Homocysteine Leads to Decreased Acylcarnitine Levels in the Heart in a Rabbit Model of Atherosclerosis

Homocysteine Leads to Decreased Acylcarnitine Levels in the Heart in a Rabbit Model of Atherosclerosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Homocysteine Leads to Decreased Acylcarnitine Levels in the Heart in a Rabbit Model of Atherosclerosis Markus S. Brunner, Thomas Züllig, Elisa Talker, Dagmar Kolb, Gerd Leitinger, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6717713/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cardiovascular disease, the leading cause of death worldwide, is mainly a result of atherosclerosis. However, 50% of all cases of atherosclerosis still cannot be explained by known risk factors including hypercholesterolemia. Hyperhomocysteinemia, an elevation of homocysteine (Hcy) levels in the blood, is an independent risk factor for atherosclerosis, aggravates atherosclerosis in the presence of hypercholesterolemia and strongly correlates with cardiovascular mortality. We showed that a diet deficient in vitamins and choline required for Hcy degradation (VCDD) leads to cholesterol-independent atherogenic transformation of the aorta and aortic lipid accumulation in balloon-injured rabbit model of atherosclerosis (Almer et al , 2022, Biomed Pharmacother). Elevation of plasma Hcy by intravenous injections of Hcy into VCDD-fed rabbits results in further atherogenic changes, degradation of aortic lipid droplets, decreased total protein methylated arginine and altered metabolomic profiles compared to rabbits fed VCDD only (Tehlivets et al , 2024, Biomed Pharmacother). Here we show that feeding VCDD with or without intravenous injections of Hcy leads to dysregulation of lipid metabolism in blood cells, heart and aorta in rabbits. Hcy has a graded effect on lipid metabolism deregulating glycerolipid, cholesterol, ceramide, acylcarnitine and fatty acid metabolism in different tissues. Accumulation of triglycerides in response to VCDD and their decrease in response to VCDD in combination with Hcy injections in blood cells and heart, accumulation of cholesterol in blood cells, heart and aorta, accumulation of ceramides and fatty acids in blood cells and drastic drop in myocardial acylcarnitines suggest mechanisms how elevated Hcy may contribute to development of CVD. General Biochemistry cardiovascular disease atherosclerosis rabbits homocysteine lipids Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Cardiovascular disease (CVD) is the leading cause of death worldwide. In 2019 17.9 million people died from CVD, representing 32% of all global deaths ( 1 ). In most cases, CVD is due to the development of atherosclerosis, a chronic, progressive, inflammatory disease of the aorta characterized by dyslipidemia and an accumulation of lipids in the aortic wall ( 2 ). However, established risk factors including hypercholesterolemia can explain only about 50% of all cases of atherosclerosis ( 2 ). Homocysteine (Hcy) is a sulfur-containing, non-proteinogenic amino acid involved in intermediary metabolism of methionine. It is produced from S-adenosyl-L-homocysteine, a strong product inhibitor of S-adenosyl-L-methionine-dependent methyltransferases, and is degraded either by its remethylation to methionine within the methylation cycle or by its transsulfuration to cysteine resulting in its removal from the methylation cycle ( 3 ). An accumulation of Hcy in the blood, termed hyperhomocysteinemia (HHcy), is found in 5–10% of the general population, in up to 30% of the elderly and even in 70% of men over 80 years of age ( 4 – 6 ). In most cases HHcy is associated with low status of one or more vitamins required for Hcy degradation ( 5 ). HHcy is an independent risk factor for atherosclerosis, leads to increased cardiovascular risk in combination with hypercholesterolemia ( 4 ), is linked to cardiac pathologies ( 7 – 11 ) and is strongly associated with cardiovascular as well as non-cardiovascular mortality ( 4 , 12 , 13 ). In addition to age, elevated Hcy is linked to further cardiovascular risk factors such as physical inactivity ( 14 ), high fat diet ( 15 ), low skeletal muscle mass ( 16 – 20 ) and has been also associated with obesity and fatty liver disease ( 19 – 22 ). In mice elevation of plasma Hcy levels due to dietary deprivation of folate, vitamins B 6 and B 12 , high methionine intake, or genetic block in Hcy metabolization exacerbates atherosclerosis development ( 23 , 24 ). Furthermore, genetic block in Hcy degradation in mice leads to deregulation of lipid metabolism, including reduced fat mass ( 25 , 26 ), aortic lipid deposition ( 27 ) as well as deregulation of phospholipid and sphingomyelin metabolism ( 28 ). Moreover, elevated Hcy leads to an upregulation of the sterol regulatory element–binding proteins (SREBPs) in cultured human hepatocytes as well as vascular endothelial and aortic smooth muscle cells, which is associated with increased expression of genes responsible for cholesterol/triglyceride biosynthesis and uptake, and with intracellular accumulation of cholesterol ( 29 ). Similarly, in yeast elevated Hcy is associated with triglyceride and fatty acid accumulation ( 30 ). In contrast to rodents, rabbits spontaneously develop foam cell-rich plaques (fatty streaks) on a high-fat diet ( 31 ). Previously we have shown that a diet free from vitamin B 12 and reduced in folate (20%), vitamin B 6 (20%), and choline (10%) required for Hcy degradation (VCDD) leads to an accumulation of macrophages and lipids in the aorta, aortic stiffening and disorganization of aortic collagen in a balloon-injured rabbit model of atherosclerosis ( 31 ). Furthermore, combination of VCDD with high cholesterol diet (HCD) results in further thickening of the aorta, altered lipoprotein profile as well as impaired aortic vascular reactivity compared to HCD alone ( 31 ). Elevation of plasma Hcy levels by intravenous injections of Hcy into rabbits fed VCDD in the absence of hypercholesterolemia results in alteration of lipoprotein profile, impairment of vascular reactivity of the aorta, disorganization of aortic elastin and collagen, decreased total protein methylated arginine and altered metabolomic profiles compared to rabbits fed VCDD only ( 32 ). Here, we analyzed lipid molecular species in blood cells and heart from rabbits fed VCDD in the presence or absence of additional intravenous injections of Hcy and hypercholesterolemia as well as in the aorta in the presence or absence of hypercholesterolemia. Our data show that elevation of plasma Hcy levels by intravenous injections of Hcy into rabbits fed VCDD in the absence of hypercholesterolemia as well as VCDD alone leads to an alteration of lipid metabolism in these tissues. Deregulation of phospholipid, cholesterol, ceramide, fatty acid and acylcarnitine metabolism in rabbits fed VCDD in the presence of intravenous Hcy injections suggests that deregulated lipid metabolism is likely to play a central role in pathological consequences associated with elevated Hcy. Materials and Methods 1. Animal experiments All animal experiments were approved by the Austrian Federal Ministry of Education, Science and Research (BMWF-66.010_0070-V_3b_2018). For the study 4–6 month old male New Zealand White (NZW) rabbits were acclimatized for 2–4 weeks and divided into groups. All rabbits were sensitized for the development of atherosclerosis by balloon injury using the JURY device for induction of fully automated pressure- and retraction-controlled vessel wall injury (one retraction, 1.8 bar, retraction speed of 2 mm/s) ( 31 ) and fed special diets. The diets used included an unpurified complete chow diet (standard diet, SD) for full nutritional conditions and purified special diets containing 1% cholesterol (high cholesterol diet, HCD), deficient in vitamins and choline required for Hcy metabolization (no vitamin B 12 , 20% folate (2 mg/kg), 20% vitamin B 6 (6 mg/kg) and 10% choline (0.128 mg/kg), VCDD), deficient in vitamins and choline, and containing 1% cholesterol (VCD/HCD) and VCDD combined with L-Hcy intraveneous injections (total 60 µmol/kg, VCDD + Hcy). VCDD was pre-fed for two weeks and HCD for one week before the surgery to elevate the levels of plasma Hcy and cholesterol at the time of sensitization. Special purified diets were acquired from Sniff, Germany. Average daily food intake was determined by weighing the remaining food after two days once per week for each animal. Body weight was measured once a week. Eight weeks after balloon injury the rabbits were sacrificed. After euthanasia blood was withdrawn with a syringe directly from the heart and collected in EDTA and Serum vacuette tubes (Greiner). All blood tubes were inverted 5 times and serum tubes were additionally incubated for 30 min at RT. Tubes were centrifuged at 2200 g and 4°C. Blood cells were extracted from EDTA tubes. Blood cells, heart and aortic specimens (for lipidomic analysis) as well as serum and liver (for metabolomic analysis) were immediately frozen in liquid nitrogen. Aortic specimens for electronic miscroscopy were prepared as described below. 2. Lipid extraction Lipid extraction was performed in accordance with the protocol previously published by Matyash et al. ( 33 ). Briefly, one steel ball was added to 12 mg of blood cells or macerated aortic tissue sample and homogenized in 700 µL methyl- tert -butyl-ether (MTBE):MeOH (10:3, v:v) + 50 µL of internal standard (IST) master mix in a homogenizer MM 40 (Retsch) three times for 8 min at 30 Hz and 4°C. IST mastermix contained LPE 17:1 (107.5 µM), LPC 17:0 (9.8 µM), PE 34:0 (34.7 µM), PC 38:0 (73.4 µM), Cer 17:0 (9.1 µM for aorta samples and 4.5 µM for blood cell samples), TG 51:0 (177.8 µM for aorta samples and 58.9 µM for blood cell samples) and SM 17:0 (20.9 µM) in chloroform/methanol (2/1, v/v). Samples were then shaken in a thermomixer for 60 min at 1400 rpm and 4°C. Then 200 µL ddH 2 O were added to the samples and the samples were shaken again for 20 min at 1400 rpm and 4°C before centrifuging samples at 13,500 rpm for 10 min at room temperature. Supernatant was transferred to a 1.5 mL auto sampler vial, dried under nitrogen stream and resolved in 500 µL chloroform/MeOH (2/1, v/v). 100 µL were dried again under nitrogen stream, resolved in 100 µL isopropanol:MeOH/H 2 O (30/15/5, v/v/v) and directly used for mass spectrometry. For heart lipid extraction 5 mg of macerated tissue was homogenized in 700 µL MTBE/MeOH (10/3, v/v) containing 1% acetic acid, 1 µM BHT and 1 µL IST master mix in a homogenizer MM 40 (Retsch) for 3 min at 30 Hz and 4°C. IST mastermix contained BMP 28:0 (100 µM), Cer 35:1 (13.33 µM), DG 28:0 (66.67 µM), FA 17:1 (1.25 mM), FC d7 (1.5 mM), GluCer 24:1 (50 µM), LPC 17:1 (40 µM), LPE 17:1 (150 µM), LPG 17:1 (200 µM), LPS 34:0 (250 µM), MG 17:0 (266.67 µM), PA 28:0 (200 µM), PC 28:0 (250 µM), PE 34:0 (666.67 µM), PG 34:0 (80 µM), SM 34:0 (50 µM) and TG 45:0 (100 µM). Samples were shaken in a thermomixer for 20 min at 1400 rpm and 4°C before 200 µL ddH 2 O was added and mixed again in thermomixer for 20 min. Samples were centrifuged at 14000 rpm for 5 min, upper phase was transferred to a new tube, dried under nitrogen stream and resolved in 500 µL MTBE/MeOH (10/3, v/v). 100 µL were further diluted 1:1 in MTBE/MeOH/ddH 2 O (30/5/5, v/v/v) for mass spectrometry. 3. Lipidomic analysis For the analysis of total blood cellsand aorta lipids, an Acquity UPLC system coupled with a Synapt quadrupole time of flight (QTOF; Waters) tandem mass spectrometer with ESI ion source was used. In addition, an L-6200 Intelligent Pump (Merck/Hitachi) was used to pump a reference solution for calibration purposes. Reversed phase chromatography was performed with a Luna Omega 1.6 µm C18 100 Å LC column (50*2.1 mm) (Phenomenex) at a column compartment temperature of 50°C. As solvent A MeOH/ddH 2 O (1/1, v/v) + 1% NH 4 Ac + 0.1% HCOOH + 8 µM H 3 PO 4 and as solvent B isopropanol + 1% NH 4 Ac + 0.1% HCOOH + 8 µM H 3 PO 4 was used. 20% solvent B was held for 2 min and then gradually increased to 45% over the next 2 min. Subsequently solvent B was further gradually increased to 85% over 13 min and then to 100% over 1 min. 100% solvent B was held for 1 min before dropping back to 20% in 3 s where it was held for 1.95 min. Flow rate was set to 100 µL/min for the first 1.05 min, to 300 µL/min between 1.05 and 19.55 min and to 100 µL/min until the end of the run. Total run time per sample was 20 min. The QTOF was set to full scan for all m/z ratios in between 50 and 1800 alternating with fragment ion scans for m/z ratios between 50 to 1800. For the analysis of total lipids in heart a UHPLC 1290 Infinity LC system coupled to a 6560 Ion Mobility Q-TOF LC/MS with a dual AJS ESI ion source (Agilent) was used. Reversed phase chromatography was performed with a Acquity BEH C18 1.7 µm (2.1 × 150 mm) column (Waters) at a column compartment temperature of 50°C. A binary gradient of solvent A ddH 2 O and solvent B isopropanol was used. Both solvents contained 1% NH 4 Ac, 0.1% HCOOH and 8 µM H 3 PO 4 . 