Mitochondrial lipidomes are tissue specific - low cholesterol contents relate to UCP1 activity.

Lipid composition is conserved within sub-cellular compartments to maintain cell function. Lipidomic analyses of liver, muscle, white and brown adipose tissue (BAT) mitochondria revealed substantial differences in their glycerophospholipid (GPL) and free cholesterol (FC) contents. The GPL to FC ratio was 50-fold higher in brown than white adipose tissue mitochondria. Their purity was verified by comparison of proteomes with ER and mitochondria-associated membranes. A lipid signature containing PC and FC, calculated from the lipidomic profiles, allowed differentiation of mitochondria from BAT of mice housed at different temperatures. Elevating FC in BAT mitochondria prevented uncoupling protein (UCP) 1 function, whereas increasing GPL boosted it. Similarly, STARD3 overexpression facilitating mitochondrial FC import inhibited UCP1 function in primary brown adipocytes, whereas a knockdown promoted it. We conclude that the mitochondrial GPL/FC ratio is key for BAT function and propose that targeting it might be a promising strategy to promote UCP1 activity.

Phospholipase PLA2G7 is complementary to GPX4 in mitigating punicic-acid-induced ferroptosis in prostate cancer cells.

Ferroptosis is a cell death pathway that can be promoted by peroxidizable polyunsaturated fatty acids in cancer cells. Here, we investigated the mechanisms underlying the toxicity of punicic acid (PunA), an isomer of conjugated linolenic acids (CLnAs) bearing three conjugated double bonds highly prone to peroxidation, on prostate cancer (PCa) cells. PunA induced ferroptosis in PCa cells and triggered massive lipidome remodeling, more strongly in PC3 androgen-negative cells than in androgen-positive cells. The greater sensitivity of androgen-negative cells to PunA was associated with lower expression of glutathione peroxidase 4 (GPX4). We then identified the phospholipase PLA2G7 as a PunA-induced ferroptosis suppressor in PCa cells. Overexpressing PLA2G7 decreased lipid peroxidation levels, suggesting that PLA2G7 hydrolyzes hydroperoxide-containing phospholipids, thus preventing ferroptosis. Importantly, overexpressing both PLA2G7 and GPX4 strongly prevented PunA-induced ferroptosis in androgen-negative PCa cells. This study shows that PLA2G7 acts complementary to GPX4 to protect PCa cells from CLnA-induced ferroptosis.

Exploration of the Lipid Profile of Edible Oleaginous Microgreens by Mass Spectrometry-Based Lipidomics.

The spreading awareness of the health benefits associated with the consumption of plant-based foods is fueling the market of innovative vegetable products, including microgreens, recognized as a promising source of bioactive compounds. To evaluate the potential of oleaginous plant microgreens as a source of bioactive fatty acids, gas chromatography-mass spectrometry was exploited to characterize the total fatty acid content of five microgreens, namely, chia, flax, soy, sunflower, and rapeseed (canola). Chia and flax microgreens appeared as interesting sources of α-linolenic acid (ALA), with total concentrations of 2.6 and 2.9 g/100 g of dried weight (DW), respectively. Based on these amounts, approximately 15% of the ALA daily intake recommended by the European Food Safety Authority can be provided by 100 g of the corresponding fresh products. Flow injection analysis with high-resolution Fourier transform single and tandem mass spectrometry enabled a semi-quantitative profiling of triacylglycerols (TGs) and sterol esters (SEs) in the examined microgreen crops, confirming their role as additional sources of fatty acids like ALA and linoleic acid (LA), along with glycerophospholipids. The highest amounts of TGs and SEs were observed in rapeseed and sunflower microgreens (ca. 50 and 4-5 µmol/g of DW, respectively), followed by flax (ca. 20 and 3 µmol/g DW). TG 54:9, 54:8, and 54:7 prevailed in the case of flax and chia, whereas TG 54:3, 54:4, and 54:5 were the most abundant TGs in the case of rapeseed. ß-Sitosteryl linoleate and linolenate were generally prevailing in the SE profiles, although campesteryl oleate, linoleate, and linolenate exhibited a comparable amount in the case of rapeseed microgreens.

Mitochondrial and Cellular Function in Fibroblasts, Induced Neurons, and Astrocytes Derived from Case Study Patients: Insights into Major Depression as a Mitochondria-Associated Disease.

