The mechanisms responsible for the inverse relationship between plasma high-density lipoprotein (HDL) levels and atherosclerotic cardiovascular disease are poorly understood. The ATP-binding cassette transporter A1 (ABCA1) mediates efflux of cellular cholesterol to lipid-poor apolipoproteins but not to HDL particles that constitute the bulk of plasma HDL. We show that two ABC transporters of unknown function, ABCG1 and ABCG4, mediate isotopic and net mass efflux of cellular cholesterol to HDL. In transfected 293 cells, ABCG1 and ABCG4 stimulate cholesterol efflux to both smaller (HDL-3) and larger (HDL-2) subclasses but not to lipid-poor apoA-I. Treatment of macrophages with an liver X receptor activator results in up-regulation of ABCG1 and increases cholesterol efflux to HDL. RNA interference reduced the expression of ABCG1 in liver X receptor-activated macrophages and caused a parallel decrease in cholesterol efflux to HDL. These studies indicate that ABCG1 and ABCG4 promote cholesterol efflux from cells to HDL. ABCG1 is highly expressed in macrophages and probably mediates cholesterol efflux from macrophage foam cells to the major HDL fractions, providing a mechanism to explain the relationship between HDL levels and atherosclerosis risk.A major theory to account for the inverse relationship between high-density lipoprotein (HDL) levels and cardiovascular risk is that HDL promotes the efflux of cholesterol from arterial wall macrophage foam cells and decrease atherosclerosis. This hypothesis appeared to be supported by the discovery that Tangier disease, a disorder characterized by very low HDL levels, macrophage foam cell accumulation, and increased atherosclerosis, is caused by mutations in the ATP-binding cassette transporter, ABCA1 (1-4). ABCA1 mediates efflux of cellular phospholipids and cholesterol to lipid-poor apolipoproteins, such as apoA-I and apoE (5, 6), initiating the formation of HDL. However, ABCA1 interacts poorly with HDL-2 and HDL-3 particles (5, 7) that constitute the bulk of the plasma HDL, and ABCA1 variants are not likely to account for a major part of the genetic variation in HDL levels in the general population (8). Thus, the activity of ABCA1 does not readily account for cholesterol efflux from foam cells to HDL, and the mechanism underlying the inverse relationship between HDL levels and atherosclerosis risk remains uncertain.The oxysterol-activated transcription factors liver X receptor͞ retinoid X receptor (LXR͞RXR) induce the expression of ABCA1, as well as a number of other molecules involved in cellular cholesterol efflux, transport, and excretion (9, 10). Treatment of macrophages with LXR activators increased net cholesterol efflux to HDL-2, suggesting the presence of unique LXR target genes mediating cholesterol efflux to HDL (11). Some ABCG family members are also LXR͞RXR targets, such as ABCG5 and ABCG8, the defective genes in sitosterolemia (12)(13)(14). ABCG family members are half-transporters, largely of unknown function. These considerations led us to investigate the ...

