Biomedical subjects

H Bryan Brewer

Publications and source records attributed to H Bryan Brewer.

47 records · Page 3Linked to original sources

PubMed2002

Developmental regulation of apolipoprotein B mRNA editing is an autonomous function of small intestine involving homeobox gene Cdx1.

Apolipoprotein B mRNA editing is developmentally regulated in the human and rodent small intestine, changing from <1% at day 14 to approximately 90% by day 20 in the rat fetus. This regulation is coincident with the developmental formation of the crypt-to-villus axis functional unit, a continuous and rapidly renewing system involving cell generation, migration, and differentiation. Utilizing small intestine isografts implanted into the subcutaneous tissue of adult recipients, apolipoprotein B mRNA editing was developmentally up-regulated, parallel to that seen with an intact control. In contrast, apoB mRNA expression remains nearly constant in the isograft, unlike the normal intact small intestine. Immunohistochemical analyses demonstrated that apoB-48 protein existed predominantly in well differentiated enterocytes along the villus surface whereas apoB-100 was in the lamina propria and crypts. ApoB mRNA editing levels were very low in the crypt-like rat intestinal cell line, IEC-6 ( approximately 0.3%), but very high in well differentiated enterocytes ( approximately 91.5%). The expression of homeobox gene Cdx1 increased 18-fold in small intestine in vivo during the same time course when apoB mRNA editing increased from approximately 2 to approximately 90%. The overexpression of Cdx1 in IEC-6 cells increased apoB mRNA editing over 10-fold compared with the vector control. This increase was associated with a significant increase of activating factor ACF, a component of the apoB mRNA editing complex. Taken together, these data suggest that the developmental regulation of apoB mRNA editing is an autonomous cytodifferentiation function of small intestine for which homeobox gene Cdx1 may play an important role.

Age Factors↗

PubMed2002

The farnesoid X-receptor is an essential regulator of cholesterol homeostasis.

To address the importance of the farnesoid X-receptor (FXR; NR1H4) for normal cholesterol homeostasis, we evaluated the major pathways of cholesterol metabolism in the FXR-deficient (-/-) mouse model. Compared with wild-type, FXR(-/-) mice have increased plasma high density lipoprotein (HDL) cholesterol and a markedly reduced rate of plasma HDL cholesterol ester clearance. Concomitantly, FXR(-/-) mice exhibit reduced expression of hepatic genes involved in reverse cholesterol transport, most notably, that for scavenger receptor BI. FXR(-/-) mice also have increased: (i) plasma non-HDL cholesterol and triglyceride levels, (ii) apolipoprotein B-containing lipoprotein synthesis, and (iii) intestinal cholesterol absorption. Surprisingly, biliary cholesterol elimination was increased in FXR(-/-) mice, despite decreased expression of hepatic genes thought to be involved in this process. These data demonstrate that FXR is a critical regulator of normal cholesterol metabolism and that genetic changes affecting FXR function have the potential to be pro-atherogenic.

Animals↗

PubMed2002

Role of the hepatic ABCA1 transporter in modulating intrahepatic cholesterol and plasma HDL cholesterol concentrations.

The current model for reverse cholesterol transport proposes that HDL transports excess cholesterol derived primarily from peripheral cells to the liver for removal. However, recent studies in ABCA1 transgenic mice suggest that the liver itself may be a major source of HDL cholesterol (HDL-C). To directly investigate the hepatic contribution to plasma HDL-C levels, we generated an adenovirus (rABCA1-GFP-AdV) that targets expression of mouse ABCA1-GFP in vivo to the liver. Compared with mice injected with control AdV, infusion of rABCA1-GFP-AdV into C57Bl/6 mice resulted in increased expression of mouse ABCA1 mRNA and protein in the liver. ApoA-I-dependent cholesterol efflux was increased 2.6-fold in primary hepatocytes isolated 1 day after rABCA1-GFP-AdV infusion. Hepatic ABCA1 expression in C57Bl/6 mice (n = 15) raised baseline levels of TC, PL, FC, HDL-C, apoE, and apoA-I by 150-300% (P < 0.05 all). ABCA1 expression led to significant compensatory changes in expression of genes that increase hepatic cholesterol, including HMG-CoA reductase (3.5-fold), LDLr (2.1-fold), and LRP (5-fold) in the liver. These combined results demonstrate that ABCA1 plays a key role in hepatic cholesterol efflux, inducing pathways that modulate cholesterol homeostasis in the liver, and establish the liver as a major source of plasma HDL-C.

