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Circulating levels of the soluble ligand-binding ectodomain of the LDL receptor (sLDLR) that is proteolytically cleaved from the cell surface have been shown to correlate with plasma triglycerides, but the lipid and lipoprotein effects of longitudinal changes in sLDLR have not been examined. We sought to assess associations between changes in sLDLR and detailed lipoprotein measurements between baseline and 6 months in participants in the DIETFITS (Diet Intervention Examining The Factors Interacting with Treatment Success) weight loss trial who were randomly assigned to the low-fat (n = 225) or low-carbohydrate (n = 236) diet arms. sLDLR was assayed using a proteomic procedure, lipids and apoprotein (apo) B and apoAI were measured by standard assays, and lipoprotein particle subfractions were quantified by ion mobility methodology. Changes in sLDLR were significantly positively associated with changes in plasma cholesterol, triglycerides, apoB, large-sized and medium-sized VLDL, and small and very small LDL, and inversely with changes in large LDL and HDL. The lipoprotein subfraction associations with sLDLR were independent of age, sex, diet, and BMI, but all except for large LDL were reduced to insignificance when adjusted for triglyceride change. Principal component analysis identified three independent clusters of changes in lipoprotein subfractions that accounted for 78% of their total variance. Change in sLDLR was most strongly correlated with change in the principal component that was loaded positively with large VLDL and small and very small LDL and negatively with large LDL and HDL. In conclusion, sLDLR is a component of a cluster of lipids and lipoproteins that are characteristic of atherogenic dyslipidemia.

Although a high amount of brown adipose tissue (BAT) is associated with low plasma triglyceride concentration, the mechanism responsible for this relationship in people is not clear. Here, we evaluate the interrelationships among BAT, very-low-density lipoprotein triglyceride (VLDL-TG), and free fatty acid (FFA) plasma kinetics during thermoneutrality in women with overweight/obesity who had a low (

Despite great progress in understanding lipoprotein physiology, there is still much to be learned about the genetic drivers of lipoprotein abundance, composition, and function. We used ion mobility spectrometry to survey 16 plasma lipoprotein subfractions in 500 Diversity Outbred mice maintained on a Western-style diet. We identified 21 quantitative trait loci (QTL) affecting lipoprotein abundance. To refine the QTL and link them to disease risk in humans, we asked if the human homologs of genes located at each QTL were associated with lipid traits in human genome-wide association studies. Integration of mouse QTL with human genome-wide association studies yielded candidate gene drivers for 18 of the 21 QTL. This approach enabled us to nominate the gene encoding the neutral ceramidase, Asah2, as a novel candidate driver at a QTL on chromosome 19 for large HDL particles (HDL-2b). To experimentally validate Asah2, we surveyed lipoproteins in Asah2-/- mice. Compared to wild-type mice, female Asah2-/- mice showed an increase in several lipoproteins, including HDL. Our results provide insights into the genetic regulation of circulating lipoproteins, as well as mechanisms by which lipoprotein subfractions may affect cardiovascular disease risk in humans.

Over the past 40 years, there has been considerable research into the management and treatment of atherogenic lipid disorders. Although the majority of treatments and management strategies for cardiovascular disease (CVD) center around targeting low-density lipoprotein cholesterol (LDL-C), there is mounting evidence for the residual CVD risk attributed to high triglyceride (TG) and lipoprotein(a) (Lp(a)) levels despite the presence of lowered LDL-C levels. Among the biological mechanisms for clearing TG-rich lipoproteins, the VLDL receptor (VLDLR) plays a key role in the trafficking and metabolism of lipoprotein particles in multiple tissues, but it is not ordinarily expressed in the liver. Since VLDLR is capable of binding and internalizing apoE-containing TG-rich lipoproteins as well as Lp(a), hepatic VLDLR expression has the potential for promoting clearance of these atherogenic particles from the circulation and managing the residual CVD risk not addressed by current lipid lowering therapies. This review provides an overview of VLDLR function and the potential for developing a genetic medicine based on liver-targeted VLDLR gene expression.

Background and aimsWe previously identified subsets of patients with NAFLD with different metabolic phenotypes. Here we align metabolomic signatures with cardiovascular disease (CVD) and genetic risk factors.Approach and resultsWe analyzed serum metabolome from 1154 individuals with biopsy-proven NAFLD, and from four mouse models of NAFLD with impaired VLDL-triglyceride (TG) secretion, and one with normal VLDL-TG secretion. We identified three metabolic subtypes: A (47%), B (27%), and C (26%). Subtype A phenocopied the metabolome of mice with impaired VLDL-TG secretion; subtype C phenocopied the metabolome of mice with normal VLDL-TG; and subtype B showed an intermediate signature. The percent of patients with NASH and fibrosis was comparable among subtypes, although subtypes B and C exhibited higher liver enzymes. Serum VLDL-TG levels and secretion rate were lower among subtype A compared with subtypes B and C. Subtype A VLDL-TG and VLDL-apolipoprotein B concentrations were independent of steatosis, whereas subtypes B and C showed an association with these parameters. Serum TG, cholesterol, VLDL, small dense LDL5,6 , and remnant lipoprotein cholesterol were lower among subtype A compared with subtypes B and C. The 10-year high risk of CVD, measured with the Framingham risk score, and the frequency of patatin-like phospholipase domain-containing protein 3 NAFLD risk allele were lower in subtype A.ConclusionsMetabolomic signatures identify three NAFLD subgroups, independent of histological disease severity. These signatures align with known CVD and genetic risk factors, with subtype A exhibiting a lower CVD risk profile. This may account for the variation in hepatic versus cardiovascular outcomes, offering clinically relevant risk stratification.

