Chromatin regulator SMARCAL1 modulates cellular lipid metabolism.

Biallelic mutations of the chromatin regulator SMARCAL1 cause Schimke Immunoosseous Dysplasia (SIOD), characterized by severe growth defects and premature mortality. Atherosclerosis and hyperlipidemia are common among SIOD patients, yet their onset and progression are poorly understood. Using an integrative approach involving proteomics, mouse models, and population genetics, we investigated SMARCAL1's role. We found that SmarcAL1 interacts with angiopoietin-like 3 (Angptl3), a key regulator of lipoprotein metabolism. In vitro and in vivo analyses demonstrate SmarcAL1's vital role in maintaining cellular lipid homeostasis. The observed translocation of SmarcAL1 to cytoplasmic peroxisomes suggests a potential regulatory role in lipid metabolism through gene expression. SmarcAL1 gene inactivation reduces the expression of key genes in cellular lipid catabolism. Population genetics investigations highlight significant associations between SMARCAL1 genetic variations and body mass index, along with lipid-related traits. This study underscores SMARCAL1's pivotal role in cellular lipid metabolism, likely contributing to the observed lipid phenotypes in SIOD patients.

Specificity of ABCA7-mediated cell lipid efflux.

Adenosine triphosphate-binding cassette transporter subfamily A member 7 (ABCA7) performs incompletely understood biochemical functions that affect pathogenesis of Alzheimer's disease. ABCA7 is most similar in primary structure to ABCA1, the protein that mediates cell lipid efflux and formation of high-density lipoprotein (HDL). Lipid metabolic labeling/tracer efflux assays were employed to investigate lipid efflux in BHK-ABCA7(low expression), BHK-ABCA7(high expression) and BHK-ABCA1 cells. Shotgun lipid mass spectrometry was used to determine lipid composition of HDL synthesized by BHK-ABCA7 and BHK-ABCA1 cells. BHK-ABCA7(low) cells exhibited significant efflux only of choline-phospholipid and phosphatidylinositol. BHK-ABCA7(high) cells had significant cholesterol and choline-phospholipid efflux to apolipoprotein (apo) A-I, apo E, the 18A peptide, HDL, plasma and cerebrospinal fluid and significant efflux of sphingosine-lipid, serine-lipid (which is composed of phosphatidylserine and phosphatidylethanolamine in BHK cells) and phosphatidylinositol to apo A-I. In efflux assays to apo A-I, after adjustment to choline-phospholipid, ABCA7-mediated efflux removed ~4 times more serine-lipid and phosphatidylinositol than ABCA1-mediated efflux, while ABCA1-mediated efflux removed ~3 times more cholesterol than ABCA7-mediated efflux. Shotgun lipidomic analysis revealed that ABCA7-HDL had ~20 mol% less phosphatidylcholine and 3-5 times more serine-lipid and phosphatidylinositol than ABCA1-HDL, while ABCA1-HDL contained only ~6 mol% (or ~1.1 times) more cholesterol than ABCA7-HDL. The discrepancy between the tracer efflux assays and shotgun lipidomics with respect to cholesterol may be explained by an underestimate of ABCA7-mediated cholesterol efflux in the former approach. Overall, these results suggest that ABCA7 lacks specificity for phosphatidylcholine and releases significantly but not dramatically less cholesterol in comparison with ABCA1.

A novel approach to measuring macrophage-specific reverse cholesterol transport in vivo in humans.

Reverse cholesterol transport (RCT) is thought to be an atheroprotective function of HDL, and macrophage-specific RCT in mice is inversely associated with atherosclerosis. We developed a novel method using 3H-cholesterol nanoparticles to selectively trace macrophage-specific RCT in vivo in humans. Use of 3H-cholesterol nanoparticles was initially tested in mice to assess the distribution of tracer and response to interventions known to increase RCT. Thirty healthy subjects received 3H-cholesterol nanoparticles intravenously, followed by blood and stool sample collection. Tracer counts were assessed in plasma, nonHDL, HDL, and fecal fractions. Data were analyzed by using multicompartmental modeling. Administration of 3H-cholesterol nanoparticles preferentially labeled macrophages of the reticuloendothelial system in mice, and counts were increased in mice treated with a liver X receptor agonist or reconstituted HDL, as compared with controls. In humans, tracer disappeared from plasma rapidly after injection of nanoparticles, followed by reappearance in HDL and nonHDL fractions. Counts present as free cholesterol increased rapidly and linearly in the first 240 min after nadir; counts in cholesteryl ester increased steadily over time. Estimates of fractional transfer rates of key RCT steps were obtained. These results support the use of 3H-cholesterol nanoparticles as a feasible approach for the measurement of macrophage RCT in vivo in humans.

