Received for publication April 4, 2008.
Revised May 26, 2008.
Accepted for publication June 10, 2008.

The glucosylceramide synthase inhibitor AMP-DNM induces SREBP regulated gene expression and cholesterol synthesis in HepG2 cells

Abstract

Recent findings have implicated glycosphingolipids as modulators of insulin receptor activity. Studies with C57BL/6J ob/ob mice have shown that insulin sensitivity is enhanced by the synthetic hydrophobic iminosugar AMP-DNM (N-(5-adamantane-1-yl-methoxy-pentyl)-deoxynojirimycin) that inhibits glucosylceramide synthase. Here we treated the liver hepatoma cell line HepG2 with AMP-DNM, resulting in a 70% reduction of glycosphingolipids, and analysed the effect on gene expression. Using Agilent whole human genome 44K oligo arrays, we identified 89 genes that were significantly (p<0.01) up- or down regulated by AMP-DNM treated treatment. Of the 56 up-regulated genes, 17 were direct target genes for transcription factors sterol regulatory element-binding protein 1 (SREBP1) or SREBP2, which activate genes in the sterol biosynthesis pathway. An increase in cholesterol production rate confirmed that the induction of SREBP target genes seen at the mRNA level resulted in activation of the cholesterol biosynthesis pathway. Interestingly, the cholesterol content of the cells did not increase. Importantly, no effects were found on expression of genes related to cell receptor signalling pathways, neither on toxicity or cell growth. Our findings indicate that inhibition of glucosylceramide synthase with AMP-DNM leads to activation of SREBP target genes and synthesis of cholesterol in HepG2 cells.

Key words: HepG2, cholesterol, glycosphingolipid, iminosugar, microarray, sterol regulatory element binding protein