Browsing by Author "Weaver, Staci A."
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Item 68722 Role of ER calcium in beta cell senescence and diabetes pathophysiology(Cambridge University Press, 2021) Weaver, Staci A.; Kono, Tatsuyoshi; Syed, Farooq; Bone, Robert; Evans-Molina, Carmella; Biochemistry and Molecular Biology, School of MedicineABSTRACT IMPACT: The proposed study has the potential to inform new paradigms of type 1 diabetes prevention and therapy with the overall goal of improving β cell health during autoimmunity. OBJECTIVES/GOALS: Type 1 diabetes (T1D) results from immune-mediated destruction of pancreatic βcells. Recent data suggest that activation of senescence and acquisition of a senescence associated secretory phenotype (SASP) by βcells may contribute to T1D pathogenesis. However, the molecular mechanisms responsible for this phenotype are not well understood. METHODS/STUDY POPULATION: We hypothesize that loss of endoplasmic reticulum (ER) Ca2+ induces βcell senescence, SASP as well as mitochondrial dysfunction which drive T1D development. The current study utilizes SERCA2 KO INS-1 βcells (S2KO) exhibiting loss of ER Ca2+ and a SERCA2 haploinsufficient mice on a non-obese diabetic background (NOD-S2+/-) to test the role of ER Ca2+ loss during T1D development. Senescence associated βgalactosidase staining (SA-βgal), expression of senescence markers (RT-qPCR), mitochondrial function (Seahorse, TMRM) and mitochondrial copy number (qPCR) were all measured in S2KO versus WT βcells and are currently being measured in the NOD-S2+/- mouse model at 6, 8, 12, 14, and 16wks of age. RESULTS/ANTICIPATED RESULTS: RT-qPCR assays detecting senescence markers cdkn1a and cdkn2a and mitochondrial specific genes cox1 and nd1 were developed and validated in both INS-1 βcells and mouse islets. Mitochondrial function assay (Seahorse) was optimized for use in INS-1 βcells and is currently under development for use in intact mouse islets. S2KO βcells displayed increased SA- βgal staining as well as increased mitochondrial coupling efficiency (p=0.0146) and baseline mitochondrial copy number (p=0.0053) compared to WT βcells, suggesting a senescence phenotype and altered mitochondrial function. NOD-S2+/- mice exhibited increased expression of the senescence marker cdkn2a in the islet at 12wks (p=0.0117) compared to control mice, whereas cdkn1a remained unchanged across all timepoints tested. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our results suggest that loss of SERCA2 and reduced ER Ca2+ alter βcell mitochondrial function and are associated with features of senescence. Future studies will test whether SERCA2 activation and/or senolytic/senomorphic drugs are able to prevent or delay diabetes onset in NOD-S2+/- mice.Item Advanced Imaging Techniques for the Characterization of Subcellular Organelle Structure in Pancreatic Islet β Cells(Wiley, 2023-12-29) McLaughlin, Madeline R.; Weaver, Staci A.; Syed, Farooq; Evans-Molina, Carmella; Pediatrics, School of MedicineType 2 diabetes (T2D) affects more than 32.3 million individuals in the United States, creating an economic burden of nearly $966 billion in 2021. T2D results from a combination of insulin resistance and inadequate insulin secretion from the pancreatic β cell. However, genetic and physiologic data indicate that defects in β cell function are the chief determinant of whether an individual with insulin resistance will progress to a diagnosis of T2D. The subcellular organelles of the insulin secretory pathway, including the endoplasmic reticulum, Golgi apparatus, and secretory granules, play a critical role in maintaining the heavy biosynthetic burden of insulin production, processing, and secretion. In addition, the mitochondria enable the process of insulin release by integrating the metabolism of nutrients into energy output. Advanced imaging techniques are needed to determine how changes in the structure and composition of these organelles contribute to the loss of insulin secretory capacity in the β cell during T2D. Several microscopy techniques, including electron microscopy, fluorescence microscopy, and soft X-ray tomography, have been utilized to investigate the structure-function relationship within the β cell. In this overview article, we will detail the methodology, strengths, and weaknesses of each approach.Item The Chd4 subunit of the NuRD complex regulates Pdx1-controlled genes involved in β-cell function(Bioscientifica, 2022-06-14) Davidson, Rebecca K.; Weaver, Staci A.; Casey, Nolan; Kanojia, Sukrati; Hogarth, Elise; Schneider Aguirre, Rebecca; Sims, Emily K.; Evans-Molina, Carmella; Spaeth, Jason M.; Biochemistry and Molecular Biology, School of MedicineType 2 diabetes (T2D) is associated with loss of transcription factors (TFs) from