Ethanol Consumption Decreases the Synthesis of the Mannose 6-Phosphatelinsulin-Like Growth Factor II Receptor but Does Not Decrease its Messenger RNA

The mannose 6-phosphatelinsulin-like growth factor I1 receptor (M6PlIGF-IIR) is a protein that facilitates the transport of acid hydrolases into the lysosome. We have shown that chronic ethanol consumption lowers the M6PAGF-IIR content in rat hepatocytes. Here, we determined the steady-state level of mRNA encoding M6PlIGF-IIR, as well as the rate of receptor synthesis, to ascertain whether the ethanol-elicited reduction in receptor protein content is related to changes in either or both of these parameters. Rats were pair-fed the normal carbohydrate (NC) or low carbohydrate high-fat (LC) liquid diets containing either ethanol or isocaloric maltose-dextrin for 7-8 weeks. RNA was isolated from hepatocytes and from whole livers of these animals and subjected to reverse transcription-polymerase chain reaction (RT-PCR) to determine the mRNA levels encoding M6PlIGF-IIR. Hepatocytes isolated from these animals were also radiolabeled with Pro-mix L-[?'S] in vitvo cell labeling mix to measure incorporation into total cellular protein and the immunoprecipitated M6PlIGF-IIR protein. The steady-state levels of M6PAGF-IIR mRNA in both hepatocytes and whole livers from ethanol-fed rats were the same as those from their respective controls regardless of whether they were fed the NC or the LC diets. Hepatocytes from ethanol-fed rats showed a 36% lower rate of total protein synthesis and an even greater reduction (70%) in receptor synthesis. When the relative rate of receptor synthesis was calculated, hepatocytes from ethanol-fed rats had a 53% lower relative rate of receptor synthesis compared with controls. Autoradiographic analysis of the immunoprecipitated receptor protein from ethanol-fed rats also indicated a 79% decline in the total M6PlIGF-IIR protein synthetic rate compared with pair-fed controls. We conclude that the ethanol-elicited reduction of M6PlIGFIIR content was, in part, related to a concomitant reduction of receptor protein synthesis but not to a decline in its mRNA level. Thus, the ethanol-elicited decline in receptor protein synthesis may be due to defective M6PlIGF-IIR mRNA translation. Krq'word.~: Messenger RNA; RT-PCR; Liver; Low carbohydrate diet Corresponding author. Tel.: +I-402-346-8800, Ext. 3556; fax: +I-402-449-0604. E-inail ucldrc.s.se.s: [email protected] (T.M. Donohue Jr.), [email protected] (J. Haorah). ' Co-corresponding author. Tel.: +I-402-346-8800, Ext. 3037; fax: +I-402-449-0604. Ahhrrviation.~: M6PlIGF-IIR, mannose 6-phosphatelinsulin-like growth factor I1 receptor; NC, normal carbohydrate; LC, low carbohydrate high-fat; RT-PCR, reverse transcription-polymerase chain reaction; dNTP, deoxynucleotide triphosphate; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; ASGPR, asialoglycoprotein receptor; LBP, lipopolysaccharide binding protein; TGF-p1, transforming growth factor-01.