Structure-toxicity relationships of saturated and unsaturated free fatty acids for elucidating the lipotoxic effects in human EndoC-ßH1 beta-cells.

Lipotoxicity has been considered a major cause for beta-cell dysfunction in type 2 diabetes mellitus. However, the underlying mechanisms are still unclear. To achieve a better understanding of the toxicity a wide range of structurally different free fatty acids (FFAs) has been analyzed in human EndoC-ßH1 beta-cells. Exposure of human EndoC-ßH1 beta-cells to physiological saturated and monounsaturated long-chain FFAs induced apoptosis. Particularly noteworthy was that the toxicity increased more rapidly with increasing chain length of saturated than of unsaturated FFAs. The highest toxicity was observed in the presence of very long-chain FFAs (C20-C22), whereas polyunsaturated FFAs were not toxic. Long-chain FFAs increased peroxisomal hydrogen peroxide generation slightly, while very long-chain FFAs increased hydrogen peroxide generation more potently in both peroxisomes and mitochondria. The greater toxicity of very long-chain FFAs was accompanied by hydroxyl radical formation, along with cardiolipin peroxidation and ATP depletion. Intriguingly, only saturated very long-chain FFAs activated ER stress. On the other hand saturated very long-chain FFAs did not induce lipid droplet formation in contrast to long-chain FFAs and unsaturated very long-chain FFAs. The present data highlight the importance of structure-activity relationship analyses for the understanding of the mechanisms of lipotoxicity. Chain length and degree of saturation of FFAs are crucial factors for the toxicity of FFAs, with peroxisomal, mitochondrial, and ER stress representing the major pathogenic factors for induction of lipotoxicity. The results might provide a guide for the composition of a healthy beta-cell protective diet.

The monounsaturated fatty acid oleate is the major physiological toxic free fatty acid for human beta cells.

Free fatty acids (FFAs) can cause glucose intolerance and diabetes. Lipotoxicity to the pancreatic beta cells is considered to be a major underlying cause for this phenomenon. The aim of this study was to analyse the toxicity profile of FFAs in the human EndoC-ßH1 beta-cell line and to compare the results with isolated rat and human islets with special reference to the physiologically most prevalent FFAs palmitic acid (PA) and oleic acid (OA). Toxicity after a 2-day incubation with the different FFAs was analysed by the caspase-3 assay and confirmed by the propidium iodide and annexin V staining tests. The long-chain saturated PA (C16:0) and the monounsaturated OA (C18:1) were both toxic to human EndoC-ßH1 beta cells and pseudoislets, as well as to rat islets, and, as confirmed in a pilot experiment, also to human islets. Furthermore, OA provided no protection against the toxicity of PA. Likewise, elaidic acid (EA, the trans isomer of OA; trans-OA) was significantly toxic, in contrast to the non-metabolisable analogues methylated PA (MePA) and methylated OA (MeOA). Fatty acids with a chain length < C16 were not toxic in EndoC-ßH1 beta cells. Caspase-3 was also activated by linoleic acid (LA)(C18:2) but not by γ-linolenic acid (γ-LNA)(C18:3). Overall, only long-chain FFAs with chain lengths > C14, which generate hydrogen peroxide in the peroxisomal beta-oxidation, were toxic. This conclusion is also supported by the toxicity of the branched-chain FFA pristanic acid, which is exclusively metabolised in the peroxisomal beta-oxidation. The lack of a protective effect of the monounsaturated fatty acid OA has important consequences for a beta-cell protective lipid composition of a diet. A cardioprotective diet with a high OA content does not fulfil this requirement.

Effect of substrate rigidity in tissue culture on the function of insulin-secreting INS-1E cells.