a subset of failing β-cells. Among these TFs is Pdx1, which controls the expression of numerous genes involved in maintaining β-cell function and identity. Pdx1 activity is modulated by transcriptional coregulators and has recently been shown, through an unbiased screen, to interact with the Chd4 ATPase subunit of the nucleosome remodeling and deacetylase complex. Chd4 contributes to the maintenance of cellular identity and functional status of numerous different cell types. Here, we demonstrated that Pdx1 dynamically interacts with Chd4 under physiological and stimulatory conditions within islet β-cells and established a fundamental role for Chd4 in regulating insulin secretion and modulating numerous Pdx1-bound genes in vitro, including the MafA TF, where we discovered Chd4 is bound to the MafA region 3 enhancer. Furthermore, we found that Pdx1:Chd4 interactions are significantly compromised in islet β-cells under metabolically induced stress in vivo and in human donor tissues with T2D. Our findings establish a fundamental role for Chd4 in regulating insulin secretion and modulating Pdx1-bound genes in vitro, and disruption of Pdx1:Chd4 interactions coincides with β-cell dysfunction associated with T2D.Item Cigarette smoke exposure impairs β-cell function through activation of oxidative stress and ceramide accumulation(Elsevier, 2020-07) Tong, Xin; Chaudhry, Zunaira; Lee, Chih-Chun; Bone, Robert N.; Kanojia, Sukrati; Maddatu, Judith; Sohn, Paul; Weaver, Staci A.; Robertson, Morgan A.; Petrache, Irina; Evans-Molina, Carmella; Kono, Tatsuyoshi; Medicine, School of MedicineObjectives Epidemiological studies indicate that first- and second-hand cigarette smoke (CS) exposure are important risk factors for the development of type 2 diabetes (T2D). Additionally, elevated diabetes risk has been reported to occur within a short period of time after smoking cessation, and health risks associated with smoking are increased when combined with obesity. At present, the mechanisms underlying these associations remain incompletely understood. The objective of this study was to test the impact of CS exposure on pancreatic β-cell function using rodent and in vitro models. Methods Beginning at 8 weeks of age, C57BL/6 J mice were concurrently fed a high-fat diet (HFD) and exposed to CS for 11 weeks, followed by an additional 11 weeks of smoking cessation with continued HFD. Glucose tolerance testing was performed during CS exposure and during the cessation period. Cultured INS-1 β-cells and primary islets were exposed ex vivo to CS extract (CSE), and β-cell function and viability were tested. Since CS increases ceramide accumulation in the lung and these bioactive sphingolipids have been implicated in pancreatic β-cell dysfunction in diabetes, islet and β-cell sphingolipid levels were measured in islets from CS-exposed mice and in CSE-treated islets and INS-1 cells using liquid chromatography-tandem mass spectrometry. Results Compared to HFD-fed, ambient air-exposed mice, HFD-fed and CS-exposed mice had reduced weight gain and better glucose tolerance during the active smoking period. Following smoking cessation, CS-mice exhibited rapid weight gain and had accelerated worsening of their glucose tolerance. CS-exposed mice had higher serum proinsulin/insulin ratios, indicative of β-cell dysfunction, significantly lower β-cell mass (p = 0.017), reduced β-cell proliferation (p = 0.006), and increased islet ceramide content compared to non-smoking control mice. Ex vivo exposure of isolated islets to CSE was sufficient to increase islet ceramide levels, which was correlated with reduced insulin gene expression and glucose-stimulated insulin secretion, and increased β-cell oxidative and endoplasmic reticulum (ER) stress. Treatment with the antioxidant N-acetylcysteine markedly attenuated the effects of CSE on ceramide levels, restored β-cell function and survival, and increased cyclin D2 expression, while also reducing activation of β-cell ER and oxidative stress. Conclusions Our results indicate that CS exposure leads to impaired insulin production, processing, secretion and reduced β-cell viability and proliferation. These effects were linked to increased β-cell oxidative and ER stress and ceramide accumulation. Mice fed HFD continued to experience detrimental effects of CS exposure even during smoking cessation. Elucidation of the mechanisms by which CS exposure impairs β-cell function in synergy with obesity will help design therapeutic and preventive interventions for both active and former smokers.Item Histone Deacetylase Inhibitors Prevent Cytokine-Induced β Cell Dysfunction Through Restoration of Stromal Interaction Molecule 1 Expression and Activation of Store-Operated Calcium Entry(bioRxiv, 2023-12-08) Lee, Chih-Chun; Kono, Tatsuyoshi; Syed, Farooq; Weaver, Staci