Glucose Concentration in Regulating Induced Pluripotent Stem Cells Differentiation Toward Insulin-Producing Cells.

The generation of insulin-producing cells from human-induced pluripotent stem cells holds great potential for diabetes modeling and treatment. However, existing protocols typically involve incubating cells with un-physiologically high concentrations of glucose, which often fail to generate fully functional IPCs. Here, we investigated the influence of high (20 mM) versus low (5.5 mM) glucose concentrations on IPCs differentiation in three hiPSC lines. In two hiPSC lines that were unable to differentiate to IPCs sufficiently, we found that high glucose during differentiation leads to a shortage of NKX6.1+ cells that have co-expression with PDX1 due to insufficient NKX6.1 gene activation, thus further reducing differentiation efficiency. Furthermore, high glucose during differentiation weakened mitochondrial respiration ability. In the third iPSC line, which is IPC differentiation amenable, glucose concentrations did not affect the PDX1/NKX6.1 expression and differentiation efficiency. In addition, glucose-stimulated insulin secretion was only seen in the differentiation under a high glucose condition. These IPCs have higher KATP channel activity and were linked to sufficient ABCC8 gene expression under a high glucose condition. These data suggest high glucose concentration during IPC differentiation is necessary to generate functional IPCs. However, in cell lines that were IPC differentiation unamenable, high glucose could worsen the situation.

Simultaneous LC-MS determination of glucose regulatory peptides secreted by stem cell-derived islet organoids.

For studying stem cell-derived islet organoids (SC-islets) in an organ-on-chip (OoC) platform, we have developed a reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) method allowing for simultaneous determination of insulin, somatostatin-14, and glucagon, with improved matrix robustness compared to earlier methodology. Combining phenyl/hexyl-C18 separations using 2.1 mm inner diameter LC columns and triple quadrupole mass spectrometry, identification and quantification were secured with negligible variance in retention time and quantifier/qualifier ratios, negligible levels of carryover (<2%), and sufficient precision (±10% RSD) and accuracy (±15% relative error) with and without use of an internal standard. The obtained lower limits of quantification were 0.2 µg/L for human insulin, 0.1 µg/L for somatostatin-14, and 0.05 µg/L for glucagon. The here-developed RPLC-MS/MS method showed that the SC-islets have an insulin response dependent on glucose concentration, and the SC-islets produce and release somatostatin-14 and glucagon. The RPLC-MS/MS method for these peptide hormones was compatible with an unfiltered offline sample collection from SC-islets cultivated on a pumpless, recirculating OoC (rOoC) platform. The SC-islets background secretion of insulin was not significantly different on the rOoC device compared to a standard cell culture well-plate. Taken together, RPLC-MS/MS method is well suited for multi-hormone measurements of SC-islets on an OoC platform.

Intracorneal Implantation of 3D Bioprinted Scaffolds Containing Mesenchymal Stromal Cells Using Femtosecond-Laser-Assisted Intrastromal Keratoplasty.

Injury of the cornea is a complex biological process. Regeneration of the corneal stroma can be facilitated by the presence of mesenchymal stromal cells (MSCs) and application of tissue equivalents. A new tissue-engineering strategy for corneal stroma regeneration is presented using cellularized 3D bioprinted hydrogel constructs implanted into organ cultured porcine corneas using femtosecond laser-assisted intrastromal keratoplasty. The ex vivo cultured, MSC-loaded 3D bioprinted structures remain intact, support cell survival, and contain de novo synthesized extracellular matrix components and migrating cells throughout the observation period. At day 14 postimplantation, the cellularized tissue equivalents contain few or no cells, as demonstrated by optical coherence tomography imaging and immunofluorescent staining. This study successfully combines a laboratory-based method with modern, patient-care practice to produce a cell-laden tissue equivalent for corneal implantation. Optimal bioink composition and cellularization of tissue equivalents are essential in fine-tuning a method to promote the current technique as a future treatment modality.

Determination of insulin secretion from stem cell-derived islet organoids with liquid chromatography-tandem mass spectrometry.

