Browsing by Subject "β cell"
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Item Dynamic regulation of pancreatic β cell function and gene expression by the SND1 coregulator in vitro(Taylor & Francis, 2023) Kanojia, Sukrati; Davidson, Rebecca K.; Conley, Jason M.; Xu, Jerry; Osmulski, Meredith; Sims, Emily K.; Ren, Hongxia; Spaeth, Jason M.; Biochemistry and Molecular Biology, School of MedicineThe pancreatic β cell synthesizes, packages, and secretes insulin in response to glucose-stimulation to maintain blood glucose homeostasis. Under diabetic conditions, a subset of β cells fail and lose expression of key transcription factors (TFs) required for insulin secretion. Among these TFs is Pancreatic and duodenal homeobox 1 (PDX1), which recruits a unique subset of transcriptional coregulators to modulate its activity. Here we describe a novel interacting partner of PDX1, the Staphylococcal Nuclease and Tudor domain-containing protein (SND1), which has been shown to facilitate protein-protein interactions and transcriptional control through diverse mechanisms in a variety of tissues. PDX1:SND1 interactions were confirmed in rodent β cell lines, mouse islets, and human islets. Utilizing CRISPR-Cas9 gene editing technology, we deleted Snd1 from the mouse β cell lines, which revealed numerous differentially expressed genes linked to insulin secretion and cell proliferation, including limited expression of Glp1r. We observed Snd1 deficient β cell lines had reduced cell expansion rates, GLP1R protein levels, and limited cAMP accumulation under stimulatory conditions, and further show that acute ablation of Snd1 impaired insulin secretion in rodent and human β cell lines. Lastly, we discovered that PDX1:SND1 interactions were profoundly reduced in human β cells from donors with type 2 diabetes (T2D). These observations suggest the PDX1:SND1 complex formation is critical for controlling a subset of genes important for β cell function and is targeted in diabetes pathogenesis.Item High-resolution analysis of the cytosolic Ca2+ events in β cell collectives in situ(American Physiological Society, 2023) Postić, Sandra; Sarikas, Srdjan; Pfabe, Johannes; Pohorec, Viljem; Križančić Bombek, Lidija; Sluga, Nastja; Skelin Klemen, Maša; Dolenšek, Jurij; Korošak, Dean; Stožer, Andraž; Evans-Molina, Carmella; Johnson, James D.; Rupnik, Marjan Slak; Pediatrics, School of MedicineThe release of peptide hormones is predominantly regulated by a transient increase in cytosolic Ca2+ concentration ([Ca2+]c). To trigger exocytosis, Ca2+ ions enter the cytosol from intracellular Ca2+ stores or from the extracellular space. The molecular events of late stages of exocytosis, and their dependence on [Ca2+]c, were extensively described in isolated single cells from various endocrine glands. Notably, less work has been done on endocrine cells in situ to address the heterogeneity of [Ca2+]c events contributing to a collective functional response of a gland. For this, β cell collectives in a pancreatic islet are particularly well suited as they are the smallest, experimentally manageable functional unit, where [Ca2+]c dynamics can be simultaneously assessed on both cellular and collective level. Here, we measured [Ca2+]c transients across all relevant timescales, from a subsecond to a minute time range, using high-resolution imaging with a low-affinity Ca2+ sensor. We quantified the recordings with a novel computational framework for automatic image segmentation and [Ca2+]c event identification. Our results demonstrate that under physiological conditions the duration of [Ca2+]c events is variable, and segregated into three reproducible modes, subsecond, second, and tens of seconds time range, and are a result of a progressive temporal summation of the shortest events. Using pharmacological tools we show that activation of intracellular Ca2+ receptors is both sufficient and necessary for glucose-dependent [Ca2+]c oscillations in β cell collectives, and that a subset of [Ca2+]c events could be triggered even in the absence of Ca2+ influx across the plasma membrane. In aggregate, our experimental and analytical platform was able to readily address the involvement of intracellular Ca2+ receptors in shaping the heterogeneity of [Ca2+]c responses in collectives of endocrine cells in situ. NEW & NOTEWORTHY: Physiological glucose or ryanodine stimulation of β cell collectives generates a large number of [Ca2+]c events, which can be rapidly assessed with our newly developed automatic image segmentation and [Ca2+]c event identification pipeline. The event durations segregate into three reproducible modes produced by a progressive temporal summation. Using pharmacological tools, we show that activation of ryanodine intracellular Ca2+ receptors is both sufficient and necessary for glucose-dependent [Ca2+]c oscillations in β cell collectives.Item Inhibition of polyamine biosynthesis preserves β cell function in type 1 diabetes(Elsevier, 2023) Sims, Emily K.; Kulkarni, Abhishek; Hull, Audrey; Woerner, Stephanie E.; Cabrera, Susanne; Mastrandrea, Lucy D.; Hammoud, Batoul; Sarkar, Soumyadeep; Nakayasu, Ernesto S.; Mastracci, Teresa L.; Perkins, Susan M.; Ouyang, Fangqian; Webb-Robertson, Bobbie-Jo; Enriquez, Jacob R.; Tersey, Sarah A.; Evans-Molina, Carmella; Long, S. Alice; Blanchfield, Lori; Gerner, Eugene W.; Mirmira, Raghavendra G.; DiMeglio, Linda A.; Pediatrics, School of MedicineIn preclinical models, α-difluoromethylornithine (DFMO), an ornithine decarboxylase (ODC) inhibitor, delays the onset of type 1 diabetes (T1D) by reducing β cell stress. However, the mechanism of DFMO action and its human tolerability remain unclear. In this study, we show that mice with β cell ODC deletion are protected against toxin-induced diabetes, suggesting a cell-autonomous role of ODC during β cell stress. In a randomized controlled trial (ClinicalTrials.gov: NCT02384889) involving 41 recent-onset T1D subjects (3:1 drug:placebo) over a 3-month treatment period with a 3-month follow-up, DFMO (125-1,000 mg/m2) is shown to meet its primary outcome of safety and tolerability. DFMO dose-dependently reduces urinary putrescine levels and, at higher doses, preserves C-peptide area under the curve without apparent immunomodulation. Transcriptomics and proteomics of DFMO-treated human islets exposed to cytokine stress reveal alterations in mRNA translation, nascent protein transport, and protein secretion. These findings suggest that DFMO may preserve β cell function in T1D through islet cell-autonomous effects.Item Loss of ARC Worsens High Fat Diet-Induced Hyperglycemia in Mice(Bioscientifica, 2021-09-20) Templin, Andrew T.; Schmidt, Christine; Hogan, Meghan F.; Esser, Nathalie; Kitsis, Richard N.; Hull, Rebecca L.; Zraika, Sakeneh; Kahn, Steven E.; Medicine, School of MedicineApoptosis repressor with caspase recruitment domain (ARC) is an endogenous inhibitor of cell death signaling that is expressed in insulin-producing β cells. ARC has been shown to reduce β-cell death in response to diabetogenic stimuli in vitro, but its role in maintaining glucose homeostasis in vivo has not been fully established. Here we examined whether loss of ARC in FVB background mice exacerbates high fat diet (HFD)-induced hyperglycemia in vivo over 24 weeks. Prior to commencing 24-week HFD, ARC−/− mice had lower body weight than wild type (WT) mice. This body weight difference was maintained until the end of the study and was associated with decreased epididymal and inguinal adipose tissue mass in ARC−/− mice. Non-fasting plasma glucose was not different between ARC−/− and WT mice prior to HFD feeding, and ARC−/− mice displayed a greater increase in plasma glucose over the first 4 weeks of HFD. Plasma glucose remained elevated in ARC−/− mice after 16 weeks of HFD feeding, at which time it had returned to baseline in WT mice. Following 24 weeks of HFD, non-fasting plasma glucose in ARC−/− mice returned to baseline and was not different from WT mice. At this final time point, no differences were observed between genotypes in plasma glucose or insulin under fasted conditions or following IV glucose administration. However, HFD-fed ARC−/− mice exhibited significantly decreased β-cell area compared to WT mice. Thus, ARC deficiency delays, but does not prevent, metabolic adaptation to HFD feeding in mice, worsening transient HFD-induced hyperglycemia.