Culture Of Pancreatic Islets Research Articles

Trophic effect of adipose tissue–derived stem cells on porcine islet cells

BackgroundAdipose tissue–derived stem cells (ADSCs), which are widely known as multipotent progenitor cells, release several cytokines that support cell survival and repair. The aim of this study was to investigate whether ADSC-secreted molecules could induce a trophic effect in pancreatic islet culture conditions in vitro. Materials and methodsWe cocultured porcine islet cells with ADSCs using a transwell system for 48 h and evaluated the viability of islet cells. We also determined the concentration levels of cytokines and insulin in the supernatant of the culture medium. We used anti-vascular endothelial growth factor (VEGF) and anti-interleukin (IL)-6 receptor antibodies to investigate the effect of VEGF and IL-6 on islet cells. ResultsADSCs improved the viability of islet cells in the absence of cell-cell contact (P < 0.05). VEGF and IL-6 levels in the culture medium increased when islet cells were cocultured with ADSCs (P < 0.05). Furthermore, inhibition of VEGF decreased the viability of islet cells (P < 0.05); however, inhibition of IL-6 did not affect islet cell viability. ConclusionsThese results suggested that trophic factors, particularly VEGF, secreted by human ADSCs enhanced the survival and function of porcine islet cells.

Proteins differentially expressed in human beta-cells-enriched pancreatic islet cultures and human insulinomas

In view of the great demand for human beta-cells for physiological and medical studies, we generated cell lines derived from human insulinomas which secrete insulin, C-peptide and express neuroendocrine and islet markers. In this study, we set out to characterize their proteomes, comparing them to those of primary beta-cells using DIGE followed by MS. The results were validated by Western blotting. An average of 1800 spots was detected with less than 1% exhibiting differential abundance. Proteins more abundant in human islets, such as Caldesmon, are involved in the regulation of cell contractility, adhesion dependent signaling, and cytoskeletal organization. In contrast, almost all proteins more abundant in insulinoma cells, such as MAGE2, were first described here and could be related to cell survival and resistance to chemotherapy. Our proteomic data provides, for the first time, a molecular snapshot of the orchestrated changes in expression of proteins involved in key processes which could be correlated with the altered phenotype of human beta-cells. Collectively our observations prompt research towards the establishment of bioengineered human beta-cells providing a new and needed source of cultured human beta-cells for beta-cell research, along with the development of new therapeutic strategies for detection, characterization and treatment of insulinomas.

Peptidomic Profiling of Secreted Products from Pancreatic Islet Culture Results in a Higher Yield of Full-length Peptide Hormones than Found using Cell Lysis Procedures

Peptide Hormone Acquisition through Smart Sampling Technique-Mass Spectrometry (PHASST-MS) is a peptidomics platform that employs high resolution liquid chromatography-mass spectrometry (LC-MS) techniques to identify peptide hormones secreted from in vitro or ex vivo cultures enriched in endocrine cells. Application of the methodology to the study of murine pancreatic islets has permitted evaluation of the strengths and weaknesses of the approach, as well as comparison of our results with published islet studies that employed traditional cellular lysis procedures. We found that, while our PHASST-MS approach identified fewer peptides in total, we had greater representation of intact peptide hormones. The technique was further refined to improve coverage of hydrophilic as well as hydrophobic peptides and subsequently applied to human pancreatic islet cultures derived from normal donors or donors with type 2 diabetes. Interestingly, in addition to the expected islet hormones, we identified alpha-cell-derived bioactive GLP-1, consistent with recent reports of paracrine effects of this hormone on beta-cell function. We also identified many novel peptides derived from neurohormonal precursors and proteins related to the cell secretory system. Taken together, these results suggest the PHASST-MS strategy of focusing on cellular secreted products rather than the total tissue peptidome may improve the probability of discovering novel bioactive peptides and also has the potential to offer important new insights into the secretion and function of known hormones.

Systematic prevention of bubble formation and accumulation for long-term culture of pancreatic islet cells in microfluidic device.

