Adenoviral vector-mediated glucagon-like peptide 1 gene therapy improves glucose homeostasis in Zucker diabetic fatty rats

BACKGROUND: Glucagon-like peptide-1 (GLP-1) is a gut-derived incretin hormone that plays an important role in glucose homeostasis. Its functions include glucose-stimulated insulin secretion, suppression of glucagon secretion, deceleration of gastric emptying, and reduction in appetite and food intake. Despite the numerous antidiabetic properties of GLP-1, its therapeutic potential is limited by its short biological half-life due to rapid enzymatic degradation by dipeptidyl peptidase IV. The present study aimed to demonstrate the therapeutic effects of constitutively expressed GLP-1 in an overt type 2 diabetic animal model using an adenoviral vector system. METHODS: A novel plasmid (pAAV-ILGLP-1) and recombinant adenoviral vector (Ad-ILGLP-1) were constructed with the cytomegalovirus promoter and insulin leader sequence followed by GLP-1(7-37) cDNA. RESULTS: The results of an enzyme-linked immunosorbent assay showed significantly elevated levels of GLP-1(7-37) secreted by human embryonic kidney cells transfected with the construct containing the leader sequence. A single intravenous administration of Ad-ILGLP-1 into 12-week-old Zucker diabetic fatty (ZDF) rats, which have overt type 2 diabetes mellitus (T2DM), achieved near normoglycemia for 3 weeks and improved utilization of blood glucose in glucose tolerance tests. Circulating plasma levels of GLP-1 increased in GLP-1-treated ZDF rats, but diminished 21 days after treatment. When compared with controls, Ad-ILGLP-1-treated ZDF rats had a lower homeostasis model assessment for insulin resistance score indicating amelioration in insulin resistance. Immunohistochemical staining showed that cells expressing GLP-1 were found in the livers of GLP-1-treated ZDF rats. CONCLUSIONS: These data suggest that GLP-1 gene therapy can improve glucose homeostasis in fully developed diabetic animal models and may be a promising treatment modality for T2DM in humans.     

Effect of polyethylenimine on recombinant adeno-associated virus mediated insulin gene therapy

BACKGROUND: Recombinant adeno-associated virus (rAAV) is becoming a promising vector for gene therapy for type I diabetes. The objective of this study was to investigate the effect of incorporation of polyethylenimine (PEI) on rAAV-mediated insulin gene therapy in vitro and in vivo. METHODS: Recombinant AAV vector, harboring the furin-mutated human insulin and enhanced green fluorescent protein (EGFP) genes, was constructed. The effect of complexation with PEI on rAAV-mediated gene transfer was examined in Huh7 human hepatoma cells. The transgene expression was also examined in streptozotocin (STZ)-induced diabetic C57BL/6J mice by direct administration of rAAV into the livers of the animals, followed by monitoring changes in body weight and blood glucose levels. Secretion of human insulin was determined by radioimmunoassay (RIA) and immunohistochemical staining in the livers. RESULTS: Complexation with PEI was shown to enhance rAAV-mediated transgene expression in Huh7 cells, resulting in higher transduction efficiency and enhanced production of immunoreactive human insulin. Heparin competition assay demonstrated that endocytosis of rAAV-PEI was partially inhibited by heparin. The enhancement of rAAV-mediated transgene expression was also demonstrated in the animals, showing lowering of blood glucose and longer duration of normoglycemia. Immunofluorescent staining of the liver sections demonstrated that PEI increased the uptake of rAAV and enhanced insulin secretion. The enhancement of PEI on rAAV-mediated insulin gene therapy was further confirmed by glucose challenge and a 10-h fasting blood glucose test. CONCLUSIONS: Results obtained in this study demonstrated that incorporation of PEI augmented rAAV-mediated insulin gene transfer and enhanced amelioration of hyperglycemia in the STZ-induced diabetic animals.     

Adenoviral insulin gene therapy prolongs survival of IDDM model BB rats by improving hyperlipidemia.

