Hepatic apoB mRNA Editing Research Articles

Why are humans LDL-animals and mice are not? Hepatic APOB mRNA editing and plasma PLTP activity are the cause, not CETP

Why are humans LDL-animals and mice are not? Hepatic APOB mRNA editing and plasma PLTP activity are the cause, not CETP

Abstract 313: Perinatal Growth Restriction Decreases Hepatic RNA Editing of Apolipoprotein B in Male Rat Offspring

Introduction: Individuals born with intrauterine growth restriction develop cardiovascular disease. We employed hepatic microarray-based expression profiling of male rat offspring born to mothers subjected to combined intrauterine and postnatal calorie restriction (IPCR) as a discovery driven experiment to interrogate intrauterine growth restriction. These studies identified that the expression of Apobec1 and Apobec1 complementation factor (A1CF), genes known to edit apolipoprotein B (ApoB) mRNA through the epigenetic process of cytosine deamination, are decreased in IPCR. Editing of ApoB mRNA at position 666 changes the ribonucleotide base from a cytosine to a uracil (U) and controls the production of ApoB100 and ApoB48 lipoproteins. Hypothesis: IPCR decreases hepatic ApoB mRNA editing in male offspring through transcriptional regulation of Apobec1 and A1CF. Methods: Pyrosequencing designed to interrogate ApoB mRNA editing was used to quantify hepatic ApoB100 and ApoB48 transcripts in an experiment employing the male offspring (litters culled to 6 male offspring for each mother) from 4 control fed mothers and 4 mothers subjected to 50% calorie restriction from embryonic day 11 to postnatal day 21 (IPCR). RT-qPCR and western blotting were employed to quantify the hepatic expression of Apobec1 and A1CF. Serum ApoB100 and Apo48 were quantified by ELISA. Results: ApoB transcripts in the control fed offspring were edited, with a U base call of 55 ± 2% at position 666, whereas IPCR offspring possessed decreased editing with a U base call of 12 ± 1% (n=12 for each group; F=368; p<0.0001). The RT-qPCR relative quotient (RQ) in the IPCR group was 0.2 ± 0.1 (F=107;p<0.0001) and 0.4 ± 0.1 (F=25;p<0.0001) for Apobec1 and A1CF, respectively (RQ=1 in the control fed offspring for both Apobec1 and A1CF). Relative to control, IPCR treatment significantly decreased hepatic Apobec1 and A1CF proteins by 45% ± 10 and 27 % ± 9.2 respectively. IPCR treatment increased serum ApoB100 by 1.5 fold ± 0.43 (p= 0.051) and significantly decreased ApoB48 by 0.8 fold ± 0.1 (n=6) compared to control. Conclusion: These data define a novel association of IPCR and ApoB editing through a nutritionally controlled epigenetic process working through transcriptional control of Apobec1 and A1CF.

Rapid Development of Atherosclerosis in the World’s Smallest Microminipig Fed a High-Fat/High-Cholesterol Diet

Experimental studies of human atherogenesis require an appropriate animal model that mimics human physiology and pathology. Because swine physiology is similar to human physiology, we developed a hyperlipidemia-induced atherosclerosis model using the recently developed world's smallest Microminipig(TM). These animals weigh only 5kg at 3months of age, much smaller than any other miniature pig. We found that the administration of a high-fat/high-cholesterol diet containing at least 0.2% cholesterol without cholic acid for as little as eight weeks induces hypercholesterolemia and subsequent atherosclerosis in these animals. The serum levels of low-density lipoprotein cholesterol(LDL-C) and the percent distribution of cholesterol in the LDL fractions were markedly increased. The hepatic expression of LDL receptor and hydroxymethylglutaryl-CoA reductase was coordinately decreased. The cholesteryl ester transfer protein activity, which plays a role in reverse cholesterol transport, was detected in the serum of the Microminipigs. Niemann-Pick C1-like 1 protein was expressed in both the liver and small intestine; however, hepatic apoB mRNA editing enzyme was not expressed. As in humans, and in contrast to that observed in mice, most of the hepatic lipase activity was localized in the liver. These results suggest that the hyperlipidemia-induced gene expression profile linked to cholesterol homeostasis and atherogenesis is similar in Microminipigs and humans. We conclude that the characteristics of the Microminipig, including its easy handling size, make it an appropriate model for studies of atherosclerosis and related conditions.

