Glucose-Modulated Transgene Expression via Recombinant Adeno-Associated Virus

Purpose. The objective of this study was to examine glucose-modulated reporter gene expression via recombinant adeno-associated viral vectors both in vitro and in vivo. Methods. Huh7 human hepatoma cells were transduced by recombinant adeno-associated virus (rAAV) vectors containing the luciferase gene under control of the rat insulin I gene promoter and a cytomegalovirus immediate-early promoter driving-enhanced green fluorescence protein gene. The reporter gene expression was evaluated by glucose stimulation either in the absence or presence of insulin secretagogues, including phorbol-12-myristate-13-acetate, dibutyryl cyclic AMP, and forskolin. In vivo studies were performed by injecting rAAV into the livers of streptozotocin-induced diabetic C57BL/6J mice followed by measurements of blood glucose concentration and luciferase activity assays 2 weeks after rAAV injection. Results. At a multiplicity of infection of 500, approximately 66-69% of cells expressed enhanced green fluorescence protein at 48 h post-transduction. Luciferase activities, driven by the insulin gene promoter, in the rAAV-transduced hepatoma cells responded to millimolars of glucose. The addition of phorbol-12-myristate-13-acetate, dibutyryl cyclic AMP, and forskolin increased luciferase expression in the presence of either 1 mM or 25 mM glucose. The stimulation of luciferase activities by these substances was inhibited by the presence of 100 nM staurosporine. Exposure to increments of exogenous insulin up to 10^-7 M inhibited luciferase gene expression in rAAV-transduced Huh7 cells. The in vivo experiments demonstrated good correlation between luciferase activities and blood glucose levels in streptozotocin-induced diabetic animals. Conclusion. rAAV is a promising vector for hepatic gene therapy for diabetes. Glucose and insulin secretagogues modulated transgene expression in rAAV-transduced hepatoma cells, suggesting that conditions affecting insulin gene promoter function in pancreatic islet beta cells also affect transgene expression in human hepatoma cells conferred with insulin gene promoter. Results obtained from in vivo experiments demonstrated that glucose modulated transgene expression can be obtained in rAAV-treated diabetic C57BL/6J mice.     

In vivo transfection rat small intestine K-cell with pGIP/Ins plasmid by DOTAP liposome

Gene therapies to produce insulin in diabetic patients have been considered for several years. Genetic engineering of ectopic insulin production and secretion in autologous non-beta-cells has been tested in different tissues. Recently, gut K-cells have been shown to express glucokinase, the glucose sensor of pancreatic beta-cells. K-cells are responsible for the secretion of the glucose-dependent insulinotropic peptide (GIP). Transfection of K-cells by a specific plasmid to produce insulin correlated to glucose level is being considered. Cationic liposomes are non-viral gene delivery to lung, spleen, liver and intestinal cells. DOTAP–GIP/Ins plasmid complex was used for transfection of K-cells in vivo. RT-PCR assay of human insulin mRNA revealed that the transfection of insulin gene by DOTAP liposome is an efficient tool. The genetic engineering of ectopic insulin production and secretion in autologous non-beta-cells is an appropriate method. The potential of the transmission of a constructed plasmid, which contains human insulin gene under the control of GIP promoter, to gut K-cells could be considered for treatment of diabetes.     

Effects of sulfhydryl inhibition on the regulation of basal lipolysis and glucose uptake in human adipose tissue.

Stimulation of basal lipolysis and inhibition of glucose uptake by N-ethylmaleimide (NEM) was demonstrated in vitro in human omental adipose tissue. NEM, at concentrations ranging from 4 to 5 × 10^?4 M, produced a maximal stimulation of basal lipolysis as well as a 50 per cent inhibition of the rate of glucose uptake, whereas concentrations above 1 × 10^-3M reduced the basal rate of glycerol release. In contrast, under the conditions in which it stimulated basal lipolysis, NEM strongly inhibited theophylline induced lipolysis. NEM-stimulated lipolysis was not directly related to decreased glucose uptake to α- or β-adrenergic mechanisms or to inhibition of phosphodiesterase. The lipolytic response of human adipose tissue to NEM was, however, markedly reduced by insulin and nicotinate, suggesting that adenylcyclase activation could be responsible for the lipolytic effect of NEM. The inhibitory effect exerted by NEM on glucose uptake was similarly reversed by the addition of insulin. The rate of basal lipolysis and glucose uptake by fat pads removed from diabetic patients was unaffected by NEM. A possible involvement of insulin in the effects induced by NEM on both basal lipolysis and glucose uptake in normal human adipose tissue is discussed.     