Insulin-secreting INS-1E cells are a useful tool in diabetes research. However, during permanent culture the cells tend to lose their ß cell phenotype, with resultant loss of insulin-secretory responsiveness. This can be at least partially attributed to inappropriate cell culture conditions. One of the important causative factors is the rigidity of the extracellular matrix. We have therefore systematically studied the performance of INS-1E insulin-secreting cells cultured on polyacrylamide gels of different stiffnesses and analysed changes in insulin content and secretion, glucokinase enzyme activity, gene expression of ß cell transcription factors and cell death and proliferation rates. INS-1E cells were cultured on polyacrylamide gels with a wide range of rigidities, including the one that simulates the stiffness of the pancreas. We detected changes in insulin content and the insulin-secretory response to glucose stimulation in parallel to the increasing stiffness of the polyacrylamide gels in the range 1700-111 000 Pa. On substrates with the highest and lowest rigidities, 322 and 111 000 Pa, the cells mainly formed pseudo-islets, while at rigidities of 1700-64800 Pa, including the rigidity of native pancreas tissue (3100 Pa), cells grew as a monolayer attached to the polyacrylamide gel surface. These observations provide evidence for an apparent mechanosensitivity of insulin-secreting INS-1E cells affecting morphology and cellular functions. The results can also provide practical advice regarding a selection of the materials appropriate for successful cell culture of insulin-secreting cells. Copyright © 2014 John Wiley & Sons, Ltd.

Impact of scavenging hydrogen peroxide in the endoplasmic reticulum for ß cell function.

Oxidative folding of nascent proteins in the endoplasmic reticulum (ER), catalysed by one or more members of the protein disulfide isomerase family and the sulfhydryl oxidase ER oxidoreductin 1 (ERO1), is accompanied by generation of hydrogen peroxide (H2O2). Because of the high rate of insulin biosynthesis and the low expression of H2O2-inactivating enzymes in pancreatic ß cells, it has been proposed that the luminal H2O2 concentration might be very high. As the role of this H2O2 in ER stress and proinsulin processing is still unsolved, an ER-targeted and luminal-active catalase variant, ER-Catalase N244, was expressed in insulin-secreting INS-1E cells. In these cells, the influence of ER-specific H2O2 removal on cytokine-mediated cytotoxicity and ER stress, insulin gene expression, insulin content and secretion was analysed. The expression of ER-Catalase N244 reduced the toxicity of exogenously added H2O2 significantly with a threefold increase of the EC50 value for H2O2. However, the expression of cytokine-induced ER stress genes and viability after incubation with ß cell toxic cytokines (IL1ß alone or together with TNFα+IFNγ) was not affected by ER-Catalase N244. In control and ER-Catalase N244 expressing cells, insulin secretion and proinsulin content was identical, while removal of luminal H2O2 reduced insulin gene expression and insulin content in ER-Catalase N244 expressing cells. These data show that ER-Catalase N244 reduced H2O2 toxicity but did not provide protection against pro-inflammatory cytokine-mediated toxicity and ER stress. Insulin secretion was not affected by decreasing H2O2 in the ER in spite of a reduced insulin transcription and processing.

N-glycosylation-negative catalase: a useful tool for exploring the role of hydrogen peroxide in the endoplasmic reticulum.

Disulfide bond formation during protein folding of nascent proteins is associated with the generation of H2O2 in the endoplasmic reticulum (ER). Approaches to quantifying H2O2 directly within the ER failed because of the oxidative environment in the ER lumen, and ER-specific catalase expression to detoxify high H2O2 concentrations resulted in an inactive protein owing to N-glycosylation. Therefore, the N-glycosylation motifs at asparagine-244 and -439 of the human catalase protein were deleted by site-directed mutagenesis. The ER-targeted expression of these variants revealed that the deletion of the N-glycosylation motif only at asparagine-244 (N244) was associated with the maintenance of full enzymatic activity in the ER. Expression of catalase N244 in the ER (ER-Catalase N244) was ER-specific and protected the cells significantly against exogenously added H2O2. With the expression of ER-Catalase N244, a highly effective H2O2 inactivation within the ER was achieved for the first time. Catalase has a high H2O2-inactivation capacity without the need of reducing cofactors, which might interfere with the ER redox homeostasis, and is not involved in protein folding. With these characteristics ER-Catalase N244 is an ideal tool to explore the impact of ER-generated H2O2 on the generation of disulfide bonds or to study the induction of ER-stress pathways through protein folding overload and accumulation of H2O2.

Influence of cytokines on Dmt1 iron transporter and ferritin expression in insulin-secreting cells.

