Improving the function of islet and beta-cell grafts

Background: Human islet transplantation would offer a less invasive and more physiological alternative than whole pancreas transplantation and insulin injections respectively for the treatment of diabetes mellitus if islet graft survival can be improved. Initial recipient post-transplant insulin independence declines to <10% after 5 years. Factors contributing to graft failure include enzymatic disruption of the islet microenvironment during isolation, diabetogenic effects of immunosuppressants and metabolic stress resulting from slow revascularisation. Aims: To investigate the effect of co-culture in both static (SC) and rotational culture (RC) of BRINBDII beta-cells (Dl1) and human umbilical vein endothelial cells (HUVEC) on Dl1 insulin secretion; and the effect of a thiazolidinedione (TZD) on DII function and HUVEC proliferation. To assess the effect of culture media, SC, RC and a TZD on human islet morphology, insulin secretion and VEGF production. To initiate in vivo protocol development for assessment of revascularisation of human islet grafts. Methods: D11 cells were cultured +/-TZD and co-cultured with HUVEC +/-TZD in SC and RC. Dl1 insulin secretion was induced by static incubation with low glucose (1.67mM), high glucose (l6.7mM: and high glucose with 10mM theophylline (G+T) and determined by ELISA. HUVEC were cultured +/-TZD in SC and RC and proliferation was assessed by ATP luminescence assay and VEGF ELISA. D II and HUVEC morphology was determined by immunocytochemistry. Human islets were cultured in SC and RC in various media +/-TZD. Insulin secretion was determined as above and VEGF production by fluorescence immunocytochemistry (FI) and ELISA. Revascularisation of islet grafts was assessed by vascular corrosion cast and FI. Results: Dll cultures showed significantly increased insulin secretion in response to 16.7mM and G+T over basal; this was enhanced by RC and further improved by adding 10mM TZD. Untreated Dll/HUVEC co-cultures displayed significantly increased insulin secretion in response to 16.7mM and G+T over basal, again enhanced by RC and improved with 10mM TZD. 10mM TZD significantly increased HUVEC proliferation over control. Human islets maintained in medium 199 (mI99) in SC and RC exhibited comparable maintenance of morphology and insulin secretory profiles compared to islets maintained in RPMI, endothelial growth media and dedicated islet medium Miami# I. All cultures showed significantly increased insulin secretion in response to 16.7mM and G+T over basal; this was enhanced by RC and in certain instances further improved by adding 25mM TZD. TZD increased VEGF production and release as determined by ELISA. Post-implant vascular corrosion casts of mouse kidneys analysed by x-ray micro tomography indicates a possible TZD enhancement of microvessel growth via VEGF upregulation. Conclusions: D II /HUVEC co-culture in SC or RC does not alter the morphology of either cell type and supports D 11 function. TZD improves 0 I I and D I I/HUVEC SC and RC co-culture insulin secretion while increasing HUVEC proliferation. Human islet RC supports islet functional viability and structural integrity compared to SC while the addition of TZD occasionally further improves secretagogue induced insulin secretion. Expensive, 'dedicated' islet media showed no advantage over ml99 in terms of maintaining islet morphology or function. TZD upregulates VEGF in islets as shown by ELISA and suggested by x-ray micro tomography analysis of vascular corrosion casts. Maintenance of islets in RC and treatment with TZD prior to transplant may improve the functional viability and revascularisation rate of islet grafts.

The role of adipokines in beta-cell failure of type 2 diabetes

Beta-cell failure coupled with insulin resistance is a key factor in the development of type 2 diabetes. Changes in circulating levels of adipokines, factors released from adipose tissue, form a significant link between excessive adiposity in obesity and both aforementioned factors. In this review we consider the published evidence for the role of individual adipokines on the function, proliferation, death and failure of beta-cells, focusing on those reported to have the most significant effects (leptin, adiponectin, TNFa, resistin, visfatin, DPP-IV and apelin). It is apparent that some adipokines have beneficial effects whereas others have detrimental properties; the overall contribution to beta-cell failure of changed concentrations of adipokines in the blood of obese pre-diabetic subjects will be highly dependent on the balance between these effects and the interactions between the adipokines which act on the beta-cell via a number of intersecting intracellular signalling pathways. We emphasise the importance, and comparative dearth, of studies into the combined effects of adipokines on beta-cells.

