Functional LCAT deficiency in human apolipoprotein A-I transgenic, SR-BI knockout mice

Reduction of plasma LCAT activity has been observed in several conditions in which the size of HDL particles is increased; however, the mechanism of this reduction remains elusive. We investigated the plasma activity, mass, and in vivo catabolism of LCAT and its association with HDL particles in human apolipoprotein A-I transgenic, scavenger receptor class B type I knockout (hA-ITg SR-BI-/-) mice. Compared with hA-ITg mice, hA-ITg SR-BI-/- mice had a 4-fold higher total plasma cholesterol concentration, which occurred predominantly in 13-18 nm diameter HDL particles, a significant reduction in plasma esterified cholesterol-total cholesterol (EC/TC) ratio, and significantly lower plasma LCAT activity, suggesting a decrease in LCAT protein. However, LCAT protein in plasma, hepatic mRNA for LCAT, and in vivo turnover of 35S-radiolabeled LCAT were similar in both genotypes of mice. HDL from hA-ITg SR-BI-/- mice was enriched in sphingomyelin (SM), relative to phosphatidylcholine, and had less associated [35S]LCAT radiolabel and endogenous LCAT activity compared with HDL from hA-ITg mice. We conclude that the decreased EC/TC ratio in the plasma of hA-ITg SR-BI-/- mice is attributed to a reduction in LCAT reactivity with SM-enriched HDL particles.     

Initial interaction of apoA-I with ABCA1 impacts in vivo metabolic fate of nascent HDL

We investigated the in vivo metabolic fate of pre-beta HDL particles in human apolipoprotein A-I transgenic (hA-I (Tg)) mice. Pre-beta HDL tracers were assembled by incubation of [(125)I]tyramine cellobiose-labeled apolipoprotein A-I (apoA-I) with HEK293 cells expressing ABCA1. Radiolabeled pre-beta HDLs of increasing size (pre-beta1, -2, -3, and -4 HDLs) were isolated by fast-protein liquid chromatography and injected into hA-I (Tg)-recipient mice, after which plasma decay, in vivo remodeling, and tissue uptake were monitored. Pre-beta2, -3, and -4 had similar plasma die-away rates, whereas pre-beta1 HDL was removed 7-fold more rapidly. Radiolabel recovered in liver and kidney 24 h after tracer injection suggested increased (P < 0.001) liver and decreased kidney catabolism as pre-beta HDL size increased. In plasma, pre-beta1 and -2 were rapidly (<5 min) remodeled into larger HDLs, whereas pre-beta3 and -4 were remodeled into smaller HDLs. Pre-beta HDLs were similarly remodeled in vitro with control or LCAT-immunodepleted plasma, but not when incubated with phospholipid transfer protein knockout plasma. Our results suggest that initial interaction of apoA-I with ABCA1 imparts a unique conformation that partially determines the in vivo metabolic fate of apoA-I, resulting in increased liver and decreased kidney catabolism as pre-beta HDL particle size increases.     

Increased cellular free cholesterol in macrophage-specific Abca1 knock-out mice enhances pro-inflammatory response of macrophages

Macrophage-specific Abca1 knock-out (Abca1(-)(M)(/-)(M)) mice were generated to determine the role of macrophage ABCA1 expression in plasma lipoprotein concentrations and the innate immune response of macrophages. Plasma lipid and lipoprotein concentrations in chow-fed Abca1(-)(M)(/-)(M) and wild-type (WT) mice were indistinguishable. Compared with WT macrophages, Abca1(-)(M)(/-)(M) macrophages had a >95% reduction in ABCA1 protein, failed to efflux lipid to apoA-I, and had a significant increase in free cholesterol (FC) and membrane lipid rafts without induction of endoplasmic reticulum stress. Lipopolysaccharide (LPS)-treated Abca1(-)(M)(/-)(M) macrophages exhibited enhanced expression of pro-inflammatory cytokines and increased activation of the NF-kappaB and MAPK pathways, which could be diminished by silencing MyD88 or by chemical inhibition of NF-kappaB or MAPK. In vivo LPS injection also resulted in a higher pro-inflammatory response in Abca1(-)(M)(/-)(M) mice compared with WT mice. Furthermore, cholesterol depletion of macrophages with methyl-beta-cyclodextrin normalized FC content between the two genotypes and their response to LPS; cholesterol repletion of macrophages resulted in increased cellular FC accumulation and enhanced cellular response to LPS. Our results suggest that macrophage ABCA1 expression may protect against atherosclerosis by facilitating the net removal of excess lipid from macrophages and dampening pro-inflammatory MyD88-dependent signaling pathways by reduction of cell membrane FC and lipid raft content.     

