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We have identified a novel gene referred to as activation-induced deaminase (AID) by subtraction of cDNAs derived from switch-induced and uninduced murine B lymphoma CH12F3-2 cells, more than 80% of which switch exclusively to IgA upon stimulation. The amino acid sequence encoded by AID cDNA is homologous to that of apolipoprotein B (apoB) mRNA-editing enzyme, catalytic polypeptide 1 (APOBEC-1), a type of cytidine deaminase that constitutes a catalytic subunit for the apoB mRNA-editing complex. In vitro experiments using a glutathione S-transferase AID fusion protein revealed significant cytidine deaminase activity that is blocked by tetrahydrouridine and by zinc chelation. However, AID alone did neither demonstrate activity in C to U editing of apoB mRNA nor bind to AU-rich RNA targets. AID mRNA expression is induced in splenic B cells that were activated in vitro or by immunizations with sheep red blood cells. In situ hybridization of immunized spleen sections revealed the restricted expression of AID mRNA in developing germinal centers in which modulation of immunoglobulin gene information through somatic hypermutation and class switch recombination takes place. Taken together, these findings suggest that AID is a new member of the RNA-editing deaminase family and may play a role in genetic events in the germinal center B cell.

Apolipoprotein B (apoB) biosynthesis by rat liver was studied following thyroid hormone (3,5,3′-triiodo-L-thyronine) administration to hypothyroid rats. Pharmacologic doses of 3,5,3′-triiodo-L-thyronine caused suppression of apoB100 synthesis but did not affect apoB48 levels. There was no detectable apoB100 synthesis in hyperthyroid rats. To examine whether these results were mediated by the previously demonstrated mechanism of RNA modification (Powell, L. M., Wallis, S. C., Pease, R. J., Edwards, Y. H., Knott, T. J., and Scott, J. (1987) Cell 50, 831-840), the DNA sequence corresponding to the C-terminal end of rat apoB48 was determined from rat liver cDNA clones. Rat cDNAs contained a stop codon at an identical position to that found in human and rabbit apoB48 intestinal cDNA. To quantitate the relative amounts of apoB100 and apoB48 message, cDNA was synthesized from hepatic and intestinal apoB RNA and a 207-base pair fragment amplified using the polymerase chain reaction. The products were then differentially hybridized with oligonucleotides specific for apoB100 (containing CAA) or apoB48 (TAA). Control and hypothyroid liver contained approximately equal amounts of CAA and TAA, while hyperthyroid liver contained greater than 90% TAA. All gut samples contained 94-98% TAA. Genomic DNA from rat liver contained only CAA. The results demonstrate that apoB mRNA modification can be hormonally modulated in the adult rat by induction of a mechanism involving substitution of a stop codon into hepatic apoB100 mRNA.

Apolipoprotein(a) (apo(a)) is synthesized and secreted from liver cells and represents one of the two major protein components of the atherogenic lipoprotein, Lp(a). Little is known, however, of the factors that regulate the secretion of this protein. We have undertaken an analysis of the response to oleate supplementation in stable clones of HepG2 and McA-RH7777 cells expressing either a 6 K-IV or 17 K-IV isoform of apo(a). These cell lines were examined by pulse-chase analysis and each demonstrated an increase (range 2-6-fold) in apo(a) secretion following supplementation with 0.8 mM oleate. Microsomal membranes, prepared from HepG2 cells expressing a 6 K-IV apo(a) isoform, demonstrated that oleate supplementation increased the apparent protection of apo(a) from protease digestion, suggesting that alterations in the translocation efficiency of apo(a) may accompany the addition of oleate. Cells incubated with brefeldin A demonstrated increased recovery of the precursor form of apo(a) with oleate supplementation, suggesting that alterations in post-translational degradation may also contribute to the observed increase in apo(a) secretion following oleate addition. To further characterize the oleate-dependent increase in apo(a) secretion, cells were incubated with an inhibitor of the microsomal triglyceride transfer protein. These experiments demonstrated a dose-dependent decrease in apo(a) secretion from both cell lines. Furthermore, addition of either the microsomal triglyceride transfer protein inhibitor or triacsin C, an inhibitor of acyl-CoA synthase, completely abrogated the oleate-dependent increase in apo(a) secretion. Taken together, these data provide evidence that apo(a) secretion from hepatoma cells may be linked to elements of cellular triglyceride assembly and secretion.

