Browsing by Subject "Beta cell"

Now showing 1 - 7 of 7
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    Circulating Unmethylated Insulin DNA As a Biomarker of Human Beta Cell Death: A Multi-laboratory Assay Comparison
    (Endocrine Society, 2020-03-01) Speake, Cate; Ylescupidez, Alyssa; Neiman, Daniel; Shemer, Ruth; Glaser, Benjamin; Tersey, Sarah A.; Usmani-Brown, Sahar; Clark, Pamela; Wilhelm, Joshua J.; Bellin, Melena D.; Herold, Kevan C.; Mirmira, Raghavendra G.; Dor, Yuval; Evans-Molina, Carmella; Pediatrics, School of Medicine
    Context: There is an unmet need for biomarkers of pancreatic beta-cell death to improve early diagnosis of type 1 diabetes, enroll subjects into clinical trials, and assess treatment response. To address this need, several groups developed assays measuring insulin deoxyribonucleic acid (DNA) with unmethylated CpG sites in cell-free DNA. Unmethylated insulin DNA should be derived predominantly from beta-cells and indicate ongoing beta-cell death. Objective: To assess the performance of three unmethylated insulin DNA assays. Design and participants: Plasma or serum samples from 13 subjects undergoing total pancreatectomy and islet autotransplantation were coded and provided to investigators to measure unmethylated insulin DNA. Samples included a negative control taken post-pancreatectomy but pretransplant, and a positive control taken immediately following islet infusion. We assessed technical reproducibility, linearity, and persistence of detection of unmethylated insulin DNA for each assay. Results: All assays discriminated between the negative sample and samples taken directly from the islet transplant bag; 2 of 3 discriminated negative samples from those taken immediately after islet infusion. When high levels of unmethylated insulin DNA were present, technical reproducibility was generally good for all assays. Conclusions: The measurement of beta cell cell-free DNA, including insulin, is a promising approach, warranting further testing and development in those with or at-risk for type 1 diabetes, as well as in other settings where understanding the frequency or kinetics of beta cell death could be useful.
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    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.
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    A novel Cre-enabled tetracycline-inducible transgenic system for tissue-specific cytokine expression in the zebrafish: CETI-PIC3
    (The Company of Biologists, 2020-06-26) Ibrahim, Sara; Harris-Kawano, Arianna; Haider, Isra; Mirmira, Raghavendra G.; Sims, Emily K.; Anderson, Ryan M.; Pediatrics, School of Medicine
    Maladaptive signaling by pro-inflammatory cytokines (PICs), such as TNFα, IL1β and IFNɣ, can activate downstream signaling cascades that are implicated in the development and progression of multiple inflammatory diseases. Despite playing critical roles in pathogenesis, the availability of in vivo models in which to model tissue-specific induction of PICs is limited. To bridge this gap, we have developed a novel multi-gene expression system dubbed Cre-enabled and tetracycline-inducible transgenic system for conditional, tissue-specific expression of pro-inflammatory cytokines (CETI-PIC3). This binary transgenic system permits the stoichiometric co-expression of proteins Tumor necrosis factor a (Tnfa), Interleukin-1 beta (Il1b) and Interferon gamma (Ifng1), and H2B-GFP fluorescent reporter in a dose-dependent manner. Furthermore, cytokine misexpression is enabled only in tissue domains that can be defined by Cre recombinase expression. We have validated this system in zebrafish using an insulin:cre line. In doubly transgenic fish, quantitative real-time polymerase chain reaction demonstrated increased expression levels of tnfa, il1b and ifng1 mRNA. Moreover, specific expression in pancreatic β cells was demonstrated by both Tnfa immunofluorescence and GFP fluorescence. Cytokine-overexpressing islets elicited specific responses: β cells exhibited increased expression of genes associated with reactive oxidative species-mediated stress and endoplasmic reticulum stress, surveilling and infiltrating macrophages were increased, and β cell death was promoted. This powerful and versatile model system can be used for modeling, analysis and therapy development of diseases with an underlying inflammatory etiology.