Recapture of hepatic apolipoprotein B mRNA editing may be a promising strategy to relieve nephrotic dyslipidemia

A high total plasma cholesterol concentration is the most common abnormality found in patients with kidney disease, which may be associated with the increased hepatic synthesis of apoB containing lipoproteins. ApoB mRNA editing plays an important physiological role in mammalian lipid metabolism by modifying the distribution of apoB-100 and apoB-48. However, it is regretful that apoB mRNA editing cannot be found in human liver because of the absence of apobec-1 expression. In this context, we hypothesize that the recapture of hepatic apoB mRNA editing may be a promising strategy to relieve nephrotic dyslipidemia. The data presented below focus on those which support this hypothesis with regards to evidence in vitro and in vivo. (1) Human wild-type apoB mRNA can be edited only when both apobec-1 and ACF proteins are presented simultaneously in vitro. (2) Adenoviral vectors can produce short-term expression of exogenous apobec-1 in the livers and lower plasma apoB-100 and LDL levels transiently. (3) Apobec-1 transgenic animals exhibit massive hepatic editing of apoB mRNA and fundamental decreased plasma levels of apoB-100 and LDL, but are exposed to high risk of liver dysplasia and hepatocellular carcinomas. In summary, taking into account the therapeutic security, we put forward that apobec-1 recombinant adenoviral vectors can be used for the recapture of hepatic apoB mRNA editing with a transient low-level manner and may achieve satisfactory lipid-lowing effect in nephropathic animals.

Metabolic regulation of ApoB mRNA editing is associated with phosphorylation of APOBEC-1 complementation factor

Apolipoprotein B (apoB) mRNA editing is a nuclear event that minimally requires the RNA substrate, APOBEC-1 and APOBEC-1 Complementation Factor (ACF). The co-localization of these macro-molecules within the nucleus and the modulation of hepatic apoB mRNA editing activity have been described following a variety of metabolic perturbations, but the mechanism that regulates editosome assembly is unknown. APOBEC-1 was effectively co-immunoprecipitated with ACF from nuclear, but not cytoplasmic extracts. Moreover, alkaline phosphatase treatment of nuclear extracts reduced the amount of APOBEC-1 co-immunoprecipitated with ACF and inhibited in vitro editing activity. Ethanol stimulated apoB mRNA editing was associated with a 2- to 3-fold increase in ACF phosphorylation relative to that in control primary hepatocytes. Significantly, phosphorylated ACF was restricted to nuclear extracts where it co-sedimented with 27S editing competent complexes. Two-dimensional phosphoamino acid analysis of ACF immunopurified from hepatocyte nuclear extracts demonstrated phosphorylation of serine residues that was increased by ethanol treatment. Inhibition of protein phosphatase I, but not PPIIA or IIB, stimulated apoB mRNA editing activity coincident with enhanced ACF phosphorylation in vivo. These data demonstrate that ACF is a metabolically regulated phosphoprotein and suggest that this post-translational modification increases hepatic apoB mRNA editing activity by enhancing ACF nuclear localization/retention, facilitating the interaction of ACF with APOBEC-1 and thereby increasing the probability of editosome assembly and activity.

The Peroxisome Proliferator-activated Receptor α (PPARα) Agonist Ciprofibrate Inhibits Apolipoprotein B mRNA Editing in Low Density Lipoprotein Receptor-deficient Mice: EFFECTS ON PLASMA LIPOPROTEINS AND THE DEVELOPMENT OF ATHEROSCLEROTIC LESIONS

