Browsing by Subject "Islet"
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Item Circulating unmethylated CHTOP and INS DNA fragments provide evidence of possible islet cell death in youth with obesity and diabetes(BMC, 2020-07-31) Syed, Farooq; Tersey, Sarah A.; Turatsinze, Jean-Valery; Felton, Jamie L.; Kang, Nicole Jiyun; Nelson, Jennifer B.; Sims, Emily K.; Defrance, Mathieu; Bizet, Martin; Fuks, Francois; Cnop, Miriam; Bugliani, Marco; Marchetti, Piero; Ziegler, Anette-Gabriele; Bonifacio, Ezio; Webb-Robertson, Bobbie-Jo; Balamurugan, Appakalai N.; Evans-Molina, Carmella; Eizirik, Decio L.; Mather, Kieren J.; Arslanian, Silva; Mirmira, Raghavendra G.; Pediatrics, School of MedicineBackground Identification of islet β cell death prior to the onset of type 1 diabetes (T1D) or type 2 diabetes (T2D) might allow for interventions to protect β cells and reduce diabetes risk. Circulating unmethylated DNA fragments arising from the human INS gene have been proposed as biomarkers of β cell death, but this gene alone may not be sufficiently specific to report β cell death. Results To identify new candidate genes whose CpG sites may show greater specificity for β cells, we performed unbiased DNA methylation analysis using the Infinium HumanMethylation 450 array on 64 human islet preparations and 27 non-islet human tissues. For verification of array results, bisulfite DNA sequencing of human β cells and 11 non-β cell tissues was performed on 5 of the top 10 CpG sites that were found to be differentially methylated. We identified the CHTOP gene as a candidate whose CpGs show a greater frequency of unmethylation in human islets. A digital PCR strategy was used to determine the methylation pattern of CHTOP and INS CpG sites in primary human tissues. Although both INS and CHTOP contained unmethylated CpG sites in non-islet tissues, they occurred in a non-overlapping pattern. Based on Naïve Bayes classifier analysis, the two genes together report 100% specificity for islet damage. Digital PCR was then performed on cell-free DNA from serum from human subjects. Compared to healthy controls (N = 10), differentially methylated CHTOP and INS levels were higher in youth with new onset T1D (N = 43) and, unexpectedly, in healthy autoantibody-negative youth who have first-degree relatives with T1D (N = 23). When tested in lean (N = 32) and obese (N = 118) youth, increased levels of unmethylated INS and CHTOP were observed in obese individuals. Conclusion Our data suggest that concurrent measurement of circulating unmethylated INS and CHTOP has the potential to detect islet death in youth at risk for both T1D and T2D. Our data also support the use of multiple parameters to increase the confidence of detecting islet damage in individuals at risk for developing diabetes.Item Deoxyhypusine synthase promotes differentiation and proliferation of T helper type 1 (Th1) cells in autoimmune diabetes(ASBMB, 2013-12-20) Colvin, Stephanie C.; Maier, Bernhard; Morris, David L.; Tersey, Sarah A.; Mirmira, Raghavendra G.; Department of Pediatrics, IU School of MedicineIn type 1 diabetes, cytokines arising from immune cells cause islet β cell dysfunction even before overt hyperglycemia. Deoxyhypusine synthase catalyzes the crucial hypusine modification of the factor eIF5A, which promotes the translation of a subset of mRNAs involved in cytokine responses. Here, we tested the hypothesis that deoxyhypusine synthase and, secondarily, hypusinated eIF5A contribute to the pathogenesis of type 1 diabetes using the non-obese diabetic (NOD) mouse model. Pre-diabetic NOD mice that received injections of the deoxyhypusine inhibitor N1-guanyl-1,7-diaminoheptane (GC7) demonstrated significantly improved glucose tolerance, more robust insulin secretion, and reduced insulitis compared with control animals. Analysis of tissues from treated mice revealed selective reductions in diabetogenic T helper type 1 (Th1) cells in the pancreatic lymph nodes, a primary site of antigen presentation. Isolated mouse CD90.2(+) splenocytes stimulated in vitro with anti-CD3/anti-CD28 and IL-2 to mimic autoimmune T cell activation exhibited proliferation and differentiation of CD4(+) T cell subsets (Th1, Th17, and Treg), but those treated with the deoxyhypusine synthase inhibitor GC7 showed a dose-dependent block in T cell proliferation with selective reduction in Th1 cells, similar to that observed in NOD mice. Inhibition of