Glucose-responsive artificial promoter-mediated insulin gene transfer improves glucose control in diabetic mice

Abstract

AIM: To investigate the effect of insulin gene therapy using a glucose-responsive synthetic promoter in type 2 diabetic obese mice.

METHODS: We employed a recently developed novel insulin gene therapy strategy using a synthetic promoter that regulates insulin gene expression in the liver in response to blood glucose level changes. We intravenously administered a recombinant adenovirus expressing furin-cleavable rat insulin under the control of the synthetic promoter (rAd-SP-rINSfur) into diabetic Lepr^db/db mice. A recombinant adenovirus expressing β-galactosidase under the cytomegalovirus promoter was used as a control (rAd-CMV-βgal). Blood glucose levels and body weights were monitored for 50 d. Glucose and insulin tolerance tests were performed. Immunohistochemical staining was performed to investigate islet morphology and insulin content.

RESULTS: Administration of rAd-SP-rINSfur lowered blood glucose levels and normoglycemia was maintained for 50 d, whereas the rAd-CMV-βgal control virus-injected mice remained hyperglycemic. Glucose tolerance tests showed that rAd-SP-rINSfur-treated mice cleared exogenous glucose from the blood more efficiently than control virus-injected mice at 4 wk [area under the curve (AUC): 21  508.80 ± 2248.18 vs 62  640.00 ± 5014.28, P < 0.01] and at 6 wk (AUC: 29  956.60 ± 1757.33 vs 60  016.60 ± 3794.47, P < 0.01). In addition, insulin sensitivity was also significantly improved in mice treated with rAd-SP-rINSfur compared with rAd-CMV-βgal-treated mice (AUC: 9150.17 ± 1007.78 vs 11  994.20 ± 474.40, P < 0.05). The islets from rAd-SP-rINSfur-injected mice appeared to be smaller and to contain a higher concentration of insulin than those from rAd-CMV-βgal-injected mice.

CONCLUSION: Based on these results, we suggest that insulin gene therapy might be one therapeutic option for remission of type 2 diabetes.

Key Words: Insulin gene therapy, Synthetic promoter, Glucose-responsive element, Liver-specific promoter, Type 2 diabetes

INTRODUCTION

Type 2 diabetes is characterized by high blood glucose levels in the context of insulin resistance[1]. Insulin resistance indicates that the body does not respond appropriately to serum insulin to regulate blood glucose levels[2]. This phenomenon is mainly caused by metabolic disorders such as obesity, hypertension, non-alcoholic fatty liver disease and elevated serum lipid levels[3-7]. Several peripheral organs contribute to an inappropriate insulin response in the body, which include the liver, muscle, hypothalamus and adipose tissues[8]. For example, the liver regulates blood glucose levels by storing glucose as glycogen and releasing glucose in response to serum insulin changes; this regulation is lost in the insulin-resistant state[9]. In adipocytes, insulin acts to inhibit lipolysis and the release of fatty acids from adipose tissue by decreasing the activity of hormone-sensitive lipase and adipose triglyceride lipase, which is attenuated in the insulin resistant state[10]. Muscle is another big player in regulating blood glucose levels by taking up serum glucose through GLUT4, a glucose transporter, in response to insulin[11]. Due to insulin resistance in the peripheral organs, more insulin is required to maintain blood glucose levels in physiological range, which is evidenced by hyperinsulinemia and impaired glucose tolerance prior to overt type 2 diabetes[12].