Protamine sulfate enhances the transduction efficiency of recombinant adeno-associated virus-mediated gene delivery

Purpose. The purpose of this study was to evaluate glucose responsiveness in HepG2 human hepatoma cells transduced by a recombinant adeno-associated virus (rAAV) vector containing the insulin gene promoter, and to investigate the effect of protamine sulfate on rAAV-mediated gene delivery. Methods. Recombinant AAV vector, AAV.Ins.Luc. EGFP, was employed to transduce HepG2 hepatoma cells. Virus infection was carried out either in the absence or presence of protamine sulfate, followed by fluorescence microscopic examination, luciferase activity assay, and flow cytometric analysis. Electrokinetic measurements were carried out to determine the effect of protamine sulfate on zeta potential of the cells and the virus. Results. Glucose-responsive luciferase gene expression was obtained in rAAV-transduced HepG2 cells. Addition of 5 g/ml protamine reversed the zeta potential of the cells and the virus particles, leading to enhanced transgene expression in rAAV-transduced HepG2 cells. Enhancement of protamine sulfate on rAAV-mediated gene transfer was dose-dependent. Addition of more than 5 g/ml protamine resulted in a reduction of infectability of the virus. Conclusions. Glucose responsiveness in the millimolar concentration range can be obtained in rAAV-transduced HepG2 cells. Protamine sulfate, up to 5 g/ml, enhanced the rAAV transduction efficiency in HepG2 cells. The enhancement was correlated with zeta potential of the cells and the virus.     

Aryl hydrocarbon receptor‐mediated effects of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin on glucose‐stimulated insulin secretion in mice

Epidemiological and laboratory studies suggested that exposure to 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) affects glucose homeostasis and increases the incidence of type 2 diabetes mellitus. To evaluate the effects of TCDD on insulin secretion from islets of Langerhans (islets), we designed in vivo, ex vivo and in vitro experiments. For the in vivo experiment, male C57BL/6J and aryl hydrocarbon receptor (AhR)‐null mice were injected intraperitoneally with TCDD (10 μg kg^?1 b.w.), fasted for 12 h and administered glucose 24 h post‐administration. TCDD exposure significantly decreased the plasma insulin concentration at 60 and 120 min after a glucose challenge in C57BL/6J mice but not in AhR‐null mice. In contrast, the plasma glucose concentration was not changed by TCDD exposure in both C57BL/6J and AhR‐null mice. For the ex vivo experiment, we isolated islets 24 h after TCDD administration and determined the glucose‐stimulated insulin secretion from the islets. The insulin secretion level was found to be significantly decreased by TCDD exposure at 60 min after glucose treatment. For the in vitro experiment, islets harvested from untreated C57BL/6J mice were exposed to 0.1, 1, 10 or 100 nM TCDD for 24 h and analyzed for glucose‐stimulated insulin secretion. Insulin secretion from the islets remained unchanged regardless of TCDD dose. In conclusion, TCDD exposure impaired the second phase of glucose‐stimulated secretion of insulin from the islets via the AhR signaling pathway. Copyright © 2009 John Wiley & Sons, Ltd.     

Antidiabetic activity of alcoholic stem extract of Gymnema montanum in streptozotocin-induced diabetic rats

In the present study, the effect of alcoholic stem extract of Gymnema montanum (GMSt) on blood glucose, plasma insulin, and carbohydrate metabolic enzymes were studied in experimental diabetes. Diabetes mellitus was induced by a single intraperitoneal injection of STZ (60 mg/kg bw). Five days after STZ induction, diabetic rats received GMSt orally at the doses of 25, 50, 100 and 200 mg/kg daily for 3 weeks. Graded doses of stem extract showed a significant reduction in blood glucose levels and improvement in plasma insulin levels. The effect was more pronounced in 100 and 200 mg/kg than 50 mg/kg. GMSt showed significant increase in hexokinase, Glucose-6-phosphate dehydrogenase and glycogen content in liver of diabetic rats while there was significant reduction in the levels of glucose-6-phosphatase and fructose-1,6-bisphosphatase. The present study clearly indicated significant antidiabetic effect with the stem extract of G. montanum and lends support for its traditional usage.     

