Introduction

GLP-1 is a peptide hormone secreted from the intestinal L cells in response to nutrient ingestion ¹. GLP-1 administration has been shown to reduce hyperglycemia with type 2 diabetic patients ². GLP-1 is a potent insulinotropic hormone, and its insulinotropic effect is glucose-dependent ³. It stimulates not only insulin gene transcription, but also all steps of insulin biosynthesis ⁴. GLP-1 has been shown to be capable of inducing new cells in subjects with an insufficient number of these cells ⁵. Recent results on the actions of GLP-1 show that it stimulates cell differentiation and proliferation and also inhibits apoptosis in cells ^6,7. These actions would increase the cell mass and function over a long-term perspective. In addition to its effect on the cells, GLP-1 reduces glucagon secretion ⁸. Also, it inhibits gastrointestinal motility, especially gastric emptying ⁹. All these effects render GLP-1 extremely desirable as a therapeutic agent for type 2 diabetes.

For the treatment of type 2 diabetes, sulfonylurea has been most commonly used because it is potent, effective, and inexpensive ¹⁰. However, sulfonylurea stimulation of insulin secretion is not glucose-dependent, and hence hypoglycemia is an adverse effect of the treatment ¹¹. Other current therapies also have significant side effects and have limited efficacy ¹². By using GLP-1, it is possible to prevent the risk of hypoglycemia by potentiating insulin secretion in a glucose-dependent manner.

Despite numerous advantages as a diabetes treatment, there is a problem limiting the usefulness of GLP-1. GLP-1 is degraded rapidly due to the presence of a ubiquitously expressed enzyme, DPP-IV ¹³. The conversion of intact, biologically active GLP-1 to its inactive metabolites occurs within 2 min ¹⁴. Therefore, continuous infusion or multiple injections are required for clinical application of GLP-1.

To delay the degradation of GLP-1, the properties of the injectable form have been modified with protamine or zinc ¹⁵. However, the results from using various methods have shown that the half-life is still too short. A number of new approaches are currently under investigation for using GLP-1 as a therapeutic agent. Since the extremely short half-life is due to DPP-IV activity, DPP-IV-resistant analogs and DPP-IV inhibitors have been developed as alternatives ¹⁶. Even though they have shown considerable promise, they still require at least everyday injection to get the therapeutic effect.

If GLP-1 plasmid can be delivered and the genetic information can be effectively processed, it would allow enough GLP-1 to stay in the body for treatment of type 2 diabetes. Nonviral gene delivery is more desirable than viral gene delivery because of its excellent safety profile and ability to carry large amounts of DNA. However, the efficiency of nonviral gene delivery is not sufficient for therapeutic application. In this study, a GLP-1 plasmid was designed to achieve enough expression of GLP-1. The GLP-1 plasmid was constructed by using optimal transcriptional regulatory elements. Also, the GLP-1 plasmid was further modified with nuclear factor B (NFB) binding sites to enhance nuclear import. One way to enhance gene expression efficiency is to increase nuclear transport of plasmids into cells ¹⁷. Nuclear factor B is a family of transcription factors present in every cell type ¹⁸. In one study, under nonstimulatory conditions, transfection was enhanced by 2.6- to 5.8-fold by incorporating NFB binding sites into plasmids ¹⁹. In addition, it has been suggested that the activation of NFB occurs in chronic disorders, including diabetes ²⁰.

Results

Construction of GLP-1 plasmid with NFB binding sites

Previously, a GLP-1 plasmid with a -actin promoter and enhancer was constructed ²¹. Similarly, we constructed the pSIGLP-1 plasmid. The expression was driven by a simian virus 40 (SV40) promoter and enhancer from the pSI vector. Briefly, we synthesized the GLP1 (7–37) gene using a DNA synthesizer. We inserted a furin recognition site (RGRR) after the GLP1 cDNA to produce the active form of GLP-1. The empty plasmid contains only a SV40 promoter and enhancer, a multicloning site, and an ampicillin-resistance gene. We constructed pSIGLP1NFB by inserting the NFB binding sites. Fig. 1 shows the structure of the pSIGLP1 plasmid and construction of the pSIGLP1NFB plasmid. As control plasmids, we constructed pCIGLP1 and pGLP1 with a CMV promoter/enhancer and -actin promoter/enhancer, respectively. The constructed plasmids were confirmed by DNA sequencing.