Diabetes is frequently associated with hyperlipidemia, which results in atherogenic complications. Insulin-dependent diabetes mellitus (IDDM) model BB/Wor//Tky (BB) rats exhibit both hyperglycemia and hyperlipidemia and die within 3 weeks after the onset of diabetes unless insulin therapy is given. We performed insulin gene therapy in BB rats with adenovirus vectors through the tail vein. After infusion, plasma triglyceride levels dropped quickly and maintained low levels for 1 week, whereas blood glucose levels showed a slight decrease. The survival period of diabetic BB rats was prolonged to up to 75 days by infusing insulin gene-expressing adenoviral vectors. We suggest that the control of hyperlipidemia can be a life-saving measure when combined with hyperglycemia control in the treatment of diabetes.     

Heat tolerance in rats following administration of streptozotocin,glucose, insulin and glucose plus insulin

Rise in rectal temperature (T_re) and survival time was determined on exposure to 38°C in adult normoglycemic and diabetic (streptozotocin treated) rats and 1 h following glucose feeding or insulin administration or both, and in young rats with and without glucose feeding or insulin treatment. The heat tolerance of adult animals treated with streptozotocin and insulin plus glucose and of adult and young animals treated with glucose feeding or insulin was less than that of their respective normoglycemic controls. The rectal temperature on exposure to heat in the treated animals was significantly higher than that of controls in the adult, but not in young rats. Exposure to heat of the normoglycemic and glucose-fed animals resulted in a rise in blood glucose in the adults and a fall in the young. The already raised blood glucose level in the streptozotocin-treated animals rose further on exposure to heat. The rate of recovery of the blood glucose was not significantly altered by exposure of the animals to heat 60 min after administration of insulin or insulin plus glucose.     

Long-term modifications of blood pressure in normotensive and spontaneously hypertensive rats by gene delivery of rAAV-mediated cytochrome P450 arachidonic acid hydroxylase

INTRODUCTION It is well known that arachidonic acid (AA) exists ex- tensively in eukaryotic cells and it is metabolized by cycloxygenases and lipoxygenases to produce prostag- landins (PGs) and hydroperoxyeicosatetraenoic acid(HPETE) [1-3]. In the 1980’s…     

Effects of soy protein and genistein on blood glucose, antioxidant enzyme activities, and lipid profile in streptozotocin-induced diabetic rats

In the current study, the effect of soy protein and genistein, one of the main isoflavones in soybeans, on blood glucose, lipid profile, and antioxidant enzyme activities in streptozotocin (STZ)-induced diabetic rats was investigated. Male Sprague-Dawley rats were divided into nondiabetic control, STZ, STZ-genistein supplemented group (STZ-G; 600 mg/kg diet), and STZ-isolated soy protein supplemented group (STZ-ISP; 200 g/kg diet). Diabetes was induced by a single injection of STZ (50 mg/kg BW) freshly dissolved in 0.1 mol/L citrate buffer (pH 4.5) into the intraperitonium. Diabetes was confirmed by measuring the fasting blood glucose concentration 48-h post-injection. The rats with blood glucose level above 350 mg/dL were considered to be diabetic. Genistein and ISP were supplemented in the diet for 3 weeks. The supplementation of genistein and ISP increased the plasma insulin level but decreased the HbA(IC) level of the STZ-induced diabetic rats. The supplementation of genistein and ISP increased the glucokinase level of the STZ-induced diabetic rats. A significant reduction in glucose-6-phosphatase was observed in the groups treated with genistein and ISP in comparison with the diabetic control group. Hepatic superoxide dismutase, catalase, and glutathione peroxidase activities of the STZ-induced diabetic rats were significantly decreased in comparison with the control rats. Administering genistein and ISP to the STZ-induced diabetic rats significantly increased those enzyme activities. The concentration of thiobarbituric acid reactive substances in the STZ-induced diabetic rats was significantly elevated, while the genistein and ISP supplement decreased it to the control concentration. Genistein and ISP supplements seem to be beneficial for correcting the hyperglycemia and preventing diabetic complications.     