Therapeutic potential of octyl gallate isolated from fruits of Terminalia bellerica in streptozotocin-induced diabetic rats

Abstract

Context: Medicinal plants are a potential source of antidiabetic drugs. Terminalia bellerica Roxb. (Combretaceae) is used in Indian traditional systems of medicine to treat diabetes mellitus.

Objective: The aim of this study was to isolate and identify antihyperglycemic principle(s) from the fruits of T. bellerica and assess the bioactivity in streptozotocin (STZ)-induced diabetic rats.

Materials and methods: Bioassay-guided fractionation was followed to isolate the active compound(s), structure was elucidated using ¹H and ¹³C NMR, IR and mass spectrometry and administered intragastrically to diabetic Wistar rats at different doses (5, 10 and 20?mg/kg, body weight) for 28?d. Plasma glucose, insulin, C-peptide and other biochemical parameters were studied.

Results: Octyl gallate (OG) isolated first time from the fruit rind of T. bellerica significantly (p?<?0.05) reduced plasma glucose to near normal values (108.47?±?6.9?mg/dl) after 14?d at the dose of 20?mg/kg. In addition, OG significantly increased plasma insulin, C-peptide, total protein, albumin, tissue glycogen, body weight and markedly decreased serum total cholesterol, triglyceride, LDL-cholesterol, urea, uric acid and creatinine in diabetic rats. Also OG restored the altered regulatory enzymes of carbohydrate metabolism.

Discussion and conclusion: OG might have augmented the secretion of insulin by the modulation of cAMP and intracellular calcium levels in the β cells of the pancreas. Our findings indicate that OG isolated first time from the fruit rind of T. bellerica has potential antidiabetic effect as it augments insulin secretion and normalizes the altered biochemical parameters in experimental diabetic rat models.     

Effect of some β-adrenoceptor blocking drugs on insulin secretion in the rat

In pentobarbitone anaesthetized rats (±)-propranolol reduced the stimulatory effect of glucose, sulphonylureas, isoprenaline or phentolamine on insulin secretion. (+)-Propranolol produced similar effects to those produced by the racemate. Sotalol reduced only the insulin secretion stimulated by isoprenaline or phentolamine but not that stimulated by glucose or sulphonylureas. Complete inhibition of isoprenaline-induced hyperinsulinaemia was obtained with practolol, in a dose which was without effect on the plasma insulin elevations produced by glucose or glibenclamide. (±)-Propranolol inhibited glucose or tolbutamide-stimulated insulin secretion from chopped pancreas in vitro indicating a direct action of the drug on the pancreas. It is suggested that propranolol-induced inhibition of insulin secretion may not be entirely due to the β-adrenoceptor blocking activity of the drug.     

Interactions of Diabetogenic Compounds: Cyproheptadine and Alloxan

Pretreatment with an oral dose (45 mg/kg) of cyproheptadine(CPH), a drug that inhibits secretion and synthesis of insulin,3 hr before alloxan (100 mg/kg, iv) protects mice from the permanentdiabetes produced by alloxan. Pretreated animals at the timeof alloxan adminisisation were hyperglycemic. Therefore, thepossibility that CPH-induced hyperglycemia protected mice fromalloxan was investigated. This was accomplished by giving mannoheptulose(a glucose antagonist) or insulin (to lower blood glucose) afterCPH and before alloxan. These interventions eliminated CPH-inducedprotection from alloxan, indicating a role for CPH-induced hyperglycemiain the protective effect. To confirm that CPH does not protectmice from alloxan-induced diabetes by a direct action, in vitroexperiments using isolated pancreatic islets were conducted.Mouse islets were pretreated with CPH, its metabolite desmethylcyproheptadine(DMCPH), or an equal mixture of the two and/or various concentrationsof glucose prior to an acute exposure to a toxic concentrationof alloxan. Glucose- stimulated insulin release was used asa measure of pancreatic ß-cell function after alloxanexposure. CPH or DMCPH (alone or in combination) pretreatmentdid not provide protection against alloxan-induced inhibitionof insulin release nor did pretreatments potentiate the protectiveaction of glucose against in vitro alloxan toxicity. The resultsindicate that the protective action of CPH when given to micebefore alloxan is due to drug-induced hyperglycemia and notto a direct effect of CPH or its metabolite.     

