Β-cell Viability Research Articles

Acute and chronic effects of the organophosphate malathion on the pancreatic α and β cell viability, cell structure, and voltage-gated K+ currents.

Studies indicate that the pesticide malathion may have a role in diabetes. Herein, we determined the effects of different concentrations of malathion on survival, ultrastructure, and electrophysiologic islet cell parameters. Acutely, high concentrations of malathion (0.5 or 1 mM) increased cell death in rat islet cells, while low concentrations (0.1 mM) caused signs of cell damage in pancreatic α and β cells. Exposure of RINm5F cells to malathion for 24 or 48 h confirmed the reduction in β-cell viability at lower concentrations (0.001–100 µM). Chronic exposure of mouse pancreatic α and β cells to 3 nM of malathion led to increased voltage-gated K+ (Kv) currents in α-cells. Our findings show a time and concentration dependency for the malathion effect on the reduction of islet cell viability and indicate that pancreatic α cells are more sensitive to malathion effects on Kv currents and cell death.

Fas Ligand-Modified Scaffolds Protect Stem Cell Derived β-Cells by Modulating Immune Cell Numbers and Polarization.

Stem cell derived β-cells have demonstrated the potential to control blood glucose levels and represent a promising treatment for Type 1 diabetes (T1D). Early engraftment post-transplantation and subsequent maturation of these β-cells are hypothesized to be limited by the initial inflammatory response, which impacts the ability to sustain normoglycemia for long periods. We investigated the survival and development of immature hPSC-derived β-cells transplanted on poly(lactide-co-glycolide) (PLG) microporous scaffolds into the peritoneal fat, a site being considered for clinical translation. The scaffolds were modified with biotin for binding of a streptavidin-FasL (SA-FasL) chimeric protein to modulate the local immune cell responses. The presence of FasL impacted infiltration of monocytes and neutrophils and altered the immune cell polarization. Conditioned media generated from SA-FasL scaffolds explanted at day 4 post-transplant did not impact hPSC-derived β-cell survival and maturation in vitro, while these responses were reduced with conditioned media from control scaffolds. Following transplantation, β-cell viability and differentiation were improved with SA-FasL modification. A sustained increase in insulin positive cell ratio was observed with SA-FasL-modified scaffolds relative to control scaffolds. These results highlight that the initial immune response can significantly impact β-cell engraftment, and modulation of cell infiltration and polarization may be a consideration for supporting long-term function at an extrahepatic site.

Vanillic acid potentiates insulin secretion and prevents pancreatic β-cells cytotoxicity under H2O2-induced oxidative stress.

Oxidative stress is known to impair cellular functions and, therefore, plays a significant role in the pathophysiology of various diseases, including diabetes. The persistently elevated glucose levels may cause enhanced mitochondrial reactive oxygen species generation, which in turn can damage the pancreatic β-cells. In this study, we have investigated the effect of vanillic acid on preventing H2O2-induced β-cells death and retaining its insulin secretion potentiating effect in the presence of H2O2. The insulin secretion from the BRIN-BD11 cells was quantified using ELISA-based assays. The viability of the cells was assessed by estimated by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) colorimetric assay and DAPI staining. The expression levels of apoptotic and antioxidant proteins were estimated by western blot experiments. Vanillic acid protected pancreatic β-cells viability and function under the H2O2 oxidative stress condition. The Erk1/2 activation appears to play an important role in vanillic acid potentiated insulin secretion and protection of the β-cells in the presence of H2O2. Vanillic acid pretreated cells exhibited enhanced expression of antioxidant enzymes such as catalase and SOD-2 and reduced the expression of proapoptotic markers such as BAX and BAD. In addition, it also enhanced the expression of oxidative stress-sensitive transcription factor Nrf-2 and cell survival protein Akt. The present study shows that vanillic acid potentiates insulin secretion and protects pancreatic β-cells from H2O2-induced oxidative stress.

N-terminal BET bromodomain inhibitors disrupt a BRD4-p65 interaction and reduce inducible nitric oxide synthase transcription in pancreatic β-cells.

