Elsevier

Peptides

Volume 32, Issue 7, July 2011, Pages 1408-1414
Peptides

A novel GLP-1 analog exhibits potent utility in the treatment of type 2 diabetes with an extended half-life and efficient glucose clearance in vivo

https://doi.org/10.1016/j.peptides.2011.05.026Get rights and content

Abstract

The multiple physiological characterizations of glucagon-like peptide-1 (GLP-1) make it a promising drug candidate for the therapy of type 2 diabetes. However, the half-life of GLP-1 is short in vivo due to degradation by dipeptidyl peptidase-IV (DPP-IV) and renal clearance. Therefore, the stabilization of GLP-1 is critical for its utility in drug development. Based on our previous research, a GLP-1 analog that contained an intra-disulfide bond exhibited a prolonged biological half-life. In this study, we improved upon previous analogs with a novel GLP-1 analog that contained a tryptophan cage-like sequence for an improved binding affinity to the GLP-1 receptor. The binding capacities and the stabilities of GLP715a were investigated, and the physiological functions of the GLP715a were compared to those of the wild-type GLP-1 in animals. The results demonstrated that the new GLP-1 analog (GLP715a) increased its biological half-life to approximately 48 h in vivo; GLP715a also exhibited a higher binding affinity to the GLP-1 receptor than the wild-type GLP-1. The increased binding capacity of GLP715a to its receptor resulted in a quick response to glucose administration. The long-acting anti-diabetic property of GLP715a was revealed by its increased glucose tolerance, higher HbA1c reduction, more efficient glucose clearance and quicker insulin stimulation upon glucose administration compared to the wild-type GLP-1 in rodents. The improved physiological characterizations of GLP715a make it a possible potent anti-diabetic drug in the treatment of type 2 diabetes mellitus.

Highlights

► GLP-1 analog (GLP715a) containing an inter-disulfide bond and a Trp-cage extended its half-life. ► The analog (GLP715a) remained the biological activity of GLP-1. ► GLP715a showed better glucose tolerance and higher HbA1c reduction than GLP-1. ► GLP715a might be utilized as a long-lasting drug for type 2 diabetes.

Introduction

Glucagon-like peptide 1 (GLP-1) is a gut hormone released from intestinal L cells following oral glucose administration [1]. GLP-1 is vital for insulin secretion, suggesting that it can be applied as a therapeutic strategy for type 2 diabetes treatments [17]. GLP-1 is a potent anti-hyperglycemic hormone [6] that stimulates the secretion of insulin in a glucose-dependent manner and suppresses glucagon secretion, which minimizes the risk of hypoglycemia [16], [25]. It was also confirmed that GLP-1 inhibits apoptosis (programmed cell death) in β cells, consequently improving their survival [23]. The deficiency of GLP-1 secretion in type 2 diabetes suggests that GLP-1 could be a potential therapy for type 2 diabetes [14]. Data have shown that the infusion of GLP-1 induces decreasing levels of blood glucose [22]. GLP-1 was shown to play a crucial role in the regulation of glucose metabolism [21]. However, it was reported that GLP-1 has an exceptionally short half-life of less than 2 min in vivo [19], due to the rapid degradation by the enzyme dipeptidyl peptidase IV (DPP-IV) [5]. This degradation causes the therapeutic administration of GLP-1 to seem impractical; thus, many efforts have focused on amending the pharmacokinetic properties of GLP-1 in a series of derivatives and analogs [20]. Two GLP-1 analogs, exenatide and liraglutide, were approved by the FDA for the treatment of type 2 diabetes in 2005 and 2010, respectively [2], [7]. Exendin-4 shares 53% amino acid sequence similarity with GLP-1, which is a 39-amino acid peptide produced in the salivary glands of the Gila monster (Heloderma suspectum) [9]. No specific exendin-4 receptor was detected; the effect of exendin-4 was expressed through the GLP-1 receptor [13]. Exendin-4 possesses a higher binding affinity to the GLP-1 receptor than GLP-1. The tryptophan cage (Trp-cage) in the C-terminal region of exendin-4 contributed to its unique high binding affinity [15].

It was reported that an intra-disulfide bond caused an extension in the biological half-life of GLP-1 [12], [26] due to the formation of a hairpin conformation. This hairpin conformation resulted from the linked GLP-1 peptide and the inserted tail next to the disulfide bond [12], [26]. It was considered that the substitution of this tail by a Trp-cage-like sequence caused an improved stability and binding capacity to the GLP-1 receptor.

The aim of this study was to seek a long-acting anti-diabetic GLP-1 analog that has improved binding capacity and physiological activities, such as insulin secretion stimulation and glucose tolerance. To achieve this aim, GLP-1 analogs are considered to be potent candidates in this research.

Section snippets

Materials

The DPP-IV enzyme (0.1 mg/ml; purity ∼95%) was purchased from Sigma. A human GLP-1 (7-37) ELISA kit was purchased from Millipore Inc. The rat insulin detection kit was purchased from Phoenix Technology, Inc. A One-Touch Blood Glucose Meter and filters were purchased from Abbott. An HTRF-AMP kit was purchased from Cisbio Inc. All other chemicals were purchased from Sigma unless otherwise specified.

Animals

All studies were carried out with permits from the Animal Experiments Inspectorate, China. Male

GLP715a exhibited improved binding capacity to GLP-1R as compared to GLP-1

GLP715c did not exhibit an improved binding capacity to GLP-1R as predicted, the binding constant of GLP715c to GLP-1R was determined as 23.45 ± 2.64 nM. It was presumed that a flexible conformation was essential for the correct formation of Trp-cage. Two GLP-1 analogs (GLP715a and GLP715d) were prepared in order to form a flexible C-terminus conformation. Results indicated that the insertion of five additional glycine residues after Trp-cage-like sequence was beneficial for an improved binding

Discussion

The known physiological functions of GLP-1 imply that GLP-1 plays a critical role in the regulation of glucose homeostasis and suggest that GLP-1 is a feasible candidate in the treatment of type 2 diabetes mellitus. Despite its attractive anti-diabetic action, the therapeutic potential of using native GLP-1 is limited by its short lifetime (<2 min) in vivo, mainly due to the rapid enzymatic inactivation by DPP-IV [11] and its renal clearance within 10 min [18]. It is, therefore, essential to

Conclusion

In this study, we found that the GLP-1 analog GLP715a exerted effects similar to that of GLP-1, including the glucoregulatory and anti-diabetic effects in vivo. Also, the advantage of the utility of GLP715a was the rapid response and long-lasting effect on insulin secretion and glucose clearance. Our data also suggest that GLP715a acts as a form of long-lasting GLP-1. GLP715a provides a safe and efficient means for pre-clinical and clinical research for the treatment of type 2 diabetes.

Acknowledgment

This study was supported by the Funds for Creative Research Groups of China (Grant No. 2009ZX09301-008-P-05).

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