40% solvent B was held for 30 s before gradualy increasing to 80% solvent B over the course of 8.5 min and further to 100% solvent B over 13 more min. 100% solvent B was held for 2.5 min and then dropped back to 40% solvent B in 30 s where it was held for 5 more min. Total run time per sample was 30 min at a constant flow rate of 150 µL/min. Per sample 1 µL was injected for positive ion mode and 5 µL for negative ion mode. Lipidomic data were annotated with the Lipid Data Analyzer (LDA version 2.8.3) software. 4. Metabolomics Metabolomics was performed as previously described ( 32 ). In brief, for metabolite analyses, liver samples were suspended in pre-mixed 400 µL of ice-cold methanol and 200 µL of MilliQ H 2 O, and transferred to Precellys tubes with 1.4 mm diameter zirconium oxide beads (Bertin Technologies). This suspension was homogenized two times for 20 s by Precellys24 tissue homogenizer at 25°C (Bertin Technologies). 400 µL MeOH were directly added to 200 µL of serum. Afterwards, the homogenized samples were stored at -20°C for at least 30 min and centrifuged at 10000 g for 30 min at 4°C. For metabolite analyses, the supernatants were transferred to new tubes and lyophilized at < 1 Torr, 850 rpm, 25°C for 10 hours in a vacuum-drying chamber (Savant Speedvac SPD210 vacuum concentrator), with an attached cooling trap (Savant RVT450 refrigerated vapor trap) and vacuum pump (VLP120) (Thermo Scientific). For NMR experiments, samples were dissolved in 50 mM phosphate buffer (pH 7.4, prepared in D 2 O) and measured at 310 K using a 600 MHz Avance Neo NMR spectrometer (Bruker) equipped with a TXI 600S3 probe head. The Carr–Purcell– Meiboom–Gill pulse sequence was used to acquire 1H 1D NMR spectra with a pre-saturation for water suppression (cpmgpr1d, 128 scans, 73728 points in F1, 12019.230 Hz spectral width, recycle delay 4 s) ( 34 , 35 ). The data were processed in Topspin version 4.4 (Bruker) using one-dimensional exponential window multiplication of the FID, Fourier transformation, and phase correction. NMR data analyses: For metabolite analyses, NMR data were imported into Matlab2014b, TSP was used as the internal standard for chemical-shift referencing (set to 0 ppm), regions around the water, TSP and methanol signals were excluded, the NMR spectra were aligned, and a probabilistic quotient normalization was performed. Metabolite identification was carried out using Chenomx NMR Suite 8.4 (Chenomx Inc.) and reference compounds. Quantification of metabolites was carried out by signal integration of normalized spectra. For each metabolite, a representative peak with no overlapping signals was identified, the start and endpoints of the integration were chosen to revolve around that peak, and the area of the peak was integrated by summing up the value of each data point. Orthogonal partial least squares discriminant analysis (OPLS) and sparse partial least squares-discriminant analysis (SPLS) were performed in Matlab 2014b and MetaboAnalyst 5.0, as well as all associated data consistency checks and cross-validation ( 36 – 38 ). The statistical significance of the determined differences was validated by the quality assessment statistic Q 2 and presented as specific p-values. For visualization of the metabolite data, heatmaps and variable importance during projection (VIP) scores were calculated with MetaboAnalyst 5.0. Normalized peak integrals were presented as scatter box plots. 5. Electron microscopy Electron microscopy of aortic tissue was performed as previously described ( 32 ). In brief, aortic tissue was fixed in 2.5% (w/v) glutaraldehyde and 2% PFA (w/v) in 0.1 M cacodylate buffer, pH 7.4, for 2 hours, and then post-fixed in 2% (w/v) osmium tetroxide for 2 hours at RT. After dehydration (in graded series of ethanol), tissues were infiltrated (ethanol and TAAB Embedding Resin, pure TAAB Embedding Resin) and placed in TAAB Embedding Resin (8 hours), transferred into embedding molds, and polymerized (48 hours, 60°C). Ultrathin sections (70 nm) were cut with a UC 7 Ultramicrotome (Leica Microsystems) and stained with lead citrate for 5 min and platinum blue for 15 min. Electron micrographs were taken using a Tecnai G2 transmission electron microscope (FEI) with a Gatan Ultrascan 1000 charge coupled device (CCD) camera (-20°C, acquisition software Digital Micrograph, Gatan, and Serial EM, University of Colorado). Acceleration voltage was 120 kV. 6. Data processing and statistical analysis All data were processed with R/Rstudio (versions 4.3/2023.03.1–446). In addition to the basic R functions, the tidyverse packages ( 39 ) were used for data transformation. For multivariate analysis (OPLS) the lipidr package ( 40 ) was used. The rstatix package was used for outlier detection and statistical analysis ( https://rpkgs.datanovia.com/rstatix/ ). Exclusion of outliers was based on values above the third quartile plus three times the interquartile range or below the first quartile minus three times the interquartile range. Statistics were calculated with Mann-Whitney U test and corrected with false discovery rate for multiple testing. The significance levels are indicated with asterisks (* = p ≤ 0.05, ** = p < 0.01, *** = p < 0.001). Sample sizes in figure legends refer to biological replicates (independent animals) and in case of box plots are depicted as individual spots. Results of statistical analyses are shown in Supplementary Material. Results 1. Hcy injections into VCDD-fed rabbits lead to deregulation of lipid metabolism in blood cells and heart. Previously we have shown that a diet deficient in vitamins and choline required for Hcy degradation (VCDD) leads to aortic lipid accumulation in balloon-injured rabbit model of atherosclerosis in the absence of hypercholesterolemia ( 31 ) and elevation of plasma Hcy levels by intravenous injections of Hcy into these rabbits (VCDD + Hcy) results in their degradation ( 41 ). Furthermore, combination of high cholesterol diet (HCD) with VCDD (VCD/HCD) led to 40% increase in LDL-cholesterol ( 31 ) and further elevation of plasma Hcy levels by Hcy injections in VCDD-fed rabbits dramatically increased VLDL-triglycerides compared to VCDD alone ( 41 ). This observation prompted us to analyze lipidome composition in our rabbit model. Balloon-injured rabbits were fed VCDD, HCD, VCD/HCD and VCDD in combination with intravenous injections of Hcy. While VCDD resulted in doubling of plasma Hcy levels, rabbits from the VCDD + Hcy group exhibited more than 3-fold elevation of plasma Hcy levels ( 41 ). OPLS analysis revealed distinct lipid profile in blood cells, but not in hearts from rabbits in the VCDD + Hcy group compared to SD, VCDD, HCD or VCD/HCD (Fig. 1). The most dysregulated lipid species in blood cells included triglycerides (TG), TG 56:0, TG 56:1, TG 58:0, TG 58:1, TG 60:1 and TG 62:1, phosphatidylethanolamines (PE), PE 34:3 and PE 36:1, as well as phosphatidylcholines (PC), PC 32:0 and 36:2 (Fig. 1A). In heart, the most dysregulated lipid species included PCs, PC 31:0, PC 34:3, PC 35:2, PC 36:5, PC 37:4 and PC 38:7 as well as acylcarnitines (CARs), CAR 16:0, CAR 16:1, CAR 18:1 and CAR 18:2 (Fig. 1B). In blood cells volcano plot analysis showed a decrease of TG species in VCDD compared to VCDD + Hcy as well as increased TG species in HCD compared to VCD/HCD group (Fig. 1A). Volcano plot analysis in blood cells also showed decreased ceramides (Cer) as well as increased CAR in VCDD compared to SD, decreased Cer, PC, PE and CAR in VCDD + Hcy compared to VCDD and increased CAR, Cer and PE in VCD/HCD compared to HCD (Fig. 1A). In the heart, volcano plot analysis revealed a decrease of CAR and increase of TG in VCDD compared to SD. Furthermore, in heart VCDD + Hcy led to dysregulation of CAR and PC species as well as decreased TG and elevated PE compared to VCDD, while VCD/HCD exhibited decreased TG and SM in addition to increased PC in hearts compared to HCD (Fig. 1B). 2. Hcy injections into VCDD-fed rabbits lead to decreased TG levels in blood cells, but not in the heart While TG levels in blood cells from VCDD-fed rabbits exhibited a significant increase compared to SD, elevation of plasma Hcy by intravenous injections of Hcy into VCDD-fed rabbits resulted in a significant decrease of total TG levels in blood cells compared to rabbits fed VCDD only (Fig. 2A). In blood cells rabbits from the VCDD group accumulated primarily TG species with 2–7 double bonds and containing fatty acids (FAs) with a total of 50–56 carbons compared to SD (Fig. 2A). Similarly, rabbits from the VCDD + Hcy group, while exhibiting decrease over all analyzed TG species compared to VCDD alone, the decrease of TG species containing 2–7 double bounds and total fatty acid chain lengths of 50–56 carbons was less pronounced compared to other species (Fig. 2A and S1A). Feeding rabbits VCD/HCD, similarly to VCDD, led to accumulation of total TG levels in blood cells, albeit tendentially (Fig. 2A). However, in contrast to rabbits from the VCDD and VCDD + Hcy groups, blood cells from rabbits fed VCD/HCD accumulated TG species containing polyunsaturated FAs with 6 or more double bonds compared to either SD, HCD and VCDD, and similarly to the VCDD + Hcy group exhibited decreased levels of saturated TG species (TG X:0) (Fig. 2A and S1A). In contrast to blood cells, hearts from VCDD-fed rabbits showed only a tendential increase of total TG levels compared to SD, however similarly to blood cells intravenous Hcy injections into VCDD-fed rabbits also resulted in tendential decrease of total TG levels in hearts compared to VCDD alone (Fig. 2B). Furthermore, similarly to blood cells VCD/HCD resulted in a drop of total TG levels in the heart compared to VCDD group, but in contrast to blood cells it led to their decrease also compared to HCD (Fig. 2B). Additionally, in contrast to blood cells, both the VCDD + Hcy and VCD/HCD groups exhibited a decrease of all TG species in the heart regardless of saturation or chain lengths (Fig. 2B and S1B). 3. Hcy injections into VCDD-fed rabbits lead to phospholipid remodeling both in blood cells and heart VCDD feeding also resulted in dysregulation of phospholipid (PL) metabolism in blood cells. VCDD both in combination with either Hcy injections or high cholesterol led to increased total PC levels, in particular an increase in PC 34:2, PC 36:3 and PC 36:4 species compared to either VCDD alone or HCD alone respectively (Fig. 2A and S1A). Furthermore, rabbits from the VCD/HCD group also displayed an increase in PC 32:1, PC 32:2, PC 38:5, PC 38:6, PC 40:4, PC 40:5 and PC 40:6 species compared to HCD, which were not affected in blood cells from rabbits in the VCDD + Hcy group. Moreover, rabbits from the VCDD + Hcy group showed increased levels of saturated PC species and species with lower number of double bonds, namely PC 32:0, PC 34:0, PC 36:1 and PC 36:2 compared to rabbits fed VCDD alone (Fig. 2A and S1A). Interestingly, HCD with or without VCDD led to a decrease in these species compared to SD (Fig. 2A and S1A). While in blood cells VCDD in combination with Hcy injections resulted in an accumulation of PC species containing lower number of double bonds and shorter fatty acid chain lengths compared to VCDD alone, VCDD in combination with HCD led to an accumulation of PC species containing more unsaturated and longer fatty acids compared to HCD alone (Fig. 2A). In contrast to PC, elevation of total PE levels in blood cells was detected only in response to VCDD + Hcy, but not in response to VCDD, HCD or VCD/HCD (Fig. 2A). In the VCDD + Hcy group all analyzed PE species with total FA chain lengths of 32–36 carbons as well as PE 38:6 were increased compared to VCDD alone (Fig. 2A and S1A). Noteworthy, longer and more unsaturated PE species, namely PE 40:4 and PE 40:5, were increased in blood cells from rabbits in the VCD/HCD group compared to HCD alone similarly to PC, but due to low abundance of these species their increase did not affect overall total PE levels in this group (Fig. 2A and S1A). Noteworthy, while levels of total PC and PE were significantly elevated in blood cells, but not in hearts of rabbits from the VCDD + Hcy group compared to rabbits fed VCDD alone, PC/PE ratios were unchanged both in blood cells and hearts of rabbits in response to both VCDD + Hcy and VCDD groups compared to SD (Fig. 2). While HCD led to an increase of total lyso-PC (LPC) levels in blood cells compared to SD as well as in VCD/HCD compared to HCD, total LPC levels were unchanged in response to VCDD compared to SD or VCDD + Hcy compared to VCDD (Fig. 2A). Furthermore, both rabbits from HCD and VCD/HCD groups exhibited an accumulation of all analyzed LPC species with exception of LPC 14:1 in blood cells compared to either SD or HCD respectively (Fig. 2A and S1A). Interestingly, while VCDD alone did not lead to any changes in blood cell LPC levels compared to SD, combination of HCD and VCDD led to even stronger accumulation of LPCs rather than HCD alone (Fig. 2A). In contrast, in heart none of the groups exhibited changes in total PC or PE levels (Fig. 2B). Regardless of unchanged total PC levels, both HCD and VCD/HCD resulted in elevation of PC species containing longer fatty acids or a higher number of double bonds and in the heart, specifically PC species containing fatty acids with a total of 5–8 double bonds or total chain lengths of 37–40 carbons compared to SD (Fig. 2B and S1B). Similarly, rabbits from the VCDD + Hcy group exhibited significantly increased PC species containing 4 and 7 double bonds in the heart as well as tendency towards an accumulation of PC species containing longer fatty acids with total chain lengths of 34–40 carbons, whereas PC species containing shorter fatty acids with total chain lengths of 24–28 carbons were decreased compared to VCDD alone (Fig. 2B and S1B). Furthermore, similar to blood cells HCD and VCD/HCD feeding led to significant accumulation of total LPC levels in hearts compared to non-HCD groups (Fig. 2B). However, in contrast to blood cells VCD/HCD did not lead to higher total LPC levels in heart compared to HCD alone (Fig. 2B). Increase of LPC species in HCD and VCD/HCD groups was distributed equally (Fig. 2B and S1B). 