SEAP activity serves for demonstrating ER stress induction by glucolipotoxicity as well as testing ER stress inhibitory potential of therapeutic agents

Raji Lenin, Viswanathan Mohan, Muthuswamy Balasubramanyam

Research output: Contribution to journalArticle

2 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)271-279
Number of pages9
JournalMolecular and Cellular Biochemistry
Volume404
Issue number1-2
DOIs
StatePublished - Jun 18 2015

Fingerprint

Endoplasmic Reticulum Stress
Alkaline Phosphatase
Testing
Therapeutics
Insulin
Tunicamycin
Assays
Medical problems
Messenger RNA
High-Throughput Screening Assays
Pyruvaldehyde
Monitoring
Diabetes Complications
Drug Discovery
Real-Time Polymerase Chain Reaction
Screening

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Clinical Biochemistry
  • Cell Biology

Cite this

SEAP activity serves for demonstrating ER stress induction by glucolipotoxicity as well as testing ER stress inhibitory potential of therapeutic agents. / Lenin, Raji; Mohan, Viswanathan; Balasubramanyam, Muthuswamy.

In: Molecular and Cellular Biochemistry, Vol. 404, No. 1-2, 18.06.2015, p. 271-279.

Research output: Contribution to journalArticle

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