Resistin down-regulates insulin receptor expression, and modulates cell viability in rodent pancreatic beta-cells

The adipokine resistin is known to induce insulin resistance in rodent tissues. Increases in adipose tissue mass are known to have a negative effect on pancreatic beta-cell function, although the mechanisms are poorly understood. This study investigated the effects of resistin on insulin secretion, insulin receptor expression and cell viability in pancreatic beta-cells. BTC-6 or BRIN-BD11 cells were treated for 24h with resistin, and insulin receptor expression, insulin secretion and cell viability were measured. Incubation with 40ng/ml resistin caused significant decreases in insulin receptor mRNA and protein expression, but did not affect insulin secretion. At low concentrations, resistin caused significant increases in cell viability. These data implicate resistin as a factor that may regulate beta-cell function/viability, and suggests a potential mechanism by which increased adiposity causes beta-cell dysfunction.

Regulation of beta-cell viability and gene expression by distinct agonist fragments of adiponectin

Obesity is an established risk factor for type 2 diabetes. Activation of the adiponectin receptors has a clear role in improving insulin resistance although conflicting evidence exists for its effects on pancreatic beta-cells. Previous reports have identified both adiponectin receptors (ADR-1 and ADR-2) in the beta-cell. Recent evidence has suggested that two distinct regions of the adiponectin molecule, the globular domain and a small N-terminal region, have agonist properties. This study investigates the effects of two agonist regions of adiponectin on insulin secretion, gene expression, cell viability and cell signalling in the rat beta-cell line BRIN-BD11, as well as investigating the expression levels of adiponectin receptors (ADRs) in these cells. Cells were treated with globular adiponectin and adiponectin (15-36) +/-leptin to investigate cell viability, expression of key beta-cell genes and ERK1/2 activation. Both globular adiponectin and adiponectin (15-36) caused significant ERK1/2 dependent increases in cell viability. Leptin co-incubation attenuated adiponectin (15-36) but not globular adiponectin induced cell viability. Globular adiponectin, but not adiponectin (15-36), caused a significant 450% increase in PDX-1 expression and a 45% decrease in LPL expression. ADR-1 was expressed at a higher level than ADR-2, and ADR mRNA levels were differentially regulated by non-esterified fatty acids and peroxisome-proliferator-activated receptor agonists. These data provide evidence of roles for two distinct adiponectin agonist domains in the beta-cell and confirm the potentially important role of adiponectin receptor agonism in maintaining beta-cell mass.

Sustained insulin secretory response in human islets co-cultured with pancreatic duct-derived epithelial cells within a rotational cell culture system

Aims/hypothesis - Loss of the trophic support provided by surrounding non-endocrine pancreatic cell populations underlies the decline in beta cell mass and insulin secretory function observed in human islets following isolation and culture. This study sought to determine whether restoration of regulatory influences mediated by ductal epithelial cells promotes sustained beta cell function in vitro. Methods - Human islets were isolated according to existing protocols. Ductal epithelial cells were harvested from the exocrine tissue remaining after islet isolation, expanded in monolayer culture and characterised using fluorescence immunocytochemistry. The two cell types were co-cultured under conventional static culture conditions or within a rotational cell culture system. The effect of co-culture on islet structural integrity, beta cell mass and insulin secretory capacity was observed for 10 days following isolation. Results - Human islets maintained under conventional culture conditions exhibited a characteristic loss in structural integrity and functional viability as indicated by a diminution of glucose responsiveness. By contrast, co-culture of islets with ductal epithelial cells led to preserved islet morphology and sustained beta cell function, most evident in co-cultures held within the rotational cell culture system, which showed a significantly (p<0.05) greater insulin secretory response to elevated glucose compared with control islets. Similarly, insulin/protein ratio data suggested that the presence of ductal epithelial cells is beneficial for the maintenance of beta cell mass. Conclusions/interpretation - The data indicate a supportive role for ductal epithelial cells in islet viability. Further characterisation of the regulatory influences may lead to novel strategies to improve long-term beta cell function both in vitro and following islet transplantation.

