Targeted Deletion of Hepatocyte Abca1 Increases Plasma HDL (High-Density Lipoprotein) Reverse Cholesterol Transport via the LDL (Low-Density Lipoprotein) Receptor.

OBJECTIVE: The role of hepatocyte Abca1 (ATP binding cassette transporter A1) in trafficking hepatic free cholesterol (FC) into plasma versus bile for reverse cholesterol transport (RCT) is poorly understood. We hypothesized that hepatocyte Abca1 recycles plasma HDL-C (high-density lipoprotein cholesterol) taken up by the liver back into plasma, maintaining the plasma HDL-C pool, and decreasing HDL-mediated RCT into feces. Approach and Results: Chow-fed hepatocyte-specific Abca1 knockout (HSKO) and control mice were injected with human HDL radiolabeled with 125I-tyramine cellobiose (125I-TC; protein) and 3H-cholesteryl oleate (3H-CO). 125I-TC and 3H-CO plasma decay, plasma HDL 3H-CO selective clearance (ie, 3H-125I fractional catabolic rate), liver radiolabel uptake, and fecal 3H-sterol were significantly greater in HSKO versus control mice, supporting increased plasma HDL RCT. Twenty-four hours after 3H-CO-HDL injection, HSKO mice had reduced total hepatic 3H-FC (ie, 3H-CO hydrolyzed to 3H-FC in liver) resecretion into plasma, demonstrating Abca1 recycled HDL-derived hepatic 3H-FC back into plasma. Despite similar liver LDLr (low-density lipoprotein receptor) expression between genotypes, HSKO mice treated with LDLr-targeting versus control antisense oligonucleotide had slower plasma 3H-CO-HDL decay, reduced selective 3H-CO clearance, and decreased fecal 3H-sterol excretion that was indistinguishable from control mice. Increased RCT in HSKO mice was selective for 3H-CO-HDL, since macrophage RCT was similar between genotypes. CONCLUSIONS: Hepatocyte Abca1 deletion unmasks a novel and selective FC trafficking pathway that requires LDLr expression, accelerating plasma HDL-selective CE uptake by the liver and promoting HDL RCT into feces, consequently reducing HDL-derived hepatic FC recycling into plasma.

Targeted Deletion of Adipocyte Abca1 (ATP-Binding Cassette Transporter A1) Impairs Diet-Induced Obesity.

OBJECTIVE: Adipose tissue cholesterol increases with adipocyte triglyceride content and size during development of obesity. However, how adipocyte cholesterol affects adipocyte function is poorly understood. The aim of this study was to evaluate the role of the cellular cholesterol exporter, Abca1 (ATP-binding cassette transporter A1), on adipose tissue function during diet-induced obesity. APPROACH AND RESULTS: Adiponectin Cre recombinase transgenic mice were crossed with Abca1flox/flox mice to generate ASKO (adipocyte-specific Abca1 knockout) mice. Control and ASKO mice were then fed a high-fat, high-cholesterol (45% calories as fat and 0.2% cholesterol) diet for 16 weeks. Compared with control mice, ASKO mice had a 2-fold increase in adipocyte plasma membrane cholesterol content and significantly lower body weight, epididymal fat pad weight, and adipocyte size. ASKO versus control adipose tissue had decreased PPARγ (peroxisome proliferator-activated receptor γ) and CCAAT/enhancer-binding protein expression, nuclear SREBP1 (sterol regulatory element-binding protein 1) protein, lipogenesis, and triglyceride accretion but similar Akt activation after acute insulin stimulation. Acute siRNA-mediated Abca1 silencing during 3T3L1 adipocyte differentiation reduced adipocyte Abca1 and PPARγ protein expression and triglyceride content. Systemic stimulated triglyceride lipolysis and glucose homeostasis were similar between control and ASKO mice. CONCLUSIONS: Adipocyte Abca1 is a key regulator of adipocyte lipogenesis and lipid accretion, likely because of increased adipose tissue membrane cholesterol, resulting in decreased activation of lipogenic transcription factors PPARγ and SREBP1.

In vivo activation of leukocyte GPR120/FFAR4 by PUFAs has minimal impact on atherosclerosis in LDL receptor knockout mice.