Evidence for a deficient pancreatic beta-cell response in a rat model of hyperthyroidism

To clarify mechanism behind the abnormal glucose tolerance, observed in hyperthyroidism, we studied genomic and nongenomic effects of thyroid hormone on insulin secretion using a rat model of hyperthyroidism. Male Sprague-Dawley rats were intraperitoneally injected with vehicle, low (100 microg/kg) or high dose (600 microg/kg) of thyroxin (T(4)) for 2 weeks. Rats treated with high dose, but not low dose, of T(4), showed an increase in serum T(3) levels, and a decrease in body weight as compared to control rats. In rats treated with either dose of T(4), fasting blood glucose levels were increased, but serum insulin levels were similar to those of controls. After an oral glucose load, blood glucose levels were increased in rats treated with high dose, but not low dose, of T(4). Serum insulin levels after the oral glucose load were decreased in rats treated with either dose of T(4). After an intravenous glucose load, blood glucose levels were comparable among groups, but serum insulin levels tended to be low in T(4)-treated rats. Steady-state blood glucose levels were comparable among groups. The insulin secretory responses to high glucose (20mM) or arginine (10mM) of the isolated pancreas was decreased in rats treated with high dose, but not low dose, of T(4). Mean insulin secretory response to glucose and arginine were decreased by 40.1% and by 60.4% in high-dose-T(4)-treated rats. Addition of T(3) in the perfusion medium decreased glucose-induced insulin release. Ratios of proinsulin mRNA levels to beta-actin mRNA were decreased in the islets of T(4)-treated rats (0.45 +/- 0.07 vs control 0.61 +/- 0.03, p < 0.05). Levels of TR (thyroid hormone nuclear receptor) alpha1 + cErb Aalpha2 mRNA, but not TRbeta1, were decreased in the pancreatic islets of T(4)-treated rats. Calculated islet area was increased, but the number of beta-cells determined immunohistochemically was not increased in T(4)-treated rats, nor the volume density of insulin positive islets. We concluded that a deficient pancreatic beta-cell response to glucose, rather than insulin resistance, was responsible for abnormal glucose tolerance in this model of hyperthyroidism. Thyroid hormone causes a decrease in glucose-induced insulin secretion. We observed nongenomic and genomic effects of thyroid hormone on glucose-induced insulin secretion.     

Cold tolerance in rats following administration of streptozotocin,glucose, insulin and glucose plus insulin

Fall in rectal temperature (T_re) and survival time was determined on exposure to–20°C in adult normoglycemic and diabetic (streptozotocin treated) rats and 1 h following glucose feeding or insulin administration or both and on exposure to–10°C in young rats with and without glucose feeding. The susceptibility to frostbite was determined by exposure of the limbs to freezing mixture of–19°C or–23°C. The rate of fall of T_re was less and the survival time more in glucose and insulin plus glucose treated animals. On the other hand, the rate of fall of T_re was more and the survival time less, in dia betic and insulin-treated animals. The rectal temperature at which the animal died was the same in the control and the treated animals. The susceptibility to frost bite was more in insulin treated and diabetic animals and less in glucose-fed animals. Exposure to cold during the second h after glucose or glucose plus insulin injection did not alter the blood glucose from that obtained at room temperature. In insulin-treated animals the rate of rise of blood glucose during the second h was much higher at low temperature than at room temperature. The rise in blood glucose in diabetic animals was much higher than in normoglycemic animals exposed to cold.     

Glucose transporter-2 (GLUT2) promoter mediated transgenic insulin production reduces hyperglycemia in diabetic mice

Insulin production afforded by hepatic gene therapy (HGT) retains promise as a potential treatment for type 1 diabetes, but successful approaches have been limited. We employed a novel and previously untested promoter for this purpose, glucose transporter-2 (GLUT2) to drive insulin production via delivery by recombinant adeno-associated virus (rAAV). In vitro, the GLUT2 promoter was capable of robust glucose-responsive expression in transduced HepG2 human hepatoma cells. Therefore, rAAV constructs were designed to express the furin-cleavable human preproinsulin B10 gene, under the control of the murine GLUT2 promoter and packaged for delivery with rAAV expressing the type 5 capsid. Streptozotocin-induced diabetic mice were subjected to hepatic portal vein injection immediately followed by implantation of a sustained-release insulin pellet to allow time for transgenic expression. All mice injected with the rAAV5-GLUT2-fHPIB10 virus remained euglycemic for up to 35 days post-injection, with 50% euglycemic after 77 days post-injection. In contrast, mock-injected mice became hyperglycemic within 15 days post-injection following dissolution of the insulin pellet. Serum levels of both human insulin and C-peptide further confirmed successful transgenic delivery by the rAAV5-GLUT2-fHPIB10 virus. These findings indicate that the GLUT2 promoter may be a potential candidate for regulating transgenic insulin production for hepatic insulin gene therapy in the treatment of type I diabetes.     