Free intracellular ferrous iron (Fe(2+)) is essential for the generation of the extremely toxic hydroxyl radicals, which contribute to ß-cell destruction by cytokines. Therefore the expression of the different divalent metal transporter 1 (Dmt1) isoforms and ferritin (Ft) subunits, responsible for iron import and chelation, was analyzed under pro-inflammatory conditions (IL1ß alone or together with TNFα+IFNγ). The Dmt1 isoforms (1A/1B and +IRE/-IRE) and the total Dmt1 expression in insulin-producing cells (RINm5F and INS-1E), in primary rat islets and, for comparison, in the neuroendocrine PC12 cell line were quantified by qRT-PCR. In addition, the expression of the light (L-Ft) and heavy Ft (H-Ft) subunits and the mitochondrial Ft isoform (Mtft) in insulin-producing cells under control conditions and after cytokine treatment was estimated. The 1B isoform was the predominant Dmt1 mRNA in all insulin-producing cells, accounting for almost 100% of the 1A/1B isoform expression. For the IRE variants, +IRE expression was higher than -IRE expression. Pro-inflammatory cytokines accelerated the expression of Dmt1 isoforms significantly with an overall 2.5- to 3-fold increase in the total Dmt1 expression. In contrast, the expression of the iron-buffering ferritin subunits L- and H-Ft was unaffected by IL1ß and only slightly induced by the cytokine mixture. Mtft expression was also not increased. Dmt1 expression was significantly elevated through pro-inflammatory cytokines, whereas Ft expression was marginally increased. This imbalance between the increased iron transport capacity and the almost unaffected iron storage capacity can foster cytokine-mediated formation of hydroxyl radicals and thus pro-inflammatory cytokine toxicity through elevated free iron concentrations.

Variable immune cell frequencies in peripheral blood of LEW.1AR1-iddm rats over time compared to other congenic LEW strains.

The LEW.1AR1-iddm rat is an animal model of human type 1 diabetes (T1D), which arose through a spontaneous mutation within the major histocompatibility complex (MHC)-congenic background strain LEW.1AR1. The LEW.1AR1-iddm rat is characterized by two phenotypes: diabetes development with a diabetes incidence of 60% and a variable T cell frequency in peripheral blood. In this study the immune cell repertoire of LEW.1AR1-iddm rats was analysed over time from days 30 to 90 of life and compared to the background strain LEW.1AR1 and the LEW rat strain as well as the LEW.1WR1 rat strain. The LEW.1AR1-iddm rats are characterized by a high variability of CD3(+), CD4(+) and CD8(+) T cell frequencies in peripheral blood over time, and the frequency is unique for each animal. The variability within the frequencies resulted in changes of the CD4(+) : CD8(+) T cell ratio. The other three rat strains studied were characterized by a stable but nevertheless strain-specific T cell frequency resulting in a specific CD4(+) : CD8(+) T cell ratio. The frequency of natural killer (NK) cells and B cells in LEW.1AR1-iddm rats was increased, with a higher variability compared to the other strains. Only monocytes showed no differences in frequency and variability between all strains studied. These variabilities of immune cell frequencies in the LEW.1AR1-iddm rats might lead to imbalances between autoreactive and regulatory T cells in peripheral blood as a prerequisite for diabetes development.

Progesterone induces apoptosis of insulin-secreting cells: insights into the molecular mechanism.

Progesterone has been associated with the development of gestational diabetes (GD) due to the enhancement of insulin resistance. As ß-cell apoptosis participates in type 1 and type 2 diabetes pathophysiology, we proposed the hypothesis that progesterone might contribute to the development of GD through a mechanism that also involves ß-cell death. To address this question, RINm5F insulin-producing cells were incubated with progesterone (25-100 µM), in the presence or absence of α-tocopherol (40 µM). After 24 or 48 h, membrane integrity and DNA fragmentation were analyzed by flow cytometry. Caspase activity was used to identify the mode of cell death. The involvement of endoplasmic reticulum stress in the action of progesterone was investigated by western blotting. Oxidative stress was measured by 2',7'-dichlorofluorescein diacetate (DCFDA) oxidation. Isolated rat islets were used in similar experiments in order to confirm the effect of progesterone in primary ß-cells. Incubation of RINm5F cells with progesterone increased the number of cells with loss of membrane integrity and DNA fragmentation. Progesterone induced generation of reactive species. Pre-incubation with α-tocopherol attenuated progesterone-induced apoptosis. Western blot analyses revealed increased expression of CREB2 and CHOP in progesterone-treated cells. Progesterone caused apoptotic death of rat islet cells and enhanced generation of reactive species. Our results show that progesterone can be toxic to pancreatic ß-cells through an oxidative-stress-dependent mechanism that induces apoptosis. This effect may contribute to the development of GD during pregnancy, particularly under conditions that require administration of pharmacological doses of this hormone.

