The TWEAK/Fn14/CD163 axis-implications for metabolic disease.

TWEAK (tumor necrosis factor-like weak inducer of apoptosis) is a member of the TNF superfamily that controls a multitude of cellular events including proliferation, migration, differentiation, apoptosis, angiogenesis, and inflammation. TWEAK control of these events is via an expanding list of intracellular signalling pathways which include NF-κB, ERK/MAPK, Notch, EGFR and AP-1. Two receptors have been identified for TWEAK - Fn14, which targets the membrane bound form of TWEAK, and CD163, which scavenges the soluble form of TWEAK. TWEAK appears to elicit specific events based on the receptor to which it binds, tissue type in which it is expressed, specific extrinsic conditions, and the presence of other cytokines. TWEAK signalling is protective in healthy tissues, but in chronic inflammatory states become detrimental to the tissue. Consistent data show a role for the TWEAK/FN14/CD163 axis in metabolic disease, chronic autoimmune diseases, and acute ischaemic stroke. Low circulating concentrations of soluble TWEAK are predictive of poor cardiovascular outcomes in those with and without diabetes. This review details the current understanding of the TWEAK/Fn14/CD163 axis as one of the chief regulators of immune signalling and its cell-specific role in metabolic disease development and progression.

Connectivity mapping (ssCMap) to predict A20-inducing drugs and their antiinflammatory action in cystic fibrosis.

Cystic fibrosis (CF) lung disease is characterized by chronic and exaggerated inflammation in the airways. Despite recent developments to therapeutically overcome the underlying functional defect in the cystic fibrosis transmembrane conductance regulator, there is still an unmet need to also normalize the inflammatory response. The prolonged and heightened inflammatory response in CF is, in part, mediated by a lack of intrinsic down-regulation of the proinflammatory NF-κB pathway. We have previously identified reduced expression of the NF-κB down-regulator A20 in CF as a key target to normalize the inflammatory response. Here, we have used publicly available gene array expression data together with a statistically significant connections' map (sscMap) to successfully predict drugs already licensed for the use in humans to induce A20 mRNA and protein expression and thereby reduce inflammation. The effect of the predicted drugs on A20 and NF-κB(p65) expression (mRNA) as well as proinflammatory cytokine release (IL-8) in the presence and absence of bacterial LPS was shown in bronchial epithelial cells lines (16HBE14o-, CFBE41o-) and in primary nasal epithelial cells from patients with CF (Phe508del homozygous) and non-CF controls. Additionally, the specificity of the drug action on A20 was confirmed using cell lines with tnfαip3 (A20) knockdown (siRNA). We also show that the A20-inducing effect of ikarugamycin and quercetin is lower in CF-derived airway epithelial cells than in non-CF cells.

Correction to: Islet-intrinsic effects of CFTR mutation.

Unfortunately, due to a tagging error, Dr Fiona N. Manderson Koivula's name is shown incorrectly as Koivula FN on PubMed. Her name appears correctly in the html and pdf versions of the paper.

Islet-intrinsic effects of CFTR mutation.

Cystic fibrosis-related diabetes (CFRD) is the most significant extra-pulmonary comorbidity in cystic fibrosis (CF) patients, and accelerates lung decline. In addition to the traditional view that CFRD is a consequence of fibrotic destruction of the pancreas as a whole, emerging evidence may implicate a role for cystic fibrosis transmembrane-conductance regulator (CFTR) in the regulation of insulin secretion from the pancreatic islet. Impaired first-phase insulin responses and glucose homeostasis have also been reported in CF patients. CFTR expression in both human and mouse beta cells has been confirmed, and recent studies have shown differences in endocrine pancreatic morphology from birth in CF. Recent experimental evidence suggests that functional CFTR channels are required for insulin exocytosis and the regulation of membrane potential in the pancreatic beta cell, which may account for the impairments in insulin secretion observed in many CF patients. These novel insights suggest that the pathogenesis of CFRD is more complicated than originally thought, with implications for diabetes treatment and screening in the CF population. This review summarises recent emerging evidence in support of a primary role for endocrine pancreatic dysfunction in the development of CFRD. Summary • CF is an autosomal recessive disorder caused by mutations in the CFTR gene • The vast majority of morbidity and mortality in CF results from lung disease. However CFRD is the largest extra-pulmonary co-morbidity and rapidly accelerates lung decline • Recent experimental evidence shows that functional CFTR channels are required for normal patterns of first phase insulin secretion from the pancreatic beta cell • Current clinical recommendations suggest that insulin is more effective than oral glucose-lowering drugs for the treatment of CFRD. However, the emergence of CFTR corrector and potentiator drugs may offer a personalised approach to treating diabetes in the CF population.