Cardiomyocyte-specific gene expression following recombinant adeno-associated viral vector transduction

Recombinant adeno-associated viral (rAAV) vectors hold promise for delivering genes for heart diseases, but cardiac-specific expression by the use of rAAV has not been demonstrated. To achieve this goal rAAV vectors were generated expressing marker or potentially therapeutic genes under the control of the cardiac muscle-specific alpha myosin heavy chain (MHC) gene promoter. The rAAV-MHC vectors expressed in primary cardiomyocytes with similar kinetics to rAAV-CMV; however, expression by the rAAV-MHC vectors was restricted to cardiomyocytes. rAAV vectors have low cytotoxicity, and it is demonstrated here that rAAV fails to induce apoptosis in cardiomyocytes compared with a recombinant adenoviral vector. rAAV-MHC or rAAV-CMV vectors were administered to mice to determine the specificity of expression in vivo. The rAAV-MHC vectors expressed specifically in cardiomyocytes, whereas the control rAAV-CMV vector expressed in heart, skeletal muscle, and brain. rAAV-MHC transduction resulted in long term (16 weeks) expression of human growth hormone following intracardiac, yet not intramuscular, injection. Finally, we defined the minimal MHC enhancer/promoter sequences required for specific and robust in vivo expression in the context of a rAAV vector. For the first time we describe a panel of rAAV vectors capable of long term cardiac specific expression of intracellular and secreted proteins.     

Heat acclimation causes some alterations in serum enzymes activities in streptozotocin-diabetic rats

The present study investigated the effect of long-term heat acclimation and experimental diabetes on serum activity of transaminases (AST, ALT), ALP, LDH and elastase complex, as well as blood glucose and HbA1c level in Wistar rats. The heat acclimation model was established with the artificial heat chamber (35±1 °C and 30-40% humidity) for a period of 28 days, while the control groups were held on 20±2 °C. Experimental diabetes was induced by single streptozotocine (STZ) injection (55 mg/kg bodyweight) The changes caused by insulin treatment (2 IU/100 g body weight, 14 days, twice daily) in both thermal groups were also investigated.STZ-diabetes leads to significant increase in blood glucose and HbA1c level, AST, ALT and ALP activities in both thermal groups (normothermic and heat acclimated), decrease in LDH activity in normothermic animals and increase in heat-acclimated ones. Treatment with insulin restores the blood glucose, HbA1c and enzymes activities regardless of the previous thermal exposure.Prolonged acclimation of control animals to elevated ambient temperature resulted in significant decrease in blood glucose level, AST, ALT, ALP and LDH activities and non-significant changes in HbA1c. Compared to diabetic rats from room temperature, heat-acclimated diabetic ones have significantly higher blood glucose, AST, ALP and LDH activity, lower HbA1c concentration and no significant changes in ALT. Most of the changes observed in heat-acclimated insulin-treated diabetic rats did not significantly differ compared to those from room temperature.The overall two-way ANOVA analyses showed that diabetic state causes significant changes in the blood glucose, HbA1c, AST, ALT and ALP activity, while heat acclimation causes significant changes only in HbA1c level and AST activity. Both of the factors (diabetic state and heat acclimation) have significant common effects on AST, ALP and LDH activity.     

Normalization of cytoplasmic calcium response in pancreatic beta-cells of spontaneously diabetic GK rat by the treatment with T-1095, a specific inhibitor of renal Na+-glucose co-transporters.

