)-Asp(13))GIP, containing a negatively charged Asp residue in place of an Ala in position 13, was synthesised and evaluated for in vitro biological activity. Incubation with dipeptidyl peptidase IV (DPP IV) showed the half-lives of GIP and (Ser(2)-Asp(13))GIP to be 2.3 and >4h, respectively. Insulin releasing studies in clonal pancreatic BRIN-BD11 cells demonstrated that (Ser(2)-Asp(13))GIP (10(-12)to 10(-7)mol/l) was significantly less potent (60-90%; P<0.05 to P<0.001) than native GIP. The peptide failed to display antagonistic properties as it did not significantly alter insulin secretion when incubated in the presence of GIP (10(-7)mol/l). These results demonstrate that despite increased resistance to DPP IV, substituting Ala in position 13 with a negatively charged Asp, thus producing the di-substituted analogue (Ser(2)-Asp(13))GIP, significantly reduces biological activity, most likely due to modifications within the secondary structure.

Effects of the novel (Pro3)GIP antagonist and exendin(9-39)amide on GIP- and GLP-1-induced cyclic AMP generation, insulin secretion and postprandial insulin release in obese diabetic (ob/ob) mice: evidence that GIP is the major physiological incretin.

AIMS/HYPOTHESIS: This study examined the biological effects of the GIP receptor antagonist, (Pro3)GIP and the GLP-1 receptor antagonist, exendin(9-39)amide. METHODS: Cyclic AMP production was assessed in Chinese hamster lung fibroblasts transfected with human GIP or GLP-1 receptors, respectively. In vitro insulin release studies were assessed in BRIN-BD11 cells while in vivo insulinotropic and glycaemic responses were measured in obese diabetic ( ob/ ob) mice. RESULTS: In GIP receptor-transfected fibroblasts, (Pro(3))GIP or exendin(9-39)amide inhibited GIP-stimulated cyclic AMP production with maximal inhibition of 70.0+/-3.5% and 73.5+/-3.2% at 10(-6) mol/l, respectively. In GLP-1 receptor-transfected fibroblasts, exendin(9-39)amide inhibited GLP-1-stimulated cyclic AMP production with maximal inhibition of 60+/-0.7% at 10(-6) mol/l, whereas (Pro(3))GIP had no effect. (Pro(3))GIP specifically inhibited GIP-stimulated insulin release (86%; p<0.001) from clonal BRIN-BD11 cells, but had no effect on GLP-1-stimulated insulin release. In contrast, exendin(9-39)amide inhibited both GIP and GLP-1-stimulated insulin release (57% and 44%, respectively; p<0.001). Administration of (Pro(3))GIP, exendin(9-39)amide or a combination of both peptides (25 nmol/kg body weight, i.p.) to fasted (ob/ob) mice decreased the plasma insulin responses by 42%, 54% and 49%, respectively (p<0.01 to p<0.001). The hyperinsulinaemia of non-fasted (ob/ob) mice was decreased by 19%, 27% and 18% (p<0.05 to p<0.01) by injection of (Pro3)GIP, exendin(9-39)amide or combined peptides but accompanying changes of plasma glucose were small. CONCLUSIONS/INTERPRETATION: These data show that (Pro(3))GIP is a specific GIP receptor antagonist. Furthermore, feeding studies in one commonly used animal model of obesity and diabetes, (ob/ob) mice, suggest that GIP is the major physiological component of the enteroinsular axis, contributing approximately 80% to incretin-induced insulin release.

Improved biological activity of Gly2- and Ser2-substituted analogues of glucose-dependent insulinotrophic polypeptide.

The therapeutic potential of glucagon-like peptide-1 (GLP-1) in improving glycaemic control in diabetes has been widely studied, but the potential beneficial effects of glucose-dependent insulinotropic polypeptide (GIP) have until recently been almost overlooked. One of the major problems, however, in exploiting either GIP or GLP-1 as potential therapeutic agents is their short duration of action, due to enzymatic degradation in vivo by dipeptidylpeptidase IV (DPP IV). Therefore, this study examined the plasma stability, biological activity and antidiabetic potential of two novel NH2-terminal Ala2-substituted analogues of GIP, containing glycine (Gly) or serine (Ser). Following incubation in plasma, (Ser2)GIP had a reduced hydrolysis rate compared with native GIP, while (Gly2)GIP was completely stable. In Chinese hamster lung fibroblasts stably transfected with the human GIP receptor, GIP, (Gly2)GIP and (Ser2)GIP stimulated cAMP production with EC(50) values of 18.2, 14.9 and 15.0 nM respectively. In the pancreatic BRIN-BD11 beta-cell line, (Gly2)GIP and (Ser2)GIP (10(-8) M) evoked significant increases (1.2- and 1.5-fold respectively; P<0.01 to P<0.001) in insulinotropic activity compared with GIP. In obese diabetic ob/ob mice, both analogues significantly lowered (P<0.001) the glycaemic excursion in response to i.p. glucose. This enhanced glucose-lowering ability was coupled to a significantly raised (P<0.01) and more protracted insulin response compared with GIP. These data indicate that substitution of the penultimate Ala2 in GIP by Gly or Ser confers resistance to plasma DPP IV degradation, resulting in enhanced biological activity, therefore raising the possibility of their use in the treatment of type 2 diabetes.