When type 2 diabetes is clinically diagnosed, only 50% of normal beta cell function remains[13]. The United Kingdom Prospective Diabetes Study demonstrated that beta cell function continues to deteriorate over time despite treatment with diet, exercise, metformin, sulfonylurea, or insulin[14]. Normal beta cells respond to blood glucose level very efficiently; however, they lose this responsiveness when they are exposed to high glucose levels or high lipid levels for prolonged periods[15]. This loss of responsiveness is easily reversed in the early stages, so it is very important to keep blood glucose levels low to sustain beta cell function during the pathogenesis of type 2 diabetes[16]. Another consideration for gradual loss of beta cell function is hyperinsulinemia, which is a common characteristic of early stage type 2 diabetes[12]. Hyper-secretion of insulin from beta cells is beneficial to control blood glucose levels, but it is paradoxically detrimental to the beta cell itself. The increased demand for insulin drives beta cells to synthesize insulin beyond their capacity for protein folding and secretion, resulting in endoplasmic reticulum stress[17-19]. Therefore, it is very important to relieve the burden on beta cells to preserve cell function during the pathogenesis of diabetes. In this context, intensive insulin therapy is the most effective approach to achieve this goal[16]. For example, it has been shown that patients injected once a day with long lasting insulin analogues showed significant improvement in glucose control, lower HbA1c levels and fewer complications related to diabetes[20]. All of these results suggest that earlier insulin treatment might be important to maintain blood glucose levels in type 2 diabetic patients.

Insulin gene therapy is a novel approach to deliver the insulin gene into a target organ to express insulin under the control of an insulin responsive promoter, thus augmenting or replacing pancreatic insulin production. There have been several attempts to control blood glucose level using insulin gene therapy[21-27]. However, most studies were unsuccessful because of lack of an effective promoter to control insulin gene expression. Recently, we developed a novel synthetic promoter that regulates insulin gene expression in the liver in response to blood glucose level changes[28]. When diabetic mice were infected with an adenoviral vector expressing the insulin gene under this synthetic promoter, blood glucose levels were controlled in the normal physiological range for up to 1 mo[28]. In the present study, we employed the same adenoviral vector system to investigate how insulin supplementation through insulin gene therapy might preserve beta cell function and help control blood glucose levels in a type 2 diabetic animal model.

MATERIALS AND METHODS

Animals

Eight-week-old Lepr^db/db mice were obtained from The Jackson Laboratory (Bar Harbor, ME) or from the South Korea Research Institute of Bioscience and Biotechnology (Daejeon, South Korea). Animals were maintained under specific pathogen-free conditions and provided with sterile food and water ad libitum at the Animal Resource Center, Faculty of Medicine, University of Calgary, Canada and at the facility at the Lee Gil Ya Cancer and Diabetes Institute, Gachon University of Medicine and Science. The use and care of the animals used in this study were approved by the Animal Care Committee, Faculty of Medicine, University of Calgary and Lee Gil Ya Cancer and Diabetes Institute, Gachon University of Medicine and Science.

Generation of recombinant adenovirus

Recombinant adenovirus expressing furin-cleavable rat insulin cDNA under the control of the synthetic promoter (rAd-SP-rINSfur, Figure 1A) was produced as described previously[28] using Transpose-Ad^TM Adenoviral vector system (Qbiogene, Carlsbad, CA). For generation of adenoviruses expressing β-galactosidase under the control of the cytomegalovirus (CMV) promoter (rAd-CMV-βgal), the lacZ coding region from pSV-β-galactosidase vector (Promega, Madison, WI) was cloned into the PCR259 transfer vector (Qbiogene), which has the CMV promoter to drive transgene expression. Recombinant viruses were generated and amplified on a large scale in HEK293 cells and purified as previously described[28]. Viral titer was determined by measurement of optical density at 260 nm.

Figure 1 rAd-SP-rINSfur administration reduces high blood glucose levels to normal physiological range in Lepr^db/db mice. A: Schematic diagram of adenoviral construct expressing furin cleavable insulin gene under the control of synthetic promoter. The synthetic promoter is composed of nine transcription factor binding elements upstream of pyruvate kinase basal promoter (L-PK). A furin-cleavable site was introduced at the junction of the B-chain and C-peptide; B: Blood glucose levels; C: Body weights of Lepr^db/db mice treated with rAd-CMV-βgal or rAd-SP-rINSfur. Viruses were injected into experimental animals at 12 wk of age with a dose of 3 × 10¹⁰ viral particles, and blood glucose levels and body weights were monitored. Blood glucose levels and body weights were measured between 2-4 pm during the day, and food and water were given ad libitum during the experiments. All values are mean ± SE. Day 0 indicates the day of viral injection. HNF-1: Hepatocyte nuclear factor-1; C/EBP: CCAAT enhancer binding protein; GlRE: Glucose responsive elements; HNF-4: Hepatocyte nuclear factor-4.