Organoids are laboratory-grown 3D organ models, mimicking human organs for e.g. drug development and personalized therapy. Islet organoids (typically 100-200 µm), which can be grown from the patient́s own cells, are emerging as prototypes for transplantation-based therapy of diabetes. Selective methods for quantifying insulin production from islet organoids are needed, but sensitivity and carry-over have been major bottlenecks in previous efforts. We have developed a reverse phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) method for studying the insulin secretion of islet organoids. In contrast to our previous attempts using nano-scale LC columns, conventional 2.1 mm inner diameter LC column (combined with triple quadrupole mass spectrometry) was well suited for sensitive and selective measurements of insulin secreted from islet organoids with low microliter-scale samples. Insulin is highly prone to carry-over, so standard tubings and injector parts were replaced with shielded fused silica connectors. As samples were expected to be very limited, an extended Box-Behnken experimental design for the MS settings was conducted to maximize performance. The finale method has excellent sensitivity, accuracy and precision (limit of detection: ≤0.2 pg/µL, relative error: ≤±10%, relative standard deviation: <10%), and was well suited for measuring 20 µL amounts of Krebs buffer containing insulin secreted from islet organoids.

International Survey of Clinical Monitoring Practices in Pancreas and Islet Transplantation.

BACKGROUND: The long-term outcomes of both pancreas and islet allotransplantation have been compromised by difficulties in the detection of early graft dysfunction at a time when a clinical intervention can prevent further deterioration and preserve allograft function. The lack of standardized strategies for monitoring pancreas and islet allograft function prompted an international survey established by an International Pancreas and Islet Transplant Association/European Pancreas and Islet Transplant Association working group. METHODS: A global survey was administered to 24 pancreas and 18 islet programs using Redcap. The survey addressed protocolized and for-cause immunologic and metabolic monitoring strategies following pancreas and islet allotransplantation. All invited programs completed the survey. RESULTS: The survey identified that in both pancreas and islet allograft programs, protocolized clinical monitoring practices included assessing body weight, fasting glucose/C-peptide, hemoglobin A1c, and donor-specific antibody. Protocolized monitoring in islet transplant programs relied on the addition of mixed meal tolerance test, continuous glucose monitoring, and autoantibody titers. In the setting of either suspicion for rejection or serially increasing hemoglobin A1c/fasting glucose levels postpancreas transplant, Doppler ultrasound, computed tomography, autoantibody titers, and pancreas graft biopsy were identified as adjunctive strategies to protocolized monitoring studies. No additional assays were identified in the setting of serially increasing hemoglobin A1c levels postislet transplantation. CONCLUSIONS: This international survey identifies common immunologic and metabolic monitoring strategies utilized for protocol and for cause following pancreas and islet transplantation. In the absence of any formal studies to assess the efficacy of immunologic and metabolic testing to detect early allograft dysfunction, it can serve as a guidance document for developing monitoring algorithms following beta-cell replacement.

The Tankyrase Inhibitor OM-153 Demonstrates Antitumor Efficacy and a Therapeutic Window in Mouse Models.

The catalytic enzymes tankyrase 1 and 2 (TNKS1/2) alter protein turnover by poly-ADP-ribosylating target proteins, which earmark them for degradation by the ubiquitin-proteasomal system. Prominent targets of the catalytic activity of TNKS1/2 include AXIN proteins, resulting in TNKS1/2 being attractive biotargets for addressing of oncogenic WNT/ß-catenin signaling. Although several potent small molecules have been developed to inhibit TNKS1/2, there are currently no TNKS1/2 inhibitors available in clinical practice. The development of tankyrase inhibitors has mainly been disadvantaged by concerns over biotarget-dependent intestinal toxicity and a deficient therapeutic window. Here we show that the novel, potent, and selective 1,2,4-triazole-based TNKS1/2 inhibitor OM-153 reduces WNT/ß-catenin signaling and tumor progression in COLO 320DM colon carcinoma xenografts upon oral administration of 0.33-10 mg/kg twice daily. In addition, OM-153 potentiates anti-programmed cell death protein 1 (anti-PD-1) immune checkpoint inhibition and antitumor effect in a B16-F10 mouse melanoma model. A 28-day repeated dose mouse toxicity study documents body weight loss, intestinal damage, and tubular damage in the kidney after oral-twice daily administration of 100 mg/kg. In contrast, mice treated oral-twice daily with 10 mg/kg show an intact intestinal architecture and no atypical histopathologic changes in other organs. In addition, clinical biochemistry and hematologic analyses do not identify changes indicating substantial toxicity. The results demonstrate OM-153-mediated antitumor effects and a therapeutic window in a colon carcinoma mouse model ranging from 0.33 to at least 10 mg/kg, and provide a framework for using OM-153 for further preclinical evaluations. Significance: This study uncovers the effectiveness and therapeutic window for a novel tankyrase inhibitor in mouse tumor models.