Toll-like receptor 4 is not targeted to the lysosome in cystic fibrosis airway epithelial cells.

The innate immune response to bacterial infection is mediated through Toll-like receptors (TLRs), which trigger tightly regulated signaling cascades through transcription factors including NF-κB. LPS activation of TLR4 triggers internalization of the receptor-ligand complex which is directed toward lysosomal degradation or endocytic recycling. Cystic fibrosis (CF) patients display a robust and uncontrolled inflammatory response to bacterial infection, suggesting a defect in regulation. This study examined the intracellular trafficking of TLR4 in CF and non-CF airway epithelial cells following stimulation with LPS. We employed cells lines [16hBE14o-, CFBE41o- (CF), and CFTR-complemented CFBE41o-] and confirmed selected experiments in primary nasal epithelial cells from non-CF controls and CF patients (F508del homozygous). In control cells, TLR4 expression (surface and cytoplasmic) was reduced after LPS stimulation but remained unchanged in CF cells and was accompanied by a heightened inflammatory response 24 h after stimulation. All cells expressed markers of the early (EEA1) and late (Rab7b) endosomes at basal levels. However, only CF cells displayed persistent expression of Rab7b following LPS stimulation. Rab7 variants may directly internalize bacteria to the Golgi for recycling or to the lysosome for degradation. TLR4 colocalized with the lysosomal marker LAMP1 in 16 hBE14o- cells, suggesting that TLR4 is targeted for lysosomal degradation in these cells. However, this colocalization was not observed in CFBE41o- cells, where persistent expression of Rab7 and release of proinflammatory cytokines was detected. Consistent with the apparent inability of CF cells to target TLR4 toward the lysosome for degradation, we observed persistent surface and cytoplasmic expression of this pathogen recognition receptor. This defect may account for the prolonged cycle of chronic inflammation associated with CF.

Expression of the nuclear factor-κB inhibitor A20 is altered in the cystic fibrosis epithelium.

A20 is a lipopolysaccharide (LPS)-inducible, cytoplasmic zinc finger protein, which inhibits Toll-like receptor-activated nuclear factor (NF)-κB signalling by deubiquitinating tumour necrosis factor receptor-associated factor (TRAF)-6. The action of A20 is facilitated by complex formation with ring finger protein (RNF)-11, Itch and TAX-1 binding protein-1 (TAX1BP1). This study investigated whether the expression of A20 is altered in the chronically inflamed cystic fibrosis (CF) airway epithelium. Nasal epithelial cells from CF patients (F508del homozygous), non-CF controls and immortalised epithelial cells (16HBE14o- and CFBE41o-) were stimulated with LPS. Cytoplasmic expression of A20 and expression of NF-κB subunits were analysed. Formation of the A20 ubiquitin editing complex was also investigated. In CFBE41o-, peak LPS-induced A20 expression was delayed compared with 16HBE14o- and fell significantly below basal levels 12-24 h after LPS stimulation. This was confirmed in primary CF airway cells. Additionally, a significant inverse relationship between A20 and p65 expression was observed. Inhibitor studies showed that A20 does not undergo proteasomal degradation in CFBE41o-. A20 interacted with TAX1BP1, RNF11 and TRAF6 in 16HBE14o- cells, but these interactions were not observed in CFBE41o-. The expression of A20 is significantly altered in CF, and important interactions with complex members and target proteins are lost, which may contribute to the state of chronic NF-κB-driven inflammation.

