ABCA1 mediates release of cellular cholesterol and phospholipid to form high density lipoprotein (HDL).The three different mutants in the first extracellular domain of human ABCA1 associated with Tangier disease, R587W, W590S, and Q597R, were examined for their subcellular localization and function by using ABCA1-GFP fusion protein stably expressed in HEK293 cells. ABCA1-GFP expressed in HEK293 was fully functional for apoA-I-mediated HDL assembly. Immunostaining and confocal microscopic analyses demonstrated that ABCA1-GFP was mainly localized to the plasma membrane (PM) but also substantially in intracellular compartments. All three mutant ABCA1-GFPs showed no or little apoA-I-mediated HDL assembly. R587W and Q597R were associated with impaired processing of oligosaccharide from high mannose type to complex type and failed to be localized to the PM, whereas W590S did not show such dysfunctions. Vanadate-induced nucleotide trapping was examined to elucidate the mechanism for the dysfunction in the W590S mutant. Photoaffinity labeling of W590S with 8-azido-[␣-32 P]ATP was stimulated by adding ortho-vanadate in the presence of Mn 2؉ as much as in the presence of wildtype ABCA1. These results suggest that the defect of HDL assembly in R587W and Q597R is due to the impaired localization to the PM, whereas W590S has a functional defect other than the initial ATP binding and hydrolysis.Cholesterol is not catabolized in the peripheral cells and therefore mostly released and transported to the liver for conversion to bile acids to maintain cholesterol homeostasis. The same pathway may also remove cholesterol that has pathologically accumulated in the cells such as an initial stage of atherosclerosis. Assembly of high density lipoprotein (HDL) 1 particles by helical apolipoproteins with cellular lipid has been recognized as one of the major mechanisms for cellular cholesterol release (1, 2). The importance of this active cholesterolreleasing pathway in regulating cholesterol homeostasis became apparent by the finding that it is impaired in the cells from patients with Tangier disease, a genetic deficiency of circulating HDL (3, 4). Mutations were identified in ATP-binding cassette transporter A1 (ABCA1) of the Tangier disease (TD) patients (5-7), but the molecular mechanism of ABCA1 in the apolipoprotein-mediated HDL assembly remains unclear. Although direct interaction between ABCA1 and apoA-I at the cell surface has been suggested on the basis of chemical crosslinking experiments (8, 9), an indirect role of ABCA1 in the apoA-I binding to the cell was also proposed by a model that ABCA1 induces phosphatidylserine exofacial flopping to generate the microenvironment required for the docking of apoA-I at the cell surface (10). The predominant substrates of the ABCA1-mediated lipid release reaction are still to be determined for the HDL assembly reaction (11, 12). More than 30 mutations have been mapped in the ABCA1 gene in patients with familial hypoalphalipoproteinemia (FHA) and TD (5-7, 13-15). Many mutations have been ...
Cholesterol is an essential component of eukaryotic cells; at the same time, however, hyperaccumulation of cholesterol is harmful. Therefore, the ABCA1 gene, the product of which mediates secretion of cholesterol, is highly regulated at both the transcriptional and post-transcriptional levels. The transcription of ABCA1 is regulated by intracellular oxysterol concentration via the nuclear liver X receptor (LXR)/retinoid X receptor (RXR); once synthesized, ABCA1 protein turns over rapidly with a half-life of 1-2 h. Here, we show that the LXR/RXR complex binds directly to ABCA1 on the plasma membrane of macrophages and modulates cholesterol secretion. When cholesterol does not accumulate, ABCA1-LXR/RXR localizes on the plasma membrane, but is inert. When cholesterol accumulates, oxysterols bind to LXR, and the LXR/RXR complex dissociates from ABCA1, restoring ABCA1 activity and allowing apoA-I-dependent cholesterol secretion. LXR can exert an immediate post-translational response, as well as a rather slow transcriptional response, to changes in cellular cholesterol accumulation. Thus, we provide the first demonstration that protein-protein interaction suppresses ABCA1 function. Furthermore, we show that LXR is involved in both the transcriptional and post-transcriptional regulation of the ABCA1 transporter.Maintenance of cellular cholesterol homeostasis is important for normal human physiology. Disruption of cellular cholesterol homeostasis leads to a variety of pathological conditions, including cardiovascular disease (1). ABCA1 (ATPbinding cassette protein A1), one of the key proteins in cholesterol homeostasis, mediates secretion of cellular free cholesterol and phospholipids to an extracellular acceptor in the plasma, apoA-I, to form high density lipoprotein (HDL) 3 (2, 3). HDL formation is the only known pathway that can eliminate excess cholesterol from peripheral cells. Defects in ABCA1 cause Tangier disease (4 -6), in which patients have a near absence of circulating HDL, prominent cholesterol ester accumulation in tissue macrophages, and premature atherosclerotic vascular disease (1, 7).ABCA1-mediated cholesterol efflux is highly regulated at both the transcriptional and post-transcriptional levels. When cholesterol accumulates in cells, intracellular concentrations of oxysterols increase; subsequently, the liver X receptor (LXR), activated via binding of oxysterols, stimulates the transcription of ABCA1 (8 -10). ABCA1 protein eliminates excess cellular cholesterol and turns over rapidly with a half-life of 1-2 h (11-15). Several proteins, including apoA-I, ␣1-syntrophin, and 1-syntrophin, have been reported to interact with ABCA1 and reduce the rate of ABCA1 protein degradation (13-16). Syntrophins play critical roles in regulating the apoA-I-dependent cholesterol efflux (and thus in lipid homeostasis) by suppressing protein degradation in brain (14) and liver (15). Because cholesterol is an essential component of cells, however, excessive elimination of cholesterol could result in cell death. Con...
