Browsing by Author "Nakayasu, Ernesto S."
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Item A proteomic meta-analysis refinement of plasma extracellular vesicles(Springer Nature, 2023-11-28) Vallejo, Milene C.; Sarkar, Soumyadeep; Elliott, Emily C.; Henry, Hayden R.; Powell, Samantha M.; Diaz Ludovico, Ivo; You, Youngki; Huang, Fei; Payne, Samuel H.; Ramanadham, Sasanka; Sims, Emily K.; Metz, Thomas O.; Mirmira, Raghavendra G.; Nakayasu, Ernesto S.; Pediatrics, School of MedicineExtracellular vesicles play major roles in cell-to-cell communication and are excellent biomarker candidates. However, studying plasma extracellular vesicles is challenging due to contaminants. Here, we performed a proteomics meta-analysis of public data to refine the plasma EV composition by separating EV proteins and contaminants into different clusters. We obtained two clusters with a total of 1717 proteins that were depleted of known contaminants and enriched in EV markers with independently validated 71% true-positive. These clusters had 133 clusters of differentiation (CD) antigens and were enriched with proteins from cell-to-cell communication and signaling. We compared our data with the proteins deposited in PeptideAtlas, making our refined EV protein list a resource for mechanistic and biomarker studies. As a use case example for this resource, we validated the type 1 diabetes biomarker proplatelet basic protein in EVs and showed that it regulates apoptosis of β cells and macrophages, two key players in the disease development. Our approach provides a refinement of the EV composition and a resource for the scientific community.Item Comprehensive Proteomics Analysis of Stressed Human Islets Identifies GDF15 as a Target for Type 1 Diabetes Intervention(Elsevier, 2020-02-04) Nakayasu, Ernesto S.; Syed, Farooq; Tersey, Sarah A.; Gritsenko, Marina A.; Mitchell, Hugh D.; Chan, Chi Yuet; Dirice, Ercument; Turatsinze, Jean-Valery; Cui, Yi; Kulkarni, Rohit N.; Eizirik, Decio L.; Qian, Wei-Jun; Webb-Robertson, Bobbie-Jo M.; Evans-Molina, Carmella; Mirmira., Raghavendra G.; Metz, Thomas O.; Pediatrics, School of MedicineType 1 diabetes (T1D) results from the progressive loss of β cells, a process propagated by pro-inflammatory cytokine signaling that disrupts the balance between pro- and anti-apoptotic proteins. To identify proteins involved in this process, we performed comprehensive proteomics of human pancreatic islets treated with interleukin-1β and interferon-γ, leading to the identification of 11,324 proteins, of which 387 were significantly regulated by treatment. We then tested the function of growth/differentiation factor 15 (GDF15), which was repressed by the treatment. We found that GDF15 translation was blocked during inflammation, and it was depleted in islets from individuals with T1D. The addition of exogenous GDF15 inhibited interleukin-1β+interferon-γ-induced apoptosis of human islets. Administration of GDF15 reduced by 53% the incidence of diabetes in NOD mice. Our approach provides a unique resource for the identification of the human islet proteins regulated by cytokines and was effective in discovering a potential target for T1D therapy.Item Deoxyhypusine Synthase Promotes a Pro-Inflammatory Macrophage Phenotype(Elsevier, 2021) Anderson-Baucum, Emily; Piñeros, Annie R.; Kulkarni, Abhishek; Webb-Robertson, Bobbie-Jo; Maier, Bernhard; Anderson, Ryan M.; Wu, Wenting; Tersey, Sarah A.; Mastracci, Teresa L.; Casimiro, Isabel; Scheuner, Donalyn; Metz, Thomas O.; Nakayasu, Ernesto S.; Evans-Molina, Carmella; Mirmira, Raghavendra G.; Biology, School of ScienceThe metabolic inflammation (meta-inflammation) of obesity is characterized by proinflammatory macrophage infiltration into adipose tissue. Catalysis by deoxyhypusine synthase (DHPS) modifies the translation factor eIF5A to generate a hypusine (Hyp) residue. Hypusinated eIF5A (eIF5AHyp) controls the translation of mRNAs involved in inflammation, but its role in meta-inflammation has not been elucidated. Levels of eIF5AHyp were found to be increased in