Co-Expression of Vascular Endothelial Growth Factor and Interleukin-1 Receptor Antagonist Improves Human Islet Survival and Function

Purpose Ex vivo gene therapy approaches can improve the outcome of islet transplantation for treating type I diabetes. We have previously shown the improvement in islet function and vascularization following ex vivo transfection for human vascular endothelial growth factor (hVEGF) gene expression. In this study, we tested the hypothesis that co-expression of two genes, which target different challenges faced by islets post-transplantation, supplement each other to improve the survival and function of islets. We determined whether there is an additive effect of hVEGF and human interleukin-1 receptor antagonist (hIL-1Ra) gene expression in human islets.Materials and Methods Human islets were co-infected with adenoviral vectors encoding hVEGF and hIL-1Ra. Islets were then incubated with a cocktail of inflammatory cytokines (IL-1β+TNFα+IFNγ), and islet viability and function were determined. In vivo function was evaluated by transplanting islets under the kidney capsules of streptozotocin-induced non-obese diabetic severe combined immunodeficient (NOD-SCID) mice.Results Infection of human islets with Adv-hVEGF and/or Adv-hIL-1Ra inhibited expression of inducible nitric oxide synthase (iNOS), decreased the production of nitric oxide (NO), and prevented the loss of in vitro glucose-stimulated insulin response and viability. Moreover, co-expression of hVEGF and hIL-1Ra reduced the blood glucose level of mice, and increased the level of blood insulin and c-peptide upon glucose challenge.Conclusions Our results indicated that co-expression of genes that target different insults to transplanted islets can improve the outcome of islet transplantation better than either gene alone.     

Poly(N-vinylcaprolactam-co-methacrylic acid) hydrogel microparticles for oral insulin delivery

pH-sensitive copolymeric hydrogels prepared from N-vinylcaprolactam and methacrylic acid monomers by free radical polymerization offered 52% encapsulation efficiency and evaluated for oral delivery of human insulin. The in vitro experiments performed on insulin-loaded microparticles in pH 1.2 media (stomach condition) demonstrated no release of insulin in the first 2?h, but almost 100% insulin was released in pH 7.4 media (intestinal condition) in 6?h. The carrier was characterized by Fourier transform infrared, differential scanning calorimeter, thermogravimetry and nuclear magnetic resonance techniques to confirm the formation of copolymer, while scanning electron microscopy was used to assess the morphology of hydrogel microparticles. The in vivo experiments on alloxan-induced diabetic rats showed the biological inhibition up to 50% and glucose tolerance tests exhibited 44% inhibition. The formulations of this study are the promising carriers for oral delivery of insulin.     

Antihyperglycemic,antilipid peroxidation,and insulin secretory activities of Otostegia persica shoot extract in streptozotocin-induced diabetic rats and in vitro C187 pancreatic β-cells

Context: Otostegia persica Boiss (Lamiaceae) contains antioxidant agents and is used in traditional medicine for treatment of diabetes mellitus complications.

Objectives: The acute antihyperglycemic, antilipid peroxidation, and insulin secretory activities of methanol extract of O. persica aerial parts were investigated.

Materials and methods: The extract [200, 300, 400?mg/kg body weight (b.w.)] was given orally to rats and glucose (2?g/kg b.w. orally) was administered 30?min later. Glucose and insulin serum levels were measured before and 30, 60, 120, and 240?min after administration of the test samples in normal and diabetic rats. The in vitro insulin secretory activity of extract was evaluated in C187 pancreatic β-cells and its antilipid peroxidation effect was determined by measuring malondialdehyde (MDA) and glutathione (GSH) levels in rat livers after 240?min. The identification of the major phytoconstituents of the extract was carried out using gas chromatography-mass spectrometry.