Autocrine activity of soluble Flt-1 controls endothelial cell function and angiogenesis

Background - The negative feedback system is an important physiological regulatory mechanism controlling angiogenesis. Soluble vascular endothelial growth factor (VEGF) receptor-1 (sFlt-1), acts as a potent endogenous soluble inhibitor of VEGF- and placenta growth factor (PlGF)-mediated biological function and can also form dominant-negative complexes with competent full-length VEGF receptors. Methods and results - Systemic overexpression of VEGF-A in mice resulted in significantly elevated circulating sFlt-1. In addition, stimulation of human umbilical vein endothelial cells (HUVEC) with VEGF-A, induced a five-fold increase in sFlt-1 mRNA, a time-dependent significant increase in the release of sFlt-1 into the culture medium and activation of the flt-1 gene promoter. This response was dependent on VEGF receptor-2 (VEGFR-2) and phosphoinositide-3'-kinase signalling. siRNA-mediated knockdown of sFlt-1 in HUVEC stimulated the activation of endothelial nitric oxide synthase, increased basal and VEGF-induced cell migration and enhanced endothelial tube formation on growth factor reduced Matrigel. In contrast, adenoviral overexpression of sFlt-1 suppressed phosphorylation of VEGFR-2 at tyrosine 951 and ERK-1/-2 MAPK and reduced HUVEC proliferation. Preeclampsia is associated with elevated placental and systemic sFlt-1. Phosphorylation of VEGFR-2 tyrosine 951 was greatly reduced in placenta from preeclamptic patients compared to gestationally-matched normal placenta. Conclusion - These results show that endothelial sFlt-1 expression is regulated by VEGF and acts as an autocrine regulator of endothelial cell function.

Effects of metformin on BRIN-BD11 beta-cell insulin secretory desensitization induced by prolonged exposure to sulphonylureas

Aims: Prolonged exposure of pancreatic beta-cells in vitro to the sulphonylureas tolbutamide and glibenclamide induces subsequent desensitization of insulinotropic pathways. Clinically, the insulin-sensitizing biguanide drug metformin is often administered alongside sulphonylurea as antidiabetic therapy. The present study examines the functional effects of metformin (200 µM) on tolbutamide- and glibenclamide-induced desensitisation. Methods: Acute and prolonged (18 h) effects of exposure to tolbutamide and glibenclamide alone, or in the presence of metformin, were examined in insulin-secreting BRIN-BD11 cells. Results: In acute 20 min incubations at 1.1 mM glucose, metformin increased (1.2-1.7-fold; p <0.001) the insulin-releasing actions of tolbutamide and glibenclamide. At 16.7 mM glucose, metformin significantly enhanced glibenclamide-induced insulin release at all concentrations (50-400 µM) examined, but tolbutamide-stimulated insulin secretion was only augmented at higher concentrations (300-400 µM). Exposure for 18 h to 100 µM tolbutamide or glibenclamide significantly impaired insulin release in response to glucose and a broad range of insulin secretagogues. Concomitant culture with metformin (200 µM) prevented or partially reversed many of the adverse effects on K channel dependent and independent insulinotropic pathways. Beneficial effects of metformin were also observed in cells exposed to glibenclamide for 18 h with significant improvements in the insulin secretory responsiveness to alanine, GLP-1 and sulphonylureas. The decrease of viable cell numbers observed with glibenclamide was reversed by co-culture with metformin, but cellular insulin content was depressed. Conclusions: The results suggest that metformin can prevent the aspects of sulphonylurea-induced beta-cell desensitization. © 2010 Blackwell Publishing Ltd.

Novel glucagon-like peptide-1 (GLP-1) analog (Val8)GLP-1 results in significant improvements of glucose tolerance and pancreatic β-cell function after 3-week daily administration in obese diabetic (ob/ob) mice

This study evaluates the antidiabetic potential of an enzyme-resistant analog, (Val8)GLP-1. The effects of daily administration of a novel dipeptidyl peptidase IV-resistant glucagon-like peptide-1 (GLP-1) analog, (Val8)GLP-1, on glucose tolerance and pancreatic β-cell function were examined in obese-diabetic (ob/ob) mice. Acute intraperitoneal administration of (Val8)GLP-1 (6.25-25 nmol/kg) with glucose increased the insulin response and reduced the glycemic excursion in a dose-dependent manner. The effects of (Val8)GLP-1 were greater and longer lasting than native GLP-1. Once-daily subcutaneous administration of (Val8)GLP-1 (25 nmol/kg) for 21 days reduced plasma glucose concentrations, increased plasma insulin, and reduced body weight more than native GLP-1 without a significant change in daily food intake. Furthermore, (Val8)GLP-1 improved glucose tolerance, reduced the glycemic excursion after feeding, increased the plasma insulin response to glucose and feeding, and improved insulin sensitivity. These effects were consistently greater with (Val8)GLP-1 than with native GLP-1, and both peptides retained or increased their acute efficacy compared with initial administration. (Val8)GLP-1 treatment increased average islet area 1.2-fold without changing the number of islets, resulting in an increased number of larger islets. These data demonstrate that (Val8)GLP-1 is more effective and longer acting than native GLP-1 in obese-diabetic ob/ob mice.