4. Hcy injections into VCDD-fed rabbits lead to an accumulation of free cholesterol in blood cells and heart, as well as total fatty acids in blood cells Analysis of free cholesterol levels in balloon-injured NZW rabbits fed VCDD in the absence or presence of HCD, or with additional intravenous injections of Hcy revealed that also cholesterol metabolism was dysregulated in response to elevated Hcy. While intravenous injections of Hcy into VCDD-fed rabbits led to significant increase of free cholesterol levels in blood cells compared to VCDD, both VCDD without Hcy injections compared to SD as well as VCD/HCD compared to HCD showed only tendential increase of free cholesterol levels in blood cells (Fig. 3A). Moreover, VCDD led to tendentially increased cholesterol ester levels in blood cells compared to SD and similarly VCD/HCD resulted in an accumulation of cholesterol esters compared to HCD (Fig. 3A). In contrast, additional injections of Hcy into rabbits fed VCDD resulted in tendentially decreased cholesterol ester levels in blood cells compared to VCDD alone (Fig. 3A). Next, we analyzed total fatty acid levels in blood cells from rabbits fed VCDD in the absence or presence of HCD, or with additional intravenous injection of Hcy. While VCDD alone resulted in only minor increase of total fatty acids in blood cells compared to SD, additional injections of Hcy into rabbits fed VCDD led to twofold increase in total fatty acids compared to both SD and VCDD (Fig. 3A). Similarly, while total fatty acid levels in blood cells from rabbits fed HCD showed an increase compared to SD, combination of VCDD and HCD led to further increase in total fatty acid levels compared to HCD alone (Fig. 3A). In hearts rabbits from the HCD, VCD/HCD as well as VCDD + Hcy groups, but not rabbits fed VCDD alone, showed significantly increased free cholesterol levels compared to SD (Fig. 3B). Rabbits from the VCDD + Hcy group, similarly to rabbits from the VCDD group, also exhibited an increase of total cholesterol esters in hearts compared to SD, albeit insignificant (Fig. 3B). Furthermore, hearts from both HCD- and VCD/HCD-fed rabbits, similarly to blood cells, exhibited also a strong accumulation of total cholesterol esters compared to SD (Fig. 3B). However, in contrast to blood cells, levels of cholesterol esters in hearts from rabbits in the VCD/HCD group were not further increased compared to rabbits fed HCD alone (Fig. 3B). 5. Hcy injections into VCDD-fed rabbits lead to ceramide and sphingomyelin accumulation in blood cells Analysis of Cer and SM content revealed that elevation of Hcy levels by intravenous injections of Hcy into VCDD-fed rabbits also affected sphingolipid metabolism. While VCDD alone did not lead to an increase of total Cer levels in blood cells compared to SD, rabbits from the VCDD + Hcy group displayed a significant increase of total Cer levels in blood cells compared to VCDD (Fig. 4A). Furthermore, VCD/HCD also led to a significant increase of total Cer levels in blood cells compared to VCDD as well as a tendential increase of total Cer levels compared to HCD (Fig. 4A). Rabbits from both VCD/HCD group compared to HCD and VCDD + Hcy group compared to VCDD accumulated Cer species containing fatty acids with total chain lengths of 40–44 carbons and a total of 1–4 double bonds, namely Cer d40:1, Cer d40:2 and Cer d44:4 in the VCD/HCD group and Cer d42:2, Cer d42:3 and Cer d44:3 in VCDD + Hcy group, in addition to Cer d38:3, Cer d42:1, Cer d42:4 and Cer d44:2 accumulating in both groups (Fig. 4A and S2A). Total SM levels, similarly to Cer levels, were also significantly increased in blood cells of rabbits from the VCDD + Hcy group compared to VCDD and tendentially increased in rabbits from the VCD/HCD group compared to HCD (Fig. 4A). Particularly, rabbits from the VCDD + Hcy group exhibited an accumulation of SM species containing longer fatty acids, namely SM d40:1, SM d42:1, SM d42:2 and SM d42:3 (Fig. 4A and S2A). In contrast, SM species containing shorter fatty acids, including SM d32:1, SM d34:1, SM d34:2, SM d36:1, SM d36:2 and SM d40:1 were elevated in blood cells from rabbits in the VCD/HCD group compared to HCD, and SM d42:2 was elevated both in HCD and VCD/HCD groups compared to SD (Fig. 4A). Of note, VCDD alone did not lead to an accumulation of total SM, but displayed an elevation of SM d42:3 in comparison to SD (Fig. 4A). In heart, VCDD both with or without additional Hcy injections did not show any changes of total Cer levels (Fig. 4B). However, HCD and VCD/HCD feeding resulted in a tendential increase of total Cer levels as well as significant increase of monounsaturated Cer species such as Cer d34:1, Cer d38:1, Cer d39:1, Cer d40:1, Cer d41:1 and Cer d42:1 compared to SD (Fig. 4B and S2B), but VCD/HCD did not show further increase of Cer levels compared to HCD (Fig. 4B and S2B). Similar to Cer, HCD and VCD/HCD but not VCDD with or without Hcy injections led to an accumulation of total SM in the heart compared to SD (Fig. 4B). In particular, rabbits from the HCD and VCD/HCD groups accumulated saturated and mono-unsaturated SM species, including SM d34:0, SM d38:0, SM d40:1, SM d41:0, SM d41:1 and SM d42:1, whereas rabbits from the HCD group additionally exhibited an elevation of SM d34:1, SM d38:0, SM d38:1 and SM d42:3 in the heart compared to SD, whereas VCD/HCD did not show further elevation of SM species in hearts compared to HCD (Fig. 4B and S2B). 6. Hcy injections into VCDD-fed rabbits lead to significantly decreased acylcarnitine levels in the heart Analysis of CAR levels in balloon-injured NZW rabbits fed VCDD in the absence or presence of HCD, or with additional intravenous injections of Hcy revealed that elevation of Hcy levels also affected CAR metabolism. While VCDD did not change the levels of total CAR in blood cells, additional injections of Hcy into VCDD-fed rabbits resulted in significantly increased total CAR levels in blood cells compared to VCDD alone (Fig. 5A). Analysis of CAR species in blood cells of rabbits from the VCDD + Hcy group revealed that they accumulated CAR 16:0 and CAR 18:1 (Fig.S3A). Similar to VCDD + Hcy group, total CAR levels were also elevated in blood cells from rabbits from the HCD group, with no further increase in CAR levels in blood cells in response to VCD/HCD compared to HCD alone (Fig. 5A). However, analysis of CAR species in blood cells from rabbits from the VCD/HCD group exhibited an increase of CAR 18:2 compared to HCD (Fig.S3A). In contrast, VCDD led to significantly decreased total CAR levels in the heart compared to SD and an even stronger decrease of myocardial total CAR levels when VCDD was combined with intravenous Hcy injections compared to VCDD alone (Fig. 5A). Furthermore, VCD/HCD also resulted in decreased total CAR levels in the heart compared to HCD alone (Fig. 5B). Noteworthy, in the heart VCDD resulted in a decrease of saturated and mono-unsaturated CAR species, while rabbits from the VCDD + Hcy group showed a significant decrease of CAR species in the heart regardless of saturation level, namely Car 3:0, Car 6:0, Car 12:0, Car 14:0, Car 16:0, Car 18:1, Car 18:3, Car 19:1 and Car 22:5 (Fig. 5B and S3B). 7. Block in Hcy degradation in VCDD-fed rabbits leads to deregulation of lipid metabolism in aorta Next, we analyzed lipids in the aortas from balloon-injured rabbits fed VCDD, HCD, VCD/HCD or SD. While OPLS scoring plot did not show differences between the groups, both OPLS loading plot and volcano plot indicated a dysregulation of TG and PL metabolism in aortic tissue from rabbit fed different diets, similarly to blood cells (Fig. 6A). In line with our findings in blood cells, both VCDD and VCD/HCD resulted in an accumulation of total TG in the aortic wall compared to SD or HCD, respectively (Fig. 6B). Furthermore, analysis of individual TG species showed that in the VCDD group elevation was equally distributed, while in the VCD/HCD group preferably TG species containing longer and unsaturated fatty acids accumulated, similar to species distribution found in blood cells (Fig. S4). Interestingly, despite no differences in total PC and PE levels in response to VCDD in blood cells or in heart, in aorta VCDD, HCD and VCD/HCD groups exhibited decreased levels of total PC and increased levels of total PE compared to SD, while an increase in PE levels was the highest in the VCDD group (Fig. 6B). In aortas from rabbits from the VCDD group, PC species containing longer FAs with no or low number of double bonds were decreased, while in HCD and VCD/HCD groups also species containing 4–5 double bonds were decreased compared to SD (Fig. S4). Noteworthy, in blood cells an accumulation of similar PC species was found in response to VCD/HCD, but not VCDD alone. In contrast, while aortas from rabbits from the VCDD group exhibited elevation of PE species containing longer, highly unsaturated fatty acids, both HCD and VCD/HCD feeding resulted in an accumulation of PE species containing shorter fatty acids with a total of 1–3 double bonds compared to SD (Fig. S4). Analysis of sphingolipids in aortas from rabbits fed different diets showed a tendency towards increased levels of total Cer in response to HCD and VCD/HCD, similar to both blood cells and heart (Fig. 6D). However, in contrast to blood cells and heart total Cer levels in aorta were also found to be slightly increased in response to VCDD alone (Fig. 6D). Furthermore, in contrast to Cer levels in aorta, total SM levels were slightly decreased in response to VCDD as well as HCD and VCD/HCD feeding in comparison to SD (Fig. 6D). Noteworthy, both in blood cells and heart total SM levels were found to be increased in response to HCD and VCD/HCD in contrast to aorta, whereas VCDD did not lead to any changes in total SM levels in blood cells and aorta compared to SD. Analysis of total CAR levels in aortas from rabbits fed different diets showed a tendential increase in response to VCDD, HCD and VCD/HCD compared to SD, while HCD and VCD/HCD showed higher elevation of total CAR than VCDD (Fig. 6E), which is in line with total CAR levels in blood cells, whereas in hearts total CAR levels were decreased in response to VCDD and VCD/HCD compared to SD or HCD, respectively (Fig. 5). Analysis of total fatty acids in aortas from rabbits fed different diets showed an elevation of fatty acid levels in aortas from rabbits fed VCDD as well as in both high cholesterol-containing diet groups, HCD and VCD/HCD, compared to SD (Fig. 6C). However, in contrast to increased total fatty acid levels in blood cells from rabbits fed VCD/HCD compared to HCD fed rabbits, in aortic tissue VCD/HCD feeding did not lead to an accumulation of total fatty acids compared to HCD (Fig. 6C). Scanning transmission electron microscopy (STEM) and transmission electron microscopy (TEM) revealed an accumulation of lipid droplets (LDs) in aortic media from rabbits fed VCDD compared to SD (Fig. 7A-B), similarly as in rabbits fed HCD and, however to a lower extend compared to HCD alone, in rabbits fed VCD/HCD (Fig. 7C-D), in line with an accumulation of LDs in aortic neointima from rabbits fed either VCDD, HCD or VCD/HCD ( 41 ). Furthermore, in aortic media rabbits fed VCDD exhibited an accumulation of dilated endoplasmic reticulum (ER) and electron-dense mitochondria in smooth muscle cells, which were not observed in response to SD (Fig. 7A-B). Similarly, an accumulation of dilated ER in smooth muscle cells was also found in aortic media from rabbits fed VCD/HCD, but not HCD alone, whereas electron-dense mitochondria were observed in VCDD-fed rabbits as well as in response to both, VCD/HCD and HCD (Fig. 7C-D). Of note, dilated ER was also observed both in smooth muscle cells and fibroblasts in aortic intima of rabbits fed VCDD and VCD/HCD, but not HCD or SD( 41 ). 