G protein-coupled receptor (GPR)120/FFA receptor (FFAR)4 (GPR120/FFAR4) activation by n-3 PUFAs attenuates inflammation, but its impact on atherosclerosis is unknown. We determined whether in vivo activation of leukocyte GPR120/FFAR4 by n-3 versus n-6 PUFAs is atheroprotective. Leukocyte GPR120/FFAR4 WT or KO mice in the LDL receptor KO background were generated by bone marrow transplantation. Mice were fed one of the four atherogenic diets containing 0.2% cholesterol and 10% calories as palm oil (PO) + 10% calories as: 1) PO, 2) fish oil (FO; 20:5 n-3 and 22:6 n-3 enriched), 3) echium oil (EO; 18:4 n-3 enriched), or 4) borage oil (BO; 18:3 n-6 enriched) for 16 weeks. Compared with PO, mice fed BO, EO, and FO had significantly reduced plasma cholesterol, TG, VLDL cholesterol, hepatic neutral lipid, and atherosclerosis that were equivalent for WT and KO mice. In BO-, EO-, and FO-fed mice, but not PO-fed mice, lack of leukocyte GPR120/FFAR4 resulted in neutrophilia, pro-inflammatory Ly6Chi monocytosis, increased aortic root monocyte recruitment, and increased hepatic inflammatory gene expression. In conclusion, leukocyte GPR120 expression has minimal effects on dietary PUFA-induced plasma lipid/lipoprotein reduction and atheroprotection, and there is no distinction between n-3 versus n-6 PUFAs in activating anti-inflammatory effects of leukocyte GPR120/FFAR4 in vivo.

Dietary PUFAs attenuate NLRP3 inflammasome activation via enhancing macrophage autophagy.

Dietary PUFAs reduce atherosclerosis and macrophage inflammation, but how nucleotide-binding oligomerization domain leucine-rich repeat-containing receptor protein (NLRP3) inflammasome activation and autophagy influence PUFA-mediated atheroprotection is poorly understood. We fed Ldlr-/- mice diets containing 10% (calories) palm oil (PO) and 0.2% cholesterol, supplemented with an additional 10% of calories as PO, fish oil (FO), echium oil (EO, containing 18:4 n-3), or borage oil (BO, containing 18:3 n-6). Inflammasome activation, autophagic flux, and mitochondrial function were measured in peritoneal macrophages, blood monocytes, or liver from diet-fed mice. Compared with PO, dietary PUFAs (FO, EO, or BO) markedly inhibited inflammasome activation, shown by 1) less macrophage IL-1ß secretion and caspase-1 cleavage in response to NLRP3 inflammasome activators, 2) less IL-1ß secretion and caspase-1 cleavage from liver or hepatocytes in response to lipopolysaccharide (LPS), and 3) attenuated caspase-1 activity in blood monocytes. Furthermore, PUFA-enriched diets increased LC3-II expression in macrophage, aorta, and liver samples and reduced numbers of dysfunctional mitochondria in macrophages in response to LPS and palmitate, suggesting enhanced autophagic activation. Dietary PUFAs did not attenuate NLRP3 inflammasome activation in atg5-deficient macrophages, indicating that autophagic activation is critical for the PUFA-mediated inflammasome inactivation. In conclusion, dietary PUFAs reduce atherosclerosis, in part, by activation of macrophage autophagy and attenuation of NLRP3 inflammasome activation.

Very Low Density Lipoprotein Assembly Is Required for cAMP-responsive Element-binding Protein H Processing and Hepatic Apolipoprotein A-IV Expression.