deoxyhypusine synthase blocked post-transcriptional expression of CD25, the high affinity IL-2 receptor α chain. Our results suggest a previously unrecognized role for deoxyhypusine synthase in promoting T cell proliferation and differentiation via regulation of CD25. Inhibition of deoxyhypusine synthase may provide a strategy for reducing diabetogenic Th1 cells and preserving β cell function in type 1 diabetes.Item DOC2B enhancement of beta cell function and survival(2018-03-08) Aslamy, Arianne; Thurmond, Debbie C.; Elmendorf, Jeffrey S.; Evans-Molina, Carmella; Baucum, Anthony J.Diabetes mellitus is a complex metabolic disease that currently affects an estimated 422 million people worldwide, with incidence rates rising annually. Type 1 diabetes (T1D) accounts for 5-10% of these cases. Its complications remain a major cause of global deaths. T1D is characterized by autoimmune destruction of β-cell mass. Efforts to preserve and protect β-cell mass in the preclinical stages of T1D are limited by few blood-borne biomarkers of β-cell destruction. In healthy β-cells, insulin secretion requires soluble n-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) complexes and associated accessory regulatory proteins to promote the docking and fusion of insulin vesicles at the plasma membrane. Two target membrane (t)-SNARE proteins, Syntaxin 1/4 and SNAP25/23, and one vesicle-associated (v)-SNARE protein, VAMP2, constitute the SNARE core complex. SNARE complex assembly is also facilitated by the regulatory protein, Double C2-domain protein β (DOC2B). I hypothesized that DOC2B deficiency may underlie β-cell susceptibility to T1D damage; conversely , overexpression of DOC2B may protect β-cell mass. Indeed, with regard to DOC2B abundance, my studies show reduced levels of DOC2B in platelets and islets of prediabetic rodents and new-onset T1D humans. Remarkably, clinical islet transplantation in T1D humans restores platelet DOC2B levels, indicating a correlation With regard to protection/functional effects, DOC2B deficiency enhances susceptibility to T1D in mice, while overexpression of DOC2B selectively in β-cells protects mice from chemically induced T1D; this correlates with preservation of functional β-cell mass. Mechanistically, overexpression of DOC2B and the DOC2B peptide, C2AB, protects clonal β-cell against cytokine or thapsigargin-induced apoptosis and reduces ER stress; this is dependent on C2AB’s calcium binding capacity. C2AB is sufficient to enhance glucose stimulated insulin secretion (GSIS) and SNARE activation in clonal β-cells to the same extent as full-length DOC2B. In summary, these studies identify DOC2B as a potential biomarker and novel therapeutic target for prevention/management of T1D.Item Doc2b enrichment enhances glucose homeostasis in mice via potentiation of insulin secretion and peripheral insulin sensitivity.(Springer, 2014-07) Ramalingam, Latha; Oh, Eunjin; Thurmond, Debbie C.; Biochemistry & Molecular Biology, School of MedicineAIMS/HYPOTHESIS: Insulin secretion from pancreatic beta cells and insulin-stimulated glucose uptake into skeletal muscle are processes regulated by similar isoforms of the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) and mammalian homologue of unc-18 (Munc18) protein families. Double C2 domain β (Doc2b), a SNARE- and Munc18-interacting protein, is implicated as a crucial effector of glycaemic control. However, whether Doc2b is naturally limiting for these processes, and whether Doc2b enrichment might exert a beneficial effect upon glycaemia in vivo, remains undetermined. METHODS: Tetracycline-repressible transgenic (Tg) mice engineered to overexpress Doc2b simultaneously in the pancreas, skeletal muscle and adipose tissues were compared with wild-type (Wt) littermate mice regarding glucose and insulin tolerance, islet function in vivo and ex vivo, and skeletal muscle GLUT4 accumulation in transverse tubule/sarcolemmal surface membranes. SNARE complex formation was further assessed using Doc2b overexpressing L6-GLUT4-myc myoblasts to derive mechanisms relatable to physiological in vivo analyses. RESULTS: Doc2b Tg mice cleared glucose substantially faster than Wt mice, correlated with enhancements in both phases of insulin secretion and peripheral insulin sensitivity. Heightened peripheral insulin sensitivity correlated with elevated insulin-stimulated GLUT4 vesicle accumulation in cell surface membranes of Doc2b Tg