8. VCDD leads to phosphatidylcholine remodeling in serum, but not in the liver Next, to better understand PC metabolism in rabbits fed VCDD with or without HCD, or with additional intravenous injections of Hcy we analyzed metabolites of PC metabolism in the liver and serum by NMR. Choline levels were significantly increased in response to HCD in the liver and in response to VCD/HCD in the serum compared to SD (Fig. 8A). Feeding VCDD, despite decreased choline content in the diet, resulted in tendential increase of choline both in the liver and serum compared to SD (Fig. 8A). Similarly, combination of VCDD and HCD increased serum choline levels compared to HCD group (Fig. 8A). While, additional Hcy injections into rabbits fed VCDD did not change liver choline levels, they resulted in decreased serum choline levels compared to VCDD alone (Fig. 8A). Phosphorylcholine was similarly affected as choline in the liver, but was oppositely affected in the serum of rabbits from different groups (Fig. 8B). In particular, phosphorylcholine in serum showed a tendency towards lower levels in response to VCDD, HCD and VCD/HCD groups compared to SD, whereas additional intravenous injections of Hcy into VCDD fed rabbits did not change serum phosphorylcholine levels compared to SD and increased them compared to VCDD (Fig. 8B). Glycerophosphocholine levels in liver, similar to phosphorylcholine levels, were not affected in response to VCD/HCD or VCDD compared to SD, but in contrast to phosphorylcholine they were tendentially decreased in livers of rabbits fed HCD (Fig. 8C). Noteworthy, combination of VCDD and HCD tendentially increased liver glycerophosphocholine levels compared to HCD alone and elevation of plasma Hcy levels by intravenous Hcy injections into VCDD-fed rabbits tendentially increased liver glycerophosphocholine levels compared to VCDD (Fig. 8C). In contrast, glycerophosphocholine levels in serum showed tendential increase in response to VCDD compared to SD as well as in response to VCD/HCD compared to HCD (Fig. 8C). Discussion Elevated Hcy is linked to many human diseases including CVD ( 4 , 6 , 12 , 42 , 43 ). Although the underlying mechanisms leading to human pathology are insufficiently understood, downstream pathways triggered by elevated Hcy are likely to involve inhibition of SAM-dependent methyltransferases due to an accumulation of their product inhibitor SAH ( 3 , 30 ), interference with protein structure and function by homocysteinylation of critical protein lysines ( 44 ), induction of oxidative stress ( 45 – 48 ), deregulation of lipid metabolism ( 29 , 30 ) and activation of UPR ( 48 ). HHcy is classified into severe, moderate and mild forms based on the levels of fasting plasma Hcy. Severe HHcy characterized by plasma Hcy levels above 100 µM is due to a block in cystathionine-ß-synthase (CBS) that hinders Hcy degradation by transsulfuration and is associated with fatty liver, lean phenotype and CVD ( 3 , 24 , 26 , 49 ). Moderate HHcy characterized by plasma Hcy levels above 25 µM is in most cases due to a block in methyltetrahydrofolate reductase (MTHFR) that hinders folate-dependent remethylation of Hcy to methionine and is associated with fatty liver, CVD, aortic lipid deposition ( 27 ) and abdominal fat accumulation ( 3 , 27 , 50 ). Mild HHcy characterized by plasma Hcy levels above 15 µM is due to deficiency of vitamins required for Hcy degradation ( 12 ), represents two thirds of all HHcy cases ( 3 , 12 ) and is associated with CVD ( 4 , 7 – 11 ) and obesity ( 19 – 22 , 51 , 52 ). Our previous work in yeast showed that elevated Hcy leads to accumulation of TG and total FAs ( 30 ). In accordance, it was reported that elevated Hcy via activation of sterol regulatory element-binding proteins (SREBPs) upregulates genes responsible for cholesterol/triglyceride biosynthesis and uptake in human hepatocytes, vascular endothelial and aortic smooth muscle cells ( 29 ). Moreover, dietary induction of HHcy in mice leads to an elevation of cholesterol and TG levels in the liver and plasma, and increased secretion of VLDL-TG and VLDL-cholesterol ( 29 ). In accordance with hepatic lipid accumulation in response to elevated Hcy, both CBS and MTHFR knockout mice develop fatty liver ( 26 , 50 ). However, in contrast to CBS knockout mice, which exhibit loss of fatty tissue ( 26 ), MTHFR knockout mice had significantly increased abdominal fat mass ( 50 ) suggesting a dose-dependent effect of Hcy on lipid metabolism. In accordance, mouse models of CBS deficiency were reported to reveal significant threshold effects of elevated Hcy, however, its effects on lipid metabolism were not addressed in these mice ( 53 ). Threshold effects of elevated Hcy observed in CBS knockout mice are similar to graded CVD risk with no threshold conferred by elevated Hcy ( 4 ). Here we aimed to analyze and compare dysregulation of lipid metabolism in various tissues including blood cells, heart and aorta from rabbits fed VCDD in the absence or presence of additional intravenous Hcy injections as well as hypercholesterolemia. Feeding rabbits VCDD in the absence of hypercholesterolemia led to a significant elevation of total TG levels in blood cells and tendential TG elevation in aorta compared to SD. In line with elevation of aortic TG levels, electron microscopy of aortic media from rabbits fed VCDD, but not SD, showed an accumulation of LDs, similarly to an accumulation of LDs in aortic intima from rabbits fed VCDD ( 41 ). However, TG accumulation was not observed in blood cells of rabbits from the VCDD + Hcy group. Similarly, total TG levels in the hearts of rabbits from the VCDD group but not from the VCDD + Hcy group were elevated, albeit non-significantly. In accordance we and others have shown that elevated Hcy leads to an accumulation of total TG in yeast ( 28 ), human hepatocytes, vascular endothelial and aortic smooth muscle cells as well as in livers and plasma of mice in response to dietary caused HHcy ( 29 , 54 ). Decrease of TG levels in blood cells and hearts from rabbits from the VCDD + Hcy group compared to VCDD alone suggest an activation of lipolysis by elevated Hcy. Indeed, HHcy induced by 2% high methionine diet over 8 week or administration of 1.8 g/L in drinking water for 2 and 4 weeks in mice demonstrated that Hcy activates adipocyte lipolysis and increases release of free FAs and glycerol ( 54 ). In accordance, free FAs and glycerol were elevated in blood cells from rabbits in the VCDD + Hcy group. On the other hand, consistent with threshold effects of Hcy, supplementation of 100 to 500 µM Hcy was shown to inhibit release of glycerol and free FAs in primary adipocytes and fully differentiated 3T3-L1 adipocytes in a dose-dependent manner, and in differentiated 3T3-L1 adipocytes to elevate intracellular TG content by phosphorylating AMP-activated protein kinase and its downstream enzyme acetyl-CoA carboxylase ( 55 ). Furthermore, supplementation of 0.1% Hcy in the diet for 2 weeks was shown to lowered circulating glycerol and FA levels ( 55 ). While rabbits from the VCDD + Hcy exhibited a nonsignificant gain in weight ( 30 ), an accumulation of abdominal fat in human patient exhibiting mild HHcy ( 19 – 22 , 51 , 52 ) or MTHFR knockout mice ( 50 ), but not in CBS knockout mice ( 26 ), further demonstrates a dose-dependent effect of Hcy on lipid metabolism. Interestingly, rabbits from the VCDD + Hcy group exhibited massively elevated TG and also cholesterol levels particularly in VLDL, but also in LDL and HDL suggesting dysregulation of lipoprotein metabolism and increased hepatic lipid synthesis in response to elevated Hcy in our model ( 30 ). Noteworthy, analysis of plasma TG levels in normal population without lipid lowering treatment showed that HHcy was independently associated with hypertriglyceridemia and MTHFR knockout mice exhibit hypertriglyceridemia further indicating the central role of Hcy in TG metabolism ( 56 , 57 ). Combination of VCDD with HCD resulted in significant decrease of total TG levels in the heart compared to HCD that were even lower than in SD but not in blood cells suggesting an activation of lipolysis in the heart and sensitization towards elevated Hcy in the presence of high cholesterol. Despite VCD/HCD did not affect total TG levels in blood cells and only slight elevation of TG in aorta, it led to an increase in polyunsaturated TG species containing 6 or more double bonds compared to HCD alone. Similarly, VCDD resulted in an accumulation of polyunsaturated TG species with 2–7 double bonds in blood cells and combination of VCDD with intravenous Hcy injections resulted in particularly low levels of saturated TG species compared to SD. Similarly, while HCD did not affect total TG levels in the heart, it led to an accumulation of TG species containing polyunsaturated FAs. Serum saturated, monounsaturated and polyunsaturated FA and TG levels have been shown to be deregulated in extremely obese human patients as well as in mice fed high-fat diet ( 58 , 59 ). Furthermore, high-fat diet was shown to increase plasma Hcy levels by inhibiting hepatic transsulfuration pathway ( 15 ) and elevated Hcy to be linked to a decrease of decosahexaenoic acid in serum ( 60 ) and phospholipids ( 61 ). In line, obesity has been shown to depend on increased methionine in humans ( 62 ) and TG accumulation and deregulation of FA metabolism has been linked to elevated Hcy in yeast ( 30 ). Moreover, deficiency in phospholipid methylation in PE-methyltransferase (PEMT)-deficient mice fed choline-deficient diet that blocks an alternative Kennedy pathway for PC synthesis leads to an accumulation of polyunsaturated FAs in TG ( 63 ). Elevation of plasma Hcy levels by intravenous Hcy injections into VCDD-fed rabbits led to a significant increase of total PC in blood cells compared to both VCDD and SD as well as to a significant increase of total PE compared to VCDD alone. The increase in PC and PE levels was not observed in hearts from rabbits from the VCDD + Hcy group. Along with significant decrease in total TG levels, PC and PE accumulation in blood cells from rabbits from the VCDD + Hcy group suggests membrane proliferation in response to elevated Hcy. Of note, the ratio of PC/PE was unaltered both in blood cells and heart from rabbits from the VCDD + Hcy group compared to all other groups. Importantly, despite total PC elevation in blood cells, rabbits from the VCDD + Hcy group accumulated only short chain PC species containing low number of double bonds compared to VCDD. In line, PEMT-deficient mice also accumulate PC species containing shorter FAs with low number of double bonds in liver and plasma, while PC species containing long chain polyunsaturated FAs are depleted in these mice ( 63 ). In contrast, aortic tissue from rabbits fed VCDD exhibited tendentially decreased levels of total PC, increased levels of total PE and decreased PC/PE ratio compared to SD, suggesting an inhibition of PE to PC methylation. Similarly, elevation of plasma Hcy and SAH leads to an accumulation of PE and a drop in PC levels in human erythrocytes as well as significant depletion of docosahexaenoic acid in PC, but not PE ( 64 ). Furthermore, aortas from rabbits fed VCDD exhibited an accumulation of PE species containing long chain polyunsaturated FAs compared to SD, suggesting that an increase in polyunsaturated FAs in PE in response to elevated Hcy/SAH is due to inhibition of PE to PC methylation. Indeed, synthesis of PC by methylation-dependent pathway from PE catalyzed by PEMT produces PC species containing long chain polyunsaturated FAs, whereas PC synthesis via CDP-choline pathway mainly produces PC species containing shorter FAs with lower number of double bonds ( 65 ). Decreased PC/PE ratio as well as an accumulation of long chain polyunsaturated FAs in PE may interfere with membrane properties and lead to a lipid disbalance in the membrane. Membrane aberrancy or an accumulation of unfolded proteins ( 66 ) may be a cause for the observation of dilated ER in smooth muscle cells and fibroblasts in the aortic wall from rabbits fed VCDD. Previously, we have shown that feeding rabbits either VCDD or VCD/HCD leads to an accumulation of dilated ER in smooth muscle cells and fibroblasts in aortic neointima ( 41 ). Here we show that rabbits from the VCDD and VCD/HCD groups also exhibited an accumulation of dilated ER in smooth muscle cells in aortic media. It was shown in yeast that alterations in membrane PLs and decreased PC levels leads to induction of ER stress ( 67 ). Furthermore, Hcy was shown to lead to ER stress induction and dilated ER accumulation in human cardiac cells ( 68 ) as well as in human endothelial cells in atherosclerotic plaques ( 69 ). Interestingly, it was shown that supplementation of vitamin B12, required for Hcy re-methylation, leads to an amelioration of UV-radiation induced dilatation of ER in rat hepatocytes ( 70 ). In contrast to PC, total LPC levels in blood cells and hearts from rabbits from the VCDD + Hcy group were unaltered compared to both SD and VCDD, and increased significantly only in response to high cholesterol containing diets, HCD and VCD/HCD. Lecithin cholesterol acyl transferase (LCAT) plays a major role in reverse cholesterol transport and cholesterol metabolism, catalyzing FA transfer from PC to free cholesterol forming LPC and cholesterol esters ( 71 ). In line, feeding rabbits high cholesterol containing diets resulted not only in strongly elevated levels of LPC, but also proportionally increased total cholesterol ester levels in blood cells, hearts and aortas. Interestingly, hearts from rabbits fed VCD/HCD exhibited tendentially lower levels of LPC compared to HCD alone. In accordance, it has been reported that CBS-deficient mice, exhibiting HHcy, show decreased expression of LCAT in liver and decreased LCAT activity in serum ( 72 ). Elevation of plasma Hcy levels by