Simvastatin is associated with superior lipid and glycaemic control to atorvastatin and reduced levels of incident Type 2 diabetes, in men and women, in the UK Biobank.

INTRODUCTION: Cardiovascular disease (CVD) is the leading cause of mortality in people with Type 2 diabetes mellitus (T2DM). Statins reduce low-density lipoproteins and positively affect CVD outcomes. Statin type and dose have differential effects on glycaemia and risk of incident T2DM; however, the impact of gender, and of individual drugs within the statin class, remains unclear. AIM: To compare effects of simvastatin and atorvastatin on lipid and glycaemic control in men and women with and without T2DM, and their association with incident T2DM. METHODS: The effect of simvastatin and atorvastatin on lipid and glycaemic control was assessed in the T2DM DiaStrat cohort. Prescribed medications, gender, age, BMI, diabetes duration, blood lipid profile and HbA1c were extracted from Electronic Care Record, and compared in men and women prescribed simvastatin and atorvastatin. Analyses were replicated in the UKBiobank in those with and without T2DM. The association of simvastatin and atorvastatin with incident T2DM was also investigated in the UKBiobank. Cohorts where matched for age, BMI and diabetes duration in men and women, in the UKBioBank analysis, where possible. RESULTS: Simvastatin was associated with better LDL (1.6 ± 0.6 vs 2.1 ± 0.9 mmol/L, p < .01) and total cholesterol (3.6 ± 0.7 vs 4.2 ± 1.0 mmol/L, p < .05), and glycaemic control (62 ± 17 vs 67 ± 19 mmol/mol, p < .059) than atorvastatin specifically in women in the DiaStrat cohort. In the UKBiobank, both men and women prescribed simvastatin had better LDL (Women: 2.6 ± 0.6 vs 2.6 ± 0.7 mmol/L, p < .05; Men: 2.4 ± 0.6 vs 2.4 ± 0.6, p < .01) and glycaemic control (Women:54 ± 14 vs 56 ± 15mmol/mol, p < .05; Men, 54 ± 14 vs 55 ± 15 mmol/mol, p < .01) than those prescribed atorvastatin. Simvastatin was also associated with reduced risk of incident T2DM in both men and women (p < .0001) in the UKBiobank. CONCLUSIONS: Simvastatin is associated with superior lipid and glycaemic control to atorvastatin in those with and without T2DM, and with fewer incident T2DM cases. Given the importance of lipid and glycaemic control in preventing secondary complications of T2DM, these findings may help inform prescribing practices.

A20 regulation of nuclear factor-κB: perspectives for inflammatory lung disease.

Persistent activation of NF-κB is central to the pathogenesis of many inflammatory lung disorders, including cystic fibrosis, asthma, and chronic obstructive pulmonary disease. A20 is an endogenous negative regulator of NF-κB signaling, which has been widely described in autoimmune and inflammatory disorders, including diabetes and Crohn's disease, but which has received little attention in terms of chronic lung disorders. This review examines the existing body of research on A20 regulation of NF-κB signaling and details the mechanism and regulation of A20 action focusing, where possible, on pulmonary inflammation. A20 and its associated signaling molecules are highlighted as being of potential therapeutic interest for the treatment of inflammatory disorders, and a proposed model of A20 activity in inflammatory lung disease is provided.

A20 controls expression of beta-cell regulatory genes and transcription factors.