Low density lipoprotein receptor (LDLR)-deficient mice fed a chow diet have a mild hypercholesterolemia caused by the abnormal accumulation in the plasma of apolipoprotein B (apoB)-100- and apoB-48-carrying intermediate density lipoproteins (IDL) and low density lipoproteins (LDL). Treatment of LDLR-deficient mice with ciprofibrate caused a marked decrease in plasma apoB-48-carrying IDL and LDL but at the same time caused a large accumulation of triglyceride-depleted apoB-100-carrying IDL and LDL, resulting in a significant increase in plasma cholesterol levels. These plasma lipoprotein changes were associated with an increase in the hepatic secretion of apoB-100-carrying very low density lipoproteins (VLDL) and a decrease in the secretion of apoB-48-carrying VLDL, accompanied by a significant decrease in hepatic apoB mRNA editing. Hepatic apobec-1 complementation factor mRNA and protein abundance were significantly decreased, whereas apobec-1 mRNA and protein abundance remained unchanged. No changes in apoB mRNA editing occurred in the intestine of the treated animals. After 150 days of treatment with ciprofibrate, consistent with the increased plasma accumulation of apoB-100-carrying IDL and LDL, the LDLR-deficient mice displayed severe atherosclerotic lesions in the aorta. These findings demonstrate that ciprofibrate treatment decreases hepatic apoB mRNA editing and alters the pattern of hepatic lipoprotein secretion toward apoB-100-associated VLDL, changes that in turn lead to increased atherosclerosis.

Two Proteins Essential for Apolipoprotein B mRNA Editing Are Expressed from a Single Gene through Alternative Splicing

Apolipoprotein B (apoB) mRNA editing involves site-specific deamination of cytidine to form uridine, resulting in the production of an in-frame stop codon. Protein translated from edited mRNA is associated with a reduced risk of atherosclerosis, and hence the protein factors that regulate hepatic apoB mRNA editing are of interest. A human protein essential for apoB mRNA editing and an eight-amino acid-longer variant of no known function have been recently cloned. We report that both proteins, henceforth referred to as ACF64 and ACF65, supported APOBEC-1 (the catalytic subunit of the editosome) equivalently in editing of apoB mRNA. They are encoded by a single 82-kb gene on chromosome 10. The transcripts are encoded by 15 exons that are expressed from a tissue-specific promoter minimally contained within the -0.33-kb DNA sequence. ACF64 and ACF65 mRNAs are expressed in both liver and intestinal cells in an approximate 1:4 ratio. Exon 11 is alternatively spliced to include or exclude 24 nucleotides of exon 12, thereby encoding ACF65 and ACF64, respectively. Recognition motifs for the serine/arginine-rich (SR) proteins SC35, SRp40, SRp55, and SF2/ASF involved in alternative RNA splicing were predicted in exon 12. Overexpression of these SR proteins in liver cells demonstrated that alternative splicing of a minigene-derived transcript to express ACF65 was enhanced 6-fold by SRp40. The data account for the expression of two editing factors and provide a possible explanation for their different levels of expression.

Plasma Apolipoprotein B-48, Hepatic Apolipoprotein B mRNA Editing and Apolipoprotein B mRNA Editing Catalytic Subunit-1 mRNA Levels Are Altered in Zinc-Deficient Rats

Apolipoprotein B (apoB) exists as two major isoforms and serves as an obligatory component of lipid-rich plasma lipoprotein particles. Apolipoprotein B mRNA editing is a zinc-dependent, site-specific cytidine deamination that determines whether the apoB-100 or apoB-48 isoform is synthesized. The objective of this work was to examine whether dietary zinc levels affect apoB mRNA editing in vivo. Adult male Sprague-Dawley rats were randomly assigned to zinc-deficient (ZD, <0.5 mg Zn/kg diet), zinc-adequate (ZA, 30 mg Zn/kg diet) or zinc-replenished (ZDA, ZD rats fed the ZA diet for last 2 d) dietary groups for 18 d. The ratio of plasma apolipoprotein B-48 (apoB-48) to total apoB was significantly lower in zinc-deficient compared with zinc-adequate rats. Primer extension analysis indicated a modest but significant reduction in hepatic apoB mRNA editing in ZD rats compared with that of the ZA group. In ZDA rats, hepatic apoB mRNA editing and the percentage of plasma apoB-48 to total apoB were not different from ZA rats. The mRNA abundance of hepatic apobec-1 (apoB mRNA editing catalytic subunit 1) was significantly lower in ZD and ZDA rats than in ZA rats. In summary, the plasma ratio of apoB-48 to total apoB protein as well as hepatic apoB mRNA editing and hepatic apobec-1 mRNA levels were reduced in rats consuming a zinc-deficient diet. These data suggest that one or more components of apoB metabolism may be influenced by dietary zinc status.