Design and synthesis of boronic acid inhibitors of endothelial lipase.

Endothelial lipase (EL) and lipoprotein lipase (LPL) are homologous lipases that act on plasma lipoproteins. EL is predominantly a phospholipase and appears to be a key regulator of plasma HDL-C. LPL is mainly a triglyceride lipase regulating (V)LDL levels. The existing biological data indicate that inhibitors selective for EL over LPL should have anti-atherogenic activity, mainly through increasing plasma HDL-C levels. We report here the synthesis of alkyl, aryl, or acyl-substituted phenylboronic acids that inhibit EL. Many of the inhibitors evaluated proved to be nearly equally potent against both EL and LPL, but several exhibited moderate to good selectivity for EL.

Gene therapy in a humanized mouse model of familial hypercholesterolemia leads to marked regression of atherosclerosis.

BACKGROUND: Familial hypercholesterolemia (FH) is an autosomal codominant disorder caused by mutations in the low-density lipoprotein receptor (LDLR) gene. Homozygous FH patients (hoFH) have severe hypercholesterolemia leading to life threatening atherosclerosis in childhood and adolescence. Mice with germ line interruptions in the Ldlr and Apobec1 genes (Ldlr(-/-)Apobec1(-/-)) simulate metabolic and clinical aspects of hoFH, including atherogenesis on a chow diet. METHODS/PRINCIPAL FINDINGS: In this study, vectors based on adeno-associated virus 8 (AAV8) were used to deliver the gene for mouse Ldlr (mLDLR) to the livers of Ldlr(-/-)Apobec1(-/-) mice. A single intravenous injection of AAV8.mLDLR was found to significantly reduce plasma cholesterol and non-HDL cholesterol levels in chow-fed animals at doses as low as 3×10(9) genome copies/mouse. Whereas Ldlr(-/-)Apobec1(-/-) mice fed a western-type diet and injected with a control AAV8.null vector experienced a further 65% progression in atherosclerosis over 2 months compared with baseline mice, Ldlr(-/-)Apobec1(-/-) mice treated with AAV8.mLDLR realized an 87% regression of atherosclerotic lesions after 3 months compared to baseline mice. Immunohistochemical analyses revealed a substantial remodeling of atherosclerotic lesions. CONCLUSIONS/SIGNIFICANCE: Collectively, the results presented herein suggest that AAV8-based gene therapy for FH may be feasible and support further development of this approach. The pre-clinical data from these studies will enable for the effective translation of gene therapy into the clinic for treatment of FH.

Knockdown of acyl-CoA:diacylglycerol acyltransferase 2 with antisense oligonucleotide reduces VLDL TG and ApoB secretion in mice.

Acyl-CoA:diacylglycerol acyltransferases (DGATs) are enzymes that catalyze the formation of triglyceride (TG) from acyl-CoA and diacylglycerol. Two DGATs have been identified which belong to two distinct gene families and both are ubiquitously expressed. DGAT2 knockout mice are lipopenic and die shortly after birth. In the current study, wild type mice were treated with increasing doses (25-60 mg/kg twice weekly) of a DGAT2 gene-specific antisense oligonucleotide (ASO). Treatment resulted in a dose dependent decrease in hepatic DGAT2 gene expression (up to 80%) which was associated with a 40% decrease in hepatic DGAT2 activity and a 45% decrease in hepatic TG. Decreased levels of DGAT2 resulted in a significant dose dependent decrease in VLDL TG secretion (up to 52%) and reduced plasma TG, total cholesterol, and ApoB. Similar results were obtained when DGAT1 KO mice were treated with the DGAT2 ASO. Treatment of ob/ob mice with the DGAT2 ASO resulted in significant decreases in weight gain (10%), adipose weight (25%) and hepatic TG content (80%). Our findings indicate that the majority of TG destined for secretion by liver is synthesized by DGAT2 and suggests that DGAT2 may be a therapeutic target for treatment of hypertriglyceridemia, hepatic steatosis and obesity.

Acyl coenzyme A dependent retinol esterification by acyl coenzyme A: diacylglycerol acyltransferase 1.

