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. 2010 Jul 7;151(9):4566–4572. doi: 10.1210/en.2010-0193

Systemic Delivery of Bioactive Glucagon-Like Peptide 1 after Adenoviral-Mediated Gene Transfer in the Murine Salivary Gland

Antonis Voutetakis 1, Ana P Cotrim 1, Anne Rowzee 1, Changyu Zheng 1, Trushar Rathod 1, Tulin Yanik 1, Y Peng Loh 1, Bruce J Baum 1, Niamh X Cawley 1
PMCID: PMC2940489  PMID: 20610567

Abstract

An adenoviral (Ad) vector that expresses bioactive glucagon-like peptide 1 (GLP-1) was generated, and its effectiveness at modulating glucose homeostasis was evaluated after transduction of murine salivary glands. The construct was engineered with the signal sequence of mouse GH to direct the peptide into the secretory pathway, followed by a furin cleavage site and the GLP-1(7–37) sequence encoding an Ala to Gly substitution at position 8 to achieve resistance to degradation. When expressed in Neuro2A and COS7 cells, an active form of GLP-1 was specifically detected by RIA in the conditioned medium of transduced cells, showed resistance to degradation by dipeptidyl-peptidase IV, and induced the secretion of insulin from NIT1 pancreatic β-cells in vitro. In vivo studies demonstrated that healthy mice transduced with Ad-GLP-1 in both submandibular glands had serum GLP-1 levels approximately 3 times higher than mice transduced with the control Ad-luciferase vector. In fasted animals, serum glucose levels were similar between Ad-GLP-1 and Ad-luciferase transduced mice in keeping with GLP-1’s glucose-dependent action. However, when challenged with glucose, Ad-GLP-1 transduced mice cleared the glucose significantly faster than control mice. In an animal model of diabetes induced by alloxan, progression of hyperglycemia was significantly attenuated in mice given the Ad-GLP-1 vector compared with control mice. These studies demonstrate that the bioactive peptide hormone, GLP-1, normally secreted from endocrine cells in the gut through the regulated secretory pathway, can be engineered for secretion into the circulatory system from exocrine cells of the salivary gland to affect glucose homeostasis.

Adenoviral-mediated expression of bioactive GLP-1 in mouse salivary glands results in secretion into the circulation and protection against alloxan-induced diabetes.

Glucagon-like peptide (GLP)-1 is a peptide hormone that is expressed as part of the prohormone, proglucagon. In the intestinal endocrine L cells, proglucagon is specifically processed to generate GLP-1 (1–37) by prohormone convertase 1/3 (1), which is further cleaved to GLP-1 (7–37) by removal of its amino terminus. The carboxyl terminus can also be amidated (2); however, this posttranslational modification is not required for biological activity (3,4,5). Thus, GLP-1 (7–37) or GLP-1(7–36-amide) are the bioactive forms of GLP-1 released into circulation after a meal (6). GLP-1 acts as an incretin and functions to slow gastric emptying and enhance insulin function by increasing insulin secretion from pancreatic β-cells and increasing β-cell mass (7). Its biological half-life is approximately 2–3 min (8), and it is inactivated in the blood by the protease, dipeptidyl-peptidase IV (DPP IV) (9). Interestingly, GLP-1 modified with a glycine residue at position 8, as found in exendin-4 of the Gila monster, is resistant to degradation by DPP IV (10).

Because of its potential use in the treatment of type 2 diabetes (T2D), many approaches are being used to enhance the effectiveness of GLP-1 by generating noncleavable GLP-1 analogs (11), DPP IV inhibitors (12), GLP-1 receptor agonists (13), and sustainable and ultimately regulatable levels of GLP-1 by gene therapy (14). However, for injectable compounds to be effective, larger amounts and/or frequent injections are required to compensate for its rapid biological half-life, whereas gene therapeutic approaches rely on safe systemic delivery of GLP-1 through plasmid- or viral vector-based expression systems to produce a constant supply of bioactive GLP-1. Such approaches have proven effective in animal models, but each comes with disadvantages if used for treatment of human T2D; repeated injections may be painful and systemic gene transfer, for instance, could lead to multiple organs being transduced leaving few options available if adverse effects develop (15).

