Are we waiting too long for the cardiovascular outcome trials with the glucagon-like peptide-1 receptor agonists?

Several new medicines are in development for the treatment of type 2 diabetes, and cardiovascular outcome trials are the gold standard for these medicines. This editorial demonstrates that despite being available for over 10 years, there are no cardiovascular outcome studies for any of the glucagon-like peptide-1 (GLP-1) receptor agonists, which demonstrate cardiovascular safety or benefit in subjects with high cardiovascular risk. The author argues that the FDA should be ensuring that clinical outcome studies for subjects with type 2 diabetes and high cardiovascular risk be undertaken in a timelier manner.

Value of the radiolabelled GLP-1 receptor antagonist exendin(9-39) for targeting of GLP-1 receptor-expressing pancreatic tissues in mice and humans

Radiolabelled glucagon-like peptide 1 (GLP-1) receptor agonists have recently been shown to successfully image benign insulinomas in patients. Moreover, it was recently reported that antagonist tracers were superior to agonist tracers for somatostatin and gastrin-releasing peptide receptor targeting of tumours. The present preclinical study determines therefore the value of an established GLP-1 receptor antagonist for the in vitro visualization of GLP-1 receptor-expressing tissues in mice and humans.

Radiolabelled GLP-1 receptor antagonist binds to GLP-1 receptor-expressing human tissues.

PURPOSE Radiolabelled glucagon-like peptide 1 (GLP-1) receptor agonists have recently been shown to successfully image benign insulinomas in patients. For the somatostatin receptor targeting of tumours, however, it was recently reported that antagonist tracers were superior to agonist tracers. The present study therefore evaluated various forms of the (125)iodinated-Bolton-Hunter (BH)-exendin(9-39) antagonist tracer for the in vitro visualization of GLP-1 receptor-expressing tissues in rats and humans and compared it with the agonist tracer (125)I-GLP-1(7-36)amide. METHODS Receptor autoradiography studies with (125)I-GLP-1(7-36)amide agonist or (125)I-BH-exendin(9-39) antagonist radioligands were performed in human and rat tissues. RESULTS The antagonist (125)I-BH-exendin(9-39) labelled at lysine 19 identifies all human and rat GLP-1 target tissues and GLP-1 receptor-expressing tumours. Binding is of high affinity and is comparable in all tested tissues in its binding properties with the agonist tracer (125)I-GLP-1(7-36)amide. For comparison, (125)I-BH-exendin(9-39) with the BH labelled at lysine 4 did identify the GLP-1 receptor in rat tissues but not in human tissues. CONCLUSION The GLP-1 receptor antagonist exendin(9-39) labelled with (125)I-BH at lysine 19 is an excellent GLP-1 radioligand that identifies human and rat GLP-1 receptors in normal and tumoural tissues. It may therefore be the molecular basis to develop suitable GLP-1 receptor antagonist radioligands for in vivo imaging of GLP-1 receptor-expressing tissues in patients.

Comparison of the anti-diabetic effects of GIP- and GLP-1-receptor activation in obese diabetic (ob/ob) mice: studies with DPP IV resistant N-AcGIP and exendin(1-39)amide

Background The two major incretin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are being actively explored as anti-diabetic agents because they lower blood glucose through multiple mechanisms. The rapid inactivation of GIP and GLP-1 by the ubiquitous enzyme, dipeptidyl peptidase IV (DPP IV) makes their biological actions short-lived, but stable agonists such as N-acetylated GIP (N-AcGIP) and exendin(1-39)amide have been advocated as stable and specific GIP and GLP-1 analogues.

