Functional recombinant apolipoprotein A5 that is stable at high concentrations at physiological pH.

APOA5 is a low-abundance exchangeable apolipoprotein that plays critical roles in human triglyceride (TG) metabolism. Indeed, aberrations in the plasma concentration or structure of APOA5 are linked to hypertriglyceridemia, hyperchylomicronemia, myocardial infarction risk, obesity, and coronary artery disease. While it has been successfully produced at low yield in bacteria, the resulting protein had limitations for structure-function studies due to its low solubility under physiological buffer conditions. We hypothesized that the yield and solubility of recombinant APOA5 could be increased by: i) engineering a fusion protein construct in a codon optimized expression vector, ii) optimizing an efficient refolding protocol, and iii) screening buffer systems at physiological pH. The result was a high-yield (25 mg/l) bacterial expression system that produces lipid-free APOA5 soluble at concentrations of up to 10 mg/ml at a pH of 7.8 in bicarbonate buffers. Physical characterization of lipid-free APOA5 indicated that it exists as an array of multimers in solution, and far UV circular dichroism analyses show differences in total α-helicity between acidic and neutral pH buffering conditions. The protein was functional in that it bound and emulsified multilamellar dimyristoyl-phosphatidylcholine vesicles and could inhibit postprandial plasma TG accumulation when injected into C57BL/6J mice orally gavaged with Intralipid.

Exon 9-deleted CETP inhibits full length-CETP synthesis and promotes cellular triglyceride storage.

Cholesteryl ester transfer protein (CETP) exists as full-length (FL) and exon 9 (E9)-deleted isoforms. The function of E9-deleted CETP is poorly understood. Here, we investigated the role of E9-deleted CETP in regulating the secretion of FL-CETP by cells and explored its possible role in intracellular lipid metabolism. CETP overexpression in cells that naturally express CETP confirmed that E9-deleted CETP is not secreted, and showed that cellular FL- and E9-deleted CETP form an isolatable complex. Coexpression of CETP isoforms lowered cellular levels of both proteins and impaired FL-CETP secretion. These effects were due to reduced synthesis of both isoforms; however, the predominate consequence of FL- and E9-deleted CETP coexpression is impaired FL-CETP synthesis. We reported previously that reducing both CETP isoforms or overexpressing FL-CETP impairs cellular triglyceride (TG) storage. To investigate this further, E9-deleted CETP was expressed in SW872 cells that naturally synthesize CETP and in mouse 3T3-L1 cells that do not. E9-deleted CETP overexpression stimulated SW872 triglyceride synthesis and increased stored TG 2-fold. Expression of E9-deleted CETP in mouse 3T3-L1 cells produced a similar lipid phenotype. In vitro, FL-CETP promotes the transfer of TG from ER-enriched membranes to lipid droplets. E9-deleted CETP also promoted this transfer, although less effectively, and it inhibited the transfer driven by FL-CETP. We conclude that FL- and E9-deleted CETP isoforms interact to mutually decrease their intracellular levels and impair FL-CETP secretion by reducing CETP biosynthesis. E9-deleted CETP, like FL-CETP, alters cellular TG metabolism and storage but in a contrary manner.

A genome-wide association study on lipoprotein (a) levels and coronary artery disease severity in a Chinese population.

Lipoprotein (a) [Lp(a)] is a genetically determined risk factor of coronary artery disease (CAD). Previous genome-wide association studies (GWASs), which were mostly carried out in Caucasians, have identified many Lp(a)-associated SNPs. Here, we performed a GWAS on Lp(a) levels and further explored the relationships between Lp(a)-associated SNPs and CAD severity in 1,403 Han Chinese subjects. We observed that elevated Lp(a) levels were significantly associated with the increased synergy between percutaneous coronary intervention with TAXUS and cardiac surgery (SYNTAX) score and the counts of heavily calcified lesions and long-range lesions (LRLs; P < 0.05), which are defined as lesions spanning >20 mm. Moreover, we identified four independent SNPs, namely, rs7770628, rs73596816, and rs6926458 in LPA, and rs144217738 in SLC22A2, that were significantly associated with Lp(a) levels. We also found that rs7770628 was associated with high SYNTAX scores [odds ratio (OR) (95% CI): 1.37 (1.05-1.80), P = 0.0213, false discovery rate (FDR) = 0.0852], and that rs7770628 and rs73596816 were associated with high risk of harboring LRLs [OR (95% CI): 1.53 (1.17-2.01), P = 0.0018, FDR = 0.0072 and 1.72 (1.19-2.49), P = 0.0040, FDR = 0.0080, respectively]. Our study was a large-scale GWAS to identify Lp(a)-associated variants in the Han Chinese population. Our findings highlight the importance and potential of Lp(a) intervention and expand our understanding of CAD prevention and treatment.