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    Role of DHS in translation control of islet β-cell replication during high fat induced obesity and glucose intolerance
    (2017-07-12) Levasseur, Esther Marie; Mirmira, Raghu
    Insulin resistance in liver, muscle, and adipose tissue almost invariably occurs during obesity. To compensate, the insulin-producing β-cell increases insulin production by expanding cellular mass. The inability of the β-cell to fully compensate leads to hyperglycemia and ultimately type 2 diabetes. The enzyme deoxyhypusine synthase (DHS) catalyzes the spermidine-dependent posttranslational modification of Lys50 of eukaryotic translation initiation factor 5A (eIF5A) to form hypusine (Hyp). Studies have demonstrated this modification of eIF5A to contribute to cellular proliferation in cancerous cells, but its role in the physiologic proliferation of islet β-cells is unknown. I hypothesized eIF5A-Hyp to be required for the proliferation of islet β cells during the early phase of insulin resistance, allowing the β-cell to respond to the increased demand for insulin to maintain glucose homeostasis. To test this hypothesis, deletion of DHS was induced post-developmentally in β-cells by crossing Dhs-fl/fl mice with MIP1-CreERT mice, and animals were fed for 1 or 4 weeks with a 60% kcal from fat diet (HFD) or normal chow diet (NCD, 16% kcal from fat diet). NCD-fed and HFD-fed animals had normal glucose homeostasis after one week feeding, regardless of genotype. However, after 4 weeks of HFD, KO mice had significantly worse glucose intolerance compared to control mice. eIF5A-Hyp levels increased in β-cells of control animals and as expected remained low in the KO mice. β-cell proliferation was significantly increased after 1 week of HFD as measured by PCNA staining, however KO mice showed no increase. Cyclin D2 protein, but not mRNA, was increased in control animals fed a HFD; this protein increase was not observed in KO animals. Furthermore, polyribosomal profile of isolated islets of 1 week HFD-fed mice showed the Ccnd2 mRNA bound to the monoribosome fractions in the KO animals compared to the controls, resulting in changes of global translation. Interestingly, Ccnd1 polyribosome to monoribosome ratio showed no changes in translation compared to Ccnd2. Taken together, these results suggest that DHS (and, consequently, eIF5A-Hyp) is necessary for the adaptive proliferative and functional response of β-cells during high fat diet induced obesity and glucose intolerance.
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    The roles of pancreatic hormones in regulating pancreas development and beta cell regeneration
    (2015-06-16) Ye, Lihua; Anderson, Ryan M.; Mirmira, Raghu G.; Roach, Peter J.; Fueger, Patrick T.; Skalnik, David G.
    Diabetes mellitus is a group of related metabolic diseases that share a common pathological mechanism: insufficient insulin signaling. Insulin is a hormone secreted from pancreatic β cells that promotes energy storage and consequently lowers blood glucose. In contrast, the hormone glucagon, released by pancreatic α cells, plays a critical complementary role in metabolic homeostasis by releasing energy stores and increasing blood glucose. Restoration of β cell mass in diabetic patients via β cell regeneration is a conceptually proven approach to finally curing diabetes. Moreover, in situ regeneration of β cells from endogenous sources would circumvent many of the obstacles encountered by surgical restoration of β cell mass via islet transplantation. Regeneration may occur both by β cell self-duplication and by neogenesis from non-β cell sources. Although the mechanisms regulating the β cell replication pathway have been highly investigated, the signals that regulate β cell neogenesis are relatively unknown. In this dissertation, I have used zebrafish as a genetic model system to investigate the process of β cell neogenesis following insulin signaling depletion by various modes. Specifically, I have found that after their ablation, β cells primarily regenerate from two discrete cellular sources: differentiation from uncommitted pancreatic progenitors and transdifferentiation from α cells. Importantly, I have found that insulin and glucagon play crucial roles in controlling β cell regeneration from both sources. As with metabolic regulation, insulin and glucagon play counter-balancing roles in directing endocrine cell fate specification. These studies have revealed that glucagon signaling promotes β cell formation by increasing differentiation of pancreas progenitors and by destabilizing α cell identity to promote α to β cell transdifferentiation. In contrast, insulin signaling maintains pancreatic progenitors in an undifferentiated state and stabilizes α cell identity. Finally, I have shown that insulin also regulates pancreatic exocrine cell development. Insufficient insulin signaling destabilized acinar cell fate and impairs exocrine pancreas development. By understanding the roles of pancreatic hormones during pancreas development and regeneration can provide new therapeutic targets for in vivo β cell regeneration to remediate the devastating consequences of diabetes.