The link between mitochondria and major depressive disorder (MDD) is increasingly evident, underscored both by mitochondria's involvement in many mechanisms identified in depression and the high prevalence of MDD in individuals with mitochondrial disorders. Mitochondrial functions and energy metabolism are increasingly considered to be involved in MDD's pathogenesis. This study focused on cellular and mitochondrial (dys)function in two atypical cases: an antidepressant non-responding MDD patient ("Non-R") and another with an unexplained mitochondrial disorder ("Mito"). Skin biopsies from these patients and controls were used to generate various cell types, including astrocytes and neurons, and cellular and mitochondrial functions were analyzed. Similarities were observed between the Mito patient and a broader MDD cohort, including decreased respiration and mitochondrial function. Conversely, the Non-R patient exhibited increased respiratory rates, mitochondrial calcium, and resting membrane potential. In conclusion, the Non-R patient's data offered a new perspective on MDD, suggesting a detrimental imbalance in mitochondrial and cellular processes, rather than simply reduced functions. Meanwhile, the Mito patient's data revealed the extensive effects of mitochondrial dysfunctions on cellular functions, potentially highlighting new MDD-associated impairments. Together, these case studies enhance our comprehension of MDD.

Rising Lysophosphatidylcholine Levels Post-Hepatitis C Clearance.

Hepatitis C virus (HCV) infection alters lysophosphatidylcholine (LPC) metabolism, enhancing viral infectivity and replication. Direct-acting antivirals (DAAs) effectively treat HCV and rapidly normalize serum cholesterol. In serum, LPC species are primarily albumin-bound but are also present in lipoprotein particles. This study aims to assess the impact of HCV eradication on serum LPC species levels in patients infected with HCV. Therefore, 12 different LPC species were measured by electrospray ionization tandem mass spectrometry (ESI-MS/MS) in the sera of 178 patients with chronic HCV infections at baseline, and in 176 of these patients after therapy with DAAs. All LPC species increased at 4 and 12 weeks post-initiation of DAA therapy. The serum profiles of the LPC species were similar before and after the viral cure. Patients with HCV and liver cirrhosis exhibited lower serum levels of all LPC species, except LPC 16:1, both before and after DAA treatment. Percentages of LPC 18:1 (relative to the total LPC level) were higher, and % LPC 22:5 and 22:6 were lower in cirrhotic compared to non-cirrhotic patients at baseline and at the end of therapy. LPC species levels inversely correlated with the model of end-stage liver disease score and directly with baseline and post-therapy albumin levels. Receiver operating characteristic curve analysis indicated an area under the curve of 0.773 and 0.720 for % LPC 18:1 (relative to total LPC levels) for classifying fibrosis at baseline and post-therapy, respectively. In summary, HCV elimination was found to increase all LPC species and elevated LPC 18:1 relative to total LPC levels may have pathological significance in HCV-related liver cirrhosis.

A High-Phosphorus Diet Moderately Alters the Lipidome and Transcriptome in the Skeletal Muscle of Adult Mice.

A high phosphorus intake has been associated with various metabolic disorders, including chronic kidney disease, cardiovascular disease, and osteoporosis. Recent studies have demonstrated the effects of dietary phosphorus on lipid and glucose metabolism. This study investigated the impact of a high-phosphorus diet on mouse skeletal muscle lipid composition and gene transcription. Adult male mice (n = 12/group) received either a diet with an adequate (0.3%) or a high (1.2%) phosphorus concentration for 6 weeks. The lipidome analysis showed that among the 17 analyzed lipid classes, the concentrations of three classes were reduced in the high phosphorus group compared to the adequate phosphorus group. These classes were phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and lysophosphatidylcholine (LPC) (p < 0.05). Out of the three hundred and twenty-three individual lipid species analyzed, forty-nine showed reduced concentrations, while three showed increased concentrations in the high phosphorus group compared to the adequate phosphorus group. The muscle transcriptome analysis identified 142 up- and 222 down-regulated transcripts in the high phosphorus group compared to the adequate phosphorus group. Gene set enrichment analysis identified that genes that were up-regulated in the high phosphorus group were linked to the gene ontology terms "mitochondria" and "Notch signaling pathway", whereas genes that were down-regulated were linked to the "PI3K-AKT pathway". Overall, the effects of the high-phosphorus diet on the muscle lipidome and transcriptome were relatively modest, but consistently indicated an impact on lipid metabolism.

Gender-Specific Differences in Serum Sphingomyelin Species in Patients with Hepatitis C Virus Infection-Sphingomyelin Species Are Related to the Model of End-Stage Liver Disease (MELD) Score in Male Patients.