The α/ß-hydrolase domain-containing 4- and 5-related phospholipase Pummelig controls energy storage in Drosophila.

Triglycerides (TGs) are the main energy storage form that accommodates changing organismal energy demands. In Drosophila melanogaster, the TG lipase Brummer is centrally important for body fat mobilization. Its gene brummer (bmm) encodes the ortholog of mammalian adipose TG lipase, which becomes activated by α/ß-hydrolase domain-containing 5 (ABHD5/CGI-58), one member of the paralogous gene pair, α/ß-hydrolase domain-containing 4 (ABHD4) and ABHD5 In Drosophila, the pummelig (puml) gene encodes the single sequence-related protein to mammalian ABHD4/ABHD5 with unknown function. We generated puml deletion mutant flies, that were short-lived as a result of lipid metabolism changes, stored excess body fat at the expense of glycogen, and exhibited ectopic fat storage with altered TG FA profile in the fly kidneys, called Malpighian tubules. TG accumulation in puml mutants was not associated with increased food intake but with elevated lipogenesis; starvation-induced lipid mobilization remained functional. Despite its structural similarity to mammalian ABHD5, Puml did not stimulate TG lipase activity of Bmm in vitro. Rather, Puml acted as a phospholipase that localized on lipid droplets, mitochondria, and peroxisomes. Together, these results show that the ABHD4/5 family member Puml is a versatile phospholipase that regulates Drosophila body fat storage and energy metabolism.

Lipoprotein-X fifty years after its original discovery.

AIMS: To review the formation, catabolism, and the possible atherogenic properties of Lp-X. DATA SYNTHESIS: The conversion of cholesterol to bile acids is regulated by several mechanisms including cholesterol 7 alpha hydroxylase, fibroblast growth factor 19, and farnesoid X receptors. During cholestasis these mechanisms are altered and there is an accumulation of bile acids and cholesterol in plasma. The hypercholesterolemia observed in cholestasis is due to the presence of an anomalous lipoprotein called lipoprotein-X (Lp-X). Lp-X is a lipoprotein rich in phospholipid and free cholesterol present in plasma of patients with cholestasis and, with some variations, in patients with lecithin-cholesterol-acyl-transferase deficiency (LCAT), and after lipid infusion. Lp-X is formed from a bile lipoprotein moving to the blood vessels where it incorporates small quantities of triglycerides, apo-C and esterified cholesterol and becomes a "mature" Lp-X. The activity of the phosphatidilcholine canalicular transporter Mdr2 P-glycoprotein (homologous to the human ABCB4) is essential for LpX appearance, since its suppression abolishes Lp-X formation. However, the concentration of Lp-X in plasma is determined also by the degree of the cholestasis, the residual liver function, and the LCAT deficiency. The Lp-X catabolism seems to be mediated by the reticuloendothelial system and possibly the kidney. CONCLUSIONS: Lp-X might be considered a defense mechanism against the toxic effect of free cholesterol in cholestasis. The frequency of cardiovascular events in patients affected by primary biliary cholangitis, in whom the Lp-X is present in high concentration, are not increased. Further studies could now clarify the remaining open questions on the role of Lp-X in the dyslipidemia of cholestasis.

Obstructive sleep apnea and effects of continuous positive airway pressure on triglyceride-rich lipoprotein metabolism.