Apolipoprotein M expression increases the size of nascent pre�� HDL formed by ATP binding cassette transporter A1

Apolipoprotein M (apoM) is a novel apolipoprotein that is reportedly necessary for preβ HDL formation; however, its detailed function remains unknown. We investigated the biogenesis and properties of apoM and its effects on the initial steps of nascent preβ HDL assembly by ABCA1 in HEK293 cells. Transiently transfected apoM was localized primarily in the endomembrane compartment. Pulse-chase analyses demonstrated that apoM is inefficiently secreted, relative to human serum albumin, and that ∼50% remains membrane-associated after extraction with sodium carbonate, pH 11.5. To investigate the role of apoM in nascent preβ HDL formation, ABCA1-expressing or control cells, transfected with empty vector, apoM, or C-terminal epitope-tagged apoM (apoM-C-FLAG), were incubated with ¹²⁵I-apoA-I for 24 h. Conditioned media were harvested and fractionated by fast-protein liquid chromatography (FPLC) to monitor HDL particle size. Preβ HDL particles were formed effectively in the absence of apoM expression; however, increased apoM expression stimulated the formation of larger-sized nascent preβ HDLs. Immunoprecipitation with anti-apoA-I antibody followed by apoM Western blot analysis revealed that little secreted apoM was physically associated with preβ HDL. Our results suggest that apoM is an atypical secretory protein that is not necessary for ABCA1-dependent preβ HDL formation but does stimulate the formation of larger-sized preβ HDL. We propose that apoM may function catalytically at an intracellular site to transfer lipid onto preβ HDL during or after their formation by ABCA1.     

Uncleaved ApoM Signal Peptide Is Required for Formation of Large ApoM/Sphingosine 1-Phosphate (S1P)-enriched HDL Particles

Apolipoprotein M (apoM), a plasma sphingosine 1-phosphate (S1P) carrier, associates with plasma HDL via its uncleaved signal peptide. Hepatocyte-specific apoM overexpression in mice stimulates formation of both larger nascent HDL in hepatocytes and larger mature apoM/S1P-enriched HDL particles in plasma by enhancing hepatic S1P synthesis and secretion. Mutagenesis of apoM glutamine 22 to alanine (apoM^Q22A) introduces a functional signal peptidase cleavage site. Expression of apoM^Q22A in ABCA1-expressing HEK293 cells resulted in the formation of smaller nascent HDL particles compared with wild type apoM (apoM^WT). When apoM^Q22A was expressed in vivo, using recombinant adenoviruses, smaller plasma HDL particles and decreased plasma S1P and apoM were observed relative to expression of apoM^WT. Hepatocytes isolated from both apoM^WT- and apoM^Q22A-expressing mice displayed an equivalent increase in cellular levels of S1P, relative to LacZ controls; however, relative to apoM^WT, apoM^Q22A hepatocytes displayed more rapid apoM and S1P secretion but minimal apoM^Q22A bound to nascent lipoproteins. Pharmacologic inhibition of ceramide synthesis increased cellular sphingosine and S1P but not medium S1P in both apoM^WT and apoM^Q22A hepatocytes. We conclude that apoM secretion is rate-limiting for hepatocyte S1P secretion and that its uncleaved signal peptide delays apoM trafficking out of the cell, promoting formation of larger nascent apoM- and S1P-enriched HDL particles that are probably precursors of larger apoM/S1P-enriched plasma HDL.     

Hepatic Apolipoprotein M (ApoM) Overexpression Stimulates Formation of Larger ApoM/Sphingosine 1-Phosphate-enriched Plasma High Density Lipoprotein