Hepatic apolipoprotein A-IV (apoA-IV) expression is correlated with hepatic triglyceride (TG) content in mouse models of chronic hepatosteatosis, and steatosis-induced hepatic apoA-IV gene expression is regulated by nuclear transcription factor cAMP-responsive element-binding protein H (CREBH) processing. To define what aspects of TG homeostasis regulate hepatic CREBH processing and apoA-IV gene expression, several mouse models of attenuated VLDL particle assembly were subjected to acute hepatosteatosis induced by an overnight fast or short term ketogenic diet feeding. Compared with chow-fed C57BL/6 mice, fasted or ketogenic diet-fed mice displayed increased hepatic TG content, which was highly correlated (r2 = 0.95) with apoA-IV gene expression, and secretion of larger, TG-enriched VLDL, despite a lower rate of TG secretion and a similar or reduced rate of apoB100 secretion. When VLDL particle assembly and secretion was inhibited by hepatic shRNA-induced apoB silencing or genetic or pharmacologic reduction in microsomal triglyceride transfer protein (MTP) activity, hepatic TG content increased dramatically; however, CREBH processing and apoA-IV gene expression were attenuated compared with controls. Adenovirus-mediated reconstitution of MTP expression proportionately restored CREBH processing and apoA-IV expression in liver-specific MTP knock-out mice. These results reveal that hepatic TG content, per se, does not regulate CREBH processing. Instead, TG mobilization into the endoplasmic reticulum for nascent VLDL particle assembly activates CREBH processing and enhances apoA-IV gene expression in the setting of acute steatosis. We conclude that VLDL assembly and CREBH activation play key roles in the response to hepatic steatosis by up-regulating apoA-IV and promoting assembly and secretion of larger, more TG-enriched VLDL particles.

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.

Botanical oils enriched in n-6 and n-3 FADS2 products are equally effective in preventing atherosclerosis and fatty liver.

Echium oil (EO), which is enriched in 18:4 n-3, the immediate product of fatty acid desaturase 2 (FADS2) desaturation of 18:3 n-3, is as atheroprotective as fish oil (FO). The objective of this study was to determine whether botanical oils enriched in the FADS2 products 18:3 n-6 versus 18:4 n-3 are equally atheroprotective. LDL receptor KO mice were fed one of four atherogenic diets containing 0.2% cholesterol and 10% calories as palm oil (PO) plus 10% calories as: 1) PO; 2) borage oil (BO; 18:3 n-6 enriched); 3) EO (18:4 n-3 enriched); or 4) FO for 16 weeks. Mice fed BO, EO, and FO versus PO had significantly lower plasma total and VLDL cholesterol concentrations; hepatic neutral lipid content and inflammation, aortic CE content, aortic root intimal area and macrophage content; and peritoneal macrophage inflammation, CE content, and ex vivo chemotaxis. Atheromas lacked oxidized CEs despite abundant generation of macrophage 12/15 lipooxygenase-derived metabolites. We conclude that botanical oils enriched in 18:3 n-6 and 18:4 n-3 PUFAs beyond the rate-limiting FADS2 enzyme are equally effective in preventing atherosclerosis and hepatosteatosis compared with saturated/monounsaturated fat due to cellular enrichment of ≥20 PUFAs, reduced plasma VLDL, and attenuated macrophage inflammation.

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 ATP-binding cassette transporter A1 deletion has minimal impact on atherogenesis in atherogenic diet-fed low-density lipoprotein receptor knockout mice.

OBJECTIVE: Transplantation studies suggest that bone marrow cell ATP-binding cassette transporter A1 protects against atherosclerosis development. However, the in vivo effect of macrophage ATP-binding cassette transporter A1 expression on atherogenesis is not fully understood because bone marrow contains other leukocytes and hematopoietic stem and progenitor cells. Myeloid-specific ATP-binding cassette transporter A1 knockout mice in the low-density lipoprotein (LDL) receptor knockout C57BL/6 background were developed to address this question. APPROACH AND RESULTS: Chow-fed myeloid-specific ATP-binding cassette transporter A1 knockout/LDL receptor knockout (double knockout [DKO]) versus LDL receptor knockout (single knockout [SKO]) mice had similar plasma lipid concentrations, but atherogenic diet (AD)-fed DKO mice had reduced plasma very-LDL (VLDL)/LDL concentrations resulting from decreased hepatic VLDL triglyceride secretion. Resident peritoneal macrophages from AD-fed DKO versus SKO mice had significantly higher cholesterol content but similar proinflammatory gene expression. Atherosclerosis extent was similar between genotypes after 10 to 16 weeks of AD but increased modestly in DKO mice by 24 weeks of AD. Lesional macrophage content was similar, likely because of the higher monocyte flux through aortic root lesions in DKO versus SKO mice. After transplantation of DKO or SKO bone marrow into SKO mice and 16 weeks of AD feeding, atherosclerosis extent was similar and plasma apolipoprotein B lipoproteins were reduced in mice receiving DKO bone marrow. When differences in plasma VLDL/LDL concentrations were minimized by maintaining mice on chow for 24 weeks, DKO mice had modest, but significantly more, atherosclerosis compared with SKO mice. CONCLUSIONS: Myeloid cell ATP-binding cassette transporter A1 increases hepatic VLDL triglyceride secretion and plasma VLDL/LDL concentrations in AD-fed LDL receptor knockout mice, offsetting its atheroprotective role in decreasing macrophage cholesterol content, resulting in a minimal increase in atherosclerosis.