Chronic inflammation of pancreatic islets is a key driver of β-cell damage that can lead to autoreactivity and the eventual onset of autoimmune diabetes (T1D). In the islet, elevated levels of proinflammatory cytokines induce the transcription of the inducible nitric oxide synthase (iNOS) gene, NOS2, ultimately resulting in increased nitric oxide (NO). Excessive or prolonged exposure to NO causes β-cell dysfunction and failure associated with defects in mitochondrial respiration. Recent studies showed that inhibition of the bromodomain and extraterminal domain (BET) family of proteins, a druggable class of epigenetic reader proteins, prevents the onset and progression of T1D in the non-obese diabetic mouse model. We hypothesized that BET proteins co-activate transcription of cytokine-induced inflammatory gene targets in β-cells and that selective, chemotherapeutic inhibition of BET bromodomains could reduce such transcription. Here, we investigated the ability of BET bromodomain small molecule inhibitors to reduce the β-cell response to the proinflammatory cytokine interleukin 1 beta (IL-1β). BET bromodomain inhibition attenuated IL-1β-induced transcription of the inflammatory mediator NOS2 and consequent iNOS protein and NO production. Reduced NOS2 transcription is consistent with inhibition of NF-κB facilitated by disrupting the interaction of a single BET family member, BRD4, with the NF-κB subunit, p65. Using recently reported selective inhibitors of the first and second BET bromodomains, inhibition of only the first bromodomain was necessary to reduce the interaction of BRD4 with p65 in β-cells. Moreover, inhibition of the first bromodomain was sufficient to mitigate IL-1β-driven decreases in mitochondrial oxygen consumption rates and β-cell viability. By identifying a role for the interaction between BRD4 and p65 in controlling the response of β-cells to proinflammatory cytokines, we provide mechanistic information on how BET bromodomain inhibition can decrease inflammation. These studies also support the potential therapeutic application of more selective BET bromodomain inhibitors in attenuating β-cell inflammation.

Regulation of oxaliplatin and carboplatin on the assembly behavior and cytotoxicity of human islet amyloid polypeptide

Human islet amyloid polypeptide (hIAPP) is associated with the pathology of Type II diabetes (T2DM) due to its misfolding and amyloid deposition. The peptide is widely concerned as a potential drug target, and the prevention of hIAPP fibrillation is a rational therapeutic strategy for T2DM. Platinum complexes are promising anticancer agents with good biocompatibility, they can resist the aggregation of amyloid peptides, while the effects of oxaliplatin and carboplatin on hIAPP fibrillation are unknown. In the present work, we selected the two platinum drugs to reveal their inhibition and disaggregation against hIAPP fibrillation by various biophysical methods. The two complexes impeded hIAPP fibril formation and dispersed the aggregates into small oligomers and most monomers. They also reduced peptides oligomerization and promoted rat insulinoma β-cells viability. They bound to hIAPP mainly through metal coordination and hydrophobic interactions. Moreover, oxaliplatin showed better inhibition and regulation on peptides aggregation and cytotoxicity than carboplatin. This work is of important biomedical values for clinical platinum drugs against T2DM and other amyloidosis related diseases.

Involvement of Ferroptosis in Diabetes-Induced Liver Pathology.

Cell death plays an important role in diabetes-induced liver dysfunction. Ferroptosis is a newly defined regulated cell death caused by iron-dependent lipid peroxidation. Our previous studies have shown that high glucose and streptozotocin (STZ) cause β-cell death through ferroptosis and that ferrostatin-1 (Fer-1), an inhibitor of ferroptosis, improves β-cell viability, islet morphology, and function. This study was aimed to examine in vivo the involvement of ferroptosis in diabetes-related pathological changes in the liver. For this purpose, male C57BL/6 mice, in which diabetes was induced with STZ (40 mg/kg/5 consecutive days), were treated with Fer-1 (1 mg/kg, from day 1–21 day). It was found that in diabetic mice Fer-1 improved serum levels of ALT and triglycerides and decreased liver fibrosis, hepatocytes size, and binucleation. This improvement was due to the Fer-1-induced attenuation of ferroptotic events in the liver of diabetic mice, such as accumulation of pro-oxidative parameters (iron, lipofuscin, 4-HNE), decrease in expression level/activity of antioxidative defense-related molecules (GPX4, Nrf2, xCT, GSH, GCL, HO-1, SOD), and HMGB1 translocation from nucleus into cytosol. We concluded that ferroptosis contributes to diabetes-related pathological changes in the liver and that the targeting of ferroptosis represents a promising approach in the management of diabetes-induced liver injury.

