title: In vitro identification of nonalcoholic fatty liver disease-related protein hnRNPM.
abstract: OBJECTIVE
  To study the formation of intracellular glyceraldehyde-derived advanced glycation end products (Glycer-AGEs) in the presence of high concentrations of fructose.
  
  
  METHODS
  Cells of the human hepatocyte cell line Hep3B were incubated with or without fructose for five days, and the corresponding cell lysates were separated by two-dimensional gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Glycer-AGEs were detected with the anti-Glycer-AGEs antibody. Furthermore, the identification of the proteins that are modified by glyceraldehyde in the presence of high concentrations of fructose was conducted using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The protein and mRNA levels were determined by Western blotting and real-time reverse transcription PCR, respectively.
  
  
  RESULTS
  The results of the two-dimensional gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a greater amount of Glycer-AGEs in the sample exposed to high concentrations of fructose than in the control. The detected Glycer-AGEs showed isoelectric points in the range of 8.0-9.0 and molecular weights in the range of 60-80 kDa. The heterogeneous nuclear ribonucleoprotein M (hnRNPM), which plays an important role in regulating gene expression by processing heterogeneous nuclear RNAs to form mature mRNAs, was identified as a modified protein using MALDI-TOF-MS. Increasing the concentration of fructose in the medium induced a concentration-dependent increase in the generated Glycer-AGEs. Furthermore, in an experiment using glyceraldehyde, which is a precursor of Glycer-AGEs, hnRNPM was found to be more easily glycated than the other proteins.
  
  
  CONCLUSIONS
  The results suggest that glyceraldehyde-modified hnRNPM alters gene expression. This change may cause adverse effects in hepatocytes and may serve as a target for therapeutic intervention.
authors: Jun-ichi Takino, Kentaro Nagamine, Takamitsu Hori, Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hiroshima International University, Hiroshima 737-0112, Japan.
authors: Jun-ichi
authors: J
authors: Takino
authors: Jun-ichi Takino, Kentaro Nagamine, Takamitsu Hori, Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hiroshima International University, Hiroshima 737-0112, Japan.
authors: Kentaro
authors: K
authors: Nagamine
authors: Jun-ichi Takino, Kentaro Nagamine, Takamitsu Hori, Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hiroshima International University, Hiroshima 737-0112, Japan.
authors: Masayoshi
authors: M
authors: Takeuchi
authors: Jun-ichi Takino, Kentaro Nagamine, Takamitsu Hori, Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hiroshima International University, Hiroshima 737-0112, Japan.
authors: Takamitsu
authors: T
authors: Hori
mesh_terms: D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005632:Fructose / Q000494:pharmacology; D017127:Glycation End Products, Advanced / Q000378:metabolism; D005985:Glyceraldehyde / Q000378:metabolism; D006031:Glycosylation; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D034761:Heterogeneous-Nuclear Ribonucleoprotein Group M / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D065626:Non-alcoholic Fatty Liver Disease / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods
chemical_list: D017127:Glycation End Products, Advanced; C440104:HNRNPM protein, human; D034761:Heterogeneous-Nuclear Ribonucleoprotein Group M; D005632:Fructose; D005985:Glyceraldehyde
keywords: Advanced glycation end-products; Fructose; Glycation; Glyceraldehyde; Heterogeneous nuclear ribonucleoprotein M; Nonalcoholic fatty liver disease; Nonalcoholic steatohepatitis