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 Pancreatic and Islet Development and Function: The Role of Thyroid Hormone(JSciMed Central, 2014) Mastracci, Teresa L.; Evans-Molina, Carmella; Department of Pediatrics, IU School of MedicineA gradually expanding body of literature suggests that Thyroid Hormone (TH) and Thyroid Hormone Receptors (TRs) play a contributing role in pancreatic and islet cell development, maturation, and function. Studies using a variety of model systems capable of exploiting species-specific developmental paradigms have revealed the contribution of TH to cellular differentiation, lineage decisions, and endocrine cell specification. Moreover, in vitro and in vivo evidence suggests that TH is involved in islet β cell proliferation and maturation; however, the signaling pathway(s) connected with this function of TH/TR are not well understood. The purpose of this review is to discuss the current literature that has defined the effects of TH and TRs on pancreatic and islet cell development and function, describe the impact of hyper- and hypothyroidism on whole body metabolism, and highlight future and potential applications of TH in novel therapeutic strategies for diabetes.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.Item β-Cell autophagy in the pathogenesis of type 1 diabetes(American Physiological Society, 2021-09-01) Muralidharan, Charanya; Linnemann, Amelia K.; Biochemistry and Molecular Biology, School of MedicineType 1 diabetes is an insulin-dependent, autoimmune disease where the pancreatic β cells are destroyed resulting in hyperglycemia. This multifactorial disease involves multiple environmental and genetic factors, and has no clear etiology. Accumulating evidence suggests that early signaling defects within the β cells may promote a change in the local immune milieu leading to autoimmunity. Therefore, many studies have been focused on intrinsic β-cell mechanisms that aid in the restoration of cellular homeostasis under environmental conditions that cause dysfunction. One of these intrinsic mechanisms to promote homeostasis is autophagy, defects which are clearly linked with β-cell dysfunction in the context of type 2 diabetes. Recent studies have now also pointed towards β-cell autophagy defects in the context of type 1 diabetes. In this perspectives review, we will discuss the evidence supporting a role for β-cell autophagy in the pathogenesis of type 1 diabetes, including a potential role for unconventional secretion of autophagosomes/lysosomes in the changing dialogue between the β cell and immune cells.Item β-Cell Death in Diabetes: Past Discoveries, Present Understanding, and Potential Future Advances(MDPI, 2021-11-22) Mukherjee, Noyonika; Lin, Li; Contreras, Christopher J.; Templin, Andrew T.; Biochemistry and Molecular Biology, School of Medicineβ-cell death is regarded as a major event driving loss of insulin secretion and hyperglycemia in both type 1 and type 2 diabetes mellitus. In this review, we explore past, present, and potential future advances in our understanding of the mechanisms that promote β-cell death in diabetes, with a focus on the primary literature. We first review discoveries of insulin insufficiency, β-cell loss, and β-cell death in human diabetes. We discuss findings in humans and mouse models of diabetes related to autoimmune-associated β-cell loss and the roles of autoreactive T cells, B cells, and the β cell itself in this process. We review discoveries of the molecular mechanisms that underlie β-cell death-inducing stimuli, including proinflammatory cytokines, islet amyloid formation, ER stress, oxidative stress, glucotoxicity, and lipotoxicity. Finally, we explore recent perspectives on β-cell death in diabetes, including: (1) the role of the β cell in its own demise, (2) methods and terminology for identifying diverse mechanisms of β-cell death, and (3) whether non-canonical forms of β-cell death, such as regulated necrosis, contribute to islet inflammation and β-cell loss in diabetes. We believe new perspectives on the mechanisms of β-cell death in diabetes will provide a better understanding of this pathological process and may lead to new therapeutic strategies to protect β cells in the setting of diabetes.