All cells have a constitutive secretory pathway (CSP), whereas dedicated secretory cells such as endocrine, neuroendocrine, and exocrine cells also contain a regulated secretory pathway (RSP) (16). In endocrine cells, prohormones are sorted to and processed within the RSP whereupon their peptide hormones are secreted in a stimulated manner. In the absence of a RSP, expression of a prohormone within nonendocrine cells would thus lead to secretion of the unprocessed precursor through the CSP. Generally, this approach is not therapeutically useful because the precursor is typically devoid of bioactivity. Alternatively, expression of the bioactive peptide itself has been used. When fused with a signal peptide that directs the peptide into the endoplasmic reticulum (ER), it is trafficked through the ER into the Golgi/trans-Golgi network (TGN) from which it is secreted via the CSP.

Salivary glands are exocrine glands that contain both secretory pathways that allow release of secreted proteins through the CSP primarily into the bloodstream (endocrine) and through the RSP into the saliva (exocrine) (17). Therefore, the expression of a bioactive peptide hormone, lacking any sorting signals that would normally direct it into the RSP, should by default exit the salivary gland cells via the CSP into the bloodstream. For such molecules, salivary glands are an excellent target tissue for gene transfer with the potential application for systemic treatment of single protein deficiency syndromes such as those involving GH (18), IGF-I (19), and leptin (20).

Use of the salivary gland for gene therapy has several benefits: 1) it has the capacity to synthesize large amounts of protein; 2) the vector is not diluted after retroductal delivery so that lower numbers of vector particles can be administered to obtain sufficient levels of the protein of interest (21); 3) these glands are encapsulated so that the vector is highly contained (22,23); 4) salivary glands are not essential for life so that if a severe adverse event occurred, a gland could be removed; and 5) vector administration can theoretically be accomplished as an outpatient procedure, similar to that used for performing contrast x-rays of salivary glands by sialography (24). Herein we report the use of an adenoviral (Ad) vector encoding GLP-1 (Ad-GLP-1) that was designed for secretion through the CSP of salivary gland cells into the bloodstream in which it could elicit its biological activity in vivo.

Materials and Methods

Gene synthesis

The synthetic gene encoding GLP-1 was custom synthesized by GeneScript Corp. (Piscataway, NJ) and included a Kozak sequence and contiguous bases that encoded the signal peptide of mouse GH (mGH) (accession no. NM_008117), a mGH filler sequence, a furin cleavage site, and GLP-1 (7–37) (Fig. 1A.). An Ala to Gly substitution was made at position 8 in GLP-1 to generate a more stable molecule. The gene was subcloned by BamH1/Xho1 into the mammalian expression vector, pcDNA3.1(+) (Invitrogen, Carlsbad, CA) and used for transfection studies and as a template for the construction of the adenoviral vector expressing GLP-1. The adenoviral vector was custom engineered by Vector Biolabs, Inc. (Philadelphia, PA). Briefly, the plasmid containing the GLP-1 construct was digested by HindIII/Xho1 and the insert subcloned into the dual-CCM shuttle vector containing a cytomegalovirus (CMV) promoter and a Simian virus 40 polyadenylation site. The CMV-GLP-1-polyadenylation expression cassette was subcloned into the pAd plasmid (type 5; dE1/E3), and the resulting pAd-CMV-GLP-1 plasmid was used for viral packaging in human embryonic kidney 293 cells. The titer of the Ad vector produced (Ad-GLP-1) was determined to be about 5 × 1010 plaque-forming units (pfu)/ml by immunostaining of Hexon viral protein in the infected cells.

Figure 1.

Figure 1

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A, Sequence of the engineered GLP-1 gene. The Ala to Gly substitution is highlighted in green. B, Expression and secretion of GLP-1 [GLP-1 (7–37) and/or GLP-1(7–36-amide)] after transfection of the plasmid in Neuro2a cells. C, Conditioned media from Neuro2a cells containing expressed transgenic GLP-1 or purchased recombinant GLP-1 [GLP-1(7–36-amide)] were incubated with 0.5 mU of purified recombinant DPP IV for the times indicated (transgenic GLP-1, upper panel; recombinant GLP-1, lower panel). The levels of GLP-1 are plotted as percent of starting material remaining at the end of the reaction. 120+ indicates reactions incubated in the presence of a specific DPP IV inhibitor (Millipore). **, P < 0.01 when compared with the starting material; Tukey honestly significant difference test after ANOVA (F = 31.08; P = 0.001). D, Transgenic GLP-1 (7–37) expressed in COS7 cells and secreted into the culture medium induced the release of insulin from the NIT1 cells. ***, P < 0.001 when compared with LacZ conditioned medium (Student t test). LacZ+ indicates the conditioned medium from the Ad-LacZ transduced COS7 cells supplemented with recombinant GLP-1 (0.5 nm). Mock, Nontransfected; ND, not detected.