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    Sirtuin 6 regulates glucose-stimulated insulin secretion in mouse pancreatic beta cells
    (Springer, 2016-01) Xiong, Xiwen; Wang, Gaihong; Tao, Rongya; Wu, Pengfei; Kono, Tatsuyoshi; Li, Kevin; Ding, Wen-Xing; Tong, Xin; Tersey, Sarah A.; Harris, Robert A.; Mirmira, Raghavendra G.; Evans-Molina, Carmella; Dong, X. Charlie; Department of Biochemistry & Molecular Biology, IU School of Medicine
    AIMS/HYPOTHESIS: Sirtuin 6 (SIRT6) has been implicated in ageing, DNA repair and metabolism; however, its function in pancreatic beta cells is unclear. The aim of this study is to elucidate the role of SIRT6 in pancreatic beta cells. METHODS: To investigate the function of SIRT6 in pancreatic beta cells, we performed Sirt6 gene knockdown in MIN6 cells and generated pancreatic- and beta cell-specific Sirt6 knockout mice. Islet morphology and glucose-stimulated insulin secretion (GSIS) were analysed. Glycolysis and oxygen consumption rates in SIRT6-deficient beta cells were measured. Cytosolic calcium was monitored using the Fura-2-AM fluorescent probe (Invitrogen, Grand Island, NY, USA). Mitochondria were analysed by immunoblots and electron microscopy. RESULTS: Sirt6 knockdown in MIN6 beta cells led to a significant decrease in GSIS. Pancreatic beta cell Sirt6 knockout mice showed a ~50% decrease in GSIS. The knockout mouse islets had lower ATP levels compared with the wild-type controls. Mitochondrial oxygen consumption rates were significantly decreased in the SIRT6-deficient beta cells. Cytosolic calcium dynamics in response to glucose or potassium chloride were attenuated in the Sirt6 knockout islets. Numbers of damaged mitochondria were increased and mitochondrial complex levels were decreased in the SIRT6-deficient islets. CONCLUSIONS/INTERPRETATION: These data suggest that SIRT6 is important for GSIS from pancreatic beta cells and activation of SIRT6 may be useful to improve insulin secretion in diabetes.
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    Targeting polyamine biosynthesis to stimulate beta cell regeneration in zebrafish
    (Taylor & Francis, 2020-07-25) Robertson, Morgan A.; Padgett, Leah R.; Fine, Jonathan A.; Chopra, Gaurav; Mastracci, Teresa L.; Biology, School of Science
    Type 1 diabetes (T1D) is a disease characterized by destruction of the insulin-producing beta cells. Currently, there remains a critical gap in our understanding of how to reverse or prevent beta cell loss in individuals with T1D. Previous studies in mice discovered that pharmacologically inhibiting polyamine biosynthesis using difluoromethylornithine (DFMO) resulted in preserved beta cell function and mass. Similarly, treatment of non-obese diabetic mice with the tyrosine kinase inhibitor Imatinib mesylate reversed diabetes. The promising findings from these animal studies resulted in the initiation of two separate clinical trials that would repurpose either DFMO (NCT02384889) or Imatinib (NCT01781975) and determine effects on diabetes outcomes; however, whether these drugs directly stimulated beta cell growth remained unknown. To address this, we used the zebrafish model system to determine pharmacological impact on beta cell regeneration. After induction of beta cell death, zebrafish embryos were treated with either DFMO or Imatinib. Neither drug altered whole-body growth or exocrine pancreas length. Embryos treated with Imatinib showed no effect on beta cell regeneration; however, excitingly, DFMO enhanced beta cell regeneration. These data suggest that pharmacological inhibition of polyamine biosynthesis may be a promising therapeutic option to stimulate beta cell regeneration in the setting of diabetes.