Blood glucose monitoring and glucose tolerance tests

All mice were given ad libitum access to food and water during the experiments. Recombinant adenoviruses were administered intravenously into Lepr^db/db mice at 12 wk of age via the tail vein, and blood glucose levels were measured using a One-Touch Profile portable blood glucose monitor (Lifescan, Milpitas, CA, United States). For glucose tolerance tests, animals were fasted for 6 h and then a 1 mol/L glucose solution was administered via an intraperitoneal injection at a dose of 2 g/kg body weight. Blood samples were collected from a small cut at the tip of the tail at 0, 15, 30, 60, 90 and 120 min after the glucose load.

Insulin tolerance tests

Animals were fasted for 6 h before injection of insulin (NPH, Lilly, Indianapolis, IN, United States) at a dose of 1.5 U/kg body weight. Blood samples were collected from a small cut at the tip of the tail at 0, 15, 30, 45, 60, 90 and 120 min after the insulin challenge.

Immunohistochemistry

Pancreatic tissues were removed from virus-treated Lepr^db/db mice on day 50 after virus treatment. The samples were fixed with 10% buffered formalin, embedded in paraffin, sectioned at 4.5 μm, and mounted on glass slides. Hematoxylin and eosin staining was performed as previously described[29]. For immunostaining with anti-insulin antibody, slides were treated with xylene, dehydrated in ethanol, and washed with tap water. Slides were incubated with guinea pig anti-rat insulin antibody for 1 h (1/200 in blocking buffer; DAKO, Carpiteria, CA, United States), washed, and incubated in biotinylated anti-guinea pig antibody (1/300) for 1 h. After washing, horseradish peroxidase-conjugated streptavidin was diluted in blocking buffer (1/300) and added to cells for 1 h, followed by color development using Vector VIP (Vector Laboratories, Burlingham, CA, United States) according to the manufacturer’s instructions. After washing with tap water, samples were counterstained with Meyer’s hematoxylin solution.

Statistical analysis

All data are presented as mean ± SE. Differences between groups were evaluated using the Student t test, with significance at P < 0.05.

RESULTS

Administration of rAd-SP-rINSfur reduces blood glucose levels to normal range in Lepr^db/db mice

The Lepr^db/db mouse is a well-known type 2 diabetic animal model that is homozygous for a point mutation in the gene for the leptin receptor. The leptin hormone plays a key role in regulating energy intake and energy expenditure through its action on the leptin receptor[30]. Since the Lepr^db/db mouse does not have a functional leptin receptor, it loses its control over food intake, leading to over-nutrition and obesity. To investigate the effect of augmented insulin gene expression on type 2 diabetes, we injected a recombinant adenovirus expressing rat insulin under the control of a synthetic promoter (rAd-SP-rINSfur, 3 × 10¹⁰ VP, Figure 1A) into diabetic Lepr^db/db mice (n = 14). As a viral control, we injected adenovirus expressing β-galactosidase under the control of the CMV promoter (rAd-CMV-βgal) at same dosage into diabetic Lepr^db/db mice (n = 16). Blood glucose levels started to decrease as early as day 5 after rAd-SP-rINSfur infection, whereas there was no significant change in the blood glucose levels of rAd-CMV-βgal-infected mice. Blood glucose levels reached the normal range on day 14 after virus infection, and were maintained up to 50 d after rAd-SP-rINSfur treatment, when animals were sacrificed (Figure 1B). We also checked the body weights during the experimental period. We found that there was no significant difference between the experimental groups (Figure 1C). These results indicate that augmented insulin gene expression maintained blood glucose levels in the normal physiological range but did not affect body weight gain.