intravenous injections of Hcy into VCDD-fed rabbits lead to significant increase of free cholesterol levels in the heart and blood cells compared to both SD and VCDD. Free cholesterol levels were also significantly elevated in rabbits fed combined VCD/HCD compared to HCD, however, only in blood cells, but not in hearts. Similarly, also only in blood cells, but not in the heart, cholesterol ester levels were non-significantly elevated in rabbits fed VCD/HCD compared to rabbits fed HCD alone. Interestingly, VCDD resulted in significant elevation of cholesterol ester levels in the heart, but not in blood cells compared to rabbits fed SD. In accordance with our data, homozygous CBS knockout mice exhibit significant elevation of free cholesterol levels in serum in comparison to CBS(+/+) or heterozygous CBS knockout mice suggesting interference between Hcy and cholesterol metabolism ( 72 ). Further suggesting a crosstalk between Hcy and cholesterol metabolism our previous experiments showed increased atherogenicity and elevation of LDL- and VLDL-cholesterol levels in rabbits fed VCDD, VCDD + Hcy and VCD/HCD compared to respective controls ( 31 , 32 ). Accumulation of free cholesterol in blood cells as well as elevation of cholesterol in circulating lipoproteins is in accordance with passive equilibrium of free cholesterol pool between lipoproteins and cell membranes ( 73 ). Furthermore, Hcy was shown to stimulate production as well as secretion of cholesterol in human hepatic cells, vascular endothelial and aortic smooth muscle cells, and monocyte-derived macrophages ( 74 – 76 ), the latter being also associated with deficient DNA methylation linked to increased expression of fatty acid-binding protein 4. In accordance, plasma Hcy was shown to be independently associated with conventional atherogenic lipid profile and remnant cholesterol in adults suggesting that Hcy-related dyslipidemia risk is clinically relevant ( 77 ). Moreover, increased cholesterol ester and LPC levels as well as elevated PC and free cholesterol levels in blood cells from rabbits fed VCD/HCD compared to HCD alone along with significant accumulation of free cholesterol and, significant only for blood cells, PC in blood cells and heart in rabbits from the VCDD + Hcy group suggest that elevated Hcy may contribute to increased atherogenicity by impacting reverse cholesterol transport and cholesterol as well as phospholipid metabolism. Ceramides are cholesterol-independent biomarkers causatively linked to CVD ( 78 ). Ceramide levels are increased significantly and sphingomyelin levels non-significantly in blood cells from rabbits from the VCDD + Hcy group compared to VCDD alone. Similarly, VCDD increases ceramide and sphingomyelin levels in blood cells from rabbits fed combined VCD/HCD compared to HCD alone. HCD also increases ceremide and sphingomyelin levels in blood cells compared to SD and VCDD. Elevation of ceremide and sphingomyelin levels in response to HCD or VCDD compared to SD can also be observed in the heart. Noteworthy, while in aorta ceramide levels are increased in VCDD compared to SD as well as in VCD/HCD compared to HCD, aorta sphingomyelin levels are decreased in VCDD and VCD/HCD compared to respective controls. Cholesterol interacts with sphingolipids in plasma membranes ( 79 ). Moreover, their synthesis and regulation are tightly linked ( 80 ). Particularly it has been shown, that clinical interventions to decrease elevated LDL cholesterol leads to decreased levels of ceramides ( 81 ) suggesting a connection between cholesterol and sphingolipid metabolism. In line, our results show that free cholesterol levels in all analyzed rabbit groups strongly correlate with total levels of Cer in blood cells, hearts and aortas as well as SM levels in blood cells and hearts. Elevation of plasma Hcy levels by intravenous Hcy injections into VCDD-fed rabbits leads to significant increase of total SM levels in blood cells compared to SD. In particular, rabbits from the VCDD + Hcy group accumulated SM d40:1, d42:1, d42:2 and d42:3 and exhibited decreased levels of SM 34:1, 36:1 and 36:2 in blood cells compared to SD. In contrast, combination of VCDD and HCD resulted in accumulation of SM d32:1, d34:1, d34:2 in addition to d40:1, but not SM d42:1, d42:2 and d42:3 compared to HCD in blood cells. In heart, HCD similar to VCD/HCD led to an accumulation of most SM molecular lipid species with exception of SM d33:1, d36:2, d40:2, d41:2, d44:1 and significantly decreased SM d38:2 compared to SD. In accordance with critical role of SM in atherosclerosis development, total SM were shown to be increased in coronary artery disease (CAD) patients and to have a predictive value for CAD after adjusting for other risk factors, including remnants ( 82 , 83 ). Moreover, circulating SM 34:1, which accumulated both in blood cells and in the heart in rabbits fed VCD/HCD, was shown to predict CVD mortality risk and be associated with higher risk for sudden cardiac death in humans ( 84 , 85 ) suggesting that dysregulated sphingolipid metabolism may contribute to CVD development in response to combination of HHcy and hypercholesterolemia. Similarly to SM, total Cer levels were significantly increased in blood cells but not in hearts from rabbits from the VCDD + Hcy group compared to both SD and VCDD. Highest Cer levels in blood cells were observed for Cer species containing longer fatty acids such as Cer d42:2, d42:3, d42:4, d44:2 and d44:3. While HCD also led to an increase in total Cer levels in blood cells, combination of VCDD and HCD resulted even in further elevation of total Cer levels in blood cells compared to HCD alone, with the highest accumulation observed for Cer d34:1, d40:1, d42:1 and d44:4. In accordance, circulating Cer 34:1 was shown to be associated with higher risk for sudden cardiac death in humans ( 85 ). In hearts, however, elevated total Cer levels were observed only in response to HCD alone, but not in combination with VCDD. In aorta, in contrast to total SM levels, which were decreased in HCD-fed rabbits compared to SD and even more pronounced decreased in VCD/HCD compared to HCD, total Cer levels were tendentially increased in response to VCDD compared to SD as well as VCD/HCD compared to HCD alone. In line with our results, elevated Hcy was shown to be linked to activation of ceramide de novo synthesis in rat mesangial cells ( 45 ) as well as in liver and brain of HHcy mice ( 86 ). Furthermore, folate deficient rats were shown to exhibit an accumulation of ceramides in the renal cortex ( 87 ). Cer accumulation was shown to promote inflammation and cell death, and to be associated with adverse cardiovascular outcomes ( 88 ) as well as mortality in patients with chronic heart failure ( 89 ). In particular, patients who died due to cardiovascular causes, had significantly higher levels of plasma Cer(d18:1/16:0) and Cer(d18:1/24:1) specially when used in ratios with Cer(d18:1/24:0) ( 90 ). In accordance, our data showed that Cer d42:2 was dramatically increased in blood cells from rabbits from the VCDD + Hcy group even if used in ratio with Cer d42:1, suggesting the role of elevated Hcy in dysregulation of ceramide species linked to cardiovascular disease. Higher plasma levels of Cer and SM containing palmitic acid were also associated with increased risk of heart failure, while on contrary higher levels of Cer and SM containing very long chain and/or unsaturated FAs, Cer-22, SM-20, SM-22, and SM-24, were associated with decreased risk of heart failure ( 91 ). Our data show that both SM d34:1 and Cer d34:1 were elevated in blood cells from rabbits fed VCD/HCD compared to HCD, but VCD/HCD feeding also led to an accumulation of Cer species containing very long fatty acids with a total of 40–44 carbons compared to HCD. However, it has to be noted that very long chain FA-containing ceramides were also reported to induce mitochondrial dysfunction leading to oxidative stress and cell death in cardiomyocytes in mouse models of diabetic cardiomyopathy ( 92 ). Of note, electron microscopy showed that rabbits fed VCDD, HCD or VCD/HCD showed darker, electron-dense mitochondria in the aortic media in contrast to SD, suggesting potential effects of elevated Hcy on mitochondrial function likely due to changes in membrane ultrastructure and/or paracrystalline protein accumulations ( 93 ). Importantly, structurally abnormal mitochondria, next to dilated ER, have also been found in endothelial in human atherosclerotic plaques ( 69 ). Increased CVD risk, cardiac hypertrophy and heart failure are also reported in association with dysregulated CAR metabolism ( 94 , 95 ). Suggesting that elevated Hcy may contribute to CVD via impaired CAR metabolism, our data show that feeding rabbits VCDD with or without intravenous Hcy injections both leads to significant decrease of total CAR levels in the heart compared to SD. Moreover, total heart CAR levels in rabbits from the VCDD + Hcy group were significantly lower than in rabbits fed VCDD alone. Combination of VCDD and HCD also led to a significant decrease of total CAR levels compared to HCD alone. Depletion of CAR suggest impaired FA transport into mitochondria, mitochondrial dysfunction and disruption of energy metabolism in the heart ( 95 ) in response to elevated Hcy. In contrast to heart, blood cells from rabbits from the VCDD + Hcy group exhibited significantly higher CAR levels compared to VCDD alone. Similarly, rabbits from the VCDD group exhibited tendential increase of total CAR levels compared to SD in both blood cells and aortic tissue. In line, medium- and long-chain acylcarnitines, preferably used for fatty acid oxidation, are markedly lower in myocardium, but exhibit no difference or were shown to be increased in plasma of human patients suffering from heart failure ( 96 ). Altered blood CAR levels in heart failure patients are linked to mitochondrial dysfunction and are independently associated with disease severity ( 97 ). Furthermore, in humans elevated serum Hcy levels, in response to deficiency of folate and vitamin B 12 , are positively correlated to increased CAR levels in plasma and are linked to myocardium hypertrophy, impaired mitochondrial FA oxidation and increased brain natriuretic peptide ( 98 ). FAs are preferred substrate in adult myocardium supplying about 70% of total ATP and are derived from circulating TG-rich lipoproteins and albumin-bound non-esterified FAs ( 99 ). While we have not analyzed serum free FAs in our model, we observed dramatically increased TG-VLDL levels in serum from rabbits fed VCDD in the presence of additional intravenous injections ( 41 ) in line with limited up-take of FA by cardiomyocytes suggesting TGs accumulation in circulating lipoproteins in particular VLDL. Increased levels of choline and glycerophosphocholine in serum in VCDD fed rabbits compared to SD, similar to rabbits from VCD/HCD group compared to HCD, suggest choline synthesis via catabolism of PC in line with dietary choline deficiency in VCDD. Furthermore, VCDD fed rabbits show a tendential decrease of phosphorylcholine in serum compared to SD in accordance with decreased PC de novo synthesis. Interestingly, further elevation of plasma Hcy by intravenous injections into VCDD fed rabbits resulted in a decrease of choline and to elevation of phosphorylcholine levels in serum compared to VCDD alone, suggesting inhibition of PC catabolism in response to elevated Hcy. In accordance we have observed a significant accumulation of total PC in blood cells as well as a tendential increase of total PC in hearts compared to VCDD. In line with an adaptive response of the liver to keep choline homeostasis during choline depletion ( 100 ), rabbits fed VCDD compared to SD, similar to VCD/HCD compared to HCD alone, did not show alterations of choline, phosphorylcholine or glycerophosphocholine in the livers. However, altered levels of choline in serum have been associated with the development of hepatic steatosis, atherosclerosis, cardiovascular disease and increased cardiovascular mortality( 101 , 102 ). Study limitation It was not possible to analyze lipidome in the aortic specimens from rabbits fed VCDD in combination with intravenous injections of Hcy. During preparation of the aorta it was not possible to quantitatively remove the surrounding fat layer from the aorta. In conclusion, here we show that Hcy leads to dysregulation of lipid metabolism in blood cells, heart and aorta in rabbits having a graded effect on glycerolipid, cholesterol, ceramide, acylcarnitine and fatty acid metabolism in different tissues. Accumulation of triglycerides in response to VCDD and their decrease in response to VCDD in combination with Hcy injections in blood cells and heart, accumulation of cholesterol in blood cells, heart and aorta, accumulation of ceramides and fatty acids in blood cells and a drastic drop in myocardial acylcarnitines suggest mechanisms how elevated Hcy may contribute to development of CVD. Abbreviations Hcy – homocysteine; HHcy – hyperhomocysteinemia; CVD – cardiovascular disease; SD – standard diet; VCDD – vitamin and choline deficient diet; HCD – high cholesterol diet; VCD/HCD – vitamin and choline deficient high cholesterol diet; VCDD+Hcy – vitamin and choline deficient diet with intravenous Hcy injections; TG – triglyceride; PL – phospholipid; PC – phosphatidylcholine; PE – phosphatidylethanolamine; LPE – lysophosphatidylethanolamine, LPC – lysophosphatidylcholine; FA – fatty acid; Cer – ceramide; SM – sphingomyelin; CAR – acylcarnitine; SREBP – sterol regulatory element binding protein; CBS – cystathionine-β-synthase; MTHFR – methyl-tetrahydrofolate reductase; PEMT – phosphatidylethanolamine methyl transferase; LCAT – lecithine-cholesterol acyl transferase Declarations Conflict of Interest statement The authors declare that there are no conflicts of interest. Funding This work was funded by the Austrian Science Fund (FWF) (P31105 and P33672) and BioTechMed-Graz. 