Mini-organs forum: how to advance organoid technology to organ transplant community.

The generation of human mini-organs, the so-called organoids, is one of the biggest scientific advances in regenerative medicine. This technology exploits traditional three-dimensional culture techniques that support cell-autonomous self-organization responses of stem cells to derive micrometer to millimeter size versions of human organs. The convergence of the organoid technology with organ transplantation is still in its infancy but this alliance is expected to open new venues to change the way we conduct both transplant and organoid research. In this Forum we provide a summary on early achievements facilitating organoid derivation and culture. We further discuss on early advances of organoid transplantation also offering a comprehensive overview of current limitations and challenges to instruct organoid maturation. We expect that this Forum sets the ground for initial discussions between stem cell biologists, bioengineers, and the transplant community to better direct organoid basic research to advance the organ transplantation field.

Pancreas-on-a-Chip Technology for Transplantation Applications.

PURPOSE OF REVIEW: Human pancreas-on-a-chip (PoC) technology is quickly advancing as a platform for complex in vitro modeling of islet physiology. This review summarizes the current progress and evaluates the possibility of using this technology for clinical islet transplantation. RECENT FINDINGS: PoC microfluidic platforms have mainly shown proof of principle for long-term culturing of islets to study islet function in a standardized format. Advancement in microfluidic design by using imaging-compatible biomaterials and biosensor technology might provide a novel future tool for predicting islet transplantation outcome. Progress in combining islets with other tissue types gives a possibility to study diabetic interventions in a minimal equivalent in vitro environment. Although the field of PoC is still in its infancy, considerable progress in the development of functional systems has brought the technology on the verge of a general applicable tool that may be used to study islet quality and to replace animal testing in the development of diabetes interventions.

Proteomic Profiling Reveals the Ambivalent Character of the Mesenchymal Stem Cell Secretome: Assessing the Effect of Preconditioned Media on Isolated Human Islets.

Previous studies in rodents have indicated that function and survival of transplanted islets can be substantially improved by mesenchymal stem cells (MSC). The few human islet studies to date have confirmed these findings but have not determined whether physical contact between MSC and islets is required or whether the benefit to islets results from MSC-secreted proteins. This study aimed to investigate the protective capacity of MSC-preconditioned media for human islets. MSC were cultured for 2 or 5 days in normoxia or hypoxia before harvesting the cell-depleted media for human islet culture in normoxia or hypoxia for 6-8 or 3-4 days, respectively. To characterize MSC-preconditioned media, proteomic secretome profiling was performed to identify angiogenesis- and inflammation-related proteins. A protective effect of MSC-preconditioned media on survival and in vitro function of hypoxic human islets was observed irrespective of the atmosphere used for MSC preconditioning. Islet morphology changed markedly when media from hypoxic MSC were used for culture. However, PDX-1 and insulin gene expression did not confirm a change in the genetic phenotype of these islets. Proteomic profiling of preconditioned media revealed the heterogenicity of the secretome comprising angiogenic and antiapoptotic as well as angiostatic or proinflammatory mediators released at an identical pattern regardless whether MSC had been cultured in normoxic or hypoxic atmosphere. These findings do not allow a clear discrimination between normoxia and hypoxia as stimulus for protective MSC capabilities but indicate an ambivalent character of the MSC angiogenesis- and inflammation-related secretome. Nevertheless, culture of human islets in acellular MSC-preconditioned media resulted in improved morphological and functional islet integrity suggesting a disbalance in favor of protective factors. Further approaches should aim to eliminate potentially detrimental factors to enable the production of advanced clinical grade islet culture media with higher protective qualities.

Inhibition of the prostaglandin D2-GPR44/DP2 axis improves human islet survival and function.