Cell culture

COS7 cells, an African green monkey kidney cell line, Neuro2a cells, a mouse neuroblastoma cell line, and NIT1 cells, a mouse pancreatic β-cell cell line, were grown at 37 C and 5% CO2, in complete DMEM supplemented with 10% fetal bovine serum and 1× penicillin/streptomycin. Neuro2a cells were transfected with the pcDNA3.1-GLP-1 plasmid using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions or transduced with the Ad-GLP-1 vector. COS7 cells were also transduced with the Ad-GLP-1 vector. GLP-1 was secreted into the culture medium in all cases and assayed using an RIA kit specific for active GLP-1 (Millipore, Billerica, MA). This kit is specific for the free N terminus of GLP-1 and so does not distinguish whether the GLP-1 is amidated. For GLP-1 expression in COS7 cells, a nonendocrine cell devoid of the amidating enzyme, we speculated that our construct is expressing GLP-1 (7–37). For the in vitro bioactivity assay, conditioned media from COS7 cells transduced with either Ad-GLP-1 or the control β-galactosidase vector (Ad-LacZ; Vector Biolabs, Philadelphia, PA) were incubated for 20 min at 37 C with NIT1 cells, which releases insulin in response to bioactive GLP-1. Insulin in the media was measured by RIA using the kit from Millipore.

DPP IV resistance

Purified, recombinant DPP IV was purchased from ProSpec (Rehovot, Israel). GLP-1 (7–37) was expressed in 6-cm dishes of COS7 cells after transduction by Ad-GLP-1 (∼5 × 107 pfu/dish). The culture medium containing the GLP-1 (7–37) was quantified and saved. Human GLP-1 standard [GLP-1(7–36-amide)] was purchased from Bachem (Torrance, CA) and diluted with conditioned medium from nontransduced COS7 cells. The conditioned media were incubated in duplicate with DPP IV (0.5 mU) for up to 2 h and assayed by RIA for residual levels of active GLP-1.

Glucose tolerance tests (GTTs)

Both submandibular glands of naïve male BALB/c mice were transduced by retroductal infusion with either Ad-GLP-1 or Ad-luciferase [Ad-Luc (25)] (5 × 108 pfu/gland, n = 10/group). After 24 h, the mice were subjected to an overnight fast. In previous tests, levels of GLP-1 in the saliva could not be determined accurately due to interference in two different assay systems by a component(s) in the saliva; hence, we focused only on the serum in the subsequent experiments. Sera were then obtained from five mice per group and assayed for GLP-1, insulin, and glucose. Glucose was measured by a handheld glucose meter; insulin was assayed using an RIA kit (Millipore), and GLP-1 was assayed using an enzyme immunoassay kit from ALPCO Diagnostics (Salem, NH), which specifically detects the amidated form of GLP-1 [GLP-1 (7–36)-amide] and cross-reacts 100% with mouse GLP-1. The remaining five mice per group, after determining fasting levels of glucose, were subjected to a GTT by an ip injection of glucose (2 g/kg body weight). Thirty minutes after injection, sera were collected and assayed for the same molecules described above.

In a separate, extended GTT experiment, naïve mice were fasted overnight and randomly divided into two groups (n = 5/group). All mice were then subjected to a GTT as described in Fig. 2, and blood glucose levels were measured over a 2-h period (30, 60, and 120 min) after injection. After 24 h, the mice were transduced with either Ad-GLP-1 or Ad-Luc as described in Fig. 2, and after an additional 24 h, the mice were subjected to another overnight fast followed by another GTT.

Figure 2.

Figure 2

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Serum levels of glucose, insulin, and GLP-1 after Ad-GLP-1 transduction. Two groups of mice were transduced (n = 10/group) and subjected to an overnight fast. Sera were obtained from five mice per group and assayed for GLP-1(7–36-amide) (A), insulin (B), and glucose (C). The remaining five mice per group were subjected to an ip injection of glucose and at 30 min after injection, and sera were collected and assayed for the same molecules (D–F). * and **, P < 0.05 and P ≤ 0.01, respectively, determined by Student’s t test; #, P = 0.032 (ANOVA; F = 6.71).

