Pancreatic INS-1 β-Cell Response to Thapsigargin and Rotenone: A Comparative Proteomics Analysis Uncovers Key Pathways of β-Cell Dysfunction

Abstract

Insulin-secreting β-cells in the pancreatic islets are exposed to various endogenous and exogenous stressing conditions, which may lead to β-cell dysfunction or apoptosis and ultimately to diabetes mellitus. However, the detailed molecular mechanisms underlying β-cell’s inability to survive under severe stresses remain to be explored. This study used two common chemical stressors, thapsigargin and rotenone, to induce endoplasmic reticulum (ER) and mitochondria stress in a rat insuloma INS-1 832/13 β-cell line, mimicking the conditions experienced by dysfunctional β-cells. Proteomic changes of cells upon treatment with stressors at IC50 were profiled with TMT-based quantitative proteomics and further verified using label-free quantitive proteomics. The differentially expressed proteins under stress conditions were selected for in-depth bioinformatic analysis. Thapsigargin treatment specifically perturbed unfolded protein response (UPR) related pathways; in addition, 58 proteins not previously linked to the UPR related pathways were identified with consistent upregulation under stress induced by thapsigargin. Conversely, rotenone treatment resulted in significant proteome changes in key mitochondria regulatory pathways such as fatty acid β-oxidation, cellular respiration, citric acid cycle, and respiratory electron transport. Our data also demonstrated that both stressors increased reactive oxygen species production and depleted adenosine triphosphate synthesis, resulting in significant dysregulation of oxidative phosphorylation signaling pathways. These novel dysregulated proteins may suggest an alternative mechanism of action in β-cell dysfunction and provide potential targets for probing ER- and mitochondria stress-induced β-cell death.

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

“…On the other hand, L13a-mediated translational silencing of ceruloplasmin expression was among the dysregulated pathways induced by both stressors (Rot and ThaGlc). This observation was also consistent with our recent reports in rotenone-treated INS-1 cells [16]. Many of these associated proteins relating to RNA binding that contribute to structural integrity of ribosomes were reduced in these stress responses, reflecting the acute conditions leading to accumulation of misfolded proteins in ER.…”

“…In line with our previous observations in INS-1 cells [16], we also found that OXPHOS proteins dysregulated by both rotenone and the combined ThaGlc in human islets. Despite recent studies demonstrated the critical regulatory link between oxidative protein folding in ER and mitochondrial dysfunction through OXPHOS, the molecular mechanism of how these oxidative stresses interact in variety of disease progression is still elusive.…”

“…Interestingly, only minor alterations were observed upon exposure of human islets to either glucose or thapsigargin alone, whereas their combination (ThaGlc) resulted in a dramatic increase in pro- cells model [16] except some additional pathways perturbed in the present data, which may reflect the use of higher rotenone dose (∼10 times) in human islets treatment. What is unique about rotenone is when it is combined with high concentration glucose, the damaging effect was dramatically reduced, resulting in merely 69 DEPs.…”

“…We recently reported that thapsigargin and rotenone induce distinct proteomic profile changes to INS-1 β-cell line [16]. In addition to the stresses in ER and mitochondria, β-cells in vivo are exposed to additional stresses during the initiation and progression stages of diabetes.…”

Vitamin K-dependent γ-carboxylation regulates calcium flux and adaptation to metabolic stress in β-cells

“…To determine if VK and γ-carboxylation can protect β-cells from the acute effects of ER stress, INS-1 832/3 β-cells were cultured for 24h in media containing 25mM glucose in the presence or absence of thapsigargin, an inhibitor of the sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA), and a pharmacological inducer of ER stress (Sharma et al, 2015). Doses of thapsigargin (10-40nM) inducing moderate to elevated cell death in INS-1 cell cultures were selected based on previously published data (Weldemariam et al, 2022). Induction of Grp78/BiP and phospho(Ser724)-IRE following thapsigargin treatment confirmed the presence of ER stress and UPR (Fig.…”