Liver ABCA1 deletion in LDLrKO mice does not impair macrophage reverse cholesterol transport or exacerbate atherogenesis.

OBJECTIVE: Hepatic ATP binding cassette transporter A1 (ABCA1) expression is critical for maintaining plasma high-density lipoprotein (HDL) concentrations, but its role in macrophage reverse cholesterol transport and atherosclerosis is not fully understood. We investigated atherosclerosis development and reverse cholesterol transport in hepatocyte-specific ABCA1 knockout (HSKO) mice in the low-density lipoprotein (LDL) receptor KO (LDLrKO) C57BL/6 background. APPROACH AND RESULTS: Male and female LDLrKO and HSKO/LDLrKO mice were switched from chow at 8 weeks of age to an atherogenic diet (10% palm oil, 0.2% cholesterol) for 16 weeks. Chow-fed HSKO/LDLrKO mice had HDL concentrations 10% to 20% of LDLrKO mice, but similar very low-density lipoprotein and LDL concentrations. Surprisingly, HSKO/LDLrKO mice fed the atherogenic diet had significantly lower (40% to 60%) very low-density lipoprotein, LDL, and HDL concentrations (50%) compared with LDLrKO mice. Aortic surface lesion area and cholesterol content were similar for both genotypes of mice, but aortic root intimal area was significantly lower (20% to 40%) in HSKO/LDLrKO mice. Although macrophage (3)H-cholesterol efflux to apoB lipoprotein-depleted plasma was 24% lower for atherogenic diet-fed HSKO/LDLrKO versus LDLrKO mice, variation in percentage efflux among individual mice was <2-fold compared with a 10-fold variation in plasma HDL concentrations, suggesting that HDL levels, per se, were not the primary determinant of plasma efflux capacity. In vivo reverse cholesterol transport, resident peritoneal macrophage sterol content, biliary lipid composition, and fecal cholesterol mass were similar between both genotypes of mice. CONCLUSIONS: The markedly reduced plasma HDL pool in HSKO/LDLrKO mice is sufficient to maintain macrophage reverse cholesterol transport, which, along with reduced plasma very low-density lipoprotein and LDL concentrations, prevented the expected increase in atherosclerosis.

LXR signaling pathways link cholesterol metabolism with risk for prediabetes and diabetes.

BACKGROUNDPreclinical studies suggest that cholesterol accumulation leads to insulin resistance. We previously reported that alterations in a monocyte cholesterol metabolism transcriptional network (CMTN) - suggestive of cellular cholesterol accumulation - were cross-sectionally associated with obesity and type 2 diabetes (T2D). Here, we sought to determine whether the CMTN alterations independently predict incident prediabetes/T2D risk, and correlate with cellular cholesterol accumulation.METHODSMonocyte mRNA expression of 11 CMTN genes was quantified among 934 Multi-Ethnic Study of Atherosclerosis (MESA) participants free of prediabetes/T2D; cellular cholesterol was measured in a subset of 24 monocyte samples.RESULTSDuring a median 6-year follow-up, lower expression of 3 highly correlated LXR target genes - ABCG1 and ABCA1 (cholesterol efflux) and MYLIP (cholesterol uptake suppression) - and not other CMTN genes, was significantly associated with higher risk of incident prediabetes/T2D. Lower expression of the LXR target genes correlated with higher cellular cholesterol levels (e.g., 47% of variance in cellular total cholesterol explained by ABCG1 expression). Further, adding the LXR target genes to overweight/obesity and other known predictors significantly improved prediction of incident prediabetes/T2D.CONCLUSIONThese data suggest that the aberrant LXR/ABCG1-ABCA1-MYLIP pathway (LAAMP) is a major T2D risk factor and support a potential role for aberrant LAAMP and cellular cholesterol accumulation in diabetogenesis.FUNDINGThe MESA Epigenomics and Transcriptomics Studies were funded by NIH grants 1R01HL101250, 1RF1AG054474, R01HL126477, R01DK101921, and R01HL135009. This work was supported by funding from NIDDK R01DK103531 and NHLBI R01HL119962.