Figure 1.

GLP-1 plasmid. (A) The structure of the pSIGLP1 plasmid. The locations of the SV40 promoter and the simian virus 40 (SV40) polyadenylation sequences are shown. The furin cleavage site is between the start codon and the GLP1 cDNA. (B) Construction of the pSIGLP1NFB plasmid. Five repeats of NFB binding sites were inserted downstream of the SV40 polyadenylation sequences.

Full figure and legend (113K)

Production of GLP-1 in transfected HepG2 cells

The expressed GLP-1 should be secreted in its active form for therapeutic purposes. To assess this, we performed an enzyme-linked immunosorbent assay (ELISA) for the active form of GLP-1 48 h after transfection. Fig. 2A shows the amount of GLP-1 in the medium after transfection with each plasmid. We used 6 g of plasmid DNA complexed with polyethylenimine (PEI) at the N:P ratio 5:1 to transfect the cells in six-well culture plates. When we assayed 6 g of pSIGLP1NFB-transfected HepG2 cells for GLP-1 production, they produced 1.5-fold more GLP-1 than 6 g of pSIGLP1-transfected HepG2 cells (130 ng/L/24 h vs 201 ng/L/24 h, P < 0.05).

Figure 2.

In vitro transfection assay in HepG2 cells for GLP-1 expression. (A) The GLP-1 levels after transfection of the PEI/pSIGLP1 complex into HepG2 cells. (B) Insulin production in isolated rat islets cocultured with pSIGLP1-transfected HepG2 cells. The graph represents the averages SE of six experiments. *P < 0.05 compared to control. **P < 0.05 compared to pSIGLP1.

Full figure and legend (64K)

Coculture of isolated rat islets with transfected HepG2 cells

We studied whether the secreted GLP-1 from the transfected HepG2 cells can stimulate secretion of insulin from isolated rat islets. We cultured isolated rat islets with the pSIGLP-1- and pSIGLP1NFB-transfected HepG2 cells. There was no enhancement of insulin secretion under low glucose concentrations (50 mg/dl). However, a remarkable increment of insulin secretion occurred under high glucose concentrations (300 mg/dl) (Fig. 2B). The transfected HepG2 cells in each well produced 13.2 and 16.3 g/L GLP-1 over 4 h, respectively. These results showed that GLP-1 significantly stimulated the secretion of insulin under high glucose conditions but not under low glucose conditions.

Delivery of pSIGLP1NFB/PEI complex to type 2 diabetic animals

We injected the pSIGLP1/PEI and pSIGLP1NFB/PEI (N:P ratio 5:1) complexes into mice via the tail vein. All mice tolerated the injections well, and no injection-related deaths occurred. We studied whether the delivered pSIGLP1NFB plasmid can decrease blood glucose levels in insulin-resistant diet-induced obese (DIO) mice and the effect of incorporating NFB binding sites into the plasmid. After intravenous administration of 200 g of the PEI/pSIGLP1NFB complex, the blood glucose levels began to decrease (Fig. 3A). This decrease continued until the 2nd day following administration, after which the blood glucose levels increased until the 21st day after injection. However, the blood glucose levels did not return to the preadministration baseline until the 17th day after injection. The control groups showed no significant change in their blood glucose levels during a similar time period. The second group of animals that received pSIGLP1/PEI showed a pattern of glucose level change similar to that of the pSIGLP1NFB/PEI group, but the change of blood glucose in the pSIGLP1 group was relatively smaller than that in the pSIGLP1NFB group.

Figure 3.

