Metabolic stability, receptor binding, cAMP generation, insulin secretion and antihyperglycaemic activity of novel N-terminal Glu(9)-substituted analogues of glucagon-like peptide-1

Glucagonlike peptide-1(7 36)amide (GLP-1) is an incretin hormone with therapeutic potential for type 2 diabetes. Rapid removal of the Nterminal dipeptide, His7-Ala8, by the ubiquitous enzyme dipeptidyl peptidase IV (DPP IV) curtails the biological activity of GLP-1. Chemical modifications or substitutions of GLP-1 at His7 or Ala8 improve resistance to DPPIV action, but this often reduces potency. Little attention has focused on the metabolic stability and functional activity of GLP-1 analogues with amino acid substitution at Glu9, adjacent to the DPP IV cleavage site. We generated three novel Glu9-substituted GLP-1 analogues, (Pro9)GLP-1, (Phe9)GLP-1 and (Tyr9)GLP-1 and show for the first time that Glu9 of GLP-1 is important in DPP IV degradation, since replacing this amino acid, particularly with proline, substantially reduced susceptibility to degradation. All three novel GLP-1 analogues showed similar or slightly enhanced insulinotropic activity compared with native GLP-1 despite a moderate 4 10-fold reduction in receptor binding and cAMP generation. In addition, (Pro9)GLP 1 showed significant ability to moderate the plasma glucose excursion and increase circulating insulin concentrations in severely insulin resistant obese diabetic (ob/ob) mice. These observations indicate the importance of Glu9 for the biological activity of GLP-1 and susceptibility to DPP IVmediated degradation.

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

Enhanced cAMP generation and insulin-releasing potency of two novel Tyr(1)-modified enzyme-resistant forms of glucose-dependent insulinotropic polypeptide is associated with significant antihyperglycaemic activity in spontaneous obesity-diabetes

Glucose-dependent insulinotropic polypeptide (GIP) is an important incretin hormone, which potentiates glucose-induced insulin secretion. Antihyperglycaemic actions of GIP provide significant potential in Type 11 diabetes therapy. However, inactivation of GIP by the enzyme dipeptidyl peptidase IV (DPP IV) and its consequent short circulating half-life limit its therapeutic use. Therefore two novel Tyr(1)-Modified analogues of GIP, N-Fmoc-GIP (where Fmoc is 9-fluorenylmethoxycarbonyl) and N-palmitate-GIP, were synthesized and tested for metabolic stability and biological activity. Both GIP analogues were resistant to degradation by DPP IV and human plasma. In Chinese hamster lung (CHL) cells expressing the cloned human GIP receptor, both analogues exhibited a 2-fold increase in cAMP-generating potency compared with native GIP (EC50 values of 9.4, 10.0 and 18.2 nM respectively). Using clonal BRIN-BD11 cells, both analogues demonstrated strong insulinotropic activity compared with native GIP (P <0.01 to P <0.001). In obese diabetic (ob/ob) mice, administration of N-Fmoc-GIP or N-palmitate-GIP (25 nmol/kg) together with glucose (18 mmol/kg) significantly reduced the peak 15 min glucose excursion (1.4- and 1.5-fold respectively; P <0.05 to P <0.01) compared with glucose alone. The area under the curve (AUC) for glucose was significantly lower after administration of either analogue compared with glucose administered alone or in combination with native GIP (1.5-fold; P <0.05). This was associated with a significantly greater AUC for insulin (2.1-fold; P <0.001) for both analogues compared with native GIP. A similar pattern of in vivo responsiveness was evident in lean control mice. These data indicate that novel N-terminal Tyr(1) modification of GIP with an Fmoc or palmitate group confers resistance to degradation by DPP IV in plasma, which is reflected by increased in vitro potency and greater insulinotropic and antihyperglycaemic activities in an animal model of Type 11 diabetes mellitus.

Increased expression of fibroblast activation protein-alpha in keloid fibroblasts: implications for development of a novel treatment option.