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Int J Cardiol 167:768–775 Huss JM, Kelly DP (2005) Mitochondrial energy metabolism in heart failure: a question of balance. J Clin Investig 115:547–555 Li Z, Agellon LB, Vance DE (2005) Phosphatidylcholine homeostasis and liver failure. J Biol Chem 280:37798–37802 Song M, Xu BP, Liang Q, Wei Y, Song Y, Chen P, Zhou Z, Zhang N, He Q, Liu L, Liu T, Zhang K, Hu C, Wang B, Xu X, Shi H (2021) Association of serum choline levels and all-cause mortality risk in adults with hypertension: a nested case-control study. Nutr Metab (Lond) 18:108 Zeisel SH, da Costa KA (2009) Choline: an essential nutrient for public health. Nutr Rev 67:615–623 Additional Declarations The authors declare no competing interests. Supplementary Files SupplementaryFigureS1.pdf Figure S1: Distribution of glycerolipid species in blood cells and heart from rabbits fed different diets in the absence or presence of intravenous Hcy injections. Analysis of individual TAG-, PC-, PE- and LPC species in blood cells and hearts from rabbits fed SD, VCDD, HCD or HCD/VCD as well as VCDD in the presence of intravenous Hcy injections (n = 3 – 10). All statistics were calculated by Mann-Whitney-U pairwise comparisons and Benjamini-Hochberg correction for multiple testing (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001). All significances are shown in supplementary material. SupplementaryFigureS2.pdf Figure S2: Distribution of sphingolipid species in blood cells and heart from rabbits fed different diets in the absence or presence of intravenous Hcy injections. Analysis of individual Cer- and SM species in blood cells and hearts from rabbits fed SD, VCDD, HCD or HCD/VCD as well as VCDD in the presence of intravenous Hcy injections (n = 3 – 10). All statistics were calculated by Mann-Whitney-U pairwise comparisons and Benjamini-Hochberg correction for multiple testing (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001). All significances are shown in supplementary material. SupplementaryFigureS3.pdf Figure S3: Distribution of acylcarnitine species in blood cells and heart from rabbits fed different diets in the absence or presence of intravenous Hcy injections. Analysis of individual CAR species in blood cells and hearts from rabbits fed SD, VCDD, HCD or HCD/VCD as well as VCDD in the presence of intravenous Hcy injections (n = 3 – 10). All statistics were calculated by Mann-Whitney-U pairwise comparisons and Benjamini-Hochberg correction for multiple testing (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001). All significances are shown in supplementary material. SupplementaryFigureS4.pdf Figure S4: Distribution of species in all analyzed lipid classes from injured aorta from rabbits fed different diets. Analysis of individual TAG-, PC-, PE-, LPC-, Cer-, SM- and CAR species in aortic tissue from rabbits fed SD, VCDD, HCD or HCD/VCD (n = 3 – 4). All statistics were calculated by Mann-Whitney-U pairwise comparisons and Benjamini-Hochberg correction for multiple testing (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001). All significances are shown in supplementary material. SupplementaryMaterialStatistics.xlsx Statistical analysis Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6717713","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":459981484,"identity":"20679e9f-4304-4e03-a4fe-e65683f72db0","order_by":0,"name":"Markus S. 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(B) Heart lipidome of rabbits fed SD, VCDD, HCD or HCD/VCD as well as VCDD in the presence of intravenous Hcy injections is shown as OPLS scoring- and loading plots as well as groupwise comparisons between SD and VCDD, VCDD and VCDD+Hcy, and HCD and VCD/HCD shown as volcano plots (n = 8 – 10).\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6717713/v1/4fc6f2a7885e8cefff081790.jpg"},{"id":83300793,"identity":"1a4076c3-443b-487a-9d64-72e854fa34d9","added_by":"auto","created_at":"2025-05-22 14:55:35","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3237092,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGlycerolipids and their species distribution in blood cells and heart from\u003c/strong\u003e \u003cstrong\u003erabbits fed different diets in the absence or presence of intravenous Hcy injections. \u003c/strong\u003e(A) Total TAG, PC, PE and LPC, and their species distribution grouped by levels of total fatty acid saturation and chain length in blood cells from rabbits fed SD, VCDD, HCD or HCD/VCD as well as VCDD in the presence of intravenous Hcy injections (n = 3 – 7). (B) Total TAG, PC, PE and LPC, and their species distribution grouped by levels of total fatty acid saturation and chain length in heart from rabbits fed SD, VCDD, HCD or HCD/VCD as well as VCDD in the presence of intravenous Hcy injections (n = 8 – 10). All statistics were calculated by Mann-Whitney-U pairwise comparisons and Benjamini-Hochberg correction for multiple testing (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001). All significances are shown in supplementary material.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6717713/v1/a119f5ea77c139374ef1c43c.jpg"},{"id":83299604,"identity":"4c42c37e-925f-4345-98c7-33c4f653b407","added_by":"auto","created_at":"2025-05-22 14:39:35","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":128555,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTotal cholesterol, cholesterol ester and fatty acid levels in blood cells and heart from\u003c/strong\u003e \u003cstrong\u003erabbits fed different diets in the absence or presence of intravenous Hcy injections. \u003c/strong\u003e(A) Total free cholesterol, cholesterol ester and fatty acids in blood cells from rabbits fed SD, VCDD, HCD or HCD/VCD as well as VCDD in the presence of intravenous Hcy injections (n = 3 – 7). (B) Total free cholesterol and cholesterol esters in heart from rabbits fed SD, VCDD, HCD or HCD/VCD as well as VCDD in the presence of intravenous Hcy injections (n = 8 – 10). All statistics were calculated by Mann-Whitney-U pairwise comparisons and Benjamini-Hochberg correction for multiple testing (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001). All significances are shown in supplementary material.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6717713/v1/7770db33f2f4823e2cd2b45b.jpg"},{"id":83299608,"identity":"5177f5a0-889b-4153-974f-1cd98aec86d6","added_by":"auto","created_at":"2025-05-22 14:39:35","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":283398,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSphingolipids and their species distribution in blood cells and heart from\u003c/strong\u003e \u003cstrong\u003erabbits fed different diets in the absence or presence of intravenous Hcy injections. \u003c/strong\u003e(A) Total ceramides and sphingomyelins, and their species distribution grouped by levels of total fatty acid saturation and chain length in blood cells from rabbits fed SD, VCDD, HCD or HCD/VCD as well as VCDD in the presence of intravenous Hcy injections (n = 3 – 7). (B) Total ceramides and sphingomyelins, and their species distribution grouped by levels of total fatty acid saturation and chain length in heart from rabbits fed SD, VCDD, HCD or HCD/VCD as well as VCDD in the presence of intravenous Hcy injections (n = 8 – 10). All statistics were calculated by Mann-Whitney-U pairwise comparisons and Benjamini-Hochberg correction for multiple testing (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001). All significances are shown in supplementary material.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6717713/v1/d34e1445dac43b04a6161c79.jpg"},{"id":83299609,"identity":"470952d7-ca0d-46dc-9ebf-0c1ad347e36d","added_by":"auto","created_at":"2025-05-22 14:39:35","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":144322,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAcylcarnitines and their species distribution in blood cells and heart from\u003c/strong\u003e \u003cstrong\u003erabbits fed different diets in the absence or presence of intravenous Hcy injections. \u003c/strong\u003e(A) Total acylcarnitines and their species distribution grouped by levels of total fatty acid saturation and chain length in blood cells from rabbits fed SD, VCDD, HCD or HCD/VCD as well as VCDD in the presence of intravenous Hcy injections (n = 3 – 7). (B) Total acylcarnitines and their species distribution grouped by levels of total fatty acid saturation and chain lengths in heart from rabbits fed SD, VCDD, HCD or HCD/VCD as well as VCDD in the presence of intravenous Hcy injections (n = 8 – 10). All statistics were calculated by Mann-Whitney-U pairwise comparisons and Benjamini-Hochberg correction for multiple testing (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001). All significances are shown in supplementary material.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6717713/v1/626770e5a113bd4a3958922c.jpg"},{"id":83300016,"identity":"07a1bfc8-c0de-40d0-8790-5a42ba73145d","added_by":"auto","created_at":"2025-05-22 14:47:35","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":708837,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLipidome of aortic tissue from rabbits fed different diets.\u003c/strong\u003e (A) Lipidome of injured aorta from rabbits fed SD, VCDD, HCD or HCD/VCD shown as OPLS scoring- and loading plots as well as groupwise comparisons between SD and VCDD, and HCD and VCD/HCD shown as volcano plots (n = 3 – 4). (B) Total TAG, PC, PE and LPC in aortic tissue from rabbits fed SD, VCDD, HCD or HCD/VCD (n = 3 – 4). (C) Total free cholesterol, cholesterol ester and fatty acids in aortic tissue from rabbits fed SD, VCDD, HCD or HCD/VCD (n = 3 – 4). (D) Total ceramides and sphingomyelins in aortic tissue from rabbits fed SD, VCDD, HCD or HCD/VCD (n = 3 – 4). (E) Total acylcarnitines in aortic tissue from rabbits fed SD, VCDD, HCD or HCD/VCD (n = 3 – 4). All statistics were calculated by Mann-Whitney-U pairwise comparisons and Benjamini-Hochberg correction for multiple testing (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001). All significances are shown in supplementary material.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6717713/v1/9e6e70c6a0c88d51626f0d53.jpg"},{"id":83300795,"identity":"0152f2ac-278b-4e49-a759-b39b6dafe929","added_by":"auto","created_at":"2025-05-22 14:55:35","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":294600,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative Scanning Transmission Electron Micrographs (STEM) and Transmission Electron Micrographs (TEM) of aortic media sections from rabbits fed different diets.\u003c/strong\u003e (A – D) Representative sections of the aortic tunica media shown as STEM, their enlargement (A1 – D1) and TEM images (right) of rabbits fed SD (A), VCDD (B), HCD (C) or VCD/HCD (D). LD, lipid droplets; asterisk, mitochondria; arrow head, endoplasmic reticulum. Corresponding scale bars as indicated in the figure.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6717713/v1/7232569dc87ffc5c01535ac2.jpg"},{"id":83299606,"identity":"58c578ae-046c-4b2a-8422-7b48401ab3b4","added_by":"auto","created_at":"2025-05-22 14:39:35","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":157991,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMetabolites of PC metabolism in liver and serum from\u003c/strong\u003e \u003cstrong\u003erabbits fed different diets in the absence or presence of intravenous Hcy injections. \u003c/strong\u003e(A) Choline (n = 8 – 9), (B) phosphorylcholine (n = 8 – 9) and (C) glycerophosphocholine (n = 8 – 9) in liver and serum from rabbits fed SD, VCDD, HCD or HCD/VCD as well as VCDD in the presence of intravenous Hcy injections. All statistics were calculated by Mann-Whitney-U pairwise comparisons and Benjamini-Hochberg correction for multiple testing (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001). All significances are shown in supplementary material.