De novo lipogenesis (DNL) converts carbon substrates to lipids. Increased hepatic DNL could contribute to pathogenic liver triglyceride accumulation in nonalcoholic steatohepatitis (NASH) and therefore may be a potential target for pharmacological intervention. Here, we measured hepatic DNL using heavy water in 123 patients with NASH with fibrosis or cirrhosis, calculated the turnover of hepatic triglycerides to allow repeat labeling studies, and determined the associations of hepatic DNL with metabolic, fibrotic, and imaging markers. We found that hepatic DNL was higher in patients with fibrotic NASH [median (IQR), 40.7% contribution to palmitate (32.1, 47.5), n=103] than has been previously reported in healthy volunteers and remained elevated [median (IQR), 36.8% (31.0, 44.5), n=20] in patients with cirrhosis, despite lower liver fat content. We also showed that turnover of intrahepatic triglyceride pools was slow (t½ >10 days). Furthermore, DNL contribution was determined to be independent of liver stiffness by magnetic resonance imaging but was positively associated with the number of large very low density lipoprotein (VLDL) particles, the size of VLDL, the lipoprotein insulin resistance score, and levels of ApoB100, and trended toward negative associations with the fibrosis markers FIB-4, FibroSure, and APRI. Finally, we found treatment with the acetyl-CoA carboxylase inhibitor firsocostat reduced hepatic DNL at 4 and 12 weeks, using a correction model for residual label that accounts for hepatic triglyceride turnover. Taken together, these data support an important pathophysiological role for elevated hepatic DNL in NASH and demonstrate that response to pharmacological agents targeting DNL can be correlated with pretreatment DNL.

AIMS: Fibroblast growth factor (FGF) 21, a key regulator of energy metabolism, is currently evaluated in humans for treatment of type 2 diabetes and non-alcoholic steatohepatitis. However, the effects of FGF21 on cardiovascular benefit, particularly on lipoprotein metabolism in relation to atherogenesis, remain elusive. METHODS AND RESULTS: Here, the role of FGF21 in lipoprotein metabolism in relation to atherosclerosis development was investigated by pharmacological administration of a half-life extended recombinant FGF21 protein to hypercholesterolaemic APOE*3-Leiden.CETP mice, a well-established model mimicking atherosclerosis initiation and development in humans. FGF21 reduced plasma total cholesterol, explained by a reduction in non-HDL-cholesterol. Mechanistically, FGF21 promoted brown adipose tissue (BAT) activation and white adipose tissue (WAT) browning, thereby enhancing the selective uptake of fatty acids from triglyceride-rich lipoproteins into BAT and into browned WAT, consequently accelerating the clearance of the cholesterol-enriched remnants by the liver. In addition, FGF21 reduced body fat, ameliorated glucose tolerance and markedly reduced hepatic steatosis, related to up-regulated hepatic expression of genes involved in fatty acid oxidation and increased hepatic VLDL-triglyceride secretion. Ultimately, FGF21 largely decreased atherosclerotic lesion area, which was mainly explained by the reduction in non-HDL-cholesterol as shown by linear regression analysis, decreased lesion severity, and increased atherosclerotic plaque stability index. CONCLUSION: FGF21 improves hypercholesterolaemia by accelerating triglyceride-rich lipoprotein turnover as a result of activating BAT and browning of WAT, thereby reducing atherosclerotic lesion severity and increasing atherosclerotic lesion stability index. We have thus provided additional support for the clinical use of FGF21 in the treatment of atherosclerotic cardiovascular disease.

Oxylipins are metabolites of polyunsaturated fatty acids that mediate cardiovascular health by attenuation of inflammation, vascular tone, hemostasis, and thrombosis. Very low-density lipoproteins (VLDL) contain oxylipins, but it is unknown whether the liver regulates their concentrations. In this study, we used a perfused liver model to observe the effect of inflammatory lipopolysaccharide (LPS) challenge and soluble epoxide hydrolase inhibition (sEHi) on VLDL oxylipins. A compartmental model of deuterium-labeled linoleic acid and palmitic acid incorporation into VLDL was also developed to assess the dependence of VLDL oxylipins on fatty acid incorporation rates. LPS decreased the total fatty acid VLDL content by 30% [6%,47%], and decreased final concentration of several oxylipins by a similar amount (13-HOTrE, 35% [4%,55%], -1.3 nM; 9(10)-EpODE, 29% [3%,49%], -2.0 nM; 15(16)-EpODE, 29% [2%,49%], -1.6 nM; AA-derived diols, 32% [5%,52%], -2.4 nM; 19(20)-DiHDPA, 31% [7%,50%], -1.0 nM). However, the EPA-derived epoxide, 17(18)-EpETE, was decreased by 75% [49%,88%], (-0.52 nM) with LPS, double the suppression of other oxylipins. sEHi increased final concentration of DHA epoxide, 16(17)-EpDPE, by 99% [35%,193%], (2.0 nM). Final VLDL-oxylipin concentrations with LPS treatment were not correlated with linoleic acid kinetics, suggesting they were independently regulated under inflammatory conditions. We conclude that the liver regulates oxylipin incorporation into VLDL, and the oxylipin content is altered by LPS challenge and by inhibition of the epoxide hydrolase pathway. This provides evidence for delivery of systemic oxylipin signals by VLDL transport.

"Quantile-dependent expressivity" is a dependence of genetic effects on whether the phenotype (e.g., triglycerides) is high or low relative to its distribution in the population. Quantile-specific offspring-parent regression slopes (βOP) were estimated by quantile regression for 6227 offspring-parent pairs. Quantile-specific heritability (h2), estimated by 2βOP/(1 + rspouse), decreased 0.0047 ± 0.0007 (P = 2.9 × 10-14) for each one-percent decrement in fasting triglyceride concentrations, i.e., h2 ± SE were: 0.428 ± 0.059, 0.230 ± 0.030, 0.111 ± 0.015, 0.050 ± 0.016, and 0.033 ± 0.010 at the 90th, 75th, 50th, 25th, and 10th percentiles of the triglyceride distribution, respectively. Consistent with quantile-dependent expressivity, 11 drug studies report smaller genotype differences at lower (post-treatment) than higher (pre-treatment) triglyceride concentrations. This meant genotype-specific triglyceride changes could not move in parallel when triglycerides were decreased pharmacologically, so that subtracting pre-treatment from post-treatment triglyceride levels necessarily created a greater triglyceride decrease for the genotype with a higher pre-treatment value (purported precision-medicine genetic markers). In addition, sixty-five purported gene-environment interactions were found to be potentially attributable to triglyceride's quantile-dependent expressivity, including gene-adiposity (APOA5, APOB, APOE, GCKR, IRS-1, LPL, MTHFR, PCSK9, PNPLA3, PPARγ2), gene-exercise (APOA1, APOA2, LPL), gene-diet (APOA5, APOE, INSIG2, LPL, MYB, NXPH1, PER2, TNFA), gene-alcohol (ALDH2, APOA5, APOC3, CETP, LPL), gene-smoking (APOC3, CYBA, LPL, USF1), gene-pregnancy (LPL), and gene-insulin resistance interactions (APOE, LPL).