Acetyl coenzyme A (acetyl-CoA) carboxylase (ACC) catalyzes carboxylation of acetyl-CoA to form malonylCoA. In mammals, two isozymes exist with distinct physiological roles: cytosolic ACC1 participates in de novo lipogenesis (DNL), and mitochondrial ACC2 is involved in negative regulation of mitochondrial -oxidation. Since systemic ACC1 null mice were embryonic lethal, to clarify the physiological role of ACC1 in hepatic DNL, we generated the liver-specific ACC1 null mouse by crossbreeding of an Acc1 lox(ex46) mouse, in which exon 46 of Acc1 was flanked by two loxP sequences and the liver-specific Cre transgenic mouse. In liver-specific ACC1 null mice, neither hepatic Acc1 mRNA nor protein was detected. However, to compensate for ACC1 function, hepatic ACC2 protein and activity were induced 1.4 and 2.2 times, respectively. Surprisingly, hepatic DNL and malonyl-CoA were maintained at the same physiological levels as in wild-type mice. Furthermore, hepatic DNL was completely inhibited by an ACC1/2 dual inhibitor, 5-tetradecyloxyl-2-furancarboxylic acid. These results strongly demonstrate that malonyl-CoA from ACC2 can access fatty acid synthase and become the substrate for the DNL pathway under the unphysiological circumstances that result with ACC1 disruption. Therefore, there does not appear to be strict compartmentalization of malonyl-CoA from either of the ACC isozymes in the liver.Acetyl coenzyme A (acetyl-CoA) carboxylase (ACC) catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, which is a key molecule in the control of intracellular fatty acid metabolism (13,16,27). ACC has two major isozymes that have different physiological roles based on their distinct subcellular distributions (2). A cytosolic enzyme, ACC1 (ACC␣; molecular mass, 265 kDa), supplies malonyl-CoA to fatty acid synthase (FAS) and is committed to de novo lipogenesis (DNL) in many tissues via subsequent nutritional and hormonal regulation (3,16,27). In contrast, ACC2 (ACC; molecular mass, 280 kDa) is anchored to the mitochondrial surface via a unique N-terminal domain that includes 20 hydrophobic amino acids (1, 2). ACC2 produces malonyl-CoA on the mitochondrial surface. It is well known that malonylCoA is a potent endogenous inhibitor of carnitine palmitoyl transferase 1 (CPT-1), which is also located on the mitochondrial surface (21, 26). Thus, ACC2 indirectly prevents the influx of fatty acids into the mitochondria and their subsequent -oxidation (4).ACC1 is ubiquitously expressed in many tissues, but higher levels occur in lipogenic tissues, including the liver and adipose tissue (8). In fact, in animals, Acc1 gene expression and ACC activity are markedly induced either by high carbohydrate feeding or hyperinsulinemia in animals and result in increases in adiposity, lipoprotein secretion, and hepatic fat content (16). It is expected that ACC1 blockade should reduce flux through the DNL pathway in lipogenic tissues and thus reduce adiposity, lipoprotein secretion, and fatty liver (11,23). It is therefore plausible that ACC1 inh...
Aims/IntroductionTo evaluate the incidence rate of and identify factors associated with severe hypoglycemic episodes in patients with treated type 2 diabetes mellitus.Materials and MethodsUsing Diagnosis Procedure Combination hospital‐based medical database, we carried out a retrospective cohort study to assess the incidence rate of severe hypoglycemia in treated type 2 diabetes mellitus patients. We evaluated the associations between severe hypoglycemia and age, sex, complications, and current use of insulin or sulfonylurea (SU) in a nested case–control study.ResultsOf 166,806 eligible patients, 1,242 had episodes of severe hypoglycemia during the observational period. The incidence rate of the first hypoglycemic events was 3.70/1,000 patient years. Based on the nested case–control analysis, age was associated with hypoglycemic events with adjusted odds ratios (ORs) of 1.64 or 65–74‐year‐old patients and 3.79 for ≥75‐year‐old patients in comparison with 20–64‐year‐old patients. Comorbidities, such as cognitive impairment, cancer, macrovascular disease and diabetic complications (retinopathy, nephropathy and neuropathy), were associated with severe hypoglycemia, with adjusted ORs ranging from 1.25 to 3.80. Severe hypoglycemic events also increased in patients with current use of both SU and insulin, either SU or insulin, with adjusted ORs of 18.36, 6.31 or 14.07, respectively, compared with patients with other antihyperglycemic agents. In patients with an SU glimepiride, adjusted ORs increased dose‐dependently from 3.65 (≤1 mg) to 13.34 (>2 mg).ConclusionsThe incidence rate of severe hypoglycemia in this cohort was 3.70/1,000 patient years. Age, cognitive impairment, cancer, diabetic complications, current use of insulin + SU and SU dosage were identified as risk factors for severe hypoglycemia.
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