Overexpression of the antioxidant enzyme catalase does not interfere with the glucose responsiveness of insulin-secreting INS-1E cells and rat islets.

AIMS/HYPOTHESIS: Hydrogen peroxide (H2O2)-inactivating enzymes such as catalase are produced in extraordinarily low levels in beta cells. Whether this low expression might be related to a signalling function of H2O2 within the beta cell is unknown. A high level of H2O2-inactivating enzymes could potentially be incompatible with glucose-induced insulin secretion. Therefore the effect of catalase overexpression on mitochondrial function and physiological insulin secretion was studied in insulin-secreting INS-1E and primary islet cells. METHODS: INS-1E and rat islet cells were lentivirally transduced to overexpress catalase in the cytosol (CytoCat) or in mitochondria (MitoCat). Cell viability and caspase-3 activation were assessed after cytokine incubation and hypoxia. Insulin secretion was quantified and expression of the gene encoding the mitochondrial uncoupling protein 2 (Ucp2) was measured in parallel to mitochondrial membrane potential and reactive oxygen species (ROS) formation. RESULTS: The ability to secret insulin in a glucose-dependent manner was not suppressed by catalase overexpression, although the glucose-dependent increase in the mitochondrial membrane potential was attenuated in MitoCat cells along with an increased Ucp2 expression and reduced mitochondrial ROS formation. In addition, MitoCat overexpressing cells were significantly more resistant against pro-inflammatory cytokines and hypoxia than CytoCat and control cells. CONCLUSIONS/INTERPRETATION: The results demonstrate that an improved antioxidative defence status of insulin-secreting cells allowing efficient H2O2 inactivation is not incompatible with proper insulin secretory responsiveness to glucose stimulation and provide no support for a signalling role of H2O2 in insulin-secreting cells. Interestingly, the results also document for the first time that the decreased ROS formation with increasing glucose concentrations is of mitochondrial origin.

Enhancement of homocysteine toxicity to insulin-secreting BRIN-BD11 cells in combination with alloxan.

Previous studies have shown that homocysteine (HC) has a detrimental impact on insulin secretion and pancreatic beta cell function. The aim of the present study was to determine the role of reactive oxygen species (ROS) in the in vitro toxic effects of HC on insulin secretion and function of BRIN-BD11 insulin-secreting cells. In this study, insulin secretion from BRIN-BD11 cells was determined radioimmunologically, cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and glucokinase activity by a glucose phosphorylation assay following culture with HC plus alloxan (Alx). Treatment with HC resulted in concentration-dependent inhibition of insulin secretion induced by glucose and other insulinotropic agents. HC in combination with Alx resulted in a more pronounced decline in insulin secretion, including that induced by 20  mM alanine, by 43% (P<0.001) and 30  mM KCl by 60% (P<0.001), compared with control culture. The glucokinase phosphorylating capacity in cells cultured with HC plus Alx was significantly lower, compared with control cells. The cells also displayed a significant 84% (P<0.001) decline in cell viability. Prolonged, 72-h culture of insulin-secreting cells with HC followed by 18-h culture without HC did not result in full restoration of beta cell responses to insulinotropic agents. In vitro oxygen consumption was enhanced by a combination of Alx with HC. The study arrived at the conclusion that HC generates ROS in a redox-cycling reaction with Alx that explains the decline in viability of insulin-secreting cells, leading to reduced glucokinase phosphorylating ability, diminished insulin secretory responsiveness and cell death.

[Pluripotent stem cells for cell replacement therapy of diabetes]. / Pluripotente Stammzellen zur Zellersatztherapie des Diabetes mellitus.

The use of pluripotent stem cells (PSCs) harbours great potential for a future use in the cell replacement therapy of diabetes mellitus. The in vitro differentiation of human or mouse embryonic stem cells has yielded pancreatic progenitor cells, but not authentic insulin-producing beta cells. Induced pluripotent cells are a class of a pluripotent stem cells potentially suited as a cell source for cell replacement therapy. These patient specific pluripotent cells are generated by reprogramming but unfortunately accumulate genetic and epigenetic errors during reprogramming, precluding their use for therapeutic purposes in humans.