Hepatitis C virus (HCV) replication depends on cellular sphingomyelin (SM), but serum SM composition in chronic HCV infection has been hardly analyzed. In this work, 18 SM species could be quantified in the serum of 178 patients with chronic HCV infection before therapy with direct-acting antivirals (DAAs) and 12 weeks later, when therapy was completed. Six SM species were higher in the serum of females than males before therapy and nine at the end of therapy; thus, sex-specific analysis was performed. Type 2 diabetes was associated with lower serum levels of SM 36:2;O2 and 38:2;O2 in men. Serum SM species did not correlate with the viral load in both sexes. Of note, three SM species were lower in males infected with HCV genotype 3 in comparison to genotype 1 infection. These SM species normalized after viral cure. SM 38:1;O2, 40:1;O2, 41:1;O2, and 42:1;O2 (and, thus, total SM levels) were higher in the serum of both sexes at the end of therapy. In males, SM 39:1;O2 was induced in addition, and higher levels of all of these SM species were already detected at 4 weeks after therapy has been started. Serum lipids are related to liver disease severity, and in females 15 serum SM species were low in patients with liver cirrhosis before initiation of and after treatment with DAAs. The serum SM species did not correlate with the model of end-stage liver disease (MELD) score in the cirrhosis and the non-cirrhosis subgroups in females. In HCV-infected male patients, nine SM species were lower in the serum of patients with cirrhosis before DAA treatment and eleven at the end of the study. Most of the SM species showed strong negative correlations with the MELD score in the male cirrhosis patients before DAA treatment and at the end of therapy. Associations of SM species with the MELD score were not detected in the non-cirrhosis male subgroup. In summary, the current analysis identified sex-specific differences in the serum levels of SM species in HCV infection, in liver cirrhosis, and during DAA therapy. Correlations of SM species with the MELD score in male but not in female patients indicate a much closer association between SM metabolism and liver function in male patients.

The ubiquitin ligase Uhrf2 is a master regulator of cholesterol biosynthesis and is essential for liver regeneration.

Fibroblast growth factors (FGFs) are key regulators of the remarkable regenerative capacity of the liver. Mice lacking FGF receptors 1 and 2 (Fgfr1 and Fgfr2) in hepatocytes are hypersensitive to cytotoxic injury during liver regeneration. Using these mice as a model for impaired liver regeneration, we identified a critical role for the ubiquitin ligase Uhrf2 in protecting hepatocytes from bile acid accumulation during liver regeneration. During regeneration after partial hepatectomy, Uhrf2 expression increased in an FGFR-dependent manner, and Uhrf2 was more abundant in the nuclei of liver cells in control mice compared with FGFR-deficient mice. Hepatocyte-specific Uhrf2 knockout or nanoparticle-mediated Uhrf2 knockdown caused extensive liver necrosis and impaired hepatocyte proliferation after partial hepatectomy, resulting in liver failure. In cultured hepatocytes, Uhrf2 interacted with several chromatin remodeling proteins and suppressed the expression of cholesterol biosynthesis genes. In vivo, the loss of Uhrf2 resulted in cholesterol and bile acid accumulation in the liver during regeneration. Treatment with a bile acid scavenger rescued the necrotic phenotype, hepatocyte proliferation, and the regenerative capacity of the liver in Uhrf2-deficient mice subjected to partial hepatectomy. Our results identify Uhrf2 as a key target of FGF signaling in hepatocytes and its essential function in liver regeneration and highlight the importance of epigenetic metabolic regulation in this process.

Replacement of soybean oil by Hermetia illucens larvae fat in broiler diets alters the breast muscle lipidome and reduces lipid oxidation of the breast muscle during heat-processing.