Tissue-specific Inhibition of Apolipoprotein B mRNA Editing in the Liver by Adenovirus-mediated Transfer of a Dominant Negative Mutant APOBEC-1 Leads to Increased Low Density Lipoprotein in Mice

APOBEC-1 is a catalytic subunit of an apolipoprotein B (apoB) mRNA editing enzyme complex. In humans it is expressed only in the intestine, whereas in mice it is expressed in both the liver and intestine. APOBEC-1 exists as a spontaneous homodimer (Lau, P. P., Zhu, H.-J., Baldini, A., Charnsangavej, C., and Chan, L. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8522-8526). We tested the editing activity and dimerization potential of three different mouse APOBEC-1 mutants using in vitro editing activity assay and immunoprecipitation in the presence of epitope-tagged APOBEC-1. One catalytically inactive mutant, mu1 (H61K/C93S/C96S), that retains its capacity to dimerize with wild-type APOBEC-1 was found to inhibit the editing activity of the latter and was thus a dominant negative mutant. Two other inactive mutants that dimerized poorly with APOBEC-1 failed to inhibit its activity. Intravenous injection of a mu1 adenovirus, Admu1, in C57BL/6J mice in vivo resulted in liver-specific expression of mu1 mRNA. On days 4 and 9 after virus injection, endogenous hepatic apoB mRNA editing was 23.3 +/- 5.0 and 36.8 +/- 5.7%, respectively, compared with 65.3 +/- 11 and 71.3 +/- 5.2%, respectively, for luciferase adenovirus-treated animals. Plasma apoB-100 accounted for 95 and 93% of total plasma apoB in Admu1 animals on days 4 and 9, respectively, compared with 78 and 72% in luciferase adenovirus animals. Plasma cholesterol on day 9 was 98 +/- 17 mg/dl in the mu1-treated animals, substantially higher than phosphate-buffered saline-treated (57 +/- 9 mg/dl) or luciferase-treated (71 +/- 12 mg/dl) controls. Fast protein liquid chromatography analysis of mouse plasma showed that the intermediate density/low density lipoprotein fractions in the animals treated with the dominant negative mutant adenovirus were much higher than those in controls. We conclude that active APOBEC-1 functions as a dimer and its activity is inhibited by a dominant negative mutant. Furthermore, apoB mRNA editing determines the availability of apoB-100, which in turn limits the amount of intermediate density/low density lipoprotein that can be formed in mice. Liver-specific inhibition of apoB mRNA editing is an important component of any strategy to enhance the value of mice as a model for human lipoprotein metabolism.

Hepatic gene transfer of the catalytic subunit of the apolipoprotein B mRNA editing enzyme results in a reduction of plasma LDL levels in normal and watanabe heritable hyperlipidemic rabbits