Non-alcoholic steatohepatitis (NASH) is characterized by the accumulation of hepatic fat in an inflammatory/fibrotic background. Herein, we show that the hepatic high-activity glutaminase 1 isoform (GLS1) is overexpressed in NASH. Importantly, GLS1 inhibition reduces lipid content in choline and/or methionine deprivation-induced steatotic mouse primary hepatocytes, in human hepatocyte cell lines, and in NASH mouse livers. We suggest that under these circumstances, defective glutamine fueling of anaplerotic mitochondrial metabolism and concomitant reduction of oxidative stress promotes a reprogramming of serine metabolism, wherein serine is shifted from the generation of the antioxidant glutathione and channeled to provide one-carbon units to regenerate the methionine cycle. The restored methionine cycle can induce phosphatidylcholine synthesis from the phosphatidylethanolamine N-methyltransferase-mediated and CDP-choline pathways as well as by base-exchange reactions between phospholipids, thereby restoring hepatic phosphatidylcholine content and very-low-density lipoprotein export. Overall, we provide evidence that hepatic GLS1 targeting is a valuable therapeutic approach in NASH.

Background & aimsObeticholic acid (OCA), a farnesoid X receptor agonist, increases total and low-density lipoprotein cholesterol (LDL-C) in patients with non-alcoholic steatohepatitis. In the present study, we aimed to evaluate the impact of OCA therapy on lipoprotein sub-particles.MethodThis study included 196 patients (99 OCA group and 97 placebo group) who were enrolled in the FLINT trial and had samples available for lipid analysis and liver biopsies at enrollment and end-of-treatment (EOT) at 72 weeks. Very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) particles were evaluated at baseline, 12 and 72 weeks after randomization, and 24 weeks following EOT.ResultsBaseline lipoprotein profiles were similar among OCA and placebo groups. OCA did not affect total VLDL particle concentrations, but OCA vs. placebo treatment was associated with decreased large VLDL particle concentration at 12 weeks (baseline-adjusted mean: 6.8 vs. 8.9 nmol/L; p = 0.002), mirrored by an increase in less atherogenic, small VLDL particle concentration (33.9 vs. 28.0 nmol/L; p = 0.02). After 12 weeks, total LDL particle concentration was higher in the OCA group than the placebo group (1,667 vs. 1,329 nmol/L; p

BackgroundAlmonds have been shown to lower LDL cholesterol but there is limited information regarding their effects on the dyslipidemia characterized by increased levels of very low density lipoproteins (VLDL) and small, dense low-density lipoprotein (LDL) particles that is associated with abdominal adiposity and high carbohydrate intake. The objective of the present study was to test whether substitution of almonds for other foods attenuates carbohydrate-induced increases in small, dense LDL in individuals with increased abdominal adiposity.MethodsThis was a randomized cross-over study of three 3wk diets, separated by 2wk washouts: a higher-carbohydrate (CHO) reference diet (CHOhigh), a higher-CHO diet with isocaloric substitution of 20% kcal (E) from almonds (CHOhigh + almonds), and a lower-CHO reference diet (CHOlow) in 9 men and 15 women who were overweight or obese. The two CHOhigh diets contained 50% carbohydrate, 15% protein, 35% fat (6% saturated, 21% monounsaturated, 8% polyunsaturated), while the CHOlow diet contained 25% carbohydrate, 28% protein, 47% fat (8% saturated, 28% monounsaturated, 8% polyunsaturated). Lipoprotein subfraction concentrations were measured by ion mobility.ResultsRelative to the CHOlow diet: 1) the CHOhigh + almonds diet significantly increased small, dense LDLIIIa (mean difference ± SE: 28.6 ± 10.4 nmol/L, P = 0.008), and reduced LDL-peak diameter (- 1.7 ± 0.6 Å, P = 0.008); 2) the CHOhigh diet significantly increased medium-sized LDLIIb (24.8 ± 11.4 nmol/L, P = 0.04) and large VLDL (3.7 ± 1.8 nmol/L, P = 0.05). Relative to CHOlow, the effects of CHOhigh on LDLIIIa (17.7 ± 10.6 nmol/L) and LDL-peak diameter (- 1.1 ± 0.6 Å) were consistent with those of CHOhigh + almonds, and the effects of CHOhigh + almonds on LDLIIb (21.0 ± 11.2 nmol/L) and large VLDL (2.8 ± 1.8 nmol/L) were consistent with those of CHOhigh, but did not achieve statistical significance (P > 0.05). None of the variables examined showed a significant difference between the CHOhigh + almonds and CHOhigh diets (P > 0.05).ConclusionOur analyses provided no evidence that deriving 20% E from almonds significantly modifies increases in levels of small, dense LDL or other plasma lipoprotein changes induced by a higher carbohydrate low saturated fat diet in individuals with increased abdominal adiposity.Trial registrationClinicaltrials.gov NCT01792648 .

BACKGROUND AND AIMS: Heparin-binding EGF-like growth factor (HB-EGF) is a representative EGF family member that interacts with EGFR under diverse stress environment. Previously, we reported that the HB-EGF-targeting using antisense oligonucleotide (ASO) effectively suppressed an aortic aneurysm in the vessel wall and circulatory lipid levels. In this study, we further examined the effects of the HB-EGF ASO administration on the development of hyperlipidemia-associated atherosclerosis using an atherogenic mouse model. METHODS AND RESULTS: The male and female LDLR deficient mice under Western diet containing 21% fat and 0.2% cholesterol content were cotreated with control and HB-EGF ASOs for 12 weeks. We observed that the HB-EGF ASO administration effectively downregulated circulatory VLDL- and LDL-associated lipid levels in circulation; concordantly, the HB-EGF targeting effectively suppressed the development of atherosclerosis in the aorta. An EGFR blocker BIBX1382 administration suppressed the hepatic TG secretion rate, suggesting a positive role of the HB-EGF signaling for the hepatic VLDL production. We newly observed that there was a significant improvement of the insulin sensitivity by the HB-EGF ASO administration in a mouse model under the Western diet as demonstrated by the improvement of the glucose and insulin tolerances. CONCLUSION: The HB-EGF ASO administration effectively downregulated circulatory lipid levels by suppressing hepatic VLDL production rate, which leads to effective protection against atherosclerosis in the vascular wall.

