Immunological quantitation and localization of ACAT-1 and ACAT-2 in human liver and small intestine

By using specific anti-ACAT-1 antibodies in immunodepletion studies, we previously found that ACAT-1, a 50-kDa protein, plays a major catalytic role in the adult human liver, adrenal glands, macrophages, and kidneys but not in the intestine. Acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity in the intestine may be largely derived from a different ACAT protein. To test this hypothesis, we produced specific polyclonal anti-ACAT-2 antibodies that quantitatively immunodepleted human ACAT-2, a 46-kDa protein expressed in Chinese hamster ovary cells. In hepatocyte-like HepG2 cells, ACAT-1 comprises 85-90% of the total ACAT activity, with the remainder attributed to ACAT-2. In adult intestines, most of the ACAT activity can be immunodepleted by anti-ACAT-2. ACAT-1 and ACAT-2 do not form hetero-oligomeric complexes. In differentiating intestinal enterocyte-like Caco-2 cells, ACAT-2 protein content increases by 5-10-fold in 6 days, whereas ACAT-1 protein content remains relatively constant. In the small intestine, ACAT-2 is concentrated at the apices of the villi, whereas ACAT-1 is uniformly distributed along the villus-crypt axis. In the human liver, ACAT-1 is present in both fetal and adult hepatocytes. In contrast, ACAT-2 is evident in fetal but not adult hepatocytes. Our results collectively suggest that in humans, ACAT-2 performs significant catalytic roles in the fetal liver and in intestinal enterocytes.     

The influence of the acyl-CoA:cholesterol acyltransferase-1 gene (-77G-->A) polymorphisms on plasma lipid and apolipoprotein levels in normolipidemic and hyperlipidemic subjects

BACKGROUND: Acyl-CoA:cholesterol acyltransferase (ACAT) plays important roles in cellular cholesterol homeostasis. Two isoforms of ACAT have been reported (ACAT-1 and ACAT-2). ACAT inhibitors cannot only prevent atherosclerosis formation, but may also induce its regression in animals. In humans, an ACAT inhibitor was shown to have a lipid-lowering effect. The present study was carried out to clarify the relationship between ACAT-1 gene variants and hyperlipidemia. METHODS AND RESULTS: To identify genetic variants, we screened 30 subjects with hyperlipidemia by direct sequencing. As a result, a missense variant (R526G) and a variant in the 5' untranslated region (-77G-->A) were identified. The genotype frequencies of each variant were determined in 178 unrelated normolipidemic and 441 unrelated hyperlipidemic subjects. The alleles frequencies of the R526G variant in normolipidemic and hyperlipidemic subjects were 0.676 and 0.633, respectively. The alleles frequencies of the -77G-->A variant in normolipidemic and hyperlipidemic subjects were 0.503 and 0.515, respectively. Differences in allele frequencies between normolipidemic and hyperlipidemic subjects were not significant in both variants. R526G variant did not affect plasma concentrations of lipids or apolipoproteins in subjects studied. However, among hyperlipidemic subjects, plasma concentrations of HDL-C and apoA-I in subjects with -77G-->A variant were significantly higher than those in subjects without variant. CONCLUSION: Two variants in ACAT-1 gene were identified in subjects with hyperlipidemia. -77G-->A variant affects plasma HDL concentrations only in hyperlipidemic subjects. These data suggest that the intracellular FC concentration might modulate plasma HDL concentrations.     

Absence of ACAT-1 attenuates atherosclerosis but causes dry eye and cutaneous xanthomatosis in mice with congenital hyperlipidemia

Acyl-CoA:cholesterol acyltransferase (ACAT) catalyzes esterification of cellular cholesterol. To investigate the role of ACAT-1 in atherosclerosis, we have generated ACAT-1 null (ACAT-1-/-) mice. ACAT activities were present in the liver and intestine but were completely absent in adrenal, testes, ovaries, and peritoneal macrophages in our ACAT-1-/- mice. The ACAT-1-/- mice had decreased openings of the eyes because of atrophy of the meibomian glands, a modified form of sebaceous glands normally expressing high ACAT activities. This phenotype is similar to dry eye syndrome in humans. To determine the role of ACAT-1 in atherogenesis, we crossed the ACAT-1-/- mice with mice lacking apolipoprotein (apo) E or the low density lipoprotein receptor (LDLR), hyperlipidemic models susceptible to atherosclerosis. High fat feeding resulted in extensive cutaneous xanthomatosis with loss of hair in both ACAT-1-/-:apo E-/- and ACAT-1-/-:LDLR-/- mice. Free cholesterol content was significantly increased in their skin. Aortic fatty streak lesion size as well as cholesteryl ester content were moderately reduced in both double mutant mice compared with their respective controls. These results indicate that the local inhibition of ACAT activity in tissue macrophages is protective against cholesteryl ester accumulation but causes cutaneous xanthomatosis in mice that lack apo E or LDLR.     