Replacement of soybean oil by insect fat from Hermetia illucens (HI) has been reported to increase the proportions of saturated fatty acids (SFA) and decrease those of polyunsaturated fatty acids (PUFA) in total lipids of breast and thigh meat in broilers. Since the susceptibility of meat to oxidation is strongly dependent on its PUFA content, the present study hypothesised that replacement of soybean oil by HI larvae fat in broiler diets reduces the formation of lipid oxidation products, including oxidation products of cholesterol and phytosterols, in heat-processed breast muscle of broilers. To test this hypothesis, 100 male, 1-day-old Cobb 500 broilers were assigned to three groups and fed three different nutrient adequate diets, which varied only in the fat source (group HI-0: 0% HI larvae fat and 5% soybean oil; group HI-2.5: 2.5% HI larvae fat and 2.5% soybean oil; group HI-5.0: 5.0% HI larvae fat and 0% soybean oil), in a three-phase feeding system for 35 days. While the growth performance of the broilers was not different, the absolute and relative breast muscle weights were higher in group HI-5.0 than in group HI-0 (p < 0.05). The proportions of C12:0, C14:0, C14:1, C16:0, C16:1 and total SFA were higher and those of C18:1, C18:2 n-6, C18:3 n-3 and total PUFA were lower in breast muscle total lipids of group HI-5.0 than in groups HI-2.5 and HI-0 (p < 0.05). Lipidomic analysis of breast muscle revealed that the concentration of triacylglycerols was 46% and 53% lower in groups HI-2.5 and HI-5.0, respectively, than in group HI-0 (p < 0.05), whereas all other lipid classes detected did not differ among groups. Concentrations of thiobarbituric acid-reactive substances, 7α-hydroxycholesterol, 7ß-hydroxycholesterol and total cholesterol oxidation products in heat-processed breast muscle were lower in group HI-5.0 than in group HI-0 (p < 0.05). Concentrations of oxidation products of phytosterols in heat-processed breast muscle were generally much lower than those of cholesterol oxidation products and did not differ between the three groups of broilers. In conclusion, complete replacement of soybean oil with HI larvae fat in broiler diets strongly alters the fatty acid composition of breast muscle total lipids and reduce lipid oxidation of the breast muscle during heat-processing.

Trib1 Deficiency Promotes Hyperlipidemia, Inflammation, and Atherosclerosis in LDL Receptor Knockout Mice.

BACKGROUND: Genetic variants at the TRIB1 gene locus are strongly associated with plasma lipid traits and the risk of coronary artery disease in humans. Here, we analyzed the consequences of Trib1 deficiency on lipid metabolism and atherosclerotic lesion formation in atherosclerosis-susceptible Ldlr-/- mice. METHODS: Trib1-/- mice were crossed onto the Ldlr-/- background to generate double-knockout mice (Trib1-/-Ldlr-/-) and fed a semisynthetic, modified AIN76 diet (0.02% cholesterol and 4.3% fat) until 20 weeks of age. RESULTS: Trib1-/-Ldlr-/- mice had profoundly larger (5.8-fold) and more advanced atherosclerotic lesions at the aortic root as compared with Trib1+/+Ldlr-/- controls. Further, we observed significantly elevated plasma total cholesterol and triglyceride levels in Trib1-/-Ldlr-/- mice, resulting from higher VLDL (very-low-density lipoprotein) secretion. Lipidomics analysis revealed that loss of Trib1 altered hepatic lipid composition, including the accumulation of cholesterol and proinflammatory ceramide species, which was accompanied by signs of hepatic inflammation and injury. Concomitantly, we detected higher plasma levels of IL (interleukin)-6 and LCN2 (lipocalin 2), suggesting increased systemic inflammation in Trib1-/-Ldlr-/- mice. Hepatic transcriptome analysis demonstrated significant upregulation of key genes controlling lipid metabolism and inflammation in Trib1-/-Ldlr-/- mice. Further experiments suggested that these effects may be mediated through pathways involving a C/EPB (CCAAT/enhancer binding protein)-PPARγ (peroxisome proliferator-activated receptor γ) axis and JNK (c-Jun N-terminal kinase) signaling. CONCLUSIONS: We provide experimental evidence that Trib1 deficiency promotes atherosclerotic lesion formation in a complex manner that includes the modulation of lipid metabolism and inflammation.

Quantification of bulk lipid species in human platelets and their thrombin-induced release.

Lipids play a central role in platelet physiology. Changes in the lipidome have already been described for basal and activated platelets. However, quantitative lipidomic data of platelet activation, including the released complex lipids, are unavailable. Here we describe an easy-to-use protocol based on flow-injection mass spectrometry for the quantitative analysis of bulk lipid species in basal and activated human platelets and their lipid release after thrombin activation. We provide lipid species concentrations of 12 healthy human donors, including cholesteryl ester (CE), ceramide (Cer), free cholesterol (FC), hexosylceramide (HexCer), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SM) and triglycerides (TG). The assay exhibited good technical repeatability (CVs < 5% for major lipid species in platelets). Except for CE and TG, the inter-donor variability of the majority of lipid species concentrations in platelets was < 30% CV. Balancing of concentrations revealed the generation of LPC and loss of TG. Changes in lipid species concentrations indicate phospholipase-mediated release of arachidonic acid mainly from PC, PI, and PE but not from PS. Thrombin induced lipid release was mainly composed of FC, PS, PC, LPC, CE, and TG. The similarity of the released lipidome with that of plasma implicates that lipid release may originate from the open-canalicular system (OCS). The repository of lipid species concentrations determined with this standardized platelet release assay contribute to elucidating the physiological role of platelet lipids and provide a basis for investigating the platelet lipidome in patients with hemorrhagic or thrombotic disorders.