Keloid scars are common benign fibroproliferative reticular dermal lesions with unknown etiology and ill-defined management with high rate of recurrence post surgery. The progression of keloids is characterized by increased deposition of extracellular matrix proteins, invasion into the surrounding healthy skin and inflammation. Fibroblasts are considered to be the key cellular mediators of fibrogenesis in keloid scars. Fibroblast activation protein alpha (FAP-a) and dipeptidyl peptidase IV (DPPIV) are proteases located at the plasma membrane promoting cell invasiveness and tumor growth and have been previously associated with keloid scars. Therefore, in this study we analyzed in further detail the expression of FAP-a in keloid fibroblasts compared to control skin fibroblasts. Dermal fibroblasts were obtained from punch-biopsies from the active margin of four keloids and four control skin samples. Flow cytometry was used to analyze FAP-a expression and the CytoSelect(®) 24-Well Collagen I Cell Invasion Assay was applied to study fibroblast invasion. Secretion of extracellular matrix (ECM) proteins was investigated by multiplexed particle-based flow cytometric assay and enzyme-linked immunosorbent assay. We found an increased expression of FAP-a in keloid fibroblasts compared to control skin fibroblasts (p

Degradation and glycemic effects of His(7)-glucitol glucagon-like peptide-1(7-36)amide in obese diabetic ob/ob mice

Glucagon-like peptide-1(7-36)amide (tGLP-1) has attracted considerable potential as a possible therapeutic agent for type 2 diabetes. However, tGLP-1 is rapidly inactivated in vivo by the exopeptidase dipeptidyl peptidase IV (DPP IV), thereby terminating its insulin releasing activity. The present study has examined the ability of a novel analogue, His(7)-glucitol tGLP-1 to resist plasma degradation and enhance the insulin-releasing and antihyperglycemic activity of the peptide in 20-25-week-old obese diabetic ob/ob mice. Degradation of native tGLP-1 by incubation at 37 degreesC with obese mouse plasma was clearly evident after 3 h (35% intact). After 6 h, more than 87% of tGLP-1 was converted to GLP-1(9-36)amide and two further N-terminal fragments, GLP-1(7-28) and GLP-1(9-28). In contrast, His7-glucitol tGLP-1 was completely resistant to N-terminal degradation. The formation of GLP-1(9-36)amide from native tGLP-1 was almost totally abolished by addition of diprotin A, a specific inhibitor of DPP IV. Effects of tGLP-1 and His7-glucitol tGLP-1 were examined in overnight fasted obese mice following i.p. injection of either peptide (30 nmol/kg) together with glucose (18 mmol/kg) or in association with feeding. Plasma glucose was significantly lower and insulin response greater following administration of His7-glucitol tGLP-1 as compared to glucose alone. Native tGLP-1 lacked antidiabetic effects under the conditions employed, and neither peptide influenced the glucose-lowering action of exogenous insulin (50 units/kg). Twice daily s.c. injection of ob/ob mice with His(7)-glucitol tGLP-1 (10 nmol/kg) for 7 days reduced fasting hyperglycemia and greatly augmented the plasma insulin response to the peptides given in association with feeding. These data demonstrate that His(7)-glucitol tGLP-1 displays resistance to plasma DPP IV degradation and exhibits antihyperglycemic activity and substantially enhanced insulin-releasing action in a commonly used animal model of type 2 diabetes. (C) 2001 Elsevier Science B.V. All rights reserved.

Investigation on key molecular targets responsible for antidiabetic properties of Symplocos cochinchinensis (Lour.) S. Moore