Upregulation of acyl-CoA: cholesterol acyltransferase in chronic renal failure

Chronic renal failure (CRF) is associated with profound abnormalities of lipid metabolism and accelerated arteriosclerotic cardiovascular disease. In a recent study, we found marked downregulation of hepatic lecithin-cholesterol acyltransferase, or LCAT, expression, which can account for impaired HDL maturation and depressed HDL cholesterol concentration in CRF. Here, we report on the effect of CRF on acyl-CoA:cholesterol acyltransferase (ACAT) expression. ACAT is an intracellular enzyme that catalyzes esterification of free cholesterol to cholesterol ester for storage or secretion. ACAT plays a major role in hepatic production and release of VLDL, intestinal absorption of cholesterol, foam cell formation, and atherogenesis. We examined hepatic expression of ACAT-1 and ACAT-2 mRNA (Northern blot) and protein (Western blot) abundance and total ACAT activity in male CRF rats (6 wk after 5/6 nephrectomy) and sham-operated controls. The CRF animals showed a significant reduction in creatinine clearance, marked hypertriglyceridemia, modest hypercholesterolemia, and significant upregulation of hepatic tissue ACAT-2 protein and mRNA abundance. In contrast, hepatic ACAT-1 mRNA and protein abundance were unaffected by CRF. Upregulation of ACAT-2 expression was accompanied by a significant increase in hepatic ACAT activity and a significant decrease in hepatic microsomal and whole liver free cholesterol concentration. Thus CRF results in significant upregulation of hepatic ACAT-2 (but not ACAT-1) expression and ACAT activity, which may, in part, contribute to the associated lipid disorders.     

Three novel partial deletions of the low-density lipoprotein (LDL) receptor gene in familial hypercholesterolemia

Summary The low-density lipoprotein (LDL) receptor genes from 18 unrelated Japanese heterozygotes and 1 homozygote with classical familial hypercholesterolemia were analyzed by Southern blot hybridization using fragments of the human LDL receptor cDNA as probes. Four different deletion mutations were detected among 20 mutant LDL receptor genes (20%); they were characterized by restriction mapping. None of these mutations has previously been reported in Caucasian patients with FH: three of the mutations were novel and one was similar to the detetion mutation of FH-Tonami described previously in Japanese patients. In three of the four deletion mutations, the rearrangements were related to intron 15 of the LDL receptor gene, in which many Alu sequences exist. The data suggest that a wide range of molecular heterogeneity exists even in major rearrangements resulting in deletions in the LDL receptor gene. The data also support the hypothesis that there are preferential sites within the LDL receptor gene for major rearrangements resulting in deletions. The possibility that a higher frequency of deletion mutations occurs in classical FH than previously suspected is discussed.     

Screening for ferrochelatase mutations: molecular heterogeneity of erythropoietic protoporphyria

The DNA of 21 patients from 19 unrelated families with erythropoietic protoporphyria (EPP) were screened for the 6 ferrochelatase point mutations so far described. The mutation previously described by us (A ? t transversion at position ?3 of the donor site of intron 10, causing exon 10 skipping) was detected in two additional unrelated EPP patients: in these patients, cDNA lacking exon 10 was also detected. The mutation described by Nakahashi et al. as responsible for exon 2 skipping (C ? T transition at position ?23 of the acceptor site of intron 1), although also observed in some normal individuals, was invariably observed in all EPP patients tested and may thus play some role in the pathognesis of EPP. Thus, it does not appear that this mutation is the primary mechanism underlying exon 2 skipping. None of the other four previously described mutations were detected. These data demonstrate the heterogeneity of the ferrochelatase locus and of the genetic defect in EPP.     

Two human ACAT2 mRNA variants produced by alternative splicing and coding for novel isoenzymes

Acyl coenzyme A：cholesterol acyltransferase 2 （ACAT2） plays an important role in cholesterol absorption. Human ACAT2 is highly expressed in small intestine and fetal liver, but its expression is greatly diminished in adult liver. The full-length human ACAT2 mRNA encodes a protein, designated ACAT2a, with 522 amino acids. We have previously reported the organization of the human ACAT2 gene and the differentiation-dependent promoter activity in intestinal Caco-2 cells. In the current work, two human ACAT2 mRNA variants produced by alternative splicing are cloned and predicted to encode two novel ACAT2 isoforms, named ACAT2b and ACAT2c, with 502 and 379 amino acids, respectively. These mRNA variants differ from ACAT2a mRNA by lack of the exon 4 （ACAT2b mRNA） and exons 4-5 plus 8-9-10 （ACAT2c mRNA）. Significantly, comparable amounts of the alternatively spliced ACAT2 mRNA variants were detected by RT- PCR, and Western blot analysis confirmed the presence of their corresponding proteins in human liver and intestine cells. Furthermore, phosphorylation and enzymatic activity analyses demonstrated that the novel isoenzymes ACAT2b and ACAT2c lacked the phosphorylatable site SLLD, and their enzymatic activities reduced to 25%-35% of that of ACAT2a. These evidences indicate that alternative splicing produces two human ACAT2 mRNA variants that encode the novel ACAT2 isoenzymes. Our findings might help to understand the regulation of the ACAT2 gene expression under certain physiological and pathological conditions.     