Reliable long-term cell culture in microfluidic system is limited by air bubble formation and accumulation. In this study, we developed a bubble removal system capable of both trapping and discharging air bubbles in a consistent and reliable manner. Combined with PDMS (Polydimethylsiloxane) hydrophilic surface treatment and vacuum filling, a microfluidic perifusion system equipped with the bubble trap was successfully applied for long-term culture of mouse pancreatic islets with no bubble formation and no flow interruption. In addition to demonstrating normal cell viability and islet morphology, post-cultured islets exhibited normal insulin secretion kinetics, intracellular calcium signaling, and changes in mitochondrial potentials in response to glucose challenge. This design could be easily adapted by other microfluidic systems due to its simple design, ease of fabrication, and portability.

Pathogen Inactivation of Human Serum Facilitates its Clinical Use for Islet Cell Culture and Subsequent Transplantation

Serum is regarded as an essential supplement to promote survival and growth of cells during culture. However, the potential risk of transmitting diseases disqualifies the use of serum for clinical cell therapy in most countries. Hence, most clinical cell therapy programs have replaced human serum with human serum albumin, which can result in inferior quality of released cell products. Photochemical treatment of different blood products utilizing Intercept® technology has been shown to inactivate a broad variety of pathogens of RNA and DNA origin. The present study assesses the feasibility of using pathogen-inactivated, blood group-compatible serum for use in human pancreatic islet culture. Isolated human islets were cultured at 37°C for 3-4 days in CMRL 1066 supplemented with 10% of either pathogen-inactivated or nontreated human serum. Islet quality assessment included glucose-stimulated insulin release (perifusion), ADP/ATP ratio, cytokine expression, and posttransplant function in diabetic nude mice. No differences were found between islets cultured in pathogen-inactivated or control serum regarding stimulated insulin release, intracellular insulin content, and ADP/ATP ratio. Whether media was supplemented with treated or nontreated serum, islet expression of IL-6, IL-8, MCP-1, or tissue factor was not affected. The final diabetes-reversal rate of mice receiving islets cultured in pathogen-inactivated or nontreated serum was 78% and 87%, respectively (NS). As reported here, pathogen-inactivated human serum does not affect viability or functional integrity of cultured human islets. The implementation of this technology for RNA- and DNA-based pathogen inactivation should enable reintroduction of human serum for clinical cell therapy.

Use of Perfluorodecalin for Pancreatic Islet Culture Prior to Transplantation: A Liquid-Liquid Interface Culture System—Preliminary Report

Although the issue remains controversial, short-term culture is probably beneficial for islet graft quality. However, significant islet loss is invariably observed. This is related to reduced survival of large islets, which is compromised by hypoxia under standard culture conditions. We aimed to develop a method of culture, which would avoid exposure to relative hypoxia and hence maintain the quality of islets. Isolated rat islets cultured for 48 h in a liquid-liquid interface culture system (LICS) with a perfluorocarbon were compared to islets cultured under standard (C1) and suboptimal conditions (C2). Islets were tested for viability and response to a glucose challenge, and a marginal mass was transplanted into syngeneic diabetic recipients. The viability of islets after 24-h culture in LICS was higher than in C1 and C2 groups (89.0% vs. 77.5% and 64.6%, respectively) and decreased with time to reach 79.0%, 62.9%, and 53.4% after 72-h culture. The stimulation index in LICS-cultured islets was also significantly higher than in C1 and C2 groups (12.3 ± 0.4 vs. 5.8 ± 0.5 and 4.1 ± 0.2, respectively). Following transplantation of LICS-cultured islets 50% of recipients were rendered normoglycemic compared with 14.3% and 31.3% for C2 and fresh islets, respectively. Our liquid-liquid interface culture system using perfluorodecalin provides optimized culture conditions, which preserve both islet viability and their ability to engraft successfully after intraportal transplantation and could be used for islet transportation.