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6717713/v1/716541f39d853d8d478b8f18.jpg"},{"id":83301107,"identity":"ceb7dd49-7f9b-4050-a5ab-9224843bf4c9","added_by":"auto","created_at":"2025-05-22 15:03:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6703426,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6717713/v1/8bd1d254-05e8-4a7f-8dfe-e6674e0c493b.pdf"},{"id":83300794,"identity":"c9ec86dd-52e6-430c-b753-7cf08740022a","added_by":"auto","created_at":"2025-05-22 14:55:35","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1630395,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1: Distribution of glycerolipid species in blood cells and heart from\u003c/strong\u003e \u003cstrong\u003erabbits fed different diets in the absence or presence of intravenous Hcy injections. \u003c/strong\u003eAnalysis of individual TAG-, PC-, PE- and LPC species in blood cells and hearts from rabbits fed SD, VCDD, HCD or HCD/VCD as well as VCDD in the presence of intravenous Hcy injections (n = 3 – 10). All statistics were calculated by Mann-Whitney-U pairwise comparisons and Benjamini-Hochberg correction for multiple testing (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001). All significances are shown in supplementary material.\u003c/p\u003e","description":"","filename":"SupplementaryFigureS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6717713/v1/2f6865bfa0bdba2154df1617.pdf"},{"id":83299617,"identity":"4e26fdbe-715b-416c-9d70-378c3187b0f7","added_by":"auto","created_at":"2025-05-22 14:39:35","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":331080,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S2: Distribution of sphingolipid species in blood cells and heart from\u003c/strong\u003e \u003cstrong\u003erabbits fed different diets in the absence or presence of intravenous Hcy injections. \u003c/strong\u003eAnalysis of individual Cer- and SM species in blood cells and hearts from rabbits fed SD, VCDD, HCD or HCD/VCD as well as VCDD in the presence of intravenous Hcy injections (n = 3 – 10). All statistics were calculated by Mann-Whitney-U pairwise comparisons and Benjamini-Hochberg correction for multiple testing (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001). All significances are shown in supplementary material.\u003c/p\u003e","description":"","filename":"SupplementaryFigureS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6717713/v1/8f5a8eefbb3770416bf07a04.pdf"},{"id":83300015,"identity":"b6764bc3-69da-48f1-869c-83b1ffe5b5ec","added_by":"auto","created_at":"2025-05-22 14:47:35","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":197827,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S3: Distribution of acylcarnitine species in blood cells and heart from\u003c/strong\u003e \u003cstrong\u003erabbits fed different diets in the absence or presence of intravenous Hcy injections. \u003c/strong\u003eAnalysis of individual CAR species in blood cells and hearts from rabbits fed SD, VCDD, HCD or HCD/VCD as well as VCDD in the presence of intravenous Hcy injections (n = 3 – 10). All statistics were calculated by Mann-Whitney-U pairwise comparisons and Benjamini-Hochberg correction for multiple testing (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001). All significances are shown in supplementary material.\u003c/p\u003e","description":"","filename":"SupplementaryFigureS3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6717713/v1/a35e9ee266d9e24df72d0517.pdf"},{"id":83300012,"identity":"59b0edc5-1a83-49fb-a0e3-05e5c0da0690","added_by":"auto","created_at":"2025-05-22 14:47:35","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":638267,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S4:\u003c/strong\u003e \u003cstrong\u003eDistribution of species in all analyzed lipid classes from injured aorta from\u003c/strong\u003e \u003cstrong\u003erabbits fed different diets. \u003c/strong\u003eAnalysis of individual TAG-, PC-, PE-, LPC-, Cer-, SM- and CAR species in aortic tissue from rabbits fed SD, VCDD, HCD or HCD/VCD (n = 3 – 4). All statistics were calculated by Mann-Whitney-U pairwise comparisons and Benjamini-Hochberg correction for multiple testing (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001). All significances are shown in supplementary material.\u003c/p\u003e","description":"","filename":"SupplementaryFigureS4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6717713/v1/f062f70b42b71734f717c830.pdf"},{"id":83299615,"identity":"660b5f8a-837b-44c4-b924-16214c3764c2","added_by":"auto","created_at":"2025-05-22 14:39:35","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":341974,"visible":true,"origin":"","legend":"\u003cp\u003eStatistical analysis\u003c/p\u003e","description":"","filename":"SupplementaryMaterialStatistics.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6717713/v1/5062331028c9c8e0ff31ddfc.xlsx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eHomocysteine Leads to Decreased Acylcarnitine Levels in the Heart in a Rabbit Model of Atherosclerosis\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCardiovascular disease (CVD) is the leading cause of death worldwide. In 2019 17.9\u0026nbsp;million people died from CVD, representing 32% of all global deaths (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). In most cases, CVD is due to the development of atherosclerosis, a chronic, progressive, inflammatory disease of the aorta characterized by dyslipidemia and an accumulation of lipids in the aortic wall (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). However, established risk factors including hypercholesterolemia can explain only about 50% of all cases of atherosclerosis (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHomocysteine (Hcy) is a sulfur-containing, non-proteinogenic amino acid involved in intermediary metabolism of methionine. It is produced from S-adenosyl-L-homocysteine, a strong product inhibitor of S-adenosyl-L-methionine-dependent methyltransferases, and is degraded either by its remethylation to methionine within the methylation cycle or by its transsulfuration to cysteine resulting in its removal from the methylation cycle (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). An accumulation of Hcy in the blood, termed hyperhomocysteinemia (HHcy), is found in 5\u0026ndash;10% of the general population, in up to 30% of the elderly and even in 70% of men over 80 years of age (\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). In most cases HHcy is associated with low status of one or more vitamins required for Hcy degradation (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). HHcy is an independent risk factor for atherosclerosis, leads to increased cardiovascular risk in combination with hypercholesterolemia (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e), is linked to cardiac pathologies (\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) and is strongly associated with cardiovascular as well as non-cardiovascular mortality (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). In addition to age, elevated Hcy is linked to further cardiovascular risk factors such as physical inactivity (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), high fat diet (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), low skeletal muscle mass (\u003cspan additionalcitationids=\"CR17 CR18 CR19\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e) and has been also associated with obesity and fatty liver disease (\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn mice elevation of plasma Hcy levels due to dietary deprivation of folate, vitamins B\u003csub\u003e6\u003c/sub\u003e and B\u003csub\u003e12\u003c/sub\u003e, high methionine intake, or genetic block in Hcy metabolization exacerbates atherosclerosis development (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Furthermore, genetic block in Hcy degradation in mice leads to deregulation of lipid metabolism, including reduced fat mass (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), aortic lipid deposition (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) as well as deregulation of phospholipid and sphingomyelin metabolism (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Moreover, elevated Hcy leads to an upregulation of the sterol regulatory element\u0026ndash;binding proteins (SREBPs) in cultured human hepatocytes as well as vascular endothelial and aortic smooth muscle cells, which is associated with increased expression of genes responsible for cholesterol/triglyceride biosynthesis and uptake, and with intracellular accumulation of cholesterol (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Similarly, in yeast elevated Hcy is associated with triglyceride and fatty acid accumulation (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn contrast to rodents, rabbits spontaneously develop foam cell-rich plaques (fatty streaks) on a high-fat diet (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Previously we have shown that a diet free from vitamin B\u003csub\u003e12\u003c/sub\u003e and reduced in folate (20%), vitamin B\u003csub\u003e6\u003c/sub\u003e (20%), and choline (10%) required for Hcy degradation (VCDD) leads to an accumulation of macrophages and lipids in the aorta, aortic stiffening and disorganization of aortic collagen in a balloon-injured rabbit model of atherosclerosis (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Furthermore, combination of VCDD with high cholesterol diet (HCD) results in further thickening of the aorta, altered lipoprotein profile as well as impaired aortic vascular reactivity compared to HCD alone (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Elevation of plasma Hcy levels by intravenous injections of Hcy into rabbits fed VCDD in the absence of hypercholesterolemia results in alteration of lipoprotein profile, impairment of vascular reactivity of the aorta, disorganization of aortic elastin and collagen, decreased total protein methylated arginine and altered metabolomic profiles compared to rabbits fed VCDD only (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Here, we analyzed lipid molecular species in blood cells and heart from rabbits fed VCDD in the presence or absence of additional intravenous injections of Hcy and hypercholesterolemia as well as in the aorta in the presence or absence of hypercholesterolemia. Our data show that elevation of plasma Hcy levels by intravenous injections of Hcy into rabbits fed VCDD in the absence of hypercholesterolemia as well as VCDD alone leads to an alteration of lipid metabolism in these tissues. Deregulation of phospholipid, cholesterol, ceramide, fatty acid and acylcarnitine metabolism in rabbits fed VCDD in the presence of intravenous Hcy injections suggests that deregulated lipid metabolism is likely to play a central role in pathological consequences associated with elevated Hcy.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1. Animal experiments\u003c/h2\u003e \u003cp\u003e All animal experiments were approved by the Austrian Federal Ministry of Education, Science and Research (BMWF-66.010_0070-V_3b_2018). For the study 4\u0026ndash;6 month old male New Zealand White (NZW) rabbits were acclimatized for 2\u0026ndash;4 weeks and divided into groups. All rabbits were sensitized for the development of atherosclerosis by balloon injury using the JURY device for induction of fully automated pressure- and retraction-controlled vessel wall injury (one retraction, 1.8 bar, retraction speed of 2 mm/s) (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) and fed special diets. The diets used included an unpurified complete chow diet (standard diet, SD) for full nutritional conditions and purified special diets containing 1% cholesterol (high cholesterol diet, HCD), deficient in vitamins and choline required for Hcy metabolization (no vitamin B\u003csub\u003e12\u003c/sub\u003e, 20% folate (2 mg/kg), 20% vitamin B\u003csub\u003e6\u003c/sub\u003e (6 mg/kg) and 10% choline (0.128 mg/kg), VCDD), deficient in vitamins and choline, and containing 1% cholesterol (VCD/HCD) and VCDD combined with L-Hcy intraveneous injections (total 60 \u0026micro;mol/kg, VCDD\u0026thinsp;+\u0026thinsp;Hcy). VCDD was pre-fed for two weeks and HCD for one week before the surgery to elevate the levels of plasma Hcy and cholesterol at the time of sensitization. Special purified diets were acquired from Sniff, Germany. Average daily food intake was determined by weighing the remaining food after two days once per week for each animal. Body weight was measured once a week. Eight weeks after balloon injury the rabbits were sacrificed. After euthanasia blood was withdrawn with a syringe directly from the heart and collected in EDTA and Serum vacuette tubes (Greiner). All blood tubes were inverted 5 times and serum tubes were additionally incubated for 30 min at RT. Tubes were centrifuged at 2200 g and 4\u0026deg;C. Blood cells were extracted from EDTA tubes. Blood cells, heart and aortic specimens (for lipidomic analysis) as well as serum and liver (for metabolomic analysis) were immediately frozen in liquid nitrogen. Aortic specimens for electronic miscroscopy were prepared as described below.