Insulin increases expression of apobec-1, the catalytic subunit of the apolipoprotein B mRNA editing complex in rat hepatocytes.

We have previously shown that chronic insulin treatment of rat hepatocytes increases the fraction of edited apolipoprotein B (apoB) mRNA from approximately 50% to as much as 90%. We have now examined the effect of insulin on apobec-1 mRNA abundance and demonstrate that increased editing of apoB mRNA following insulin treatment is accompanied by elevated apobec-1 mRNA levels in primary rat hepatocytes. Time-course measurements of the effects of insulin on apoB mRNA editing and apobec-1 mRNA abundance showed that both were elevated almost maximally within 48 hours and sustained for at least 5 days of insulin treatment.

A test of the cytosolic apolipoprotein E hypothesis fails to detect the escape of apolipoprotein E from the endocytic pathway into the cytosol and shows that direct expression of apolipoprotein E in the cytosol is cytotoxic.

Genetic evidence indicates that apolipoprotein E4 (apoE4) is a risk factor for the development of Alzheimer's disease. A controversial hypothesis proposes that apoE, a typical secretory protein, accesses the neuronal cytosol in which apoE3, but not apoE4, protects tau from hyperphosphorylation. However, no conclusive evidence for the presence of apoE in the cytosolic compartment has been presented. We designed a novel assay to test whether apoE can access the cytosol via escape from the endocytic pathway by incorporating a nuclear localization signal (NLS) into apoE. Control experiments demonstrated that apoE plus NLS (apoE+NLS) is chaperoned to the nucleus if it reaches the cytosolic compartment. When exogenous apoE+NLS was endocytosed by neuronal cells, no nuclear apoE was detected, indicating that apoE remains within the endocytic pathway and does not escape into the cytosol. Furthermore, we show that direct cytosolic expression of apoE is cytotoxic. These data argue that effects of apoE on the neuronal cytoskeleton and on neurite outgrowth are not mediated via cytosolic interactions but rather by actions originating at the cell surface.

Low levels of extrahepatic nonmacrophage ApoE inhibit atherosclerosis without correcting hypercholesterolemia in ApoE-deficient mice.

The prevention of atherosclerosis by apolipoprotein E (apoE) is generally attributed to the removal of plasma lipoprotein remnant particles. We developed transgenic apoE-knockout mice expressing apoE specifically in the adrenal gland and found that only 3% of the wild-type plasma level of apoE was sufficient to normalize plasma cholesterol levels in the apoE-deficient mouse. As expected, mice expressing apoE at levels that correct hypercholesterolemia had almost no cholesteryl ester deposition in their aortas. In contrast, their nontransgenic siblings had significant atherosclerosis. Unexpectedly, we found that atherosclerosis was also reduced in 2 transgenic lines expressing too little apoE (<1% to 2% of wild type) to correct their hypercholesterolemia. Gel exclusion chromatographic profiles of plasma lipoproteins and the size distributions of lipoproteins with density <1.063 (low density and very low density lipoproteins), as determined by dynamic laser light scattering, were the same in mice expressing <2 microg/mL plasma apoE and their nontransgenic littermates. We conclude that the antiatherogenic action of low levels of plasma apoE is not due to the clearance of remnant lipoproteins. Thus, low levels of apoE provided systemically, but not made in the liver or in macrophages, can block atherogenesis in the vascular wall independently of normalizing the plasma concentration of atherogenic remnant lipoprotein particles.

Insulin promotes the biosynthesis and secretion of apolipoprotein B-48 by altering apolipoprotein B mRNA editing.

Long-term insulin treatment selectively stimulates secretion of the truncated form of apolipoprotein B (apoB), apoB-48, from primary rat hepatocytes in culture. Chronic treatment with insulin at 400 ng/ml causes a 3-fold increase in total apoB secretion, with apoB-48 making up about 75% of that increase. apo-B-48 is the protein product generated by translation of full-length apoB mRNA which has been modified by a posttranscriptional editing mechanism. Editing changes codon 2153 in the middle of the apoB-100 coding region from CAA, coding for glutamine, to UAA, a translation stop signal. We therefore examined the effect of insulin treatment on the ratio of edited to nonedited apoB mRNA in RNA isolated from primary rat hepatocyte cultures. There was a dramatic shift in the ratio of edited versus nonedited forms of apoB mRNA, from about 1:1 in untreated cells to 7:1 in insulin-treated cells. Insulin exerted a dose-dependent effect on apoB secretion and apoB mRNA editing over the range of insulin concentrations studied (0.4-400 ng/ml). In contrast, oleic acid, which also increased apoB (B-48 and B-100) secretion, had no significant effect on the ratio of apoB-48 to apoB-100 particles secreted and no effect on the proportion of edited apoB mRNA. Neither insulin nor oleic acid affects total apoB mRNA levels as assayed by Northern blot analysis. These data strongly suggest that insulin stimulates biosynthesis and secretion of apoB-48 in rat hepatocytes by regulating the proportion of edited apoB mRNA.

Changes in carnitine palmitoyltransferase-I mRNA abundance produced by hyperthyroidism and hypothyroidism parallel changes in activity.

To study the regulation of carnitine palmitoyltransferase-I by thyroid hormone, a cDNA was obtained by PCR amplification of DNA obtained by reverse transcription of rat liver RNA. CPT-I mRNA abundance was measured in livers of hyperthyroid, euthyroid and hypothyroid rats. In hypothyroid rats, the CPT-I mRNA levels decreased 40-fold relative to that of the hyperthyroid animals. These changes paralleled alterations in enzyme activity. These data suggest that CPT-I is regulated at the transcriptional level by thyroid hormone.