AIMS/HYPOTHESIS: Inflammatory signals and increased prostaglandin synthesis play a role during the development of diabetes. The prostaglandin D2 (PGD2) receptor, GPR44/DP2, is highly expressed in human islets and activation of the pathway results in impaired insulin secretion. The role of GPR44 activation on islet function and survival rate during chronic hyperglycaemic conditions is not known. In this study, we investigate GPR44 inhibition by using a selective GPR44 antagonist (AZ8154) in human islets both in vitro and in vivo in diabetic mice transplanted with human islets. METHODS: Human islets were exposed to PGD2 or proinflammatory cytokines in vitro to investigate the effect of GPR44 inhibition on islet survival rate. In addition, the molecular mechanisms of GPR44 inhibition were investigated in human islets exposed to high concentrations of glucose (HG) and to IL-1ß. For the in vivo part of the study, human islets were transplanted under the kidney capsule of immunodeficient diabetic mice and treated with 6, 60 or 100 mg/kg per day of a GPR44 antagonist starting from the transplantation day until day 4 (short-term study) or day 17 (long-term study) post transplantation. IVGTT was performed on mice at day 10 and day 15 post transplantation. After termination of the study, metabolic variables, circulating human proinflammatory cytokines, and hepatocyte growth factor (HGF) were analysed in the grafted human islets. RESULTS: PGD2 or proinflammatory cytokines induced apoptosis in human islets whereas GPR44 inhibition reversed this effect. GPR44 inhibition antagonised the reduction in glucose-stimulated insulin secretion induced by HG and IL-1ß in human islets. This was accompanied by activation of the Akt-glycogen synthase kinase 3ß signalling pathway together with phosphorylation and inactivation of forkhead box O-1and upregulation of pancreatic and duodenal homeobox-1 and HGF. Administration of the GPR44 antagonist for up to 17 days to diabetic mice transplanted with a marginal number of human islets resulted in reduced fasting blood glucose and lower glucose excursions during IVGTT. Improved glucose regulation was supported by increased human C-peptide levels compared with the vehicle group at day 4 and throughout the treatment period. GPR44 inhibition reduced plasma levels of TNF-α and growth-regulated oncogene-α/chemokine (C-X-C motif) ligand 1 and increased the levels of HGF in human islets. CONCLUSIONS/INTERPRETATION: Inhibition of GPR44 in human islets has the potential to improve islet function and survival rate under inflammatory and hyperglycaemic stress. This may have implications for better survival rate of islets following transplantation.

In vivo Environment Swiftly Restricts Human Pancreatic Progenitors Toward Mono-Hormonal Identity via a HNF1A/HNF4A Mechanism.

Generating insulin-producing ß-cells from human induced pluripotent stem cells is a promising cell replacement therapy for improving or curing insulin-dependent diabetes. The transplantation of end-stages differentiating cells into living hosts was demonstrated to improve ß-cell maturation. Nevertheless, the cellular and molecular mechanisms outlining the transplanted cells' response to the in vivo environment are still to be properly characterized. Here we use global proteomics and large-scale imaging techniques to demultiplex and filter the cellular processes and molecular signatures modulated by the immediate in vivo effect. We show that in vivo exposure swiftly confines in vitro generated human pancreatic progenitors to single hormone expression. The global proteome landscape of the transplanted cells was closer to native human islets, especially in regard to energy metabolism and redox balance. Moreover, our study indicates a possible link between these processes and certain epigenetic regulators involved in cell identity. Pathway analysis predicted HNF1A and HNF4A as key regulators controlling the in vivo islet-promoting response, with experimental evidence suggesting their involvement in confining islet cell fate following xeno-transplantation.

Encapsulation boosts islet-cell signature in differentiating human induced pluripotent stem cells via integrin signalling.

Cell replacement therapies hold great therapeutic potential. Nevertheless, our knowledge of the mechanisms governing the developmental processes is limited, impeding the quality of differentiation protocols. Generating insulin-expressing cells in vitro is no exception, with the guided series of differentiation events producing heterogeneous cell populations that display mixed pancreatic islet phenotypes and immaturity. The achievement of terminal differentiation ultimately requires the in vivo transplantation of, usually, encapsulated cells. Here we show the impact of cell confinement on the pancreatic islet signature during the guided differentiation of alginate encapsulated human induced pluripotent stem cells (hiPSCs). Our results show that encapsulation improves differentiation by significantly reshaping the proteome landscape of the cells towards an islet-like signature. Pathway analysis is suggestive of integrins transducing the encapsulation effect into intracellular signalling cascades promoting differentiation. These analyses provide a molecular framework for understanding the confinement effects on hiPSCs differentiation while confirming its importance for this process.