A.; Sohn, Paul; Wu, Wenting; Chang, Garrick; Liu, Jing; Slak Rupnik, Marjan; Evans-Molina, Carmella; Pediatrics, School of MedicineHistone deacetylase inhibitors (HDIs) modulate β cell function in preclinical models of diabetes; however, the mechanisms underlying these beneficial effects have not been determined. In this study, we investigated the impact of the HDI sodium butyrate (NaB) on β cell function and calcium (Ca2+) signaling using ex vivo and in vitro models of diabetes. Our results show that NaB significantly improved glucose-stimulated insulin secretion in islets from human organ donors with type 2 diabetes and in cytokine-treated INS-1 β cells. Consistently, NaB partially rescued glucose-stimulated Ca2+ oscillations in mouse islets treated with proinflammatory cytokines. Because the oscillatory phenotype of Ca2+ in the β cell is governed by changes in endoplasmic reticulum (ER) Ca2+ levels, next we explored the relationship between NaB and store-operated calcium entry (SOCE), a rescue mechanism that acts to refill ER Ca2+ levels through STIM1-mediated gating of plasmalemmal Orai channels. We found that NaB treatment preserved basal ER Ca2+ levels and restored SOCE in IL-1β-treated INS-1 cells. Furthermore, we linked these changes with the restoration of STIM1 levels in cytokine-treated INS-1 cells and mouse islets, and we found that NaB treatment was sufficient to prevent β cell death in response to IL-1β treatment. Mechanistically, NaB counteracted cytokine-mediated reductions in phosphorylation levels of key signaling molecules, including AKT, ERK1/2, glycogen synthase kinase-3α (GSK-3α), and GSK-3β. Taken together, these data support a model whereby HDI treatment promotes β cell function and Ca2+ homeostasis under proinflammatory conditions through STIM1-mediated control of SOCE and AKT-mediated inhibition of GSK-3.Item Inhibiting Glycogen Synthesis Prevents Lafora Disease in a Mouse Model(Wiley, 2013) Pederson, Bartholomew A.; Turnbull, Julie; Epp, Jonathan R.; Weaver, Staci A.; Zhao, Xiaochu; Pencea, Nela; Roach, Peter J.; Frankland, Paul; Ackerley, Cameron A.; Minassian, Berge A.; Biochemistry and Molecular Biology, School of MedicineLafora disease (LD) is a fatal progressive myoclonus epilepsy characterized neuropathologically by aggregates of abnormally structured glycogen and proteins (Lafora bodies [LBs]), and neurodegeneration. Whether LBs could be prevented by inhibiting glycogen synthesis and whether they are pathogenic remain uncertain. We genetically eliminated brain glycogen synthesis in LD mice. This resulted in long-term prevention of LB formation, neurodegeneration, and seizure susceptibility. This study establishes that glycogen synthesis is requisite for LB formation and that LBs are pathogenic. It opens a therapeutic window for potential treatments in LD with known and future small molecule inhibitors of glycogen synthesis.Item miR-146a-5p mediates inflammation-induced β cell mitochondrial dysfunction and apoptosis(bioRxiv, 2024-03-19) Krishnan, Preethi; Branco, Renato Chaves Souto; Weaver, Staci A.; Chang, Garrick; Lee, Chih-Chun; Syed, Farooq; Evans-Molina, Carmella; Medicine, School of MedicineWe previously showed that miR-146a-5p is upregulated in pancreatic islets treated with pro-inflammatory cytokines. Others have reported that miR-146a-5p overexpression is associated with β cell apoptosis and impaired insulin secretion. However, the molecular mechanisms mediating these effects remain elusive. To investigate the role of miR-146a-5p in β cell function, we developed stable MIN6 cell lines to either overexpress or inhibit the expression of miR-146a-5p. Monoclonal cell populations were treated with pro-inflammatory cytokines (IL-1β, IFNγ, and TNFα) to model T1D in vitro. We found that overexpression of miR-146a-5p increased cell death under conditions of inflammatory stress, whereas inhibition of miR-146a-5p reversed these effects. Additionally, inhibition of miR-146a-5p increased mitochondrial DNA copy number, respiration rate, and ATP production. Further, RNA sequencing data showed enrichment of pathways related to insulin secretion, apoptosis, and mitochondrial function when the expression levels of miR-146a-5p were altered. Finally, a temporal increase in miR-146a-5p expression levels and a decrease in mitochondria function markers was observed in islets derived from NOD mice. Collectively, these data suggest that miR-146a-5p may promote β cell dysfunction and death during inflammatory stress by suppressing mitochondrial function.Item miR-146a-5p mediates inflammation-induced β cell mitochondrial dysfunction and apoptosis(Elsevier, 2024) Krishnan, Preethi; Branco, Renato Chaves Souto; Weaver, Staci A.; Chang, Garrick; Lee, Chih-Chun; Syed, Farooq; Evans-Molina, Carmella; Medicine, School