rAd-SP-rINSfur administration improves glucose and insulin tolerance in Lepr^db/db mice

Next, we investigated the ability of rAd-SP-rINSfur-treated mice to clear serum glucose during a glucose tolerance test. At 4 and 6 wk after virus injection, there was a significant difference between the groups in the ability to remove challenged glucose. Mice injected with the control virus showed glucose intolerance, whereas mice treated with rAd-SP-rINSfur showed improved glucose clearance in response to glucose challenge (Figure 2A and B). Since this result indicated improved glucose responsiveness, we also checked insulin sensitivity. In addition to an enhanced ability to clear loaded glucose, the rAd-SP-rINSfur-treated Lepr^db/db mice showed significantly improved insulin sensitivity compared with the Lepr^db/db mice treated with control virus, which showed relative insulin resistance (Figure 3).

Figure 2 rAd-SP-rINSfur administration improves glucose tolerance in Lepr^db/db mice. Glucose (2 g/kg body weight) was administered by intraperitoneal injection, and blood glucose levels were measured at (A) 4 wk and (B) 6 wk (n = 4-6) after virus injection. The area under the curve (AUC) for each glucose tolerance test was quantified. All values are mean ± SE. ^bP < 0.01 vs rAd-CMV-βgal group.

Figure 3 rAd-SP-rINSfur administration improves insulin tolerance in Lepr^db/db mice. Insulin (1.5 U/kg body weight, ip) was administered to mice injected with rAd-CMV-βgal or rAd-SP-rINSfur at 5 wk after virus injection (n = 4-6), and blood glucose levels were measured. The area under the curve (AUC) for each insulin tolerance test was quantified. All values are mean ± SE. ^aP < 0.05 vs rAd-CMV-βgal group.

rAd-SP-rINSfur administration preserves endogenous islets in Lepr^db/db mice

Since it is likely that the additional insulin expression from the liver in rAd-SP-rINSfur treated Lepr^db/db mice might relieve the burden that was imposed on the beta cells in diabetic insulin-resistant Lepr^db/db mice, we expected that beta cells would be protected in rAd-SP-rINSfur treated mice. In Lepr^db/db mice, it is known that the increased demand for insulin causes beta cell hypertrophy to generate more insulin[31], which is a typical characteristic of this type 2 diabetic animal model. Consistently, the islets in Lepr^db/db mice injected with rAd-CMV-βgal virus were larger than those in rAd-SP-rINSfur-treated mice (Figure 4A). This result suggests that there is less demand for insulin, which will relieve the beta cells from overproduction of insulin. In addition to the size, there seemed to be greater insulin content in the islets from rAd-SP-rINSfur-treated mice (Figure 4B), as shown by darker immunocytochemical staining for insulin, indicating that beta cell function is also preserved by augmented insulin gene expression by insulin gene therapy.

Figure 4 rAd-SP-rINSfur administration preserves endogenous islets in Lepr^db/db mice. Pancreatic samples were obtained from rAd-CMV-βgal and rAd-SP-rINSfur virus-treated animals on day 50 after virus injection. Tissue sections were prepared and stained with hematoxylin and eosin (HE) or stained with anti-insulin antibody. (A) is 20 × and (B) is 40 × magnification.

DISCUSSION

The prevalence of type 2 diabetes is increasing in developed and especially in developing countries including South Korea[32]. In the early period of diagnosis, blood glucose levels can be managed by exercise and oral medication such as rosiglitazone[13]. However, during progression of disease, these approaches do not control blood glucose levels, and eventually diabetic patients need insulin treatment, as in type 1 diabetes, because the beta cells do not respond to glucose change or the beta cell mass is significantly reduced[13]. This phenomenon is likely due to prolonged high blood glucose levels (glucotoxicity) and/or high blood lipid levels (lipotoxicity) under insulin resistance status. In addition, beta cells over-produce insulin to compensate for insulin resistance, resulting in endoplasmic reticulum stress, which is known to be one of the main causes of beta cell death in type 2 diabetes. In this context, it is important to find an approach to preserve beta cell function and mass during the early stage of type 2 diabetes. In fact, it was reported that in newly diagnosed type 2 diabetic patients with elevated fasting glucose levels, a short period of intensive insulin therapy helped patients control blood glucose levels in an acceptable range for a long time[33].