Intermediate-density lipoproteins (IDLs), the remnants of very-low-density lipoproteins via lipolysis, are rich in cholesteryl ester and are associated with cardiovascular disease. Despite pharmacological interest in IDLs, their three-dimensional (3D) structure is still undetermined due to their variation in size, composition, and dynamic structure. To explore the 3D structure of IDLs, we reconstructed 3D density maps from individual IDL particles using cryo-electron microscopy (cryo-EM) and individual-particle electron tomography (IPET, without averaging from different molecules). 3D reconstructions of IDLs revealed an unexpected polyhedral structure that deviates from the generally assumed spherical shape model (Frias et al., 2007; Olson, 1998; Shen et al., 1977). The polyhedral-shaped IDL contains a high-density shell formed by flat surfaces that are similar to those of very-low-density lipoproteins but have sharper dihedral angles between nearby surfaces. These flat surfaces would be less hydrophobic than the curved surface of mature spherical high-density lipoprotein (HDL), leading to a lower binding affinity of IDL to hydrophobic proteins (such as cholesteryl ester transfer protein) than HDL. This is the first visualization of the IDL 3D structure, which could provide fundamental clues for delineating the role of IDL in lipid metabolism and cardiovascular disease.

COPII-coated vesicles are the primary mediators of ER-to-Golgi trafficking. Sar1, one of the five core COPII components, is a highly conserved small GTPase, which, upon GTP binding, recruits the other COPII proteins to the ER membrane. It has been hypothesized that the changes in the kinetics of SAR1 GTPase may allow for the secretion of large cargoes. Here we developed a cell-free assay to recapitulate COPII-dependent budding of large lipoprotein cargoes from the ER. We identified fatty-acid binding protein 5 (FABP5) as an enhancer of this budding process. We found that FABP5 promotes the budding of particles ∼150 nm in diameter and modulates the kinetics of the SAR1 GTPase cycle. We further found that FABP5 enhances the trafficking of lipoproteins and of other cargoes, including collagen. These data identify a novel regulator of SAR1 GTPase activity and highlight the importance of this activity for trafficking of large cargoes.

Genetic studies of metabolites have identified thousands of variants, many of which are associated with downstream metabolic and obesogenic disorders. However, these studies have relied on univariate analyses, reducing power and limiting context-specific understanding. Here we aim to provide an integrated perspective of the genetic basis of metabolites by leveraging the Finnish Metabolic Syndrome In Men (METSIM) cohort, a unique genetic resource which contains metabolic measurements, mostly lipids, across distinct time points as well as information on statin usage. We increase effective sample size by an average of two-fold by applying the Covariates for Multi-phenotype Studies (CMS) approach, identifying 588 significant SNP-metabolite associations, including 228 new associations. Our analysis pinpoints a small number of master metabolic regulator genes, balancing the relative proportion of dozens of metabolite levels. We further identify associations to changes in metabolic levels across time as well as genetic interactions with statin at both the master metabolic regulator and genome-wide level.

Human phospholipid transfer protein (PLTP) mediates the transfer of phospholipids among atheroprotective high-density lipoproteins (HDL) and atherogenic low-density lipoproteins (LDL) by an unknown mechanism. Delineating this mechanism would represent the first step towards understanding PLTP-mediated lipid transfers, which may be important for treating lipoprotein abnormalities and cardiovascular disease. Here, using various electron microscopy techniques, PLTP is revealed to have a banana-shaped structure similar to cholesteryl ester transfer protein (CETP). We provide evidence that PLTP penetrates into the HDL and LDL surfaces, respectively, and then forms a ternary complex with HDL and LDL. Insights into the interaction of PLTP with lipoproteins at the molecular level provide a basis to understand the PLTP-dependent lipid transfer mechanisms for dyslipidemia treatment.

Lipoprotein lipase (LPL) mediates hydrolysis of triglycerides (TGs) to supply free fatty acids (FFAs) to tissues. Here, we show that LPL activity is also required for hematopoietic stem progenitor cell (HSPC) maintenance. Knockout of Lpl or its obligatory cofactor Apoc2 results in significantly reduced HSPC expansion during definitive hematopoiesis in zebrafish. A human APOC2 mimetic peptide or the human very low-density lipoprotein, which carries APOC2, rescues the phenotype in apoc2 but not in lpl mutant zebrafish. Creating parabiotic apoc2 and lpl mutant zebrafish rescues the hematopoietic defect in both. Docosahexaenoic acid (DHA) is identified as an important factor in HSPC expansion. FFA-DHA, but not TG-DHA, rescues the HSPC defects in apoc2 and lpl mutant zebrafish. Reduced blood cell counts are also observed in Apoc2 mutant mice at the time of weaning. These results indicate that LPL-mediated release of the essential fatty acid DHA regulates HSPC expansion and definitive hematopoiesis.

Metabolic syndrome (MetSyn) is characterized by chronic inflammation which mediates the associated high risk for cardiovascular and other diseases. Oxylipins are a superclass of lipid mediators with potent bioactivities in inflammation, vascular biology, and more. While their role as locally produced agents is appreciated, most oxylipins in plasma are found in lipoproteins suggesting defective regulation of inflammation could be mediated by the elevated VLDL and low HDL levels characteristic of MetSyn. Our objective was to compare the oxylipin composition of VLDL, LDL, and HDL in 14 optimally healthy individuals and 31 MetSyn patients, and then to determine the effects of treating MetSyn subjects with 4g/day of prescription omega-3 fatty acids (P-OM3) on lipoprotein oxylipin profiles. We compared oxylipin compositions of healthy (14) and MetSyn (31) subjects followed by randomization and assignment to 4g/d P-OM3 for 16 weeks using LC/MS/MS. Compared to healthy subjects, MetSyn is characterized by abnormalities of (1) pro-inflammatory, arachidonate-derived oxylipins from the lipoxygenase pathway in HDL; and (2) oxylipins mostly not derived from arachidonate in VLDL. P-OM3 treatment corrected many components of these abnormalities, reducing the burden of inflammatory mediators within peripherally circulating lipoproteins that could interfere with, or enhance, local effectors of inflammatory stress. We conclude that MetSyn is associated with a disruption of lipoprotein oxylipin patterns consistent with greater inflammatory stress, and the partial correction of these dysoxylipinemias by treatment with omega-3 fatty acids could explain some of their beneficial effects.