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.

Omega-3 fatty acids ameliorate atherosclerosis by favorably altering monocyte subsets and limiting monocyte recruitment to aortic lesions.

OBJECTIVE: Fish oil, containing omega-3 fatty acids, attenuates atherosclerosis. We hypothesized that omega-3 fatty acid-enriched oils are atheroprotective through alteration of monocyte subsets and their trafficking into atherosclerotic lesions. METHODS AND RESULTS: Low-density lipoprotein receptor knockout and apolipoprotein E(-/-) mice were fed diets containing 10% (calories) palm oil and 0.2% cholesterol, supplemented with an additional 10% palm oil, echium oil (containing 18:4 n-3), or fish oil. Compared with palm oil-fed low-density lipoprotein receptor knockout mice, echium oil and fish oil significantly reduced plasma cholesterol, splenic Ly6C(hi) monocytosis by ≈50%, atherosclerosis by 40% to 70%, monocyte trafficking into the aortic root by ≈50%, and atherosclerotic lesion macrophage content by 30% to 44%. In contrast, atherosclerosis and monocyte trafficking into the artery wall was not altered by omega-3 fatty acids in apolipoprotein E(-/-) mice; however, Ly6C(hi) splenic monocytes positively correlated with aortic root intimal area across all diet groups. In apolipoprotein E(-/-) mice, fish oil reduced the percentage of blood Ly6C(hi) monocytes, despite an average 2-fold higher plasma cholesterol relative to palm oil. CONCLUSIONS: The presence of splenic Ly6C(hi) monocytes parallels the appearance of atherosclerotic disease in both low-density lipoprotein receptor knockout and apolipoprotein E(-/-) mice. Furthermore, omega-3 fatty acids favorably alter monocyte subsets independently from effects on plasma cholesterol and reduce monocyte recruitment into atherosclerotic lesions.

Hepatocyte ABCA1 deficiency is associated with reduced HDL sphingolipids.

ATP binding cassette transporter A1 (ABCA1) limits the formation of high density lipoproteins (HDL) as genetic loss of ABCA1 function causes virtual HDL deficiency in patients with Tangier disease. Mice with a hepatocyte-specific ABCA1 knockout (Abca1 HSKO) have 20% of wild type (WT) plasma HDL-cholesterol levels, suggesting a major contribution of hepatic ABCA1 to the HDL phenotype. Whether plasma sphingolipids are reduced in Tangier disease and to what extent hepatic ABCA1 contributes to plasma sphingolipid (SL) levels is unknown. Here, we report a drastic reduction of total SL levels in plasma of a Tangier patient with compound heterozygosity for mutations in ABCA1. Compared to mutation-free controls, heterozygous mutations in ABCA1 had no significant effect on total SLs in plasma; however, apoB-depleted plasma showed a reduction in total SL also in het carriers. Similarly, liver specific Abca1 KO mice (Abca1 HSKO) showed reduced total sphingolipids in plasma and liver. In parallel, apoM and sphingosine-1-phosphate (S1P) levels were reduced in plasma of Abca1 HSKO mice. Primary hepatocytes from Abca1 HSKO mice showed a modest, but significant reduction in total SLs concentration compared to WT hepatocytes, although SL de novo synthesis and secretion were slightly increased in Abca1 HSKO hepatocytes. We conclude that hepatic ABCA1 is a signficant contributor to maintaining total plasma pool of HDL sphingolipids, including sphingomyelins and S1P.

Macrophage 12/15 lipoxygenase expression increases plasma and hepatic lipid levels and exacerbates atherosclerosis.