Results: The extract (300?mg/kg b.w.) significantly decreased the serum glucose level in diabetic rats at 1 h (494?±?13.4 vs. 426?±?12.9), 2 h (472.8?±?17.8 vs. 396?±?22), and 4?h (438.8?±?25 vs. 346?±?19) after treatment. Accordingly, the serum insulin level increased at the same times. The extract significantly increased glucose-induced insulin secretion in C187 β-cells. Moreover, the extract significantly decreased MDA and increased GSH levels in the liver of diabetic rats. Phytochemical analysis revealed thymol as the major phytoconstituent in the extract.

Discussion and conclusion: O. persica shoot extract has antihyperglycemic, antilipid peroxidation, and insulin secretory properties.     

In vitro and in vivo evaluation of thermosensitive chitosan hydrogel for sustained release of insulin

Injectable In situ gel-forming chitosan/β-glycerol phosphate (CS/β-Gp) solution can be introduced into the body in a minimally invasive manner prior to solidifying within the target tissue. This hydrogel is a good candidate for achieving a prolonged drug delivery system for insulin considering its high molecular weight. In addition to the physicochemical characterization of this hydrogel, in vitro and in vivo applications were studied as a sustained insulin delivery system. In the in vitro release studies, 19–63% of total insulin was released from the CS/β-Gp hydrogel within 150?h at different β-Gp and insulin concentrations. The best formulation was selected for in vivo experimentation to control the plasma glucose of diabetic mice models. The hypoglycemic effect of this formulation following subcutaneous injection in diabetic mice lasted 5?d, significantly longer than that of free insulin solution which lasted several hours.     

Upregulation of insulin secretion and downregulation of pro-inflammatory cytokines,oxidative stress and hyperglycemia in STZ-nicotinamide-induced type 2 diabetic rats by Pseuduvaria monticola bark extract

The current study aimed to ascertain the antidiabetic potential of Pseuduvaria monticola bark methanolic extract (PMm) using in vitro mechanistic study models. In particular, the study determined the effect of PMm on cellular viability, 2-NBDG glucose uptake, insulin secretion, and NF-κB translocation in mouse pancreatic insulinoma cells (NIT-1). Furthermore, in vivo acute toxicity and antidiabetic studies were performed using streptozotocin (STZ)-induced type 1 and STZ-nicotinamide-induced type 2 diabetic rat models to evaluate various biochemical parameters and markers of oxidative stress and pro-inflammatory cytokines. Five isoquinoline alkaloids and three phenolic compounds were tentatively identified in the PMm by LC/MS Triple TOF. The study results showed that PMm is non-toxic to NIT-1 cells and significantly increased the glucose uptake and insulin secretion without affecting the translocation of NF-κB. Moreover, the non-toxic effects of PMm were confirmed through an in vivo acute toxicity study, which revealed that the serum insulin and C-peptide levels were significantly upregulated in type 2 diabetic rats and that no significant changes were observed in type 1 diabetic rats. Similarly, PMm was found to downregulate the levels of oxidative stress and pro-inflammatory cytokines in type 2 diabetic rats by alleviating hyperglycemia. Therefore, we conclude that PMm may be developed as an antidiabetic agent for the treatment of type 2 diabetes-associated conditions.     

Co-administration of paroxetine and pravastatin causes deregulation of glucose homeostasis in diabetic rats via enhanced paroxetine exposure