Delivery of PEI/pSIGLP1 in DIO mice. (A) Blood glucose level, (B) plasma GLP-1 level, and (C) plasma insulin concentration changes after PEI/pSIGLP1 injection. The DIO mice received intravenous injection of PEI only or PEI with empty plasmid or PEI with pSIGLP1 or PEI/pSIGLP1. Each group was composed of six rats, and the graphs represent the averages SE. *P < 0.05 compared to control.

Full figure and legend (127K)

We also monitored the plasma level of GLP-1 from each group. In conjunction with the changes in blood glucose levels, the PEI/pSIGLP1NFB group plasma GLP-1 concentration began to increase at the 2nd day after injection (Fig. 3B). The GLP-1 level steadily decreased through the 28th day of the study. The control groups showed almost no changes in GLP-1 concentration.

We assayed the plasma insulin levels to see the insulinotropic effect of GLP-1. The pattern of the insulin concentration change in the PEI/pSIGLP1NFB group mirrored the temporal profile of the plasma GLP-1 levels. This group's plasma insulin concentration value increased 2.5-fold above the baseline (Fig. 3C). The control groups showed minimal changes in plasma insulin concentration.

Two days after the complex was injected, plasma GLP-1 values increased dramatically, and these values gradually returned to baseline levels after 3 weeks (Fig. 3B). Blood glucose levels also decreased and returned to preinjection levels after 3 weeks (Fig. 3A). Plasma insulin levels also increased and gradually returned to baseline 17 days after injection (Fig. 3C). We performed an intraperitoneal glucose tolerance test (IPGTT) to verify the improvement of glucose tolerance. The blood glucose level showed marked decrease (Fig. 4A).

Figure 4.

Results of PEI/pSIGLP1 delivery in DIO mice. (A) Blood glucose level of DIO mice after intraperitoneal glucose loading. At day 2, blood glucose levels in mice treated with nothing or with PEI/pSIGLP1NFB were assayed for the indicated times after intraperitoneal glucose injection. (B) Daily amount of food consumption. (C) Body weight of DIO mice after injection. Each group was composed of six mice, and the graphs represent the averages SE. *P < 0.05 compared to control.

Full figure and legend (93K)

We also monitored the amount of food consumption and the change in body weight after injection. Food consumption decreased 2 days after injection and gradually returned to preinjection values after 14 days (Fig. 4B). Body weight also decreased after injection (Fig. 4C). The time sequences of the changes in parameters showed close correlation with the changes in the plasma GLP-1 levels. After continuous subcutaneous infusion of GLP-1, patients with type 2 diabetes reported a reduction in appetite, which led to significant reductions in body weight ²². Several reasons seem to be involved with reductions in body weight. First, GLP-1 decreases gastric emptying rates. However, the reduced sensation of appetite was reported not only in the postprandial state, but also in the fasting state and before meal ingestion in humans ²³. This suggests that mechanisms other than decreased gastric emptying also contribute to body weight reduction. It has been shown that the central administration of GLP-1 inhibits food intake in rodents ²⁴. Since circulating GLP-1 can access GLP-1 receptors in brain, it will participate in the regulation of appetite ²⁵. It is also possible that gastric distension activates GLP-1-containing neurons, so it can act as an inhibitor of food intake ²⁶.

Discussion

Despite its many remarkable advantages as a therapeutic agent for diabetes, GLP-1 is not immediately clinically applicable because of its extremely short half-life ²⁷. One way to overcome this drawback is GLP1 gene delivery, which enables GLP-1 production in the body. The plasmid containing the GLP1 minigene was tested for in vitro transfection of mouse insulinoma cells ²⁸. A DNA fragment of the human glucagon gene encoding GLP-1 was used for plasmid construction. The GLP-1 plasmid with GLP1 cDNA was also tested in vivo as well as in vitro ²¹. In that study, the expression level of GLP-1 was not high enough for normalizing blood glucose level. The main aim of this study was to design an effective GLP1 delivery system for therapeutic use for type 2 diabetes treatment based upon construction of an efficient GLP-1 plasmid and its delivery.