This study aimed to explore lipoprotein metabolism in obstructive sleep apnea (OSA) and the effects of continuous positive airway pressure (CPAP). We studied 15 men with severe OSA [apnea-hypopnea index (AHI) ≥30 events/hour] and 12 age-, BMI-, and waist circumference-matched volunteers without OSA (AHI <5 events/hour). Carotid intima-media thickness (CIMT) was determined by a blind examiner. After 12 h fasting, a triglyceride-rich chylomicron-like emulsion, labeled with [14C]cholesteryl oleate and [3H]triolein, was injected intravenously followed by blood sample collection at preestablished times. Fractional clearance rate (FCR) of the radiolabeled lipids was estimated by compartmental analysis of radioisotope decay curves. Compared with controls, patients with OSA showed a significant delay in both cholesteryl ester FCR (0.0126 ± 0.0187 vs. 0.0015 ± 0.0025 min-1; P = 0.0313) and triglycerides FCR (0.0334 ± 0.0390 vs. 0.0051 ± 0.0074 min-1; P = 0.0001). CIMT was higher in the OSA group: 620 ± 17 vs. 725 ± 29 µm; P = 0.004. Cholesteryl ester FCRs were inversely related to total sleep time <90% (r = -0.463; P = 0.029) and CIMT (r = -0.601; P = 0.022). The triglyceride FCR was inversely correlated with AHI (r = -0.537; P = 0.04). In a subgroup of patients treated with CPAP for 3 months (n = 7), triglyceride FCR increased 5-fold (P = 0.025), but the cholesteryl ester FCR was unchanged. In conclusion, severe OSA decreased lipolysis of triglyceride-rich lipoproteins and delayed removal of remnants. CPAP treatment may be effective to restore the lipolysis rates.

Efficacy of two vitamin E formulations in patients with abetalipoproteinemia and chylomicron retention disease.

Abetalipoproteinemia (ABL) and chylomicron retention disease (CMRD) are extremely rare recessive forms of hypobetalipoproteinemia characterized by intestinal lipid malabsorption and severe vitamin E deficiency. Vitamin E is often supplemented in the form of fat-soluble vitamin E acetate, but fat malabsorption considerably limits correction of the deficiency. In this crossover study, we administered two different forms of vitamin E, tocofersolan (a water-soluble derivative of RRR-α-tocopherol) and α-tocopherol acetate, to three patients with ABL and four patients with CMRD. The aims of this study were to evaluate the intestinal absorption characteristics of tocofersolan versus α-tocopherol acetate by measuring the plasma concentrations of α-tocopherol over time after a single oral load and to compare efficacy by evaluating the ability of each formulation to restore vitamin E storage after 4 months of treatment. In patients with ABL, tocofersolan and α-tocopherol acetate bioavailabilities were extremely low (2.8% and 3.1%, respectively). In contrast, bioavailabilities were higher in patients with CMRD (tocofersolan, 24.7%; α-tocopherol acetate, 11.4%). Plasma concentrations of α-tocopherol at 4 months were not significantly different by formulation type in ABL or CMRD. This study provides new insights about vitamin E status in ABL and CMRD and suggests the potential of different formulations as treatment options.

Genome-wide association study identifies novel recessive genetic variants for high TGs in an Arab population.

Abnormal blood lipid levels are influenced by genetic and lifestyle/dietary factors. Although many genetic variants associated with blood lipid traits have been identified in Europeans, similar data in Middle Eastern populations are limited. We performed a genome-wide association study with Arab individuals (discovery cohort: 1,353; replication cohort: 1,176) from Kuwait to identify possible associations of genetic variants with high lipid levels. We used Illumina HumanOmniExpress BeadChip and candidate SNP genotyping in the discovery and replication phases, respectively. For association tests, we used genetic models that were based on additive and recessive modes of inheritance. High triglycerides (TGs) were recessively associated with six risk variants (rs1002487/RPS6KA1, rs11805972/LAD1) rs7761746/Or5v1, rs39745/CTTNBP2-LSM8, rs2934952/PGAP3, and rs9626773/RP11-191L9.4-CERK) at genome-wide significance (P  6.12E-09), and another six variants (rs10873925/ST6GALNAC5, rs4663379/SPP2-ARL4C, rs10033119/NPY1R, rs17709449/LINC00911-FLRT2, rs11654954/CDK12-NEUROD2, and rs9972882/STARD3) were associated at borderline significance (P  5.0E-08). High TG was also additively associated with rs11654954. All of the 12 identified markers are novel and are harbored in runs of homozygosity. Literature evidence supports the involvement of these gene loci in lipid-related processes. This study in an Arab population augments international efforts to identify genetic regulation of lipid traits.

Arginine 123 of apolipoprotein A-I is essential for lecithin:cholesterol acyltransferase activity.