ATP Binding Cassette Transporter 1↗

PubMed2002

The ABCA1 transporter functions on the basolateral surface of hepatocytes.

ABCA1 on the cell surface and in endosomes plays an essential role in the cell-mediated lipidation of apoA-I to form nascent HDL. Our previous studies of transgenic mice overexpressing ABCA1 suggested that ABCA1 in the liver plays a major role in regulating plasma HDL levels. The site of function of ABCA1 in the polarized hepatocyte was currently assessed by expression of an adenoviral construct encoding a human ABCA1-GFP fusion protein in the polarized hepatocyte-like WIF-B cell line. Consistent with localization of ABCA1 at the basolateral (vascular) cell surface, expression of ABCA1-GFP stimulated apoA-I mediated efflux of WIF-B cell cholesterol into the culture medium. Confocal fluorescence microscopy revealed that ABCA1-GFP was expressed solely on the basolateral surface and associated endocytic vesicles. These findings suggest an important role for hepatocyte basolateral membrane ABCA1 in the regulation of the levels of intracellular hepatic cholesterol, as well as plasma HDL.

Apolipoprotein A-I↗

PubMed2002

Comparative genome analysis of potential regulatory elements in the ABCG5-ABCG8 gene cluster.

The excretion of sterols from the liver and intestine is regulated by the ABCG5 and ABCG8 transporters. To identify potential regulatory elements, 152 kb of the human ABCG5-ABCG8 gene cluster was sequenced and comparative genome analysis was performed. The two genes are oriented in a head-to-head configuration and are separated by a 374-bp intergenic region, which is highly conserved among several species. Using a reporter construct, the intergenic region was found to act as a bidirectional promoter. A conserved GATA site in the intergenic region was shown by site-directed mutagenesis to act as a repressor for the ABCG5 promoter. The intergenic region was also shown to be partially responsive to treatment by LXR agonists. In summary, several potential regulatory elements were found for the ABCG5 and ABCG8 genes, and the intergenic region was found to act as a bidirectional promoter.

Animals↗

PubMed2002

In vivo metabolism of apolipoprotein E within the HDL subpopulations LpE, LpE:A-I, LpE:A-II and LpE:A-I:A-II.

High-density lipoproteins can be separated into distinct particles based on their apolipoprotein content. In the present study, the in vivo metabolism of apoE within the apoE-containing HDL particles LpE, LpE:A-I, LpE:A-II and LpE:A-I:A-II was assessed in control subjects and in patients with abetalipoproteinemia (ABL), in whom HDL are the sole plasma lipoproteins. The metabolism of apoE within these HDL subspecies was investigated in three separate studies which differed by donor or recipient status: (1) particles purified from normolipidemic plasma and reassociated with 125I or 131I-labeled apoE injected into normolipidemic subjects (study 1); (2) particles purified from ABL plasma injected into normolipidemic subjects (study 2); and (3) particles purified from ABL plasma injected into ABL subjects (study 3). The plasma residence times (RT, hours) in study 1 were 14.3+/-2.9, 11.3+/-3.4, and 9.1+/-1.2 for apoE within LpE:A-I:A-II, LpE:A-II and LpE:A-I, respectively, while those in study 2 were 10.1+/-2.2, 9.7+/-2.4, 7.9+/-1.0 and 7.3+/-0.8 for apoE within LpE:A-I:A-II, LpE:A-II, LpE:A-I and LpE, respectively. In study 3, RTs for apoE within LpE:A-I:A-II and LpE were 8.7+/-0.9 and 6.8+/-0.9, respectively. In comparison, RT for apoA-I on LpA-I:A-II has been reported to be 124.1+/-5.5 h and that for apoA-I on LpA-I 105.8+/-6.2 h. Thus, apoE within the different apoE-containing HDL particles was metabolized rapidly and at a similar rate in control and ABL subjects. The plasma RT of apoE was longest when injected on LpE:A-I:A-II particles and shortest when injected on LpE. In summary, our data show that: (1) the plasma RT of apoE within HDL is approximately ten times shorter than that of apoA-I within HDL, and (2) apoE within HDL is metabolized at a slower rate when apoproteins A-I and A-II are present (LpE:A-I:A-II RT>LpE:A-II>LpE:A-I>LpE). These differences were related to the lipid and apolipoprotein composition of the HDL subspecies, and, in control subjects, to the transfer of apoE from HDL subspecies to apoB-containing lipoproteins as well.