Cholesteryl ester transfer protein (CETP) inhibitors are a new class of therapeutics for dyslipidemia that simultaneously improve two major cardiovascular disease (CVD) risk factors: elevated low-density lipoprotein (LDL) cholesterol and decreased high-density lipoprotein (HDL) cholesterol. However, the detailed molecular mechanisms underlying their efficacy are poorly understood, as are any potential mechanistic differences among the drugs in this class. Herein, we used electron microscopy (EM) to investigate the effects of three of these agents (Torcetrapib, Dalcetrapib and Anacetrapib) on CETP structure, CETP-lipoprotein complex formation and CETP-mediated cholesteryl ester (CE) transfer. We found that although none of these inhibitors altered the structure of CETP or the conformation of CETP-lipoprotein binary complexes, all inhibitors, especially Torcetrapib and Anacetrapib, increased the binding ratios of the binary complexes (e.g., HDL-CETP and LDL-CETP) and decreased the binding ratios of the HDL-CETP-LDL ternary complexes. The findings of more binary complexes and fewer ternary complexes reflect a new mechanism of inhibition: one distal end of CETP bound to the first lipoprotein would trigger a conformational change at the other distal end, thus resulting in a decreased binding ratio to the second lipoprotein and a degraded CE transfer rate among lipoproteins. Thus, we suggest a new inhibitor design that should decrease the formation of both binary and ternary complexes. Decreased concentrations of the binary complex may prevent the inhibitor was induced into cell by the tight binding of binary complexes during lipoprotein metabolism in the treatment of CVD.

Hepatitis C virus (HCV) exists as a lipoprotein-virus hybrid lipoviroparticle (LVP). In vitro studies have demonstrated the importance of apolipoproteins in HCV secretion and infectivity, leading to the notion that HCV coopts the secretion of very-low-density lipoprotein (VLDL) for its egress. However, the mechanisms involved in virus particle assembly and egress are still elusive. The biogenesis of VLDL particles occurs in the endoplasmic reticulum (ER), followed by subsequent lipidation in the ER and Golgi compartment. The secretion of mature VLDL particles occurs through the Golgi secretory pathway. HCV virions are believed to latch onto or fuse with the nascent VLDL particle in either the ER or the Golgi compartment, resulting in the generation of LVPs. In our attempt to unravel the collaboration between HCV and VLDL secretion, we studied HCV particles budding from the ER en route to the Golgi compartment in COPII vesicles. Biophysical characterization of COPII vesicles fractionated on an iodixanol gradient revealed that HCV RNA is enriched in the highly buoyant COPII vesicle fractions and cofractionates with apolipoprotein B (ApoB), ApoE, and the HCV core and envelope proteins. Electron microscopy of immunogold-labeled microsections revealed that the HCV envelope and core proteins colocalize with apolipoproteins and HCV RNA in Sec31-coated COPII vesicles. Ultrastructural analysis also revealed the presence of HCV structural proteins, RNA, and apolipoproteins in the Golgi stacks. These findings support the hypothesis that HCV LVPs assemble in the ER and are transported to the Golgi compartment in COPII vesicles to embark on the Golgi secretory route.IMPORTANCE HCV assembly and release accompany the formation of LVPs that circulate in the sera of HCV patients and are also produced in an in vitro culture system. The pathway of HCV morphogenesis and secretion has not been fully understood. This study investigates the exact site where the association of HCV virions with host lipoproteins occurs. Using immunoprecipitation of COPII vesicles and immunogold electron microscopy (EM), we characterize the existence of LVPs that cofractionate with lipoproteins, viral proteins, RNA, and vesicular components. Our results show that this assembly occurs in the ER, and LVPs thus formed are carried through the Golgi network by vesicular transport. This work provides a unique insight into the HCV LVP assembly process within infected cells and offers opportunities for designing antiviral therapeutic cellular targets.

Lipoprotein lipase (LPL) is responsible for the hydrolysis of triglycerides from circulating lipoproteins. Whereas most identified mutations in the LPL gene are deleterious, one mutation, LPLS447X, causes a gain of function. This mutation truncates two amino acids from LPL's C-terminus. Carriers of LPLS447X have decreased VLDL levels and increased HDL levels, a cardioprotective phenotype. LPLS447X is used in Alipogene tiparvovec, the gene therapy product for individuals with familial LPL deficiency. It is unclear why LPLS447X results in a serum lipid profile more favorable than that of LPL. In vitro reports vary as to whether LPLS447X is more active than LPL. We report a comprehensive, biochemical comparison of purified LPLS447X and LPL dimers. We found no difference in specific activity on synthetic and natural substrates. We also did not observe a difference in the Ki for ANGPTL4 inhibition of LPLS447X relative to that of LPL. Finally, we analyzed LPL-mediated uptake of fluorescently labeled lipoprotein particles and found that LPLS447X enhanced lipoprotein uptake to a greater degree than LPL did. An LPL structural model suggests that the LPLS447X truncation exposes residues implicated in LPL binding to uptake receptors.

Human VLDLs assembled in the liver and secreted into the circulation supply energy to peripheral tissues. VLDL lipolysis yields atherogenic LDLs and VLDL remnants that strongly correlate with CVD. Although the composition of VLDL particles has been well-characterized, their 3D structure is elusive because of their variations in size, heterogeneity in composition, structural flexibility, and mobility in solution. Here, we employed cryo-electron microscopy and individual-particle electron tomography to study the 3D structure of individual VLDL particles (without averaging) at both below and above their lipid phase transition temperatures. The 3D reconstructions of VLDL and VLDL bound to antibodies revealed an unexpected polyhedral shape, in contrast to the generally accepted model of a spherical emulsion-like particle. The smaller curvature of surface lipids compared with HDL may also reduce surface hydrophobicity, resulting in lower binding affinity to the hydrophobic distal end of the N-terminal β-barrel domain of cholesteryl ester transfer protein (CETP) compared with HDL. The directional binding of CETP to HDL and VLDL may explain the function of CETP in transferring TGs and cholesteryl esters between these particles. This first visualization of the 3D structure of VLDL could improve our understanding of the role of VLDL in atherogenesis.

