Contribution of the toxic advanced glycation end-productsreceptor axis in nonalcoholic steatohepatitis-related hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is one of the most common malignancies worldwide. The main etiologies of HCC are hepatitis B virus and hepatitis C virus(HCV), and non-hepatitis B/non-hepatitis C HCC(NBNCHCC) has also been identified as an etiological factor. Although the incidence of HCV-related HCC in Japan has decreased slightly in recent years, that of NBNC-HCC has increased. The onset mechanism of NBNC-HCC, which has various etiologies, remains unclear; however, nonalcoholic steatohepatitis(NASH), a severe form of nonalcoholic fatty liver disease, is known to be an important risk factor for NBNC-HCC. Among the different advanced glycation end-products(AGEs) formed by the Maillard reaction, glyceraldehyde-derived AGEs, the predominant components of toxic AGEs(TAGE), have been associated with NASH and NBNC-HCC, including NASH-related HCC. Furthermore, the expression of the receptor for AGEs(RAGE) has been correlated with the malignant progression of HCC. Therefore, TAGE induce oxidative stress by binding with RAGE may, in turn, lead to adverse effects, such as fibrosis and malignant transformation, in hepatic stellate cells and tumor cells during NASH or NASH-related HCC progression. The aim of this review was to examine the contribution of the TAGE-RAGE axis in NASH-related HCC.

In vitro identification of nonalcoholic fatty liver diseaserelated protein hnRNPM

AIM: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 matrixassisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS).The protein and m RNA levels were determined by Western blotting and realtime reverse transcription PCR,respectively.RESULTS:The results of the two-dimensional gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a greater amount of GlycerAGEs in the sample exposed to high concentrations of fructose than in the control.The detected GlycerAGEs showed isoelectric points in the range of 8.0-9.0and molecular weights in the range of 60-80 k Da.The heterogeneous nuclear ribonucleoprotein M(hn RNPM),which plays an important role in regulating gene expression by processing heterogeneous nuclear RNAs to form mature m RNAs,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,hn RNPM was found to be more easily glycated than the other proteins.CONCLUSION:The results suggest that glyceraldehyde-modified hn RNPM alters gene expression.This change may cause adverse effects in hepatocytes and may serve as a target for therapeutic intervention.

Gene Expression Changes Associated with the Loss of Heterogeneous Nuclear Ribonucleoprotein M Function

Advanced glycation endproducts (AGEs) are formed by the nonenzymatic reaction of sugars with proteins. Glycation may adversely affect proteins, such as by inducing a loss of function. It has been shown that glyceraldehyde-derived AGEs (Glycer-AGEs) accumulate in the liver of patients with nonalcoholic steatohepatitis (NASH). Previously, we showed the formation of intracellular Glycer-AGEs upon exposure of hepatocytes to fructose in vitro, and identified an RNA-binding protein, heterogeneous nuclear ribonucleoprotein M (HNRNPM), as a target for glycation. However, the impact of glycated HNRNPM in NASH remains poorly understood. In this study, we examined gene expression changes caused by HNRNPM knockdown, and investigated the up- and down-regulated genes as noninvasive biomarker candidates for NASH. Microarray analysis after HNRNPM knockdown showed that the levels of 138 transcripts were increased, while those of 100 transcripts were decreased as compared with those in the control. Gene Ontology-based functional analysis showed that 14 upregulated and 9 downregulated genes were associated with the extracellular space, which may enable their detection using blood tests. Among these, six of the up- and down-regulated genes were associated with the extracellular exosome. These results suggest that the loss of HNRNPM function by glycation is reflected extracellularly. Therefore, the identified genes may serve as noninvasive biomarkers for Glycer-AGEs-related NASH.