In vivo hyperglycaemia exposure elicits distinct period-dependent effects on human pancreatic progenitor differentiation, conveyed by oxidative stress.

AIM: The loss of insulin-secreting ß-cells, ultimately characterizing most diabetes forms, demands the development of cell replacement therapies. The common endpoint for all ex vivo strategies is transplantation into diabetic patients. However, the effects of hyperglycaemia environment on the transplanted cells were not yet properly assessed. Thus, the main goal of this study was to characterize global effect of brief and prolonged in vivo hyperglycaemia exposure on the cell fate acquisition and maintenance of transplanted human pancreatic progenitors. METHODS: To rigorously study the effect of hyperglycaemia, in vitro differentiated human-induced pluripotent stem cells (hiPSC)-derived pancreatic progenitors were xenotransplanted in normoglycaemic and diabetic NSG rat insulin promoter (RIP)-diphtheria toxin receptor (DTR) mice. The transplants were retrieved after 1-week or 1-month exposure to overt hyperglycaemia and analysed by large-scale microscopy or global proteomics. For this study we pioneer the use of the NSG RIP-DTR system in the transplantation of hiPSC, making use of its highly reproducible specific and absolute ß-cell ablation property in the absence of inflammation or other organ toxicity. RESULTS: Here we show for the first time that besides the presence of an induced oxidative stress signature, the cell fate and proteome landscape response to hyperglycaemia was different, involving largely different mechanisms, according to the period spent in the hyperglycaemic environment. Surprisingly, brief hyperglycaemia exposure increased the bihormonal cell number by impeding the activity of specific islet lineage determinants. Moreover, it activated antioxidant and inflammation protection mechanisms signatures in the transplanted cells. In contrast, the prolonged exposure was characterized by decreased numbers of hormone + cells, low/absent detoxification signature, augmented production of oxygen reactive species and increased apoptosis. CONCLUSION: Hyperglycaemia exposure induced distinct, period-dependent, negative effects on xenotransplanted human pancreatic progenitor, affecting their energy homeostasis, cell fate acquisition and survival.

Interleukin-22 reverses human islet dysfunction and apoptosis triggered by hyperglycemia and LIGHT.

Interleukin (IL)-22 has recently been suggested as an anti-inflammatory cytokine that could protect the islet cells from inflammation- and glucose-induced toxicity. We have previously shown that the tumor necrosis factor family member, LIGHT, can impair human islet function at least partly via pro-apoptotic effects. Herein, we aimed to investigate the protective role of IL-22 on human islets exposed to the combination of hyperglycemia and LIGHT. First, we found upregulation of LIGHT receptors (LTßR and HVEM) in engrafted human islets exposed to hyperglycemia (>11 mM) for 17 days post transplantation by using a double islet transplantation mouse model as well as in human islets cultured with high glucose (HG) (20 mM glucose) + LIGHT in vitro, and this latter effect was attenuated by IL-22. The effect of HG + LIGHT impairing glucose-stimulated insulin secretion was reversed by IL-22. The harmful effect of HG + LIGHT on human islet function seemed to involve enhanced endoplasmic reticulum stress evidenced by upregulation of p-IRE1α and BiP, elevated secretion of pro-inflammatory cytokines (IL-6, IL-8, IP-10 and MCP-1) and the pro-coagulant mediator tissue factor (TF) release and apoptosis in human islets, whereas all these effects were at least partly reversed by IL-22. Our findings suggest that IL-22 could counteract the harmful effects of LIGHT/hyperglycemia on human islet cells and potentially support the strong protective effect of IL-22 on impaired islet function and survival.

ß Cell Replacement Therapy: The Next 10 Years.

