ApoE mRNA Expression Research Articles

Lipid Level and Type Alter Stearoyl CoA Desaturase mRNA Abundance Differently in Mice with Distinct Susceptibilities to Diet-Influenced Diseases ,

Chronic diseases develop in susceptible individuals following exposure to environmental conditions including high fat diets. Inbred strains of mice differing in susceptibility to atherosclerosis, diabetes, obesity and certain cancers are models for understanding the genetic basis and molecular mechanisms whereby diet influences these polygenic and multifactorial disorders. Expression sequence tags (EST) and disease quantitative trait loci (QTL) are also being identified with these strains. Reported here are comparisons of food intake, growth, nonfasting serum lipids and expression of mRNA for hepatic apolipoprotein E (ApoE), hepatic stearoyl CoA desaturase (Scd1) and heart lipoprotein lipase (Lpl) in a 2 × 2 × 2 design with C57BL/6J and BALB/cByJ mice fed semipurified diets with 4 or 20% saturated (coconut) or unsaturated (corn) oils for 4 mo. Histological studies of aortas and coronary arteries are also reported for these animals. After 4 mo, BALB/cByJ mice were significantly heavier and had significantly higher total serum cholesterol, HDL cholesterol and triglyceride concentrations in the fed state than C57BL/6J mice. Efficiency of utilizing dietary energy did not differ consistently between strains. Oil level affected serum total cholesterol, triglycerides and HDL cholesterol, which were significantly greater in mice fed high fat diets. Lpl and ApoE mRNA expression levels were not significantly affected by mouse strain, oil source or oil level. Scd1 mRNA expression, however, was significantly higher in C57BL/6J than in BALB/cByJ mice and was lower in all mice fed 20% compared with those fed 4% fat diets. Genes regulated differently by diet among strains with distinct susceptibility to diet-influenced disease may be associated with molecular pathways contributing to incidence or severity.

Astrocytes and Microglia Respond to Estrogen with Increased apoE mRNAin Vivoandin Vitro

This study examined the regulation of apolipoprotein E (apoE) by 17β-estradiol (E2) in brain glia, using rats with regular ovulatory cycles as anin vivomodel and cultured astrocytes and mixed glia asin vitromodels. Two brain regions were examined which had demonstrated transient synaptic remodeling during the estrous cycle. In the hippocampal CA1 region and the hypothalamic arcuate nucleus, apoE mRNA was elevated at proestrus when plasma E2 was high and synaptic density was increasing. Both astrocytes and microglia contributed to this increase in apoE mRNA.In vitro,E2 treatment had no effect on apoE mRNA levels in monotypic cultures of either astrocytes or microglia. In contrast, mixed glial cultures responded to E2 with increased apoE mRNA and protein, suggesting that heterotypic cellular interactions are important in the brain response to estrogens.In situhybridization in combination with cell-specific markers showed that E2 increased apoE mRNA levels in both astrocytes and microglia. These results, which are the first evidence of apoE mRNA localization to microgliain vivoand the control of apoE expression in brain cells by estrogens, are discussed in terms of the possible protective role of E2 in Alzheimer's disease and prior findings that emphasize the expression of apoE mRNA in astrocytes within the brain.

Expression of apolipoprotein E mRNA in rat microglia

Apolipoprotein E (apoE) is a major risk factor for Alzheimer disease (AD), which is the most common cause of progressive dementing illness. ApoE has been postulated to be synthesized by astrocytes and taken up by microglia and neuronal cells. However, it remains unknown whether apoE is also produced by microglia in the brain. We analyzed apoE mRNA expression of microglia using a rat primary culture system. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed expression of apoE mRNA in cultured rat microglia. By RT-in situ-PCR, microglia showed positive staining for the PCR product of apoE mRNA. These results indicated that apoE was biosynthesized in rat microglia. We suggest that microglia might be one of the sources of apoE in the brain, and that apoE synthesized in microglia might be closely related to the pathogenesis of AD.

