John M. Taylor

Apolipoprotein E associated with astrocytic glia of the central nervous system and with nonmyelinating glia of the peripheral nervous system.

The plasma protein apolipoprotein (apo) E is an important determinant of lipid transport and metabolism in mammals. In the present study, immunocytochemistry has been used to identify apo E in specific cells of the central and peripheral nervous systems of the rat. Light microscopic examination revealed that all astrocytes, including specialized astrocytic cells (Bergmann glia of the cerebellum, tanycytes of the third ventricle, pituicytes of the neurohypophysis, and Müller cells of the retina), possessed significant concentrations of apo E. In all of the major subdivisions of the central nervous system, the perinuclear region of astrocytic cells, as well as their cell processes that end on basement membranes at either the pial surface or along blood vessels, were found to be rich in apo E. Extracellular apo E was present along many of these same surfaces. The impression that apo E is secreted by astrocytic cells was confirmed by electron microscopic immunocytochemical studies, which demonstrated the presence of apo E in the Golgi apparatus. Apo E was not present in neurons, oligodendroglia, microglia, ependymal cells, and choroidal cells. In the peripheral nervous system, apo E was present within the glia surrounding sensory and motor neurons; satellite cells of the dorsal root ganglia and superior cervical sympathetic ganglion as well as the enteric glia of the intestinal ganglia were reactive. Apo E was also present within the non-myelinating Schwann cells but not within the myelinating Schwann cells of peripheral nerves. These results suggest that apo E has an important, previously unsuspected role in the physiology of nervous tissue.

Overexpression and Accumulation of Apolipoprotein E as a Cause of Hypertriglyceridemia

The molecular mechanisms of hypertriglyceridemia (HTG), a common lipid metabolic disorder in humans, often of genetic origin, are not well understood. In studying the effect of apolipoprotein (apo) E on the metabolism of triglyceride-rich lipoproteins, we found that expressing high plasma levels of human apoE3 in transgenic mice lacking endogenous mouse apoE caused HTG. These transgenic animals had 3-fold higher plasma triglyceride levels, higher very low density lipoproteins (VLDL), and lower high density lipoproteins than did nontransgenics. Removing one or both low density lipoprotein receptor alleles in the apoE3-overexpressing mice caused severe HTG (8–11-fold over nontransgenics) and increased VLDL and decreased low and high density lipoproteins, and apoE3-enriched VLDL were markedly depleted in apoC-II. At least two mechanisms could explain HTG associated with apoE3 overexpression: stimulated VLDL triglyceride production and impaired VLDL lipolysis. The apoE3 mice with HTG had a 50% increase in hepatic VLDL triglyceride production. Furthermore, overexpression of apoE (E2, E3, or E4) in cultured hepatocytes (McA-RH7777 cells) correlated positively with secretion of VLDL into the medium. However, apoE3 overexpression–associated HTG was only partially explained by VLDL overproduction, as lipoprotein lipase–mediated VLDL lipolysis was also decreased 20–86% depending on apoE3 levels, most likely by displacing or masking apoC-II on the particles. In human subjects, HTG correlated positively with increased VLDL triglyceride and plasma and VLDL apoE levels. However, plasma and VLDL apoE correlated negatively with VLDL apoC-II levels and lipoprotein lipase-mediated VLDL lipolysis. Thus, optimal expression of apoE is crucial for normal metabolism of triglyceride-rich lipoproteins, and overexpression and/or accumulation of apoE may contribute to HTG by stimulating VLDL triglyceride production and by impairing VLDL lipolysis. The apoE3-overexpressing mice will be useful for studying the pathophysiology of this disorder.

Overexpression of Hepatic Lipase in Transgenic Mice Decreases Apolipoprotein B-containing and High Density Lipoproteins EVIDENCE THAT HEPATIC LIPASE ACTS AS A LIGAND FOR LIPOPROTEIN UPTAKE