Chronic hyperglycemia is known to lead to a progressively further impaired insulin response and to hasten the development of complications in patients with type 2 diabetes, a notion referred as glucose toxicity. T-1095, a derivative of phlorizin, is a newly developed oral hypoglycemic agent that acts as a specific inhibitor of renal Na(+)-glucose co-transporters, reducing circulating blood glucose levels by promoting glucose excretion into urine. The effects of glycemic improvement by T-1095 on secretory function and cytoplasmic calcium response in pancreatic beta-cells were investigated using spontaneously diabetic GK rats. After four weeks of treatment with T-1095 (age 4 to 8 week rats), serum glucose and HbA1c levels were significantly improved (serum glucose level, GK vs. GK T-1095, 277.3 +/- 11.8 vs. 204.7 +/- 6.4 mg/dl; HbA1c level, GK vs. GK T-1095, 6.2 +/- 0.2 vs. 4.8 +/- 0.1 %). Insulin secretion induced by 16.7 mM glucose was also significantly increased in the T-1095-treated group compared to the untreated group. The [Ca(2+)]i response induced by 16.7 mM glucose in GK beta-cells was characterized by the loss of the steep first peak of [Ca(2+)]i elevation, and the lost first peak of [Ca(2+)]i reappeared in T-1095-treated beta-cells in 32 of 34 observations. In T-1095-treated beta-cells, the time lag to peak [Ca(2+)]i levels in the 16.7 mM glucose stimulation was significantly reduced (259.1 +/- 15.3 sec, p < 0.01) compared to untreated GK rats (524.7 +/- 52.9 sec). Thus, improvement of hyperglycemia by T-1095 ameliorates beta-cell function by relieving [Ca(2+)]i response.     

Hypoglycemic effect of Sclerocarya birrea {(A. Rich.) Hochst.} [Anacardiaceae] stem-bark aqueous extract in rats

This study was undertaken to evaluate the hypoglycemic effect of Sclerocarya birrea [(A. Rich.) Hochst.] subspecies caffra (Sond.) Kokwaro [family: Anacardiaceae] stem-bark aqueous extract in normal (normoglycemic) and in streptozotocin (STZ)-treated, diabetic Wistar rats. In one set of experiments, graded doses of S. birrea stem-bark aqueous extract (SB, 100-800 mg/kg p.o.) were separately administered to groups of fasted normal and fasted diabetic rats. In another set of experiments, a single dose of the plant aqueous extract (SB, 800 mg/kg p.o.) was used. The hypoglycemic effect of this single dose (SB, 800 mg/kg p.o.) of S. birrea stem-bark aqueous extract was compared with that of chlorpropamide (250 mg/kg p.o.) in both fasted normal and fasted diabetic rats. Following acute treatment, relatively moderate to high doses of S. birrea stem-bark extract (SB, 100-800 mg/kg p.o.) produced dose-dependent, significant reductions (P < 0.05-0.001) in the blood glucose concentrations of both fasted normal and fasted diabetic rats. Chlorpropamide (250 mg/kg p.o.) also produced significant reductions (P < 0.05-0.001) in the blood glucose concentrations of the fasted normal and fasted diabetic rats. Administrations of the single dose of S. birrea stem-bark aqueous extract (SB, 800 mg/kg p.o.) significantly reduced (P 0.01 < 0.001) the blood glucose levels of both fasted normal (normoglycemic) and fasted STZ-treated, diabetic rats. The results of this experimental animal study indicate that aqueous extract of Sclerocarya birrea possesses hypoglycemic activity, and thus lend credence to the suggested folkloric use of the plant in the management and/or control of adult-onset, type-2 diabetes mellitus in some African communities.     

Dose dependent hypoglycemic effect of aqueous extract of Enicostemma littorale blume in alloxan induced diabetic rats.

Previous studies in our lab had confirmed the blood glucose lowering effect of E. littorale Blume in alloxan induced diabetic rats with no change in normoglycemic control rats. Present paper deals with dose dependent (0.5, 1.0, 1.5, 2.5, 3.5 g dry plant equivalent extract/100 g body wt., p.o.) blood glucose lowering effect of aqueous extract of E. littorale Blume in alloxan induced diabetic rats. The effective dose was found to be 1.5 g dry plant equivalent extract/100 g body wt.. The above dose caused significant decrease in glycosylated haemoglobin, liver glucose-6-phosphatase activity and significant increase in serum insulin levels of the diabetic rats. No significant changes were observed in the toxicity parameters of extract treated diabetic rats as compared to diabetic control rats. The above results suggest that E. littorale is a potent antidiabetic agent without any toxic effect at this particular dose (1.5 g dry plant equivalent extract/100 g body wt.).     