Diabetes mellitus is a heterogeneous metabolic disorder characterized by hyperglycemia with disturbances in carbohydrate, protein and lipid metabolism resulting from defects in insulin secretion, insulin action or both. Currently there are 387 million people with diabetes worldwide and is expected to affect 592 million people by 2035. Insulin resistance in peripheral tissues and pancreatic beta cell dysfunction are the major challenges in the pathophysiology of diabetes. Diabetic secondary complications (like liver cirrhosis, retinopathy, microvascular and macrovascular complications) arise from persistent hyperglycemia and dyslipidemia can be disabling or even life threatening. Current medications are effective for control and management of hyperglycemia but undesirable effects, inefficiency against secondary complications and high cost are still serious issues in the present prognosis of this disorder. Hence the search for more effective and safer therapeutic agents of natural origin has been found to be highly demanding and attract attention in the present drug discovery research. The data available from Ayurveda on various medicinal plants for treatment of diabetes can efficiently yield potential new lead as antidiabetic agents. For wider acceptability and popularity of herbal remedies available in Ayurveda scientific validation by the elucidation of mechanism of action is very much essential. Modern biological techniques are available now to elucidate the biochemical basis of the effectiveness of these medicinal plants. Keeping this idea the research programme under this thesis has been planned to evaluate the molecular mechanism responsible for the antidiabetic property of Symplocos cochinchinensis, the main ingredient of Nishakathakadi Kashayam, a wellknown Ayurvedic antidiabetic preparation. A general introduction of diabetes, its pathophysiology, secondary complications and current treatment options, innovative solutions based on phytomedicine etc has been described in Chapter 1. The effect of Symplocos cochinchinensis (SC), on various in vitro biochemical targets relevant to diabetes is depicted in Chapter 2 including the preparation of plant extract. Since diabetes is a multifactorial disease, ethanolic extract of the bark of SC (SCE) and its fractions (hexane, dichloromethane, ethyl acetate and 90 % ethanol) were evaluated by in vitro methods against multiple targets such as control of postprandial hyperglycemia, insulin resistance, oxidative stress, pancreatic beta cell proliferation, inhibition of protein glycation, protein tyrosine phosphatase-1B (PTP-1B) and dipeptidyl peptidase-IV (DPPxxi IV). Among the extracts, SCE exhibited comparatively better activity like alpha glucosidase inhibition, insulin dependent glucose uptake (3 fold increase) in L6 myotubes, pancreatic beta cell regeneration in RIN-m5F and reduced triglyceride accumulation in 3T3-L1 cells, protection from hyperglycemia induced generation of reactive oxygen species in HepG2 cells with moderate antiglycation and PTP-1B inhibition. Chemical characterization by HPLC revealed the superiority of SCE over other extracts due to presence of bioactives (beta-sitosterol, phloretin 2’glucoside, oleanolic acid) in addition to minerals like magnesium, calcium, potassium, sodium, zinc and manganese. So SCE has been subjected to oral sucrose tolerance test (OGTT) to evaluate its antihyperglycemic property in mild diabetic and diabetic animal models. SCE showed significant antihyperglycemic activity in in vivo diabetic models. Chapter 3 highlights the beneficial effects of hydroethanol extract of Symplocos cochinchinensis (SCE) against hyperglycemia associated secondary complications in streptozotocin (60 mg/kg body weight) induced diabetic rat model. Proper sanction had been obtained for all the animal experiments from CSIR-CDRI institutional animal ethics committee. The experimental groups consist of normal control (NC), N + SCE 500 mg/kg bwd, diabetic control (DC), D + metformin 100 mg/kg bwd, D + SCE 250 and D + SCE 500. SCEs and metformin were administered daily for 21 days and sacrificed on day 22. Oral glucose tolerance test, plasma insulin, % HbA1c, urea, creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, total protein etc. were analysed. Aldose reductase (AR) activity in the eye lens was also checked. On day 21, DC rats showed significantly abnormal glucose response, HOMA-IR, % HbA1c, decreased activity of antioxidant enzymes and GSH, elevated AR activity, hepatic and renal oxidative stress markers compared to NC. DC rats also exhibited increased level of plasma urea and creatinine. Treatment with SCE protected from the deleterious alterations of biochemical parameters in a dose dependent manner including histopathological alterations in pancreas. SCE 500 exhibited significant glucose lowering effect and decreased HOMA-IR, % HbA1c, lens AR activity, and hepatic, renal oxidative stress and function markers compared to DC group. Considerable amount of liver and muscle glycogen was replenished by SCE treatment in diabetic animals. Although metformin showed better effect, the activity of SCE was very much comparable with this drug. xxii The possible molecular mechanism behind the protective property of S. cochinchinensis against the insulin resistance in peripheral tissue as well as dyslipidemia in in vivo high fructose saturated fat diet model is described in Chapter 4. Initially animal were fed a high fructose saturated fat (HFS) diet for a period of 8 weeks to develop insulin resistance and dyslipidemia. The normal diet control (ND), ND + SCE 500 mg/kg bwd, high fructose saturated fat diet control (HFS), HFS + metformin 100 mg/kg bwd, HFS + SCE 250 and HFS + SCE 500 were the experimental groups. SCEs and metformin were administered daily for the next 3 weeks and sacrificed at the end of 11th week. At the end of week 11, HFS rats showed significantly abnormal glucose and insulin tolerance, HOMA-IR, % HbA1c, adiponectin, lipid profile, liver glycolytic and gluconeogenic enzyme activities, liver and muscle triglyceride accumulation compared to ND. HFS rats also exhibited increased level of plasma inflammatory cytokines, upregulated mRNA level of gluconeogenic and lipogenic genes in liver. HFS exhibited the increased expression of GLUT-2 in liver and decreased expression of GLUT-4 in muscle and adipose. SCE treatment also preserved the architecture of pancreas, liver, and kidney tissues. Treatment with SCE reversed the alterations of biochemical parameters, improved insulin sensitivity by modifying gene expression in liver, muscle and adipose tissues. Overall results suggest that SC mediates the antidiabetic activity mainly via alpha glucosidase inhibition, improved insulin sensitivity, with antiglycation and antioxidant activities.