Background and aimsDietary fructose may play a role in the pathogenesis of metabolic syndrome (MetS). In a recently published study of obese children with MetS, we showed that isocaloric fructose restriction reduced fasting triglyceride (TG) and LDL-cholesterol (LDL-C). In these ancillary analyses, we tested the hypothesis that these effects were also accompanied by improved quantitative and qualitative changes in LDL and HDL subclasses and their apolipoproteins; as well as change in VLDL, particularly apoC-III.MethodsObese children with MetS (n = 37) consumed a diet that matched self-reported macronutrient composition for nine days, with the exception that dietary fructose was reduced from 11.7 ± 4.0% to 3.8 ± 0.5% of daily calories and substituted with glucose (in starch). Participants underwent fasting biochemical analyses on Days 0 and 10. HDL and LDL subclasses were analyzed using the Lipoprint HDL and LDL subfraction analysis systems from Quantimetrix.ResultsSignificant reductions in apoB (78 ± 24 vs. 66 ± 24 mg/dl) apoC-III (8.7 ± 3.5 vs. 6.5 ± 2.6 mg/dl) and apoE (4.6 ± 2.3 vs. 3.6 ± 1.1 mg/dl), all p

BackgroundThe DASH (Dietary Approaches to Stop Hypertension) dietary pattern, which is high in fruit, vegetables, and low-fat dairy foods, significantly lowers blood pressure as well as low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol.ObjectiveThe study was designed to test the effects of substituting full-fat for low-fat dairy foods in the DASH diet, with a corresponding increase in fat and a reduction in sugar intake, on blood pressure and plasma lipids and lipoproteins.DesignThis was a 3-period randomized crossover trial in free-living healthy individuals who consumed in random order a control diet, a standard DASH diet, and a higher-fat, lower-carbohydrate modification of the DASH diet (HF-DASH diet) for 3 wk each, separated by 2-wk washout periods. Laboratory measurements, which included lipoprotein particle concentrations determined by ion mobility, were made at the end of each experimental diet.ResultsThirty-six participants completed all 3 dietary periods. Blood pressure was reduced similarly with the DASH and HF-DASH diets compared with the control diet. The HF-DASH diet significantly reduced triglycerides and large and medium very-low-density lipoprotein (VLDL) particle concentrations and increased LDL peak particle diameter compared with the DASH diet. The DASH diet, but not the HF-DASH diet, significantly reduced LDL cholesterol, HDL cholesterol, apolipoprotein A-I, intermediate-density lipoprotein and large LDL particles, and LDL peak diameter compared with the control diet.ConclusionsThe HF-DASH diet lowered blood pressure to the same extent as the DASH diet but also reduced plasma triglyceride and VLDL concentrations without significantly increasing LDL cholesterol. This trial was registered at clinicaltrials.gov as NCT01404897.

BackgroundEstradiol, testosterone (T), and sex hormone binding globulin (SHBG) levels are associated with lipid subfractions in men and women. Our objective was to determine if associations are independent from adipose tissue area among Asian Indians.MethodsWe used data from 42 women and 57 Asian Indian men who did not use exogenous steroids or lipid-lowering medications. Lipoprotein subfractions including low-density lipoprotein cholesterol (LDL), very low-density lipoprotein cholesterol (VLDL), and intermediate density lipoprotein (IDL) were assessed by ion mobility spectrometry. Intra-abdominal adiposity was assessed by computed tomography. Multivariable regression models estimated the association between sex hormones with lipoprotein subfractions before and after adjustment for adiposity.ResultsAmong women, lower logSHBG levels were associated with smaller logLDL particle size and higher logtriglycerides, logVLDL, and logIDL, although these associations were attenuated with adjustment for visceral adiposity in particular. Among women, lower logSHBG levels was significantly associated with lower logmedium LDL and logsmall LDL concentrations even after consideration of visceral and hepatic adiposity and insulin resistance as represented by the homeostasis model assessment of insulin resistance (HOMA-IR). Among men, lower logSHBG was also associated with smaller logLDL peak diameter size and higher logtriglycerides and logVLDL, even after adjustment for HOMA-IR and adiposity. Relationships between sex steroids and lipid subfractions were not significant among women. Among men, higher total testosterone was associated with higher logHDL and logLDL particle size, and lower logtriglycerides and logVLDL, but these associations were partially attenuated with adjustment for adiposity and HOMA-IR.ConclusionsAmong Asian Indians, SHBG is associated with more favorable lipid subfraction concentrations, independent of hepatic and visceral fat.

Fatty acid and fat synthesis in the liver is a highly regulated metabolic pathway that is important for very low-density lipoprotein (VLDL) production and thus energy distribution to other tissues. Having common features at their promoter regions, lipogenic genes are coordinately regulated at the transcriptional level. Transcription factors, such as upstream stimulatory factors (USFs), sterol regulatory element-binding protein 1C (SREBP1C), liver X receptors (LXRs) and carbohydrate-responsive element-binding protein (ChREBP) have crucial roles in this process. Recently, insights have been gained into the signalling pathways that regulate these transcription factors. After feeding, high blood glucose and insulin levels activate lipogenic genes through several pathways, including the DNA-dependent protein kinase (DNA-PK), atypical protein kinase C (aPKC) and AKT-mTOR pathways. These pathways control the post-translational modifications of transcription factors and co-regulators, such as phosphorylation, acetylation or ubiquitylation, that affect their function, stability and/or localization. Dysregulation of lipogenesis can contribute to hepatosteatosis, which is associated with obesity and insulin resistance.

ObjectivePericardial fat and lipoprotein abnormalities contribute to increased risk of cardiovascular disease (CVD). We investigated the relationship between pericardial fat volume and lipoprotein distribution, and whether the association of pericardial fat volume with subclinical atherosclerosis and incident CVD events differs according to lipoprotein distribution.MethodsWe analyzed data from 5407 participants from the Multi-Ethnic Study of Atherosclerosis who had measurements of pericardial fat volume, lipoprotein distribution, carotid intima-media thickness (IMT), and coronary artery calcium (CAC). All participants were free of clinically apparent CVD at baseline. Incident CVD was defined as any adjudicated CVD event.ResultsAfter adjusting for demographic factors, traditional risk factors, and biomarkers of inflammation and hemostasis, a larger pericardial fat volume was associated with higher large VLDL particle (VLDL-P) concentration and small HDL particle (HDL-P) concentration, and smaller HDL-P size (regression coefficients = 0.585 nmol/L, 0.366 μmol/L, and -0.025 nm per SD increase in pericardial fat volume respectively, all P 0.05).ConclusionPericardial fat is associated with atherogenic lipoprotein abnormalities. However, its relationship with subclinical atherosclerosis and incident CVD events does not differ according to lipoprotein distribution.

