apoE Gene Expression Research Articles

Abundance of mRNA of Apolipoprotein B100, Apolipoprotein E, and Microsomal Triglyceride Transfer Protein in Liver from Periparturient Dairy Cows

Limited secretion of very low density lipoproteins (VLDL) in dairy cows is strongly related to fatty liver and other metabolic disorders in the early postpartum. Currently, there is limited information on which roles apolipoprotein B100 (ApoB100), apolipoprotein E (ApoE), and microsomal triglyceride transfer protein (MTP) play in that VLDL limitation. To our knowledge, no studies have simultaneously measured ApoB100, ApoE, and MTP mRNA in periparturient dairy cows. Therefore, a trial was conducted to assess liver gene expression of these proteins in transition dairy cows and to evaluate the relationships between their expression and metabolic status. Eight multiparous Holstein cows were monitored during the transition period. To evaluate metabolic and nutritional status, body condition score was registered, and plasma indexes of energy metabolism and VLDL were determined from 35 d before to 35 d after calving. Liver biopsies were performed on d −35, 3, and 35 relative to day of calving, and gene expression of ApoB100, ApoE, and MTP were determined on liver tissue. Body condition, plasma glucose and VLDL decreased, and plasma NEFA and BHBA increased after calving. Compared with values of d −35, on d 3 after calving the ApoB100 mRNA synthesis was lower, whereas MTP and ApoE mRNA abundance were higher. Negative correlation (r=−0.57) between plasma NEFA concentration and ApoB100 mRNA abundance, and positive correlation between ApoB100 mRNA abundance and plasma cholesterol (r=0.65) and plasma albumins (r=0.52) were detected at 3 d postpartum. Data on changes of gene expression of the 3 main proteins involved in the regulation of synthesis and secretion of VLDL in the liver suggest that decreased mRNA for ApoB100 may be consistent with decreased synthesis and/or secretion of VLDL from liver during the peripart-urient period.

The role and mode of action of apolipoproteins CIII and AV: synergistic actors in triglyceride metabolism?

Apolipoprotein (apo)CIII and apoAV play an important role in triglyceride metabolism as evidenced by the unambiguous and opposing phenotypes of transgenic and knockout mouse models. In this review we discuss studies on the genetics, protein structure, and regulation of apoCIII and apoAV and compare their potential molecular mechanisms of action in triglyceride metabolism. We examine the hypothesis that apoCIII and apoAV synergistically affect triglyceride metabolism. It has now been firmly established that variation in plasma triglyceride levels in a wide range of human populations is strongly associated with genetic variation at the chromosomal locus encoding both the APOC3 and APOA5 genes, the APOA1/C3/A4/A5 gene cluster. The close physical linkage of these genes and the frequent concurrence of genetic variants, however, complicate the assignment of specific metabolic defects to specific polymorphisms. Recent insight into the regulation of APOC3 and APOA5 gene expression and structural modeling studies on the apoAV protein have provided novel clues for the potential molecular mechanisms responsible for the effects of apoCIII and apoAV on triglyceride metabolism. Hypertriglyceridemia is a major independent risk factor in the development of cardiovascular disease. Moreover, triglyceride-derived fatty acids are thought to play a key role in the development and progression of the metabolic syndrome. As modulators of triglyceride metabolism, apoCIII and apoAV are key players and potential therapeutic targets. However, little is known of their molecular mechanism and potential cooperativity. Rational therapeutic application will require the filling of this hiatus in our knowledge.