The loss of pancreatic islet NADPH oxidase (NOX)2 improves islet transplantation

Islet transplantation is a promising treatment strategy for type 1 diabetes mellitus (T1DM) patients. However, oxidative stress-induced graft failure due to an insufficient revascularization is a major problem of this therapeutic approach. NADPH oxidase (NOX)2 is an important producer of reactive oxygen species (ROS) and several studies have already reported that this enzyme plays a crucial role in the endocrine function and viability of β-cells. Therefore, we hypothesized that targeting islet NOX2 improves the outcome of islet transplantation. To test this, we analyzed the cellular composition and viability of isolated wild-type (WT) and Nox2−/− islets by immunohistochemistry as well as different viability assays. Ex vivo, the effect of Nox2 deficiency on superoxide production, endocrine function and anti-oxidant protein expression was studied under hypoxic conditions. In vivo, we transplanted WT and Nox2−/− islets into mouse dorsal skinfold chambers and under the kidney capsule of diabetic mice to assess their revascularization and endocrine function, respectively. We found that the loss of NOX2 does not affect the cellular composition and viability of isolated islets. However, decreased superoxide production, higher glucose-stimulated insulin secretion as well as expression of nuclear factor erythroid 2-related factor (Nrf)2, heme oxygenase (HO)-1 and superoxide dismutase 1 (SOD1) was detected in hypoxic Nox2−/− islets when compared to WT islets. Moreover, we detected an early revascularization, a higher take rate and restoration of normoglycemia in diabetic mice transplanted with Nox2−/− islets. These findings indicate that the suppression of NOX2 activity represents a promising therapeutic strategy to improve engraftment and function of isolated islets.

A New Berberine Preparation Protects Pancreatic Islet Cells from Apoptosis Mediated by Inhibition of Phospholipase A2/p38 MAPK Pathway.

We studied an amorphous solid dispersion of berberine with absorption enhancer sodium caprate (Huang-Gui solid dispersion preparations, HGSD). A therapeutic effect of HGSD was revealed in mice with type 2 diabetes mellitus and palmitate-induced injury to MIN6 β-cells. HGSD treatment (150 mg/kg) improved glucose metabolism and decreased β-cell apoptosis in diabetic mice. Furthermore, the effective component of HGSD berberine significantly attenuated the palmitate-induced decrease in MIN6 β-cells viability and insulin secretion. Moreover, molecular docking analysis and Western blotting showed that berberine decreased cell apoptosis and expression of group VIA phospholipase A2 (iPLA2), p38 mitogen-activated protein kinase (p38 MAPK), and caspase-3. These data suggest that HGSD treatment protected β-cells via inhibiting the iPLA2/p38 MAPK pathway.

247-LB: Pancreatic Elastase Regulates ß-Cell Viability by Impairing the Mechanosignaling Pathway