Glucokinase mediates coupling of glycolysis to mitochondrial metabolism but not to beta cell damage at high glucose exposure levels.

AIMS/HYPOTHESIS: Glucose is the main stimulus of insulin secretion in pancreatic beta cells. However, high glucose has also been considered to damage beta cells. In this study we examined, with special emphasis on the role of the glucose sensor enzyme glucokinase, whether elevated glucose metabolism evokes toxicity to beta cells. METHODS: RINm5F-R-EYFP-GK cells, producing glucokinase in response to a synthetic inducer, and rat beta cells were incubated at different glucose concentrations. Glucokinase enzyme activity, insulin secretion, cell viability and mitochondrial metabolism were analysed. RESULTS: Glucokinase production evoked a concentration-dependent increase in glucose-induced insulin secretion from RINm5F-R-EYFP-GK cells without reducing cell viability. Pre-culture at high glucose (30 mmol/l) in the absence of high concentrations of NEFA neither reduced viability nor significantly increased apoptosis in RINm5F-R-EYFP-GK cells and rat beta cells. The integrity of the mitochondrial respiratory chain and mitochondrial dynamics, namely fusion and fission, were not impaired by high glucose pre-culture. As previously demonstrated in mouse beta cells, pre-culture at high glucose significantly decreased the mitochondrial membrane potential heterogeneity in RINm5F-R-EYFP-GK cells. Indeed, after starvation, in response to glucose, rat beta cells and RINm5F-R-EYFP-GK cells with glucokinase production pre-cultured for 48 h at high glucose showed the fastest increase in the mitochondrial membrane potential. CONCLUSIONS/INTERPRETATION: Our experiments do not support the hypothesis that glucokinase and the glucose metabolism on its own act as a mediator of beta cell toxicity. By contrast, rather a beneficial effect on glucose-induced insulin secretion after glucokinase production was observed, based on an improved coupling of the glucose stimulus to the mitochondrial metabolism.

Role of metabolically generated reactive oxygen species for lipotoxicity in pancreatic ß-cells.

Chronically elevated concentrations of non-esterified fatty acids (NEFAs) in type 2 diabetes may be involved in ß-cell dysfunction and apoptosis. It has been shown that long-chain saturated NEFAs exhibit a strong cytotoxic effect upon insulin-producing cells, while short-chain as well as unsaturated NEFAs are well tolerated. Moreover, long-chain unsaturated NEFAs counteract the toxicity of palmitic acid. Reactive oxygen species (ROS) formation and gene expression analyses together with viability assays in different ß-cell lines showed that the G-protein-coupled receptors 40 and 120 do not mediate lipotoxicity. This is independent from the role, which these receptors, specifically GPR40, play in the potentiation of glucose-induced insulin secretion by saturated and unsaturated long-chain NEFAs. Long-chain NEFAs are not only metabolized in the mitochondria but also in peroxisomes. In contrast to mitochondrial ß-oxidation, the acyl-coenzyme A (CoA) oxidases in the peroxisomes form hydrogen peroxide and not reducing equivalents. As ß-cells almost completely lack catalase, they are exceptionally vulnerable to hydrogen peroxide generated in peroxisomes. ROS generation in the respiratory chain is less important because overexpression of catalase and superoxide dismutase in the mitochondria do not provide protection. Thus, peroxisomally generated hydrogen peroxide is the likely ROS that causes pancreatic ß-cell dysfunction and ultimately ß-cell death.

Sustained production of spliced X-box binding protein 1 (XBP1) induces pancreatic beta cell dysfunction and apoptosis.