We provide biochemical evidence that enzymes involved in the synthesis of triacylglycerol, namely acyl coenzyme A:diacylglycerol acyltransferase (DGAT) and acyl coenzyme A:monoacylglycerol acyltransferase (MGAT), are capable of carrying out the acyl coenzyme A:retinol acyltransferase (ARAT) reaction. Among them, DGAT1 appears to have the highest specific activity. The apparent K(m) values of recombinant DGAT1/ARAT for retinol and palmitoyl coenzyme A were determined to be 25.9+/-2.1 microM and 13.9+/-0.3 microM, respectively, both of which are similar to the values previously determined for ARAT in native tissues. A novel selective DGAT1 inhibitor, XP620, inhibits recombinant DGAT1/ARAT at the retinol recognition site. In the differentiated Caco-2 cell membranes, XP620 inhibits approximately 85% of the Caco-2/ARAT activity indicating that DGAT1/ARAT may be the major source of ARAT activity in these cells. Of the two most abundant fatty acyl retinyl esters present in the intact differentiated Caco-2 cells, XP620 selectively inhibits retinyl-oleate formation without influencing the retinyl-palmitate formation. Using this inhibitor, we estimate that approximately 64% of total retinyl ester formation occurs via DGAT1/ARAT. These studies suggest that DGAT1/ARAT is the major enzyme involved in retinyl ester synthesis in Caco-2 cells.

Determining hepatic triglyceride production in mice: comparison of poloxamer 407 with Triton WR-1339.

Triglyceride (TG), a water-insoluble energy-rich lipid, is secreted by the liver as part of very low density lipoproteins (VLDLs) to supply energy to extrahepatic tissues. Overproduction of VLDL is associated with increased risk of cardiovascular heart disease; this has renewed an interest in factors that affect hepatic TG production. The TG production rate is determined by measuring temporal increases in plasma TG under conditions in which TG hydrolysis by lipoprotein lipase (LPL) is inhibited. The nonionic detergent, Triton WR-1339 (Triton), has commonly been used to inhibit LPL for this purpose. Triton, in addition to inhibition of TG hydrolysis, has properties that have the potential to adversely influence lipoprotein metabolism. Another nonionic detergent, poloxamer 407 (P-407), also inhibits LPL. In these studies, we demonstrate that P-407 is comparable to Triton in the determination of TG production but without the unwanted side effects of Triton.

Identification of two novel human acyl-CoA wax alcohol acyltransferases: members of the diacylglycerol acyltransferase 2 (DGAT2) gene superfamily.

The esterification of alcohols such as sterols, diacylglycerols, and monoacylglycerols with fatty acids represents the formation of both storage and cytoprotective molecules. Conversely, the overproduction of these molecules is associated with several disease pathologies, including atherosclerosis and obesity. The human acyl-CoA:diacylglycerol acyltransferase (DGAT) 2 gene superfamily comprises seven members, four of which have been previously implicated in the synthesis of di- or triacylglycerol. The remaining 3 members comprise an X-linked locus and have not been characterized. We describe here the expression of DGAT2 and the three X-linked genes in Saccharomyces cerevisiae strains virtually devoid of neutral lipids. All four gene products mediate the synthesis of triacylglycerol; however, two of the X-linked genes act as acyl-CoA wax alcohol acyltransferases (AWAT 1 and 2) that predominantly esterify long chain (wax) alcohols with acyl-CoA-derived fatty acids to produce wax esters. AWAT1 and AWAT2 have very distinct substrate preferences in terms of alcohol chain length and fatty acyl saturation. The enzymes are expressed in many human tissues but predominate in skin. In situ hybridizations demonstrate a differentiation-specific expression pattern within the human sebaceous gland for the two AWAT genes, consistent with a significant role in the composition of sebum.

Raising HDL cholesterol without inducing hepatic steatosis and hypertriglyceridemia by a selective LXR modulator.

Liver X receptors (LXRs) are ligand-activated transcription factors that belong to the nuclear receptor superfamily. LXRs activate transcription of a spectrum of genes that regulate reverse cholesterol transport, including the ATP binding cassette transporter A1 (ABCA1), and raise HDL cholesterol (HDL-C) levels. However, LXR agonists also induce genes that stimulate lipogenesis, including the sterol response element binding protein (SREBP1-c) and fatty acid synthetase (FAS). The induction of these genes in the liver cause increased hepatic triglyceride synthesis, hypertriglyceridemia, and hepatic steatosis. As LXR response elements have been identified in these promoters, it is not clear if these two processes can be separated. Herein, we demonstrate that plasma HDL-C elevation and intestinal ABCA1 induction can occur with relatively little induction of FAS and SREBP1-c in mouse liver via a selective LXR modulator GW3965. This is in contrast to the strong induction of hepatic lipogenic genes by the well-characterized LXR agonist T0901317 (T317). Consistent with the in vivo results, GW3965 is a very weak LXR activator compared with T317 in human hepatoma cells. GW3965-liganded LXR recruits selected coactivators less effectively than T317 and may explain in part the tissue selective gene induction. This demonstration that tissue and gene selective modulation is possible with selective LXR modulators has positive implications for the development of this class of antiatherosclerotic agents.