Apolipoprotein gene and its interaction with the environmentally driven risk factors: molecular, genetic and epidemiological studies of Alzheimer’s disease

Herein we review the role of apolipoprotein E (ApoE) in Alzheimer’s disease (AD) and how ApoE interacts with various risk factors. ApoE is localized with the major pathological hallmarks of AD, extracellular amyloid deposits and intracellular neurofibrillary tangles. The ApoEε4 allele is associated with the development of late-onset familial and sporadic AD. ApoEε4 has a gene dose effect on the risk and age of onset of AD. ApoE mRNA and protein are found predominantly in astrocytes within the CNS. There is also a high expression of ApoE mRNA in the brains of people with sporadic AD. ApoE acts as a cholesterol transporter in the brain. Cholesterol controls amyloid production and deposition by regulating β-secretase. In transgenic animal studies, ApoEε4 expression causes neuropathology and behavioral deficits. We also discuss data from three different cohorts for AD in the general population, in different racial and ethnic groups and the role of the ε4 allele in the clinical onset of the disease. Although the ε4 allele is an important genetic risk factor for AD, it accounts for a fairly small fraction of disease in the population. The effect of the ε4 allele on annual decline in episodic memory is significantly stronger than its effect on decline in other cognitive systems. Notably, the ε2 allele has an equal and opposite effect. Thus, ApoE allele status influences risk of AD by a relatively selective effect on episodic memory. Mechanistically, the role of APoE in AD needs to be established in terms of its gene expression, which ultimately controls levels of various ApoE isoforms. Transcriptional regulation suggests complex regulation of this gene and the resultant ApoE protein in injured neurons. We discuss the characteristics of ApoE regulatory elements, including their interactions with different transcription factors, to understand ApoE gene expression. Thus, ApoEε4 contributes to the pathogenesis of AD, but additional environmental risk factors will also be identified independent of ApoE and other genetic polymorphisms.

Apolipoprotein E as a target for developing new therapeutics for Alzheimer's disease based on studies from protein, RNA, and regulatory region of the gene.

Alzheimer's disease (AD), the most common form of dementia, is a progressive, degenerative disorder of the central nervous system. The major hallmarks of AD include selective neuronal cell death and the presence of amyloid deposits and neurofibrillary tangles. Apolipoprotein E (ApoE) has also been shown to colocalize with these neuropathological lesions. Here is reviewed the role of ApoE in AD. The human ApoE gene has three alleles (epsilon2, epsilon3, epsilon4)-all products of the same gene. The epsilon3-allele accounts for the majority of the ApoE gene pool (approximately 70-80%), the epsilon4-allele accounts for 10-15% and the epsilon2 allele for 5-10%. Inheritance of the epsilon4-allele strongly increases the risk for developing AD at an earlier age. Functions of ApoE include cholesterol transport, neuronal repair, dendritic growth and anti-inflammatory activities. Putative pathological functions or "risk-factor activities" of ApoE-epsilon4 include its role in promoting amyloid accumulation, neurotoxicity, oxidative stress and neuro fibrillary tangles.ApoE mRNA is most abundant in the liver followed by the brain, where it is synthesized and secreted primarily by astrocytes. ApoE protein and mRNA are further detected in cortical and hippocampal neurons in humans. ApoE gene expression is induced by brain injury in some neurons and upregulated in astrocytes during aging. In AD, an increased ApoE mRNA was reported in the hippocampus. The risk for AD has been reported to correlate with transcriptional activity of the ApoE gene. Binding sites for putative transcriptional factors (TF), such as AP-1, AP-2 and NF-kappaB, are present in the ApoE promoter. The promoter also contains sites for the inflammatory response transcription factors IL-6 RE-BP, MED1, STAT1 and STAT2. A functional peroxisome-proliferator-activated receptor gamma (PPARgamma) has been detected in the ApoE/ApoCI intergenic region. ApoE mRNA levels were shown to be regulated by ciglitazone, a PPARgamma inducer. Certain statin drugs may also affect ApoE promoter activity. Two distal enhancers that specify ApoE gene expression in macrophages were identified. These results have implications for the regulation of ApoE gene expression, which plays an important role in the development of AD. The interaction of different transcription factors with the regulatory region of the ApoE gene is important to understand the neuroinflammatory process seen in AD.

