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Aim: Primary human hepatocytes (PHHs) undergo dedifferentiation upon the two-dimensional (2D) culture, which particularly hinders their utility in long-term in vitro studies. Lipids, as a major class of biomolecules, play crucial roles in cellular energy storage, structure, and signaling. Here, for the first time, we mapped the alterations in the lipid profile of the dedifferentiating PHHs and studied the possible role of lipids in the loss of the phenotype of PHHs. Simultaneously, differentially expressed miRNAs associated with changes in the lipids and fatty acids (FAs) of the dedifferentiating PHHs were investigated., Methods: PHHs were cultured in monolayer and their phenotype was monitored morphologically, genetically, and biochemically for five days. The lipid and miRNA profile of the PHHs were analyzed by mass spectrometry and Agilent microarray, respectively. In addition, 24 key genes involved in the metabolism of lipids and FAs were investigated by qPCR., Results: The typical morphology of PHHs was lost from day 3 onward. Additionally, ALB and CYP genes were downregulated in the cultured PHHs. Lipidomics revealed a clear increase in the saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) containing lipids, but a decrease in the polyunsaturated fatty acids (PUFA) containing lipids during the dedifferentiation of PHHs. In line with this, FASN , SCD , ELOVL1 , ELOVL3 , and ELOVL7 were upregulated but ELOVL2 was downregulated in the dedifferentiated PHHs. Furthermore, differentially expressed miRNAs were identified, and the constantly upregulated miR-27a and miR-21, and downregulated miR-30 may have regulated the synthesis, accumulation and secretion of PHH lipids during the dedifferentiation., Conclusion: Our results showed major alterations in the molecular lipid species profiles, lipid-metabolizing enzyme expression as wells as miRNA profiles of the PHHs during their prolonged culture, which in concert could play important roles in the PHHs' loss of phenotype. These findings promote the understanding from the dedifferentiation process and could help in developing optimal culture conditions, which better meet the needs of the PHHs and support their original phenotype.

Hepatocyte-like cells (HLCs) differentiated from human-induced pluripotent stem cells offer an alternative platform to primary human hepatocytes (PHHs) for studying the lipid metabolism of the liver. However, despite their great potential, the lipid profile of HLCs has not yet been characterized. Here, we comprehensively studied the lipid profile and fatty acid (FA) metabolism of HLCs and compared them with the current standard hepatocyte models: HepG2 cells and PHHs. We differentiated HLCs by five commonly used methods from three cell lines and thoroughly characterized them by gene and protein expression. HLCs generated by each method were assessed for their functionality and the ability to synthesize, elongate, and desaturate FAs. In addition, lipid and FA profiles of HLCs were investigated by both mass spectrometry and gas chromatography and then compared with the profiles of PHHs and HepG2 cells. HLCs resembled PHHs by expressing hepatic markers: secreting albumin, lipoprotein particles, and urea, and demonstrating similarities in their lipid and FA profile. Unlike HepG2 cells, HLCs contained low levels of lysophospholipids similar to the content of PHHs. Furthermore, HLCs were able to efficiently use the exogenous FAs available in their medium and simultaneously modify simple lipids into more complex ones to fulfill their needs. In addition, we propose that increasing the polyunsaturated FA supply of the culture medium may positively affect the lipid profile and functionality of HLCs. In conclusion, our data showed that HLCs provide a functional and relevant model to investigate human lipid homeostasis at both molecular and cellular levels., (© 2018 Wiley Periodicals, Inc.)