Regulation of Na(+)/H(+) exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells

Carraro-Lacroix LR, Malnic G, Girardi AC. Regulation of Na(+)/H(+) exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells. Am J Physiol Renal Physiol 297: F1647-F1655, 2009. First published September 23, 2009; doi:10.1152/ajprenal.00082.2009.-The gut incretin hormone glucagon-like peptide 1 (GLP-1) is released in response to ingested nutrients and enhances insulin secretion. In addition to its insulinotropic properties, GLP-1 has been shown to have natriuretic actions paralleled by a diminished proton secretion. We therefore studied the role of the GLP-1 receptor agonist exendin-4 in modulating the activity of Na(+)/H(+) exchanger NHE3 in LLC-PK(1) cells. We found that NHE3-mediated Na(+)-dependent intracellular pH (pH(i)) recovery decreased similar to 50% after 30-min treatment with 1 nM exendin-4. Pharmacological inhibitors and cAMP analogs that selectively activate protein kinase A (PKA) or the exchange protein directly activated by cAMP (EPAC) demonstrated that regulation of NHE3 activity by exendin-4 requires activation of both cAMP downstream effectors. This conclusion was based on the following observations: 1) the PKA antagonist H-89 completely prevented the effect of the PKA activator but only partially blocked the exendin-4-induced NHE3 inhibition; 2) the MEK1/2 inhibitor U-0126 abolished the effect of the EPAC activator but only diminished the exendin-4-induced NHE3 inhibition; 3) combination of H-89 and U-0126 fully prevented the effect of exendin-4 on NHE3; 4) no additive effect in the inhibition of NHE3 activity was observed when exendin-4, PKA, and EPAC activators were used together. Mechanistically, the inhibitory effect of exendin-4 on pHi recovery was associated with an increase of NHE3 phosphorylation. Conversely, this inhibition took place without changes in the surface expression of the transporter. We conclude that GLP-1 receptor agonists modulate sodium homeostasis in the kidney, most likely by affecting NHE3 activity.

Cooperative effects of aminopeptidase N (CD13) expressed by nonmalignant and cancer cells within the tumor microenvironment

Processes that promote cancer progression such as angiogenesis require a functional interplay between malignant and nonmalignant cells in the tumor microenvironment. The metalloprotease aminopeptidase N (APN; CD13) is often overexpressed in tumor cells and has been implicated in angiogenesis and cancer progression. Our previous studies of APN-null mice revealed impaired neoangiogenesis in model systems without cancer cells and suggested the hypothesis that APN expressed by nonmalignant cells might promote tumor growth. We tested this hypothesis by comparing the effects of APN deficiency in allografted malignant (tumor) and nonmalignant (host) cells on tumor growth and metastasis in APN-null mice. In two independent tumor graft models, APN activity in both the tumors and the host cells cooperate to promote tumor vascularization and growth. Loss of APN expression by the host and/or the malignant cells also impaired lung metastasis in experimental mouse models. Thus, cooperation in APN expression by both cancer cells and nonmalignant stromal cells within the tumor microenvironment promotes angiogenesis, tumor growth, and metastasis.

Proteolytic processing of chemokines: implications in physiological and pathological conditions

Chemokines are small, secreted proteins that orchestrate the migration of cells, which are involved in immune defence, immune surveillance and haematopoiesis. However, chemokines are also implicated in the pathology of various inflammatory diseases, cancers and HIV. The chemokine system is considerably large and has a redundancy in the repertoire of its inflammatory mediators. Therefore, strict regulation of chemokine activity is crucial. Chemokines are the substrate for various proteases including the serine protease CD26/dipeptidyl-peptidase IV and matrix metalloproteinases. Regulation by proteolytic cleavage controls and fine-tunes chemokine function by either enhancing or reducing its chemotactic activity or receptor selectivity. Often chemokines and the proteases that regulate them are produced in the same microenvironment and expression of both may be simultaneously induced by a common stimulus enabling the rapid regulation of chemokine activity. The overall impact of cleaved chemokines in cellular responses is very complex. In this review, we will give an overview on chemokine modification and the respective chemokine modifying proteases. Furthermore, we will summarize the emerging literature describing the consequences in inflammation, haematopoiesis, cancer and HIV infection upon proteolytic chemokine processing.