Disruption of TR4 Orphan Nuclear Receptor Reduces the Expression of Liver Apolipoprotein E/C-I/C-II Gene Cluster

Apolipoprotein E (apoE) is synthesized in many tissues, and the liver is the primary site from which apoE redistributes cholesterol and other lipids to peripheral tissues. Here we demonstrate that the TR4 orphan nuclear receptor (TR4) can induce apoE expression in HepG2 cells. This TR4-mediated regulation of apoE gene expression was further confirmed in vivo using TR4 knockout mice. Both serum apoE protein and liver apoE mRNA levels were significantly reduced in TR4 knockout mice. Gel shift and luciferase reporter gene assays further demonstrated that TR4 can induce apoE gene expression via a TR4 response element located in the hepatic control region that is 15 kb downstream of the apoE gene. Furthermore our in vivo data from TR4 knockout mice prove that TR4 can also regulate apolipoprotein C-I and C-II gene expression via the TR4 response element within the hepatic control region. Together our data show that loss of TR4 down-regulates expression of the apoE/C-I/C-II gene cluster in liver cells, demonstrating important roles of TR4 in the modulation of lipoprotein metabolism.

Apolipoprotein A5, a Crucial Determinant of Plasma Triglyceride Levels, Is Highly Responsive to Peroxisome Proliferator-activated Receptor α Activators

The recently discovered APOA5 gene has been shown in humans and mice to be important in determining plasma triglyceride levels, a major cardiovascular disease risk factor. apoAV represents the first described apolipoprotein where overexpression lowers triglyceride levels. Since fibrates represent a commonly used therapy for lowering plasma triglycerides in humans, we investigated their ability to modulate APOA5 gene expression and consequently influence plasma triglyceride levels. Human primary hepatocytes treated with Wy 14,643 or fenofibrate displayed a strong induction of APOA5 mRNA. Deletion and mutagenesis analyses of the proximal APOA5 promoter firmly demonstrate the presence of a functional peroxisome proliferator-activated receptor response element. These findings demonstrate that APOA5 is a highly responsive peroxisome proliferator-activated receptor alpha target gene and support its role as a major mediator for how fibrates reduce plasma triglycerides in humans.

Feed Restriction Significantly Alters Lipogenic Gene Expression in Broiler Breeder Chickens

Broiler breeder pullets were divided into two groups at 21 wk of age. One group was given free access to feed (ad libitum) and the other fed a limited amount of feed (restricted). At 22 wk, all birds were photostimulated and maintained throughout an egg-laying cycle ending at 36 wk. Samples of liver and abdominal fat pad were collected just before photostimulation (prelight), after photostimulation at first egg and at peak egg production (plateau). Hepatic expression of sterol regulatory element binding protein-1, ATP-citrate lyase, fatty acid synthase, malic enzyme, acetyl-CoA carboxylase and stearoyl-CoA (Delta9) desaturase 1 genes in ad libitum birds declined from their highest levels just before photostimulation as the birds came into and maintained egg production. In contrast, the restricted birds had significant (P < 0.05) increases in the expression of these genes after photostimulation at first egg with a subsequent decline as they reached peak egg production. Hepatic expression of fatty acid binding protein, VLDL apolipoprotein (apoVLDL-II) and apoB genes increased significantly (P < 0.05) in both ad libitum and restricted breeders after photostimulation, whereas apoA1 gene expression declined during this time. Abdominal fat pad weights were significantly (P < 0.05) higher in the ad libitum compared with restricted birds after photostimulation. Lipoprotein lipase in this tissue showed a pattern of expression similar to that observed for the hepatic lipogenic enzyme genes. In conclusion, feed restriction during the pullet-to-breeder transition period significantly (P < 0.05) altered hepatic lipogenic gene expression in broiler breeders.

Apolipoprotein A-I expression in rats is not altered by troglitazone.