The impact of cigarette smoke (CS), a major cause of lung diseases, on the composition and metabolism of lung lipids is incompletely understood. Here, we integrated quantitative lipidomics and proteomics to investigate exposure effects on lung lipid metabolism in a C57BL/6 and an Apolipoprotein E-deficient (Apoe(-/-)) mouse study. In these studies, mice were exposed to high concentrations of 3R4F reference CS, aerosol from potential modified risk tobacco products (MRTPs) or filtered air (Sham) for up to 8 months. The 2 assessed MRTPs, the prototypical MRTP for C57BL/6 mice and the Tobacco Heating System 2.2 for Apoe(-/-) mice, utilize "heat-not-burn" technologies and were each matched in nicotine concentrations to the 3R4F CS. After 2 months of CS exposure, some groups were either switched to the MRTP or underwent cessation. In both mouse strains, CS strongly affected several categories of lung lipids and lipid-related proteins. Candidate surfactant lipids, surfactant proteins, and surfactant metabolizing proteins were increased. Inflammatory eicosanoids, their metabolic enzymes, and several ceramide classes were elevated. Overall, CS induced a coordinated lipid response controlled by transcription regulators such as SREBP proteins and supported by other metabolic adaptations. In contrast, most of these changes were absent in the mice exposed to the potential MRTPs, in the cessation group, and the switching group. Our findings demonstrate the complex biological response of the lungs to CS exposure and support the benefits of cessation or switching to a heat-not-burn product using a design such as those employed in this study., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology.)

Purpose: This systematic review was performed to summarize published data on lipidomic and metabolomic risk markers of coronary artery disease., Methods: Studies were identified from a literature search of PubMed., Results: Published data shows that analysis of metabolites and lipids offers an opportunity to increase our knowledge of the biological processes related to development and progression of atherosclerotic coronary disease. It is evident that advanced analytical technologies are able to detect and identify a large number of molecules that may have important structural and functional roles over and above currently used biomarkers in the cardiovascular field. It is suggested in a number of reports that the novel biomarkers can be used to improve risk stratification and patient selection for different treatments. Also, monitoring treatment efficacy and safety as well as lifestyle changes should be facilitated by such novel markers., Conclusion: Until now a plethora of biomarker candidates associated with cardiovascular event risk have been identified, but very few have passed through clinical and analytical validation and found their way into clinical use. Consequently, the appetite of physicians to use these novel tests in daily clinical routine has not yet been truly tested.

The importance of lipids for cell function and health has been widely recognized, e.g., a disorder in the lipid composition of cells has been related to atherosclerosis caused cardiovascular disease (CVD). Lipidomics analyses are characterized by large yet not a huge number of mutually correlated variables measured and their associations to outcomes are potentially of a complex nature. Differential network analysis provides a formal statistical method capable of inferential analysis to examine differences in network structures of the lipids under two biological conditions. It also guides us to identify potential relationships requiring further biological investigation. We provide a recipe to conduct permutation test on association scores resulted from partial least square regression with multiple imputed lipidomic data from the LUdwigshafen RIsk and Cardiovascular Health (LURIC) study, particularly paying attention to the left-censored missing values typical for a wide range of data sets in life sciences. Left-censored missing values are low-level concentrations that are known to exist somewhere between zero and a lower limit of quantification. To make full use of the LURIC data with the missing values, we utilize state of the art multiple imputation techniques and propose solutions to the challenges that incomplete data sets bring to differential network analysis. The customized network analysis helps us to understand the complexities of the underlying biological processes by identifying lipids and lipid classes that interact with each other, and by recognizing the most important differentially expressed lipids between two subgroups of coronary artery disease (CAD) patients, the patients that had a fatal CVD event and the ones who remained stable during two year follow-up.

Upstream transcription factor 1 (USF1) allelic variants significantly influence future risk of cardiovascular disease and overall mortality in females. We investigated sex-specific effects of USF1 gene allelic variants on serum indices of lipoprotein metabolism, early markers of asymptomatic atherosclerosis and their changes during six years of follow-up. In addition, we investigated the cis-regulatory role of these USF1 variants in artery wall tissues in Caucasians. In the Cardiovascular Risk in Young Finns Study, 1,608 participants (56% women, aged 31.9 ± 4.9) with lipids and cIMT data were included. For functional study, whole genome mRNA expression profiling was performed in 91 histologically classified atherosclerotic samples. In females, serum total, LDL cholesterol and apoB levels increased gradually according to USF1 rs2516839 genotypes TT < CT < CC and rs1556259 AA < AG < GG as well as according to USF1 H3 (GCCCGG) copy number 0 < 1 < 2. Furthermore, the carriers of minor alleles of rs2516839 (C) and rs1556259 (G) of USF1 gene had decreased USF1 expression in atherosclerotic plaques (P = 0.028 and 0.08, respectively) as compared to non-carriers. The genetic variation in USF1 influence USF1 transcript expression in advanced atherosclerosis and regulates levels and metabolism of circulating apoB and apoB-containing lipoprotein particles in sex-dependent manner, but is not a major determinant of early markers of atherosclerosis.

MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression at post-transcriptional level. Dysregulation of miRNA expression may lead to severe pathophysiologies in human cells. Niemann-Pick type C (NPC) disease is a complex lipid storage disease characterized by late endosomal-lysosomal accumulation of multiple lipid molecules. Our aim was to characterize the miRNA profile in NPC fibroblasts as they may play an active role in the NPC disease associated changes in the cellular physiology. To investigate the miRNA expression, total RNAs were isolated from cultured human NPC fibroblasts and healthy fibroblasts and then, TaqMan Low-Density Array system containing 365 mature human miRNAs was used. Expression differences between the healthy and NPC cells were detected according to the relative quantification values. Target genes were predicted by using three different algorithms and classified regarding NPC related biological processes and cellular components. We found that three miRNAs, miR-196a, miR-196b and miR-296 were up-regulated (>3.5-fold increase, p<0.05) whereas 38 miRNAs were significantly down-regulated in NPC cells (>3.5-fold decrease, p<0.05). Among these non-coding RNAs, miR-98 was the most down-regulated (-33.3-fold) miRNA and miR-143, the lipid biosynthesis associated miRNA, had a 20-fold decreased expression in the NPC cells. Additionally, gene ontology analyses of the target genes suggested a distinct role for each miRNA. Our results show that NPC fibroblasts have an altered miRNA expression profile and certain miRNAs have importance in disease pathogenesis as well as the therapeutic capacity to correct lipid related pathophysiologies in the NPC cells., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Plasma concentrations of total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglycerides are among the most important risk factors for coronary artery disease (CAD) and are targets for therapeutic intervention. We screened the genome for common variants associated with plasma lipids in >100,000 individuals of European ancestry. Here we report 95 significantly associated loci (P < 5 x 10(-8)), with 59 showing genome-wide significant association with lipid traits for the first time. The newly reported associations include single nucleotide polymorphisms (SNPs) near known lipid regulators (for example, CYP7A1, NPC1L1 and SCARB1) as well as in scores of loci not previously implicated in lipoprotein metabolism. The 95 loci contribute not only to normal variation in lipid traits but also to extreme lipid phenotypes and have an impact on lipid traits in three non-European populations (East Asians, South Asians and African Americans). Our results identify several novel loci associated with plasma lipids that are also associated with CAD. Finally, we validated three of the novel genes-GALNT2, PPP1R3B and TTC39B-with experiments in mouse models. Taken together, our findings provide the foundation to develop a broader biological understanding of lipoprotein metabolism and to identify new therapeutic opportunities for the prevention of CAD.

Background: The number of patients eligible for cardiovascular therapies in general is forecast to increase substantially in the coming decades. However, the current list of potential future cardiovascular blockbuster drugs is alarmingly short. There is thus a clear need for innovative strategies to increase the efficiency of drug development pipelines by establishing new sensitive biomarkers to monitor drug efficacy and safety in the context of complexity of lipoprotein metabolism targeted by the cardiovascular drugs., Methods: Metabolomics is a discipline dedicated to the systematic study of small molecules in cells, tissues and biofluids. Since lipids (including cholesterol), as well as other metabolites, are key constituents of lipoprotein particles and are thus part of the complex lipoprotein metabolism that includes exchange of lipids and metabolites with peripheral tissues, cardiovascular drug safety and efficacy needs to be addressed in the context of systemic lipid metabolism., Results/conclusion: Metabolomics, lipidomics in particular, is expected to make an important impact on the discovery and development of cardiovascular therapies.