mouse skeletal muscle. Mechanistic studies demonstrated Doc2b enrichment to enhance syntaxin-4-SNARE complex formation in skeletal muscle cells. CONCLUSIONS/INTERPRETATION: Doc2b is a limiting factor in SNARE exocytosis events pertinent to glycaemic regulation in vivo. Doc2b enrichment may provide a novel means to simultaneously boost islet and skeletal muscle function in vivo in the treatment and/or prevention of diabetes.Item Episodic β-cell death and dedifferentiation during diet-induced obesity and dysglycemia in male mice(Federation of American Societies for Experimental Biology, 2018-05-29) Tersey, Sarah A.; Levasseur, Esther M.; Syed, Farooq; Farb, Thomas B.; Orr, Kara S.; Nelson, Jennifer B.; Shaw, Janice L.; Bokvist, Krister; Mather, Kieren J.; Mirmira, Raghavendra G.; Pediatrics, School of MedicineLoss of functional islet β-cell mass through cellular death or dedifferentiation is thought to lead to dysglycemia during the progression from obesity to type 2 diabetes. To assess these processes in a mouse model of obesity, we performed measures of circulating cell-free differentially methylated insulin II ( Ins2) DNA as a biomarker of β-cell death and aldehyde dehydrogenase 1 family member A3 (ALDH1A3) and forkhead box 01 (Foxo1) immunostaining as markers of β-cell dedifferentiation. Eight-week-old, C57BL/6J mice were fed a low-fat diet (LFD; 10% kcal from fat) or a high-fat diet (HFD; 60% kcal from fat) and were followed longitudinally for up to 13 wk to measure glycemic control and β-cell mass, death, and dedifferentiation. Compared with LFD controls, β-cell mass increased during the feeding period in HFD animals, and statistically greater β-cell death (unmethylated Ins2) was detectable at 2 and 6 wk after diet initiation. Those times correspond to periods when significant step increases in fasting glucose and glucose intolerance, respectively, were detected. ALDH1A3 and Foxo1 immunostaining of the pancreas revealed evidence of β-cell dedifferentiation by 13 wk when fed an HFD, but not in LFD controls. In conclusion, early episodic β-cell death may be a feature of cellular turnover correlated with changes in glycemia during β-cell mass accrual in obesity, whereas β-cell dedifferentiation may be a feature seen later in established disease.-Tersey, S. A., Levasseur, E. M., Syed, F., Farb, T. B., Orr, K. S., Nelson, J. B., Shaw, J. L., Bokvist, K., Mather, K. J., Mirmira, R. G. Episodic β-cell death and dedifferentiation during diet-induced obesity and dysglycemia in male mice.Item Interleukin 6 protects pancreatic β cells from apoptosis by stimulation of autophagy(Federation of American Societies for Experimental Biology, 2017-09) Linnemann, Amelia K.; Blumer, Joseph; Marasco, Michelle R.; Battiola, Therese J.; Umhoefer, Heidi M.; Han, Jee Young; Lamming, Dudley W.; Davis, Dawn Belt; Pediatrics, School of MedicineIL-6 is a pleiotropic cytokine with complex roles in inflammation and metabolic disease. The role of IL-6 as a pro- or anti-inflammatory cytokine is still unclear. Within the pancreatic islet, IL-6 stimulates secretion of the prosurvival incretin hormone glucagon-like peptide 1 (GLP-1) by α cells and acts directly on β cells to stimulate insulin secretion in vitro. Uncovering physiologic mechanisms promoting β-cell survival under conditions of inflammation and stress can identify important pathways for diabetes prevention and treatment. Given the established role of GLP-1 in promoting β-cell survival, we hypothesized that IL-6 may also directly protect β cells from apoptosis. Herein, we show that IL-6 robustly activates signal transducer and activator of transcription 3 (STAT3), a transcription factor that is involved in autophagy. IL-6 stimulates LC3 conversion and autophagosome formation in cultured β cells. In vivo IL-6 infusion stimulates a robust increase in lysosomes in the pancreas that is restricted to the islet. Autophagy is critical for β-cell homeostasis, particularly under conditions of stress and increased insulin demand. The stimulation of autophagy by IL-6 is regulated via multiple complementary mechanisms including inhibition of mammalian target of rapamycin complex 1 (mTORC1) and activation of Akt, ultimately leading to increases in autophagy enzyme production. Pretreatment with IL-6 renders β cells resistant to apoptosis induced by proinflammatory cytokines, and inhibition of autophagy with chloroquine prevents the ability of IL-6 to protect from apoptosis. Importantly, we find that IL-6 can activate STAT3 and the autophagy enzyme GABARAPL1 in human islets. We also see evidence of decreased IL-6 pathway signaling in islets from donors with type 2 diabetes. On the basis of our results, we propose direct stimulation of autophagy as a novel mechanism for IL-6-mediated protection of β cells from stress-induced apoptosis.