ApoA-I activates LCAT that converts lipoprotein cholesterol to cholesteryl ester (CE). Molecular dynamic simulations suggested earlier that helices 5 of two antiparallel apoA-I molecules on discoidal HDL form an amphipathic tunnel for migration of acyl chains and unesterified cholesterol to the active sites of LCAT. Our recent crystal structure of Δ(185-243)apoA-I showed the tunnel formed by helices 5/5, with two positively charged residues arginine 123 positioned at the edge of the hydrophobic tunnel. We hypothesized that these uniquely positioned residues Arg123 are poised for interaction with fatty acids produced by LCAT hydrolysis of the sn-2 chains of phosphatidylcholine, thus positioning the fatty acids for esterification to cholesterol. To test the importance of Arg123 for LCAT phospholipid hydrolysis and CE formation, we generated apoA-I[R123A] and apoA-I[R123E] mutants and made discoidal HDL with the mutants and WT apoA-I. Neither mutation of Arg123 changed the particle composition or size, or the protein conformation or stability. However, both mutations of Arg123 significantly reduced LCAT catalytic efficiency and the apparent Vmax for CE formation without affecting LCAT phospholipid hydrolysis. A control mutation, apoA-I[R131A], did not affect LCAT phospholipid hydrolysis or CE formation. These data suggest that Arg123 of apoA-I on discoidal HDL participates in LCAT-mediated cholesterol esterification.

Targeting the lipid metabolic axis ACSL/SCD in colorectal cancer progression by therapeutic miRNAs: miR-19b-1 role.

An abnormal acyl-CoA synthetase/stearoyl-CoA desaturase (ACSL/SCD) lipid network fuels colon cancer progression, endowing cells with invasive and migratory properties. Therapies against this metabolic network may be useful to improve clinical outcomes. Because micro-RNAs (miRNAs/miRs) are important epigenetic regulators, we investigated novel miRNAs targeting this pro-tumorigenic axis; hence to be used as therapeutic or prognostic miRNAs. Thirty-one putative common miRNAs were predicted to simultaneously target the three enzymes comprising the ACSL/SCD network. Target validation by quantitative RT-PCR, Western blotting, and luciferase assays showed miR-544a, miR-142, and miR-19b-1 as major regulators of the metabolic axis, ACSL/SCD Importantly, lower miR-19b-1 expression was associated with a decreased survival rate in colorectal cancer (CRC) patients, accordingly with ACSL/SCD involvement in patient relapse. Finally, miR-19b-1 regulated the pro-tumorigenic axis, ACSL/SCD, being able to inhibit invasion in colon cancer cells. Because its expression correlated with an increased survival rate in CRC patients, we propose miR-19b-1 as a potential noninvasive biomarker of disease-free survival and a promising therapeutic miRNA in CRC.

Effect of LSR polymorphism on blood lipid levels and age-specific epistatic interaction with the APOE common polymorphism.

The lipolysis stimulated lipoprotein receptor (LSR) is an apolipoprotein (Apo) B and ApoE receptor that participates in the removal of triglyceride-rich lipoproteins during the postprandial phase. LSR gene is located upstream of APOE, an important risk factor for cardiovascular disease (CVD). Since the APOE common polymorphism significantly affects the variability of lipid metabolism, this study aimed to determine the potential impact of a functional SNP rs916147 in LSR gene on lipid traits in healthy subjects and to investigate potential epistatic interaction between LSR and APOE. Unrelated healthy adults (N = 432) and children (N = 328, <18 years old) from the STANISLAS Family Study were used. Age-specific epistasis was observed between APOE and LSR, reversing the protective effect of APOE ε2 allele on cholesterol, ApoE and low-density lipoprotein levels (ß: .114, P: .777 × 10-8 , ß: .125, P: .639 × 10-3 , ß: .059, P: .531 × 10-3 , respectively). This interaction was verified in an independent adult population (n = 1744). These results highlight the importance of the LSR polymorphism and reveal the existence of complex molecular links between LSR and ApoE for the regulation of lipid levels, revealing potential new pathways of interest in type III hyperlipidemia and its involvement in CVD pathology.

Ceramide activation of RhoA/Rho kinase impairs actin polymerization during aggregated LDL catabolism.