Pioglitazone reverses down-regulation of cardiac PPARgamma expression in Zucker diabetic fatty rats

Peroxisome proliferator-activated receptor-gamma (PPARgamma) plays a critical role in peripheral glucose homeostasis and energy metabolism, and inhibits cardiac hypertrophy in non-diabetic animal models. The functional role of PPARgamma in the diabetic heart, however, is not fully understood. Therefore, we analyzed cardiac gene expression, metabolic control, and cardiac glucose uptake in male Zucker diabetic fatty rats (ZDF fa/fa) and lean ZDF rats (+/+) treated with the high affinity PPARgamma agonist pioglitazone or placebo from 12 to 24 weeks of age. Hyperglycemia, hyperinsulinemia, and hypertriglyceridemia as well as lower cardiac PPARgamma, glucose transporter-4 and alpha-myosin heavy chain expression levels were detected in diabetic ZDF rats compared to lean animals. Pioglitazone increased body weight and improved metabolic control, cardiac PPARgamma, glut-4, and alpha-MHC expression levels in diabetic ZDF rats. Cardiac [(18)F]fluorodeoxyglucose uptake was not detectable by micro-PET studies in untreated and pioglitazone treated ZDF fa/fa rats but was observed after administration of insulin to pioglitazone treated ZDF fa/fa rats. PPARgamma agonists favorably affect cardiac gene expression in type-2 diabetic rats via activation and up-regulation of cardiac PPARgamma expression whereas improvement of impaired cardiac glucose uptake in advanced type-2 diabetes requires co-administration of insulin.     

Insulin secretion and islet endocrine cell content at onset and during the early stages of diabetes in the BB rat: effect of the level of glycemic control.

Although it is agreed that autoimmune destruction of pancreatic islets in diabetic BB rats is rapid, reports of endocrine cell content of islets from BB diabetic rats at the time of onset of diabetes vary considerably. Because of the rapid onset of the disease (hours) and the attendant changes in islet morphology and insulin secretion, it was the aim of this study to compare islet beta-cell numbers to other islet endocrine cells as close to the time of onset of hyperglycemia as possible (within 12 h). As it has been reported that hyperglycemia renders the beta cell insensitive to glucose, the early effects of different levels of insulin therapy (well-controlled vs. poorly controlled glycemia) on islet morphology and insulin secretion were examined. When measured within 12 h of onset, insulin content of BB diabetic islets, measured by morphometric analysis or pancreatic extraction, was 60% of insulin content of control islets. Despite significant amounts of insulin remaining in the pancreas, 1-day diabetic rats exhibited fasting hyperglycemia and were glucose intolerant. The insulin response from the isolated perfused pancreas to glucose and the glucose-dependent insulinotropic hormone, gastric inhibitory polypeptide (GIP), was reduced by 95%. Islet content of other endocrine peptides, glucagon, somatostatin, and pancreatic polypeptide, was normal at onset and at 2 weeks post onset. A group of diabetic animals, maintained in a hyperglycemic state for 7 days with low doses of insulin, were compared with a group kept normoglycemic by appropriate insulin therapy. No insulin could be detected in islets of poorly controlled diabetics, while well-controlled animals had 30% of the normal islet insulin content.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential rAAV2 transduction efficiencies and insulin secretion profiles in pure and co-culture models of human enteroendocrine L-cells and enterocytes

BACKGROUND: Cell-based therapies for treating insulin-dependent diabetes (IDD) can provide a more physiologic regulation of blood glucose levels in a less invasive fashion than insulin injections. Previously, we developed an engineered human enteroendocrine L-cell model for regulated insulin release via recombinant adeno-associated virus serotype 2, or rAAV2, transduction. The aim of this study was to evaluate the efficiency and selectivity of rAAV2-mediated insulin gene delivery to enteroendocrine L-cells in co-culture with a prevailing number of enterocytes, which are the predominant cell type in intestinal epithelium. METHODS: We tested rAAV2 transduction in pure and co-culture models of human cell lines of enterocytes (Caco-2 and T84 cell lines) and enteroendocrine L-cells (NCI-H716 cell line). Non-viral, chemical-mediated transfection was used as a control. Transduced and transfected co-cultures were subjected to insulin secretion studies. RESULTS: In pure cultures, rAAV2 exhibited a low transduction efficiency towards both Caco-2 and T84 enterocytes, as opposed to a strong reporter expression in permissive NCI-H716 L-cells. In co-cultures of NCI-H716 L-cells and Caco-2 or T84 enterocytes, rAAV2 exhibited differential transduction efficiency with a strong preference towards NCI-H716 L-cells. The rAAV2-transduced co-culture achieved regulated insulin release against stimulation, whereas the chemically transfected co-culture failed to respond. CONCLUSIONS: This study demonstrated that rAAV2-mediated insulin gene transfer can differentiate human intestinal cell types in vitro, in particular enterocyte and enteroendocrine L-cell lines. We consider the AAV2 vector a useful tool in developing enteroendocrine L-cell-specific insulin gene delivery for IDD treatment, in terms of AAV2 avoiding enterocytes and targeting selectively L-cells.     

