The Anti-diabetic Effects And Its Mechanism Of EXf, An Exendin-4 Analogue

Exendin-4 (EX-4) is an incretin mimetic that has been developed for the treatment of patients with type 2 diabetes. EX-4 binds to and activates the known GLP-1 receptor (GLP-1R) with a potency comparable to that of the mammalian incretin GLP-1, thereby acting as a glucoregulatory agent. EXf is a fragment of EX-4 with three quarters of amino acids. The purpose of these studies was to evaluate the biological activity of EXf in vivo and in vitro.EXf at the dose of 0.125-2.0 mcg/kg, administered as a single SC injection, lowered modestly plasma glucose concentration in non-diabetic (ICR) and obese (MSG-treated) and spontaneous diabetic KK^Ay mice. Even in the animal model of T1DM, the alloxan diabetic mice, EXf produced dose-related decreases in plasma glucose concentration. EXf significantly decreased glucose excursion following the administration of an oral glucose challenge in ICR and MSG-treated mice. The magnitude of glucose lowering of EXf was comparable to EX-4 at the same dose. EXf has glucose-dependently insulinotropic activity in vivo. Administration of EXf resulted in an increase of first-phase insulin secretion in normal mice (3-fold) and KK^Ay mice (2-fold) following an oral glucose challenge. Although having no effect on gastric emptying, EXf was shown to inhibit dose-dependently the transit of test meal in small intestine in rodent animals. Unexpectedly, long-term treatment with EXf failed to ameliorate the non-fasting blood glucose in KK^Ay and alloxan diabetic mice.GLP-1, a hormonal activator of adenyl cyclase, has been shown to have an important role in keeping the functional activity ofβ-cells, such as stimulates insulin gene transcription, an effect mediated by the cAMP response element (CRE) of the rat insulin I gene promoter (RIP1). EXf produced a concentration-dependent activation of a RIP1-CRE GFP construct (RIP1-CRE-GFP) in a cultured mouse insulinoma cell line, termed NIT-1, and agonist effects were blocked by the GLP-1R antagonist exendin (9-39). Although EXf stimulates cAMP production in NIT-1 cells, we find accompanying stimulation of the RIP1-CRE-driven transcriptional activity to exist independently of this second messenger.The activation of the GLP-1R has been shown to have an important role in the expansion ofβ-cell mass. Constant remodeling ofβ-cell mass is mediated in vivo by proliferative and apoptotic stimuli to ensure a dynamic response to a changing demand for insulin. The present study was undertaken to investigate the biological activity of EXf when cells were challenged by proapoptotic stimulus, actinomycin D and H₂O₂. It has been shown that activation of the GLP-1R should prevent proapoptotic events in NIT-1 cells. EXf improved cell viabilty and reduced DNA fragmentation. This was mediated by an increased expression of the prosurvival proteins PKB and reduced expression of caspase-3. Interestedly, the antiapoptotic action of EXf did not require pretreatment cells with this agent.In summary, EXf is an analog of EX-4 that shares many of the biological and glucoregulatory activities of EX-4 in animals. In vitro, EXf contributes to ameliorate the secretion function ofβ-cell, by stimulation of the RIP1-CRE-driven transcriptional activity, and increaseβ-cell mass, by protectionβ-cell from apoptosis. The present results demonstrate that the substituted peptide has a therapeutic potential for diabetes.