Macrophages use an extracellular, hydrolytic compartment formed by local actin polymerization to digest aggregated LDL (agLDL). Catabolism of agLDL promotes foam cell formation and creates an environment rich in LDL catabolites, including cholesterol and ceramide. Increased ceramide levels are present in lesional LDL, but the effect of ceramide on macrophage proatherogenic processes remains unknown. Here, we show that macrophages accumulate ceramide in atherosclerotic lesions. Using macrophages from sphingosine kinase 2 KO (SK2KO) mice to mimic ceramide-rich conditions of atherosclerotic lesions, we show that SK2KO macrophages display impaired actin polymerization and foam cell formation in response to contact with agLDL. C16-ceramide treatment impaired wild-type but not SK2KO macrophage actin polymerization, confirming that this effect is due to increased ceramide levels. We demonstrate that knockdown of RhoA or inhibition of Rho kinase restores agLDL-induced actin polymerization in SK2KO macrophages. Activation of RhoA in macrophages was sufficient to impair actin polymerization and foam cell formation in response to agLDL. Finally, we establish that during catabolism, macrophages take up ceramide from agLDL, and inhibition of ceramide generation modulates actin polymerization. These findings highlight a critical regulatory pathway by which ceramide impairs actin polymerization through increased RhoA/Rho kinase signaling and regulates foam cell formation.

Lipid and lipoprotein abnormalities in acute lymphoblastic leukemia survivors.

Survivors of acute lymphoblastic leukemia (ALL), the most common cancer in children, are at increased risk of developing late cardiometabolic conditions. However, the mechanisms are not fully understood. This study aimed to characterize the plasma lipid profile, Apo distribution, and lipoprotein composition of 80 childhood ALL survivors compared with 22 healthy controls. Our results show that, despite their young age, 50% of the ALL survivors displayed dyslipidemia, characterized by increased plasma triglyceride (TG) and LDL-cholesterol, as well as decreased HDL-cholesterol. ALL survivors exhibited lower plasma Apo A-I and higher Apo B-100 and C-II levels, along with elevated Apo C-II/C-III and B-100/A-I ratios. VLDL fractions of dyslipidemic ALL survivors contained more TG, free cholesterol, and phospholipid moieties, but less protein. Differences in Apo content were found between ALL survivors and controls for all lipoprotein fractions except HDL3 HDL2, especially, showed reduced Apo A-I and raised Apo A-II, leading to a depressed Apo A-I/A-II ratio. Analysis of VLDL-Apo Cs disclosed a trend for higher Apo C-III1 content in dyslipidemic ALL survivors. In conclusion, this thorough investigation demonstrates a high prevalence of dyslipidemia in ALL survivors, while highlighting significant abnormalities in their plasma lipid profile and lipoprotein composition. Special attention must, therefore, be paid to these subjects given the atherosclerotic potency of lipid and lipoprotein disorders.

Aromatic residues in the C terminus of apolipoprotein C-III mediate lipid binding and LPL inhibition.

Plasma apoC-III levels correlate with triglyceride (TG) levels and are a strong predictor of CVD outcomes. ApoC-III elevates TG in part by inhibiting LPL. ApoC-III likely inhibits LPL by competing for lipid binding. To probe this, we used oil-drop tensiometry to characterize binding of six apoC-III variants to lipid/water interfaces. This technique monitors the dependence of lipid binding on surface pressure, which increases during TG hydrolysis by LPL. ApoC-III adsorption increased surface pressure by upward of 18 mN/m at phospholipid/TG/water interfaces. ApoC-III was retained to high pressures at these interfaces, desorbing at 21-25 mN/m. Point mutants, which substituted alanine for aromatic residues, impaired the lipid binding of apoC-III. Adsorption and retention pressures decreased by 1-6 mN/m in point mutants, with the magnitude determined by the location of alanine substitutions. Trp42 was most critical to mediating lipid binding. These results strongly correlate with our previous results, linking apoC-III point mutants to increased LPL binding and activity at lipid surfaces. We propose that aromatic residues in the C-terminal half of apoC-III mediate binding to TG-rich lipoproteins. Increased apoC-III expression in the hypertriglyceridemic state allows apoC-III to accumulate on lipoproteins and inhibit LPL by preventing binding and/or access to substrate.

Microsomal triglyceride transfer protein in the ectoparasitic crustacean salmon louse (Lepeophtheirus salmonis).