12/15 lipoxygenase (12/15LO) oxidizes polyunsaturated fatty acids (PUFAs) to form bioactive lipid mediators. The role of 12/15LO in atherosclerosis development remains controversial. We evaluated atherosclerosis development and lipid metabolism in 12/15LO-LDL receptor (LDLr) double knockout (DK) vs. LDLr knockout (SK) mice fed a PUFA-enriched diet to enhance production of 12/15LO products. Compared with SK controls, DK mice fed a PUFA-enriched diet had decreased plasma and liver lipid levels, hepatic lipogenic gene expression, VLDL secretion, and aortic atherosclerosis and increased VLDL turnover. Bone marrow transplantation and Kupffer cell ablation studies suggested both circulating leukocytes and Kupffer cells contributed to the lipid phenotype in 12/15LO-deficient mice. Conditioned medium from in vitro incubation of DK vs. SK macrophages reduced triglyceride secretion in McArdle 7777 hepatoma cells. Our results suggest that, in the context of dietary PUFA enrichment, macrophage 12/15LO expression adversely affects plasma and hepatic lipid metabolism, resulting in exacerbated atherosclerosis.

Hematopoietic Cell-Specific SLC37A2 Deficiency Accelerates Atherosclerosis in LDL Receptor-Deficient Mice.

Macrophages play a central role in the pathogenesis of atherosclerosis. Our previous study demonstrated that solute carrier family 37 member 2 (SLC37A2), an endoplasmic reticulum-anchored phosphate-linked glucose-6-phosphate transporter, negatively regulates macrophage Toll-like receptor activation by fine-tuning glycolytic reprogramming in vitro. Whether macrophage SLC37A2 impacts in vivo macrophage inflammation and atherosclerosis under hyperlipidemic conditions is unknown. We generated hematopoietic cell-specific SLC37A2 knockout and control mice in C57Bl/6 Ldlr-/- background by bone marrow transplantation. Hematopoietic cell-specific SLC37A2 deletion in Ldlr-/- mice increased plasma lipid concentrations after 12-16 wks of Western diet induction, attenuated macrophage anti-inflammatory responses, and resulted in more atherosclerosis compared to Ldlr-/- mice transplanted with wild type bone marrow. Aortic root intimal area was inversely correlated with plasma IL-10 levels, but not total cholesterol concentrations, suggesting inflammation but not plasma cholesterol was responsible for increased atherosclerosis in bone marrow SLC37A2-deficient mice. Our in vitro study demonstrated that SLC37A2 deficiency impaired IL-4-induced macrophage activation, independently of glycolysis or mitochondrial respiration. Importantly, SLC37A2 deficiency impaired apoptotic cell-induced glycolysis, subsequently attenuating IL-10 production. Our study suggests that SLC37A2 expression is required to support alternative macrophage activation in vitro and in vivo. In vivo disruption of hematopoietic SLC37A2 accelerates atherosclerosis under hyperlipidemic pro-atherogenic conditions.

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

Apolipoprotein M (apoM) is a novel apolipoprotein that is reportedly necessary for pre beta 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 pre beta 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 approximately 50% remains membrane-associated after extraction with sodium carbonate, pH 11.5. To investigate the role of apoM in nascent pre beta HDL formation, ABCA1-expressing or control cells, transfected with empty vector, apoM, or C-terminal epitope-tagged apoM (apoM-C-FLAG), were incubated with (125)I-apoA-I for 24 h. Conditioned media were harvested and fractionated by fast-protein liquid chromatography (FPLC) to monitor HDL particle size. Pre beta HDL particles were formed effectively in the absence of apoM expression; however, increased apoM expression stimulated the formation of larger-sized nascent pre beta HDLs. Immunoprecipitation with anti-apoA-I antibody followed by apoM Western blot analysis revealed that little secreted apoM was physically associated with pre beta HDL. Our results suggest that apoM is an atypical secretory protein that is not necessary for ABCA1-dependent pre beta HDL formation but does stimulate the formation of larger-sized pre beta HDL. We propose that apoM may function catalytically at an intracellular site to transfer lipid onto pre beta HDL during or after their formation by ABCA1.

Myeloid atg5 deletion impairs n-3 PUFA-mediated atheroprotection.