TNFAIP3 encodes a zinc finger protein called A20, which has potent anti-inflammatory and anti-apoptotic properties. A20 promotes beta-cell survival and protects against islet graft rejection in experimental models. The current study sought to investigate the mechanisms underlying the protective role of A20 in the pancreatic beta-cell. Two islet cell types were used for experiments: the insulin-secreting BRIN-BD11 cell line and human islet cells. A20 was silenced using siRNA against TNFAIP3, and knockdown was confirmed by qPCR and immunostaining of cells. Cell viability, cytotoxicity and apoptosis were assessed using the ApotoxGlo assay. Glucose-stimulated insulin secretion and production of inflammatory cytokines (TNFa, IL1b and IFNg) were measured by ELISA. Expression of beta-cell regulatory genes (Abcc8, Kcnj11, Kcnq1, Gck, Scl2a2) and transcription factors (Hnf1a, Pdx1, Nkx6.1, Ngn3) was determined by qPCR. A20 deficiency increased apoptosis, impaired glucose-induced insulin secretion, and reduced expression of beta-cell regulatory genes and transcription factors. Addition of recombinant A20 normalized gene expression profiles. TNFa, IL1b and IFNg were elevated in A20 deficient cells and found to independently elicit changes in gene expression. Analysis of PCR array data suggests that A20 action in the beta cell is largely, although not exclusively, driven by the P65 subunit of NF-kB. The current report demonstrates a role for A20 in controlling beta-cell integrity and survival, which likely results from the regulation of inflammatory signalling. Of particular note is the impact that A20 deficiency has on the expression of transcription factors regulating the maturation and normal function of beta cells.

The Interdependency and Co-Regulation of the Vitamin D and Cholesterol Metabolism.

Vitamin D and cholesterol metabolism overlap significantly in the pathways that contribute to their biosynthesis. However, our understanding of their independent and co-regulation is limited. Cardiovascular disease is the leading cause of death globally and atherosclerosis, the pathology associated with elevated cholesterol, is the leading cause of cardiovascular disease. It is therefore important to understand vitamin D metabolism as a contributory factor. From the literature, we compile evidence of how these systems interact, relating the understanding of the molecular mechanisms involved to the results from observational studies. We also present the first systems biology pathway map of the joint cholesterol and vitamin D metabolisms made available using the Systems Biology Graphical Notation (SBGN) Markup Language (SBGNML). It is shown that the relationship between vitamin D supplementation, total cholesterol, and LDL-C status, and between latitude, vitamin D, and cholesterol status are consistent with our knowledge of molecular mechanisms. We also highlight the results that cannot be explained with our current knowledge of molecular mechanisms: (i) vitamin D supplementation mitigates the side-effects of statin therapy; (ii) statin therapy does not impact upon vitamin D status; and critically (iii) vitamin D supplementation does not improve cardiovascular outcomes, despite improving cardiovascular risk factors. For (iii), we present a hypothesis, based on observations in the literature, that describes how vitamin D regulates the balance between cellular and plasma cholesterol. Answering these questions will create significant opportunities for advancement in our understanding of cardiovascular health.

Cell-to-cell communication and cellular environment alter the somatostatin status of delta cells.

INTRODUCTION: Somatostatin, released from pancreatic delta cells, is a potent paracrine inhibitor of insulin and glucagon secretion. Islet cellular interactions and glucose homeostasis are essential to maintain normal patterns of insulin secretion. However, the importance of cell-to-cell communication and cellular environment in the regulation of somatostatin release remains unclear. METHODS: This study employed the somatostatin-secreting TGP52 cell line maintained in DMEM:F12 (17.5mM glucose) or DMEM (25mM glucose) culture media. The effect of pseudoislet formation and culture medium on somatostatin content and release in response to a variety of stimuli was measured by somatostatin EIA. In addition, the effect of pseudoislet formation on cellular viability (MTT and LDH assays) and proliferation (BrdU ELISA) was determined. RESULTS: TGP52 cells readily formed pseudoislets and showed enhanced functionality in three-dimensional form with increased E-cadherin expression irrespective of the culture environment used. However, culture in DMEM decreased cellular somatostatin content (P<0.01) and increased somatostatin secretion in response to a variety of stimuli including arginine, calcium and PMA (P<0.001) when compared with cells grown in DMEM:F12. Configuration of TGP52 cells as pseudoislets reduced the proliferative rate and increased cellular cytotoxicity irrespective of culture medium used. CONCLUSIONS: Somatostatin secretion is greatly facilitated by cell-to-cell interactions and E-cadherin expression. Cellular environment and extracellular glucose also significantly influence the function of delta cells.