Two novel mutations in the lipoprotein lipase gene in a family with marked hypertriglyceridemia in heterozygous carriers. Potential interaction with the polymorphic marker D1S104 on chromosome 1q21-q23

Two novel mutations in the lipoprotein lipase (LPL) gene are described in an Austrian family: a splice site mutation in intron 1 (3 bp deletion of nucleotides -2 to -4) which results in skipping of exon 2, and a missense mutation in exon 5 which causes an asparagine for histidine substitution in codon 183 and complete loss of enzyme activity. A 5-year-old boy who exhibited all the clinical features of primary hyperchylomicronemia was a compound heterozygote for these two mutations. Nine other family members were investigated: seven were heterozygotes for the splice site mutation, one was a heterozygote for the missense mutation, and one had two wild-type alleles of the LPL gene. LPL activity in the post-heparin plasma of the heterozygotes was reduced to 49;-79% of the mean observed in normal individuals. Two of the heterozygotes had extremely high plasma triglyceride levels; in three of the other heterozygotes the plasma triglycerides were also elevated. As plasma triglycerides in carriers of one defective LPL allele can be normal or elevated, the heterozygotes of this family have been studied for a possible additional cause of the expression of hypertriglyceridemia in these subjects. Body mass index, insulin resistance, mutations in other candidate genes (Asn291Ser and Asp9Asn in the LPL gene, apoE isoforms, polymorphisms in the apoA-II gene and in the apoAI-CIII-AIV gene cluster, and in the IRS-1 gene) could be ruled out as possible factors contributing to the expression of hypertriglyceridemia in this family. A linkage analysis using the allelic marker D1S104 on chromosome 1q21;-q23 suggested that a gene in this region could play a role in the expression of hypertriglyceridemia in the heterozygous carriers of this family, but the evidence was not sufficiently strong to prove this assumption. Nevertheless, this polymorphic marker seems to be a good candidate for further studies.     

Human acyl-CoA:cholesterol acyltransferase-1 in the endoplasmic reticulum contains seven transmembrane domains.

Acyl-CoA:cholesterol acyltransferase (ACAT) plays important roles in cellular cholesterol homeostasis and is involved in atherosclerosis. ACAT-1 protein is located mainly in the ER. The hydropathy plot suggests that ACAT-1 protein contains multiple transmembrane segments. We inserted either the hemagglutinin tag or the HisT7 tag at various hydrophilic regions within the human ACAT-1 protein and used immunofluorescence microscopy to determine the topography of the tagged proteins expressed in mutant Chinese hamster ovary cells lacking endogenous ACAT. All of the tagged proteins are located mainly in the ER and retain full or partial enzyme activities. None of the tagged proteins produces detectable intracellular degradation intermediates. Treating cells with digitonin at 5 micrograms/ml permeabilizes the plasma membranes while leaving the ER membranes sealed; in contrast, treating cells with 0.25% Triton X-100 or with cold methanol permeabilizes both the plasma membranes and the ER membranes. After appropriate permeabilization, double immunostaining using antibodies against the N-terminal region and against the inserted tag were used to visualize various regions of the tagged protein. The results show that human ACAT-1 in the ER contains seven transmembrane domains.     

Human peroxisome assembly factor-2 (PAF-2): a gene responsible for group C peroxisome biogenesis disorder in humans.

Peroxisome-biogenesis disorders (PBD) are genetically heterogeneous and can be classified into at least ten complementation groups. We recently isolated the cDNA for rat peroxisome assembly factor-2 (PAF-2) by functional complementation using the peroxisome-deficient Chinese-hamster-ovary cell mutant, ZP92. To clarify the novel pathogenic gene of PBD, we cloned the full-length human PAF-2 cDNA that morphologically and biochemically restores peroxisomes of group C Zellweger fibroblasts (the same as group 4 in the Kennedy-Krieger Institute) and identified two pathogenic mutations in the PAF-2 gene in two patients with group C Zellweger syndrome. The 2,940-bp open reading frame of the human PAF-2 cDNA encodes a 980-amino-acid protein that shows 87.1% identity with rat PAF-2 and also restored the peroxisome assembly after gene transfer to fibroblasts of group C patients. Direct sequencing of the PAF-2 gene revealed a homozygous 1-bp insertion at nucleotide 511 (511 insT) in one patient with group C Zellweger syndrome (ZS), which introduces a premature termination codon in the PAF-2 gene, and, in the second patient, revealed a splice-site mutation in intron 3 (IVS3+1G-->A), which skipped exon 3, an event that leads to peroxisome deficiency. Chromosome mapping utilizing FISH indicates that PAF-2 is located on chromosome 6p21.1. These results confirm that human PAF-2 cDNA restores peroxisome of group C cells and that defects in the PAF-2 produce peroxisome deficiency of group C PBD.     