ß cell replacement with either pancreas or islet transplantation has progressed immensely over the last decades with current 1- and 5-year insulin independence rates of approximately 85% and 50%, respectively. Recent advances are largely attributed to improvements in immunosuppressive regimen, donor selection, and surgical technique. However, both strategies are compromised by a scarce donor source. Xenotransplantation offers a potential solution by providing a theoretically unlimited supply of islets, but clinical application has been limited by concerns for a potent immune response against xenogeneic tissue. ß cell clusters derived from embryonic or induced pluripotent stem cells represent another promising unlimited source of insulin producing cells, but clinical application is pending further advances in the function of the ß cell like clusters. Exciting developments and rapid progress in all areas of ß cell replacement prompted a lively debate by members of the young investigator committee of the International Pancreas and Islet Transplant Association at the 15th International Pancreas and Islet Transplant Association Congress in Melbourne and at the 26th international congress of The Transplant Society in Hong Kong. This international group of young investigators debated which modality of ß cell replacement would predominate the landscape in 10 years, and their arguments are summarized here.

Human Adipose-Derived Mesenchymal Stem Cells Respond to Short-Term Hypoxia by Secreting Factors Beneficial for Human Islets In Vitro and Potentiate Antidiabetic Effect In Vivo.

Adipose-derived mesenchymal stem cells (ASCs) release factors beneficial for islets in vitro and protect against hyperglycemia in rodent models of diabetes. Oxygen tension has been shown to induce metabolic changes and alter ASCs' release of soluble factors. The effects of hypoxia on the antidiabetic properties of ASCs have not been explored. To investigate this, we incubated human ASCs for 48 h in 21% (normoxia) or 1% O2 (hypoxia) and compared viability, cell growth, surface markers, differentiation capability, and soluble factors in the conditioned media (CM). Human islets were exposed to CM from ASCs incubated in either normoxia or hypoxia, and islet function and apoptosis after culture with or without proinflammatory cytokines were measured. To test hypoxic preconditioned ASCs' islet protective effects in vivo, ASCs were incubated for 48 h in normoxia or hypoxia before being injected into Balb/c Rag 1-/- immunodeficient mice with streptozotocin-induced insulitis. Progression of diabetes and insulin content of pancreas were measured. We found that incubation in hypoxia was well tolerated by ASCs and that levels of VEGF-A, FGF-2, and bNGF were elevated in CM from ASCs incubated in hypoxia compared to normoxia, while levels of HGF, IL-8, and CXCL1 were reduced. CM from ASCs incubated in hypoxia significantly improved human islet function and reduced apoptosis after culture, and reduced cytokine-induced apoptosis. In our mouse model, pancreas insulin content was higher in both groups receiving ASCs compared to control, but the mice receiving preconditioned ASCs had lower random and fasting blood glucose, as well as improved oral glucose tolerance compared to untreated mice. In conclusion, our in vitro results indicate that the islet protective potential of ASCs improves in hypoxia, and we give insight into factors involved in this. Finally we show that hypoxic preconditioning potentiates ASCs' antidiabetic effect in vivo.

Glial cell-line derived neurotrophic factor protects human islets from nutrient deprivation and endoplasmic reticulum stress induced apoptosis.

One of the key limitations to successful human islet transplantation is loss of islets due to stress responses pre- and post-transplantation. Nutrient deprivation and ER stress have been identified as important mechanisms leading to apoptosis. Glial Cell-line Derived Neurotrophic Factor (GDNF) has recently been found to promote islet survival after isolation. However, whether GDNF could rescue human islets from nutrient deprivation and ER stress-mediated apoptosis is unknown. Herein, by mimicking those conditions in vitro, we have shown that GDNF significantly improved glucose stimulated insulin secretion, reduced apoptosis and proinsulin:insulin ratio in nutrient deprived human islets. Furthermore, GDNF alleviated thapsigargin-induced ER stress evidenced by reduced expressions of IRE1α and BiP and consequently apoptosis. Importantly, this was associated with an increase in phosphorylation of PI3K/AKT and GSK3B signaling pathway. Transplantation of ER stressed human islets pre-treated with GDNF under kidney capsule of diabetic mice resulted in reduced expressions of IRE1α and BiP in human islet grafts with improved grafts function shown by higher levels of human C-peptide post-transplantation. We suggest that GDNF has protective and anti-apoptotic effects on nutrient deprived and ER stress activated human islets and could play a significant role in rescuing human islets from stress responses.