Apolipoprotein (apo) B exists in two forms, the full length protein apoB-100 and the carboxyterminal-truncated apoB-48 that is synthesized in the intestine due to editing of the apoB mRNA which generates a premature stop codon. To determine whether gene transfer of the catalytic subunit of the apoB mRNA editing enzyme APOBEC-1 (apoB mRNA editing enzyme catalytic polypeptide 1) into the liver of rabbits reconstitutes hepatic apoB mRNA editing and how this affects the plasma levels of apoB-containing lipoproteins, we constructed an APOBEC-1 recombinant adenovirus (Ad APOBEC-1). After injection of Ad APOBEC-1 into normal New Zealand White (NZW) or Watanabe heritable hyperlipidemic (WHHL) rabbits, up to 50% of the hepatic apoB mRNA was edited and freshly isolated hepatocytes secreted predominantly apoB-48-containing lipoproteins. VLDL isolated from Ad APOBEC-1-treated NZW and WHHL rabbits contained both apoB-100 and apoB-48, whereas that from control rabbits infected with a beta-galactosidase recombinant adenovirus (Ad LacZ) contained exclusively apoB-100. VLDL from WHHL rabbits treated with Ad APOBEC-1 had the same particle size, lipid composition, and content of apolipoprotein E as VLDL from Ad LacZ-infected control animals. An increase of VLDL was observed in NZW and WHHL rabbits after infection with Ad APOBEC-1 as well as Ad LacZ. After injection of Ad APOBEC-1, LDL became undetectable in the plasma of NZW rabbits and was reduced by an average of 65% in the plasma of WHHL rabbits compared to Ad LacZ-infected controls. LDL from Ad APOBEC-1-infected WHHL rabbits contained only apoB-100. VLDL isolated from Ad APOBEC-1-infected WHHL rabbits were rapidly cleared from the circulation after injection into NZW rabbits. These results provide further evidence that the switch in the hepatic synthesis from exclusively apoB-100 to partly apoB-48 can result in a reduction of LDL formation that requires the full-length apoB-100.

Copper deficiency increases hepatic apolipoprotein B secretion and mRNA editing in rats

Male weanling Sprague-Dawley rats were assigned to copper-deficient (9.0 mumol Cu/kg diet) or copper-adequate (102 mumol Cu/kg diet) dietary treatments for 6 wk. Pulse-chase studies using freshly isolated rat liver parenchymal cells demonstrated that apolipoprotein B (apoB)-48 and apoB-100 syntheses were not altered, but secretion was increased twofold in hepatocytes derived from copper-deficient rats. Both plasma apoB-48 and apoB-100 levels were increased by copper deficiency, but only the apoB-48 increase was significant. Hepatic apoB mRNA editing, expressed as a ratio of apoB-48 mRNA to apoB-48 plus apoB-100 mRNA, was significantly increased from 60.8% in copper-adequate to 70.2% in copper-deficient rats. Moreover, hepatic apoB mRNA abundance was not significantly altered by copper deficiency. Thus the increased amount of nascent apoB-48 secreted into the medium as well as the enhanced apoB mRNA editing may have contributed to the differential increase in plasma apoB-48 over apoB-100 level in copper-deficient rats.

Hepatic Expression of the Catalytic Subunit of the Apolipoprotein B mRNA Editing Enzyme (apobec-1) Ameliorates Hypercholesterolemia in LDL Receptor-Deficient Rabbits

Apolipoprotein (apo) B48, a protein contained in intestinally derived lipoprotein particles, is synthesized by post-transcriptional editing of apoB100 mRNA. This reaction is mediated by an enzyme complex that includes the catalytic subunit, apobec-1. The liver of most mammals, by contrast, contains only unedited apoB mRNA and secretes apoB100, the major protein component of plasma low-density lipoprotein (LDL). Because rabbits, like humans, fail to edit hepatic apoB100 mRNA, we introduced a recombinant adenovirus encoding apobec-1 into the livers of LDL receptor-defective rabbits to determine the impact on lipoprotein metabolism of hepatic apoB48 secretion. Transgene expression was mainly confined to the liver and was sustained for up to 3 weeks following virus administration, as evidenced by the presence of apobec-1 mRNA and the ability of hepatic S100 extracts to edit a synthetic apoB RNA template in vitro. The transient induction of hepatic apoB mRNA editing accompanied alterations in very-low-density lipoprotein (VLDL) size, the presence of apoB48 in fractions spanning the VLDL and LDL range, and modest reductions in total plasma cholesterol levels.

