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.

Acute and long-term effects of metformin on the function and insulin secretory responsiveness of clonal β-cells

Functional effects of acute and prolonged (48 h) exposure to the biguanide drug metformin were examined in the clonal pancreatic ß-cell line, BRIN-BD11. Effects of metformin on prolonged exposure to excessive increased concentrations of glucose and palmitic acid were also assessed. In acute 20-min incubations, 12.5-50 µm metformin did not alter basal (1.1 mm glucose) or glucose-stimulated (16.7 mm glucose) insulin secretion. However, higher concentrations of metformin (100-1000 µm) increased (1.3-1.5-fold; p

Interactions among secretory immunoglobulin A, CD71, and transglutaminase-2 affect permeability of intestinal epithelial cells to gliadin peptides

BACKGROUND & AIMS: The transferrin receptor (CD71) is up-regulated in duodenal biopsy samples from patients with active celiac disease and promotes retrotransport of secretory immunolglobulin A (SIgA)-gliadin complexes. We studied intestinal epithelial cell lines that overexpress CD71 to determine how interactions between SIgA and CD71 promote transepithelial transport of gliadin peptides. METHODS: We analyzed duodenal biopsy specimens from 8 adults and 1 child with active celiac disease. Caco-2 and HT29-19A epithelial cell lines were transfected with fluorescence-labeled small interfering RNAs against CD71. Interactions among IgA, CD71, and transglutaminase 2 (Tgase2) were analyzed by flow cytometry, immunoprecipitation, and confocal microscopy. Transcytosis of SIgACD71 complexes and intestinal permeability to the gliadin 3H-p3149 peptide were analyzed in polarized monolayers of Caco-2 cells. RESULTS: Using fluorescence resonance energy transfer and in situ proximity ligation assays, we observed physical interactions between SIgA and CD71 or CD71 and Tgase2 at the apical surface of enterocytes in biopsy samples and monolayers of Caco-2 cells. CD71 and Tgase2 were co-precipitated with SIgA, bound to the surface of Caco-2 cells. SIgACD71 complexes were internalized and localized in early endosomes and recycling compartments but not in lysosomes. In the presence of celiac IgA or SIgA against p3149, transport of intact 3H-p3149 increased significantly across Caco-2 monolayers; this transport was inhibited by soluble CD71 or Tgase2 inhibitors. CONCLUSIONS: Upon binding to apical CD71, SIgA (with or without gliadin peptides) enters a recycling pathway and avoids lysosomal degradation; this process allows apicalbasal transcytosis of bound peptides. This mechanism is facilitated by Tgase2 and might be involved in the pathogenesis of celiac disease.

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.

Acute and long-term effects of peroxisome proliferator-activated receptor-γ activation on the function and insulin secretory responsiveness of clonal beta-cells

Thiazolidinediones (TZDs) are used as antidiabetic therapy. The purpose of the present study was to examine whether the TZD rosiglitazone has direct actions on pancreatic beta-cells that contribute to its overall effects. Effects of acute and prolonged (48 h) exposure to rosiglitazone, as a model glitazone compound, were assessed in clonal pancreatic BRIN-BD11 beta-cells maintained in standard, glucotoxic and lipotoxic cultures. In acute 20-min incubations, rosiglitazone (0.2-100 M) did not alter basal or glucose-stimulated insulin secretion. However, rosiglitazone (6.25 M) enhanced (p

Regulation of the expression of secretory component by human airway epithelial cells in vitro

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Analysis of secretory, immunostaining and clinical characteristics of human "functionless" pituitary adenomas:transdifferentiation or gonadotropinomas?

In this study, the central technique of in vitro culture has been used to further investigate whether LH/FSH-expressing, but clinically "functionless" pituitary adenomas are gonadotropinomas or whether their hormone secretion is due to transdifferentiation events. 664 "functionless" pituitary adenomas were examined for hormone secretion by in vitro culture and for hormone content by immunostaining. The results were correlated with the clinical findings. 40% of the tumours (n = 263) secreted at least one of the gonadotropins alone, 8% (n = 53) exhibited various patterns of anterior pituitary hormones, whilst the remaining 52% of tumours were not associated with any hormone. In the secretory tumours, immunostaining revealed only a few scattered hormone-containing cells (5 to 15%). Mild hyperprolactinaemia was observed in some cases, presumably because of pressure effects of the tumours. The majority of the patients suffered clear cut hypopituitarism (p < 0.05). Pre-operatively, gonadotropin hypersecretion was observed in 3 cases, but only one of these secreted hormones in culture. Interestingly, a higher proportion of tumours removed from patients with hypopituitarism showed secretory activity in vitro than those tumours removed from patients showing no hormonal dysfunction or hyperprolactinaemia. We conclude that the term "gonadotropinoma" to describe functionless pituitary tumours associated with LH and/or FSH secretion is a misnomer, because the presence of LH and/or FSH confirmed by in vitro methods in the present series is a result of only a few scattered cells. We suggest that primary pituitary tumour cells differentiate into a secretory type (transdifferentiation), possibly in response to altered serum hormone levels such as decreased steroids. Further work is required to identify the factors which trigger the altered cells' characteristics. © J. A. Barth Verlag in Georg Thieme Verlag KG.