In this study, we demonstrated that additional insulin production by insulin gene therapy improved serum glucose management and glucose tolerance in a type 2 diabetic animal model. In addition to these beneficial effects, there was improved insulin tolerance, indicating that insulin gene therapy is also beneficial for insulin sensitivity. Better glucose management can be explained by increased insulin sensitivity in the liver because of the autocrine action of insulin produced by liver per se. In addition, relieving the burden of insulin production would enable beta cells to store more insulin in the vesicles for later needs. We showed that islets from mice injected with rAd-SP-rINSfur appear smaller and appear to contain more insulin compared with control mice, which suggests that glucose tolerance is improved due to enough insulin reservoirs. In addition, lower blood glucose levels are beneficial to preserve beta cell function to secrete insulin in response to serum glucose level changes[34].

Although there was improved glucose tolerance and insulin sensitivity, these effects were attenuated in the later period of treatment. It is most likely that insulin gene expression gradually decreases due to clearance by the host’s immune system[28]. Our previous study clearly showed that an insulin gene delivered by gene therapy was cleared by immune surveillance and thus the effect of the gene was transient[28]. This transient effect can be overcome by using the new generation of adenoviral vectors that lack most of the viral components, enabling them to escape the host’s immune system[35]. Using lentivirus is another option for prolonging the expression period. This viral vector enables the transgene to integrate into the host chromosome for long-term expression of the transgene[35].

In this study, we successfully improved glucose management in a type 2 diabetic animal model through preservation of beta cells; however, there are several considerations to improve the current study. First, we need to extend the insulin gene expression for a longer time. Second, we need to consider safety. Even though expression of an insulin gene is beneficial to manage the blood glucose level, there is potential risk of hypoglycemia[36,37]. Therefore, precautions must be taken to avoid any risk that can be encountered in a future clinical study.

ACKNOWLEDGMENTS

We thank Dr. Ann Kyle for editorial assistance.

COMMENTS

Background

The prevalence of type 2 diabetes is increasing quickly across the world, and it is widely recognized as one of the leading causes of death in the United States. It is closely related to insulin resistance and subsequent loss of beta cell function and mass.

Research frontiers

When type 2 diabetes is clinically diagnosed, there is still a portion of beta cells with normal function. However, this function continues to deteriorate over time despite treatment with diet, exercise or therapeutic drugs. However, studies have found that intensive insulin therapy improves glucose control and preserves beta cell function in the pathogenesis of diabetes. In this study, the authors demonstrate that early insulin treatment using gene therapy improves glucose levels and insulin resistance in obese diabetic mice.

Innovations and breakthroughs

Recent reports have suggested the importance of intensive insulin therapy in the early stages of type 2 diabetes to improve outcomes by preserving beta cell mass and function. In this study, the authors employed insulin gene therapy to provide extra insulin to the body. The therapy induced the liver to produce insulin, driven by a newly developed synthetic promoter which is glucose responsive. The authors successfully improved glucose management in a type 2 diabetic animal model through preservation of beta cells.

Applications

By showing the efficacy of insulin gene therapy, this study may provide a future strategy for therapeutic intervention in the treatment of type 2 diabetic patients.

Terminology

Insulin gene therapy: Delivering an insulin gene into a target organ in the body to express insulin under the control of a glucose-responsive promoter. The insulin required to maintain blood glucose is then provided by insulin gene expression by the target organ, replacing the need for exogenous insulin injections.

Peer review

The authors demonstrated that administration of adenovirus expressing insulin under the control of synthetic promoter that is responsive to glucose change improved serum glucose management and tolerance as well as insulin tolerance in a genetically obese animal model.