The role of glucagon- and somatostatin-secreting cells in the regulation of insulin release and beta-cell function in heterotypic pseudoislets.

BACKGROUND: Pseudoislet studies have concentrated on single beta-cell lines or a combination of insulin and glucagon-secreting cells, overlooking the potential role of somatostatin in insulin release. This study sought to evaluate a heterotypic pseudoislet model containing insulin- (MIN6), glucagon- (αTC1.9) and somatostatin (TGP52)-secreting cells of mouse origin and to compare these pseudoislets with traditional monolayer preparations. METHODS: Cellular viability (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and lactate dehydrogenase assays), proliferation (5-bromo-2-deoxyuridine ELISA), hormone content and functional insulin release in response to a variety of stimuli were measured. Differential expression of E-cadherin, connexin 36 and connexin 43 was assessed by reverse transcriptase-polymerase chain reaction and Western blot to determine a possible role for adherens in insulin release from these pseudoislets. RESULTS: All pseudoislet cells displayed reduced proliferation coupled with an increase in cell death which may contribute to their static size in culture. While MIN6 and TGP52 cells expressed E-cadherin and showed sustained or improved hormone content when configured as pseudoislets, αTC1.9 lacked E-cadherin and contained less glucagon following pseudoislet formation. MIN6 and αTC1.9 cells expressed connexin 36, but not connexin 43 and TGP52 cells expressed connexin 43 only. In the presence of Alanine, Arginine and glucagon-like peptide-1, heterotypic pseudoislet cultures secreted levels of insulin that were comparable to that of MIN6 pseudoislets. In addition, pseudoislets comprising all three cell lines released more insulin into the surrounding culture medium than MIN6 pseudoislets when studied over a 1-week period. CONCLUSIONS: The current model may prove useful in studying the role of islet cell interactions in the release of insulin from pancreatic islets.

Cystic Fibrosis-Related Diabetes: Pathophysiology and Therapeutic Challenges.

Cystic fibrosis-related diabetes (CFRD) is among the most common extrapulmonary co-morbidity associated with cystic fibrosis (CF), affecting an estimated 50% of adults with the condition. Cystic fibrosis is prevalent in 1 in every 2500 Caucasian live births and is caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Mutated CFTR leads to dehydrated epithelial surfaces and a build-up of mucus in a variety of tissues including the lungs and pancreas. The leading cause of mortality in CF is repeated respiratory bacterial infections, which prompts a decline in lung function. Co-morbid diabetes promotes bacterial colonisation of the airways and exacerbates the deterioration in respiratory health. Cystic fibrosis-related diabetes is associated with a 6-fold higher mortality rate compared with those with CF alone. The management of CFRD adds a further burden for the patient and creates new therapeutic challenges for the clinical team. Several proposed hypotheses on how CFRD develops have emerged, including exocrine-driven fibrosis and destruction of the entire pancreas and contrasting theories on the direct or indirect impact of CFTR mutation on islet function. The current review outlines recent data on the impact of CFTR on endocrine pancreatic function and discusses the use of conventional diabetic therapies and new CFTR-correcting drugs on the treatment of CFRD.

Short-term CFTR inhibition reduces islet area in C57BL/6 mice.