Evidence that the major degradation product of glucose-dependent insulinotropic polypeptide, GIP(3-42), is a GIP receptor antagonist in vivo.

The incretin hormone glucose-dependent insulinotropic polypeptide (GIP) is rapidly degraded in the circulation by dipeptidyl peptidase IV forming the N-terminally truncated peptide GIP(3-42). The present study examined the biological activity of this abundant circulating fragment peptide to establish its possible role in GIP action. Human GIP and GIP(3-42) were synthesised by Fmoc solid-phase peptide synthesis, purified by HPLC and characterised by electrospray ionisation-mass spectrometry. In GIP receptor-transfected Chinese hamster lung fibroblasts, GIP(3-42) dose dependently inhibited GIP-stimulated (10(-7) M) cAMP production (up to 75.4+/-5.4%; P<0.001). In BRIN-BD11 cells, GIP(3-42) was significantly less potent at stimulating insulin secretion (1.9- to 3.2-fold; P<0.001), compared with native GIP and significantly inhibited GIP-stimulated (10(-7) M) insulin secretion with maximal inhibition (48.8+/-6.2%; P<0.001) observed at 10(-7) M. In (ob/ob) mice, administration of GIP(3-42) significantly inhibited GIP-stimulated insulin release (2.1-fold decrease; P<0.001) and exaggerated the glycaemic excursion (1.4-fold; P<0.001) induced by a conjoint glucose load. These data indicate that the N-terminally truncated GIP(3-42) fragment acts as a GIP receptor antagonist, moderating the insulin secreting and metabolic actions of GIP in vivo.

Improved stability, insulin-releasing activity and antidiabetic potential of two novel N-terminal analogues of gastric inhibitory polypeptide: N-acetyl-GIP and pGlu-GIP.

AIMS/HYPOTHESIS: This study examined the plasma stability, biological activity and antidiabetic potential of two novel N-terminally modified analogues of gastric inhibitory polypeptide (GIP). METHODS: Degradation studies were carried out on GIP, N-acetyl-GIP (Ac-GIP) and N-pyroglutamyl-GIP (pGlu-GIP) in vitro following incubation with either dipeptidylpeptidase IV or human plasma. Cyclic adenosine 3'5' monophosphate (cAMP) production was assessed in Chinese hamster lung fibroblast cells transfected with the human GIP receptor. Insulin-releasing ability was assessed in vitro in BRIN-BD11 cells and in obese diabetic ( ob/ ob) mice. RESULTS: GIP was rapidly degraded by dipeptidylpeptidase IV and plasma (t(1/2) 2.3 and 6.2 h, respectively) whereas Ac-GIP and pGlu-GIP remained intact even after 24 h. Both Ac-GIP and pGlu-GIP were extremely potent ( p<0.001) at stimulating cAMP production (EC(50) values 1.9 and 2.7 nmol/l, respectively), almost a tenfold increase compared to native GIP (18.2 nmol/l). Both Ac-GIP and pGlu-GIP (10(-13)-10(-8) mmol/l) were more potent at stimulating insulin release compared to the native GIP ( p<0.001), with 1.3-fold and 1.2-fold increases observed at 10(-8) mol/l, respectively. Administration of GIP analogues (25 nmol/kg body weight, i.p.) together with glucose (18 mmol/kg) in ( ob/ ob) mice lowered ( p<0.001) individual glucose values at 60 min together with the areas under the curve for glucose compared to native GIP. This antihyperglycaemic effect was coupled to a raised ( p<0.001) and more prolonged insulin response after administration of Ac-GIP and pGlu-GIP (AUC, 644+/-54 and 576+/-51 ng.ml(-1) x min, respectively) compared with native GIP (AUC, 257+/-29 ng.ml(-1) x min). CONCLUSION/INTERPRETATION: Ac-GIP and pGlu-GIP, show resistance to plasma dipeptidylpeptidase IV degradation, resulting in enhanced biological activity and improved antidiabetic potential in vivo, raising the possibility of their use in therapy of Type II (non-insulin-dependent) diabetes mellitus.