Aim： Clinical evidence shows that co-administration of pravastatin and paroxetine deregulates glucose homeostasis in diabetic patients. The aim of this study was to verify this phenomenon in diabetic rats and to elucidate the underlying mechanisms.
Methods： Diabetes mellitus was induced in male SD rats by a high-fat diet combined with a low-dose streptozotocin injection. The rats were orally administered paroxetine （10 mg/kg） and pravastatin （10 mg/d） or both the drugs daily for 28 d. The pharmacokinetics of paroxetine and pravastatin were examined on d 1 and d 28. Biochemical parameters including serum insulin, glucose and lipids were monitored during the treatments. An insulin-secreting cell line （INS-1） was used for measuring insulin secretion.
Results： In diabetic rats, co-administration of paroxetine and pravastatin markedly increased the concentrations of both the drugs compared with administration of each drug alone. Furthermore, co-administration severely impaired glucose homeostasis in diabetic rats, as demonstrated by significantly increased serum glucose level, decreased serum and pancreatic insulin levels, and decreased pancreatic Insulin-2 mRNA and tryptophan hydroxylase-1 （Tph-1） mRNA levels. Treatment of INS-1 cells with paroxetine （5 and 10 μmol/L） significantly inhibited insulin secretion, decreased the intracellular insulin, 5-HT, Insulin-2 mRNA and Tph-1 mRNA levels. Treatment of the cells with pravastatin （10 μmol/L） significantly stimulated insulin secretion, which was weakened by co-treatment with paroxetine.
Conclusion： Paroxetine inhibits insulin secretion at least via decreasing intracellular 5-HT and insulin biosynthesis. The deregulation of glucose homeostasis by co-administration of paroxetine and pravastatin in diabetic rats can be attributed to enhanced paroxetine exposure.     

Self-assembly pH-sensitive chitosan/alginate coated polyelectrolyte complexes for oral delivery of insulin

Polyelectrolyte complexes (PEC) provide new opportunities for controlled release system of drugs, and have potentials to address challenges on the way to effective oral insulin delivery. Here, an innovative pH-sensitive PEC for insulin oral administration was developed, which was formed by self-assembly of two oppositely charged nanoparticles (chitosan-coated nanoparticles and alginate-coated nanoparticles) through electrostatic interaction via optimised double emulsion method. The encapsulation efficiency of insulin-loaded alginate-coated and chitosan-coated nanoparticles were 81.5?±?7.4% and 55.2?±?7.0%, respectively, and the particle size of these nanoparticles were in 200–300?nm range. The pH-dependent morphology of PEC was observed by transmission electron microscopy. The PEC exhibited insulin release profile triggered by pH in vitro and was non-cytotoxicity against Caco-2 cell. The insulin-loaded PEC could decrease blood glucose levels effectively and prolong insulin release after oral administration to diabetic rats. The results illustrated that the as-prepared PEC may be employed as a potential oral insulin delivery system.     

Alteration of Pancreatic B-Cells in Wistar Rats Treated with Non-Diabetogenic Doses of Cyclosporin A

Abstract: We have investigated the effect of non-immunosuppressive cyclosporin A (CS-A) doses on glucose tolerance, pancreatic insulin content, insulin content of isolated islets and insulin secretion in vitro in response to glucose. Within 12 weeks animals treated permanently with a dose of 2.5 mg CS-A/kg b.wt. developed glucose intolerance (but not hyperglycaemia) accompanied by a decrease of pancreatic insulin content due to a decrease of islet insulin content, whereas the relative B-cell volume density was.not changed. Isolated islets obtained from rats treated for 4 weeks had a diminished sensitivity for glucose, whereas islets obtained from animals treated for > 4 weeks showed a diminished half-maximum and maximum secretion rate. Rats treated for 12 weeks with 1.25 mg CS-A/kg b.wt. developed an impaired glucose tolerance after 8 weeks accompanied by a diminished pancreatic insulin content. Despite continued treatment the pancreatic insulin content was able to increase and the glucose tolerance to normalize, indicating an adaptation of pancreatic B-cells to CS-A. The results support the theory that a potential toxic effect of cyclosporin A can be diagnosed by functional tests (e.g. insulin secretion in response to a stepwise increase of glucose) before the irreversible (e.g. morphological) alterations occur.     

Polymeric Gene Transfection on Insulin-Secreting Cells: Sulfonylurea Receptor-Mediation and Transfection Medium Effect