Abetalipoproteinemia↗

PubMed2002

Comparative in vivo metabolism of apolipoproteins E2 and E4 in heterozygous apoE2/4 subjects.

Apolipoprotein E (apoE) exists in three common forms in humans: the wild-type apoE3 and two common genetic variants, apoE2 and apoE4. Although previous studies have examined the metabolism of the different apoE isoforms in human subjects, they have not involved direct comparison of two different isoforms in subjects heterozygous for the same two isoforms. We conducted this study to directly compare the catabolism of apoE2 and apoE4 in heterozygous E2/4 subjects in vivo. Iodine 131-labeled apoE2 and iodine 125-labeled apoE4 were simultaneously injected into three E4/2 heterozygous subjects. The mean residence time of apoE4 (0.40 +/- 0.01 day) was found to be one-third that of apoE2 (1.20 +/- 0.18 day). ApoE2 was present primarily in high-density lipoprotein, whereas apoE4 was present equally in very low density and high-density lipoprotein. In all lipoprotein subfractions, apoE4 was catabolized at a much faster rate than apoE2. In conclusion, E4 is catabolized three times faster than apoE2 in heterozygous E2/4 subjects, indicating that these two apoE isoproteins have distinct metabolic pathways.

Adult↗

PubMed2002

Conditional disruption of the peroxisome proliferator-activated receptor gamma gene in mice results in lowered expression of ABCA1, ABCG1, and apoE in macrophages and reduced cholesterol efflux.

Disruption of the peroxisome proliferator-activated receptor gamma (PPAR gamma) gene causes embryonic lethality due to placental dysfunction. To circumvent this, a PPAR gamma conditional gene knockout mouse was produced by using the Cre-loxP system. The targeted allele, containing loxP sites flanking exon 2 of the PPAR gamma gene, was crossed into a transgenic mouse line expressing Cre recombinase under the control of the alpha/beta interferon-inducible (MX) promoter. Induction of the MX promoter by pIpC resulted in nearly complete deletion of the targeted exon, a corresponding loss of full-length PPAR gamma mRNA transcript and protein, and marked reductions in basal and troglitazone-stimulated expression of the genes encoding lipoprotein lipase, CD36, LXR alpha, and ABCG1 in thioglycolate-elicited peritoneal macrophages. Reductions in the basal levels of apolipoprotein E (apoE) mRNA in macrophages and apoE protein in total plasma and high-density lipoprotein (HDL) were also observed in pIpC-treated PPAR gamma-MXCre(+) mice. Basal cholesterol efflux from cholesterol-loaded macrophages to HDL was significantly reduced after disruption of the PPAR gamma gene. Troglitazone selectively inhibited ABCA1 expression (while rosiglitazone, ciglitazone, and pioglitazone had little effect) and cholesterol efflux in both PPAR gamma-deficient and control macrophages, indicating that this drug can exert paradoxical effects on cholesterol homeostasis that are independent of PPAR gamma. Together, these data indicate that PPAR gamma plays a critical role in the regulation of cholesterol homeostasis by controlling the expression of a network of genes that mediate cholesterol efflux from cells and its transport in plasma.

ATP Binding Cassette Transporter 1↗

PubMed2002

Rising to the challenge of the new NCEP ATP III guidelines: exceeding current therapeutic limitations.