Pancreatic Islet Culture and Preservation Strategies: Advances, Challenges, and Future Outlook

Postisolation islet survival is a critical step for achieving successful and efficient islet transplantation. This involves the optimization of islet culture in order to prolong survival and functionality in vitro. Many studies have focused on different strategies to culture pancreatic islets in vitro through manipulation of culture media, surface modified substrates, and the use of various techniques such as encapsulation, embedding, scaffold, and bioreactor culture strategies. This review aims to present and discuss the different methodologies employed to optimize pancreatic islet culture in vitro as well as address their respective advantages and drawbacks.

Long-term in vitro human pancreatic islet culture using three-dimensional microfabricated scaffolds

Human pancreatic islet in vitro culture is very challenging and requires the presence of various extra cellular matrix (ECM) components in a three-dimensional environment, which provides mechanical and biological support. The development of such an environment is vital in providing favourable conditions to preserve human islets in long-term culture. In this study, we investigated the effects of human islet culture within various three-dimensional environments; collagen I gel, collagen I gel supplemented with ECM components fibronectin and collagen IV, and microfabricated scaffold with ECM-supplemented gel. The cultured human islets were analyzed for functionality, gene expression and hormone content following long-term in vitro culture. It was clear the incorporation of ECM components within the three-dimensional support improved prolonged culture. However, long-term and highly uniform human islet culture within a microfabricated scaffold, with controlled pore structures, coupled with the presence of ECM components, displayed an insulin release profile similar to freshly isolated islets, yielding a stimulation index of ∼1.8. Moreover, gene expression was markedly increased for all pancreatic genes, giving a ∼50-fold elevation of insulin gene expression with respect to suspension culture. The distribution and presence of pancreatic hormones was also highly elevated. These findings provide a platform for the long-term maintenance and preservation of human pancreatic islets in vitro.

Small-molecule inducers of insulin expression in pancreatic α-cells

High-content screening for small-molecule inducers of insulin expression identified the compound BRD7389, which caused alpha-cells to adopt several morphological and gene expression features of a beta-cell state. Assay-performance profile analysis suggests kinase inhibition as a mechanism of action, and we show that biochemical and cellular inhibition of the RSK kinase family by BRD7389 is likely related to its ability induce a beta-cell-like state. BRD7389 also increases the endocrine cell content and function of donor human pancreatic islets in culture.

Trophic Molecules Derived From Human Mesenchymal Stem Cells Enhance Survival, Function, and Angiogenesis of Isolated Islets After Transplantation

Mesenchymal stem cells (MSCs), also known as multipotent progenitor cells, release several factors that support cell survival and enhance wound healing. We hypothesized that MSC-secreted molecules would induce a trophic effect in pancreatic islet culture conditions. Pancreatic islets were co-cultured with MSCs, and ADP/ATP ratios, glucose stimulated insulin secretion (GSIS), and DNA fragmentation were evaluated to measure islet quality and viability in vitro. The induction of signal molecules related to the control of survival, function, and angiogenesis was also analyzed. Cell quality assays, DNA fragmentation assays, and islet transplantation into streptozotocin-induced diabetic mice were performed using MSC-conditioned medium (CM)-cultured islets. Furthermore, we identified soluble molecules within MSC-CM. Islets co-cultured with MSCs demonstrated lower ADP/ATP ratios, and higher GSIS indexes and viability. Furthermore, co-cultured islets revealed higher levels of anti-apoptotic signal molecules (X-linked inhibitor of apoptosis protein, Bcl-xL, Bcl-2, and heat shock protein-32) and demonstrated increased vascular endothelial growth factor receptor 2 and Tie-2 mRNA expression and increased levels of phosphorylated Tie-2 and focal adhesion kinase protein. Islets cultured in MSC-CM demonstrated lower ADP/ATP ratios, less apoptosis, and a higher GSIS indexes. Diabetic mice that received islet transplants (200 islet equivalent) cultured in MSC-CM for 48 hr demonstrated significantly lower blood glucose levels and enhanced blood vessel formation. In addition, interleukin-6, interleukin-8, vascular endothelial growth factor-A, hepatocyte growth factor, and transforming growth factor-beta were detected at significant levels in MSC-CM. These results suggest that the trophic factors secreted by human MSCs enhance islet survival and function after transplantation.