Combined effect of ACE inhibitor and exercise training on insulin resistance in type 2 diabetic rats

The aim of this study was to investigate whether a combined treatment of ACE inhibitor and exercise training is more effective than either treatment alone in alleviating the insulin resistant states in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat, a model of type 2 diabetes. OLETF rats (25 weeks old) were randomly divided into 5 groups; sedentary control, exercise-trained, temocapril (ACE inhibitor; 2 mg/kg/day)-treated, with and without exercise, and losartan (AT1 receptor antagonist; 1 mg/kg/day)-treated. Long-Evans Tokushima Otsuka rats were used as a non-diabetic control. Body weight, the amount of abdominal fat and blood pressure were higher for OLETF rats than for control rats. However, glucose infusion rate (GIR), an index of insulin resistance, was decreased greatly in OLETF rats. The fasting levels of blood glucose, insulin and lipids were also increased in the diabetic strain. In OLETF rats, both temocapril and losartan reversed hypertensive states significantly, whereas GIR and hyperlipidemia were improved when rats were treated with ACE inhibitors, but not with the AT1 receptor antagonist. Exercise training decreased body weight and the amount of abdominal fat, and also increased GIR in parallel with improved dislipidemia. The combination of the ACE inhibitor with exercise training also improved obesity, hyperinsulinemia, dislipidemia and fasting level of blood glucose, and this combination resulted in the greatest improvement of insulin resistance. These results suggest that the combination of ACE inhibitor and exercise training may be a beneficial treatment for mixed diabetic and hypertensive conditions.     

Central leptin gene therapy suppresses body weight gain, adiposity and serum insulin without affecting food consumption in normal rats: a long-term study

The weight-reducing effects of leptin are predominantly mediated through the hypothalamus in the brain. Gene therapy strategies designed for weight control have so far tested the short-term effect of peripherally delivered viral vectors encoding the leptin gene. In order to circumvent the multiple peripheral effects of hyperleptinemia and to overcome the age-related development of leptin resistance due to multiple factors, including defective leptin transport across the blood brain barrier, we determined whether delivery of viral vectors directly into the brain is a viable therapeutic strategy for long-term weight control in normal wild-type rats. A recombinant adeno-associated virus (rAAV) vector encoding rat leptin (Ob) cDNA was generated (rAAV-betaOb). When administered once intracerebroventricularly (i.c.v.), rAAV-betaOb suppressed the normal time-related weight gain for extended periods of time in adult Sprague-Dawley rats. The vector expression was confirmed by immunocytochemical localization of GFP and RT-PCR analysis of leptin in the hypothalamus. This sustained restraint on weight gain was not due to shifts in caloric consumption because food-intake was similar in rAAV-betaOb-treated and rAAV-GFP-treated control rats throughout the experiment. Weight gain suppression, first apparent after 2 weeks, was a result of reduced white fat depots and was accompanied by drastically reduced serum leptin and insulin concentrations in conjunction with normoglycemia. Additionally, there was a marked increase in uncoupling protein-1 (UCP1) mRNA expression in brown adipose tissue, thereby indicating increased energy expenditure through thermogenesis. Seemingly, a selective enhancement in energy expenditure following central delivery of the leptin gene is a viable therapeutic strategy to control the age-related weight gain and provide protection from the accompanying multiple peripheral effects of hyperleptinemia and hyperinsulinemia.