The third set of guidelines recently issued by the National Cholesterol Education Program (NCEP) differs from the second set issued in 1993 in several ways. The third set introduced a quantitative risk scoring system and identified and/or reclassified certain groups of patients at high or moderate risk for a coronary event. Among these groups are patients with type 2 diabetes, and patients with multiple risk factors other than coronary heart disease or diabetes that cumulatively confer high risk for a coronary event. However, the new guidelines also present physicians with the major challenges of identifying these patients, determining their true risk, and implementing the recommended approaches to treatment in clinical practice settings. Although reducing elevated levels of low-density lipoprotein (LDL) cholesterol remains the primary focus of therapy, the new NCEP guidelines also include strategies to identify and treat patients with low levels of high-density lipoprotein (HDL) cholesterol and/or elevated triglyceride levels. Just as there is "good" cholesterol (HDL) and "bad" cholesterol (LDL), there are also "good" triglycerides, which contain high concentrations of triglyceride remnants and are associated with low risk, and "bad" triglycerides, which contain high concentrations of cholesterol remnants and are associated with increased risk. The mechanisms by which "bad" triglycerides develop explain why elevated triglycerides and low HDL--and patients with the metabolic syndrome--warrant special attention. These mechanisms and others also suggest new targets for therapeutic intervention and the development of new drugs that will correct lipid and lipoprotein abnormalities through a number of different metabolic pathways.

Anticholesteremic Agents↗

PubMed2002

The E-box motif in the proximal ABCA1 promoter mediates transcriptional repression of the ABCA1 gene.

To identify regulatory elements in the proximal human ATP-binding cassette transporter A1 (hABCA1) gene promoter we transfected RAW cells with plasmids containing mutations in the E-box, AP1, and liver X receptor (LXR) elements as well as the two Sp1 motifs. Point mutations in either Sp1 site or in the AP1 site had only a minor effect whereas mutation of the LXR element decreased promoter activity. In contrast, mutation or deletion of the E-box motif caused a 3-fold increase in transcriptional activity under basal conditions. Gel shift and DNaseI footprint analysis showed binding of a protein or protein complex to this region. Preincubation of nuclear extracts with antibodies established that USF1, USF2, and fos related antigen (Fra) 2 bind to DNA sequences in the human ABCA1 promoter that contains the intact E-box but not the mutant or deleted E-box. Co-transfection of USF1 and USF2 enhanced, but Fra2 repressed, ABCA1 promoter activity. Thus, a complex consisting of USF1, USF2, and Fra2 binds the E-box motif 147 bp upstream of the transcriptional start site and facilitates repression of the human ABCA1 promoter. These combined studies identify a novel site in the human ABCA1 promoter involved in the regulation of ABCA1 gene expression.

ATP Binding Cassette Transporter 1↗

PubMed2001

The ATP binding cassette transporter A1 (ABCA1) modulates the development of aortic atherosclerosis in C57BL/6 and apoE-knockout mice.

Identification of mutations in the ABCA1 transporter (ABCA1) as the genetic defect in Tangier disease has generated interest in modulating atherogenic risk by enhancing ABCA1 gene expression. To investigate the role of ABCA1 in atherogenesis, we analyzed diet-induced atherosclerosis in transgenic mice overexpressing human ABCA1 (hABCA1-Tg) and spontaneous lesion formation in hABCA1-Tg x apoE-knockout (KO) mice. Overexpression of hABCA1 in C57BL/6 mice resulted in a unique anti-atherogenic profile characterized by decreased plasma cholesterol (63%), cholesteryl ester (63%), free cholesterol (67%), non-high density lipoprotein (HDL)-cholesterol (53%), and apolipoprotein (apo) B (64%) but markedly increased HDL-cholesterol (2.8-fold), apoA-I (2.2-fold), and apoE (2.8-fold) levels. These beneficial changes in the lipid profile led to significantly lower (65%) aortic atherosclerosis in hABCA1-Tg mice. In marked contrast, ABCA1 overexpression had a minimal effect on the plasma lipid profile of apoE-KO mice and resulted in a 2- to 2.6-fold increase in aortic lesion area. These combined results indicate that overexpression of ABCA1 in C57BL/6 mice on a high cholesterol diet results in an atheroprotective lipoprotein profile and decreased atherosclerosis, and thus provide previously undocumented in vivo evidence of an anti-atherogenic role for the ABCA1 transporter. In contrast, overexpression of ABCA1 in an apoE-KO background led to increased atherosclerosis, further substantiating the important role of apoE in macrophage cholesterol metabolism and atherogenesis. In summary, these results establish that, in the presence of apoE, overexpression of ABCA1 modulates HDL as well as apoB-containing lipoprotein metabolism and reduces atherosclerosis in vivo, and indicate that pharmacological agents that will increase ABCA1 expression may reduce atherogenic risk in humans.

ATP Binding Cassette Transporter 1↗