The thyroid hormone T3 improves function and survival of rat pancreatic islets during in vitro culture

Ex vivo islet cell culture in the presence of stimulating factors prior to transplantation is considered a good strategy in contrast to the short conclusion of islets transplantation. Previously, we demonstrated how T3 can increase b-cell function via specific activation of Akt; therefore we hypothesized that thyroid hormone T3 can be considered a promising candidate for the in vitro expansion of islet cell mass. Rat pancreatic islets have been isolated by the collagenase digestion and cultured in the presence or not of the thyroid hormone T3 10-7 M. Islets viability has been evaluated by the use of two different dyes, one cell-permeable green fluorescent dye and propidium iodide, and by the analysis of core cell damage upcoming. Moreover, islets function has been evaluated by insulin secretion. The ability of b-cells to counteract apoptosis induced by streptozotocin has been analyzed by TUNEL assay. We demonstrated that treatment of primary cultures of rat pancreatic islets with T3 results in augmented β-cell vitality with an increase of their functional properties. In addition, a sensible reduction of the core cell damage has been observed in the T3 treated islets, suggesting the preservation of the β-cells integrity during the culture period. Nonetheless, the insulin secretion is sensibly augmented after T3 stimulation. The strong increment shown in Akt activation suggests the involvement of this pathway in the observed phenomena. In conclusion we indicate T3 as a good factor to improve ex vivo islets cell culture.

The effect of extracellular matrix components on the preservation of human islet function in vitro

Human islet isolation leads to the loss of the ECM basement membrane which contributes to eventual apoptosis in vitro. The reestablishment of this environment is vital in understanding the mechanism of islet interaction with its surroundings in order to arrive at conditions favourable to islet culture in vitro. In this study, we investigated the effects of the main ECM components collagen I and IV, fibronectin, and laminin on human islet adhesion, survival, and functionality. Results have provided insight into integrin-mediated effects and behaviour. Collagen I/IV and fibronectin induced adhesion, while fibronectin was the only ECM protein capable of maintaining islet structural integrity and insulin content distribution. Furthermore, islet phenotype was eventually lost, but insulin gene expression was highest in islets cultured on collagen I and IV. However, insulin release was highest on fibronectin, along with a decrease in SUR1 expression, while glucose metabolism, along with GLUT2 and GCK expression, was highest on collagen I and IV surfaces. These findings provide a basis for the future establishment of a modified three-dimensional construct for the culture of human pancreatic islets in vitro.

Hyper- and hypoinsulinemia in type-2 diabetes: what may be wrong in the secretory mechanism of the B-cell.

Type-2 diabetes frequently is the consequence of overnutrition causing overweight, which then produces insulin resistance. The following hyperglycemia induces permanent overstimulation of the insulin secretory machinery of the B-cell, which results in hyperinsulinemia and/or hypoinsulinemia. The mechanisms, however, of these disturbances are not understood so far. Animal models, which can be used to solve these questions, are, among others, long-term incubation of pancreatic islets in culture in the presence of high glucose and/or long-term infusion of rats with glucose. Using these models sensitization [Bedoya and Jeanrenaud, 1991; Leahy et al., 1987; Purrello et al., 1992; Thams, 1991; Thibault et al., 1993; Timmers et al., 1990] and/or desensitization of insulin release [Bedoya and Jeanrenaud, 1991; Leahy et al., 1987; Davalli et al., 1992; Eizirik et al., 1992; Bolaffi et al., 1988; Timmers et al., 1990] have been reported. Whether or not there is sensitization or desensitization depends on the glucose concentration [Eizirik et al., 1992; Leahy et al., 1986; Sako and Grill, 1990] and the duration of glucose action [Bedoya and Jeanrenaud, 1991; Bolaffi et al., 1988]. Both, sensitization and desensitization, appear to be reversible on return to normal glucose or mild hypoglycemia [Leahy and Weir, 1991; Svenson and Hellerström, 1991]. It seems, however, that there is a fluent change from the first occurring hyperinsulinemia to hypoinsulinemia. In this lecture I first want to talk about the possible mechanism of sensitization of insulin secretion discussing previous results we have obtained from rat islets isolated after a 48 hours period of i.v. infusion with 50% glucose. In the second part I will discuss some of the findings coming from the respective literature concerning desensitization of insulin secretion.

