Hepatic lipid accumulation, altered very low density lipoprotein formation and apolipoprotein E deposition in apolipoprotein E3-Leiden transgenic mice.

BACKGROUND/AIM: Apolipoprotein (apo) E-deficiency leads to hepatic steatosis and impaired Very Low Density Lipoprotein (VLDL)-triglyceride production rates in mice. A mutant apoE isoform, apoE3-Leiden, is associated with a dominantly inherited form of dysbetalipoproteinemia in humans. The aim of this study was to evaluate the effects of APOE*3-Leiden expression on hepatic lipid content, VLDL formation and liver morphology in mice. METHODS: Comparison of lipid parameters and liver morphology in mouse strains with different expression of the APOE*3-Leiden transgene with and without co-expression of human APOCI. RESULTS: Hepatic triglyceride content was increased to maximally 233% of control values, depending on hepatic APOE*3-Leiden expression. Hepatic secretion of VLDL-associated triglycerides was impaired (-20%) in high-expressing transgenics, with a concomitant increase from 1.6 to 8.1 of the apoB48/ apoB100 ratio in newly-formed VLDL. Hepatocytes of the transgenic mice contained characteristic inclusions, up to 20 microm in diameter, in numbers dependent on APOE*3-Leiden expression and independent of APOCI expression. These inclusions contained material positively reacting with antihuman apoE antibodies. Immunogold-labeling confirmed the presence of apoE3-Leiden within these inclusions and also revealed the presence of the mutant protein on sinusoidal membranes, in multivesicular bodies and in peroxisomes, i.e., a distribution pattern similar to that of endogenous apoE in rodents. Nascent VLDL particles associated with the Golgi apparatus were also labeled. CONCLUSION: This study has demonstrated that introduction of human apoE3-Leiden in mice, in addition to its reported effects on lipolysis and lipoprotein clearance, leads to hepatic deposition of the mutant apolipoprotein, development of fatty liver and to altered hepatic VLDL secretion. The latter findings are consistent with a role of apoE in the regulation of intrahepatic lipid metabolism.

Purification and molecular cloning of a novel essential component of the apolipoprotein B mRNA editing enzyme-complex.

Editing of apolipoprotein B (apoB) mRNA requires the catalytic component APOBEC-1 together with "auxiliary" proteins that have not been conclusively characterized so far. Here we report the purification of these additional components of the apoB mRNA editing enzyme-complex from rat liver and the cDNA cloning of the novel APOBEC-1-stimulating protein (ASP). Two proteins copurified into the final active fraction and were characterized by peptide sequencing and mass spectrometry: KSRP, a 75-kDa protein originally described as a splicing regulating factor, and ASP, a hitherto unknown 65-kDa protein. Separation of these two proteins resulted in a reduction of APOBEC-1-stimulating activity. ASP represents a novel type of RNA-binding protein and contains three single-stranded RNA-binding domains in the amino-terminal half and a putative double-stranded RNA-binding domain at the carboxyl terminus. Purified recombinant glutathione S-transferase (GST)-ASP, but not recombinant GST-KSRP, stimulated recombinant GST-APOBEC-1 to edit apoB RNA in vitro. These data demonstrate that ASP is the second essential component of the apoB mRNA editing enzyme-complex. In rat liver, ASP is apparently associated with KSRP, which may confer stability to the editing enzyme-complex with its substrate apoB RNA serving as an additional auxiliary component.

Absence of APOBEC-1 mediated mRNA editing in human carcinomas.

The transgene expression of the catalytic subunit APOBEC-1 of the apo B mRNA editing enzyme-complex can cause hepatocellular carcinoma in mice and rabbits. It has been proposed that aberrant editing of mRNA may represent a novel oncogenic principle. This investigation aimed to define whether such aberrant hyperediting mediated by APOBEC-1 occurs in human carcinomas. Editing and hyperediting of apo B, NAT1 or NF1 mRNA was not identified in any of 28 resected tumor specimens, including hepatocellular, bile duct, gastric, colorectal, pancreatic adeno- and neuroendocrine, lung adeno-, medullary thyroid and breast carcinoma, soft tissue sarcoma and neuroblastoma. In most types of carcinoma, significant levels for full-length APOBEC-1 mRNA could not be detected. Low level expression of APOBEC-1 was found in colorectal and gastric carcinoma where most of the APOBEC-1 mRNA is inactivated by alternate splicing. The 'auxiliary' components of the apo B mRNA editing enzyme-complex are missing in many tumors including colorectal and gastric carcinoma, but are highly expressed in hepatocellular, lung adeno- and breast carcinoma all of which lack APOBEC-1. Taken together, either APOBEC-1 or the 'auxiliary' components of the apo B mRNA editing enzyme-complex or both are missing in human carcinomas resulting in the absence of mRNA editing. Currently, there is no evidence that aberrant editing mediated by APOBEC-1 contributes to the tumorigenesis of natural human carcinomas.