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The lipidation of apoA-I in liver greatly influences HDL biogenesis and plasma HDL levels by stabilizing the secreted apoA-I. Niacin is the most effective lipid-regulating agent clinically available to raise HDL. This study was undertaken to identify regulatory mechanisms of niacin action in hepatic lipidation of apoA-I, a critical event involved in HDL biogenesis. In cultured human hepatocytes (HepG2), niacin increased: association of apoA-I with phospholipids and cholesterol by 46% and 23% respectively, formation of lipid-poor single apoA-I molecule-containing particles up to ∼ 2.4-fold, and pre β 1 and α migrating HDL particles. Niacin dose-dependently stimulated the cell efflux of phospholipid and cholesterol and increased transcription of ABCA1 gene and ABCA1 protein. Mutated DR4, a binding site for nuclear factor liver X receptor alpha (LXR α ) in the ABCA1 promoter, abolished niacin stimulatory effect. Further, knocking down LXR α or ABCA1 by RNA interference eliminated niacin-stimulated apoA-I lipidation. Niacin treatment did not change apoA-I gene expression. The present data indicate that niacin increases apoA-I lipidation by enhancing lipid efflux through a DR4-dependent transcription of ABCA1 gene in HepG2 cells. A stimulatory role of niacin in early hepatic formation of HDL particles suggests a new mechanism that contributes to niacin action to increase the stability of newly synthesized circulating HDL.

Pioglitazone, a hypoglycemic agent, has been shown to increase plasma HDL cholesterol, but the mechanism is incompletely understood. We further investigated effects of pioglitazone on transcriptional regulation of apolipoprotein (apo)A-I gene and functional properties of pioglitazone-induced apoA-I-containing particles. Pioglitazone dose-dependently stimulated apoA-I promoter activities in HepG2 cells. A peroxisome proliferator-activated receptor (PPAR)-response element located in site A (−214 to −192 bp, upstream of the transcription start site) of the promoter is required for pioglitazone-induced apoA-I gene transcription. Deletion of site A (−214 to −192 bp), B (−169 to −146 bp), or C (−134 to −119 bp), which clusters a number of cis-acting elements for binding of different transcription factors, reduced the basal apoA-I promoter activities, and no additional pioglitazone-sensitive elements were found within this region. Overexpression or knock-down of liver receptor homolog-1, a newly identified nuclear factor with strong stimulatory effect on apoA-I transcription, did not alter pioglitazone-induced apoA-I transcription. Pioglitazone-induced apoA-I transcription is mainly mediated through PPARα but not PPARγ in hepatocytes. Pioglitazone induced production of HDL enriched in its subfraction containing apoA-I without apoA-II, which inhibited monocyte adhesion to endothelial cells in vitro. In conclusion, pioglitazone increases apoA-I production by directly enhancing PPAR-response element-dependent transcription, resulting in generation of apoA-I-containing HDL particles with increased anti-inflammatory property.

Niacin is an effective agent for raising HDL, but its cellular target sites are largely unknown. We examined effects of niacin on the surface expression of ATP synthase β chain, a newly described HDL/apolipoprotein A-I (apoA-I) receptor for HDL endocytosis, in HepG2 cells. A significant amount of immunodetectable β chain was observed on the surface of HepG2 cells, which was competitively displaced by apoA-I. Niacin treatment reduced the surface expression of β chain in HepG2 cells by ∼27%, and decreased 125I-labeled HDL uptake up to ∼35%. However, nicotinamide, a niacin metabolite that does not have clinical lipid effects, exhibited weaker inhibition on the β chain cell surface expression, and failed to show inhibitory action on 125I-labeled HDL uptake. Furthermore, anti-β chain antibody significantly reduced 125I-labeled HDL uptake and abolished the inhibitory effect of niacin. Niacin did not change β chain mRNA expression. These data suggest that niacin inhibits cell surface expression of the ATP synthase β chain, leading to reduced hepatic removal of HDL protein, thus implicating a potential cellular target for niacin action to raise HDL.

Mutations in ABCA1 uniformly decrease plasma HDL-cholesterol (HDL-C) and reduce cholesterol efflux, yet different mutations in ABCA1 result in different phenotypic effects in heterozygotes. For example, truncation mutations result in significantly lower HDL-C and apoliprotein A-I (apoA-I) levels in heterozygotes compared with nontruncation mutations, suggesting that truncation mutations may negatively affect the wild-type allele. To specifically test this hypothesis, we examined ABCA1 protein expression in response to 9-cis-retinoic acid (9-cis-RA) and 22-R-hydroxycholesterol (22-R-OH-Chol) in a collection of human fibroblasts representing eight different mutations and observed that truncation mutations blunted the response to oxysterol stimulation and dominantly suppressed induction of the remaining full-length allele to 5–10% of wild-type levels. mRNA levels between truncation and nontruncation mutations were comparable, suggesting that ABCA1 expression was suppressed at the protein level. Dominant negative activity of truncated ABCA1 was recapitulated in an in vitro model using transfected Cos-7 cells. Our results suggest that the severe reduction of HDL-C in patients with truncation mutations may be at least partly explained by dominant negative suppression of expression and activity of the remaining full-length ABCA1 allele.These data suggest that ABCA1 requires a physical association with itself or other molecules for normal function and has important pharmacogenetic implications for individuals with truncation mutations.

The Wisconsin hypoalpha mutant (WHAM) chicken has a >90% reduction in plasma HDL due to hypercatabolism by the kidney of lipid-poor apoA-I. The WHAM chickens have a recessive white skin phenotype caused by a single-gene mutation that maps to the chicken Z-chromosome. This corresponds to human 9q31.1, a chromosomal segment that contains the ATP-binding cassette protein-1 (ABCA1) gene, which is mutated in Tangier Disease and familial hypoalphalipoproteinemia. Complete sequencing of the WHAM ABCA1 cDNA identified a missense mutation near the N-terminus of the protein (E89K). The substitution of this evolutionary conserved glutamate residue for lysine in the mouse ABCA1 transporter leads to complete loss of function, resulting principally from defective intracellular trafficking and very little ABCA1 reaching the plasma membrane.The WHAM chicken is a naturally occurring animal model for Tangier Disease.