Effect of replacement of soybean oil by Hermetia illucens fat on performance, digestibility, cecal microbiome, liver transcriptome and liver and plasma lipidomes of broilers.

BACKGROUND: In contrast to protein-rich insect meal, the feed potential of insect fat is generally less explored and knowledge about the suitability of insect fat as a fat source specifically in broiler diets is still limited. In view of this, the present study aimed to comprehensively investigate the effect of partial (50%) and complete replacement of soybean oil with insect fat from Hermetia illucens (HI) larvae in broiler diets on performance, fat digestibility, cecal microbiome, liver transcriptome and liver and plasma lipidomes. Thus, 100 male, 1-day-old Cobb 500 broilers were randomly assigned to three groups and fed three different diets with either 0 (group HI-0, n = 30), 2.5% (group HI-2.5, n = 35) or 5.0% (HI-5.0, n = 35) Hermetia illucens (HI) larvae fat for 35 d. RESULTS: Body weight gain, final body weight, feed intake, and feed:gain ratio during the whole period and apparent ileal digestibility coefficient for ether extract were not different between groups. Cecal microbial diversity did not differ between groups and taxonomic analysis revealed differences in the abundance of only four low-abundance bacterial taxa among groups; the abundances of phylum Actinobacteriota, class Coriobacteriia, order Coriobacteriales and family Eggerthellaceae were lower in group HI-5.0 compared to group HI-2.5 (P < 0.05). Concentrations of total and individual short-chain fatty acids in the cecal digesta were not different between the three groups. Liver transcriptomics revealed a total of 55 and 25 transcripts to be differentially expressed between groups HI-5.0 vs. HI-0 and groups HI-2.5 vs. HI-0, respectively (P < 0.05). The concentrations of most lipid classes, with the exception of phosphatidylethanolamine, phosphatidylglycerol and lysophosphatidylcholine in the liver and cholesterylester and ceramide in plasma (P < 0.05), and of the sum of all lipid classes were not different between groups. CONCLUSIONS: Partial and complete replacement of soybean oil with HI larvae fat in broiler diets had no effect on growth performance and only modest, but no adverse effects on the cecal microbiome and the metabolic health of broilers. This suggests that HI larvae fat can be used as an alternative fat source in broiler diets, thereby, making broiler production more sustainable.

A low-carbohydrate diet induces hepatic insulin resistance and metabolic associated fatty liver disease in mice.

OBJECTIVES: Metabolic-associated fatty liver disease (MAFLD) is the most common chronic liver disease that can range from hepatic steatosis to non-alcoholic steatohepatitis (NASH), which can lead to fibrosis and cirrhosis. Recently, ketogenic diet (KD), a low carbohydrate diet, gained popularity as a weight-loss approach, although it has been reported to induce hepatic insulin resistance and steatosis in animal model systems via an undefined mechanism. Herein, we investigated the KD metabolic benefits and its contribution to the pathogenesis of NASH. METHODS: Using metabolic, biochemical and omics approaches, we identified the effects of a KD on NASH and investigated the mechanisms by which KD induces hepatic insulin resistance and steatosis. RESULTS: We demonstrate that KD can induce fibrosis and NASH regardless of body weight loss compared to high-fat diet (HFD) fed mice at thermoneutrality. At ambient temperature (23 °C), KD-fed mice develop a severe hepatic injury, inflammation, and steatosis. In addition, KD increases liver cholesterol, IL-6, and p-JNK and aggravates diet induced-glucose intolerance and hepatic insulin resistance compared to HFD. Pharmacological inhibition of IL-6 and JNK reverses KD-induced glucose intolerance, and hepatic steatosis and restores insulin sensitivity. CONCLUSIONS: Our studies uncover a new mechanism for KD-induced hepatic insulin resistance and NASH potentially via IL-6-JNK signaling and provide a new NASH mouse model.