Cystic fibrosis-related diabetes (CFRD) worsens CF lung disease leading to early mortality. Loss of beta cell area, even without overt diabetes or pancreatitis is consistently observed. We investigated whether short-term CFTR inhibition was sufficient to impact islet morphology and function in otherwise healthy mice. CFTR was inhibited in C57BL/6 mice via 8-day intraperitoneal injection of CFTRinh172. Animals had a 7-day washout period before measures of hormone concentration or islet function were performed. Short-term CFTR inhibition increased blood glucose concentrations over the course of the study. However, glucose tolerance remained normal without insulin resistance. CFTR inhibition caused marked reductions in islet size and in beta cell and non-beta cell area within the islet, which resulted from loss of islet cell size rather than islet cell number. Significant reductions in plasma insulin concentrations and pancreatic insulin content were also observed in CFTR-inhibited animals. Temporary CFTR inhibition had little long-term impact on glucose-stimulated, or GLP-1 potentiated insulin secretion. CFTR inhibition has a rapid impact on islet area and insulin concentrations. However, islet cell number is maintained and insulin secretion is unaffected suggesting that early administration of therapies aimed at sustaining beta cell mass may be useful in slowing the onset of CFRD.

Role of islet peptides in beta cell regulation and type 2 diabetes therapy.

The endocrine pancreas is composed of islets of Langerhans, which secrete a variety of peptide hormones critical for the maintenance of glucose homeostasis. Insulin is the primary regulator of glucose and its secretion from beta-cells is tightly regulated in response to physiological demands. Direct cell-cell communication within islets is essential for glucose-induced insulin secretion. Emerging data suggest that islet connectivity is also important in the regulating the release of other islet hormones including glucagon and somatostatin. Autocrine and paracrine signals exerted by secreted peptides within the islet also play a key role. A great deal of attention has focused on classical islet peptides, namely insulin, glucagon and somatostatin. Recently, it has become clear that islets also synthesise and secrete a range of non-classical peptides, which regulate beta-cell function and insulin release. The current review summarises the roles of islet cell connectivity and islet peptide-driven autocrine and paracrine signalling in beta-cell function and survival. The potential to harness the paracrine effects of non-classical islet peptides for the treatment of type 2 diabetes is also briefly discussed.

Hyperglycemia and Diabetes Downregulate the Functional Expression of TRPV4 Channels in Retinal Microvascular Endothelium.

Retinal endothelial cell dysfunction is believed to play a key role in the etiology and pathogenesis of diabetic retinopathy. Numerous studies have shown that TRPV4 channels are critically involved in maintaining normal endothelial cell function. In the current paper, we demonstrate that TRPV4 is functionally expressed in the endothelium of the retinal microcirculation and that both channel expression and activity is downregulated by hyperglycaemia. Quantitative PCR and immunostaining demonstrated molecular expression of TRPV4 in cultured bovine retinal microvascular endothelial cells (RMECs). Functional TRPV4 activity was assessed in cultured RMECs from endothelial Ca2+-responses recorded using fura-2 microfluorimetry and electrophysiological recordings of membrane currents. The TRPV4 agonist 4α-phorbol 12,13-didecanoate (4-αPDD) increased [Ca2+]i in RMECs and this response was largely abolished using siRNA targeted against TRPV4. These Ca2+-signals were completely inhibited by removal of extracellular Ca2+, confirming their dependence on influx of extracellular Ca2+. The 4-αPDD Ca2+-response recorded in the presence of cyclopiazonic acid (CPA), which depletes the intracellular stores preventing any signal amplification through store release, was used as a measure of Ca2+-influx across the cell membrane. This response was blocked by HC067047, a TRPV4 antagonist. Under voltage clamp conditions, the TRPV4 agonist GSK1016790A stimulated a membrane current, which was again inhibited by HC067047. Following incubation with 25 mM D-glucose TRPV4 expression was reduced in comparison with RMECs cultured under control conditions, as were 4αPDD-induced Ca2+-responses in the presence of CPA and ion currents evoked by GSK1016790A. Molecular expression of TRPV4 in the retinal vascular endothelium of 3 months' streptozotocin-induced diabetic rats was also reduced in comparison with that in age-matched controls. We conclude that hyperglycaemia and diabetes reduce the molecular and functional expression of TRPV4 channels in retinal microvascular endothelial cells. These changes may contribute to diabetes induced endothelial dysfunction and retinopathy.