The discovery of SerpinB1 as an inducer of human β-cell proliferation pointed to acinar cell-derived proteases such as pancreatic elastase (PE) being involved in regulating β-cell viability. Indeed, we observed that expression of a major form of PE, namely CELA3B, was increased in acinar cells from donors with type 2 diabetes (T2D) compared to samples from non-diabetic (ND) donors. Surprisingly, CELA3B protein levels were higher in T2D vs. ND islets, although its gene expression levels were virtually undetectable. We confirmed that increasing doses of exogenous PE is able to decrease viability of EndoC-βH3 (βH3) cells in vitro. To evaluate the effects of PE inhibition on β-cell survival, we used telaprevir, an FDA-approved drug, that we identified as a potent PE blocker using a High-Throughput Screening assays with an IC50 of 15.6 nM to inhibit PE activity. Consistent with the mitogenic effects of SerpinB1 on β-cells, telaprevir also increased human β-cell viability in in vitro and in vivo systems. By performing phospho-antibody microarrays and single-cell RNA-Seq in human islets treated with vehicle or telaprevir, we identified the mechano-signaling pathway, which integrates the signals generated by the extracellular matrix (ECM) , as a candidate pathway linking PE inhibition to β-cell viability. We validated the results by treating EndoC-βH1 (βH1) cells lacking focal adhesion kinase (siFAK) with PE +/- telaprevir. We observed that PE impaired the mechano-signaling cascade, and these effects were reversed by telaprevir in control cells, but not in siFAK cells. Furthermore, using the novel approach of Brillouin microscopy we observed that PE increased the intracellular stiffness in βH1 cells, as a readout of cytoskeleton arrangements, whereas telaprevir blunted these effects. These data point to novel pathways underlying the acinar-β-cell crosstalk that could be harnessed for therapeutic approaches to resolve diabetes in patients. Disclosure G. Basile: None. B. Wagner: Research Support; Japan Tobacco Inc., Novo Nordisk Foundation, Ono Pharmaceutical Co., Ltd., Stock/Shareholder; Moderna, Inc., Pfizer Inc. R. Kulkarni: Advisory Panel; Biomea, Novo Nordisk, REDD Pharma, Consultant; Relay Therapeutics, Research Support; Inversago, SerPlus. A. Vetere: None. J. Hu: None. O. Ijaduola: None. D. F. De jesus: n/a. K. Fukuda: None. A. M. Eltony: None. A. Yun: None. A. Choudhary: None. Funding JDRF: 3-PDF-2020-935-A-NNIH: U01 DK123717NIH: UC4 DK116255

10-OR: Combination Therapy with Anti-CD3 Antibody and Harmine plus Exendin-4 Enhances Remission from Recent-Onset Type 1 Diabetes.

Type 1 diabetes (T1D) results from loss of both immune tolerance and functional β-cells. Administration of harmine (H) plus exendin-4 (E) markedly induces human β-cell expansion. Anti-CD3 antibody treatment reduces C-peptide loss in T1D patients. Here, we tested whether combination therapy with anti-CD3 antibody and H+E enhances T1D remission in non-obese diabetic (NOD) mice. First, we tested whether H+E protects human β-cells against inflammation and ER stress. We found that H+E, but not the drugs alone, significantly reduced both thapsigargin- and cytokine-induced human β-cell apoptosis. Single-cell RNAseq of human islets treated with cytokines and H+E showed reduced IL1β and IFNγ signaling in β-cells. Apoptosis genes such as CYLD and RIPK1 were downregulated and prosurvival genes such as HIF1A and VEGFA were upregulated in β-cells of H+E-treated islets. Next, treatment of early-onset diabetic NOD mice with H+E (daily ip) for 8 weeks improved glucose homeostasis but failed to induce long-lasting immune tolerance and T1D remission. Therefore, we next treated early-onset diabetic NOD mice with low-dose anti-CD3 antibody daily for 3 days (40µg/day) followed by 8 weeks treatment with H+E or vehicle (V) (Alzet pumps) . Treatment with anti-CD3 and H+E eliminated hyperglycemia in 100% of the mice vs. only 20% remission in anti-CD3 and V-treated mice. CD3+ T cell numbers were significantly and similarly reduced (∼50%) in mice treated with H+E or V. Preliminary results indicate that activated T cells in pancreatic lymph nodes (PLNs) and islet insulitis were significantly reduced, and T regulatory cells in spleen and PLNs were increased in H+E-treated mice. TUNEL+ β-cells were decreased and Ki67+ β-cells were increased in the pancreas of H+E-treated mice. Collectively, these results indicate that combination therapy with low-dose anti-CD3 antibody and H+E enhances T1D remission in diabetic NOD mice by increasing β-cell viability and favoring immune tolerance. Disclosure G.Lu: None. R.Kang: None. Y.Li: None. P.Wang: None. C.Rosselot: None. R.J.Devita: None. A.F.Stewart: None. A.Garcia-ocana: Consultant; Sun Pharmaceutical Industries Ltd. Funding NIH DK105015

The cultivation conditions affect the aggregation and functionality of β-cell lines alone and in coculture with mesenchymal stromal/stem cells.