Gut microbiota promote liver regeneration through hepatic membrane phospholipid biosynthesis.

BACKGROUND & AIMS: Hepatocyte growth and proliferation depends on membrane phospholipid biosynthesis. Short-chain fatty acids (SCFAs) generated by bacterial fermentation, delivered through the gut-liver axis, significantly contribute to lipid biosynthesis. We therefore hypothesized that dysbiotic insults like antibiotic treatment not only affect gut microbiota, but also impair hepatic lipid synthesis and liver regeneration. METHODS: Stable isotope labeling and 70% partial hepatectomy (PHx) was carried out in C57Bl/6J wild-type mice, in mice treated with broad-spectrum antibiotics, in germ-free mice and mice colonized with minimal microbiota. The microbiome was analyzed by 16S rRNA gene sequencing and microbial culture. Gut content, liver, blood and primary hepatocyte organoids were tested by mass spectrometry-based lipidomics, quantitative reverse-transcription PCR (qRT-PCR), immunoblot and immunohistochemistry for expression of proliferative and lipogenic markers. Matched biopsies from hyperplastic and hypoplastic liver tissue of patients subjected to surgical intervention to induce hyperplasia were analyzed by qRT-PCR for lipogenic enzymes. RESULTS: Three days of antibiotic treatment induced persistent dysbiosis with significantly decreased beta-diversity and richness, but a massive increase of Proteobacteria, accompanied by decreased colonic SCFAs. After PHx, antibiotic-treated mice showed delayed liver regeneration, increased mortality, impaired hepatocyte proliferation and decreased hepatic phospholipid synthesis. Expression of the lipogenic enzyme SCD1 was upregulated after PHx but delayed by antibiotic treatment. Germ-free mice essentially recapitulated the phenotype of antibiotic treatment. Phospholipid biosynthesis, hepatocyte proliferation, liver regeneration and survival were rescued in gnotobiotic mice colonized with a minimal SCFA-producing microbial community. SCFAs induced the growth of murine hepatocyte organoids and hepatic SCD1 expression in mice. Further, SCD1 was required for proliferation of human hepatoma cells and was associated with liver regeneration in human patients. CONCLUSION: Gut microbiota are pivotal for hepatic membrane phospholipid biosynthesis and liver regeneration. IMPACT AND IMPLICATIONS: Gut microbiota affect hepatic lipid metabolism through the gut-liver axis, but the underlying mechanisms are poorly understood. Perturbations of the gut microbiome, e.g. by antibiotics, impair the production of bacterial metabolites, which normally serve as building blocks for membrane lipids in liver cells. As a consequence, liver regeneration and survival after liver surgery is severely impaired. Even though this study is preclinical, its results might allow physicians in the future to improve patient outcomes after liver surgery, by modulation of gut microbiota or their metabolites.

HCV Infection and Liver Cirrhosis Are Associated with a Less-Favorable Serum Cholesteryl Ester Profile Which Improves through the Successful Treatment of HCV.

Background: Infection with hepatitis C virus (HCV) lowers serum cholesterol levels, which rapidly recover during therapy with direct-acting antivirals (DAAs). Serum cholesterol is also reduced in patients with liver cirrhosis. Studies investigating serum cholesterol in patients with chronic liver diseases are generally based on enzymatic assays providing total cholesterol levels. Hence, these studies do not account for the individual cholesteryl ester (CE) species, which have different properties according to acyl chain length and desaturation. Methods: Free cholesterol (FC) and 15 CE species were quantified by flow injection analysis high-resolution Fourier Transform mass spectrometry (FIA-FTMS) in the serum of 178 patients with chronic HCV before therapy and during treatment with DAAs. Results: Serum CEs were low in HCV patients with liver cirrhosis and, compared to patients without cirrhosis, proportions of CE 16:0 and 16:1 were higher whereas % CE 20:4 and 20:5 were reduced. FC levels were unchanged, and the CE/FC ratio was consequently low in cirrhosis. FC and CEs did not correlate with viral load. Four CE species were reduced in genotype 3 compared to genotype 1-infected patients. During DAA therapy, 9 of the 15 measured CE species, and the CE/FC ratio, increased. Relative to total CE levels, % CE 16:0 declined and % CE 18:3 was higher at therapy end. At this time, % CE 14:0, 16:0 and 16:1 were higher and % CE 20:4 and 22:6 were lower in the cirrhosis than the non-cirrhosis patients. Viral genotype associated changes of CEs disappeared at therapy end. Conclusions: The serum CE composition differs between patients with and without liver cirrhosis, and changes through the efficient elimination of HCV. Overall, HCV infection and cirrhosis are associated with a higher proportion of CE species with a lower number of carbon atoms and double bonds, reflecting a less-favorable CE profile.