Growth hormone stimulation of serum insulin concentration in cattle: Nutritional dependency and potential mechanisms

Previous studies on the effect of growth hormone (GH) on serum insulin concentration in cattle had generated seemingly conflicting results, and little was known about the mechanism by which GH affects serum insulin concentration in cattle, if it does. In this study, we determined whether the effect of GH on serum insulin concentration in cattle could be affected by the nutritional levels of the animal and whether GH increased serum insulin concentration in cattle by directly stimulating insulin release or insulin gene expression in the pancreatic islets. Administration of recombinant bovine GH increased serum insulin concentration in nonlactating, nonpregnant beef cows fed a daily concentrate meal in addition to ad libitum hay, but it had no effect in those cows fed hay only. Both GH treatments for 1 and 24 h increased insulin concentrations in cultures of pancreatic islets isolated from growing cattle. Growth hormone treatment for 24 h increased insulin mRNA expression in cultured bovine pancreatic islets. Growth hormone treatment for 16 h increased reporter gene expression directed by a ∼1,500-bp bovine insulin gene promoter in a rat insulin-producing β cell line. Taken together, these results suggest that exogenous GH can increase serum insulin concentration in cattle, but this effect depends on the nutritional levels of fed cattle, and that GH increases serum insulin concentration in cattle by stimulating both insulin release and insulin gene expression in the pancreatic islets.

Antihyperglycemic and insulin secretory activity of Costus pictus leaf extract in streptozotocin induced diabetic rats and in in vitro pancreatic islet culture

Aim of the study The leaves of Costus pictus D. Don were used extensively for its antihyperglycemic activity by the people in Kerala, India. In the present study, the antihyperglycemic and insulin secretory activity of an aqueous extract of Costus pictus leaf extract was investigated in streptozotocin induced diabetic rats. Materials and methods Oral Glucose Tolerance Test was done to determine the effective dose of Costus pictus extract. Aqueous extract of Costus pictus leaves was given orally to the diabetic rats for 14 days. The insulin secretory action of the leaf extract was investigated using isolated pancreatic islets from rat. Liver glucose uptake activity was measured using d-[ 14C] glucose. Results The oral administration of an aqueous extract of Costus pictus at a dose of 250 mg/kg body weight significantly decreased the blood glucose with significant increase in plasma insulin level in diabetic rats at the end of 14 days treatment. The Costus pictus leaf extract significantly increased glucose induced insulin secretion at both 4 mM and 20 mM glucose concentrations which represents normal physiological and diabetic condition respectively. The decreased glucose uptake activity of the liver of diabetic rats was reverted to near normal levels after the treatment with Costus pictus leaf extract. Conclusion Our results suggest the glucose lowering effect of Costus pictus to be associated with the potentiation of insulin release from pancreatic islets and enhancement of peripheral utilization of glucose.

Short-term glucocorticoid treatment increases insulin secretion in islets derived from lean mice through multiple pathways and mechanisms