The extracellular surface of the GLP-1 receptor is a molecular trigger for biased agonism

Ligand-directed signal bias offers opportunities for sculpting molecular events, with the promise of better, safer therapeutics. Critical to the exploitation of signal bias is an understanding of the molecular events coupling ligand binding to intracellular signaling. Activation of class B G protein-coupled receptors is driven by interaction of the peptide N terminus with the receptor core. To understand how this drives signaling, we have used advanced analytical methods that enable separation of effects on pathway-specific signaling from those that modify agonist affinity and mapped the functional consequence of receptor modification onto three-dimensional models of a receptor-ligand complex. This yields molecular insights into the initiation of receptor activation and the mechanistic basis for biased agonism. Our data reveal that peptide agonists can engage different elements of the receptor extracellular face to achieve effector coupling and biased signaling providing a foundation for rational design of biased agonists.

Insulin-releasing and metabolic effects of small molecule GLP-1 receptor agonist 6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline

Much recent attention has focused on the GLP-1 receptor as a potential target for antidiabetic drugs. Enzyme resistant GLP-1 mimetics such as exenatide are now employed for the treatment of type 2 diabetes, but must be administered by injection. The present study has examined and compared the in vitro and in vivo metabolic actions of a small molecule GLP-1 receptor agonist 6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline (DMB), with native GLP-1, exenatide and liraglutide. DMB significantly stimulated in vitro insulin secretion from BRIN-BD11 cells but with decreased molar potency compared to native GLP-1 or related mimetics. Administration of DMB in combination with glucose to mice significantly (P

Dose-dependent effects of the once-daily GLP-1 receptor agonist lixisenatide in patients with Type 2 diabetes inadequately controlled with metformin:a randomized, double-blind, placebo-controlled trial

To evaluate the dose-response relationship of lixisenatide (AVE0010), a glucagon-like peptide-1 (GLP-1) receptor agonist, in metformin-treated patients with Type 2 diabetes.

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.

[Lys40(Ahx-DTPA-111In)NH2]exendin-4, a very promising ligand for glucagon-like peptide-1 (GLP-1) receptor targeting

High levels of glucagon-like peptide-1 (GLP-1) receptor expression in human insulinomas and gastrinomas provide an attractive target for imaging, therapy, and intraoperative tumor localization, using receptor-avid radioligands. The goal of this study was to establish a tumor model for GLP-1 receptor targeting and to use a newly designed exendin-4-DTPA (DTPA is diethylenetriaminepentaacetic acid) conjugate for GLP-1 receptor targeting. METHODS: Exendin-4 was modified C-terminally with Lys(40)-NH(2), whereby the lysine side chain was conjugated with Ahx-DTPA (Ahx is aminohexanoic acid). The GLP-1 receptor affinity (50% inhibitory concentration [IC(50)] value) of [Lys(40)(Ahx-DTPA)NH(2)]exendin-4 as well as the GLP-1 receptor density in tumors and different organs of Rip1Tag2 mice were determined. Rip1Tag2 mice are transgenic mice that develop insulinomas in a well-defined multistage tumorigenesis pathway. This animal model was used for biodistribution studies, pinhole SPECT/MRI, and SPECT/CT. Peptide stability, internalization, and efflux studies were performed in cultured beta-tumor cells established from tumors of Rip1Tag2 mice. RESULTS: The GLP-1 receptor affinity of [Lys(40)(Ahx-DTPA)NH(2)]exendin-4 was found to be 2.1 +/- 1.1 nmol/L (mean +/- SEM). Because the GLP-1 receptor density in tumors of Rip1Tag2 mice was very high, a remarkably high tumor uptake of 287 +/- 62 %IA/g (% injected activity per gram tissue) was found 4 h after injection. This resulted in excellent tumor visualization by pinhole SPECT/MRI and SPECT/CT. In accordance with in vitro data, [Lys(40)(Ahx-DTPA-(111)In)NH(2)]exendin-4 uptake in Rip1Tag2 mice was also found in nonneoplastic tissues such as pancreas and lung. However, lung and pancreas uptake was distinctly lower compared with that of tumors, resulting in a tumor-to-pancreas ratio of 13.6 and in a tumor-to-lung ratio of 4.4 at 4 h after injection. Furthermore, in vitro studies in cultured beta-tumor cells demonstrated a specific internalization of [Lys(40)(Ahx-DTPA-(111)In)NH(2)]exendin-4, whereas peptide stability studies indicated a high metabolic stability of the radiopeptide in beta-tumor cells and human blood serum. CONCLUSION: The high density of GLP-1 receptors in insulinomas as well as the high specific uptake of [Lys(40)(Ahx-DTPA-(111)In)NH(2)]exendin-4 in the tumor of Rip1Tag2 mice indicate that targeting of GLP-1 receptors in insulinomas may become a useful imaging method to localize insulinomas in patients, either preoperatively or intraoperatively. In addition, Rip1Tag2 transgenic mice represent a suitable animal tumor model for GLP-1 receptor targeting.