The GLP-1 plasmid was designed after optimization of transcriptional regulatory elements for higher gene expression. The SV40 early region promoter/enhancer has been used as a viral promoter and has shown very high levels of expression in muscle, liver, and pancreas ²⁹. The strength of the SV40 promoter/enhancer has been tested indirectly by monitoring the GLP-1 gene activity following introduction into the cell via transfection of the plasmid/carrier complex into the cells. Then, the GLP-1 plasmid was designed by increasing nuclear transport, which is the main barrier in nonviral gene delivery. By incorporating nuclear factor B binding sites in the constructed GLP-1 plasmids, the expression level can be increased due to the enhancement of nuclear import of plasmid DNA. For a gene delivery carrier, PEI has been widely used because its ability to form complexes with DNA within physiological pH range and to escape the endosome ³⁰.

Type 2 diabetes is characterized by excessive hepatic glucose production, decreased insulin secretion, and insulin resistance ³¹. Elevated glucose level causes increased oxidative stress due to increased production of reactive oxygen species ³². The oxidative stress leads to the activation of stress-sensitive signaling pathways and worsens insulin secretion and action. NFB is a major intracellular target of hyperglycemia and oxidative stress.

Fig. 2A shows that the production of active GLP-1 by pSIGLP1 and pSIGLP1NFB plasmids was successful. To study the in vitro effect of produced GLP-1, rat islet cells were cocultured with transfected HepG2 cells. The results showed that the produced GLP-1 could stimulate insulin secretion from rat islets only under high glucose concentrations (300 mg/dl). Under low glucose concentrations (50 mg/dl), there was no significant difference between the transfected group and the untransfected control (Fig. 2B). Therefore, the result supports that produced GLP-1 does not cause hypoglycemia.

The DIO mice were treated with the GLP1 gene expression system and showed near-normalization of blood glucose levels for more than 2 weeks (Fig. 3A). Male C57BL/6J mice were used to make a fully developed insulin-resistant DIO phenotype. Normal mice showed an average glucose level around 128 mg/dl at the same age (data not shown). The plasma level of active GLP-1 in the PEI/GLP-1 plasmid group increased nearly twofold compared with the control groups (Fig. 3B). The plasma insulin levels also increased in the PEI/GLP-1 plasmid group, similar to the increase in active GLP-1 levels (Fig. 3C). Error bars suggest that the degree of each response was different, but there was no nonresponder in this study. The DIO mice showed mild conditions of type 2 diabetes similar to early stage conditions that occur in humans. Data suggest that GLP1 gene delivery is effective for early type 2 diabetes. There was an increment of active GLP-1 and insulin levels after GLP1 gene delivery.

In conclusion, the GLP-1 plasmid was designed for high expression levels and as a result of its delivery blood glucose level was significantly lowered in a type 2 diabetic animal model. Also, delivered GLP1 gene showed insulinotropic activity. Therefore, it is proposed that the design of the GLP-1 plasmid and its delivery can be effectively used for treatment of type 2 diabetes.

Methods and materials

Plasmid construction

A plasmid containing an expression cassette for GLP-1 (7–37) was constructed as in a previous study ²¹. Briefly, the GLP1 (7–37) gene was synthesized using a DNA synthesizer. The synthesized GLP1 (7–37) was treated with KpnI and XbaI and the GLP1 (7–37) gene was inserted into the KpnI and XbaI sites of the pSI plasmid. NFB binding sites were inserted. The resulting pSIGLP1NFB was amplified in Escherichia coli JM109 cells (Promega, Madison, WI, USA). The plasmid was prepared free of endotoxins. The purity of the plasmid was confirmed by absorbance at 260 and 280 nm and the quantity was determined with absorbance at 260 nm.

Formation of DNA/carrier complex

The carrier/DNA complexes were prepared by self-assembly. PEI (25,000 Da) was dissolved in 5% glucose solution. The diluted carrier solution was slowly dripped into the prepared DNA plasmid and left for 30 min for formation of complex. The PEI/pSIGLP1NFB (25,000 Da) complex was prepared at an N:P ratio of 5:1. The formation of PEI/pSIGLP1NFB complexes was routinely monitored by 1.0% agarose gel electrophoresis.