Apolipoprotein E mRNA in the brains of patients with Alzheimer's disease

To clarify the role of apolipoprotein E (apoE) in the pathophysiology of Alzheimer's disease (AD), we used an RT-PCR method to investigate apoE and glial fibrillary acidic protein (GFAP) mRNA expression in the brain. ApoE mRNA was significantly more abundant in AD (0.379 ± 0.191, mean ± SD) than in control (0.125 ± 0.073, p < 0.05) brain tissue, but in AD it was decreased in relation to the apoE-ϵ4 gene dosage. The GFAP mRNA content also was greater in AD (5.96 ± 2.94) than in control (3.80 ± 2.78) tissue, and in AD showed an increase relative to the apoE-ϵ4 gene dosage. AD patients who had long survival times showed high expression of apoE and low expression of GFAP. These results suggest that apoE suppresses the progression of AD, including gliosis, in the brain.

Apolipoprotein E expression in Y1 adrenal cells is associated with increased intracellular cholesterol content and reduced free cholesterol efflux.

The expression of apoE mRNA in the adrenal gland is inversely correlated to steroidogenesis and directly correlated to the level of cholesteryl ester stores. To further investigate the relationship between apoE and cellular cholesterol homeostasis, several parameters of cholesterol metabolism in the murine Y1 adrenal cell line engineered to constitutively express human apoE (Y1-E cells) have been studied. It is reported here that Y1-E cells have increased cellular cholesterol content and markedly reduced efflux of free cholesterol as compared to control Y1 cells that do not express apoE. Y1-E cells have increases in both free and esterified cholesterol. However, Y1 and Y1-E cells incorporate [14C]oleate into cholesteryl ester at similar rates and have similar levels of maximal ACAT activity in isolated microsomes. Turnover of cholesteryl ester stores prelabeled with [14C]oleate occurred at similar rates in Y1-E and control Y1 cells, suggesting that increased cholesteryl ester stores in Y1-E cells do not result from reduced cholesteryl ester hydrolysis. Y1-E cells showed reduced cholesterol efflux as compared to control Y1 cells with either native high-density lipoprotein or cholesterol-free reconstituted particles as extracellular acceptors. Cholesterol efflux was not altered by inhibition of ACAT, suggesting that cholesterol esterification in Y1-E cells is not inhibiting efflux. These results suggest that reduced cholesterol efflux is responsible, at least in part, for the cholesterol accumulation in Y1-E cells. In comparison to the rat adrenal gland in vivo, Y1-E cells resemble adrenocortical cells under conditions where steroidogenesis is suppressed and apoE expression and cholesteryl ester storage are increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical significance of ApoE expression in human gastric cancer

ApoE plays a key role in various biological events. The aim of this study is to clarify its clinical significance in gastric cancer. We obtained paired clinical bulk samples of tumor tissue and corresponding normal tissue from 124 gastric cancer patients. To address ApoE mRNA expression clearly, we selected four samples, and differentially dissected gastric cancer and normal epithelium using laser microdissection (LMD) system. ApoE mRNA expression was examined by real-time reverse transcription (RT)-polymerase chain reaction (PCR). ApoE protein expression was assessed by immnunohistochemistry. The relationship between ApoE mRNA expression and clinicopathologic factors was statistically analyzed. RT-PCR assay for 124 bulk samples showed that ApoE mRNA expression was more highly expressed in gastric cancer tissue than in corresponding normal mucosa (p < 0.0001). By RT-PCR assay of four LMD samples, ApoE mRNA was overexpressed in gastric cancer. Immunohistochemistry showed that ApoE was predominantly expressed in gastric cancer. Tumors with high ApoE mRNA expression showed deeper tumor invasion into the muscle layer (p < 0.0001), the serosal layer (p < 0.01), or more positive lymph node metastasis (p < 0.05). When assessed by Kaplan-Meier analysis, patients with high ApoE expression tumor had a shorter survival than those with low ApoE expression tumor (p < 0.05). Moreover, multivariate analysis indicated that high ApoE mRNA expression was an independent indicator for muscular invasion (p < 0.01). ApoE is highly expressed in gastric cancer, contributing to shorter survival. In particular, ApoE was closely correlated with muscular invasion, and may be a possible biomarker predicting muscular invasion of gastric cancer.