Mutations in the hepatocyte nuclear factor-4alpha gene in Japanese with non-insulin-dependent diabetes: a nucleotide substitution in the polypyrimidine tract of intron 1b.

Mutations of the hepatocyte nuclear factor 4 alpha (HNF-4alpha) gene have been demonstrated in maturity-onset diabetes of the young (MODY) 1 families. To investigate the possibility that the HNF-4alpha gene contributes to the onset of non-insulin-dependent diabetes mellitus (NIDDM) in Japanese patients, we screened all exons and flanking introns of this gene for mutations in 100 patients with NIDDM diagnosed after 25 years of age. We identified two missense mutations: M49V in exon 1c and T1301 in exon 4; and two nucleotide substitutions in introns: cytosine to thymidine at -5 nt in intron 1b and adenine to thymidine at -21 nt in intron 5. We screened an additional 220 diabetic subjects for the polymorphism in intron 1b. The c/t substitution in intron 1b was associated with NIDDM. This substitution in the polypyrimidine tract, an important cis-acting element directing intron removal, is likely to influence pre-mRNA splicing of this gene. T1301 in exon 4 was observed in only two diabetic subjects. This mutation could influence the conformation of this peptide, resulting in changes in ligand binding domain function. M49V in exon 1c was found in both diabetic and non-diabetic subjects; isoforms HNF-4alpha 4, 5, and 6 with this mutation may impair glucose metabolism in tissue. In contrast to the primary cause of nonsense and missense mutations of the HNF-4alpha gene in MODY1, the nucleotide substitution in intron 1b may partially contribute to development of NIDDM in combination with other genetic and environmental factors.     

Large-scale molecular screening for galactosemia alleles in a pan-ethnic population

DNA samples from 4,796 subjects from diverse ethnic groups were screened for five frequently encountered galactose-1-phosphate uridyl transferase (GALT) mutations: S135L (cDNA nt 404C-->T, as numbered from the initiator ATG codon, with A=1); Q188R (cDNA nt 563A-->G); K285 N (cDNA nt 855G-->T); the Duarte variant, N314D (cDNA nt 940A-->G); and the Los Angeles variant, which contains L218L (cDNA nt 652C-->T) and N314D. Among Whites, the gene frequency of the Q188R mutation was 0.29%, and that of the K285 N mutation was 0.062%. Only one S135L mutation was encountered among 505 African-Americans (gene frequency 0.10%). The pan-ethnic gene frequencies of the Duarte and the Los Angeles variants were 5.1% and 2.7%, respectively. Both of these frequencies were significantly less among African-Americans and Asians than among Whites and Hispanics. Native Americans revealed a higher incidence of the both variants. Based upon the gene frequency of the Q188R mutation in the White population, the birth incidence of classic galactosemia is estimated at one patient per 47,000 in the White population. This prevalence would be increased by inbreeding. It agrees well with the results from newborn screening programs and is only minimally higher than that reported in most studies, suggesting that most, if not all, infants with the galactosemia genotype are born and survive sufficiently long to be screened.     

Glibenclamide acts as an inhibitor of acyl-CoA:cholesterol acyltransferase enzyme

Sulfonylureas are used in the treatment of non-insulin-dependent diabetes mellitus. Little is known, however, about their effects on cholesterol metabolism. We tested in the present study the effects of glibenclamide (GB) on cholesterol esterification (CE) in macrophage-derived cells. GB inhibited intracellular accumulation of CE induced by acetylated LDL or oxidized LDL in J774 cells, but no such effect on total cholesterol, suggesting that the target of GB was acyl-CoA:cholesterol acyltransferase (ACAT). In the cell-free reconstitution ACAT assay, GB inhibited the ACAT activity with an IC(50) value of 20 microM. Furthermore, GB effectively inhibited the ACAT activity of PMA-stimulated THP-1 cells to the undifferentiated level of THP-1. In the whole-cell ACAT assay using CHO cells overexpressed with ACAT-1 or ACAT-2, GB inhibited the activity of both isozymes with similar potency. Our in vitro data suggest that sulfonylurea could be a potential seed for a new generation of ACAT inhibitors.