Insulin is known to upregulate apolipoprotein A-I (apoA-I) promoter activity and to increase apoA1 gene expression in vivo. To determine if enhancement of insulin action with insulin sensitizers can also increase the apoA-I expression, we studied the in vivo effect of troglitazone, a potent insulin sensitizer, on the expression of rat hepatic and intestinal apoA-I mRNA using Northern blot analysis. The plasma, hepatic, and intestinal apoA-I content was also measured with immunoblot analysis using a specific anti-rat apoA-I antiserum. Troglitazone, given mixed with rat chow (0.2%) for 18 days, did not increase either plasma or tissue apoA-I mRNA or protein content. Intestinal apoA-I mRNA content relative to glyceraldehyde-3 phosphate dehydrogenase (G(3)PDH) mRNA was significantly lower compared with hepatic tissue content in both control and troglitazone-treated rats. The effect of troglitazone on the rat apoA-I promoter was examined using transient transfection analysis in HepG2 cells transfected with the apoA-I-chloramphenicol acetyl transferase (CAT) reporter plasmid (pAI.474.CAT). CAT activity (percentage acetylation of chloramphenicol as means +/- SEM) was not significantly different in ethanol (vehicle)-treated cells compared with cells treated with troglitazone (50.5% +/- 2.5% in control cells vs 57.7% +/- 8.2% and 53.5% +/- 4.2% in cells treated with 10 and 100 mM troglitazone, respectively). It is concluded that troglitazone doses known to achieve insulin sensitization did not enhance rat apoA-I promoter activity sufficiently to result in an increased apoA-I mRNA or protein expression in the intact rat. However, peroxisome proliferator activator receptor (PPAR) agonists that have significant PPAR alpha activity in addition to their PPAR gamma effects, may well be able to induce apoA-I expression.

Regulation of Macrophage ApoE Expression and Processing by Extracellular Matrix

Macrophage-derived apoE in the vessel wall has important effects on atherogenesis in vivo, making it important to understand factors that regulate its expression. Vessel wall macrophages are embedded in an extracellular matrix produced largely by arterial smooth muscle cells and endothelial cells. In this series of studies, we evaluated the influence of extracellular matrix on macrophage apoE expression. Subendothelial matrix, fibronectin, or collagen I stimulated macrophage apoE gene expression and apoE synthesis. Adhesion of macrophages to a polylysine substrate had no effect. An increase in apoE synthesis after plating on fibronectin could be observed by 2 h and was inhibited by blocking antibodies to the alpha(5)beta(1) integrin receptor for fibronectin. Fibronectin also regulated the post-translational processing of newly synthesized macrophage apoE by inhibiting its degradation. The increment in apoE resulting from suppressed degradation was retained in the cell-fibronectin monolayer in a pool that was resistant to release by exogenous high density lipoprotein subfraction 3. These observations establish a new pathway for the regulation of macrophage apoE expression in the vessel wall. The composition of the extracellular matrix changes after vessel wall injury and in response to locally produced cytokines and growth factors. The evolving composition of this matrix will, therefore, be important for regulating apoE expression and processing by vessel wall macrophages.

Modulation of Apolipoprotein D and Apolipoprotein E mRNA Expression by Growth Arrest and Identification of Key Elements in the Promoter

Apolipoprotein D (apoD) and apolipoprotein E (apoE) are co-expressed in many tissues, and, in certain neuropathological situations, their expression appears to be under coordinate regulation. We have previously shown that apoD gene expression in cultured human fibroblasts is up-regulated when the cells undergo growth arrest. Here, we demonstrate that, starting around day 2 of growth arrest, both apoD and apoE mRNA levels increase between 1.5- and 27-fold in other cell types, including mouse primary fibroblasts and fibroblast-like and human astrocytoma cell lines. To understand the regulatory mechanisms of apoD expression, we have used apoD promoter-luciferase reporter constructs to compare gene expression in growing cells and in cells that have undergone growth arrest. Analysis of gene expression in cells transfected with constructs with deletions and mutations in the apoD promoter and constructs with artificial promoters demonstrated that the region between nucleotides -174 and -4 is fully responsible for the basal gene expression, whereas the region from -558 to -179 is implicated in the induction of apoD expression following growth arrest. Within this region, an alternating purine-pyrimidine stretch and a pair of serum-responsive elements (SRE) were found to be major determinants of growth arrest-induced apoD gene expression. Evidence is also presented that SREs in the apoE promoter may contribute to the up-regulation of apoE gene expression following growth arrest.