GLP-1 receptor expression in human tumors and human normal tissues: potential for in vivo targeting

Peptide hormone receptors overexpressed in human tumors, such as somatostatin receptors, can be used for in vivo targeting for diagnostic and therapeutic purposes. A novel promising candidate in this field is the GLP-1 receptor, which was recently shown to be massively overexpressed in gut and lung neuroendocrine tumors--in particular, in insulinomas. Anticipating a major development of GLP-1 receptor targeting in nuclear medicine, our aim was to evaluate in vitro the GLP-1 receptor expression in a large variety of other tumors and to compare it with that in nonneoplastic tissues. METHODS: The GLP-1 receptor protein expression was qualitatively and quantitatively investigated in a broad spectrum of human tumors (n=419) and nonneoplastic human tissues (n=209) with receptor autoradiography using (125)I-GLP-1(7-36)amide. Pharmacologic competition experiments were performed to provide proof of specificity of the procedure. RESULTS: GLP-1 receptors were expressed in various endocrine tumors, with particularly high amounts in pheochromocytomas, as well as in brain tumors and embryonic tumors but not in carcinomas or lymphomas. In nonneoplastic tissues, GLP-1 receptors were present in generally low amounts in specific tissue compartments of several organs--namely, pancreas, intestine, lung, kidney, breast, and brain; no receptors were identified in lymph nodes, spleen, liver, or the adrenal gland. The rank order of potencies for receptor binding--namely, GLP-1(7-36)amide = exendin-4 >> GLP-2 = glucagon(1-29)--provided proof of specific GLP-1 receptor identification. CONCLUSION: The GLP-1 receptors may represent a novel molecular target for in vivo scintigraphy and targeted radiotherapy for a variety of GLP-1 receptor-expressing tumors. For GLP-1 receptor scintigraphy, a low-background signal can be expected, on the basis of the low receptor expression in the normal tissues surrounding tumors.

Effects of the novel (Pro(3))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.

Beneficial effects of sub-chronic activation of glucagon-like peptide-1 (GLP-1) receptors on deterioration of glucose homeostasis and insulin secretion in aging mice

Aging is associated with an increased incidence of glucose intolerance and type 2 diabetes. Glucagon-like peptide-1 (GLP-1) is an important insulinotropic peptide secreted from the gastrointestinal tract in response to nutrient absorption. The present study was designed to assess the sub-chronic glucose regulatory effects of the potent long-acting GLP-1 receptor agonist, (Val(8))GLP-1, in aging 45-49 week old mice. Daily injection of (Val$)GLP-1 (25 nmol/kg body weight) for 12 days had no significant effect on food intake, body weight, non-fasting plasma glucose and insulin concentrations. However, after 12 days, the glycaemic response to intraperitoneal glucose was improved (P <0.05) in (Val(8))GLP-1 treated mice. In keeping with this, glucose-mediated insulin secretion was enhanced (P <0.05) and insulin sensitivity improved (P <0.05) compared to controls. These data indicate that sub-chronic activation of the GLP-1 receptor by daily treatment with (Val(8))GLP-1 counters aspects of the age-related impairment of pancreatic beta-cell function and insulin sensitivity. 2006 Elsevier Inc. All rights reserved.