The manufacturing of viable and functional β-cell spheroids is required for diabetes cell therapy and drug testing. Mesenchymal stromal/stem cells (MSCs) are known to improve β-cell viability and functionality. We therefore investigated the aggregation behavior of three different β-cell lines (rat insulinoma-1 cell line [INS-1], mouse insulinoma-6 cell line [MIN6], and a cell line formed by the electrofusion of primary human pancreatic islets and PANC-1 cells [1.1B4]), two MSC types, and mixtures of β-cells and MSCs under different conditions. We screened several static systems to produce uniform β-cell and MSC spheroids, finding cell-repellent plates the most suitable. The three different β-cell lines differed in their aggregation behavior, spheroid size, and growth in the same static environment. We found no major differences in spheroid formation between primary MSCs and an immortalized MSC line, although both differed with regard to the aggregation behavior of the β-cell lines. All spheroids showed a reduced viability due to mass transfer limitations under static conditions. We therefore investigated three dynamic systems (shaking multi-well plates, spinner flasks, and shaking flasks). In shaking flasks, there were no β-cell-line-dependent differences in aggregation behavior, resulting in uniform and highly viable spheroids. We found that the aggregation behavior of the β-cell lines changed in a static coculture with MSCs. The β-cell/MSC coculture conditions must be refined to avoid a rapid segregation into distinct populations under dynamic conditions.

Adipose Tissue Secretion Pattern Influences β-Cell Wellness in the Transition from Obesity to Type 2 Diabetes.

The dysregulation of the β-cell functional mass, which is a reduction in the number of β-cells and their ability to secure adequate insulin secretion, represents a key mechanistic factor leading to the onset of type 2 diabetes (T2D). Obesity is recognised as a leading cause of β-cell loss and dysfunction and a risk factor for T2D. The natural history of β-cell failure in obesity-induced T2D can be divided into three steps: (1) β-cell compensatory hyperplasia and insulin hypersecretion, (2) insulin secretory dysfunction, and (3) loss of β-cell mass. Adipose tissue (AT) secretes many hormones/cytokines (adipokines) and fatty acids that can directly influence β-cell function and viability. As this secretory pattern is altered in obese and diabetic patients, it is expected that the cross-talk between AT and pancreatic β-cells could drive the maintenance of the β-cell integrity under physiological conditions and contribute to the reduction in the β-cell functional mass in a dysmetabolic state. In the current review, we summarise the evidence of the ability of the AT secretome to influence each step of β-cell failure, and attempt to draw a timeline of the alterations in the adipokine secretion pattern in the transition from obesity to T2D that reflects the progressive deterioration of the β-cell functional mass.

Inclusion of extracellular matrix molecules and necrostatin-1 in the intracapsular environment of alginate-based microcapsules synergistically protects pancreatic β cells against cytokine-induced inflammatory stress

Immunoisolation of pancreatic islets in alginate-based microcapsules is a promising approach for grafting of islets in absence of immunosuppression. However, loss and damage to the extracellular matrix (ECM) during islet isolation enhance susceptibility of islets for inflammatory stress. In this study, a combined strategy was applied to reduce this stress by incorporating ECM components (collagen type IV/RGD) and necroptosis inhibitor, necrostatin-1 (Nec-1) in alginate-based microcapsules in vitro. To demonstrate efficacy, viability and function of MIN6 β-cells and human islets in capsules with collagen type IV/RGD and/or Nec-1 was investigated in presence and absence of IL-1β, IFN-γ and TNF-α. The combination of collagen type IV/RGD and Nec-1 had higher protective effects than the molecules alone. Presence of collagen type IV/RGD and Nec-1 in the intracapsular environment reduced cytokine-induced overproduction of free radical species and unfavorable shifts in mitochondrial dynamics. In addition, the ECM components collagen type IV/RGD prevented a cytokine induced suppression of the FAK/Akt pathway. Our data indicate that the inclusion of collagen type IV/RGD and Nec-1 in the intracapsular environment prevents islet-cell loss when exposed to inflammatory stress, which might contribute to higher survival of β-cells in the immediate period after transplantation. This approach of inclusion of stress reducing agents in the intracapsular environment of immunoisolating devices may be an effective way to enhance the longevity of encapsulated islet grafts. Statement of significanceIslet-cells in immunoisolated alginate-based microcapsules are very susceptible to inflammatory stress which impacts long-term survival of islet grafts. Here we show that incorporation of ECM components (collagen type IV/RGD) and necrostatin-1 (Nec-1) in the intracapsular environment of alginate-based capsules attenuates this susceptibility and promotes islet-cell survival. This effect induced by collagen type IV/RGD and Nec-1 was probably due to lowering free radical production, preventing mitochondrial dysfunction and by maintaining ECM/integrin/FAK/Akt signaling and Nec-1/RIP1/RIP3 signaling. Our study provides an effective strategy to extend longevity of islet grafts which might be of great potential for future clinical application of immunoisolated cells.