Fluorescence-based, Nonradioactive Method for Efficient Detection of the Pentanucleotide Repeat (TTTTA)n Polymorphism in the Apolipoprotein(a) Gene

The apolipoprotein(a) [apo(a)] gene is a major predictor of plasma lipoprotein(a) concentrations, an independent risk factor for cardiovascular disease. The apo(a) gene contains a pentanucleotide repeat (PNR) polymorphism, 1.4 kb upstream from the apo(a) gene reading frame. This polymorphism has been suggested to be important in control of apo(a) gene expression. We developed a fluorescence-based, nonradioactive procedure to detect the PNR polymorphism. After amplification of the polymorphism by PCR, the respective PCR products were separated by denaturing polyacrylamide gel electrophoresis and detected using a 3'-end fluorescently labeled oligonucleotide as a probe. We used the method to characterize the PNR polymorphism pattern in 313 individuals, 195 Caucasians and 118 African Americans. The new method efficiently separated DNAs corresponding to the different PNR repeats. Among both ethnic groups, alleles containing eight PNRs were most common. Smaller PNRs were more common among African Americans, and larger PNRs were more common among Caucasians. We developed a nonradioactive technique that separates the PNR polymorphism in the apo(a) gene and can be used in other studies involving closely sized polymorphisms.

Regulation of apoA1 gene expression with acidosis: requirement for a transcriptional repressor.

Serum apolipoprotein A(1) (apoA(1)) concentration is inversely correlated with the risk of premature atherosclerosis. Serum apoA(1) concentrations are regulated, in part, at the transcriptional level. ApoA(1) mRNA is synthesized primarily in the liver and small intestine, under the direction of a number of signaling molecules and tissue-specific regulatory elements. Previously, we demonstrated that extracellular acidosis suppresses apoA(1) mRNA levels at the level of transcription. Here we demonstrate that intracellular acidosis, in the absence of extracellular pH changes, represses apoA(1) promoter activity. Repression occurs through a pH responsive element (pH-RE) located within the apoA(1) gene promoter. Acidosis increases the specific DNA binding activity of a putative repressor protein within the immediate 5'-flanking region of the apoA(1) gene. The cis-element that binds the putative repressor protein contains a negative thyroid hormone response element (nTRE) located 3' and adjacent to the apoA(1) TATA box. Mutation of the nTRE/pH-RE abrogates protein binding and alters the activity of reporter genes controlled by this element. Repression by acidosis did not require de novo mRNA and protein synthesis. Inhibition of tyrosine kinase activity and diacylglycerol-stimulated protein kinase C (PKC) signaling pathways with tyrophostin A47 and phorbol myristate acetate, respectively, did not affect the repression of apoA(1) promoter activity with acidosis. These results suggest that transcriptional repression of the apoA(1) gene by alterations in ambient pH is associated with enhanced DNA binding activity of a repressor protein, through a mechanism which appears to be independent of de novo mRNA and protein synthesis, tyrosine kinase activity, or PKC activation.

Identification of a peroxisome-proliferator-activated-receptor response element in the apolipoprotein E gene control region

