Role of the O-GlcNAc modification on insulin resistance and endoplasmic reticulum stress in 3T3-L1 cells
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ORIGINAL ARTICLE
Role of the O-GlcNAc modification on insulin resistance and endoplasmic reticulum stress in 3T3-L1 cells Benan Pelin Sermikli1,2,3 · Gulizar Aydogdu1,2,4 · Erkan Yilmaz1 Received: 4 May 2020 / Accepted: 11 July 2020 © Springer Nature B.V. 2020
Abstract O-linked N-acetyl-glucosamine (O-GlcNAc) is a post-translational protein modification that regulates cell signaling and involves in several pathological conditions. O-GlcNAc transferase (OGT) catalyzes the attachment, while O-GlcNAcase (OGA) splits the GlcNAc molecules from the serine or threonine residues of the nuclear and cellular proteins. The hexosamine biosynthesis pathway (HBP) is a small branch of glycolysis that provides a substrate for the OGT and serves as a nutrient sensor. In this study, we investigated the impact of external O-GlcNAc modification stimulus on the insulin signal transduction, unfolded protein response, and HBP in 3T3-L1 cells. First, we treated cells with glucosamine and PUGNAc to stimulate the O-GlcNAcylation of total proteins. Also, we treated cells with tunicamycin as a positive internal control, which is a widely-used endoplasmic reticulum stressor. We used two in vitro models to understand the impact of the cellular state of insulin sensibility on this hypothesis. So, we employed insulin-sensitive preadipocytes and insulin-resistant adipocytes to answer these questions. Secondly, the OGT-silencing achieved in the insulin-resistant preadipocyte model by using the short-hairpin RNA (shRNA) interference method. Thereafter, the cells treated with the above-mentioned compounds to understand the role of the diminished O-GlcNAc protein modification on the insulin signal transduction, unfolded protein response, and HBP. We found that elevated O-GlcNAcylation of the total proteins displayed a definite correlation in insulin resistance and endoplasmic reticulum stress. Furthermore, we identified that the degree of this correlation depends on the cellular state of insulin sensitivity. Moreover, reduced O-GlcNAcylation of the total proteins by the shRNA-mediated silencing of the OGT gene, which is the only gene to modify proteins with the O-GlcNAc molecule, reversed the insulin resistance and endoplasmic reticulum stress phenotype, even with the externally stimulated O-GlcNAc modification conditions. In conclusion, our results suggest that OGT regulates insulin receptor signaling and unfolded protein response by modulating O-GlcNAc levels of total proteins, in response to insulin resistance. Therefore, it can be a potential therapeutic target to prevent insulin resistance and endoplasmic reticulum stress. Keywords 3T3-L1 · Adipocyte · Endoplasmic reticulum stress · Insulin resistance · O-GlcNAc · Unfolded protein response · Type 2 diabetes
* Benan Pelin Sermikli benan‑[email protected] * Erkan Yilmaz [email protected] Gulizar Aydogdu [email protected] 1
Ankara University Biotechnology Institute, Ankara, Turkey
2
Department of Biology, Faculty of Science, Ankara University, Ankara, Turkey
3
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