To determine the mechanisms by which human hepatic lipase (HL) contributes to the metabolism of apolipoprotein (apo) B-containing lipoproteins and high density lipoproteins (HDL)in vivo, we developed and characterized HL transgenic mice. HL was localized by immunohistochemistry to the liver and to the adrenal cortex. In hemizygous (hHLTg +/0) and homozygous (hHLTg +/+) mice, postheparin plasma HL activity increased by 25- and 50-fold and plasma cholesterol levels decreased by 80% and 85%, respectively. In mice fed a high fat, high cholesterol diet to increase endogenous apoB-containing lipoproteins, plasma cholesterol decreased 33% (hHLTg +/0) and 75% (hHLTg +/+). Both apoB-containing remnant lipoproteins and HDL were reduced. To extend this observation, the HL transgene was expressed in human apoB transgenic (huBTg) and apoE-deficient (apoE −/−) mice, both of which have high plasma levels of apoB-containing lipoproteins. (Note that thehuBTg mice that were used in these studies were all hemizygous for the human apoB gene.) In both thehuBTg,hHLTg +/0 mice and theapoE −/−,hHLTg +/0mice, plasma cholesterol decreased by 50%. This decrease was reflected in both the apoB-containing and the HDL fractions. To determine if HL catalytic activity is required for these decreases, we expressed catalytically inactive HL (HL-CAT) in apoE −/−mice. The postheparin plasma HL activities were similar in theapoE −/− and theapoE −/−,HL-CAT +/0mice, reflecting the activity of the endogenous mouse HL and confirming that the HL-CAT was catalytically inactive. However, the postheparin plasma HL activity was 20-fold higher in theapoE −/−,hHLTg +/0mice, indicating expression of the active human HL. Immunoblotting demonstrated high levels of human HL in postheparin plasma of bothapoE −/−,hHLTg +/0and apoE −/−,HL-CAT +/0mice. Plasma cholesterol and apoB-containing lipoprotein levels were ∼60% lower inapoE −/−,HL-CAT +/0mice than in apoE −/− mice. However, the HDL were only minimally reduced. Thus, the catalytic activity of HL is critical for its effects on HDL but not for its effects on apoB-containing lipoproteins. These results provide evidence that HL can act as a ligand to remove apoB-containing lipoproteins from plasma.

Duplicated Downstream Enhancers Control Expression of the Human Apolipoprotein E Gene in Macrophages and Adipose Tissue

Two distal enhancers that specify apolipoprotein (apo) E gene expression in isolated macrophages and adipose tissue were identified in transgenic mice that were generated with constructs of the human apoE/C-I/C-I′/C-IV/C-II gene cluster. One of these enhancers, multienhancer 1, consists of a 620-nucleotide sequence located 3.3 kilobases (kb) downstream of the apoE gene. The second enhancer, multienhancer 2, is a 619-nucleotide sequence located 15.9 kb downstream of the apoE gene and 5.9 kb downstream of the apoC-I gene. The two enhancers are 95% identical in sequence, and they are likely to have arisen as a consequence of the gene duplication event that yielded the apoC-I gene and the apoC-I′ pseudogene. Both enhancer sequences appear to have equivalent activity in directing apoE gene expression in peritoneal macrophages and in adipocytes, suggesting that their activity in specific cell types may be determined by common regulatory elements.

Two Hepatic Enhancers, HCR.1 and HCR.2, Coordinate the Liver Expression of the Entire Human Apolipoprotein E/C-I/C-IV/C-II Gene Cluster

We show that the liver-specific expression of all four genes in the human apolipoprotein (apo) E/C-I/C-IV/C-II gene cluster in transgenic mice is determined by the coordinate action of two distinct hepatic control regions (HCR). These enhancers are positioned 15 kilobases (kb) (HCR.1) and 26 kb (HCR.2) downstream of the apoE gene. To investigate the action of each HCR, transgenic mice were generated with a 70-kb human genomic fragment that contained the complete apoE gene cluster or with this fragment modified by the specific deletion of HCR.1, HCR.2, or both HCR domains. Hepatic expression of all four apolipoprotein genes was observed in transgenic mice in which either HCR.1 or HCR.2 was deleted, but no transgene expression was found in the liver in the absence of both HCR domains. The overall patterns of transgene expression suggested that HCR.2 was the dominant element for apoC-IV and apoC-II expression and that HCR.1 was dominant for the apoE/C-I expression. No liver-specific transcriptional activity was identified for the proximal promoter of any gene in the cluster; all liver-specific activity was associated with HCR.1 and HCR.2. Thus, the HCRs of the apoE gene cluster constitute unique regulatory domains for determining the requirements for apolipoprotein gene expression in the liver.