Targeted Disruption of the Mouse apobec-1 Gene Abolishes Apolipoprotein B mRNA Editing and Eliminates Apolipoprotein B48

A site-specific C to U editing reaction modifies nuclear apolipoprotein B100 (apoB100) mRNA, producing apolipoprotein B48 in the mammalian small intestine. This reaction is mediated by a multicomponent enzyme complex, which contains a catalytic subunit, Apobec-1. We have used gene targeting to disrupt mouse apobec-1 in order to establish its requisite importance in apoB mRNA editing and also, in view of its widespread tissue distribution in rodents, as a preliminary indication of other potential roles. Both heterozygous (apobec-1+/-) and homozygous (apobec-1-/-) gene-targeted mice appear healthy and fertile with no alterations in serum cholesterol or triglyceride concentrations. The apobec-1+/- mice demonstrated reduced levels of hepatic apoB mRNA editing. By contrast, levels of small intestinal apoB mRNA editing were indistinguishable in wild-type and apobec-1+/- animals, suggesting that Apobec-1 is expressed in limited quantities in the liver but not in the small intestine. The apobec-1-/- mice lacked detectable levels of Apobec-1 mRNA, expressed only unedited apoB mRNA in all tissues, and contained no apoB48 in their serum, demonstrating that there is no functional duplication of this gene.

REPR and complementation factor(s) interact to modulate rat apolipoprotein B mRNA editing in response to alterations in cellular cholesterol flux.

Apolipoprotein B (apoB) mRNA editing is a post-transcriptional cytidine deamination involving several protein factor(s), one of which has recently been cloned. We have examined the effects of alterations in cellular cholesterol flux in the rat liver and small intestine as a means of dissecting the physiologic mechanisms regulating apoB mRNA editing, both in vivo and in isolated S-100 extracts. Hepatic cholesteryl ester accumulation was produced by feeding rats a high cholesterol diet, alone, or in combination with either ethinyl estradiol treatment, or after induction of hypothyroidism. Endogenous hepatic apoB mRNA editing decreased in parallel with the increase in cellular cholesteryl ester content (r = -0.948, P < 0.001). None of these conditions altered endogenous intestinal apoB mRNA editing. Hepatic S-100 extracts demonstrated decreased in vitro apoB RNA editing activity, in parallel with the changes observed in vivo. By contrast, the activity of intestinal S-100 extracts demonstrated a paradoxical increase in hypothyroid rats and a similar, paradoxical decrease in hyperthyroid rats, when compared to controls. Hepatic REPR mRNA, quantitated by RNase protection assay, showed a 25-50% decrease in cholesterol-fed rats. The editing activity of hepatic S-100 extracts prepared from cholesterol-fed, hypothyroid rats was restored to control levels with REPR supplementation but not with chicken intestinal S-100 extracts, suggesting that changes in REPR, but not complementation activity, may play a critical role in the regulation of apoB mRNA editing in rat liver. By contrast, the editing activity of intestinal S-100 extracts prepared from hyperthyroid animals was unaltered by supplementation with REPR, but was restored to control levels after the addition of chicken intestinal S-100 extracts. Taken together, the data suggest that tissue-specific factors regulate apoB mRNA editing in the rat and that the complex interplay of REPR and complementation factor(s) may be modulated in response to alterations in cholesterol flux, in vivo.

Apolipoprotein B messenger RNA editing in rat liver: developmental and hormonal modulation is divergent from apolipoprotein A-IV gene expression despite increased hepatic lipogenesis.

Rat hepatic apolipoprotein B (apoB) mRNA editing is regulated developmentally as well as by hormonal and nutritional modulation of hepatic lipogenesis, changes previously associated with coordinate modulation of hepatic apoA-IV gene expression. We have examined the effects of dexamethasone administration on apoB mRNA editing and the expression of other apolipoprotein genes in both neonatal and adult rats. Administration of dexamethasone increased hepatic triglyceride content in neonatal rats and increased hepatic but not intestinal apoA-IV mRNA abundance. However, neither the developmental profile nor the extent of hepatic apoB mRNA editing was changed after hormone administration. Dexamethasone produced a dose-dependent increase in adult hepatic triglyceride content and a coordinate fourfold increase in hepatic but not intestinal apoA-IV mRNA abundance, and hepatic and serum apoA-IV protein concentrations. Immunocytochemical localization revealed apoA-IV to be expressed in hepatocytes around the central vein while dexamethasone treatment produced a dose-dependent appearance of fat-filled hepatocytes throughout the lobule that were immunoreactive for apoA-IV. Despite these changes in hepatic triglyceride accumulation there was no change in the extent of hepatic apoB mRNA editing at any dose of dexamethasone. The data suggest that hormonal and metabolic modulation of hepatic apoB mRNA editing may be independent of factors that modulate apoA-IV gene expression despite alterations in hepatic triglyceride content.

