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Oral delivery of bioencapsulated exendin-4 expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta-TC6 cells.

ABSTRACT: Glucagon-like peptide (GLP-1) increases insulin secretion but is rapidly degraded (half-life: 2 min in circulation). GLP-1 analogue, exenatide (Byetta) has a longer half-life (3.3-4 h) with potent insulinotropic effects but requires cold storage, daily abdominal injections with short shelf life. Because patients with diabetes take >60 000 injections in their life time, alternative delivery methods are highly desired. Exenatide is ideal for oral delivery because insulinotropism is glucose dependent, with reduced risk of hypoglycaemia even at higher doses. Therefore, exendin-4 (EX4) was expressed as a cholera toxin B subunit (CTB)-fusion protein in tobacco chloroplasts to facilitate bioencapsulation within plant cells and transmucosal delivery in the gut via GM1 receptors present in the intestinal epithelium. The transgene integration was confirmed by PCR and Southern blot analysis. Expression level of CTB-EX4 reached up to 14.3% of total leaf protein (TLP). Lyophilization of leaf material increased therapeutic protein concentration by 12- to 24-fold, extended their shelf life up to 15 months when stored at room temperature and eliminated microbes present in fresh leaves. The pentameric structure, disulphide bonds and functionality of CTB-EX4 were well preserved in lyophilized materials. Chloroplast-derived CTB-EX4 showed increased insulin secretion similar to the commercial EX4 in beta-TC6, a mouse pancreatic cell line. Even when 5000-fold excess dose of CTB-EX4 was orally delivered, it stimulated insulin secretion similar to the intraperitoneal injection of commercial EX4 but did not cause hypoglycaemia in mice. Oral delivery of the bioencapsulated EX4 should eliminate injections, increase patient compliance/convenience and significantly lower their cost.

SUBMITTER: Kwon KC

PROVIDER: S-EPMC3535676 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Oral delivery of bioencapsulated exendin-4 expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta-TC6 cells.

Kwon Kwang-Chul KC Nityanandam Ramya R New James S JS Daniell Henry H

Plant biotechnology journal 20121018 1

Glucagon-like peptide (GLP-1) increases insulin secretion but is rapidly degraded (half-life: 2 min in circulation). GLP-1 analogue, exenatide (Byetta) has a longer half-life (3.3-4 h) with potent insulinotropic effects but requires cold storage, daily abdominal injections with short shelf life. Because patients with diabetes take >60 000 injections in their life time, alternative delivery methods are highly desired. Exenatide is ideal for oral delivery because insulinotropism is glucose depende ...[more]

PMID: 23078126

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Project description:Background: Glucose oxidation is a major contributor to myocardial energy production and its contribution is orchestrated by insulin. While insulin can increase glucose oxidation indirectly by enhancing glucose uptake and glycolysis, it also directly stimulates mitochondrial glucose oxidation, independent of increasing glucose uptake or glycolysis, through activating mitochondrial pyruvate dehydrogenase (PDH), the rate-limiting enzyme of glucose oxidation. However, how insulin directly stimulates PDH is not known. To determine this, we characterized the impacts of modifying mitochondrial insulin signaling kinases, namely protein kinase B (Akt), protein kinase C-delta (PKC-?) and glycogen synthase kinase-3 beta (GSK-3?), on the direct insulin stimulation of glucose oxidation.Methods: We employed an isolated working mouse heart model to measure the effect of insulin on cardiac glycolysis, glucose oxidation and fatty acid oxidation and how that could be affected when mitochondrial Akt, PKC-? or GSK-3? is disturbed using pharmacological modulators. We also used differential centrifugation to isolate mitochondrial and cytosol fraction to examine the activity of Akt, PKC-? and GSK-3? between these fractions. Data were analyzed using unpaired t-test and two-way ANOVA.Results: Here we show that insulin-stimulated phosphorylation of mitochondrial Akt is a prerequisite for transducing insulin's direct stimulation of glucose oxidation. Inhibition of mitochondrial Akt completely abolishes insulin-stimulated glucose oxidation, independent of glucose uptake or glycolysis. We also show a novel role of mitochondrial PKC-? in modulating mitochondrial glucose oxidation. Inhibition of mitochondrial PKC-? mimics insulin stimulation of glucose oxidation and mitochondrial Akt. We also demonstrate that inhibition of mitochondrial GSK3? phosphorylation does not influence insulin-stimulated glucose oxidation.Conclusion: We identify, for the first time, insulin-stimulated mitochondrial Akt as a prerequisite transmitter of the insulin signal that directly stimulates cardiac glucose oxidation. These novel findings suggest that targeting mitochondrial Akt is a potential therapeutic approach to enhance cardiac insulin sensitivity in condition such as heart failure, diabetes and obesity.

Dataset Information

Oral delivery of bioencapsulated exendin-4 expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta-TC6 cells.

Publications

Oral delivery of bioencapsulated exendin-4 expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta-TC6 cells.

Similar Datasets

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