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.

Over the past 18 months different lots of [1,2-3H]cholesterol and [24,25-3H]cholesterol were found to be radiochemically acceptable by conventional chemical and other in vitro tests, yet, when co-administered with [4-14C]cholesterol to human subjects, an abrupt fall in the 3H/14C specific activity ratio in plasma cholesterol was discovered in every case. We have concluded that all batches of [3H]cholesterol should be regarded as radiochemically unreliable unless they are shown to behave identically in all respects to [4-14C]cholesterol in an appropriate in vivo assay system.

Apolipoprotein[a] (apo[a]) is a highly polymorphic glycoprotein that forms a covalent complex with apolipoprotein B-100 (apoB-100), producing a lipoprotein species referred to as lipoprotein[a] (Lp[a]). We have studied the effects of alterations in glycosylation of apo[a] on its intracellular processing and secretion as well as its ability to associate with low density lipoprotein (LDL) apoB-100. HepG2 cells transfected with a 6 kringle IV (6 K-IV) apo[a] minigene were treated with tunicamycin, an inhibitor of N-linked glycosylation, which eliminated apo[a]-B-100 complexes from the media. Tunicamycin treatment also reduced secretion of the 6 K-IV apo[a] protein from transfected McA-RH7777 cells by approximately 50%, but completely eliminated secretion of apo[a] species containing 9 and 17 K-IV repeats. Mixing experiments, performed with radiolabeled media (+/-tunicamycin) from transfected McA-RH7777 cells, demonstrated no alteration in the extent of association of apo[a] with human LDL. Similar mixing experiments using culture media from glycosylation-defective mutant chinese hamster ovary (CHO) cells transfected with the same apo[a] minigene showed identical results. Apo[a] secretion was demonstrated in all mutant cell lines in the absence of either N- or O-linked (or both) glycosylation. The mechanisms underlying the reduced secretion of apo[a] from transfected hepatoma cells were examined by pulse-chase radiolabeling and apo[a] immunoprecipitation. Tunicamycin treatment altered the efficiency of precursor apo[a] processing from the ER by increasing its ER retention time. The increased accumulation of precursor apo[a] in the ER was associated with alterations in the kinetics of association with two resident endoplasmic reticulum (ER) chaperone proteins, calnexin and BiP. These findings suggest that the glycosylation state and size of apo[a] appear to play a role in regulating its efficient exit from the endoplasmic reticulum. However, neither N- nor O-linked glycosylation of apo[a] exerts a major regulatory role in its covalent association with apoB-100.

The rate of incorporation of [2-14C]acetate into cholesterol has been measured in freshly isolated peripheral blood mononuclear leukocytes from patients on various hypolipidemic drugs that affect whole body cholesterol synthesis. These studies have demonstrated a significant two-fold increase in mononuclear cell cholesterol synthesis rates in patients receiving cholestyramine, a response measurable after 10 days of drug treatment. Mononuclear cell cholesterol synthesis rates were also measured in four groups of patients on the following drug regimens: 1) no medication, 2) clofibrate (2 g/day), 3) cholestyramine (16 g/day) or 4) both clofibrate and cholestyramine. The results demonstrated that the rate of acetate incorporation into cholesterol was significantly greater in the mononuclear cells from patients receiving either cholestyramine (P less than 0.005) or clofibrate plus cholestyramine (P less 0.001), as compared to controls. Patients receiving clofibrate alone did not differ significantly from controls in their rates of mononuclear cell cholesterol synthesis. Factors other than plasma lipoprotein and lipid levels appeared to be responsible for the elevated sterol synthesis rates observed in all patients receiving cholestyramine.