The role of specific phospholipids (PLs) in lipid transport has been difficult to assess due to an inability to selectively manipulate membrane composition in vivo. Here we show that the phospholipid remodeling enzyme lysophosphatidylcholine acyltransferase 3 (Lpcat3) is a critical determinant of triglyceride (TG) secretion due to its unique ability to catalyze the incorporation of arachidonate into membranes. Mice lacking Lpcat3 in the intestine fail to thrive during weaning and exhibit enterocyte lipid accumulation and reduced plasma TGs. Mice lacking Lpcat3 in the liver show reduced plasma TGs, hepatosteatosis, and secrete lipid-poor very low-density lipoprotein (VLDL) lacking arachidonoyl PLs. Mechanistic studies indicate that Lpcat3 activity impacts membrane lipid mobility in living cells, suggesting a biophysical basis for the requirement of arachidonoyl PLs in lipidating lipoprotein particles. These data identify Lpcat3 as a key factor in lipoprotein production and illustrate how manipulation of membrane composition can be used as a regulatory mechanism to control metabolic pathways.

ImportanceThe identification of a patient with a rare form of severe dysbetalipoproteinemia allowed the study of the consequences of total absence of apolipoprotein E (apoE).ObjectivesTo discover the molecular basis of this rare disorder and to determine the effects of complete absence of apoE on neurocognitive and visual function and on lipoprotein metabolism.Design, setting, and participantsWhole-exome sequencing was performed on the patient's DNA. He underwent detailed neurological and visual function testing and lipoprotein analysis. Lipoprotein analysis was also performed in the Cardiovascular Research Institute, University of California, San Francisco, on blood samples from the proband's mother, wife, 2 daughters, and normolipidemic control participants.Main outcome measuresWhole-exome sequencing, lipoprotein analysis, and neurocognitive function.ResultsThe patient was homozygous for an ablative APOE frameshift mutation (c.291del, p.E97fs). No other mutations likely to contribute to the phenotype were discovered, with the possible exception of two, in ABCC2 (p.I670T) and LIPC (p.G137R). Despite complete absence of apoE, he had normal vision, exhibited normal cognitive, neurological, and retinal function, had normal findings on brain magnetic resonance imaging, and had normal cerebrospinal fluid levels of β-amyloid and tau proteins. He had no significant symptoms of cardiovascular disease except a suggestion of myocardial ischemia on treadmill testing and mild atherosclerosis noted on carotid ultrasonography. He had exceptionally high cholesterol content (760 mg/dL; to convert to millimoles per liter, multiply by 0.0259) and a high cholesterol to triglycerides ratio (1.52) in very low-density lipoproteins with elevated levels of small-diameter high-density lipoproteins, including high levels of prebeta-1 high-density lipoprotein. Intermediate-density lipoproteins, low-density lipoproteins, and very low-density lipoproteins contained elevated apoA-I and apoA-IV levels. The patient's apoC-III and apoC-IV levels were decreased in very low-density lipoproteins. Electron microscopy revealed large lamellar particles having electron-opaque cores attached to electron-lucent zones in intermediate-density and low-density lipoproteins. Low-density lipoprotein particle diameters were distributed bimodally.Conclusions and relevanceDespite a profound effect on lipoprotein metabolism, detailed neurocognitive and retinal studies failed to demonstrate any defects. This suggests that functions of apoE in the brain and eye are not essential or that redundant mechanisms exist whereby its role can be fulfilled. Targeted knockdown of apoE in the central nervous system might be a therapeutic modality in neurodegenerative disorders.

BackgroundOxylipins mediate inflammation, vascular tension, and more. Their presence in lipoproteins could explain why lipoproteins mediate nearly identical activities.MethodsTo determine how oxylipins are distributed in the lipoproteins of hypertriglyceridemic subjects, and whether omega-3 fatty acids alter them in a manner consistent with improved cardiovascular health, we recruited 15 dyslipidemic subjects whose levels of low density lipoprotein cholesterol (LDL-C) were at goal but who remained hypertriglyceridemic (200-499 mg/dL). They were treated them with the indicated dose of 4 g/d omega-3 acid ethyl esters (P-OM3) for 8 weeks. Measured oxylipins included mid-chain alcohols (HETEs, HEPEs and HDoHEs), ketones (KETEs), epoxides (as EpETrEs, EpETEs, and EpDPEs).ResultsAt baseline, arachidonate-oxylipins (HETEs, KETEs, and EpETrEs) were most abundant in plasma with the greatest fraction of total abundance (mean |95% CI|) being carried in high density lipoproteins (HDL); 42% |31, 57| followed by very low density lipoproteins (VLDL); 27% |20, 36|; and LDL 21% |16, 28|. EPA- and DHA-derived oxylipins constituted less than 11% of total. HDL carried alcohols and epoxides but VLDL was also rich in ketones. Treatment decreased AA-derived oxylipins across lipoprotein classes (-23% |-33, -12|, p = 0.0003), and expanded EPA-(322% |241, 422|, p

ObjectivePostprandial hyperlipemia, characterized by increased circulating very low-density lipoproteins (VLDL) and circulating lipopolysaccharide (LPS), has been proposed as a mechanism of vascular injury. Our goal was to examine the interactions between postprandial lipoproteins, LPS, and apoE3 and apoE4 on monocyte activation.Methods and resultsWe showed that apoE3 complexed to phospholipid vesicles attenuates LPS-induced THP-1 monocyte cytokine expression, while apoE4 increases expression. ELISA revealed that apoE3 binds to LPS with higher affinity than apoE4. Electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels placed on specific amino acids of apoE3 showed that LPS interferes with conformational changes normally associated with lipid binding. Specifically, compared to apoE4, apoE bearing the E3-like R112→Ser mutation displays increased self association when exposed to LPS, consistent with a stronger apoE3-LPS interaction. Additionally, lipolysis of fasting VLDL from normal human donors attenuated LPS-induced TNFα secretion from monocytes to a greater extent than postprandial VLDL, an effect partially reversed by blocking apoE. This effect was reproduced using fasting VLDL lipolysis products from e3/e3 donors, but not from e4/e4 subjects, suggesting that apoE3 on fasting VLDL prevents LPS-induced inflammation more readily than apoE4.ConclusionPostprandial apoE isoform and conformational changes associated with VLDL dramatically modulate vascular inflammation.