Apolipoprotein E (apoE) is a protein involved in reverse cholesterol transport. Among other tissues, apoE is expressed in macrophages where its expression increases when macrophages develop into foam cells. It has been recently shown that peroxisome-proliferator-activated receptor gamma (PPARγ) is involved in this conversion. Northern-blot analysis was carried out in the macrophage cell line THP1 to determine whether apoE mRNA levels were regulated by ciglitazone, a PPARγ inducer. The results indicated that treatment with ciglitazone doubled the levels of apoE mRNA. To identify a possible PPARγ response element (PPRE), several portions of apoE gene control region were used to construct luciferase reporter plasmids. In U-87 MG cells, a 185bp fragment located in the apoE/apoCI intergenic region was sufficient to induce a 10-fold increase in the luciferase activity of the extract of cells co-transfected with a PPARγ expression plasmid. Subsequent analysis revealed the presence of a sequence with a high level of sequence similarity to the consensus PPRE. Mutations in this sequence resulted in a lack of functionality both in transient transfection and in electrophoretic-mobility-shift assays. These results demonstrated the presence of a functional PPRE in the apoE/apoCI intergenic region. These results have implications for the regulation of apoE gene expression and could be relevant for understanding the anti-atherogenic effect of thiazolidinediones.

Identification of a peroxisome-proliferator-activated-receptor response element in the apolipoprotein E gene control region.

Apolipoprotein E (apoE) is a protein involved in reverse cholesterol transport. Among other tissues, apoE is expressed in macrophages where its expression increases when macrophages develop into foam cells. It has been recently shown that peroxisome-proliferator-activated receptor gamma (PPARgamma) is involved in this conversion. Northern-blot analysis was carried out in the macrophage cell line THP1 to determine whether apoE mRNA levels were regulated by ciglitazone, a PPARgamma inducer. The results indicated that treatment with ciglitazone doubled the levels of apoE mRNA. To identify a possible PPARgamma response element (PPRE), several portions of apoE gene control region were used to construct luciferase reporter plasmids. In U-87 MG cells, a 185 bp fragment located in the apoE/apoCI intergenic region was sufficient to induce a 10-fold increase in the luciferase activity of the extract of cells co-transfected with a PPARgamma expression plasmid. Subsequent analysis revealed the presence of a sequence with a high level of sequence similarity to the consensus PPRE. Mutations in this sequence resulted in a lack of functionality both in transient transfection and in electrophoretic-mobility-shift assays. These results demonstrated the presence of a functional PPRE in the apoE/apoCI intergenic region. These results have implications for the regulation of apoE gene expression and could be relevant for understanding the anti-atherogenic effect of thiazolidinediones.

Protective effects of Saiko-ka-ryukotsu-borei-to (Chai-Hu-Jia-Long-Gu-Mu-Li-Tang) against atherosclerosis in kurosawa and kusanagi-hypercholesterolemic (KHC) rabbits

We investigated the protective effects of the traditional Japanese herbal medicine Saiko-ka-ryukotsu-borei-to (Chai-Hu-Jia-Long-Gu-Mu-Li-Tang in Chinese) (SRBT) against hypercholesterolemia and atherosclerotic lesions. We focused on atherosclerosis using female heterozygous Kurosawa and Kusanagi-hypercholesterolemic (KHC) rabbits. The total plasma cholesterol levels increased for up to 12 weeks after beginning a diet containing 0.1% cholesterol and then reached a plateau of about 600 mg dl−1. When SRBT was administered at a dose of 1.0 g kg−1per day for 24 weeks, total plasma cholesterol levels were significantly decreased after 20–24 weeks. On the other hand, pravastatin at a dose of 10 mg kg−1per day produced a significant decrease in total plasma cholesterol levels from 4 to 24 weeks (about 105–130 mg dl−1). Moreover, 1.0 g kg−1per day of SRBT significantly decreased plasma low density lipoprotein (LDL) cholesterol levels but did not change either very low density lipoprotein (VLDL), or high density lipoprotein (HDL) cholesterol levels. Animals that received pravastatin had significantly decreased LDL cholesterol levels and VLDL cholesterol levels after 8 weeks and at 24 weeks. We also examined the expression of apoB, E and LDL receptor mRNA levels in the liver at 24 weeks after beginning the administration of 1.0 g kg−1per day of SRBT. Both apoE and LDL receptor mRNA levels were significantly increased compared with those in rabbits receiving the 0.1% cholesterol diet. SRBT at a dose of 1.0 g kg−1per day significantly depressed the intimal surface area of the thoracic aortae involved with atheromatous plaques. The present results suggest that SRBT may protect against hypercholesterolemia and atheromatous lesions by affecting apoE and LDL receptor mRNA gene expression in the liver.