Purpose In vitro transfection of secreting cells is regarded as one strategy for improved cell engineering/transplantation. Insulin-secreting insulinoma cell lines or pancreatic β-cells could be genetically engineered using designed polymeric vectors which are safer than viral vectors. This study investigates the effects of the constituents in transfection media on polymeric transfection.Methods Polyplexes conjugated with sulfonylurea (SU) were evaluated under different transfection conditions for gene transfection and their effects on cytotoxicity and insulin secretion. Several components in transfection media specifically associated with the insulin secretion pathway were amino acids, vitamins, Ca²⁺ and K⁺. The interactions of the polyplexes with insulin were monitored by surface charge and particle size to monitor how insulin as a protein influences transfection.Results For an insulin-secreting cell line (RINm5F), polyplexes in Ca²⁺-containing KRH medium (Ca²⁺(+)KRH) enhanced transfection and did not cause damage to biological functions. When adding amino acids, vitamins, or K⁺ or depleting Ca²⁺ from Ca²⁺(+)KRH, poly(l-lysine)/DNA complexes showed a greater reduction in transfection than SU receptor (SUR)-targeting polyplexes (SU-polyplex). Positively charged polyplexes interacted with insulin, developing a negative surface charge, and these interactions may cause a decrease in transfection.Conclusion The findings suggest that in vitro and ex vivo polymeric transfection of insulin-secreting cells can be modulated and enhanced by adjusting the transfection conditions.     

Characterization of an encapsulated insulin secreting human pancreatic beta cell line in a modular microfluidic device

Type I diabetes mellitus is characterised by the destruction of the insulin producing beta cells within the pancreas by the immune system. After the success of Edmonton protocol, islet transplantation has shown to be a promising therapy, but with the Achilles´ heel of the need of using immunosuppressive drugs. Currently, cell encapsulation technology represents a real alternative to protect transplanted islets from the host´s immune attack. Although preliminary in vitro studies with encapsulated cells have been traditionally performed under static conditions in terms of viability and efficiency, these static cultures do not represent a close approach to in vivo environments. We have developed and characterised different alginate-poly-l-lysine-alginate (APA) microcapsules loaded with the insulin producing 1.1B4 cell line. Static in vitro studies confirmed a constant insulin secretion and a boost of the secretion when the medium was enriched with glucose. Nevertheless, these results were not completely reproduced in a dynamic system by APA liquefied microcapsules containing 1.1B4 cells. The dynamic culture setting created by a microfluidic device, allowed the determination of the glucose response in APA liquefied microcapsules, showing that dynamic conditions can mimic better physiological in vivo environments.     

(d-Ser)Oxm[mPEG-PAL]: A novel chemically modified analogue of oxyntomodulin with antihyperglycaemic, insulinotropic and anorexigenic actions

Oxyntomodulin (Oxm) is a hormone which has been shown to exhibit a range of potentially beneficial actions for alleviation of obesity-diabetes. However, exploitation of Oxm-based therapies has been severely restricted due to degradation by the enzyme dipeptidylpeptidase-IV (DPP-IV). Thus, the aim of this study was to assess the glucose-lowering, insulin-releasing and anorexigenic actions of chemically modified, enzyme-resistant analogues of Oxm. Oxm, (d-Ser²)Oxm and (d-Ser²)Oxm[mPEG-PAL], were incubated with DPP-IV to assess enzyme stability and pancreatic beta-cells to evaluate insulin secretion. cAMP production was assessed using glucagon-like peptide-1 (GLP-1) and glucagon receptor transfected cells. In vivo effects of Oxm analogues on glucose homeostasis, insulin secretion, food intake and bodyweight were examined in obese diabetic (ob/ob) mice. (d-Ser²)Oxm[mPEG-PAL] displayed enhanced DPP-IV resistance compared to (d-Ser²)Oxm and Oxm. All peptides demonstrated similar in vitro cAMP and insulin-releasing actions, which was associated with dual action at GLP-1 and glucagon receptors. Acute administration of (d-Ser²)Oxm[mPEG-PAL] and (d-Ser²)Oxm reduced plasma glucose and food intake, whilst plasma insulin levels were elevated. Once-daily administration of (d-Ser²)Oxm[mPEG-PAL] for 14 days to ob/ob mice decreased food intake, bodyweight, plasma glucose and increased plasma insulin. Furthermore, daily (d-Ser²)Oxm[mPEG-PAL] improved glucose tolerance, increased glucose-mediated insulin secretion, pancreatic insulin content, adiponectin and decreased both visfatin and triglyceride levels. The ability of enzyme-resistant (d-Ser²)Oxm[mPEG-PAL] to improve glucose homeostasis, insulin secretion, satiety, bodyweight and markers of fat metabolism suggests significant promise for Oxm-based therapies for obesity-diabetes.