Chronic exposure to elevated levels of glucocorticoids leads to metabolic dysfunctions with hyperglycemia and insulin resistance. Long-term treatment with glucocorticoids induces severe impairment of glucose-stimulated insulin secretion. We analyzed the effects of short-, and medium-term (2-120h) treatment with 50-200nM glucocorticoids on primary pancreatic islet cultures derived from lean C57BL/6J mice. In contrast to animal models of insulin resistance, beta-cells from lean mice respond with an increased glucose-stimulated insulin secretion, with a peak effect around 18-24h of treatment. Analyses of the insulin secretion response reveal that early and late phase responses are dissociated upon glucocorticoid treatment. Whereas late phase responses return to basal levels after long treatment, early phase responses remain increased over several days. Increased insulin secretion is also obtained by incubation with the inactive glucocorticoid dehydrocorticosterone, pointing to an important role of the enzyme 11beta-hydroxysteroid dehydrogenase type 1 in mediating glucocorticoid effects in beta-cells. Transcript profiling revealed differential regulation of genes involved in mediation of signal transduction, insulin secretion, stress and inflammatory responses. The results show that short- to medium-term glucocorticoid treatment of pancreatic islets derived from lean mice leads to an increased insulin release and may constitute an important parameter in changing towards a pro-diabetic phenotype.

An enterovirus strain isolated from diabetic child belongs to a genetic subcluster of echovirus 11, but is also neutralised with monotypic antisera to coxsackievirus A9

An enterovirus strain (designated D207) isolated from a Slovakian diabetic child and originally serotyped as coxsackievirus A9 (CAV-9) was found to cause rapid cytolysis coinciding with severe functional damage of the surviving cells in primary cultures of human pancreatic islets. This finding prompted us to clone the isolate for full-length genome sequencing and molecular characterization as the prototype strain of CAV-9 is known to cause only minimal damage to insulin-producing beta-cells. Based on capsid-coding sequence comparisons, the isolate turned out to be echovirus 11 (E-11). Phylogenetic analyses demonstrated that E-11/D207 was closely related to a specific subgroup B of E-11 strains known to cause uveitis. To study further antigenic properties of isolate E-11/D207 and uveitis-causing E-11 strains, neutralization experiments were carried out with CAV-9- and E-11-specific antisera. Unlike the prototype strains, the isolate E-11/D207 and uveitis-causing E-11 strains were well neutralized with both CAV-9- and E-11-specific antisera. Attempts to identify recombination of the capsid coding sequences as a reason for double-reactivity using the Simplot analysis failed to reveal major transferred motifs. However, peptide scanning technique was able to identify antigenic regions of capsid proteins of E-11/D207 as well as regions cross-reacting with an antiserum raised to CAV-9. Thus, double specificity of E-11/D207 seems to be a real characteristic shared by the phylogenetically closely related virus strains in the genetic subgroup B of E-11.

Anti-proliferative effect of pro-inflammatory cytokines in cultured β cells is associated with extracellular signal-regulated kinase 1/2 pathway inhibition: protective role of glucagon-like peptide -1

Pancreatic beta-cell homeostasis is a balance between programmed cell death (apoptosis) and regeneration. Although autoimmune diabetes mellitus type 1 (DM1) is the most-studied cause of beta-cell mass loss by pro-inflammatory cytokine-induced apoptosis, influences of a pro-inflammatory environment on beta-cell regenerative response have been poorly studied. In this study, we assess the anti-proliferative effect of pro-inflammatory cytokines and glucose concentration on rat pancreatic beta cells and the potential protective role of glucagon-like peptide (GLP-1). Apoptotic and proliferating islet cells were stained using the DeadEnd Fluorimetric TUNEL System and 5-bromo-2'-deoxyuridine label respectively, in the presence-absence of varying concentrations of glucose, pro-inflammatory cytokines, and GLP-1. The potential signaling pathways involved were evaluated by western blot. Considerable anti-proliferative effects of pro-inflammatory cytokines interleukin (IL)-1beta, interferon (IFN)-gamma, and tumour necrosis factor-alpha (TNF-alpha) were observed. The effects were synergistic and independent of glucose concentration, and appeared to be mediated by the inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) activation, the signaling pathway involved in beta-cell replication. GLP-1 completely reversed the cytokine-induced inhibition of ERK phosphorylation and increased beta-cell proliferation threefold in cytokine-treated cultures. While pro-inflammatory cytokines reduced islet cell ERK1/2 activation and beta-cell proliferation in pancreatic islet culture, GLP-1 was capable of reversing this effect. These data suggest a possible pharmacological application of GLP-1 in the treatment of early stage DM1, to prevent the loss of pancreatic beta cells as well as to delay the development of overt diabetes.

