caa a22 4500 606233628 CHVBK 20210128101233.0 cr unu---uuuuu 210128e20150601xx s 000 0 eng 10.1007/s11010-015-2387-1 doi (NATIONALLICENCE)springer-10.1007/s11010-015-2387-1 SEAP activity serves for demonstrating ER stress induction by glucolipotoxicity as well as testing ER stress inhibitory potential of therapeutic agents [Elektronische Daten] [Raji Lenin, Viswanathan Mohan, Muthuswamy Balasubramanyam] Endoplasmic reticulum (ER) stress is emerging as a unifying paradigm and one of the underlying mechanisms in the genesis of diabetes and its complications. While this has prompted the development of ER stress inhibitors, there is a limitation in monitoring of ER stress in vitro and in vivo by reliable methodologies. We validated the secreted alkaline phosphatase (SEAP) activity as a surrogate marker of ER stress in mouse β-TC6 cells exposed to glucolipotoxicity or tunicamycin and studied insulin secretion along with alterations in ER stress markers. SEAP activity assay was measured using the Great EscAPe SEAP kit, insulin levels were determined by Mercodia reagents and mRNA expression of ER stress markers was quantified by real-time PCR. SEAP activity in β-cells was significantly decreased (indicating increased ER stress) on exposure either to glucolipotoxicity or tunicamycin. This was accompanied by an increased mRNA expression of ER stress markers (GRP-78, PERK, IRE1α, ATF6, XBP-1, and CHOP) and decreased insulin secretion. Treating the cells with phenylbutyric acid normalized SEAP activity, decreased mRNA expression of ER stress markers and improved insulin secretion. Interestingly, cells exposed to different classes of anti-diabetes agents or compounds such as resveratrol resisted ER stress. Methylglyoxal also induces ER stress and this was counteracted by aminoguanidine. Out study demonstrates SEAP activity as a novel ER stress monitoring assay to investigate the therapeutic value of agents with ER stress inhibitory potential. Future studies should focus on the exercise of adopting this reporter assay for high-throughput screening mode of drug discovery. Springer Science+Business Media New York, 2015 ER stress nationallicence SEAP activity nationallicence UPR nationallicence Glucolipotoxicity nationallicence PBA nationallicence Metformin nationallicence Vildagliptin nationallicence β-Cells nationallicence ATF-6 : Activating transcription factor-6 nationallicence CHOP : CCAAT/enhancer-binding homologous protein nationallicence GRP-78 : Glucose-regulated protein-78 nationallicence IRE-1 α : Inositol-requiring enzyme-1α nationallicence PERK : PKR-like ER kinase nationallicence XBP-1 : X box binding protein1 nationallicence PBA : 4-Phenylbutyric acid nationallicence SEAP : Secreted alkaline phosphatase nationallicence PDI : Protein disulfide isomerase nationallicence ERO1 : ER oxidoreductase1 nationallicence INSIG1 : Insulin-induced gene1 nationallicence SREBP : Sterol regulatory element-binding transcription factor nationallicence AGEs : Advanced Glycation Endproducts nationallicence Lenin Raji Department of Cell and Molecular Biology, Madras Diabetes Research Foundation & Dr. Mohan's Diabetes Specialties Centre, Gopalapuram, 600086, Chennai, India aut Mohan Viswanathan Department of Cell and Molecular Biology, Madras Diabetes Research Foundation & Dr. Mohan's Diabetes Specialties Centre, Gopalapuram, 600086, Chennai, India aut Balasubramanyam Muthuswamy Department of Cell and Molecular Biology, Madras Diabetes Research Foundation & Dr. Mohan's Diabetes Specialties Centre, Gopalapuram, 600086, Chennai, India aut Molecular and Cellular Biochemistry Springer US; http://www.springer-ny.com 404/1-2(2015-06-01), 271-279 0300-8177 404:1-2<271 2015 404 11010 https://doi.org/10.1007/s11010-015-2387-1 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s11010-015-2387-1 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Lenin Raji Department of Cell and Molecular Biology, Madras Diabetes Research Foundation & Dr. Mohan's Diabetes Specialties Centre, Gopalapuram, 600086, Chennai, India aut NATIONALLICENCE 700 1- Mohan Viswanathan Department of Cell and Molecular Biology, Madras Diabetes Research Foundation & Dr. Mohan's Diabetes Specialties Centre, Gopalapuram, 600086, Chennai, India aut NATIONALLICENCE 700 1- Balasubramanyam Muthuswamy Department of Cell and Molecular Biology, Madras Diabetes Research Foundation & Dr. Mohan's Diabetes Specialties Centre, Gopalapuram, 600086, Chennai, India aut NATIONALLICENCE 773 0- Molecular and Cellular Biochemistry Springer US; http://www.springer-ny.com 404/1-2(2015-06-01), 271-279 0300-8177 404:1-2<271 2015 404 11010