Recent data suggest that aggressive lipid-lowering treatment results in significant reductions of atherosclerotic complications, ie, strokes, heart attacks, or peripheral vascular diseases. Thus, more patients will be titrated to higher doses of statins in order to reach the aggressive targets of low-density lipoprotein- cholesterol reduction. However, aggressive treatment with high statin doses has increased the risk of statin-induced myopathy. The incidence of myopathy in cohort studies and in randomized trials has been low, supporting the good safety profile of statin drugs. However, muscle effects seem to be more frequent in clinical practice. Of all statin users, approximately 1% to 5% suffers from muscular symptoms caused by medication. This potentially reduces the compliance toward treatment and number of patients reaching their treatment targets due to withdrawal of therapy. Thus, novel biomarkers are needed for prediction or improved diagnoses of statin-induced side effects. This would potentially increase the quality of life of patients and improve treatment results. Using lipidomic analysis, we found that the plasma lipidomic changes following simvastatin treatment correlate with the muscle expression of the arachidonate 5-lipoxygenase-activating protein. Intriguingly, these results suggest that the plasma lipidomic profile may serve as a highly sensitive biomarker of statin-induced metabolic alterations in muscle and may thus allow us to identify patients who should be treated with a lower dose to prevent a possible toxicity.

A polymorphism at position -511 of interleukin-1B (IL-1B) gene promoter regulates IL-1B levels, immune and inflammatory responses and possible atherogenesis. We used positron emission tomography (PET) to study whether coronary reactivity or its response to pravastatin is related to this IL-1B polymorphism. The study comprised a randomized, double-blind, placebo-controlled trial with two treatment groups: (i) pravastatin (40 mg/day, n=14) and (ii) placebo (n=20) for 6 months (baseline mean cholesterol 5.5 +/- 0.8 mmol/l; age 35 +/- 4 years). Myocardial blood flow was measured by PET at rest and during adenosine infusion using 15O-labelled water. PET studies, lipid, IL-1beta and C-reactive protein analyses were performed at baseline and after 6 months of therapy. IL-1B genotype was determined by polymerase chain reaction. There were no differences between IL-1B allele 2 carriers (A2+) and non-carriers (A2-) in basal or adenosine-stimulated myocardial flow (ASMF), at baseline. Regarding the change in ASMF and coronary flow reserve, there was a significant IL-1B genotype-by-treatment group interaction (analysis of covariance, P=0.028 and P=0.002, respectively) during follow-up. In the pravastatin group, the ASMF increased by 18.0% in subjects with IL-1B A2- (n=7), but decreased by 2% in subjects with IL-1B A2+ (n=7). There were no significant changes from the baseline values in placebo recipients. After treatment, both genotype groups showed a similar decrease in serum total and low density lipoprotein cholesterol (P<0.0001 for both). In conclusion, coronary function improves after 6 months of pravastatin therapy in subjects with the IL-1B A2- allele but not in those with the IL-1B A2+ allele.

Genetic architecture of plasma lipidome provides insights into regulation of lipid metabolism and related diseases. We applied an unsupervised machine learning method, PGMRA, to discover phenotype-genotype many-to-many relations between genotype and plasma lipidome (phenotype) in order to identify the genetic architecture of plasma lipidome profiled from 1,426 Finnish individuals aged 30–45 years. PGMRA involves biclustering genotype and lipidome data independently followed by their inter-domain integration based on hypergeometric tests of the number of shared individuals. Pathway enrichment analysis was performed on the SNP sets to identify their associated biological processes. We identified 93 statistically significant (hypergeometric p-value < 0.01) lipidome-genotype relations. Genotype biclusters in these 93 relations contained 5977 SNPs across 3164 genes. Twenty nine of the 93 relations contained genotype biclusters with more than 50% unique SNPs and participants, thus representing most distinct subgroups. We identified 30 significantly enriched biological processes among the SNPs involved in 21 of these 29 most distinct genotype-lipidome subgroups through which the identified genetic variants can influence and regulate plasma lipid related metabolism and profiles. This study identified 29 distinct genotype-lipidome subgroups in the studied Finnish population that may have distinct disease trajectories and therefore could be useful in precision medicine research. [ABSTRACT FROM AUTHOR]