BACKGROUND AND AIMS: Dietary long-chain (≥20 carbons) n-3 polyunsaturated fatty acids (PUFAs) reduce atherosclerosis and enhance macrophage autophagy activation. How macrophage autophagy impacts atherosclerotic progression, particularly when comparing dietary n-3 PUFA supplementation vs. saturated fat feeding, is unknown. METHODS: We generated myeloid-specific autophagy-deficient and control mice in the Ldlr-/- background by transplanting bone marrow from myeloid-specific autophagy-related (atg) 5 knockout mice and wild type controls into irradiated Ldlr-/- recipients. After 7 weeks for recovery from radiation, mice were fed an atherogenic diet containing 0.2% cholesterol and 20% calories as palm oil (PO diet), or 10% calories as PO plus 10% calories as fish oil (FO diet) for 16 weeks. RESULTS: Compared to PO, FO significantly reduced plasma cholesterol, triglyceride, hepatic neutral lipid, and aortic caspase-1 cleavage, but increased aortic efferocytosis, leading to attenuated atherosclerosis in Ldlr-/- mice receiving wild type bone marrow. Myeloid atg5 deletion had little impact on plasma lipid concentrations and hepatic neutral lipid content, regardless of diet. Myeloid atg5 deletion increased aortic caspase-1 cleavage, decreased aortic efferocytosis and worsened atherosclerosis only in the FO-fed Ldlr-/- mice. CONCLUSIONS: Deficient myeloid autophagy significantly attenuated FO-induced atheroprotection, suggesting that dietary n-3 PUFAs reduce atherosclerosis, in part, by activation of macrophage autophagy.

Targeted inactivation of hepatic Abca1 causes profound hypoalphalipoproteinemia and kidney hypercatabolism of apoA-I.

Patients with Tangier disease exhibit extremely low plasma HDL concentrations resulting from mutations in the ATP-binding cassette, sub-family A, member 1 (ABCA1) protein. ABCA1 controls the rate-limiting step in HDL particle assembly by mediating efflux of cholesterol and phospholipid from cells to lipid-free apoA-I, which forms nascent HDL particles. ABCA1 is widely expressed; however, the specific tissues involved in HDL biogenesis are unknown. To determine the role of the liver in HDL biogenesis, we generated mice with targeted deletion of the second nucleotide-binding domain of Abca1 in liver only (Abca1(-L/-L)). Abca1(-L/-L) mice had total plasma and HDL cholesterol concentrations that were 19% and 17% those of wild-type littermates, respectively. In vivo catabolism of HDL apoA-I from wild-type mice or human lipid-free apoA-I was 2-fold higher in Abca1(-L/-L) mice compared with controls due to a 2-fold increase in the catabolism of apoA-I by the kidney, with no change in liver catabolism. We conclude that in chow-fed mice, the liver is the single most important source of plasma HDL. Furthermore, hepatic, but not extrahepatic, Abca1 is critical in maintaining the circulation of mature HDL particles by direct lipidation of hepatic lipid-poor apoA-I, slowing its catabolism by the kidney and prolonging its plasma residence time.

Echium oil reduces plasma lipids and hepatic lipogenic gene expression in apoB100-only LDL receptor knockout mice.

We tested the hypothesis that dietary supplementation with echium oil (EO), which is enriched in stearidonic acid (SDA; 18:4 n-3), the product of Delta-6 desaturation of 18:3 n-3, will decrease plasma triglyceride (TG) concentrations and result in conversion of SDA to eicosapentaenoic acid (EPA) in the liver. Mildly hypertriglyceridemic mice (apoB100-only LDLrKO) were fed a basal diet containing 10% calories as palm oil (PO) and 0.2% cholesterol for 4 weeks, after which they were randomly assigned to experimental diets consisting of the basal diet plus supplementation of 10% of calories as PO, EO or fish oil (FO) for 8 weeks. The EO and FO experimental diets decreased plasma TG and VLDL lipid concentration, and hepatic TG content compared to PO, and there was a significant correlation between hepatic TG content and plasma TG concentration among diet groups. EO fed mice had plasma and liver lipid EPA enrichment that was greater than PO-fed mice but less than FO-fed mice. Down-regulation of several genes involved in hepatic TG biosynthesis was similar for mice fed EO and FO and significantly lower compared to those fed PO. In conclusion, EO may provide a botanical alternative to FO for reduction of plasma TG concentrations.