Overexpression of Apolipoprotein E3 in Transgenic Rabbits Causes Combined Hyperlipidemia by Stimulating Hepatic VLDL Production and Impairing VLDL Lipolysis

The differential effects of overexpression of human apolipoprotein (apo) E3 on plasma cholesterol and triglyceride metabolism were investigated in transgenic rabbits expressing low (<10 mg/dL), medium (10 to 20 mg/dL), or high (>20 mg/dL) levels of apoE3. Cholesterol levels increased progressively with increasing levels of apoE3, whereas triglyceride levels were not significantly affected at apoE3 levels up to 20 mg/dL but were markedly increased at levels of apoE3 >20 mg/dL. The medium expressers had marked hypercholesterolemia (up to 3- to 4-fold over nontransgenics), characterized by an increase in low density lipoprotein (LDL) cholesterol, while the low expressers had only slightly increased plasma cholesterol levels. The medium expressers displayed an 18-fold increase in LDL but also had a 2-fold increase in hepatic very low density lipoprotein (VLDL) triglyceride production, an 8-fold increase in VLDL apoB, and a moderate decrease in the ability of the VLDL to be lipolyzed. However, plasma clearance of VLDL was increased, likely because of the increased apoE3 content. The increase in LDL appears to be due to an enhanced competition of VLDL for LDL receptor binding and uptake, resulting in the accumulation of LDL. The combined hyperlipidemia of the apoE3 high expressers (>20 mg/dL) was characterized by a 19-fold increase in LDL cholesterol but also a 4-fold increase in hepatic VLDL triglyceride production associated with a marked elevation of plasma VLDL triglycerides, cholesterol, and apoB100 (4-, 9-, and 25-fold over nontransgenics, respectively). The VLDL from the high expressers was much more enriched in apoE3 and markedly depleted in apoC-II, which contributed to a >60% inhibition of VLDL lipolysis. The combined effects of stimulated VLDL production and impaired VLDL lipolysis accounted for the increases in plasma triglyceride and VLDL concentrations in the apoE3 high expressers. The hyperlipidemic apoE3 rabbits have phenotypes similar to those of familial combined hyperlipidemia, in which VLDL overproduction is a major biochemical feature. Overall, elevated expression of apoE3 appears to determine plasma lipid levels by stimulating hepatic VLDL production, enhancing VLDL clearance, and inhibiting VLDL lipolysis. Thus, the differential expression of apoE may, within a rather narrow range of concentrations, play a critical role in modulating plasma cholesterol and triglyceride levels and may represent an important determinant of specific types of hyperlipoproteinemia.

Structure of the Hepatic Control Region of the Human Apolipoprotein E/C-I Gene Locus

The specificity of expression in the liver of the human apolipoprotein (apo) E/C-I gene locus is determined by a hepatic control region (HCR) that is located 15 kilobases downstream of the apoE gene. DNase I footprint studies of this sequence using nuclear extracts identified a region of the HCR that is enriched in nuclear protein-binding sites. Nuclease analysis of chromatin revealed liver-specific DNase I-hypersensitive sites that were associated with this region, and additional liver-specific nuclease-sensitive sites associated with the apoE gene were identified. The HCR domain has a limited binding affinity for the nuclear scaffold. The specific domain required for liver expression was tested by ligating subfragments of the HCR to the apoE gene and examining their activity in transgenic mice. A segment of 319 nucleotides that contained several potential regulatory sequences was required for full activity of liver-specific transcription with shorter segments yielding much lower levels of expression in the liver. All constructs that contained a fully active HCR were expressed in approximately a copy-dependent manner, suggesting that transgene expression was independent of integration position. Taken together, the properties of the HCR are consistent with its function as a locus control region for the liver-specific expression of the apoE gene.