Expression of the inflammatory regulator A20 correlates with lung function in patients with cystic fibrosis.

BACKGROUND: A20 and TAX1BP1 interact to negatively regulate NF-κB-driven inflammation. A20 expression is altered in F508del/F508del patients. Here we explore the effect of CFTR and CFTR genotype on A20 and TAX1BP1 expression. The relationship with lung function is also assessed. METHODS: Primary nasal epithelial cells (NECs) from CF patients (F508del/F508del, n=7, R117H/F508del, n=6) and controls (age-matched, n=8), and 16HBE14o- cells were investigated. A20 and TAX1BP1 gene expression was determined by qPCR. RESULTS: Silencing of CFTR reduced basal A20 expression. Following LPS stimulation A20 and TAX1BP1 expression was induced in control NECs and reduced in CF NECs, broadly reflecting the CF genotype: F508del/F508del had lower expression than R117H/F508del. A20, but not TAX1BP1 expression, was proportional to FEV(1) in all CF patients (r=0.968, p<0.001). CONCLUSIONS: A20 expression is reduced in CF and is proportional to FEV1. Pending confirmation in a larger study, A20 may prove a novel predictor of CF inflammation/disease severity.

Directed differentiation of progenitor cells towards an islet-cell phenotype.

Exogenous insulin administration and oral anti-diabetic drugs are the primary means of treating diabetes. However, tight glycaemic control, with its inherent risk of hypoglycaemia, is required to prevent the microvascular and macrovascular complications of the disease. While islet or pancreas transplantations offer a longer-term cure, their widespread application is not possible, primarily because of a lack of donor tissue, the burden of life-long immunosuppression, and eventual graft rejection. The rapid increase in the incidence of diabetes has promoted the search for alternative cell-based therapies. Here we review recent advances in the directed differentiation of both endocrine and non-endocrine progenitors towards an islet-like phenotype.

Configuration of electrofusion-derived human insulin-secreting cell line as pseudoislets enhances functionality and therapeutic utility.

Formation of pseudoislets from rodent cell lines has provided a particularly useful model to study homotypic islet cell interactions and insulin secretion. This study aimed to extend this research to generate and characterize, for the first time, functional human pseudoislets comprising the recently described electrofusion-derived insulin-secreting 1.1B4 human ß-cell line. Structural pseudoislets formed readily over 3-7 days in culture using ultra-low-attachment plastic, attaining a static size of 100-200 µm in diameter, corresponding to ~6000 ß cells. This was achieved by decreases in cell proliferation and integrity as assessed by BrdU ELISA, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, and lactate dehydrogenase assays. Insulin content was comparable between monolayers and pseudoislets. However, pseudoislet formation enhanced insulin secretion by 1·7- to 12·5-fold in response to acute stimulation with glucose, amino acids, incretin hormones, or drugs compared with equivalent cell monolayers. Western blot and RT-PCR showed expression of key genes involved in cell communication and the stimulus-secretion pathway. Expression of E-Cadherin and connexin 36 and 43 was greatly enhanced in pseudoislets with no appreciable connexin 43 protein expression in monolayers. Comparable levels of insulin, glucokinase, and GLUT1 were found in both cell populations. The improved secretory function of human 1.1B4 cell pseudoislets over monolayers results from improved cellular interactions mediated through gap junction communication. Pseudoislets comprising engineered electrofusion-derived human ß cells provide an attractive model for islet research and drug testing as well as offering novel therapeutic application through transplantation.