Apolipoprotein E expression in the cerebellum of normal and hypercholesterolemic rabbits

We have studied the expression of apolipoprotein E (ApoE) mRNA in the cerebella of control and experimental rabbits fed with a cholesterol-rich diet for 8 weeks. Cholesterol-treated rabbits show a dramatic increase in serum cholesterol levels; however, no significant variations in the expression level of cerebellar ApoE mRNA were found in comparison to control rabbits. In addition, no differences were observed between control and hypercholesterolemic rabbits in the in situ hybridization pattern of ApoE mRNA on cerebellar cortex sections. ApoE mRNA was localized in astroglial processes associated with Purkinje cell bodies and dendrites, granule cell clusters, blood vessels and nerve fibers of the white matter. No expression of ApoE mRNA was observed in Purkinje and granule cell neurons. Polarized light examination of cryostat cerebellar sections revealed the absence of cholesterol-rich microglia/macrophage cells induced by the hypercholesterolemia. In this way, neither reactive microglial cells nor perivascular phagocytes were found by ultrastructural analysis in hypercholesterolemic conditions. The pattern of glial fibrillary acidic protein of the astroglial cells of the cerebellar cortex as well as their nuclear size were unchanged following cholesterol treatment, indicating the absence of astroglial activation induced by hypercholesterolemia. Our results suggest that cerebellar ApoE does not contribute to the general cholesterol homeostasis outside of the brain and supports the view that this cerebellar ApoE is involved in paracrine and autocrine functions particularly related with synpase turnover and membrane remodelling of astroglial cells.

Abundant expression of apoprotein E by macrophages in human and rabbit atherosclerotic lesions.

Previous studies have demonstrated the presence of apoprotein (apo) E protein and message in arterial lesions. To determine the source of the synthesized apoE, we performed simultaneous in situ hybridization and immunocytochemistry on human and rabbit atherosclerotic tissue. Studies of serial sections of aortic atherosclerotic lesions from humans and hypercholesterolemic New Zealand White rabbits and Watanabe heritable hyperlipidemic rabbits revealed a similar pattern of macrophage-specific apoE expression in the rabbit and human lesions. In early lesions of rabbit atherosclerotic tissue, in which many macrophages were present, there was abundant expression of apoE mRNA. Northern blot analyses of total mRNA obtained from arterial macrophage-derived foam cells, freshly isolated from ballooned, cholesterol-fed New Zealand White rabbits, demonstrated positive hybridization with an apoE-specific riboprobe. Western blot analyses of conditioned media from the isolated foam cells placed in culture for up to 24 hours demonstrated the presence of secreted apoE. These studies demonstrated that in atherosclerotic lesions, arterial wall macrophages synthesize and secrete apoE and probably account for most of the apoE synthesized in the atherosclerotic artery.

Relationship between apolipoprotein E mRNA expression and tissue cholesterol content in rat adrenal gland.

Among extrahepatic tissues the adrenal gland has one of the highest concentrations of apoE mRNA and the highest rate of apoE synthesis. In the present investigation several previously described in vivo treatments were used to assess the relationship between apoE expression and cellular cholesterol in the rat adrenal gland. Treatment of rats with 4-aminopyrazolo[3,4-d]pyrimidine (4-APP) to lower serum cholesterol concentration and deplete adrenal gland cholesterol content decreased adrenal gland apoE mRNA concentration. These adrenal responses were blocked by dexamethasone (DEX) suggesting that the effect of 4-APP occurred indirectly via stimulation of the adrenal gland by endogenous adrenocorticotrophic (ACTH). Relative to control rats, DEX treatment increased both adrenal gland cholesterol content and apoE mRNA concentration. Concurrent ACTH and DEX administration reduced both adrenal gland cholesterol content and apoE mRNA concentration relative to DEX-treated rats. ACTH administration also rapidly decreased adrenal gland apoE mRNA concentration and cholesterol content in rats pretreated with DEX. In all the above experiments, adrenal gland cholesterol content and apoE mRNA concentration were positively correlated (r = 0.78, P = 0.0001). In contrast, aminoglutethimide treatment, which blocks adrenal gland steroidogenesis and greatly increases adrenal gland cholesterol content, was without effect on apoE mRNA concentration. ACTH administration to rats treated with DEX + aminoglutethimide resulted in decreased adrenal apoE mRNA despite greatly increased adrenal cholesterol content. This uncoupling of adrenal gland cholesterol content and apoE mRNA concentration suggests that apoE mRNA expression and cellular cholesterol are regulated independently by ACTH.