AIMS/HYPOTHESIS: Pro-inflammatory cytokines involved in the pathogenesis of type 1 diabetes deplete endoplasmic reticulum (ER) Ca2+ stores, leading to ER-stress and beta cell apoptosis. However, the cytokine-induced ER-stress response in beta cells is atypical and characterised by induction of the pro-apoptotic PKR-like ER kinase (PERK)-C/EBP homologous protein (CHOP) branch of the unfolded protein response, but defective X-box binding protein 1 (XBP1) splicing and activating transcription factor 6 activation. The purpose of this study was to overexpress spliced/active Xbp1 (XBP1s) to increase beta cell resistance to cytokine-induced ER-stress and apoptosis. METHODS: Xbp1s was overexpressed using adenoviruses and knocked down using small interference RNA in rat islet cells. In selected experiments, Xbp1 was also knocked down in FACS-purified rat beta cells and rat fibroblasts. Expression and production of XBP1s and key downstream genes and proteins was measured and beta cell function and viability were evaluated. RESULTS: Adenoviral-mediated overproduction of Xbp1s resulted in increased XBP1 activity and induction of several XBP1s target genes. Surprisingly, XBP1s overexpression impaired glucose-stimulated insulin secretion and increased beta cell apoptosis, whereas it protected fibroblasts against cell death induced by ER-stress. mRNA expression of Pdx1 and Mafa was inhibited in cells overproducing XBP1s, leading to decreased insulin expression. XBP1s knockdown partially restored cytokine/ER-stress-driven insulin and Pdx1 inhibition but had no effect on cytokine-induced ER-stress and apoptosis. CONCLUSIONS/INTERPRETATION: XBP1 has a distinct inhibitory role in beta cell as compared with other cell types. Prolonged XBP1s production hampers beta cell function via inhibition of insulin, Pdx1 and Mafa expression, eventually leading to beta cell apoptosis.

Prevention of spontaneous immune-mediated diabetes development in the LEW.1AR1-iddm rat by selective CD8+ T cell transfer is associated with a cytokine shift in the pancreas-draining lymph nodes.

AIMS/HYPOTHESIS: The LEW.1AR1-iddm rat is an animal model of spontaneous type 1 diabetes mellitus. This study analysed how adoptive transfer of selective T cell subpopulations affects the incidence of diabetes. METHODS: CD4(+) or CD8(+) T cells were isolated from diabetic LEW.1AR1-iddm rats or diabetes-resistant LEW.1AR1 rats. Cells were selectively transferred into athymic LEW.1AR1-Whn ( rnu ) or prediabetic LEW.1AR1-iddm rats. The animals were monitored for blood glucose, islet infiltration and immune cell composition of pancreas-draining lymph nodes. RESULTS: After adoptive transfer of CD4(+) T cells from diabetic LEW.1AR1-iddm rats into athymic LEW.1AR1-Whn ( rnu ) rats, 50% of the recipients developed diabetes. Transfer of CD8(+) T cells failed to induce diabetes. Only 10% of the athymic recipients became diabetic after co-transfer of CD4(+) and CD8(+) T cells. Adoptive transfer of CD8(+) T cells from LEW.1AR1 or diabetic LEW.1AR1-iddm rats into prediabetic LEW.1AR1-iddm rats significantly reduced the incidence of diabetes. In protected normoglycaemic animals regulatory CD8(+)/CD25(+) and CD4(+)/CD25(+) T cell subpopulations that were also FOXP3-positive accumulated in the pancreas-draining lymph nodes. In this lymphatic organ, gene expression of anti-inflammatory cytokines was significantly higher than in diabetic rats. CONCLUSIONS/INTERPRETATION: Our results show that adoptive transfer of CD4(+) but not CD8(+) T cells from diabetic LEW.1AR1-iddm rats induced diabetes development. Importantly, CD8(+) T cells from diabetic LEW.1AR1-iddm rats and diabetes-resistant LEW.1AR1 rats provided protection against beta cell destruction. The accumulation of regulatory T cells in the pancreas-draining lymph nodes from protected rats indicates that transferred CD8(+) T cells may have beneficial effects in the control of beta cell autoimmunity.

Kinetics of insulin secretion from MIN6 pseudoislets after encapsulation in a prototype device of a bioartificial pancreas.