Sex-specific changes in triglyceride profiles in liver cirrhosis and hepatitis C virus infection.

BACKGROUND: Hepatitis C virus (HCV) infection is associated with serum lipid abnormalities, which partly normalize following direct-acting antiviral (DAA) therapy. Here, associations of serum triglycerides (TGs) with viral genotype and markers of liver disease severity were evaluated in patients with chronic HCV.  METHODS: The study included the serum of 177 patients with chronic HCV. TGs were quantified by flow injection analysis Fourier transform mass spectrometry. Laboratory values and noninvasive scores for liver fibrosis assessment were determined. The nonparametric Kruskal‒Wallis test, one-way ANOVA, multiple linear regression and Student's t test were used as appropriate. P values were adjusted for multiple comparisons. RESULTS: HCV-infected women had lower serum TGs than men, and thus, a sex-specific analysis was performed. None of the 46 TG species analyzed differed in the serum of female patients with and without liver cirrhosis. In contrast, in the serum of male patients with liver cirrhosis, TGs with 53, 56 and 58 carbon atoms and three to eight double bonds were diminished. These polyunsaturated TGs were also low in males with a high fibrosis-4 score. TGs with 7 or 8 double bonds negatively correlated with the model of end-stage liver disease score in males. In addition, TGs with 49, 51 and 53 carbon atoms were reduced in male patients infected with genotype 3a in comparison to genotype 1a. TGs with 56 carbon atoms were lower in genotype 3a-infected males than in genotype 1b-infected males. TGs did not differ in females by genotype. Genotype 3-related changes disappeared at the end of therapy with DAAs. Overall, the levels of serum TGs did not change during DAA therapy in either sex. Consequently, the serum TGs of males with liver cirrhosis were lower than those of males without cirrhosis at the end of therapy. Such a difference was not apparent in females. CONCLUSIONS: The decline in TGs observed only in male patients with liver cirrhosis and male patients infected with genotype 3 illustrates sex-specific changes in lipid metabolism in chronic HCV.

Benchmarking One-Phase Lipid Extractions for Plasma Lipidomics.

A key element of successful lipidomics analysis is a sufficient extraction of lipid molecules typically by two-phase systems such as chloroform-based Bligh and Dyer (B&D). However, numerous metabolomics and lipidomics studies today apply easy to use one-phase extractions. In this work, quantitative flow injection analysis high-resolution mass spectrometry was applied to benchmark the lipid recovery of popular one-phase extraction methods for human plasma samples. The following organic solvents were investigated: methanol (MeOH), ethanol (EtOH), 2-propanol (IPA), 1-butanol (BuOH), acetonitrile (ACN) and the solvent mixtures BuOH/MeOH (3:1) and MeOH/ACN (1:1). The recovery of polar lysophospholipids was sufficient for all tested solvents. However, nonpolar lipid classes such as triglycerides (TG) and cholesteryl esters (CE) revealed extraction efficiencies less than 5% due to precipitation in polar solvents EtOH, MeOH, MeOH/ACN, and ACN. Sample pellets also contained a substantial amount of phospholipids, for example, more than 75% of total phosphatidylcholine and sphingomyelin for ACN. The loss of lipids by precipitation was directly related to the polarity of solvents and lipid classes. Although, lipid recovery increased with the volume of organic solvent, recovery in polar MeOH remains incomplete also for less polar lipid classes such as ceramides. Addition of stable isotope-labeled internal standards prior to lipid extraction could compensate for insufficient lipid recovery for polar lipid classes including lysolipids and phospholipids but not for nonpolar CE and TG. In summary, application of one-phase extractions should be limited to polar lipid classes unless sufficient recovery/solubility of nonpolar lipids has been demonstrated. The presented data reveal that appropriate lipid extraction efficiency is fundamental to achieve accurate lipid quantification.

Serum Ceramide Species Are Associated with Liver Cirrhosis and Viral Genotype in Patients with Hepatitis C Infection.