Bile acid production has been measured in 13 studies in 10 hyperlipidemic subjects by simultaneous use of isotope dilution kinetics and chemical balance methodology. When the data of all 13 studies were averaged, the correlation between the two sets of values was negative and weak (r - 0.01, NS). However, when the correlations for normoglyceridemic and hyperglyceridemic subjects were examined separately, a strong positive correlation was found between the values obtained by the two methods in normoglyceridemic subjects (r + 0.92, P less than 0.01) but not in hyperglyceridemic subjects (r - 0.25, NS). Examination of bile acid specific activity decay characteristics in eight studies, where bile was sampled up to six times within the first 24 hr after radio-labeled bile acid infusion, revealed differences in three rate of attainment of peak specific activity and in the time taken subsequently to achieve first order kinetic decay. However, analysis of the data from these eight studies by input-output analysis yielded values for primary bile acid synthesis no different that those generated by conventional isotope dilution kinetics. Thus, bile acid production in normoglyceridemic subjects may be accurately quantitated by either isotope dilution or chemical balance methodology. Our data, as well as results from other laboratories, indicate that the values obtained are strictly comparable. On the other hand, the quantitation of bile acid production in hyperglyceridemic subjects by isotope dilution kinetics gives higher values than those obtained by chemical balance methodology, and in addition, higher values than those obtained in patients with normal plasma triglyceride levels.

Apolipoprotein(a) (apo(a)), a large glycoprotein with extensive homology to plasminogen, forms a complex with apolipoprotein B100 (apoB100), which circulates in human plasma in the form of lipoprotein(a) (Lp(a)). Evidence indicates that the association of apo(a) with apoB100 occurs in the extracellular environment. We have reevaluated the possibility that apo(a)-B100 association can also occur as an intracellular event through studies with HepG2 cells stably transfected with an apo(a) minigene. Several lines of evidence support this possibility. First, continued Lp(a) production was demonstrated following incubation of transfected HepG2 cells with anti-apo(a) antisera, conditions that effectively block the fluid-phase association of apo(a) and apoB100 in vitro. Second, an apo(a)-B100 complex was detectable in Western blot analyses of transfected HepG2 lysates following immunoprecipitation with anti-apo(a) antisera. These studies incorporated precautions to eliminate cell-surface attachment of preformed apo(a)-B100 complexes to the low density lipoprotein receptor and were conducted in the presence of the lysine analog epsilon-aminocaproic acid, which precludes apo(a)-B100 association occurring during the isolation and analyses. Third, the presence of an intracellular apo(a)-B100 complex was demonstrated in lipoproteins isolated from microsomal contents. Of particular significance was the observation that this complex contained the precursor form of apo(a), which is not secreted, in addition to the mature, recombinant form. Finally, direct evidence was provided for the synthesis of a precursor form of apo(a) in a nascent intracellular complex with apoB100 following treatment of transfected HepG2 cells with brefeldin A plus N-acetyl-leucyl-leucyl-norleucinal. Taken together, these data suggest that apo(a)-B100 association can occur as an intracellular event in a human hepatoma-derived cell line, raising important implications for the regulation of Lp(a) secretion from human liver.

Measurements of the key parameters of cholesterol homeostasis and the mass of the body pools of cholesterol were carried out in two patients with familial hypercholesterolemia (FH), one homozygote and one heterozygote, before and 28 and 18 months, respectively, after portacaval anastomosis (PCA). In both patients the procedure significantly reduced the plasma concentrations of total and low density lipoprotein cholesterol and the daily rate of whole body cholesterol and bile acid synthesis. In addition, PCA caused a net efflux of accumulated tissue cholesterol as demonstrated by reductions in the rapidly exchangeable and total exchangeable masses of body cholesterol. Shunt patency was verified by demonstration of increased bile acids in serum from fasting patients and from patients 2 hr after a meal and by increased plasma glucagon before and after arginine infusion. Other than a persistently increased level of serum alkaline phosphatase, liver function tests have fallen within the normal range in both patients; there has been no evidence of hepatic encephalopathy. In the homozygous patient there has also been a striking resolution in xanthoma size and distribution. These multiple effects on cholesterol homeostasis and pool sizes strongly suggest that PCA can reverse the progressive accumulation of cholesterol in body tissues of FH patients.