LIGHT/TNFSF14 is increased in patients with type 2 diabetes mellitus and promotes islet cell dysfunction and endothelial cell inflammation in vitro.

AIMS/HYPOTHESIS: Activation of inflammatory pathways is involved in the pathogenesis of type 2 diabetes mellitus. On the basis of its role in vascular inflammation and in metabolic disorders, we hypothesised that the TNF superfamily (TNFSF) member 14 (LIGHT/TNFSF14) could be involved in the pathogenesis of type 2 diabetes mellitus. METHODS: Plasma levels of LIGHT were measured in two cohorts of type 2 diabetes mellitus patients (191 Italian and 40 Norwegian). Human pancreatic islet cells and arterial endothelial cells were used to explore regulation and relevant effects of LIGHT in vitro. RESULTS: Our major findings were: (1) in both diabetic cohorts, plasma levels of LIGHT were significantly raised compared with sex- and age-matched healthy controls (n = 32); (2) enhanced release from activated platelets seems to be an important contributor to the raised LIGHT levels in type 2 diabetes mellitus; (3) in human pancreatic islet cells, inflammatory cytokines increased the release of LIGHT and upregulated mRNA and protein levels of the LIGHT receptors lymphotoxin ß receptor (LTßR) and TNF receptor superfamily member 14 (HVEM/TNFRSF14); (4) in these cells, LIGHT attenuated the insulin release in response to high glucose at least partly via pro-apoptotic effects; and (5) in human arterial endothelial cells, glucose boosted inflammatory response to LIGHT, accompanied by an upregulation of mRNA levels of HVEM (also known as TNFRSF14) and LTßR (also known as LTBR). CONCLUSIONS/INTERPRETATION: Our findings show that patients with type 2 diabetes mellitus are characterised by increased plasma LIGHT levels. Our in vitro findings suggest that LIGHT may contribute to the progression of type 2 diabetes mellitus by attenuating insulin secretion in pancreatic islet cells and by contributing to vascular inflammation.

Mitochondrial Respiration in Insulin-Producing ß-Cells: General Characteristics and Adaptive Effects of Hypoxia.

OBJECTIVE: To provide novel insights on mitochondrial respiration in ß-cells and the adaptive effects of hypoxia. METHODS AND DESIGN: Insulin-producing INS-1 832/13 cells were exposed to 18 hours of hypoxia followed by 20-22 hours re-oxygenation. Mitochondrial respiration was measured by high-resolution respirometry in both intact and permeabilized cells, in the latter after establishing three functional substrate-uncoupler-inhibitor titration (SUIT) protocols. Concomitant measurements included proteins of mitochondrial complexes (Western blotting), ATP and insulin secretion. RESULTS: Intact cells exhibited a high degree of intrinsic uncoupling, comprising about 50% of oxygen consumption in the basal respiratory state. Hypoxia followed by re-oxygenation increased maximal overall respiration. Exploratory experiments in peremabilized cells could not show induction of respiration by malate or pyruvate as reducing substrates, thus glutamate and succinate were used as mitochondrial substrates in SUIT protocols. Permeabilized cells displayed a high capacity for oxidative phosphorylation for both complex I- and II-linked substrates in relation to maximum capacity of electron transfer. Previous hypoxia decreased phosphorylation control of complex I-linked respiration, but not in complex II-linked respiration. Coupling control ratios showed increased coupling efficiency for both complex I- and II-linked substrates in hypoxia-exposed cells. Respiratory rates overall were increased. Also previous hypoxia increased proteins of mitochondrial complexes I and II (Western blotting) in INS-1 cells as well as in rat and human islets. Mitochondrial effects were accompanied by unchanged levels of ATP, increased basal and preserved glucose-induced insulin secretion. CONCLUSIONS: Exposure of INS-1 832/13 cells to hypoxia, followed by a re-oxygenation period increases substrate-stimulated respiratory capacity and coupling efficiency. Such effects are accompanied by up-regulation of mitochondrial complexes also in pancreatic islets, highlighting adaptive capacities of possible importance in an islet transplantation setting. Results also indicate idiosyncrasies of ß-cells that do not respire in response to a standard inclusion of malate in SUIT protocols.