Acetaldehyde‐Induced Growth Retardation and Micro‐Heterogeneity of the Sugar Chain in Transferrin Synthesized by HepG2 Cells

Abstract

Background: A carbohydrate-deficient transferrin (CDT) is the most useful marker of alcohol abuse; however, the mechanism of production and the pathophysiologic roles of CDT remain obscure. The effects of alcohol and its metabolites on growth and proliferation, transferrin synthesis, and phosphomannomutase enzyme activity in a human hepatoblastoma, HepG2, were examined.Methods: HepG2 cells were treated with either ethanol at 80 mM or acetaldehyde at 400 M. Transferrin secreted by the cells was prepared from conditioned culture medium by single-step immunoaffinity column chromatography using a goat-specific antibody against human transferrin. Phosphomannomutase and some related enzyme activities in the cell extracts were determined. Reverse transcription-polymerase chain reaction analysis of phosphomannomutase mRNA expression was also determined in HepG2 cultured with or without acetaldehyde (400 M).Results: HepG2 cells usually synthesized and secreted transferrin with three separated bands: main broad bands estimated to be 78 to 82 kDa, 75 kDa, and 72 kDa. The last two bands were compatible with part or the entire N-glycans-deficient transferrin (CDT) from alcoholic liver damage. Increased secretion of CDT from HepG2 correlated well with the extent of growth retardation to the level of confluent cell density. The activity of phosphomannomutase also decreased with prolongation of cellular doubling time. Furthermore, acetaldehyde treatment at 400 M accelerated the inhibitory effect of cell growth compared with nontreated cells, and this condition facilitated CDT secretion from HepG2 cells. Determination of the enzyme activity and mRNA expression indicated that acetaldehyde showed competitive type inhibition of phosphomannomutase activity but not suppression of phosphomannomutase gene expression.Conclusions: By culturing HepG2 cells with acetaldehyde containing media, growth inhibitiondependent increase of CDT showed good correlation with reduced enzyme activity of phosphomannomutase. Acetaldehyde facilitated growth retardation, inhibition of phosphomannomutase activity, and increased secretion of CDT. The HepG2 cell line is useful as an in vitro model to investigate the pathophysiologic state of alcoholic liver damage and mechanisms of production as well as the physiologic role of CDT.

Carbohydrate-deficient transferrin (CDT) as a marker of alcohol abuse: A critical review of the literature 2001–2005

“…Sillanaukee et al, on the basis of a review of the most relevant literature on CDT, concluded that the ethanol-induced effect on glycoprotein metabolism is a multistep process involving both protein transport and enzyme activity [12]. The effects of ethanol and its metabolites on growth, proliferation and synthesis of transferrin in hepatic cells have been investigated by culturing human hepatoblastoma cells (HepG2) in acetaldeyde containing media [13]. Acetaldehyde proved to facilitate growth retardation, inhibite phosphomannomutase activity and increase secretion of CDT.…”

Serum and glucocorticoid-regulated kinase Sgk1 inhibits insulin-dependent activation of phosphomannomutase 2 in transfected COS-7 cells

“…PMM2 activity can be modulated by exogenous factors. Acetaldehyde has been shown to be able to suppress the activity of the enzyme without affecting gene expression in HepG2 cells (18). We have demonstrated for the first time that the activity of PMM2 immunoprecipitated from cells treated with insulin is increased more than twofold, suggesting that the activity of the enzyme can be regulated in vivo by hormones.…”

Glycoproteomics in Cerebrospinal Fluid Reveals Brain-Specific Glycosylation Changes

“…It is thought that ethanol and its metabolite acetaldehyde can cause Golgi remodeling and have inhibitory effects on glycosyltransferases [37][38][39]. For example, acetaldehyde reduces the activity of the phosphomannomutase PMM2, which can cause the failure of complete glycan maturation or disrupt the en bloc transfers of the oligosaccharide precursor to the asparagine side chain of the nascent polypeptide in the ER (see Figure 4) [40]. We hypothesized that this effect is more pronounced in the liver, the central location for alcohol metabolism, which can explain the strong changes in glycosylation observed for blood proteins compared to little changes on brain-derived proteins.…”