Overexpression of Vascular Endothelial Growth Factor In Vitro Using Deferoxamine: A New Drug to Increase Islet Vascularization During Transplantation

During pancreatic islet transplantation, delayed and insufficient revascularization can deprive islets of oxygen and nutrients, resulting in cell death and early graft failure. Deferoxamine (DFO), an iron chelator, increases vascular endothelial growth factor (VEGF) expression in cells. The aim of this work was to study the effect of DFO on β-cell and pancreatic islet viability as well as VEGF expression. β-cell lines from rat insulinoma (Rin m5f) and primary cultures of pancreatic islets from Wistar rats were incubated with DFO (10, 100, and 1000 μmol/L). The viability was evaluated using fluorescein diacetate/propidium iodide for dying pancreatic islets and using cell titers for Rin m5f. Expression of VEGF messenger RNA (mRNA) was quantified using reverse transcriptase polymerase chain reaction (RT-PCR). Finally, VEGF secretion was determined using enzyme-linked immunosorbent assays at 1 to 3 days after treatment. The addition of 10 μmol/L of DFO preserved Rin m5F viability at 24 hours after treatment (10 μmol/L; 101.33% ± 5.66%; n = 7). However, 100 and 1000 μmol/L of DFO induced cell death (68.92% ± 5.83% and 65.89% ± 5.83%, respectively; n = 4). In the same way, viability of pancreatic islets in the presence of DFO was preserved. RT-PCR analysis showed stimulation of VEGF mRNA in the presence of 10 μmol/L of DFO in islets at 3 days after culture. Finally, 10 μmol/L of DFO stimulated secretion of VEGF 7.95 ± 0.84 versus 1.80 ± 1.10 pg/μg total protein with 10 μmol/L of DFO in rat islets at 3 days after culture, n = 3; P < .001). The use of DFO to stimulate VEGF expression and increase islet vascularization may be a realistic approach to improve islet viability during transplantation.

Impairment of the Ubiquitin-Proteasome Pathway Is a Downstream Endoplasmic Reticulum Stress Response Induced by Extracellular Human Islet Amyloid Polypeptide and Contributes to Pancreatic β-Cell Apoptosis

Human islet amyloid polypeptide (hIAPP) aggregation plays a major role in the development of islet amyloidosis in type 2 diabetes. It is known that extracellular hIAPP oligomers are toxic to pancreatic beta-cells and associated with apoptosis. We therefore investigated the molecular mechanism by which extracellular hIAPP mediates pancreatic beta-cell apoptosis. MIN6 cells and primary cultures of human pancreatic islets were treated with freshly dissolved hIAPP peptide. Morphology of the cultures was evaluated by electron microscopy. Gene expression was analyzed by microarray, RT-PCR, and immunoblot. Calcium levels were measured in fura-2-loaded cells. Apoptosis was quantified by cytometry. Increased expression of several heat shock proteins and activation of the spliced form of XBP-1, a transcription factor for overexpression of chaperones during endoplasmic reticulum (ER) stress, were detected together with morphological evidence of ER dysfunction. Intracellular calcium overload was detected in association with this process. Moreover, reduction in the proteasome activity, which was detected over time, contributed to the intracellular accumulation of ubiquitinated proteins, leading to a functional suppression of the ubiquitin-proteasome pathway. In addition, impairment of the proteasome function contributed to apoptosis, while, despite the presence of hIAPP, cell viability improved when a proteasome activator was overexpressed. The key cytotoxic events induced by extracellular hIAPP were also observed in treated human islets. Our data suggest that ER stress responses are intracellular signaling mechanisms induced by extracellular hIAPP aggregation and that impairment of the ubiquitin-proteasome pathway is implicated in ER stress-mediated pancreatic beta-cell apoptosis.