Free secretory component from cystic fibrosis sputa displays the cystic fibrosis glycosylation phenotype

Secretory IgA contributes to humoral defense mechanisms against pathogens targeting mucosal surfaces, and secretory component (SC) fulfills multiple roles in this defense. The aims of this study were to quantify total SC and to analyze the form of free SC in sputa from normal subjects, subjects with asthma, and subjects with cystic fibrosis (CF). Significantly higher levels of SC were detected in CF compared with both other groups. Gel filtration chromatography revealed that SC in CF was relatively degraded. Free SC normally binds interleukin (IL)-8 and inhibits its function. However, in CF sputa, IL-8 binding to intact SC was reduced. Analysis of the total carbohydrate content of free SC signified overglycosylation in CF compared with normal subjects and subjects with asthma. Monosaccharide composition analysis of free SC from CF subjects revealed overfucosylation and undersialylation, in agreement with the reported CF glycosylation phenotype. SC binding to IL-8 did not interfere with the binding of IL-8 to heparin, indicating distinct binding sites on IL-8 for negative regulation of function by SC and heparin. We suggest that defective structure and function of SC contribute to the characteristic sustained inflammatory response in the CF airways.

IL-8 released constitutively by primary bronchial epithelial cells in culture forms an inactive complex with secretory component

The bronchial epithelium is a source of both α and β chemokines and, uniquely, of secretory component (SC), the extracellular ligand-binding domain of the polymeric IgA receptor. Ig superfamily relatives of SC, such as IgG and α2-macroglobulin, bind IL-8. Therefore, we tested the hypothesis that SC binds IL-8, modifying its activity as a neutrophil chemoattractant. Primary bronchial epithelial cells were cultured under conditions to optimize SC synthesis. The chemokines IL-8, epithelial neutrophil-activating peptide-78, growth-related oncogene α, and RANTES were released constitutively by epithelial cells from both normal and asthmatic donors and detected in high m.w. complexes with SC. There were no qualitative differences in the production of SC-chemokine complexes by epithelial cells from normal or asthmatic donors, and in all cases this was the only form of chemokine detected. SC contains 15% N-linked carbohydrate, and complete deglycosylation with peptide N-glycosidase F abolished IL-8 binding. In micro-Boyden chamber assays, no IL-8-dependent neutrophil chemotactic responses to epithelial culture supernatants could be demonstrated. SC dose-dependently (IC50 ∼0.3 nM) inhibited the neutrophil chemotactic response to rIL-8 (10 nM) in micro-Boyden chamber assays and also inhibited IL-8-mediated neutrophil transendothelial migration. SC inhibited the binding of IL-8 to nonspecific binding sites on polycarbonate filters and endothelial cell monolayers, and therefore the formation of haptotactic gradients, without effects on IL-8 binding to specific receptors on neutrophils. The data indicate that in the airways IL-8 may be solubilized and inactivated by binding to SC

The effect of the senescence-associated secretory phenotype (SASP) on glucose homeostasis in metabolic cells

Aim: Dysregulated glucose homeostasis is a hallmark of Type 2diabetes. A distinctive feature of ageing is the accumulation ofsenescent cells, defined as cells that have undergone irreversible lossof proliferative capacity. Characteristic of senescent cells is thesenescence-associated secretory phenotype (SASP) involving theproduction of factors which reinforce senescence arrest in neigh-bouring tissue environments. We hypothesise that SASP inducesmetabolic dysfunction in non-senescent cells, impairing glucosemetabolism and propagating insulin resistance. We sought todetermine the effect of SASP on glucose homeostasis in hepatic,adipose and skeletal muscle cell lines. Methods: Human dermal fibroblasts were subjected to a geno-toxic dose of doxorubicin to induce senescence, confirmed using ab-galactosidase assay. Conditioned media containing SASP werecollected post 24h and 48h of inducing senescence and used at20% and 40% concentrations to treat AML-12 hepatocytes, 3T3-L1 adipocytes and C2C12 myocytes for 24h and 48h. Cells andmedia were collected and glucose and lipid concentrations weremeasured before and after the respective incubation periods. Results: Cell media obtained from C2C12 myocytes exposed to40% SASP for 24h and 48h and AML-12 hepatocytes after 48hexhibited significantly higher concentrations of glucose in com-parison to control media (p < 0.0001, p < 0.05) suggesting areduced glucose uptake. Glucose utilisation remained unchanged in3T3-L1 cells. Conclusion: Our data suggest an important role for SASP inaltering glucose homeostasis and identify SASP as a potentialmediator between ageing and the increase in age-related insulinresistance.