Background and Purpose: Fenretinide, a synthetic retinoid derivative first investigated for cancer prevention and treatment, has been shown to ameliorate glucose tolerance, improve plasma lipid profile and reduce body fat mass. These effects, together with its ability to inhibit ceramide synthesis, suggest that fenretinide may have an anti-atherosclerotic action.Experimental Approach: To this aim, nine-week-old apoE-knockout (EKO) female mice were fed for twelve weeks a Western diet, without (control) or with (0.1% w/w) fenretinide. As a reference, wild-type (WT) mice were treated similarly. Growth and metabolic parameters were monitored throughout the study. Atherosclerosis development was evaluated in the aorta and at the aortic sinus. Blood and lymphoid organs were further characterized with thorough cytological/histological and immunocytofluorimetric analyses.Key Results: Fenretinide treatment significantly lowered body weight, glucose levels and plasma levels of total cholesterol, triglycerides, and phospholipids. In the liver, fenretinide remarkably reduced hepatic glycogenosis and steatosis driven by the Western diet. Treated spleens were abnormally enlarged, with severe follicular atrophy and massive extramedullary haematopoiesis. Severe renal hemosiderin deposition was observed in treated EKO mice. Treatment resulted in a threefold increase of total leukocytes (WT and EKO) and raised the activated/resting monocyte ratio in EKO mice. Finally, atherosclerosis development was markedly increased at the aortic arch, thoracic and abdominal aorta of fenretinide-treated mice.Conclusions and Implications: We provide the first evidence that, despite beneficial metabolic effects, fenretinide treatment may enhance the development of atherosclerosis. [ABSTRACT FROM AUTHOR]

The impact of cigarette smoke (CS), a major cause of lung diseases, on the composition and metabolism of lung lipids is incompletely understood. Here, we integrated quantitative lipidomics and proteomics to investigate exposure effects on lung lipid metabolismin a C57BL/6 and an Apolipoprotein E-deficient (Apoe-/-) mouse study. In these studies, mice were exposed to high concentrations of 3R4F reference CS, aerosol from potential modified risk tobacco products (MRTPs) or filtered air (Sham) for up to 8 months. The 2 assessed MRTPs, the prototypical MRTP for C57BL/6 mice and the Tobacco Heating System 2.2 for Apoe-/- mice, utilize"heat-not-burn" technologies and were each matched in nicotine concentrations to the 3R4F CS. After 2 months of CS exposure, some groups were either switched to the MRTP or underwent cessation. In both mouse strains, CS strongly affected several categories of lung lipids and lipid-related proteins. Candidate surfactant lipids, surfactant proteins, and surfactant metabolizing proteins were increased. Inflammatory eicosanoids, their metabolic enzymes, and several ceramide classes were elevated. Overall, CS induced a coordinated lipid response controlled by transcription regulators such as SREBP proteins and supported by other metabolic adaptations. In contrast, most of these changes were absent in the mice exposed to the potential MRTPs, in the cessation group, and the switching group. Our findings demonstrate the complex biological response of the lungs to CS exposure and support the benefits of cessation or switching to a heat-not-burn product using a design such as those employed in this study. [ABSTRACT FROM AUTHOR]

• Independent value over and above traditional CVD risk factors has consistently been shown. • Independent predictive value suggests a strong clinical utility for ceramides in gauging CVD risk. • Ceramides are actionable and modifiable CVD risk markers. • Ceramide score is used in assessing risk of patients deemed to be of intermediate risk. • Ceramide score is used also to assess response to interventions and to motivate patient compliance. Ceramides are bioactive lipids that act as secondary messengers for both intra- and inter-cellular signaling. Elevated plasma concentrations of ceramides are associated with multiple risk factors of atherosclerotic cardiovascular diseases and comorbidities including obesity, insulin resistance and diabetes mellitus. Furthermore, atherosclerotic plaques have been shown to be highly enriched with ceramides. Increases in ceramide content may accelerate atherosclerosis development by promoting LDL infiltration to the endothelium and aggregation within the intima of artery walls. Thus, ceramides appear to play a key role in the development of cardiometabolic disease due to their central location in major metabolic pathways that intersect lipid and glucose metabolism. Recently published data have shown that ceramides are not only of scientific interest but may also have diagnostic value. Their independent prognostic value for future cardiovascular outcomes over and above LDL cholesterol and other traditional risk factors have consistently been shown in numerous clinical studies. Thus, ceramide testing with a mass spectrometer offers a simple, reproducible and cost-effective blood test for risk stratification in atherosclerotic cardiovascular diseases. [ABSTRACT FROM AUTHOR]