adipose tissue macrophages from obese mice and in murine macrophages activated to a proinflammatory M1-like state. Global proteomics and transcriptomics revealed that DHPS deficiency in macrophages altered the abundance of proteins involved in NF-κB signaling, likely through translational control of their respective mRNAs. DHPS deficiency in myeloid cells of obese mice suppressed M1 macrophage accumulation in adipose tissue and improved glucose tolerance. These findings indicate that DHPS promotes the post-transcriptional regulation of a subset of mRNAs governing inflammation and chemotaxis in macrophages and contributes to a proinflammatory M1-like phenotype.Item Extracellular vesicles in β cell biology: Role of lipids in vesicle biogenesis, cargo, and intercellular signaling(Elsevier, 2022) Aguirre, Rebecca S.; Kulkarni, Abhishek; Becker, Matthew W.; Lei, Xiaoyong; Sarkar, Soumyadeep; Ramanadham, Sasanka; Phelps, Edward A.; Nakayasu, Ernesto S.; Sims, Emily K.; Mirmira, Raghavendra G.; Pediatrics, School of MedicineBackground: Type 1 diabetes (T1D) is a complex autoimmune disorder whose pathogenesis involves an intricate interplay between β cells of the pancreatic islet, other islet cells, and cells of the immune system. Direct intercellular communication within the islet occurs via cell surface proteins and indirect intercellular communication has traditionally been seen as occurring via secreted proteins (e.g., endocrine hormones and cytokines). However, recent literature suggests that extracellular vesicles (EVs) secreted by β cells constitute an additional and biologically important mechanism for transmitting signals to within the islet. Scope of review: This review summarizes the general mechanisms of EV formation, with a particular focus on how lipids and lipid signaling pathways influence their formation and cargo. We review the implications of EV release from β cells for T1D pathogenesis, how EVs and their cargo might be leveraged as biomarkers of this process, and how EVs might be engineered as a therapeutic candidate to counter T1D outcomes. Major conclusions: Islet β cells have been viewed as initiators and propagators of the cellular circuit giving rise to autoimmunity in T1D. In this context, emerging literature suggests that EVs may represent a conduit for communication that holds more comprehensive messaging about the β cells from which they arise. As the field of EV biology advances, it opens the possibility that intervening with EV formation and cargo loading could be a novel disease-modifying approach in T1D.Item GDF15: a potential therapeutic target for type 1 diabetes(Taylor & Francis, 2022-01) Sarkar, Soumyadeep; Melchior, John T.; Henry, Hayden R.; Syed, Farooq; Mirmira, Raghavendra G.; Nakayasu, Ernesto S.; Metz, Thomas O.; Pediatrics, School of MedicineIntroduction: Current treatment for type 1 diabetes (T1D) is centered around insulin supplementation to manage the effects of pancreatic β cell loss. GDF15 is a potential preventative therapy against T1D progression that could work to curb increasing disease incidence. Areas covered: This paper discusses the known actions of GDF15, a pleiotropic protein with metabolic, feeding, and immunomodulatory effects, connecting them to highlight the open opportunities for future research. The role of GDF15 in the prevention of insulitis and protection of pancreatic β cells against pro-inflammatory cytokine-mediated cellular stress are examined and the pharmacological promise of GDF15 and critical areas of future research are discussed. Expert opinion: GDF15 shows promise as a potential intervention but requires further development. Preclinical studies have shown poor efficacy, but this result may be confounded by the measurement of gross GDF15 instead of the active form. Additionally, the effect of GDF15 in the induction of anorexia and nausea-like behavior and short-half-life present significant challenges to its deployment, but a systems pharmacology approach paired with chronotherapy may provide a possible solution to therapy for this currently unpreventable disease.