—Linnemann, A. K., Blumer, J., Marasco, M. R., Battiola, T. J., Umhoefer, H. M., Han, J. Y., Lamming, D. W., Davis, D. B. Interleukin 6 protects pancreatic β cells from apoptosis by stimulation of autophagy.Item Measurement of Differentially Methylated INS DNA Species in Human Serum Samples as a Biomarker of Islet β Cell Death(JoVE, 2016-12-21) Tersey, Sarah A.; Nelson, Jennifer B.; Fisher, Marisa M.; Mirmira, Raghavendra G.; Department of Pediatrics, IU School of MedicineThe death of islet β cells is thought to underlie the pathogenesis of virtually all forms of diabetes and to precede the development of frank hyperglycemia, especially in type 1 diabetes. The development of sensitive and reliable biomarkers of β cell death may allow for early therapeutic intervention to prevent or delay the development of diabetes. Recently, several groups including our own have reported that cell-free, differentially methylated DNA encoding preproinsulin (INS) in the circulation is correlated to β cell death in pre-type 1 diabetes and new-onset type 1 diabetes. Here, we present a step-by-step protocol using digital PCR for the measurement of cell-free INS DNA that is differentially methylated at cytosine at position -69 bp (relative to the transcriptional start site). We demonstrate that the assay can distinguish between methylated and unmethylated cytosine at position -69 bp, is linear across several orders of magnitude, provides absolute quantitation of DNA copy numbers, and can be applied to samples of human serum from individuals with new-onset type 1 diabetes and disease-free controls. The protocol described here can be adapted to any DNA species for which detection of differentially methylated cytosines is desired, whether from circulation or from isolated cells and tissues, and can provide absolute quantitation of DNA fragments.Item Mechanisms of transcriptional regulation in the maintenance of β cell function(2015-05-08) Maganti Vijaykumar, Aarthi; Mirmira, Raghavendra G.; Thurmond, Debbie C.; Herring, Paul B.; Evans-Molina, Carmella; Mosley, Amber L.The islet β cell is central to the maintenance of glucose homeostasis as the β cell is solely responsible for the synthesis of Insulin. Therefore, better understanding of the molecular mechanisms governing β cell function is crucial to designing therapies for diabetes. Pdx1, the master transcription factor of the β cell, is required for the synthesis of proteins that maintain optimal β cell function such as Insulin and glucose transporter type 2. Previous studies showed that Pdx1 interacts with the lysine methyltransferase Set7/9, relaxing chromatin and increasing transcription. Because Set7/9 also methylates non-histone proteins, I hypothesized that Set7/9-mediated methylation of Pdx1 increases its transcriptional activity. I showed that recombinant and cellular Pdx1 protein is methylated at two lysine residues, Lys123 and Lys131. Lys131 is involved in Set7/9 mediated augmented transactivation of Pdx1 target genes. Furthermore, β cell-specific Set7/9 knockout mice displayed glucose intolerance and impaired insulin secretion, accompanied by a reduction in the expression of Pdx1 target genes. Our results indicate a previously unappreciated role for Set7/9 in the maintenance of Pdx1 activity and β cell function. β cell function is regulated on both the transcriptional and translational levels. β cell function is central to the development of type 1 diabetes, a disease wherein the β cell is destroyed by immune cells. Although the immune system is considered the primary instigator of the disease, recent studies suggest that defective β cells may initiate the autoimmune response. I tested the hypothesis that improving β cell function would reduce immune infiltration of the islet in the NOD mouse, a mouse model of spontaneous type 1 diabetes. Prediabetic NOD mice treated with pioglitazone, a drug that improves β cell function, displayed an improvement in β cell function, a reduction in β cell death, accompanied by reductions in β cell