Alloxan treatment

The submandibular glands of two groups of naïve male BALB/c mice (9 wk old) were transduced with either Ad-Luc or Ad-GLP-1 (n = 15/group, 1.6 × 108 pfu/gland). One day later and after an overnight fast, the mice were injected with alloxan (200 mg/kg in saline, ip, 0 h; Sigma, St. Louis, MO) to induce diabetes, and blood glucose levels were monitored for 3 d (0, +24, +48, +72 h). After the experiment, the submandibular glands were analyzed by hematoxylin and eosin staining. The staining revealed only the expected morphology changes due to the acute immune response that typically follows transduction with adenoviral vector, but they were similar between the two groups and appeared to be returning to normal by the end of the experiment (not shown).

Results and Discussion

Ad-GLP-1 was designed with the signal peptide sequence of mGH to achieve efficient ER translocation in mouse salivary glands (Fig. 1A). A filler sequence composed of mGH amino acids was included followed by a consensus sequence for the proprotein convertase, furin (Lys-Ala-Lys-Arg), an endoprotease found in all cells and localized to the TGN (26). The filler sequence allows for a larger substrate for furin binding, and, in addition, an Arg at P6 of the cleavage site, located within the filler sequence, is known to enhance the protease’s catalytic efficiency (27). Removal of the filler sequence by furin in the TGN would release the GLP-1 (7–37) for secretion via the CSP. Indeed, in two cell lines, Neuro2a (Fig. 1, B and C) and COS7 cells (Fig. 1D), transfection of the initial plasmid (Fig. 1B) or transduction by Ad-GLP-1 (Fig. 1, C and D) resulted in the constitutive secretion of an active form of GLP-1 into the culture medium as assayed by RIA.

We tested our transgenic GLP-1 expressed in the nonendocrine COS7 cells [likely GLP-1 (7–37)] and found it to be significantly resistant to degradation by purified recombinant DPP IV in contrast to a synthetic GLP-1 standard [human GLP-1(7–36-amide) with Ala at position 8] that was readily degraded (Fig. 1C). To demonstrate that our transgenic GLP-1 was bioactive, we incubated the COS7 conditioned medium containing the transgenic GLP-1 (7–37) with NIT1 cells, a mouse pancreatic β-cell line (28) that secretes RSP granule cargo including insulin in response to GLP-1 (29). Cells treated with the conditioned medium from COS7 cells transduced with the Ad-LacZ control virus released insulin at a baseline level. However, the medium from COS7 cells transduced with Ad-GLP-1 had significantly more insulin present, indicating a stimulated secretory response of these cells to the expressed GLP-1 (Fig. 1D). Addition of synthetic GLP-1(7–36-amide) peptide to the control LacZ medium also stimulated the release of insulin, demonstrating that there was nothing present in the control medium that was inhibiting secretion.

These results demonstrate that Ad-GLP-1 mediates expression of a bioactive form of GLP-1 that is secreted constitutively from cells in culture and is resistant to degradation by DPP IV. Thus, Ad-GLP-1 delivery to salivary glands would be expected to result in a bioactive form of GLP-1 that would be released into the circulation and have a longer biological half-life.

In accordance with an animal protocol approved by the Animal Care and Use Committee of the National Institute of Dental and Craniofacial Research, the submandibular glands of BALB/c mice were transduced with either Ad-GLP-1 or a control vector (Ad-Luc) by retroductal infusion as described (30). After 24 h, animals were subjected to an overnight fast and the sera subsequently assayed for glucose, insulin, and GLP-1. GLP-1(7–36-amide), as measured by enzyme immunoassay specific for this molecule, was approximately 2–3 times higher in the sera of Ad-GLP-1-treated mice compared with control mice (Fig. 2, A and D), demonstrating that the expressed GLP-1 was being secreted from the transduced salivary gland cells into the bloodstream.

In previous experiments we attempted to quantify the levels of GLP-1 in the saliva but were unsuccessful due presumably to interference in the assay; hence, in these next experiments, we did not try to measure the GLP-1 in the saliva. In the serum, however, and in accordance with the glucose-dependent function of GLP-1 (31), fasting levels of insulin and glucose were not different between the two groups (Fig. 2, B and C). When assayed 30 min after being challenged with a GTT, the levels of glucose and insulin increased but were significantly less compared with the control group (Fig. 2, E and F). This demonstrated that the Ad-GLP-1-treated mice cleared the injected glucose more efficiently than the control group and might suggest that they had increased insulin sensitivity. This may be the case to some extent, as has been reported (32), but it is also likely that the initial secretory burst (phase 1) of insulin secretion was enhanced due to the elevated GLP-1, resulting in a more efficient initial clearance of the glucose before the 30 min time point.

