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Role of the O-GlcNAc modification on insulin resistance and endoplasmic reticulum stress in 3T3-L1 cells.

Molecular biology reports (2020-07-22)
Benan Pelin Sermikli, Gulizar Aydogdu, Erkan Yilmaz
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
PUGNAc, ≥95% (HPLC)
Sigma-Aldrich
3-Isobutyl-1-methylxanthine, ≥99% (HPLC), powder
Sigma-Aldrich
L-Glutamine solution, 200 mM, solution, sterile-filtered, BioXtra, suitable for cell culture
Sigma-Aldrich
Insulin solution from bovine pancreas, 10 mg/mL insulin in 25  mM HEPES, pH 8.2, BioReagent, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Penicillin-Streptomycin, Solution stabilized, with 10,000 units penicillin and 10 mg streptomycin/mL, 0.1 μm filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
D-(+)-Glucosamine hydrochloride, ≥99%, BioReagent, suitable for cell culture
Sigma-Aldrich
Dulbecco′s Modified Eagle′s Medium - low glucose, With 1000 mg/L glucose, L-glutamine, and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Fetal Bovine Serum, USA origin, sterile-filtered, suitable for cell culture, suitable for hybridoma
Sigma-Aldrich
Tunicamycin from Streptomyces sp.
Sigma-Aldrich
Dexamethasone, powder, BioReagent, suitable for cell culture, ≥97%
Sigma-Aldrich
Anti-O-GlcNAc Transferase (DM-17) antibody produced in rabbit, affinity isolated antibody, buffered aqueous solution