The protein deacetylase, sirtuin 1 (SIRT1), is a proposed master regulator of exercise-induced mitochondrial biogenesis in skeletal muscle, primarily via its ability to deacetylate and activate peroxisome proliferator-activated receptor-␥ coactivator-1␣ (PGC-1␣). To investigate regulation of mitochondrial biogenesis by SIRT1 in vivo, we generated mice lacking SIRT1 deacetylase activity in skeletal muscle (mKO). We hypothesized that deacetylation of PGC-1␣ and mitochondrial biogenesis in sedentary mice and after endurance exercise would be impaired in mKO mice. Skeletal muscle contractile characteristics were determined in extensor digitorum longus muscle ex vivo. Mitochondrial biogenesis was assessed after 20 days of voluntary wheel running by measuring electron transport chain protein content, enzyme activity, and mitochondrial DNA expression. PGC-1␣ expression, nuclear localization, acetylation, and interacting protein association were determined following an acute bout of treadmill exercise (AEX) using co-immunoprecipitation and immunoblotting. Contrary to our hypothesis, skeletal muscle endurance, electron transport chain activity, and voluntary wheel running-induced mitochondrial biogenesis were not impaired in mKO versus wild-type (WT) mice. Moreover, PGC-1␣ expression, nuclear translocation, activity, and deacetylation after AEX were similar in mKO versus WT mice. Alternatively, we made the novel observation that deacetylation of PGC-1␣ after AEX occurs in parallel with reduced nuclear abundance of the acetyltransferase, general control of amino-acid synthesis 5 (GCN5), as well as reduced association between GCN5 and nuclear PGC-1␣. These findings demonstrate that SIRT1 deacetylase activity is not required for exercise-induced deacetylation of PGC-1␣ or mitochondrial biogenesis in skeletal muscle and suggest that changes in GCN5 acetyltransferase activity may be an important regulator of PGC-1␣ activity after exercise.Impaired mitochondrial function has been linked with physical inactivity, insulin resistance, and the pathogenesis of type 2 diabetes (1, 2). Importantly, aerobic exercise training increases mitochondrial biogenesis and oxidative capacity in skeletal muscle (3-7). The precise mechanisms, however, linking muscle contraction to mitochondrial plasticity are incompletely defined (8). Clearly, allosteric factors (e.g. Ca 2ϩ , AMP, NAD ϩ , P i ) that are increased during contraction are important because they activate signaling pathways that ultimately converge at the transcriptional co-activator, peroxisome proliferator-activated receptor-␥ coactivator 1-␣ (PGC-1␣) 2 (9 -12). Once active, PGC-1␣ targets an array of transcription factors and nuclear receptors to coordinate gene expression of nuclear and mitochondrial-encoded genes (13, 14). However, how perturbations in cellular energy stress are sensed during exercise and subsequently how this signal is transduced to regulate mitochondrial biogenesis in a coordinated manner are still under intense investigation.In recent years, lysine acetylation...

ATP-binding cassette transporter 1 (ABCA1), the defective transporter in Tangier disease, binds and promotes cellular cholesterol and phospholipid efflux to apolipoprotein I (apoA-I). Based on a high degree of sequence homology between ABCA1 and ABCA7, a transporter of unknown function, we investigated the possibility that ABCA7 might be involved in apolipoprotein binding and lipid efflux. Similarly to cells expressing ABCA1, HEK293 cells overexpressing ABCA7 showed specific binding and cross-linking of lipid-poor apoA-I. ABCA7 expression increased cellular phosphatidylcholine and sphingomyelin efflux to apoA-I in a manner similar to ABCA1 but had no effect on cholesterol efflux. Western analysis showed a high protein level of ABCA7 in mouse spleen, lung, adrenal, and brain but low expression in liver. In contrast to ABCA1, ABCA7 showed moderate basal mRNA and protein levels in macrophages and lymphocytes but no induction by liver X receptor activation. These studies show that ABCA7 has the ability to bind apolipoproteins and promote efflux of cellular phospholipids without cholesterol, and they suggest a possible role of ABCA7 in cellular phospholipid metabolism in peripheral tissues.

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Although many organic anion transport protein (Oatp) family members have PDZ consensus binding sites at their C termini, the functional significance is unknown. In the present study, we utilized rat Oatp1a1 (NM_017111) as a prototypical member of this family to examine the mechanism governing its subcellular trafficking. A peptide corresponding to the C-terminal 16 amino acids of rat Oatp1a1 was used to affinity-isolate interacting proteins from rat liver cytosol. Protein mass fingerprinting identified PDZK1 as the major interacting protein. This was confirmed by immunoprecipitation of an Oatp1a1-PDZK1 complex from cotransfected 293T cells as well as from native rat liver membrane extracts. Oatp1a1 bound predominantly to the first and third PDZ binding domains of PDZK1, whereas the high density lipoprotein receptor, scavenger receptor B type I binds to the first domain. Although it is possible that PDZK1 forms a complex with these two integral membrane proteins, this did not occur, suggesting that as yet undescribed factors lead to selectivity in the interaction of these protein ligands with PDZK1. Oatp1a1 protein expression was near normal in PDZK1 knock-out mouse liver. However, it was located predominantly in intracellular structures, in contrast to its normal basolateral plasma membrane distribution. Plasma disappearance of the Oatp1a1 ligand [35S]sulfobromophthalein was correspondingly delayed in knock-out mice. These studies show a critical role for oligomerization of Oatp1a1 with PDZK1 for its proper subcellular localization and function. Because its ability to transport substances into the cell requires surface expression, this must be considered in any assessment of physiologic function.