Prospective testing for drug-dependent antibodies reduces the incidence of thrombocytopenia observed with the small molecule glycoprotein IIb/IIIa antagonist roxifiban: implications for the etiology of thrombocytopenia.

Thrombocytopenia is a relatively common side effect observed during glycoprotein (GP) IIb/IIIa antagonist therapy. With the oral antagonist roxifiban, we observed thrombocytopenia, defined as 50% reduction of platelets over predose values or below 90 000/microL (9 x 10(10)/L), with a frequency of 2% (8 of 386). Thrombocytopenia occurred either early (days 2 to 4) or delayed (days 11 to 16). No additional cases were observed with up to 6 months of treatment. Retrospective analysis provided evidence for drug-dependent antibodies (DDABs) to GP IIb/IIIa in 5 of 6 subjects, suggestive of an immune etiology of thrombocytopenia. The hypothesis that excluding patients based on positive DDAB reaction would reduce the frequency of thrombocytopenia was tested. Patients were screened for DDABs during the study qualification period and, overall, 3.9% of the patients were excluded based on pre-existing DDAB concentrations above a statistically defined medical decision limit. An additional 2.6% were excluded based on therapy-related antibody production during the first 2 weeks. With antibody testing, 0.2% of patients (2 of 1044) developed immune-mediated thrombocytopenia. One case developed a rapidly increasing antibody concentration and presented with thrombocytopenia despite discontinuation of roxifiban therapy. The second case was related to a false-negative test result. The frequency of thrombocytopenia was statistically significantly reduced from 2% to 0.2% (P =.0007) comparing nonscreened and screened patients. Testing for DDABs can reduce the frequency of thrombocytopenia in patients treated with roxifiban and, by analogy, other GP IIb/IIIa antagonists. Thus, DDAB testing may be employed to increase the safety of GP IIb/IIIa antagonists.

Evidence that thrombocytopenia observed in humans treated with orally bioavailable glycoprotein IIb/IIIa antagonists is immune mediated.

Glycoprotein (GP) IIb/IIIa antagonists are effective therapeutic agents, but elicit thrombocytopenia with a frequency that approaches 2%. Here, we provide evidence that thrombocytopenia in humans treated with the GP IIb/IIIa antagonist roxifiban is immune mediated. Two patients underwent conversion to a highly positive drug-dependent antibody (DDAB) status temporally associated with thrombocytopenia. Despite the continued presence of DDABs, the fall in platelet count was reversed by discontinuation of drug treatment, pointing to the exquisite drug dependency of the immune response. DDABs appear to bind to neoepitopes in GP IIb/IIIa elicited on antagonist binding. This information was used to develop an enzyme-linked immunosorbent assay (ELISA) for DDAB using solid-phase GP IIb/IIIa. A high level of specificity is indicated by the observation that DDAB binding is dependent on the chemical structure of the GP IIb/IIIa antagonist and that only 2% to 5% of human blood donors and 5% of chimpanzees present with pre-existing DDABs. Furthermore, none of 108 nonthrombocytopenic patients from the phase II roxifiban study showed an increase in antibody titer. Absorption of thrombocytopenia plasma with platelets reduced the DDAB ELISA signal, indicating that the test detects physiologically relevant antibodies. Screening patients for pre-existing or increasing DDAB titer during treatment with GP IIb/IIIa antagonists may reduce the incidence of drug-induced thrombocytopenia.

The DGA1 gene determines a second triglyceride synthetic pathway in yeast.

Diacylglycerol esterification provides an excellent target for the pharmacological reduction of triglyceride accumulation in several human disease states. We have used Saccharomyces cerevisiae as a model system to study this critical component of triglyceride synthesis. Recent studies of an oleaginous fungus, Mortierella ramanniana, identified a new family of enzymes with in vitro acyl-CoA:diacylglycerol acyltransferase activity. We show here that DGA1, the sole member of this gene family in yeast, has a physiological role in triglyceride synthesis. Metabolic labeling of DGA1 deletion strains with triglyceride precursors detected significant reductions in triglyceride synthesis. Triglyceride synthesis was virtually abolished in four different growth conditions when DGA1 was deleted in concert with LRO1, an enzyme that esterifies diacylglycerol from a phospholipid acyl donor. The relative contributions of the two enzymes depended on growth conditions. The residual synthesis was lost when ARE2, encoding an acyl-CoA:sterol acyltransferase, was deleted. In vitro microsomal assays verified that DGA1 and ARE2 mediate acyl-CoA:diacylglycerol acyltransferase reactions. Three enzymes can thus account for diacylglycerol esterification in yeast. Yeast strains deficient in both diacylglycerol and sterol esterification showed only a slight growth defect indicating that neutral lipid synthesis is dispensable under common laboratory conditions.