Apolipoprotein (apo) B48 is synthesized by mammalian small intestine as a result of post-transcriptional RNA editing. This process is mediated by an enzyme complex containing a catalytic subunit, apobec-1, which is homologous to other cytidine deaminases, particularly in a domain (H/C)-(A/V)-E-(X)24-30-P-C-(X)2-C which coordinates zinc, apobec-1, expressed as a glutathione S-transferase fusion protein, demonstrates both apoB RNA editing and cytidine deaminase activity. His61, Cys93, and Cys96, the putative zinc-coordinating residues, were mutated to Arg, Ser, and Ser, respectively, with loss of RNA editing activity and either great reduction or abolition of cytidine deaminase activity. Mutation of the catalytically active Glu63 residue to Gln and Pro92 to Leu abolished both cytidine deaminase and RNA editing activity. The conservative His61-->Cys mutation, which should coordinate zinc, retained both editing and cytidine deaminase activity. Thus, zinc binding is required for both apoB RNA editing and cytidine deaminase activity. Mutation of the first four leucines within the heptad repeat of the leucine-rich region (LRR) of apobec-1 resulted in reduced RNA editing but preservation of wild-type cytidine deaminase activity. GST/APOBEC-1 was also demonstrated to cross-link to apoB RNA. Mutation of His61-->Arg abolished RNA binding, while the Glu63-->Gln and Cys96-->Ser mutant proteins showed wild-type levels of RNA binding. The remaining mutants had reduced levels of activity. Overexpression of wild-type apobec-1 in McA 7777 cells resulted in a 5-6-fold increase in editing of endogenous apoB. Transfection of the His61-->Cys, LRR, and Cys93-->Ser mutants increased endogenous editing 2-3-fold, while Glu63-->Gln and His61-->Arg mutants acted as dominant negatives, reducing endogenous editing. These data suggest that apobec-1 has distinct functional domains which modulate activity in the context of the apoB mRNA editing enzyme.

Apolipoprotein B (apoB) mRNA editing is mediated by an enzyme complex which includes the catalytic subunit, apobec-1. Recombinant GST/APOBEC-1 binds with high specificity to a rat apoB RNA template as demonstrated by UV cross-linking and electrophoretic mobility shift assay (EMSA). ApoB RNA binding was competed by poly(U), poly(A,U), and tRNA, but not by poly(A) or other homopolymeric ribonucleotides. UV cross-linking of GST/APOBEC-1 to an apoB RNA template was uninfluenced by the binding of proteins of approximately 60 and approximately 44 kDa, present in S100 extracts prepared from different sources. The binding of these proteins was similarly uninfluenced by the simultaneous binding of GST/APO-BEC-1. Moreover, the inclusion of heterologous S100 extracts in the RNA binding reactions completely abrogated the competitive displacement of GST/APOBEC-1 by tRNA. EMSA revealed the onset of RNA binding within 1-2 min, and its specificity was confirmed by a supershift with anti-GST/APOBEC-1 antisera. The structural specificity for apoB RNA binding, as inferred from EMSA, appears to be distinct from apoB RNA editing since wild-type chicken apoB RNA, which is not editable, and several mutant chicken apoB RNAs containing clustered mutations within the minimal apoB RNA editing cassette, bound with efficiency similar to the rat apoB RNA template. In conclusion, while the data suggest that apobec-1 binds AU-rich templates, the importance of this observation in the context of mammalian apoB mRNA editing remains unknown.