The salmon louse, Lepeophtheirus salmonis, is an endemic ectoparasite on salmonid fish that is challenging for the salmon farming industry and wild fish. Salmon lice produce high numbers of offspring, necessitating sequestration of large amounts of lipids into growing oocytes as a major energy source for larvae, most probably mediated by lipoproteins. The microsomal triglyceride transfer protein (MTP) is essential for the assembly of lipoproteins. Salmon lice have three L. salmonis MTP (LsMTP) transcript variants encoding two different protein isoforms, which are predicted to contain three ß-sheets (N, C, and A) and a central helical domain, similar to MTPs from other species. In adult females, the LsMTPs are differently transcribed in the sub-cuticular tissues, the intestine, the ovary, and in the mature eggs. RNA interference-mediated knockdown of LsMTP in mature females gave offspring with significantly fewer neutral lipids in their yolk and only 10-30% survival. The present study suggests the importance of LsMTP in reproduction and lipid metabolism in adult female L. salmonis, a possible metabolic bottleneck that could be exploited for the development of new anti-parasitic treatment methods.

Use of next-generation sequencing to detect LDLR gene copy number variation in familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is a heritable condition of severely elevated LDL cholesterol, caused predominantly by autosomal codominant mutations in the LDL receptor gene (LDLR). In providing a molecular diagnosis for FH, the current procedure often includes targeted next-generation sequencing (NGS) panels for the detection of small-scale DNA variants, followed by multiplex ligation-dependent probe amplification (MLPA) in LDLR for the detection of whole-exon copy number variants (CNVs). The latter is essential because ∼10% of FH cases are attributed to CNVs in LDLR; accounting for them decreases false negative findings. Here, we determined the potential of replacing MLPA with bioinformatic analysis applied to NGS data, which uses depth-of-coverage analysis as its principal method to identify whole-exon CNV events. In analysis of 388 FH patient samples, there was 100% concordance in LDLR CNV detection between these two methods: 38 reported CNVs identified by MLPA were also successfully detected by our NGS method, while 350 samples negative for CNVs by MLPA were also negative by NGS. This result suggests that MLPA can be removed from the routine diagnostic screening for FH, significantly reducing associated costs, resources, and analysis time, while promoting more widespread assessment of this important class of mutations across diagnostic laboratories.

Changes in LXR signaling influence early-pregnancy lipogenesis and protect against dysregulated fetoplacental lipid homeostasis.

Human pregnancy is associated with enhanced de novo lipogenesis in the early stages followed by hyperlipidemia during advanced gestation. Liver X receptors (LXRs) are oxysterol-activated nuclear receptors that stimulate de novo lipogenesis and also promote the efflux of cholesterol from extrahepatic tissues followed by its transport back to the liver for biliary excretion. Although LXR is recognized as a master regulator of triglyceride and cholesterol homeostasis, it is unknown whether it facilitates the gestational adaptations in lipid metabolism. To address this question, biochemical profiling, protein quantification, and gene expression studies were used, and gestational metabolic changes in T0901317-treated wild-type mice and Lxrab-/- mutants were investigated. Here, we show that altered LXR signaling contributes to the enhanced lipogenesis in early pregnancy by increasing the expression of hepatic Fas and stearoyl-CoA desaturase 1 (Scd1). Both the pharmacological activation of LXR with T0901317 and the genetic ablation of its two isoforms disrupted the increase in hepatic fatty acid biosynthesis and the development of hypertriglyceridemia during early gestation. We also demonstrate that absence of LXR enhances maternal white adipose tissue lipolysis, causing abnormal accumulation of triglycerides, cholesterol, and free fatty acids in the fetal liver. Together, these data identify LXR as an important factor in early-pregnancy lipogenesis that is also necessary to protect against abnormalities in fetoplacental lipid homeostasis.

NAFLD is associated with methylation shifts with relevance for the expression of genes involved in lipoprotein particle composition.