NMDA Receptor Antagonists Increase the Release of GLP-1 From Gut Endocrine Cells.

Type 2 diabetes mellitus (T2DM) remains one of the most pressing health issues facing modern society. Several antidiabetic drugs are currently in clinical use to treat hyperglycaemia, but there is a need for new treatments that effectively restore pancreatic islet function in patients. Recent studies reported that both murine and human pancreatic islets exhibit enhanced insulin release and β-cell viability in response to N-methyl-D-aspartate (NMDA) receptor antagonists. Furthermore, oral administration of dextromethorphan, an over-the-counter NMDA receptor antagonist, to diabetic patients in a small clinical trial showed improved glucose tolerance and increased insulin release. However, the effects of NMDA receptor antagonists on the secretion of the incretin hormone GLP-1 was not tested, and nothing is known regarding how NMDA receptor antagonists may alter the secretion of gut hormones. This study demonstrates for the first time that, similar to β-cells, the NMDA receptor antagonist MK-801 increases the release of GLP-1 from a murine L-cell enteroendocrine model cell line, GLUTag cells. Furthermore, we report the 3′ mRNA expression profiling of GLUTag cells, with a specific focus on glutamate-activated receptors. We conclude that if NMDA receptor antagonists are to be pursued as an alternative, orally administered treatment for T2DM, it is essential that the effects of these drugs on the release of gut hormones, and specifically the incretin hormones, are fully investigated.

Phenotypic Screening for Small Molecules that Protect β-Cells from Glucolipotoxicity.

Type 2 diabetes is marked by progressive β-cell failure, leading to loss of β-cell mass. Increased levels of circulating glucose and free fatty acids associated with obesity lead to β-cell glucolipotoxicity. There are currently no therapeutic options to address this facet of β-cell loss in obese type 2 diabetes patients. To identify small molecules capable of protecting β-cells, we performed a high-throughput screen of 20,876 compounds in the rat insulinoma cell line INS-1E in the presence of elevated glucose and palmitate. We found 312 glucolipotoxicity-protective small molecules (1.49% hit rate) capable of restoring INS-1E viability, and we focused on 17 with known biological targets. 16 of the 17 compounds were kinase inhibitors with activity against specific families including but not limited to cyclin-dependent kinases (CDK), PI-3 kinase (PI3K), Janus kinase (JAK), and Rho-associated kinase 2 (ROCK2). 7 of the 16 kinase inhibitors were PI3K inhibitors. Validation studies in dissociated human islets identified 10 of the 17 compounds, namely, KD025, ETP-45658, BMS-536924, AT-9283, PF-03814735, torin-2, AZD5438, CP-640186, ETP-46464, and GSK2126458 that reduced glucolipotoxicity-induced β-cell death. These 10 compounds decreased markers of glucolipotoxicity including caspase activation, mitochondrial depolarization, and increased calcium flux. Together, these results provide a path forward toward identifying novel treatments to preserve β-cell viability in the face of glucolipotoxicity.