Xenotransplantation of insulin-secreting cells from nonhuman sources is an alternative therapeutic approach to bypass the shortage of human pancreatic islet tissue for transplantation in order to treat insulin deficiency in type 1 diabetes mellitus. Therefore, we studied the suitability of pseudoislets generated from insulin-secreting MIN6 tissue culture cells to serve as a surrogate for replacement of pancreatic islets after encapsulation in a minicell, representing a prototype of a new bioartificial pancreas device. MIN6 pseudoislets showed an excellent insulin secretory responsiveness with a typical biphasic secretory pattern to glucose stimulation. When encapsulated in the minicell, insulin release from the pseudoislets in response to glucose stimulation was reduced. The initial first phase insulin secretory response was greatly attenuated. In contrast, the first phase insulin secretory response of the encapsulated pseudoislets was restored on stimulation with the sulfonylurea drug tolbutamide. Our results indicate that the reason for the attenuated first phase of release is the restricted permeability of the pores in the separating membrane in the minicell for the hydrophilic glucose molecule rather than a limited permeability for the secretion product insulin. The reduced release of insulin from the encapsulated pseudoislets could be compensated by overexpression of glucokinase in MIN6 cells, which resulted in an increased glucose responsiveness of the pseudoislets for stimulation with glucose. Thus, this minicell is a well-suited miniature test system for the evaluation of the feasibility of encapsulation of insulin-secreting cells and allows the testing of permeability properties of separating membranes in bioartificial pancreas devices.

An efficient experimental strategy for mouse embryonic stem cell differentiation and separation of a cytokeratin-19-positive population of insulin-producing cells.

OBJECTIVES: Embryonic stem cells are a potential source for insulin-producing cells, but existing differentiation protocols are of limited efficiency. Here, the aim has been to develop a new one, which drives development of embryonic stem cells towards insulin-producing cells rather than to neuronal cell types, and to combine this with a strategy for their separation from insulin-negative cells. MATERIALS AND METHODS: The cytokeratin-19 (CK19) promoter was used to control the expression of enhanced yellow fluorescence protein in mouse embryonic stem cells during their differentiation towards insulin-producing cells, using a new optimized four-stage protocol. Two cell populations, CK19(+) and CK19(-) cells, were successfully fluorescence sorted and analysed. RESULTS: The new method reduced neuronal progeny and suppressed differentiation into glucagon- and somatostatin-producing cells. Concomitantly, beta-cell like characteristics of insulin-producing cells were strengthened, as documented by high gene expression of the Glut2 glucose transporter and the transcription factor Pdx1. This novel protocol was combined with a cell-sorting technique. Through the combined procedure, a fraction of glucose-responsive insulin-secreting CK19(+) cells was obtained with 40-fold higher insulin gene expression and 50-fold higher insulin content than CK19(-) cells. CK19(+) cells were immunoreactive for C-peptide and had ultrastructural characteristics of an insulin-secretory cell. CONCLUSION: Differentiated CK19(+) cells reflect an endocrine precursor cell type of ductal origin, potentially suitable for insulin replacement therapy in diabetes.

Monitoring of glucose-regulated single insulin secretory granule movement by selective photoactivation.

AIMS/HYPOTHESIS: Fluorescence microscopy opens new perspectives for the analysis of insulin secretory granule movement. In this study, we examined whether recently developed photoactivatable/photoconvertible proteins are a useful tool for studying this process at the single granule level in insulin-secreting cells after glucose stimulation. METHODS: Plasmids were generated for expression of fusion proteins of the granule membrane phosphatase phogrin or the granule cargo protein neuropeptide Y (NPY) with the photoactivatable green fluorescent protein mutant A206K (PA-GFP-A206K), the photoconvertible protein Dendra2 and the fluorescent protein mCherry. Transfected insulin-secreting MIN6 cells were analysed by fluorescence microscopy. RESULTS: Point-resolved 405 nm light exposure during image acquisition of MIN6 cells transiently transfected with Phogrin-PA-GFP-A206K or NPY-PA-GFP-A206K as well as of stable MIN6-Phogrin-Dendra2 cells resulted in selective visualisation of few granules by green or red fluorescence, respectively. Movement of these granules was analysed by an automated tracking method from confocal 3D image series. The high spatiotemporal resolution facilitated an elongated tracking of single granules. Interestingly, the track speed and track displacement of granules after 1 h starvation and subsequent glucose stimulation was lower in cells pre-cultured for 48 h at 3 mmol/l glucose than in cells pre-cultured at 25 mmol/l glucose. CONCLUSIONS/INTERPRETATION: Targeting of the granule membrane or its cargo with a photoactivatable/photoconvertible protein allows in-depth visualisation and tracking of single insulin granules in dependence upon glucose. This technique may also open the way to elucidating the regulation of granule movement velocity within the pancreatic beta cell with respect to secretory defects in type 2 diabetes.