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 Inhibition of the Eukaryotic Initiation Factor-2-α Kinase PERK Decreases Risk of Autoimmune Diabetes in Mice(bioRxiv, 2024-06-03) Muralidharan, Charanya; Huang, Fei; Enriquez, Jacob R.; Wang, Jiayi E.; Nelson, Jennifer B.; Nargis, Titli; May, Sarah C.; Chakraborty, Advaita; Figatner, Kayla T.; Navitskaya, Svetlana; Anderson, Cara M.; Calvo, Veronica; Surguladze, David; Mulvihill, Mark J.; Yi, Xiaoyan; Sarkar, Soumyadeep; Oakes, Scott A.; Webb-Robertson, Bobbie-Jo M.; Sims, Emily K.; Staschke, Kirk A.; Eizirik, Decio L.; Nakayasu, Ernesto S.; Stokes, Michael E.; Tersey, Sarah A.; Mirmira, Raghavendra G.; Pediatrics, School of MedicinePreventing the onset of autoimmune type 1 diabetes (T1D) is feasible through pharmacological interventions that target molecular stress-responsive mechanisms. Cellular stresses, such as nutrient deficiency, viral infection, or unfolded proteins, trigger the integrated stress response (ISR), which curtails protein synthesis by phosphorylating eIF2α. In T1D, maladaptive unfolded protein response (UPR) in insulin-producing β cells renders these cells susceptible to autoimmunity. We show that inhibition of the eIF2α kinase PERK, a common component of the UPR and ISR, reverses the mRNA translation block in stressed human islets and delays the onset of diabetes, reduces islet inflammation, and preserves β cell mass in T1D-susceptible mice. Single-cell RNA sequencing of islets from PERK-inhibited mice shows reductions in the UPR and PERK signaling pathways and alterations in antigen processing and presentation pathways in β cells. Spatial proteomics of islets from these mice shows an increase in the immune checkpoint protein PD-L1 in β cells. Golgi membrane protein 1, whose levels increase following PERK inhibition in human islets and EndoC-βH1 human β cells, interacts with and stabilizes PD-L1. Collectively, our studies show that PERK activity enhances β cell immunogenicity, and inhibition of PERK may offer a strategy to prevent or delay the development of T1D.Item Inhibition of the eukaryotic initiation factor-2α kinase PERK decreases risk of autoimmune diabetes in mice(American Society for Clinical Investigation, 2024-06-18) Muralidharan, Charanya; Huang, Fei; Enriquez, Jacob R.; Wang, Jiayi E.; Nelson, Jennifer B.; Nargis, Titli; May, Sarah C.; Chakraborty, Advaita; Figatner, Kayla T.; Navitskaya, Svetlana; Anderson, Cara M.; Calvo, Veronica; Surguladze, David; Mulvihill, Mark J.; Yi, Xiaoyan; Sarkar, Soumyadeep; Oakes, Scott A.; Webb-Robertson, Bobbie-Jo M.; Sims, Emily K.; Staschke, Kirk A.; Eizirik, Decio L.; Nakayasu, Ernesto S.; Stokes, Michael E.; Tersey, Sarah A.; Mirmira, Raghavendra G.; Pediatrics, School of MedicinePreventing the onset of autoimmune type 1 diabetes (T1D) is feasible through pharmacological interventions that target molecular stress–responsive mechanisms. Cellular stresses, such as nutrient deficiency, viral infection, or unfolded proteins, trigger the integrated stress response (ISR), which curtails protein synthesis by phosphorylating eukaryotic translation initiation factor-2α (eIF2α). In T1D, maladaptive unfolded protein response (UPR) in insulin-producing β cells renders these cells susceptible to autoimmunity. We found that inhibition of the eIF2α kinase PKR-like ER kinase (PERK), a common component of the UPR and ISR, reversed the mRNA translation block in stressed human islets and delayed the onset of diabetes, reduced islet inflammation, and preserved β cell mass in T1D-susceptible mice. Single-cell RNA-Seq of islets from PERK-inhibited mice showed reductions in the UPR and PERK signaling pathways and alterations in antigen-processing and presentation pathways in β cells. Spatial proteomics of islets from these mice showed an increase in the immune checkpoint protein programmed death-ligand 1 (PD-L1) in β cells. Golgi membrane protein 1, whose levels increased following PERK inhibition in human islets and EndoC-βH1 human β cells, interacted with and