Associations between persistent organic pollutants and type 1 diabetes in youth

BackgroundDiabetes affects millions of people worldwide with a continued increase in incidence occurring within the pediatric population. The potential contribution of persistent organic pollutants (POPs) to diabetes in youth remains poorly known, especially regarding type 1 diabetes (T1D), generally the most prevalent form of diabetes in youth. ObjectivesWe investigated the associations between POPs and T1D in youth and studied the impacts of POPs on pancreatic β-cell function and viability in vitro. MethodsWe used data and plasma samples from the SEARCH for Diabetes in Youth Case Control Study (SEARCH-CC). Participants were categorized as Controls, T1D with normal insulin sensitivity (T1D/IS), and T1D with insulin resistance (T1D/IR). We assessed plasma concentrations of polychlorinated biphenyls (PCBs) and organochlorine pesticides and estimated the odds of T1D through multivariable logistic regression. In addition, we performed in vitro experiments with the INS-1E pancreatic β-cells. Cells were treated with PCB-153 or p,p’-DDE at environmentally relevant doses. We measured insulin production and secretion and assessed the mRNA expression of key regulators involved in insulin synthesis (Ins1, Ins2, Pdx1, Mafa, Pcsk1/3, and Pcsk2), glucose sensing (Slc2a2 and Gck), and insulin secretion (Abcc8, Kcnj11, Cacna1d, Cacna1b, Stx1a, Snap25, and Sytl4). Finally, we assessed the effects of PCB-153 and p,p’-DDE on β-cell viability. ResultsAmong 442 youths, 112 were controls, 182 were classified with T1D/IS and 148 with T1D/IR. The odds ratios (OR) of T1D/IS versus controls were statistically significant for p,p’-DDE (OR 2.0, 95% confidence interval (CI) 1.0, 3.8 and 2.4, 95% CI 1.2, 5.0 for 2nd and 3rd tertiles, respectively), trans-nonachlor (OR 2.5, 95% CI 1.3, 5.0 and OR 2.3, 95% CI 1.1, 5.1 for 2nd and 3rd tertiles, respectively), and PCB-153 (OR 2.3, 95% CI 1.1, 4.6 for 3rd tertile). However, these associations were not observed in participants with T1D/IR. At an experimental level, treatment with p,p’-DDE or PCB-153, at concentrations ranging from 1 × 10-15 M to 5 × 10-6 M, impaired the ability of pancreatic β-cells to produce and secrete insulin in response to glucose. These failures were paralleled by impaired Ins1 and Ins2 mRNA expression. In addition, among different targeted genes, PCB-153 significantly reduced Slc2a2 and Gck mRNA expression whereas p,p’-DDE mainly affected Abcc8 and Kcnj11. While treatment with PCB-153 or p,p’-DDE for 2 days did not affect β-cell viability, longer treatment progressively killed the β-cells. ConclusionThese results support a potential role of POPs in T1D etiology and demonstrate a high sensitivity of pancreatic β-cells to POPs.

MiR-25 and miR-92b regulate insulin biosynthesis and pancreatic β-cell apoptosis

Pancreatic β-cell failure is a central hallmark of the pathogenesis of diabetes mellitus; however, the molecular basis underlying chronic inflammation-caused β-cell failure remains unclear. This study reported here specifically assessed the association between miR-25/miR-92b family and β-cell failure in diabetes. IL-1β and two additional ER stress activators, palmitate and tunicamycin were applied to evaluate the expression level miR-25 by Taqman® RT-PCR. Glucose- and potassium-stimulated insulin secretion assays were performed to assess β-cell function. Dual-luciferase activity, and western blotting assays were utilized for miR-25 target gene verification. CCK-8 and TUNEL staining were used to evaluate β-cell viability and apoptosis. miRNA ChIP identified the increased level of miR-25 in INS-1 cells by IL-1β treatment. Expression levels of miR-25 were significantly upregulated with the treatment of IL-1β, palmitate or tunicamycin in both INS-1 cells and human islets. Ectopic elevation of miR-25 recapitulated most featured β-cell defects caused by IL-1β, including inhibition of insulin biosynthesis and increased β-cell apoptosis. These detrimental effects of miR-25 relied on its seed sequence recognition and repressed expression of its target genes Neurod1 and Mcl1. The miR-25/NEUROD1 axis reduced insulin biosynthesis via transcriptional regulation of β-cell specific genes. The miR-25/MCL1 axis caused β-cell apoptosis in a CASPASE-3/PARP1-dependent manner. Comparable impairments were generated by miR-92b and miR-25, emphasizing the redundant biological roles of miRNA family members with the same seed sequence. MiR-25/miR-92b family plays a major role in β-cell failure occurring under inflammation and diabetes states.

Gut Metabolite Trimethylamine N-Oxide Protects INS-1 β-Cell and Rat Islet Function under Diabetic Glucolipotoxic Conditions.