of MedicineWe previously showed that miR-146a-5p is upregulated in pancreatic islets treated with proinflammatory cytokines. Others have reported that miR-146a-5p overexpression is associated with β cell apoptosis and impaired insulin secretion. However, the molecular mechanisms mediating these effects remain elusive. To investigate the role of miR-146a-5p in β cell function, we developed stable MIN6 cell lines to either overexpress or inhibit the expression of miR-146a-5p. Monoclonal cell populations were treated with proinflammatory cytokines (interleukin-1β, interferonγ, and tumor necrosis factor α) to model type 1 diabetes in vitro. We found that overexpression of miR-146a-5p increased cell death under conditions of inflammatory stress and led to mitochondrial membrane depolarization, whereas inhibition of miR-146a-5p reversed these effects. Additionally, inhibition of miR-146a-5p increased insulin secretion, mitochondrial DNA copy number, respiration rate, and ATP production. Further, RNA-seq data showed enrichment of pathways related to insulin secretion, apoptosis, and mitochondrial function when the expression levels of miR-146a-5p were altered. Finally, a temporal increase in miR-146a-5p expression levels and a decrease in mitochondria function markers were observed in islets derived from nonobese diabetic mice. Collectively, these data suggest that miR-146a-5p may promote β cell dysfunction and death during inflammatory stress by suppressing mitochondrial function.Item A parallax view of type 1 diabetes(Portland press, 2021) Weaver, Staci A.; Felton, Jamie L.; Evans-Molina, Carmella; Biochemistry and Molecular Biology, School of MedicineThe year 2021 marks the 100th anniversary of the discovery of insulin, a therapy that transformed type 1 diabetes (T1D) from a once fatal diagnosis into a chronic condition that can be successfully managed with exogenous insulin administration. In this article, we celebrate the remarkable journey that led to the discovery of insulin and our subsequent understanding of T1D as a disease of immune-mediated β-cell destruction. Further, we discuss an alternative and parallax view of disease development, highlighting maladaptive β-cell responses that act to amplify immune responses and have been implicated in diabetes development.Item Pharmacological inhibition of tyrosine protein-kinase 2 reduces islet inflammation and delays type 1 diabetes onset in mice(bioRxiv, 2024-05-09) Syed, Farooq; Ballew, Olivia; Lee, Chih-Chun; Rana, Jyoti; Krishnan, Preethi; Castela, Angela; Weaver, Staci A.; Chalasani, Namratha Shivani; Thomaidou, Sofia F.; Demine, Stephane; Chang, Garrick; de Brachène, Alexandra Coomans; Alvelos, Maria Ines; Marselli, Lorella; Orr, Kara; Felton, Jamie L.; Liu, Jing; Marchetti, Piero; Zaldumbide, Arnaud; Scheuner, Donalyn; Eizirik, Decio L.; Evans-Molina, Carmella; Pediatrics, School of MedicineTyrosine protein-kinase 2 (TYK2), a member of the Janus kinase family, mediates inflammatory signaling through multiple cytokines, including interferon-α (IFNα), interleukin (IL)-12, and IL-23. Missense mutations in TYK2 are associated with protection against type 1 diabetes (T1D), and inhibition of TYK2 shows promise in the management of other autoimmune conditions. Here, we evaluated the effects of specific TYK2 inhibitors (TYK2is) in pre-clinical models of T1D. First, human β cells, cadaveric donor islets, and iPSC-derived islets were treated in vitro with IFNα in combination with a small molecule TYK2i (BMS-986165 or a related molecule BMS-986202). TYK2 inhibition prevented IFNα-induced β cell HLA class I up-regulation, endoplasmic reticulum stress, and chemokine production. In co-culture studies, pre-treatment of β cells with a TYK2i prevented IFNα-induced activation of T cells targeting an epitope of insulin. In vivo administration of BMS-986202 in two mouse models of T1D (RIP-LCMV-GP mice and NOD mice) reduced systemic and tissue-localized inflammation, prevented β cell death, and delayed T1D onset. Transcriptional phenotyping of pancreatic islets, pancreatic lymph nodes (PLN), and spleen during early disease pathogenesis highlighted a role for TYK2 inhibition in modulating signaling pathways associated with inflammation, translational control, stress signaling, secretory function, immunity, and diabetes. Additionally, TYK2i treatment changed the composition of innate and adaptive immune cell populations in the blood and disease target tissues, resulting in an immune phenotype with a diminished capacity for β cell destruction. Overall, these findings indicate that TYK2i has beneficial effects in both the immune and endocrine compartments in models of T1D, thus supporting a path forward for testing TYK2 inhibitors in human T1D.