Apolipoprotein E gene expression in mouse 3T3-L1 adipocytes and human adipose tissue and its regulation by differentiation and lipid content

Apolipoprotein E (apoE) is an important constituent of plasma lipoproteins and a ligand for several lipoprotein receptors. It is produced mainly in the liver but also in several peripheral tissues like brain, adrenal glands, kidney, and macrophages. Some of these tissues also coexpress lipoprotein lipase (LPL), an important enzyme in the metabolism of lipids and lipoproteins. This suggested a possible coordinate expression of these genes and led us to analyze whether adipocytes, a major source of LPL, could also synthesize apoE. Northern blotting experiments showed that apoE mRNA is found in differentiated mouse 3T3-L1 adipocytes as well as biopsies of human adipose tissue maintained in organ culture but not in undifferentiated 3T3-L1 preadipocytes. [35S]Methionine pulse-labeling experiments revealed that apoE protein is produced in human adipose tissue and differentiated mouse 3T3-L1 adipocytes but not in preadipocytes. In biosynthetic labeling experiments, most apoE was found to be cell associated even after prolonged chase periods. Heparin treatment of the cultured cells did not enhance apoE secretion. During differentiation of 3T3-L1 cells, the onset of apoE gene expression was later than that of LPL. The apoE mRNA and intracellular apoE protein concentrations increased linearly with time of differentiation, at least through day 11, whereas LPL showed highest expression at day 7 and then declined. The increase in apoE mRNA correlated with the cellular lipid content. Inhibition of lipid accumulation in differentiated cells by biotin deprivation decreased apoE expression. Cholesterol-loading experiments suggested that apoE mRNA expression is regulated by the intracellular free cholesterol content of 3T3-L1 adipocytes. In contrast, the LPL mRNA level was not influenced by biotin deprivation or cholesterol loading. Human recombinant tumor necrosis factor, a potent inhibitor of LPL gene transcription, had no effect on adipocyte apoE mRNA levels. Therefore, although apoE and LPL are both expressed in adipocytes in a differentiation-dependent manner, the time course of their expression differs as do their responses to cellular lipid content and tumor necrosis factor. We conclude that these genes are not coordinately regulated in adipocytes.

Increased levels of messenger ribonucleic acid for apolipoprotein E in the spleen of probucol-treated rabbits

To study the organ-specific effect of probucol, a potent cholesterol-lowering drug, on apolipoprotein(apo)E synthesis, rabbit apoE complementary deoxyribonucleic acid (cDNA) clones were isolated and apoE messenger ribonucleic acid (mRNA) levels were determined in various tissues isolated from probucol-treated rabbits. A 0.54 kb apoE cDNA was cloned from a rabbit liver cDNA library using a synthetic oligonucleotide probe. The amino acid sequence deduced from an apoE coding region indicated 78% homology for human apoE and 63% homology for rat apoE. RNA blot analysis showed that apoE mRNA was most abundant in the liver, then in the brain and spleen. The relative amounts of apoE mRNA were determined in tissues of rabbits fed a normal diet, or high-cholesterol (1%) diet with or without probucol (1%). ApoE mRNA levels increased 2- to 4-fold in the spleen and brain after cholesterol feeding for 4 weeks and were higher by 52 to 70% in the spleen of probucol-treated rabbits than in nontreated rabbits. This induction of apoE mRNA occurs within 7 days after the initiation of probucol treatment. However, apoE mRNA levels in the liver, a major apoE-synthesizing organ, were not enhanced after probucol treatment. These results indicate that probucol induces apoE mRNA expression specifically in the spleen and may affect apoE-mediated lipoprotein metabolism, the so-called reverse cholesterol transport.