Internalization of CD26 by mannose 6-phosphate/insulin-like growth factor II receptor contributes to T cell activation

CD26 is a T cell activation antigen known to bind adenosine deaminase and have dipeptidyl peptidase IV activity. Cross-linking of CD26 and CD3 with immobilized mAbs can deliver a costimulatory signal that contributes to T cell activation. Our earlier studies revealed that cross-linking of CD26 induces its internalization, the phosphorylation of a number of proteins involved in the signaling pathway, and subsequent T cell proliferation. Although these findings suggest the importance of internalization in the function of CD26, CD26 has only 6 aa residues in its cytoplasmic region with no known motif for endocytosis. In the present study, we have identified the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGFIIR) as a binding protein for CD26 and that mannose 6-phosphate (M6P) residues in the carbohydrate moiety of CD26 are critical for this binding. Activation of peripheral blood T cells results in the mannose 6 phosphorylation of CD26. In addition, the cross-linking of CD26 with an anti-CD26 antibody induces not only capping and internalization of CD26 but also colocalization of CD26 with M6P/IGFIIR. Finally, both internalization of CD26 and the T cell proliferative response induced by CD26-mediated costimulation were inhibited by the addition of M6P, but not by glucose 6-phosphate or mannose 1-phosphate. These results indicate that internalization of CD26 after cross-linking is mediated in part by M6P/IGFIIR and that the interaction between mannose 6-phosphorylated CD26 and M6P/IGFIIR may play an important role in CD26-mediated T cell costimulatory signaling.

Acyl glucuronide reactivity in perspective: biological consequences

The metabolic conjugation of exogenous and endogenous carboxylic acid substrates with endogenous glucuronic acid, mediated by the uridine diphosphoglucuronosyl transferase (UGT) superfamily of enzymes, leads to the formation of acyl glucuronide metabolites. Since the late 1970s, acyl glucuronides have been increasingly identified as reactive electrophilic metabolites, capable of undergoing three reactions: intramolecular rearrangement, hydrolysis, and intermolecular reactions with proteins leading to covalent drug-protein adducts. This essential dogma has been accepted for over a decade. The key question proposed by researchers, and now the pharmaceutical industry, is: does or can the covalent modification of endogenous proteins, mediated by reactive acyl glucuronide metabolites, lead to adverse drug reactions, perhaps idiosyncratic in nature? This review evaluates the evidence for acyl glucuronide-derived perturbation of homeostasis, particularly that which might result from the covalent modification of endogenous proteins and other macromolecules. Because of the availability of acyl glucuronides for test tube/in vitro experiments, there is now a substantial literature documenting their rearrangement, hydrolysis and covalent modification of proteins in vitro. It is certain from in vitro experiments that serum albumin, dipeptidyl peptidase IV, tubulin and UGTs are covalently modified by acyl glucuronides. However, these in vitro experiments have been specifically designed to amplify any interference with a biological process in order to find biological effects. The in vivo situation is not at all clear. Certainly it must be concluded that all humans taking carboxylate drugs that form reactive acyl glucuronides will form covalent drug-protein adducts, and it must also be concluded that this in itself is normally benign. However, there is enough in vivo evidence implicating acyl glucuronides, which, when backed up by in vivo circumstantial and documented in vitro evidence, supports the view that reactive acyl glucuronides may initiate toxicity/immune responses. In summary, though acyl glucuronide-derived covalent modification of endogenous macromolecules is well-defined, the work ahead needs to provide detailed links between such modification and its possible biological consequences. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

Novel glucagon-like peptide-1 (GLP-1) analog (Val8)GLP-1 results in significant improvements of glucose tolerance and pancreatic β-cell function after 3-week daily administration in obese diabetic (ob/ob) mice

This study evaluates the antidiabetic potential of an enzyme-resistant analog, (Val8)GLP-1. The effects of daily administration of a novel dipeptidyl peptidase IV-resistant glucagon-like peptide-1 (GLP-1) analog, (Val8)GLP-1, on glucose tolerance and pancreatic β-cell function were examined in obese-diabetic (ob/ob) mice. Acute intraperitoneal administration of (Val8)GLP-1 (6.25-25 nmol/kg) with glucose increased the insulin response and reduced the glycemic excursion in a dose-dependent manner. The effects of (Val8)GLP-1 were greater and longer lasting than native GLP-1. Once-daily subcutaneous administration of (Val8)GLP-1 (25 nmol/kg) for 21 days reduced plasma glucose concentrations, increased plasma insulin, and reduced body weight more than native GLP-1 without a significant change in daily food intake. Furthermore, (Val8)GLP-1 improved glucose tolerance, reduced the glycemic excursion after feeding, increased the plasma insulin response to glucose and feeding, and improved insulin sensitivity. These effects were consistently greater with (Val8)GLP-1 than with native GLP-1, and both peptides retained or increased their acute efficacy compared with initial administration. (Val8)GLP-1 treatment increased average islet area 1.2-fold without changing the number of islets, resulting in an increased number of larger islets. These data demonstrate that (Val8)GLP-1 is more effective and longer acting than native GLP-1 in obese-diabetic ob/ob mice.