Developmental and age-related changes in apolipoprotein B mRNA editing in mice.

Apolipoprotein B (apoB) mRNA is modified by a post-transcriptional editing reaction (C to U) changing a glutamine (CAA) to a translational stop codon (UAA) and producing apoB-48 mRNA in mammalian liver and intestine. Developmental and age-related changes in apoB mRNA editing were studied using two mouse strains with different aging processes (SAM-R/1 with a normal aging process and SAM-P/1 with an accelerated aging process). During growth of both strains, the proportion of unedited (apoB-100) mRNA decreased from 80% in the fetal liver at the 17th day of gestation to 30% in the liver of mature 2-month-old mice. Age-associated increase in the proportion of hepatic apoB-100 mRNA was observed from the age of 18 months in the SAM-R/1 strain. In the SAM-P/1 strain, apoB-100 mRNA in the liver continued to increase from the age of 10 months to death. The profiles of developmental and age-related changes in the proportion of two serum apoB isoproteins (apoB-100 and apoB-48) followed the extent of hepatic apoB mRNA editing. Age-related changes in the extent of apoB mRNA editing in the small intestine were not observed in either strain. A slight expression of apoB was detected by reverse transcriptase-polymerase chain reaction in the kidney, stomach, and colon, and age-associated change in the extent of editing was observed in the kidney. These correlated changes in apoB mRNA editing and serum apoB proteins suggest that RNA editing may be one mechanism involved in the regulation of lipoprotein biogenesis in biological development and in senescent mice. An age-associated decrease in the extent of hepatic apoB mRNA editing and increases of the proportion of serum apoB-100 protein were observed in senescent mice.

Modulation of apolipoprotein B-100 mRNA editing: effects on hepatic very low density lipoprotein assembly and intracellular apoB distribution in the rat.

We have studied the consequences of alterations to hepatic apoB mRNA editing on the biosynthesis and intracellular distribution of newly synthesized apoB variants together with their mass distribution in nascent Golgi very low density lipoproteins (VLDL). Radiolabeled liver membrane fractions were prepared from control or hypothyroid animals and separated by discontinuous sucrose gradient centrifugation. Hepatic apoB-100 synthesis in these groups accounted for 93-100% of total newly synthesized apoB species of Golgi fractions recovered from the sucrose gradients (G1 and G2). The analogous fractions isolated from the livers of hyperthyroid (treated with 3,3',5-triiodo-L-thyronine, T3) animals revealed that newly synthesized apoB-100 accounted for only 46 +/- 10% (G1) and 24 +/- 11% (G2), respectively, of total newly synthesized apoB. ApoB-100 mass in nascent Golgi VLDL from control and hypothyroid G1 fractions represented 70-78% total apoB as determined by Western blot analysis. By contrast, Golgi VLDL from hyperthyroid animals contained predominantly (greater than 78%) apoB-48 as the apoB species. Electron microscopy revealed that the morphology and size distribution of hyperthyroid G1 VLDL were similar to particles isolated from control animals. Thus, despite a profound reduction in the proportion of apoB-100 mRNA species containing an unmodified codon (CAA, B-GLN) at position 2153 in hyperthyroid animals (6 +/- 1% vs 50-61% in control and hypothyroid animals) apoB-100 biosynthesis was detectable in a defined membrane fraction isolated by discontinuous sucrose gradient centrifugation. However, no apoB-100 synthesis was detectable in liver samples prepared by Polytron disruption in Triton-containing buffers. These data suggest that effective hepatic VLDL assembly and secretion in the T3-treated rat continues despite a profound reduction in apoB-100 biosynthesis and implies that apoB-48 contains the requisite domains to direct this process, a situation analogous to that in the intestine.