Measurement of hepatic protein fractional synthetic rate with stable isotope labeling technique in thapsigargin stressed HepG2 cells

Juquan Song, Xiao jun Zhang, Darren Boehning, Natasha C. Brooks, David N. Herndon, Marc G. Jeschke

Research output: Contribution to journalArticle

10 Scopus citations

Abstract

Severe burn-induced liver damage and dysfunction is associated with endoplasmic reticulum (ER) stress. ER stress has been shown to regulate global protein synthesis. In the current study, we induced ER stress in vitro and estimated the effect of ER stress on hepatic protein synthesis. The aim was two-fold: (1) to establish an in vitro model to isotopically measure hepatic protein synthesis and (2) to evaluate protein fractional synthetic rate (FSR) in response to ER stress. Human hepatocellular carcinoma cells (HepG2) were cultured in medium supplemented with stable isotopes 1,2- 13C 2-glycine and L-[ring- 13C 6]phenylalanine. ER stress was induced by exposing the cells to 100 nM of thapsigargin (TG). Cell content was collected from day 0 to 14. Alterations in cytosolic calcium were measured by calcium imaging and ER stress markers were confirmed by Western blotting. The precursor and product enrichments were detected by GC-MS analysis for FSR calculation. We found that the hepatic protein FSR were 0.97±0.02 and 0.99±0.05%/hr calculated from 1,2- 13C 2-glycine and L-[ring- 13C 6]phenylalanine, respectively. TG depleted ER calcium stores and induced ER stress by upregulating p-IRE-1 and Bip. FSR dramatically decreased to 0.68±0.03 and 0.60±0.06%/hr in the TG treatment group (p<0.05, vs. control). TG-induced ER stress inhibited hepatic protein synthesis. The stable isotope tracer incorporation technique is a useful method for studying the effects of ER stress on hepatic protein synthesis.

Original languageEnglish (US)
Pages (from-to)265-271
Number of pages7
JournalInternational Journal of Biological Sciences
Volume8
Issue number2
DOIs
StatePublished - 2012

Keywords

  • Calcium
  • Endoplasmic reticulum (ER) stress
  • Gas chromatography-mass spectrometry (GC-MS)
  • Hepatic protein synthesis
  • In vitro

ASJC Scopus subject areas

  • Ecology, Evolution, Behavior and Systematics
  • Applied Microbiology and Biotechnology
  • Molecular Biology
  • Developmental Biology
  • Cell Biology

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