In a separate experiment, involving an extended GTT in which glucose was monitored for 2 h after glucose injection, Ad-GLP-1-treated mice again reproducibly cleared the glucose more quickly than controls (Fig. 3).

Figure 3.

Figure 3

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GTTs in mice before (A) and after transduction with either Ad-GLP-1 or Ad-Luc. Note that the clearance rate of glucose for mice treated with the control vector is not different before or after vector administration (B), whereas the mice administered the Ad-GLP-1 have an improved clearance rate after vector administration (C). *, P < 0.05 determined by Student’s t test and the Mann-Whitney U test when compared between the same time points from both groups. Similar results described in C were obtained in a separate experiment on mice that were not fasted overnight (n = 5/group; data not shown).

We extended our findings to include an experiment with a mouse model of inducible diabetes. Mice were transduced with either the Ad-GLP-1 or Ad-Luc vectors as described for Fig. 2. After an overnight fast, the mice were given one injection of alloxan to induce diabetes. Glucose levels were monitored for the following 3 d. In keeping with the elevated levels of transgenic GLP-1(7–36-amide), mice transduced with Ad-GLP-1 developed diabetes but at a significantly attenuated level compared with the Ad-Luc transduced mice (Fig. 4), demonstrating that the expression of the transgenic GLP-1 construct in the submandibular glands provided protection against the diabetic phenotype induced by alloxan.

Figure 4.

Figure 4

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A, Scatter plot of blood glucose levels from mice treated with alloxan and Ad-GLP-1 or Ad-Luc. Note that both groups of animals developed diabetes in a time-dependent manner; however, the group of Ad-GLP-1 transduced mice (filled squares) were significantly less affected than the group of Ad-Luc transduced mice (open diamonds). B, Line graph of data in A illustrates the protective effect of Ad expressed GLP-1 on the induction of diabetes. The results demonstrate that transduced salivary glands expressed GLP-1(7–36-amide), secreted it into the circulation and helped ameliorate the effects of alloxan-induced diabetes (ANOVA; F = 9.88, P < 0.001 between groups). Least significant differences post hoc analysis identified the groups at the 48 and 72 h time points to be statistically different (48 h: **, P < 0.01; 72 h: *, P < 0.05).

The discovery of GLP-1 and its incretin function in glucose homeostasis broadened the conceivable treatment options for T2D. Since then, the biology if GLP-1 has been extensively studied and new therapeutic approaches have been developed based on increasing the efficacy of GLP-1 by either preventing it from being inactivated or providing more to the system. Our approach used the latter in that we expressed a biologically active form of GLP-1 in salivary glands, resulting in its secretion into the circulation in which it could function under physiological conditions. Whereas the current construct is Ad-derived and used here only as a proof of concept, future studies will use adeno-associated virus-based expression strategies including ones with regulatable promoters as has been achieved previously in salivary glands (33,34). Based on the results presented herein and our previous studies, it is conceivable to envision drug-inducible expression of GLP-1 from the salivary glands, allowing circulating GLP-1 levels to be modulated by a small molecule (35). Indeed, work is underway to evaluate the use of a glucose promoter to drive expression in a glucose-dependent manner that would represent an ideal self-regulatable solution.

Acknowledgments

We thank Dr. Barton G. Weick (facility veterinarian and animal program director, the National Institute of Dental and Craniofacial Research). We also thank Mr. Milton Papa for his expert help with the animal studies.

Footnotes

This work was supported by the Intramural Research Programs of the Eunice Kennedy Shriver National Institute of Child Health and Human Development and the National Institute of Dental and Craniofacial Research, National Institutes of Health (Bethesda, MD).

Disclosure Summary: The authors have nothing to disclose.

First Published Online July 7, 2010

Abbreviations: Ad, Adenoviral; Ad-GLP-1, Ad vector encoding GLP-1; Ad-Luc, Ad-luciferase; CMV, cytomegalovirus; CSP, constitutive secretory pathway; DPP IV, dipeptidyl-peptidase IV; ER, endoplasmic reticulum; GLP, glucagon-like peptide; GTT, glucose tolerance test; mGH, mouse GH; pfu, plaque-forming units; RSP, regulated secretory pathway; T2D, type 2 diabetes; TGN, trans-Golgi network.

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