Expression of rat apolipoprotein A-IV and A-I genes: mRNA induction during development and in response to glucocorticoids and insulin.

Rat apolipoproteins (apo-) A-IV and A-I share many structural similarities, the most notable of which is a domain of repeated docosapeptides with amphipathic helical potential. Although the genes for apo-A-IV and apo-A-I probably diverged from a common ancestor, these proteins seem to have developed different functions in their evolution. In the present study, cloned cDNAs were used to characterize the expression of apo-A-IV and apo-A-I mRNAs in a wide variety of adult rat tissues, as well as in small intestine and liver obtained from fetal, suckling, and weanling animals; comparisons were made to the expression of apo-E mRNA. The apo-A-IV and apo-A-I mRNAs were most abundant in adult small intestine and liver, with trace amounts detected in other tissues. Substantial amounts of these mRNAs were detected in the yolk sac, suggesting that this fetal tissue plays an important role in lipid metabolism during gestation. Noncoordinate accumulation of apo-A-IV and apo-A-I mRNAs was observed within and between the liver and small intestine during neonatal development. The apo-A-IV mRNA levels in the developing small intestine and liver appeared to correlate with their triglyceride secretion rates, suggesting that this protein plays an important role in the metabolism of triglyceride-rich lipoproteins. When dexamethasone (0.1 microM), insulin (0.01 microM), or insulin and dexamethasone together were incubated with primary cultures of nonproliferating adult rat hepatocytes, apo-A-IV mRNA levels were 4-, 7-, and 11-fold higher, respectively, than in non-hormone-treated control hepatocytes. Hormone administration resulted in a 2-fold greater amount of apo-A-I mRNA in each case, with no significant change in the level of apo-E mRNA. The overall results suggest that these structurally related apolipoproteins are regulated in substantially different ways.

Apolipoprotein E gene mapping and expression: localization of the structural gene to human chromosome 19 and expression of ApoE mRNA in lipoprotein- and non-lipoprotein-producing tissues.

Apolipoprotein E (apoE) binds to specific cell-surface receptors and appears to be an important determinant in lipoprotein metabolism in man. Cloned human apoE cDNA (pAE155) was used as a probe in chromosome mapping studies to detect the structural gene sequences in human--Chinese hamster cell hybrids. Southern blot analysis of HincII-digested DNAs from 13 hybrids localized the gene to human chromosome 19. This observation indicates that apoE is syntenic to at least two other genes related to lipid metabolism, those for the low-density lipoprotein (LDL) receptor (the LDLR) and apoC-II. The cloned apoE cDNA was further used to detect the presence of apoE mRNA in RNA extracts of various human and baboon tissues. Northern gel analysis using the 32P-labeled pAE155 as a probe demonstrated the presence of hybridizable apoE mRNAs in human liver and in baboon liver, intestine, spleen, kidney, adrenal gland, and brain but not in baboon skeletal muscle. The apoE mRNAs appear to be intact and migrate on an agarose gel under denaturing conditions at approximately 18 S. To assay for the biological activity of the apoE mRNAs in these tissues, they were translated in a reticulocyte lysate system in vitro. Immunoprecipitation with an apoE-specific antiserum followed by sodium dodecyl sulfate gel electrophoresis and fluorography demonstrated that immunoreactive apoE with the expected apparent size was a product of translation of mRNAs from baboon liver, intestine, kidney, spleen, and brain but not that from baboon skeletal muscle.(ABSTRACT TRUNCATED AT 250 WORDS)