The role of ERK5 in endothelial cell function

Extracellular-signal-regulated kinase 5 (ERK5), also termed big MAPK1 (BMK1), is the most recently discovered member of the mitogen-activated protein kinase (MAPK) family. It is expressed in a variety of tissues and is activated by a range of growth factors, cytokines and cellular stresses. Targeted deletion of Erk5 in mice has revealed that the ERK5 signalling cascade is critical for normal cardiovascular development and vascular integrity. In vitro studies have revealed that, in endothelial cells, ERK5 is required for preventing apoptosis, mediating shear-stress signalling and regulating tumour angiogenesis. The present review focuses on our current understanding of the role of ERK5 in regulating endothelial cell function.

Expression profiling of type 2 diabetes susceptibility genes in the pancreatic islets, adipose tissue and liver of obese mice

Type 2 diabetes (T2D) is characterized by impaired beta cell function and insulin resistance. T2D susceptibility genes identified by Genome-wide association studies (GWAS) are likely to have roles in both impaired insulin secretion from the beta cell as well as insulin resistance. The aim of this study was to use gene expression profiling to assess the effect of the diabetic milieu on the expression of genes involved in both insulin secretion and insulin resistance. We measured the expression of 43 T2D susceptibility genes in the islets, adipose and liver of leptin-deficient Ob/Ob mice compared with Ob/+ littermates. The same panel of genes were also profiled in cultured rodent adipocytes, hepatocytes and beta cells in response to high glucose conditions, to distinguish expression effects due to elevated glycemia from those on the causal pathway to diabetes or induced by other factors in the diabetic microenviroment. We found widespread deregulation of these genes in tissues from Ob/Ob mice, with differential regulation of 23 genes in adipose, 18 genes in liver and one gene (Tcf7l2) in islets of diabetic animals (Ob/Ob) compared to control (Ob/+) animals. However, these expression changes were in most cases not noted in glucose-treated adipocyte, hepatocyte or beta cell lines, indicating that they may not be an effect of hyperglycemia alone. This study indicates that expression changes are apparent with diabetes in both the insulin producing beta cells, but also in peripheral tissues involved in insulin resistance. This suggests that incidence or progression of diabetic phenotypes in a mouse model of diabetes is driven by both secretory and peripheral defects. © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart New York.

Obesity in the pathogenesis of type 2 diabetes

Obesity, and especially visceral adiposity, escalates the development of insulin resistance and type 2 diabetes. Excess adipose tissue contributes to a chronic increase in circulating fatty acids reducing the usage of glucose as a source of cellular energy. Excess fatty acids also result in increased deposition of fat in muscle and liver, and increased metabolites such as diacylglycerol and ceramide which activate isoforms of protein kinase C that impede cellular insulin signalling. Chronically raised lipid levels also impair islet beta cell function, acting in conjuction with insulin resistance to aggravate hyperglycaemia. The detrimental effects of several adipokines such as TNF, IL6 and RBP4, which are produced in excess by an increased adipose mass, and reduced production of adiponectin are further mechanisms through which obesity potentiates the development of type 2 diabetes. © 2011 The Author(s).

Insulin sensitivity and secretion in obese Type 2 diabetic women after various bariatric operations

Objective: To compare the effects of biliopancreatic diversion (BPD) and laparoscopic gastric banding (LAGB) on insulin sensitivity and secretion with the effects of laparoscopic gastric plication (P). Methods: A total of 52 obese women (age 30-66 years) suffering from type 2 diabetes mellitus (T2DM) were prospectively recruited into three study groups: 16 BPD; 16 LAGB, and 20 P. Euglycemic clamps and mixed meal tolerance tests were performed before, at 1 month and at 6 months after bariatric surgery. Beta cell function derived from the meal test parameters was evaluated using mathematical modeling. Results: Glucose disposal per kilogram of fat free mass (a marker of peripheral insulin sensitivity) increased significantly in all groups, especially after 1 month. Basal insulin secretion decreased significantly after all three types of operations, with the most marked decrease after BPD compared with P and LAGB. Total insulin secretion decreased significantly only following the BPD. Beta cell glucose sensitivity did not change significantly post-surgery in any of the study groups. Conclusion: We documented similar improvement in insulin sensitivity in obese T2DM women after all three study operations during the 6-month postoperative follow-up. Notably, only BPD led to decreased demand on beta cells (decreased integrated insulin secretion), but without increasing the beta cell glucose sensitivity.

