title: SEAP activity serves for demonstrating ER stress induction by glucolipotoxicity as well as testing ER stress inhibitory potential of therapeutic agents.
abstract: 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.
authors.affiliation: Department of Cell and Molecular Biology, Madras Diabetes Research Foundation & Dr. Mohan's Diabetes Specialties Centre, Gopalapuram, Chennai, 600086, India.
authors.forename: Raji
authors.initials: R
authors.lastname: Lenin
authors.forename: Viswanathan
authors.initials: V
authors.lastname: Mohan
authors.forename: Muthuswamy
authors.initials: M
authors.lastname: Balasubramanyam
mesh_terms: D000469:Alkaline Phosphatase / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000235:genetics*; D059865:Endoplasmic Reticulum Stress / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D051379:Mice; D010654:Phenylbutyrates / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D014415:Tunicamycin / Q000633:toxicity
chemical_list: D015415:Biomarkers; D007328:Insulin; D010654:Phenylbutyrates; D012333:RNA, Messenger; D014415:Tunicamycin; D000469:Alkaline Phosphatase