In vitro experiment: production of GLP-1 from transfected HepG2 cells

HepG2 cells were cultured and transfected with pSIGLP1NFB/PEI complex as previously described ²¹. GLP-1 concentration was determined by an ELISA technique that measured the biologically active GLP-1 forms GLP-1 (7–36) amide and GLP-1 (7–37) in the sample (Linco Research).

In vitro experiment: coculture of isolated rat islets with pSIGLP1NFB/PEI-transfected HepG2 cells

Islets of Langerhans were isolated from male Sprague–Dawley rat pancreas by a collagenase digestion technique and discontinuous Ficoll density gradient centrifugation. On average, 500–700 islets were isolated from the rat pancreas. For the coculture study, the isolated islets were subcultured for 48 h.

HepG2 cells were cultured and transfected with the complexes as described ²¹. Then, the coculture study was performed with isolated islets and pSIGLP1NFB-transfected HepG2 cells. The 30 islets with small size distribution (mean size of 150 m) were carefully transferred into the pSIGLP1NFB-transfected HepG2 cell culture system. The islets and HepG2 cells were separated by a physical barrier (cell culture insert). Then, the culture medium was changed to fresh RPMI 1640 medium (1.5 ml) supplemented with 10% FBS with basal and high glucose content (50, 300 mg/dl). After 4 h of co-incubation, the insulin and GLP-1 contents in the medium were measured by insulin RIA and GLP-1 ELISA, respectively.

Animals

Male C57BL/6J mice, 4 weeks of age, were used to study the effect of GLP-1 plasmid on type 2 diabetic subjects. Mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). To make a fully developed DIO animal phenotype, 50 animals were fed with a high-fat and high-calorie diet (60 kcal% fat, D12492; Research Diet, New Brunswick, NJ, USA) for 3 months. After 3 months, these animals showed the phenotype of metabolic syndrome characterized by obesity, high blood glucose, and low plasma insulin level. Blood samples were obtained from the tail vein for glucose, GLP-1, and insulin assays. Animals were maintained under virus-antibody-free conditions in climate-controlled rooms with free access to sterilized tap water and special food for DIO mice. Experimental protocols concerning the use of laboratory animals were reviewed and approved by the University of Utah Institutional Animal Care and Use Committee.

Animal experiment

All animals were kept under specific-pathogen-free conditions in the animal facility. Anesthesia was induced by intramuscular injections of pentobarbital (6 mg/kg). The experimental animals, male DIO mice, 4 months of age, were divided into five groups. Each group consisted of 15 mice for three sets of assay. In addition to blood sampling, IPGTT was done 2 days after injection. The first group was not injected. The second group was injected with the carrier PEI only. The third group was injected with the PEI/empty plasmid complex, with the N:P ratio of 5:1. The next two groups were injected with the PEI/pSIGLP1 and PEI/pSIGLP1NfB plasmid complex. Blood samples were withdrawn at days 0, 2, 4, 7, 10, 14, 17, and 21 after injection to measure blood glucose, GLP-1, and insulin concentrations. A glucose tolerance test was used to evaluate the ability of the animals to tolerate a standard glucose load. Mice (n = 5 each at 2 days after injection) were fasted overnight before the intraperitoneal glucose tolerance test. A preload blood sample was taken and a glucose load of 1 g/kg was administered via intraperitoneal injection. Subsequent blood samples were taken 0, 15, 30, 60, and 120 min after injection. Blood samples were obtained from the tail vein and used for measuring the levels of insulin. This procedure allowed for collection without catheterization of blood vessel. All animals were anesthetized by intramuscular injections of pentobarbital (6 mg/kg) before the glucose injection. Data for the effect of GLP1 gene therapy in the type 2 diabetic animal model were analyzed by a generalized linear model. In all cases a P value of <0.05 was considered to be statistically significant. All the data are presented as means SE.

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