Nuclear factor- B regulates f-Cell death: A critical role for A20 in f-cell protection

Apoptotic f-cell death is central to the pathogenesis of type 1 diabetes and may be important in islet graft rejection. Despite this, genetic control of f-cell apoptosis is only poorly understood. We report that inhibition of gene transcription sensitized f-cells to tumor necrosis factor (TNF)-induced apoptosis, indicating the presence of a regulated antiapoptotic response. Using oligonucleotide microarrays and real-time PCR, we identified TNFAIP3/A20 as the most highly regulated antiapoptotic gene expressed in cytokine-stimulated human and mouse islets. Cytokine induction of A20 mRNA in primary islets and insulinoma cells was rapid and observed within 1 h, consistent with A20 being an immediate early response gene in f-cells. Regulation of A20 was nuclear factor-B (NF-B)-dependent, two NF-B sites within the A20 promoter were found to be necessary and sufficient for A20 expression in f-cells. Activation of NF-B by TNF receptor-associated factor (TRAF) 2, TRAF6, NF-B-inducing kinase, or protein kinase D, which transduce signals downstream of Toll-like receptors, TNF receptors, and free radicals, respectively, were all potent activators of the A20 promoter. Moreover, A20 expression was induced in transplanted islets in vivo. Finally, A20 expression was sufficient to protect f-cells from TNF-induced apoptosis. These data demonstrate that A20 is the cardinal antiapoptotic gene in f-cells. Further, A20 expression is NF-B dependent, thus linking islet proinflammatory gene responses with protection from apoptosis

The role of nuclear factor-kappa B in beta-cell survival and function

In Type 1 diabetes, beta-cells is subjected to an autoimmune-mediated apoptoticand inflammatory attack. Whilst lymphocytes are the primary contributor of beta-celldeath, exposure of beta-cells to stress signals such as cytokines, transform these cellsinto an inflammatory state activating transcripts for toxic agents. Demonstrating asignificant role for beta-cells in participating in their own destruction through theelaboration of toxins and chemotactic molecules that could contribute to increasedcellular infiltration.The Nuclear Factor-kappa B (NF-kB) is a transcription factor that provides forearly immediate stress responses governing inflammation and cell survival. In islets,NF-kB is thought to have an important role in beta-cell inflammation and apoptosis. Fewstudies however, have explored the role of NF-kB in beta-cell protection. Indeed, wefound that the expression NF-kB is responsible for the islet-intrinsic immediate-earlypro-inflammatory gene expression. Importantly however, we also found that in islets,NF-kB is responsible for the expression and regulation of anti-apoptotic genes. Wedemonstrated for the first time that similar to other cells, the expression andregulation of the anti-inflammatory/ anti-apoptotic gene A20, in islets, is regulated byNF-kB.Consequently, we found a somewhat paradoxical role for NF-kB, where on theone hand it is responsible for beta-cell death, whilst on the other hand it is alsoresponsible for beta-cell survival. In vivo however, we found that islet survival andfunction was severely impaired in the absence of NF-kB activity. We observed thatblockade of NF-kB abrogate cytokine-induced A20 expression, and inhibited theactivation of glucose-stimulated insulin secretion in vitro. In contrast, blockade ofNF-kB by over-expression of A20 resulted in an improved islet allograft survival withgood metabolic control. Thus demonstrating the importance of NF-kB-dependentanti-apoptotic genes for islet survival and function.The findings presented in this thesis demonstrate a fundamental bimodal rolefor NF-kB in maintaining the balance of survival of beta-cells in the context of T1D.These data, uncovers a sophisticated molecular mechanism in the regulation of beta-celldeath, survival, and metabolic function. Thus providing a better understanding of therole of NF-kB in beta-cells in the context of T1D

Pulsatile portal vein insulin delivery enhances hepatic insulin action and signaling.

Insulin is secreted as discrete insulin secretory bursts at ~5-min intervals into the hepatic portal vein, these pulses being attenuated early in the development of type 1 and type 2 diabetes mellitus (T2DM). Intraportal insulin infusions (pulsatile, constant, or reproducing that in T2DM) indicated that the pattern of pulsatile insulin secretion delivered via the portal vein is important for hepatic insulin action and, therefore, presumably for hepatic insulin signaling. To test this, we examined hepatic insulin signaling in rat livers exposed to the same three patterns of portal vein insulin delivery by use of sequential liver biopsies in anesthetized rats. Intraportal delivery of insulin in a constant versus pulsatile pattern led to delayed and impaired activation of hepatic insulin receptor substrate (IRS)-1 and IRS-2 signaling, impaired activation of downstream insulin signaling effector molecules AKT and Foxo1, and decreased expression of glucokinase (Gck). We further established that hepatic Gck expression is decreased in the HIP rat model of T2DM, a defect that correlated with a progressive defect of pulsatile insulin secretion. We conclude that the physiological pulsatile pattern of insulin delivery is important in hepatic insulin signaling and glycemic control. Hepatic insulin resistance in diabetes is likely in part due to impaired pulsatile insulin secretion