Apolipoprotein M (apoM), a lipocalin family member, preferentially associates with plasma HDL and binds plasma sphingosine 1-phosphate (S1P), a signaling molecule active in immune homeostasis and endothelial barrier function. ApoM overexpression in ABCA1-expressing HEK293 cells stimulated larger nascent HDL formation, compared with cells that did not express apoM; however, the in vivo role of apoM in HDL metabolism remains poorly understood. To test whether hepatic apoM overexpression increases plasma HDL size, we generated hepatocyte-specific apoM transgenic (APOM Tg) mice, which had an ∼3–5-fold increase in plasma apoM levels compared with wild-type mice. Although HDL cholesterol concentrations were similar to wild-type mice, APOM Tg mice had larger plasma HDLs enriched in apoM, cholesteryl ester, lecithin:cholesterol acyltransferase, and S1P. Despite the presence of larger plasma HDLs in APOM Tg mice, in vivo macrophage reverse cholesterol transport capacity was similar to that in wild-type mice. APOM Tg mice had an ∼5-fold increase in plasma S1P, which was predominantly associated with larger plasma HDLs. Primary hepatocytes from APOM Tg mice generated larger nascent HDLs and displayed increased sphingolipid synthesis and S1P secretion. Inhibition of ceramide synthases in hepatocytes increased cellular S1P levels but not S1P secretion, suggesting that apoM is rate-limiting in the export of hepatocyte S1P. Our data indicate that hepatocyte-specific apoM overexpression generates larger nascent HDLs and larger plasma HDLs, which preferentially bind apoM and S1P, and stimulates S1P biosynthesis for secretion. The unique apoM/S1P-enriched plasma HDL may serve to deliver S1P to extrahepatic tissues for atheroprotection and may have other as yet unidentified functions.     

Myeloid cell-specific ABCA1 deletion does not worsen insulin resistance in HF diet-induced or genetically obese mouse models

Obesity-associated low-grade chronic inflammation plays an important role in the development of insulin resistance. The membrane lipid transporter ATP-binding cassette transporter A1 (ABCA1) promotes formation of nascent HDL particles. ABCA1 also dampens macrophage inflammation by reducing cellular membrane cholesterol and lipid raft content. We tested the hypothesis that myeloid-specific ABCA1 deletion may exacerbate insulin resistance by increasing the obesity-associated chronic low-grade inflammation. Myeloid cell-specific ABCA1 knockout (MSKO) and wild-type (WT) mice developed obesity, insulin resistance, mild hypercholesterolemia, and hepatic steatosis to a similar extent with a 45% high-fat (HF) diet feeding or after crossing into the ob/ob background. Resident peritoneal macrophages and stromal vascular cells from obese MSKO mice accumulated significantly more cholesterol. Relative to chow, HF diet markedly induced macrophage infiltration and inflammatory cytokine expression to a similar extent in adipose tissue of WT and MSKO mice. Among pro-inflammatory cytokines examined, only IL-6 was highly upregulated in MSKO-ob/ob versus ob/ob mouse peritoneal macrophages, indicating a nonsignificant effect of myeloid ABCA1 deficiency on obesity-associated chronic inflammation. In conclusion, myeloid-specific ABCA1 deficiency does not exacerbate obesity-associated low-grade chronic inflammation and has minimal impact on the pathogenesis of insulin resistance in both HF diet-induced and genetically obese mouse models.     

A molecular characterization of the charismatic Faroe house mouse

Faroe house mice are a ‘classic’ system of rapid and dramatic morphological divergence highlighted by J. S. Huxley during the development of the Modern Synthesis. In the present study, we characterize these charismatic mice using modern molecular techniques, examining specimens from all Faroe islands occupied by mice. The aims were to classify the mice within the modern house mouse taxonomy (i.e. as either Mus musculus domesticus or Mus musculus musculus) using four molecular markers and a morphological feature, and to examine the genetic diversity and possible routes of colonization using mitochondrial (mt) control region DNA sequences and microsatellite data (15 loci). Mice on the most remote islands were characterized as M. m. domesticus and exhibited exceptionally low genetic diversity, whereas those on better connected islands were more genetically diverse and had both M. m. musculus and M. m. domesticus genetic elements, including one population which was morphologically M. m. musculus‐like. The mtDNA data indicate that the majority of the mice had their origins in south‐western Norway (or possibly southern Denmark/northern Germany), and probably arrived with the Vikings, earlier than suggested by Huxley. The M. m. musculus genetic component appears to derive from recent mouse immigration from Denmark. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 471–482.     

Identification of structural DNA variations in human cell cultures after long-term passage

Random amplified polymorphic DNA (RAPD) analysis was adapted for genomic identification of cell cultures and evaluation of DNA stability in cells of different origin at different culture passages. DNA stability was observed in cultures after no more than 5 passages. Adipose-derived stromal cells demonstrated increased DNA instability. RAPD fragments from different cell lines after different number of passages were cloned and sequenced. The chromosomal localization of these fragments was identified and single-nucleotide variations in RAPD fragments isolated from cell lines after 8–12 passages were revealed. Some of them had permanent localization, while most variations demonstrated random distribution and can be considered as de novo mutations.