autoimmunity, indicating that β cell dysfunction assists in the development of type 1 diabetes. Therefore, understanding the molecular mechanisms involved in β cell function is essential for the development of therapies for diabetes.Item Microrna 21 targets B Cell Lymphoma 2 (Bcl2) Mrna to increase beta cell apoptosis and exosomal Microrna 21 could serve as a biomarker of developing Type 1 Diabetes Mellitus(2018) Sims, Emily K.The role of beta cell miR-21 in Type 1 Diabetes (T1D) pathophysiology has been controversial. Here, we sought to define the context of beta cell miR-21 upregulation in T1D and the phenotype of beta cell miR-21 overexpression through target identification. Furthermore, we sought to identify whether circulating extracellular vesicle (EV) beta cell-derived miR-21 may reflect inflammatory stress within the islet during T1D development.. Results suggest that beta cell miR-21 is increased in in-vivo models of T1D and cytokine-treated cells/islets. miR-21 overexpression decreased cell count and viability, and increased cleaved caspase-3 levels, suggesting increased cell death. In silico prediction tools identified the anti-apoptotic mRNA B Cell Lymphoma 2 (BCL2) as a conserved miR-21 target. Consistent with this, miR-21 overexpression decreased BCL2 transcript and protein expression, while miR-21 inhibition increased BCL2 protein levels and reduced cleaved caspase-3 levels following cytokine-treatment. miR-21-mediated cell death was abrogated in 828/33 cells, which constitutively overexpress BCL-2. Luciferase assays suggested a direct interaction between miR-21 and the BCL2 3’untranslated region. With miR-21 overexpression, PRP revealed a shift of BCL-2 message toward monosome-associated fractions, indicating inhibition of BCL2 translation. Finally, overexpression in dispersed human islets confirmed a reduction in BCL2 transcripts and increased cleaved caspase 3 production. Analysis of EVs from human beta cells and islets exposed to cytokines revealed a 3-5-fold increase in miR-21. Nanoparticle tracking analysis showed no changes in EV quantity in response to cytokines, implicating specific changes within EV cargo as responsible for the miR-21 increase. Circulating EVs from diabetic non-obese diabetic (NOD) mice displayed progressive increases in miR-21 that preceded diabetes onset. To validate relevance to human T1D, we assayed serum samples collected from 19 pediatric T1D subjects at the time of diagnosis and 16 healthy controls. Consistent with our NOD data, EV miR-21 was increased 5-fold in T1D samples. In conclusion, in contrast to the pro-survival role reported in other systems, our results demonstrate that miR-21 increases beta cell death via BCL2 transcript degradation and inhibition of BCL2 translation. Furthermore, we propose that EV miR-21 may be a promising marker of developing T1D.Item Mouse and human islets survive and function after coating by biosilicification(American Physiological Society, 2013-11-15) Jaroch, David B.; Lu, Jing; Madangopal, Rajtarun; Stull, Natalie D.; Stensberg, Matthew; Shi, Jin; Kahn, Jennifer L.; Herrera-Perez, Ruth; Zeitchek, Michael; Sturgis, Jennifer; Robinson, J. Paul; Yoder, Mervin C.; Porterfield, D. Marshall; Mirmira, Raghavendra; Rickus, Jenna L.; Medicine, School of MedicineInorganic materials have properties that can be advantageous in bioencapsulation for cell transplantation. Our aim was to engineer a hybrid inorganic/soft tissue construct by inducing pancreatic islets to grow an inorganic shell. We created pancreatic islets surrounded by porous silica, which has potential application in the immunoprotection of islets in transplantation therapies for type 1 diabetes. The new method takes advantage of the islet capsule surface as a template for silica formation. Mouse and human islets were exposed to medium containing saturating silicic acid levels for 9-15 min. The resulting tissue constructs were then cultured for up to 4 wk under normal conditions. Scanning electron microscopy and energy dispersive X-ray spectroscopy was used to monitor the morphology and elemental composition of the material at the islet surface. A cytokine assay was used to assess biocompatibility with macrophages. Islet survival and function were assessed by confocal microscopy, glucose-stimulated insulin release assays, oxygen flux at the islet surface, expression of key genes by RT-PCR, and syngeneic transplant into diabetic mice.