Low-Dose Interleukin-2 Combined With Rapamycin Led to an Expansion of CD4+CD25+FOXP3+ Regulatory T Cells and Prolonged Human Islet Allograft Survival in Humanized Mice

© 2020 by the American Diabetes Association. Islet transplantation is an emerging therapy for type 1 diabetes and hypoglycemic unawareness. However, a key challenge for islet transplantation is cellular rejection and the requirement for long-term immunosuppression. In this study, we established a diabetic humanized NOD-scidIL2Rγnull (NSG) mouse model of T-cell-mediated human islet allograft rejection and developed a therapeutic regimen of low-dose recombinant human interleukin-2 (IL-2) combined with low-dose rapamycin to prolong graft survival. NSG mice that had received renal subcapsular human islet allografts and were transfused with 1 × 107 of human spleen mononuclear cells reconstituted human CD45+ cells that were predominantly CD3+ T cells and rejected their grafts with a median survival time of 27 days. IL-2 alone (0.3 × 106 IU/m2 or 1 × 106 IU/m2) or rapamycin alone (0.5-1 mg/kg) for 3 weeks did not prolong survival. However, the combination of rapamycin with IL-2 for 3 weeks significantly prolonged human islet allograft survival. Graft survival was associated with expansion of CD4+CD25+FOXP3+ regulatory T cells (Tregs) and enhanced transforming growth factor-β production by CD4+ T cells. CD8+ T cells showed reduced interferon-γ production and reduced expression of perforin-1. The combination of IL-2 and rapamycin has the potential to inhibit human islet allograft rejection by expanding CD4+FOXP3+ Tregs in vivo and suppressing effector cell function and could be the basis of effective tolerance-based regimens

The long noncoding RNA MALAT1 predicts human pancreatic islet isolation quality

Human islet isolation is a cost- and resource-intensive program for generating islets for cell therapy in type 1 diabetes. However, only one-third of cadaveric pancreases get to clinical transplantation because of low quality/number of islets. There is a need to identify biomarkers that predict the quality of islets, before initiating their isolation. Here, we sequenced transcriptomes from 18 human islet preparations stratified into 3 groups (group 1: best quality/transplantable islets; group 2: intermediary quality; and group 3: inferior quality/nontransplantable islets) based on routine measurements, including islet purity/viability. Machine-learning algorithms involving penalized regression analyses identified 10 long noncoding RNAs (lncRNAs) that were significantly different across all group-wise comparisons (group 1 vs. group 2, group 2 vs. group 3, and group 1 vs. group 3). Two variants of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) lncRNA were common across all comparisons. We then confirmed RNA-Seq findings in a validation set of 75 human islet preparations. Finally, in 19 pancreas samples, we demonstrated that assessing the levels of MALAT1 variants alone (receiver operator characteristic curve AUC: 0.83) offers higher specificity in predicting postisolation islet quality, further improving the predictive potential for clinical islet transplantation when combined with Edmonton Donor Points/BMI/North American Islet Donor Score. We present this resource of islet quality-stratified lncRNA transcriptome data and identify MALAT1 as a biomarker that significantly enhances current selection methods for clinical-grade (good manufacturing practice-grade) islet isolation

β Cell Hypoxia-Inducible Factor-1α Is Required for the Prevention of Type 1 Diabetes

Summary: The development of autoimmune disease type 1 diabetes (T1D) is determined by both genetic background and environmental factors. Environmental triggers include RNA viruses, particularly coxsackievirus (CV), but how they induce T1D is not understood. Here, we demonstrate that deletion of the transcription factor hypoxia-inducible factor-1α (HIF-1α) from β cells increases the susceptibility of non-obese diabetic (NOD) mice to environmentally triggered T1D from coxsackieviruses and the β cell toxin streptozotocin. Similarly, knockdown of HIF-1α in human islets leads to a poorer response to coxsackievirus infection. Studies in coxsackievirus-infected islets demonstrate that lack of HIF-1α leads to impaired viral clearance, increased viral load, inflammation, pancreatitis, and loss of β cell mass. These findings show an important role for β cells and, specifically, lack of β cell HIF-1α in the development of T1D. These data suggest new strategies for the prevention of T1D. : Lalwani et al. describe a role for β cell hypoxia-inducible factor-1α (HIF1a) in determining whether β cell injury is followed by resolution and normal function or ongoing injury, autoimmunity, and type 1 diabetes. Keywords: type 1 diabetes, coxsackievirus, beta-cell, apoptosis, autoimmunity, streptozotocin, inflammatio