ObjectivePostprandial hyperlipemia, characterized by increased circulating very low-density lipoproteins (VLDL) and circulating lipopolysaccharide (LPS), has been proposed as a mechanism of vascular injury. Our goal was to examine the interactions between postprandial lipoproteins, LPS, and apoE3 and apoE4 on monocyte activation.Methods and resultsWe showed that apoE3 complexed to phospholipid vesicles attenuates LPS-induced THP-1 monocyte cytokine expression, while apoE4 increases expression. ELISA revealed that apoE3 binds to LPS with higher affinity than apoE4. Electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels placed on specific amino acids of apoE3 showed that LPS interferes with conformational changes normally associated with lipid binding. Specifically, compared to apoE4, apoE bearing the E3-like R112→Ser mutation displays increased self association when exposed to LPS, consistent with a stronger apoE3-LPS interaction. Additionally, lipolysis of fasting VLDL from normal human donors attenuated LPS-induced TNFα secretion from monocytes to a greater extent than postprandial VLDL, an effect partially reversed by blocking apoE. This effect was reproduced using fasting VLDL lipolysis products from e3/e3 donors, but not from e4/e4 subjects, suggesting that apoE3 on fasting VLDL prevents LPS-induced inflammation more readily than apoE4.ConclusionPostprandial apoE isoform and conformational changes associated with VLDL dramatically modulate vascular inflammation.

Since Zilversmit first proposed postprandial lipemia as the most common risk of cardiovascular disease, chylomicrons (CM) and CM remnants have been thought to be the major lipoproteins which are increased in the postprandial hyperlipidemia. However, it has been shown over the last two decades that the major increase in the postprandial lipoproteins after food intake occurs in the very low density lipoprotein (VLDL) remnants (apoB-100 particles), not CM or CM remnants (apoB-48 particles). This finding was obtained using the following three analytical methods; isolation of remnant-like lipoprotein particles (RLP) with specific antibodies, separation and detection of lipoprotein subclasses by gel permeation HPLC and determination of apoB-48 in fractionated lipoproteins by a specific ELISA. The amount of the apoB-48 particles in the postprandial RLP is significantly less than the apoB-100 particles, and the particle sizes of apoB-48 and apoB-100 in RLP are very similar when analyzed by HPLC. Moreover, CM or CM remnants having a large amount of TG were not found in the postprandial RLP. Therefore, the major portion of the TG which is increased in the postprandial state is composed of VLDL remnants, which have been recognized as a significant risk for cardiovascular disease.

BackgroundThe response in levels of very-low-density (VLDL) and low-density (LDL) lipoproteins varies substantially among hyperlipidemic patients during treatment with HMGCoA reductase inhibitors. Apolipoprotein E genotype and gender are known to contribute to the regulation of steady state levels of plasma lipoproteins. This study explores the effect of these and other potential determinants of the response of VLDL and LDL to treatment with reductase inhibitors.MethodsUsing mixed linear statistical models, the response of lipoprotein lipid values was studied in 142 hyperlipidemic individuals who were treated with reductase inhibitors. Patients received one or more of the following drugs individually for a total of 623 treatment observations: lovastatin, pravastatin, simvastatin, or atorvastatin. For evaluation of the effects of treatment in the aggregate, actual doses were expressed as equivalent doses of atorvastatin, using factors based on random assignment comparisons in 16 reported studies. The analysis factors considered were apolipoprotein E genotype, baseline average triglycerides >170 mg/dL (vs less), and gender.ResultsPresence of an apo epsilon4 allele was associated with a trend toward greater reduction of triglyceride levels and a diminished ability of the reductase inhibitors to reduce LDL cholesterol levels. Gender had only minimal effect on the response of either LDL cholesterol or triglycerides. However, the effect of elevated baseline triglycerides on the response of both triglycerides and LDL cholesterol was striking and was exerted in opposite directions. The triglyceride-lowering effect of reductase inhibitors was greater in patients with initial triglyceride levels above 170 mg/dL (P=0.0001). The effect was even greater in patients with initial triglyceride levels over 250 mg/dL (P=0.015). Conversely, for LDL cholesterol levels, elevated baseline triglycerides were associated with a significantly decreased response to the drugs (P=0.0015).ConclusionsThese findings indicate that baseline triglyceride levels are an important predictor of response of plasma lipoproteins to HMGCoA reductase inhibitors, perhaps reflecting fundamental differences in mechanism underlying the hyperlipidemic phenotype.

Pure macrophage-derived foam cells (MFC) were isolated from the aortas of rabbits made atherosclerotic by balloon deendothelialization followed by diet-induced hypercholesterolemia. The MFC were isolated under sterile conditions using an enzymatic digestion procedure and discontinuous density gradient centrifugation. The purity of the MFC preparations was verified immunocytochemically with the macrophage specific monoclonal antibody RAM-11. MFC plated in medium containing 0.5% FCS for 24 h contained approximately 600 micrograms cholesterol per mg cell protein, 80% of which was esterified cholesterol. The MFC specifically degraded low density lipoprotein (LDL), acetyl-LDL, copper oxidized LDL, and beta-very low density lipoprotein (beta-VLDL) at rates comparable to mouse peritoneal macrophages (MPM) in 5-h assays. MFC within sections of the atherosclerotic lesions from the ballooned rabbits as well as the MFC isolated from the same lesions in the presence of antioxidants, exhibited positive immunoreactivity with polyclonal guinea pig antisera and mouse monoclonal antibodies directed against malondialdehyde-LDL, and 4-hydroxynonal-LDL. The MFC also exhibited the capacity to induce the oxidation of LDL at rates comparable to those exhibited by MPM and rabbit aortic endothelial cells. These data provide direct evidence that arterial wall macrophages express modified LDL receptors in vivo, contain epitopes found in oxidized-LDL and are capable of oxidizing LDL even when maximally loaded with cholesterol.