Effects of multiple stressors on pancreatic human islets proteome reveal new insights into the pathways involved

Abstract

Pancreatic β‐cell dysfunction is an early hallmark of type 1 diabetes mellitus. Among the potentially critical factors that cause β‐cell dysfunction are cytokine attack, glucotoxicity, induction of endoplasmic reticulum (ER) or mitochondria stress. However, the exact molecular mechanism underlying β‐cell's inability to maintain glucose homeostasis under severe stresses is unknown. This study used proinflammatory cytokines, thapsigargin, and rotenone in the presence of high concentration glucose to mimicking the conditions experienced by dysfunctional β‐cells in human pancreatic islets, and profiled the alterations to the islet proteome with TMT‐based proteomics. The results were further verified with label‐free quantitative proteomics. The differentially expressed proteins under stress conditions reveal that immune related pathways are mostly perturbed by cytokines, while the respiratory electron transport chains and protein processing in ER pathways by rotenone. Thapsigargin together with high glucose induces dramatic increases of proteins in lipid synthesis and peroxisomal protein import pathways, with energy metabolism and vesicle secretion related pathways downregulated. High concentration glucose, on the other hand, alleviated complex I inhibition induced by rotenone. Our results contribute to a more comprehensive understanding of the molecular events involved in β‐cell dysfunction.

Effect of PTFGRN Expression on the Proteomic Profile of A431 Cells and Determination of the PTGFRN Interactome

“…The two-tailed Student’s t -test was applied for comparisons between two conditions ( p < 0.05) to determine if each treatment group was significantly different from the control group. Enrichment of functions and signaling pathways of the differentially expressed proteins (DEPs) identified from different conditions was performed using Metascape () as described previously. , Once the DEPs were identified, we subsequently used bioinformatics pathway analysis to further infer perturbed pathways. Ingenuity Pathway Analysis (IPA) was also used to predict canonical pathways and upstream regulators. , …”

“…Cell lysis and protein digestion were performed as previously described. 11 Briefly, samples were lysed in a lysis buffer containing 5% sodium dodecyl sulfate (Sigma, L4509) and 50 mM triethylammonium bicarbonate (1 M, pH 8.0) (Sigma, 7408). Proteins were extracted and digested using S-trap micro columns (ProtiFi, NY).…”