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.

Effects of ketoacidosis on rat apolipoprotein A1 gene expression: a link with acidosis but not with ketones.

To determine if ketoacidosis contributes to reduced apolipoprotein A1 (apoA1) expression in insulin-deficient diabetic rats, we examined the regulation of apoA1 gene expression in response to changes in ambient pH or ketone body concentrations. Hepatic apoAI mRNA levels were reduced 42% in diabetic rats relative to nondiabetic controls (means+/-s.d.; 321.8+/-43.7 vs 438.7+/-58.8 arbitrary units; P<0.03). Neither endogenous apoA1 mRNA nor transcriptional activity of the rat apoA1 gene promoter (from -474 to -7) were altered by sodium butyrate or isobutyramide (0.3 mM to 10 mM) in Hep G2 or Caco-2 cells. Rat hepatic and intestinal apoA1 mRNA levels, and plasma apoA1 concentration, were not altered 24 h after isobutyramide administration (500 mg/kg by gavage). When the effect of altering ambient pH within a wide range commonly encountered in vivo was studied, acidosis (pH 6.7), relative to alkalosis (pH 7.9), decreased apoAI mRNA levels relative to glyceraldehyde-3-phosphate dehydrogenase mRNA by 47% in Hep G2 cells (P<0.025) and by 24% in Caco-2 cells (P<0.017). Acidosis did not alter cytomegalo virus (CMV)-beta-galactosidase activity, or the activity of the simian virus (SV40) early-region promoter, in either cell line transfected with the respective constructs. The lowering of ambient pH was associated with a graded reduction in apoAI promoter activity. At pH 6.7, apoAI promoter activity was reduced by 75% compared with promoter activity at pH 7.9. These observations indicate that acidosis, but not ketosis, contributes to the reduction in apoA1 expression during diabetic ketoacidosis by down-regulating apoAI promoter activity.

Apolipoprotein E gene expression is reduced in apolipoprotein A-I transgenic mice.

The levels of plasma apolipoprotein (apo) E, an anti-atherogenic protein involved in mammalian cholesterol transport, were found to be 2-3 fold lower in mice over-expressing human apoA-I gene. ApoE is mainly associated with VLDL and HDL-size particles, but in mice the majority of the apoE is associated with the HDL particles. Over-expression of the human apoA-I in mice increases the levels of human apoA-I-rich HDL particles by displacing mouse apoA-I from HDL. This results in lowering of plasma levels of mouse apoA-I. Since plasma levels of apoE also decreased in the apoA-I transgenic mice, the mechanism of apoE lowering was investigated. Although plasma levels of apoE decreased by 2-3 fold, apoB levels remained unchanged. As expected, the plasma levels of human apoA-I were almost 5-fold higher in the apoAI-Tg mice compared to mouse apoA-I in WT mice. If the over-expression of human apoA-I caused displacement of apoE from the HDL, the levels of hepatic apoE mRNA should remain the same in WT and the apoAI-Tg mice. However, the measurements of apoE mRNA in the liver showed 3-fold decreases of apoE mRNA in apoAI-Tg mice as compared to WT mice, suggesting that the decreased apoE mRNA expression, but not the displacement of the apoE from HDL, resulted in the lowering of plasma apoE in apoAI-Tg mice. As expected, the levels of hepatic apoA-I mRNA (transgene) were 5-fold higher in the apoAI-Tg mice. ApoE synthesis measured in hepatocytes also showed lower synthesis of apoE in the apoAI-Tg mice. These studies suggest that the integration of human apoA-I transgene in mouse genome occurred at a site that affected apoE gene expression. Identification of this locus may provide further understanding of the apoE gene expression.