Serum accumulation of the gut microbial metabolite trimethylamine N-oxide (TMAO) is associated with high caloric intake and type 2 diabetes (T2D). Impaired pancreatic β-cell function is a hallmark of diet-induced T2D, which is linked to hyperglycemia and hyperlipidemia. While TMAO production via the gut microbiome-liver axis is well defined, its molecular effects on metabolic tissues are unclear, since studies in various tissues show deleterious and beneficial TMAO effects. We investigated the molecular effects of TMAO on functional β-cell mass. We hypothesized that TMAO may damage functional β-cell mass by inhibiting β-cell viability, survival, proliferation, or function to promote T2D pathogenesis. We treated INS-1 832/13 β-cells and primary rat islets with physiological TMAO concentrations and compared functional β-cell mass under healthy standard cell culture (SCC) and T2D-like glucolipotoxic (GLT) conditions. GLT significantly impeded β-cell mass and function by inducing oxidative and endoplasmic reticulum (ER) stress. TMAO normalized GLT-mediated damage in β-cells and primary islet function. Acute 40µM TMAO recovered insulin production, insulin granule formation, and insulin secretion by upregulating the IRE1α unfolded protein response to GLT-induced ER and oxidative stress. These novel results demonstrate that TMAO protects β-cell function and suggest that TMAO may play a beneficial molecular role in diet-induced T2D conditions.

Exendin-4-Conjugated Manganese Magnetism-Engineered Iron Oxide Nanoparticles as a Potential Magnetic Resonance Imaging Contrast Agent for Tracking Transplanted β-Cells.

To specifically detect and trace transplanted islet β-cells by magnetic resonance imaging (MRI), we conjugated manganese magnetism-engineered iron oxide nanoparticles (MnMEIO NPs) with exendin-4 (Ex4) which specifically binds glucagon-like peptide-1 receptors on the surface of β-cells. The size distribution of MnMEIO and MnMEIO-Ex4 NPs were 67.8 ± 1.3 and 70.2 ± 2.3 nm and zeta potential 33.3 ± 0.5 and 0.6 ± 0.1 mV, respectively. MnMEIO and MnMEIO-Ex4 NPs with iron content ≤ 40 μg/mL did not affect MIN6 β-cell viability and insulin secretion. Positive iron staining was found in MIN6 β-cells loaded with MnMEIO-Ex4 NPs but not in those with MnMEIO NPs. A transmission electron microscope confirmed MnMEIO-Ex4 NPs were distributed in the cytoplasm of MIN6. In vitro MR images revealed a loss of signal intensity in MIN6 β-cells labeled with MnMEIO-Ex4 NPs but not with MnMEIO NPs. After transplantation of islets labeled with MnMEIO-Ex4, the graft under kidney capsule could be visualized on MRI as persistent hypointense areas up to 17 weeks. Moreover, histology of the islet graft showed positive staining for insulin, glucagon and iron. Our results indicate MnMEIO-Ex4 NPs are safe and effective for the detection and long-term monitoring of transplanted β-cells by MRI.

Expression of CD47 in the pancreatic β-cells of people with recent-onset type 1 diabetes varies according to disease endotype.

We are studying the dialogue between β-cells and the immune system in type 1 diabetes and have identified a cell surface receptor, signal regulatory protein-alpha (SIRPα) as an important component in the regulation of β-cell survival. SIRPα interacts with another protein, CD47, to mediate signalling. In the present work, we have studied the expression and role of CD47 in human islet cells in type 1 diabetes. Clonal EndoC-βH1 cells were employed for functional studies. Cells were exposed to pro-inflammatory cytokines and their viability monitored by flow cytometry after staining with propidium iodide. Targeted knockdown of CD47 or SIRPα was achieved with small interference RNA molecules and the expression of relevant proteins studied by Western blotting or immunocytochemistry. Human pancreas sections were selected from the Exeter Archival Diabetes Biobank and used to examine the expression of CD47 by immunofluorescence labelling. Image analysis was employed to quantify expression. CD47 is abundantly expressed in both α and β cells in human pancreas. In type 1 diabetes, the levels of CD47 are increased in α cells across all age groups, whereas the expression in β-cells varies according to disease endotype. Knockdown of either CD47 or SIRPα in EndoC-βH1 cells resulted in a loss of viability. We conclude that the CD47 plays a previously unrecognised role in the regulation of β-cell viability. This system is dysregulated in type 1 diabetes suggesting that it may be targeted therapeutically to slow disease progression.