Non-alcoholic fatty liver disease (NAFLD) is characterized by the accumulation of triglycerides, cholesterol and toxic free fatty acids and is related to low vitamin D levels. In an analysis of specific gene sets we elucidate to what extent NAFLD associates to epigenetic and related transcriptional changes in gene networks regulating lipid, energy and vitamin D balance. Two gene clusters responsible for lipid homeostasis (74 genes) and vitamin D and energy balance (31 genes) were investigated with regard to average epigenetic shifts within the first 1500bp next to the transcriptional start site. Three cohorts from two published genome wide driven studies that used a microarray approach were investigated including altogether 103 NAFLD and 75 liver healthy subjects. In the first two steps associations between NAFLD abundance, strength of fibrosis and methylation were investigated in two cohorts by multiple linear regression analyses, correcting for important clinical and demographic parameters. Methylation associated strength of transcription in genes showing significant NAFLD related methylation changes were studied in a third step using a third cohort and applying Pearson's correlation and robust linear regression analyses. 41 genes in gene cluster 1 and 14 genes in cluster 2 were significantly differentially methylated in dependency of NAFLD and hepatic fibrosis. We detect new genes significantly changed in methylation, including APO family members (lipid transport), NPC1L1, STARD (cholesterol transport) and GRHL (energy homeostasis). Our results allow novel insights into the hepatic epigenetic regulation of genes important for lipid and vitamin D balance in NAFLD.

Preservation of myocardial fatty acid oxidation prevents diastolic dysfunction in mice subjected to angiotensin II infusion.

RATIONALE: Diastolic dysfunction is a common feature in many heart failure patients with preserved ejection fraction and has been associated with altered myocardial metabolism in hypertensive and diabetic patients. Therefore, metabolic interventions to improve diastolic function are warranted. In mice with a germline cardiac-specific deletion of acetyl CoA carboxylase 2 (ACC2), systolic dysfunction induced by pressure-overload was prevented by maintaining cardiac fatty acid oxidation (FAO). However, it has not been evaluated whether this strategy would prevent the development of diastolic dysfunction in the adult heart. OBJECTIVE: To test the hypothesis that augmenting cardiac FAO is protective against angiotensin II (AngII)-induced diastolic dysfunction in an adult mouse heart. METHODS AND RESULTS: We generated a mouse model to induce cardiac-specific deletion of ACC2 in adult mice. Tamoxifen treatment (20mg/kg/day for 5days) was sufficient to delete ACC2 protein and increase cardiac FAO by 50% in ACC2 flox/flox-MerCreMer+ mice (iKO). After 4weeks of AngII (1.1mg/kg/day), delivered by osmotic mini-pumps, iKO mice showed normalized E/E' and E'/A' ratios compared to AngII treated controls (CON). The prevention of diastolic dysfunction in iKO-AngII was accompanied by maintained FAO and reduced glycolysis and anaplerosis. Furthermore, iKO-AngII hearts had a~50% attenuation of cardiac hypertrophy and fibrosis compared to CON. In addition, maintenance of FAO in iKO hearts suppressed AngII-associated increases in oxidative stress and sustained mitochondrial respiratory complex activities. CONCLUSION: These data demonstrate that impaired FAO is a contributor to the development of diastolic dysfunction induced by AngII. Maintenance of FAO in this model leads to an attenuation of hypertrophy, reduces fibrosis, suppresses increases in oxidative stress, and maintains mitochondrial function. Therefore, targeting mitochondrial FAO is a promising therapeutic strategy for the treatment of diastolic dysfunction.

Cholesteryl ester transfer protein alters liver and plasma triglyceride metabolism through two liver networks in female mice.

Elevated plasma TGs increase risk of cardiovascular disease in women. Estrogen treatment raises plasma TGs in women, but molecular mechanisms remain poorly understood. Here we explore the role of cholesteryl ester transfer protein (CETP) in the regulation of TG metabolism in female mice, which naturally lack CETP. In transgenic CETP females, acute estrogen treatment raised plasma TGs 50%, increased TG production, and increased expression of genes involved in VLDL synthesis, but not in nontransgenic littermate females. In CETP females, estrogen enhanced expression of small heterodimer partner (SHP), a nuclear receptor regulating VLDL production. Deletion of liver SHP prevented increases in TG production and expression of genes involved in VLDL synthesis in CETP mice with estrogen treatment. We also examined whether CETP expression had effects on TG metabolism independent of estrogen treatment. CETP increased liver ß-oxidation and reduced liver TG content by 60%. Liver estrogen receptor α (ERα) was required for CETP expression to enhance ß-oxidation and reduce liver TG content. Thus, CETP alters at least two networks governing TG metabolism, one involving SHP to increase VLDL-TG production in response to estrogen, and another involving ERα to enhance ß-oxidation and lower liver TG content. These findings demonstrate a novel role for CETP in estrogen-mediated increases in TG production and a broader role for CETP in TG metabolism.