Bioactivity Of Glucagon-like Peptide-1(1-37) And Oral Drug Study Of Chitin-PGA Encapsulating TGLP-1

Glucagon-like peptide-1(GLP-1) is produced by the posttranslational processing of proglucagon and mainly secreted from enteroglucagon-producing L cells in the lower gut, i.e., the ileum and colon/rectum. GLP-1can stimulate glucose-dependent insulin secretion, inhibit glucagon secretion, induce β-cell proliferation and islet neogenesis and inhibit β-cell apoptosis. In addition to these functions, GLP-1can inhibit gastrointestinal motility and reduce appetite. A prominent feature of GLP-1is its glucose-dependent insulinotropic action, so it is thought that the use of exogenous GLP-1could be a new possibility to treat type2diabetic patients without the danger of inducing hypoglycemia. In ou study GLP-1was reseached in two aspects. On one hand, most studies have focused on the effects of the truncated GLP-1form, while the role of GLP-1(1-37) in reducing the blood glucose level is still unclear. Therefore, the bioactivity of GLP-1(1-37) was studied. A expression vector pET32a-GLP-1(1-37) was designed via the method of genetic engineering. After isopropylthio-β-D-galactoside (IPTG) induction, the peptide was purified by Ni2+-affinity chromatography and administrated to mice to analyze its biological activity, the peptide stability in mouse serum and GLP-1R activation in GLP-1R-expressing HEK293cells were also examined. On the other hand, GLP-1(7-37) can be cleaved by the enzyme dipeptidyl peptidase Ⅳ (DPP-4), which results in a very short half-life and low oral availability, so the structure of GLP-1were reformed (A1a8, Lys26, Lys34and Arg36were respectively replaced with Ser8, G1n26, Asp34and Thr36) to obtain the structure of tGLP-1. Enzyme cutting sites of DPP-4and trypsogen for GLP-1were mutated for prolonging the half-life of GLP-1and resisting the clearage of enzyme. Finally the drug can be made to take orally. We made tGLP-1in series to produce concatemer recombinant expression vector for obtaining more peptide tGLP-1. To improve the oral activity of tGLP-1, Chitin-PGA particles encapsulating tGLP-1-C4were prepared and the oral activity was detected.Our studies are summarized as the followings.1. C loning, expression and purification of GLP-1(1-37) The gene encoding GLP-1(1-37) was amplified from pET32a-GLP-1(7-37) by PCR for two times. The final PCR product was cloned into pET32a(+) vector to obtain a recombinant expression plasmid pET32a-GLP-1(1-37) which was transformed into E.coli BL21(DE3) to obtain genetically engineered strain E.coli BL21(DE3)/pET32a-GLP-1(1-37). The interest fusion protein expressed after induction was soluble and the production was to reach30.74%. The fusion proteins were purified by Ni2+-affnity chromatography and cleaved by enterokinase. The digested product was further purified using Ni2+-affinity chromatography.2. Characterization of GLP-1(1-37)The bioactivities of GLP-1(1-37) were determined using an intraperitoneal glucose tolerance test (IPGTT) in normal Kunming mice. The areas under the blood glucose curve (AUCs) in the treated groups were also significantly lower than that of the control group (P<0.001). The mice were treated with GLP-1(1-37) at doses of6,12and24nmol/kg via intraperitoneal injection, the results showed that GLP-1(1-37) decreased glycemic excursion in a dose-dependent manner. To our surprise, GLP-1(1-37) is a highly potent agonist of the GLP-1R (EC50value of0.12nmol/L) by measuring the expression of the luciferase reporter gene expression in transiently transfected human embryonic kidney (HEK293) cells. Unlike the glucose lowering effect of GLP-1(7-37), the glucose-lowering effect of GLP-1(1-37) could not be blocked by the GLP-1R antagonist exendin(9-39). our findings suggest that GLP-1(1-37) might activate the GLP-1R via a different mechanism and could be a potential therapeutic drug for the treatment of type2diabetes in the future.3. Construction of tGLP-1concatemerWe compared with the oral activities of several GLP-1derivants and selected tGLP-1with the best activity. the concatemer recombinant plasmid of tGLP-1were constructed successfully and expressed in E.coli. In addition, we selected tGLP-1-C4which had more stable plasmid and more production to be considered as the following materials by analyzing the stability and expression content of eight recombinant plasmids.4. Expression optimization of tGLP-1-C4fusion protein Four fermentative conditions of IPTG induction were studied. The results showed that the optimal fermentative condition was:liquid LB medium volume was30%, IPTG was added into the culture at the concentrate of0.6mmol/L. Then the bacterium were cultured shakly in25℃for6hours. Besides, the fusion protein tGLP-1-C4existed as inclusion body.5Characterization of Chitin-PGA encapsulating tGLP-1The protein tGLP-1-C4could be clearaged by trypsogen to yield small fragment. When clearaged by trypsogen, tGLP-1-C4showed bioactivity. Compared the intraperitoneal and oral activity of tGLP-1-C4with native GLP-1, the action of tGLP-1-C4is better than that of native GLP-1and has longer potency. Chitin-PGA particles enapsulating tGLP-1-C4was prepared and the encapsulated ratio could be to reach58%. The duration of the efficacy of encapsulated tGLP-1-C4was3h which was longer than that of unencapsulated tGLP-1-C4, when the administration dosage was400nmol/kg. Then the Chitin-PGA particles were encapsulated with casein, and the duration of the efficacy could lengthen to4hours. The results indicated that Chitin-PGA encapsulating tGLP-1-C4could be a potential oral drug for the treatment of type2diabetes in the future.