Abstract
Disclosure: M. D'silva: None. B. Malachowska: None. Y. Qiu: None. J. Sepulveda: None. R. Chang: None. S. Sidoli: None. J.L. Nadler: None. I.J. Kurland: Scorpion Therapeutics. An important early hallmark of Type I Diabetes Mellitus (T1D) is beta-cell dysfunction, a critical mechanism of which is cytokine mediated. Our exploratory study defined regulatory programs controlling human islet responses to pro-inflammatory cytokines by mapping abundance proteomics , as well as metabolomics and lipidomics from the same islets exposed to inflammation-inducing cytokines TNF-alpha, IL-1β and IFN-γ over the time course at 1,6 and 24 hrs after cytokine exposure. Methods: Pancreatic islets were purchased from Prodo Labs (https://prodolabs.com/). Islet samples were extracted by the Folch method, and targeted mass spectrometric methods employing a Sciex 6500+ QTRAP was used to identified metabolites, neutral and phospholipids and oxylipins/eicosanoids,from the aqueous and chloroform fractions. The Folch protein interface was collected, redissolved and then digested using S-Trap flters (Protif), 5% SDS. Proteome raw files were searched using Proteome Discoverer software. Results: Approximately 3500 proteins were identified, of which approximately 200 were significantly changed with cytokine vs control treatment that were further identified from the RECON genome-scale metabolic reconstruction as potentially important for metabolic network flux determination. ∼100 of these metabolic proteomic changes were seen at 1 hr , and an additional 85 metabolic proteins were significantly changed at 6 hr after cytokine stimulation along 35 proteins that were still changed from 1hr. Overall, cytokine stimulation resulted in an inhibition of metabolic protein expression at these time points, however, the metabolomic analysis indicated that islet metabolism was re-directed to specific pathways rather than simply suppressed. Levels of nucleotides needed for DNA and RNA synthesis could be seen to be increased, as well as pentose and hexosamine pathway intermediates, which depleted glycolytic and TCA cycle intermediates. The inflammatory islet response was reflected by an increase in platelet-activating factor PAF and lyso PAF at 1 and 6 hours, however the brunt of the oxylipin/eicosanoid response was delayed until 24 hours after cytokine stimulation, and secreted (media) oxylipins were composed mainly of cycloxygenase (COX) products of arachidonic (AA) acid, as well as 12- and 15-lipoxygenase (LOX) products of AA and DHA. At 24 hours after cytokine stimulation, the suppression/elevation of the metabolic responses seen at 1 and 6 hrs were largely resolved, along with elevations in HLA proteins and proteosome subunit expression consistent with immune response activation. Conclusion: Interventions for arresting beta cell dysfunction need to consider the pathways which may be activated sequentially and in coordination with immune activation. Presentation: Monday, July 14, 2025