GIP(Lys16PAL) and GIP(LyS37PAL):novel long-acting acylated analogues of glucose-dependent insulinotropic polypeptide with improved antidiabetic potentia

Glucose-dependent insulinotropic polypeptide (GIP) is a physiological insulin releasing peptide. We have developed two novel fatty acid derivatized GIP analogues, which bind to serum albumin and demonstrate enhanced duration of action in vivo. GIP(Lys16PAL) and GIP(Lys37PAL) were resistant to dipeptidyl peptidase IV (DPP IV) degradation. In vitro studies demonstrated that GIP analogues retained their ability to activate the GIP receptor through production of cAMP and to stimulate insulin secretion. Intraperitoneal administration of GIP analogues to obese diabetic (ob/ob) mice significantly decreased the glycemic excursion and elicited increased and prolonged insulin responses compared to native GIP. A protracted glucose-lowering effect was observed 24 h following GIP(Lys37PAL) administration. Once a day injection for 14 days decreased nonfasting glucose, improved glucose tolerance, and enhanced the insulin response to glucose. These data demonstrate that fatty acid derivatized GIP peptides represent a novel class of long-acting stable GIP analogues for therapy of type 2 diabetes. © 2006 American Chemical Society.

Gliptins - do they increase cardiovascular risk or benefit?

INTRODUCTION: In 2008, the US FDA required all new glucose-lowering therapies to show cardiovascular safety, and this applies to the dipeptidyl peptidase-4 inhibitors ('gliptins'). AREAS COVERED: The cardiovascular safety trials of saxagliptin and alogliptin have recently been published and are the subject of this evaluation. EXPERT OPINION: The Saxagliptin Assessment of Vascular Outcomes Recorded in Patients with Diabetes Mellitus - Thrombolysis in Myocardial Infarction 53 trial and Examination of Cardiovascular Outcomes with Alogliptin versus Standard of Care were both multicentre, randomised, double-blind, placebo-controlled, Phase IV clinical trials. These trials showed that saxagliptin and alogliptin did not increase the primary end point, which was a composite of cardiovascular outcomes that did not include hospitalisations for heart failure. However, saxagliptin significantly increased hospitalisation for heart failure, which was a component of the secondary end point. The effect of alogliptin on hospitalisations for heart failure has not been reported. Neither agent improved cardiovascular outcomes. As there is no published evidence of improved outcomes with gliptins, it is unclear to us why these agents are so widely available for use. We suggest that the use of gliptins be restricted to Phase IV clinical trials until such time as cardiovascular safety and benefits/superiority are clearly established

Improved glycaemic control in obese diabetic ob/ob mice using N-terminally modified gastric inhibitory polypeptide

Gastric inhibitory polypeptide (GIP) is an important insulin-releasing hormone of the enteroinsular axis which is rapidly inactivated by the exopeptidase dipeptidyl peptidase (DPP) IV. The present study has examined the ability of Tyr(1)-glucitol GIP to be protected from plasma degradation and to enhance insulin-releasing and antihyperglycaemic activity in 20- to 25-week-old obese diabetic ob/ob mice. Degradation of GIP by incubation at 37 degrees C with obese mouse plasma was clearly evident after 3 h (35% degraded). After 6 h, more than 61% of GIP was converted to GIP(3-42) whereas N-terminally modified Tyr(1)-glucitol GIP was resistant to degradation in plasma (>99% intact after 6 h). The formation of GIP(3-42) was almost completely abolished by inhibition of plasma DPP IV with diprotin A. Effects of GIP and Tyr(1)-glucitol GIP were examined in overnight-fasted obese mice following i.p. injection of either peptide (20 nmol/kg) together with glucose (18 mmol/kg) or in association with feeding. Most prominent effects were observed in the former group where plasma glucose values at 60 min together with the area under the curve (AUC) for glucose were significantly lower following GIP (AUC, 874 +/- 72 mmol/l.min; P