Overexpression of Human Apolipoprotein B-100 in Transgenic Rabbits Results in Increased Levels of LDL and Decreased Levels of HDL

In this study, and 80-kb human genomic DNA fragment spanning the human apoB gene was used to generate transgenic New Zealand White rabbits that expressed human apoB-100. The concentration of human apoB in the plasma of the transgenic rabbits ranged between 5 and 100 mg/dL. The transgenic rabbits had nearly threefold elevations in the plasma levels of triglycerides and cholesterol compared with nontransgenic controls. Nearly all the cholesterol and human apoB in the plasma was in the LDL fraction. Pronounced triglyceride enrichment of the LDL fraction was a striking feature of human apoB overexpression in the transgenic rabbits, in which the LDL fraction contained more than 75% of the plasma triglycerides. The triglyceride-enriched LDL particles were smaller and more dense than the native rabbit LDL and contained markedly increased amounts of apoE and apoC-III. In the nontransgenic control animals most of the triglycerides were in the VLDL, and most of the apoE and apoC-III were in the VLDL and HDL fractions. In addition to increased LDL levels, overexpression of human apoB in rabbits resulted in lower plasma levels of HDL cholesterol and apoA-I. In our prior studies on transgenic mice expressing human apoB, we documented triglyceride-rich LDL and reduced levels of HDL cholesterol. These prior findings in mice, together with the present findings in transgenic rabbits, suggest that triglyceride-rich LDL and lowered levels of HDL cholesterol may be hallmark features of apoB overexpression.

RNA-directed DNA polymerase of Rous sarcoma virus: Initiation of synthesis with 70 S viral RNA as template☆

Abstract DNA synthesis by the RNA-directed DNA polymerase of Rous sarcoma virus with 70 S viral RNA as template initiates by the covalent attachment of dAMP to the 3′ terminal adenosine of an RNA molecule. Initiation continues throughout the course of a 90-minute enzymatic reaction, and chain propagation occurs on most if not all of the dAMP residues attached to primer RNA. The nature of the primer molecules was established in two ways. First, the RNA was tagged by attachment of radioactive mono- and oligodeoxynucleotides. Second, primers were isolated directly from their covalent complexes with nascent DNA. The results of both procedures indicate that DNA synthesis initiates on the 3′ termini of 4 S RNA molecules hydrogen-bonded to 70 S RNA. Purified primer RNA has a nucleotide composition (G + C = 64%) different from that (G + C = 60%) of other 4 S RNAs found hydrogen-bonded to the 70 S RNA of Rous sarcoma virus.

Increased Production of Apolipoprotein B-containing Lipoproteins in the Absence of Hyperlipidemia in Transgenic Mice Expressing Cholesterol 7α-Hydroxylase

The finding that expression of a cholesterol 7α-hydroxylase (CYP7A1) transgene in cultured rat hepatoma cells caused a coordinate increase in lipogenesis and secretion of apoB-containing lipoproteins led to the hypothesis that hepatic production of apoB-containing lipoproteins may be linked to the expression of CYP7A1 (Wang, S.-L., Du, E., Martin, T. D., and Davis, R. A. (1997) J. Biol. Chem. 272, 19351–19358). To examine this hypothesis in vivo, a transgene encoding CYP7A1 driven by the constitutive liver-specific enhancer of the human apoE gene was expressed in C56BL/6 mice. The expression of CYP7A1 mRNA (20-fold), protein (∼10-fold), and enzyme activity (5-fold) was markedly increased in transgenic mice compared with non-transgenic littermates. The bile acid pool of CYP7A1 transgenic mice was doubled mainly due to increased hydrophobic dihydroxy bile acids. In CYP7A1 transgenic mice, livers contained ∼3-fold more sterol response element-binding protein-2 mRNA. Hepatic expression of mRNAs encoding lipogenic enzymes (i.e. fatty-acid synthase, acetyl-CoA carboxylase, stearoyl-CoA desaturase, squalene synthase, farnesyl-pyrophosphate synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, and low density lipoprotein receptor) as well as microsomal triglyceride transfer protein were elevated ∼3–5-fold in transgenic mice. CYP7A1 transgenic mice also displayed a >2-fold increase in hepatic production and secretion of triglyceride-rich apoB-containing lipoproteins. Despite the increased hepatic secretion of apoB-containing lipoproteins in CYP7A1 mice, plasma levels of triglycerides and cholesterol were not significantly increased. These data suggest that the 5-fold increased expression of the low density lipoprotein receptor displayed by the livers of CYP7A1 transgenic mice was sufficient to compensate for the 2-fold increase production of apoB-containing lipoproteins. These findings emphasize the important homeostatic role that CYP7A1 plays in balancing the anabolic lipoprotein assembly/secretion pathway with the cholesterol catabolic bile acid synthetic pathway.