Visfatin regulates insulin secretion, insulin receptor signalling and mRNA expression of diabetes-related genes in mouse pancreatic beta-cells

The role of the adipocyte-derived factor visfatin in metabolism remains controversial, although some pancreatic ß-cell-specific effects have been reported. This study investigated the effects of visfatin upon insulin secretion, insulin receptor activation and mRNA expression of key diabetes-related genes in clonal mouse pancreatic ß-cells. ß-TC6 cells were cultured in RPMI 1640 and were subsequently treated with recombinant visfatin. One-hour static insulin secretion was measured by ELISA. Phospho-specific ELISA and western blotting were used to detect insulin receptor activation. Real-time SYBR Green PCR array technology was used to measure the expression of 84 diabetes-related genes in both treatment and control cells. Incubation with visfatin caused significant changes in the mRNA expression of several key diabetes-related genes, including marked up-regulation of insulin (9-fold increase), hepatocyte nuclear factor (HNF)1ß (32-fold increase), HNF4a (16-fold increase) and nuclear factor ?B (40-fold increase). Significant down-regulation was seen in angiotensin-converting enzyme (-3.73-fold) and UCP2 (-1.3-fold). Visfatin also caused a significant 46% increase in insulin secretion compared to control (P<0.003) at low glucose, and this increase was blocked by co-incubation with the specific nicotinamide phosphoribosyltransferase inhibitor FK866. Both visfatin and nicotinamide mononucleotide induced activation of both insulin receptor and extracellular signal-regulated kinase (ERK)1/2, with visfatin-induced insulin receptor/ERK1/2 activation being inhibited by FK866. We conclude that visfatin can significantly regulate insulin secretion, insulin receptor phosphorylation and intracellular signalling and the expression of a number of ß-cell function-associated genes in mouse ß-cells.

Does metabolic reprogramming underpin age-associated changes in T cell phenotype and function?

T cells are required for an effective adaptive immune response. The principal function of T cells is to promote efficient removal of foreign material by identifying and mounting a specific response to nonself. A decline in T cell function in aging is thought to contribute to reduced response to infection and vaccination and an increase in autoimmunity. This may in part be due to the age-related decrease in naïve CD4+ T cells and increase in antigen-experienced CD4+ T cells, loss of redox homeostasis, and impaired metabolic switching. Switching between subsets is triggered by the integration of extracellular signals sensed through surface receptors and the activation of discrete intracellular metabolic pathways. This article explores how metabolic programming and loss of redox homeostasis during aging may contribute to age-associated changes in T cell phenotype and function. © 2014 Elsevier Inc.

Antioxidants, reactive oxygen and nitrogen species, gene induction and mitochondrial function

Redox-sensitive cell signalling Thiol groups and the regulation of gene expression Redox-sensitive signal transduction pathways Protein kinases Protein phosphatases Lipids and phospholipases Antioxidant (electrophile) response element Intracellular calcium signalling Transcription factors NF-?B AP-1 p53 Cellular responses to oxidative stress Cellular responses to change in redox state Proliferation Cell death Immune cell function Reactive oxygen and nitrogen species – good or bad? Reactive oxygen species and cell death Reactive oxygen species and inflammation Are specific reactive oxygen species and antioxidants involved in modulating cellular responses? Specific effects of dietary antioxidants in cell regulation Carotenoids Vitamin E Flavonoids Inducers of phase II enzymes Disease states affected Oxidants, antioxidants and mitochondria Introduction Mitochondrial generation of reactive oxygen and nitrogen species Mitochondria and apoptosis Mitochondria and antioxidant defences Key role of mitochondrial GSH in the defence against oxidative damage Mitochondrial oxidative damage Direct oxidative damage to the mitochondrial electron transport chain Nitric oxide and damage to mitochondria Effects of nutrients on mitochondria Caloric restriction and antioxidants Lipids Antioxidants Techniques and approaches Mitochondrial techniques cDNA microarray approaches Proteomics approaches Transgenic mice as tools in antioxidant research Gene knockout and over expression Transgenic reporter mice Conclusions Future research needs