The Heparin-Binding Proteins Apolipoprotein E and Lipoprotein Lipase Enhance Cellular Proteoglycan Production

Apolipoprotein E (apoE) and lipoprotein lipase (LPL), key proteins in the regulation of lipoprotein metabolism, bind with high affinity to heparin and cell-surface heparan sulfate proteoglycan (HSPG). In the present study, we tested whether the expression of apoE or LPL would modulate proteoglycan (PG) metabolism in cells. Two apoE-expressing cells, macrophages and fibroblasts, and LPL-expressing Chinese hamster ovary (CHO) cells were used to study the effect of apoE and LPL on PG production. Cellular PGs were metabolically labeled with (35)[S]sulfate for 20 hours, and medium, pericellular PGs, and intracellular PGs were assessed. In all transfected cells, PG levels in the 3 pools increased 1.6- to 3-fold when compared with control cells. Initial PG production was assessed from the time of addition of radiolabeled sulfate; at 1 hour, there was no difference in PG synthesis by apoE-expressing cells when compared with control cells. After 1 hour, apoE-expressing cells had significantly greater production of PGs. Total production assessed with [(3)H]glucosamine was also increased. This was due to an increase in the length of the glycosaminoglycan chains. To assess whether the increase in PGs was due to a decrease in PG degradation, a pulse-chase experiment was performed. Loss of sulfate-labeled pericellular PGs was similar in apoE and control cells, but more labeled PGs appeared in the medium of the apoE-expressing cells. Addition of exogenous apoE and anti-human apoE antibody to both non-apoE-expressing and apoE-expressing cells did not alter PG production. Moreover, LPL addition did not alter cell-surface PG metabolism. These results show that enhanced gene expression of apoE and LPL increases cellular PG production. We postulate that such changes in vascular PGs can affect the atherogenic potential of arteries.

Modulation of apolipoprotein D and apolipoprotein E expression in rat hippocampus after entorhinal cortex lesion

Apolipoprotein (apo) D is a member of the lipocalin family of proteins. Although its physiological function is unknown, apoD is thought to transport one or more small hydrophobic ligands. A second apolipoprotein, apoE is known to play an important role in lipid transport, and apoE genetic polymorphism has been shown to be associated with susceptibility to Alzheimer's disease. Both apoD and apoE are expressed in the central nervous system (CNS) and both proteins accumulate at sites of peripheral nerve injury due to increased local synthesis. The two proteins may have overlapping or complementary functions within nervous tissue. In order to define the role of apoD within the CNS, we have studied the regional distribution of apoD and apoE mRNA and protein within the normal rat brain and the changes in apoD and apoE expression in the hippocampus of rats after entorhinal cortex lesion (EC lesion). Within the brains of normal rats, apoD expression in the hippocampus was as high as 180-fold that of the liver. ApoD mRNA levels in other areas of the rat brain ranged from 40 to 120 times the hepatic levels. The distribution of apoE gene expression within the brain was similar to that of apoD, but was much lower than hepatic apoE expression. When rats were subjected to EC lesion, the apoD message increased by 54% at 4 days post lesion (DPL) in the ipsilateral region of hippocampus while apoE mRNA levels (ipsilateral and contralateral) decreased by 43%. At 6 to 8 DPL apoD mRNA in the ipsilateral hippocampus remained elevated (42% above controls) whereas the apoE mRNA levels increased to about 15% above those of controls. At 14 and 31 DPL, both apoD and apoE expression was similar to controls. The increase in immunoreactive apoD in hippocampal extracts was more dramatic. At 1 DPL, immunoreactive apoD levels were already 16-fold higher than those in extracts of non-lesioned animals and, at 31 DPL, levels were still 8-fold higher than those of control animals. Finally, we have demonstrated that the levels of apoD in the brains of apoE-deficient mice are 50-fold those of wildtype control mice. ApoD clearly has an important function within the CNS in both normal and pathological situations.