Hepatitis C virus (HCV) infection affects ceramide metabolism, and, here, we have evaluated associations of eight serum ceramide species with viral load, viral genotype, and disease markers in 178 patients with chronic HCV. In this cohort, ceramide d18:1;O2/16:0 was higher in the serum of the 20 diabetic patients compared to the patients without this complication. Moreover, ceramide d18:1;O2/24:0 was negatively correlated with age. Of note, all but ceramide d18:1;O2/16:0 and 26:0 were diminished in the serum of patients with liver cirrhosis and, with the exception of ceramide d18:1;O2/16:0, were negatively correlated with the model for end-stage liver disease (MELD) score. Most of the serum ceramides are carried in low-density lipoprotein (LDL), which rises following effective direct-acting antiviral (DAA) therapy. Ceramide d18:1;O2/24:0 recovered in parallel with LDL, whereas ceramide d18:1;O2/18:0 declined. Genotype-3-infected patients had the lowest ceramide levels, which were comparable to other genotypes after DAA treatment. Notably, ceramide d18:1;O2/23:0 and 24:0 were negatively correlated with the MELD score in patients with liver cirrhosis at the end of DAA therapy. Long-chain (LC) ceramides show adverse effects, whereas very-long-chain (VL) species have protective functions in the liver. The ratio of VL/LC ceramides was higher in non-cirrhosis patients than cirrhosis patients and further increased at the end of therapy in this subgroup. In summary, our study shows that serum ceramide levels are related to liver cirrhosis and viral genotype. Whether the more favorable serum ceramide profile in non-cirrhosis patients, before and after DAA therapy, is of pathophysiological importance needs further investigation.

GOLM1 depletion modifies cellular sphingolipid metabolism and adversely affects cell growth.

Golgi membrane protein 1 (GOLM1) is a Golgi-resident type 2 transmembrane protein known to be overexpressed in several cancers, including hepatocellular carcinoma (HCC), as well as in viral infections. However, the role of GOLM1 in lipid metabolism remains enigmatic. In this study, we employed siRNA-mediated GOLM1 depletion in Huh-7 HCC cells to study the role of GOLM1 in lipid metabolism. Mass spectrometric lipidomic analysis in GOLM1 knockdown cells showed an aberrant accumulation of sphingolipids, such as ceramides, hexosylceramides, dihexosylceramides, sphinganine, sphingosine, and ceramide phosphate, along with cholesteryl esters. Furthermore, we observed a reduction in phosphatidylethanolamines and lysophosphatidylethanolamines. In addition, Seahorse extracellular flux analysis indicated a reduction in mitochondrial oxygen consumption rate upon GOLM1 depletion. Finally, alterations in Golgi structure and distribution were observed both by electron microscopy imaging and immunofluorescence microscopy analysis. Importantly, we found that GOLM1 depletion also affected cell proliferation and cell cycle progression in Huh-7 HCC cells. The Golgi structural defects induced by GOLM1 reduction might potentially affect the trafficking of proteins and lipids leading to distorted intracellular lipid homeostasis, which may result in organelle dysfunction and altered cell growth. In conclusion, we demonstrate that GOLM1 depletion affects sphingolipid metabolism, mitochondrial function, Golgi structure, and proliferation of HCC cells.

Dietary PUFA Preferably Modify Ethanolamine-Containing Glycerophospholipids of the Human Plasma Lipidome.

The content of polyunsaturated fatty acids (PUFA) in complex lipids essentially influences their physicochemical properties and has been linked to health and disease. To investigate the incorporation of dietary PUFA in the human plasma lipidome, we quantified glycerophospholipids (GPL), sphingolipids, and sterols using electrospray ionization coupled to tandem mass spectrometry of plasma samples obtained from a dietary intervention study. Healthy individuals received foods supplemented with different vegetable oils rich in PUFA. These included sunflower, linseed, echium, and microalgae oil as sources of linoleic acid (LA; FA 18:2 n-6), alpha-linolenic acid (ALA; FA 18:3 n-3), stearidonic acid (SDA; FA 18:4 n-3), and docosahexaenoic acid (DHA; FA 22:6 n-3). While LA and ALA did not influence the species profiles of GPL, sphingolipid, and cholesteryl ester drastically, SDA and DHA were integrated primarily in ethanolamine-containing GPL. This significantly altered phosphatidylethanolamine and plasmalogen species composition, especially those with 38-40 carbons and 6 double bonds. We speculate that diets enriched with highly unsaturated FA more efficiently alter plasma GPL acyl chain composition than those containing primarily di- and tri-unsaturated FA, most likely because of their more pronounced deviation of FA composition from typical western diets.