stabilized PD-L1. Collectively, our studies show that PERK activity enhances β cell immunogenicity and that inhibition of PERK may offer a strategy for preventing or delaying the development of T1D.Item Regulation of β-cell death by ADP-ribosylhydrolase ARH3 via lipid signaling in insulitis(Springer Nature, 2024-02-21) Sarkar, Soumyadeep; Deiter, Cailin; Kyle, Jennifer E.; Guney, Michelle A.; Sarbaugh, Dylan; Yin, Ruichuan; Li, Xiangtang; Cui, Yi; Ramos‑Rodriguez, Mireia; Nicora, Carrie D.; Syed, Farooq; Juan‑Mateu, Jonas; Muralidharan, Charanya; Pasquali, Lorenzo; Evans‑Molina, Carmella; Eizirik, Decio L.; Webb‑Robertson, Bobbie‑Jo M.; Burnum‑Johnson, Kristin; Orr, Galya; Laskin, Julia; Metz, Thomas O.; Mirmira, Raghavendra G.; Sussel, Lori; Ansong, Charles; Nakayasu, Ernesto S.; Pediatrics, School of MedicineBackground: Lipids are regulators of insulitis and β-cell death in type 1 diabetes development, but the underlying mechanisms are poorly understood. Here, we investigated how the islet lipid composition and downstream signaling regulate β-cell death. Methods: We performed lipidomics using three models of insulitis: human islets and EndoC-βH1 β cells treated with the pro-inflammatory cytokines interlukine-1β and interferon-γ, and islets from pre-diabetic non-obese mice. We also performed mass spectrometry and fluorescence imaging to determine the localization of lipids and enzyme in islets. RNAi, apoptotic assay, and qPCR were performed to determine the role of a specific factor in lipid-mediated cytokine signaling. Results: Across all three models, lipidomic analyses showed a consistent increase of lysophosphatidylcholine species and phosphatidylcholines with polyunsaturated fatty acids and a reduction of triacylglycerol species. Imaging assays showed that phosphatidylcholines with polyunsaturated fatty acids and their hydrolyzing enzyme phospholipase PLA2G6 are enriched in islets. In downstream signaling, omega-3 fatty acids reduce cytokine-induced β-cell death by improving the expression of ADP-ribosylhydrolase ARH3. The mechanism involves omega-3 fatty acid-mediated reduction of the histone methylation polycomb complex PRC2 component Suz12, upregulating the expression of Arh3, which in turn decreases cell apoptosis. Conclusions: Our data provide insights into the change of lipidomics landscape in β cells during insulitis and identify a protective mechanism by omega-3 fatty acids.Item The role of proteomics in assessing beta-cell dysfunction and death in type 1 diabetes(Taylor & Francis, 2019-06-24) Nakayasu, Ernesto S.; Qian, Wei-Jun; Evans-Molina, Carmella; Mirmira, Raghavendra G.; Eizirik, Decio L.; Metz, Thomas O.; Pediatrics, School of MedicineIntroduction: Type 1 diabetes (T1D) is characterized by autoimmune-induced dysfunction and destruction of the pancreatic beta cells. Unfortunately, this process is poorly understood, and the current best treatment for type 1 diabetes is administration of exogenous insulin. To better understand these mechanisms and to develop new therapies, there is an urgent need for biomarkers that can reliably predict disease stage. Areas covered: Mass spectrometry (MS)-based proteomics and complementary techniques play an important role in understanding the autoimmune response, inflammation and beta-cell death. MS is also a leading technology for the identification of biomarkers. This, and the technical difficulties and new technologies that provide opportunities to characterize small amounts of sample in great depth and to analyze large sample cohorts will be discussed in this review. Expert opinion: Understanding disease mechanisms and the discovery of disease-associated biomarkers are highly interconnected goals. Ideal biomarkers would be molecules specific to the different stages of the disease process that are released from beta cells to the bloodstream. However, such molecules are likely present in trace amounts in the blood due to the small number of pancreatic beta cells in the human body and the heterogeneity of the target organ and disease process.