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2. Lipid extraction\u003c/h3\u003e\n\u003cp\u003eLipid extraction was performed in accordance with the protocol previously published by Matyash et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Briefly, one steel ball was added to 12 mg of blood cells or macerated aortic tissue sample and homogenized in 700 \u0026micro;L methyl-\u003cem\u003etert\u003c/em\u003e-butyl-ether (MTBE):MeOH (10:3, v:v)\u0026thinsp;+\u0026thinsp;50 \u0026micro;L of internal standard (IST) master mix in a homogenizer MM 40 (Retsch) three times for 8 min at 30 Hz and 4\u0026deg;C. IST mastermix contained LPE 17:1 (107.5 \u0026micro;M), LPC 17:0 (9.8 \u0026micro;M), PE 34:0 (34.7 \u0026micro;M), PC 38:0 (73.4 \u0026micro;M), Cer 17:0 (9.1 \u0026micro;M for aorta samples and 4.5 \u0026micro;M for blood cell samples), TG 51:0 (177.8 \u0026micro;M for aorta samples and 58.9 \u0026micro;M for blood cell samples) and SM 17:0 (20.9 \u0026micro;M) in chloroform/methanol (2/1, v/v). Samples were then shaken in a thermomixer for 60 min at 1400 rpm and 4\u0026deg;C. Then 200 \u0026micro;L ddH\u003csub\u003e2\u003c/sub\u003eO were added to the samples and the samples were shaken again for 20 min at 1400 rpm and 4\u0026deg;C before centrifuging samples at 13,500 rpm for 10 min at room temperature. Supernatant was transferred to a 1.5 mL auto sampler vial, dried under nitrogen stream and resolved in 500 \u0026micro;L chloroform/MeOH (2/1, v/v). 100 \u0026micro;L were dried again under nitrogen stream, resolved in 100 \u0026micro;L isopropanol:MeOH/H\u003csub\u003e2\u003c/sub\u003eO (30/15/5, v/v/v) and directly used for mass spectrometry. For heart lipid extraction 5 mg of macerated tissue was homogenized in 700 \u0026micro;L MTBE/MeOH (10/3, v/v) containing 1% acetic acid, 1 \u0026micro;M BHT and 1 \u0026micro;L IST master mix in a homogenizer MM 40 (Retsch) for 3 min at 30 Hz and 4\u0026deg;C. IST mastermix contained BMP 28:0 (100 \u0026micro;M), Cer 35:1 (13.33 \u0026micro;M), DG 28:0 (66.67 \u0026micro;M), FA 17:1 (1.25 mM), FC d7 (1.5 mM), GluCer 24:1 (50 \u0026micro;M), LPC 17:1 (40 \u0026micro;M), LPE 17:1 (150 \u0026micro;M), LPG 17:1 (200 \u0026micro;M), LPS 34:0 (250 \u0026micro;M), MG 17:0 (266.67 \u0026micro;M), PA 28:0 (200 \u0026micro;M), PC 28:0 (250 \u0026micro;M), PE 34:0 (666.67 \u0026micro;M), PG 34:0 (80 \u0026micro;M), SM 34:0 (50 \u0026micro;M) and TG 45:0 (100 \u0026micro;M). Samples were shaken in a thermomixer for 20 min at 1400 rpm and 4\u0026deg;C before 200 \u0026micro;L ddH\u003csub\u003e2\u003c/sub\u003eO was added and mixed again in thermomixer for 20 min. Samples were centrifuged at 14000 rpm for 5 min, upper phase was transferred to a new tube, dried under nitrogen stream and resolved in 500 \u0026micro;L MTBE/MeOH (10/3, v/v). 100 \u0026micro;L were further diluted 1:1 in MTBE/MeOH/ddH\u003csub\u003e2\u003c/sub\u003eO (30/5/5, v/v/v) for mass spectrometry.\u003c/p\u003e\n\u003ch3\u003e3. Lipidomic analysis\u003c/h3\u003e\n\u003cp\u003eFor the analysis of total blood cellsand aorta lipids, an Acquity UPLC system coupled with a Synapt quadrupole time of flight (QTOF; Waters) tandem mass spectrometer with ESI ion source was used. In addition, an L-6200 Intelligent Pump (Merck/Hitachi) was used to pump a reference solution for calibration purposes. Reversed phase chromatography was performed with a Luna Omega 1.6 \u0026micro;m C18 100 \u0026Aring; LC column (50*2.1 mm) (Phenomenex) at a column compartment temperature of 50\u0026deg;C. As solvent A MeOH/ddH\u003csub\u003e2\u003c/sub\u003eO (1/1, v/v)\u0026thinsp;+\u0026thinsp;1% NH\u003csub\u003e4\u003c/sub\u003eAc\u0026thinsp;+\u0026thinsp;0.1% HCOOH\u0026thinsp;+\u0026thinsp;8 \u0026micro;M H\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e and as solvent B isopropanol\u0026thinsp;+\u0026thinsp;1% NH\u003csub\u003e4\u003c/sub\u003eAc\u0026thinsp;+\u0026thinsp;0.1% HCOOH\u0026thinsp;+\u0026thinsp;8 \u0026micro;M H\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e was used. 20% solvent B was held for 2 min and then gradually increased to 45% over the next 2 min. Subsequently solvent B was further gradually increased to 85% over 13 min and then to 100% over 1 min. 100% solvent B was held for 1 min before dropping back to 20% in 3 s where it was held for 1.95 min. Flow rate was set to 100 \u0026micro;L/min for the first 1.05 min, to 300 \u0026micro;L/min between 1.05 and 19.55 min and to 100 \u0026micro;L/min until the end of the run. Total run time per sample was 20 min. The QTOF was set to full scan for all m/z ratios in between 50 and 1800 alternating with fragment ion scans for m/z ratios between 50 to 1800. For the analysis of total lipids in heart a UHPLC 1290 Infinity LC system coupled to a 6560 Ion Mobility Q-TOF LC/MS with a dual AJS ESI ion source (Agilent) was used. Reversed phase chromatography was performed with a Acquity BEH C18 1.7 \u0026micro;m (2.1 \u0026times; 150 mm) column (Waters) at a column compartment temperature of 50\u0026deg;C. A binary gradient of solvent A ddH\u003csub\u003e2\u003c/sub\u003eO and solvent B isopropanol was used. Both solvents contained 1% NH\u003csub\u003e4\u003c/sub\u003eAc, 0.1% HCOOH and 8 \u0026micro;M H\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e. 40% solvent B was held for 30 s before gradualy increasing to 80% solvent B over the course of 8.5 min and further to 100% solvent B over 13 more min. 100% solvent B was held for 2.5 min and then dropped back to 40% solvent B in 30 s where it was held for 5 more min. Total run time per sample was 30 min at a constant flow rate of 150 \u0026micro;L/min. Per sample 1 \u0026micro;L was injected for positive ion mode and 5 \u0026micro;L for negative ion mode. Lipidomic data were annotated with the Lipid Data Analyzer (LDA version 2.8.3) software.\u003c/p\u003e\n\u003ch3\u003e4. Metabolomics\u003c/h3\u003e\n\u003cp\u003eMetabolomics was performed as previously described (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). In brief, for metabolite analyses, liver samples were suspended in pre-mixed 400 \u0026micro;L of ice-cold methanol and 200 \u0026micro;L of MilliQ H\u003csub\u003e2\u003c/sub\u003eO, and transferred to Precellys tubes with 1.4 mm diameter zirconium oxide beads (Bertin Technologies). This suspension was homogenized two times for 20 s by Precellys24 tissue homogenizer at 25\u0026deg;C (Bertin Technologies). 400 \u0026micro;L MeOH were directly added to 200 \u0026micro;L of serum. Afterwards, the homogenized samples were stored at -20\u0026deg;C for at least 30 min and centrifuged at 10000 g for 30 min at 4\u0026deg;C. For metabolite analyses, the supernatants were transferred to new tubes and lyophilized at \u0026lt;\u0026thinsp;1 Torr, 850 rpm, 25\u0026deg;C for 10 hours in a vacuum-drying chamber (Savant Speedvac SPD210 vacuum concentrator), with an attached cooling trap (Savant RVT450 refrigerated vapor trap) and vacuum pump (VLP120) (Thermo Scientific). For NMR experiments, samples were dissolved in 50 mM phosphate buffer (pH 7.4, prepared in D\u003csub\u003e2\u003c/sub\u003eO) and measured at 310 K using a 600 MHz Avance Neo NMR spectrometer (Bruker) equipped with a TXI 600S3 probe head. The Carr\u0026ndash;Purcell\u0026ndash; Meiboom\u0026ndash;Gill pulse sequence was used to acquire 1H 1D NMR spectra with a pre-saturation for water suppression (cpmgpr1d, 128 scans, 73728 points in F1, 12019.230 Hz spectral width, recycle delay 4 s) (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). The data were processed in Topspin version 4.4 (Bruker) using one-dimensional exponential window multiplication of the FID, Fourier transformation, and phase correction. NMR data analyses: For metabolite analyses, NMR data were imported into Matlab2014b, TSP was used as the internal standard for chemical-shift referencing (set to 0 ppm), regions around the water, TSP and methanol signals were excluded, the NMR spectra were aligned, and a probabilistic quotient normalization was performed. Metabolite identification was carried out using Chenomx NMR Suite 8.4 (Chenomx Inc.) and reference compounds. Quantification of metabolites was carried out by signal integration of normalized spectra. For each metabolite, a representative peak with no overlapping signals was identified, the start and endpoints of the integration were chosen to revolve around that peak, and the area of the peak was integrated by summing up the value of each data point. Orthogonal partial least squares discriminant analysis (OPLS) and sparse partial least squares-discriminant analysis (SPLS) were performed in Matlab 2014b and MetaboAnalyst 5.0, as well as all associated data consistency checks and cross-validation (\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). The statistical significance of the determined differences was validated by the quality assessment statistic Q\u003csup\u003e2\u003c/sup\u003e and presented as specific p-values. For visualization of the metabolite data, heatmaps and variable importance during projection (VIP) scores were calculated with MetaboAnalyst 5.0. Normalized peak integrals were presented as scatter box plots.\u003c/p\u003e\n\u003ch3\u003e5. Electron microscopy\u003c/h3\u003e\n\u003cp\u003eElectron microscopy of aortic tissue was performed as previously described (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). In brief, aortic tissue was fixed in 2.5% (w/v) glutaraldehyde and 2% PFA (w/v) in 0.1 M cacodylate buffer, pH 7.4, for 2 hours, and then post-fixed in 2% (w/v) osmium tetroxide for 2 hours at RT. After dehydration (in graded series of ethanol), tissues were infiltrated (ethanol and TAAB Embedding Resin, pure TAAB Embedding Resin) and placed in TAAB Embedding Resin (8 hours), transferred into embedding molds, and polymerized (48 hours, 60\u0026deg;C). Ultrathin sections (70 nm) were cut with a UC 7 Ultramicrotome (Leica Microsystems) and stained with lead citrate for 5 min and platinum blue for 15 min. Electron micrographs were taken using a Tecnai G2 transmission electron microscope (FEI) with a Gatan Ultrascan 1000 charge coupled device (CCD) camera (-20\u0026deg;C, acquisition software Digital Micrograph, Gatan, and Serial EM, University of Colorado). Acceleration voltage was 120 kV.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e6. Data processing and statistical analysis\u003c/h2\u003e \u003cp\u003eAll data were processed with R/Rstudio (versions 4.3/2023.03.1\u0026ndash;446). In addition to the basic R functions, the tidyverse packages (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e) were used for data transformation. For multivariate analysis (OPLS) the lipidr package (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e) was used. The rstatix package was used for outlier detection and statistical analysis (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://rpkgs.datanovia.com/rstatix/\u003c/span\u003e\u003cspan address=\"https://rpkgs.datanovia.com/rstatix/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Exclusion of outliers was based on values above the third quartile plus three times the interquartile range or below the first quartile minus three times the interquartile range. Statistics were calculated with Mann-Whitney U test and corrected with false discovery rate for multiple testing. The significance levels are indicated with asterisks (* = p\u0026thinsp;\u0026le;\u0026thinsp;0.05, ** = p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** = p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Sample sizes in figure legends refer to biological replicates (independent animals) and in case of box plots are depicted as individual spots. Results of statistical analyses are shown in Supplementary Material.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003e1. Hcy injections into VCDD-fed rabbits lead to deregulation of lipid metabolism in blood cells and heart.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePreviously we have shown that a diet deficient in vitamins and choline required for Hcy degradation (VCDD) leads to aortic lipid accumulation in balloon-injured rabbit model of atherosclerosis in the absence of hypercholesterolemia (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) and elevation of plasma Hcy levels by intravenous injections of Hcy into these rabbits (VCDD\u0026thinsp;+\u0026thinsp;Hcy) results in their degradation (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Furthermore, combination of high cholesterol diet (HCD) with VCDD (VCD/HCD) led to 40% increase in LDL-cholesterol (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) and further elevation of plasma Hcy levels by Hcy injections in VCDD-fed rabbits dramatically increased VLDL-triglycerides compared to VCDD alone (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). This observation prompted us to analyze lipidome composition in our rabbit model.\u003c/p\u003e \u003cp\u003eBalloon-injured rabbits were fed VCDD, HCD, VCD/HCD and VCDD in combination with intravenous injections of Hcy. While VCDD resulted in doubling of plasma Hcy levels, rabbits from the VCDD\u0026thinsp;+\u0026thinsp;Hcy group exhibited more than 3-fold elevation of plasma Hcy levels (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). OPLS analysis revealed distinct lipid profile in blood cells, but not in hearts from rabbits in the VCDD\u0026thinsp;+\u0026thinsp;Hcy group compared to SD, VCDD, HCD or VCD/HCD (Fig.\u0026nbsp;1). The most dysregulated lipid species in blood cells included triglycerides (TG), TG 56:0, TG 56:1, TG 58:0, TG 58:1, TG 60:1 and TG 62:1, phosphatidylethanolamines (PE), PE 34:3 and PE 36:1, as well as phosphatidylcholines (PC), PC 32:0 and 36:2 (Fig.\u0026nbsp;1A). In heart, the most dysregulated lipid species included PCs, PC 31:0, PC 34:3, PC 35:2, PC 36:5, PC 37:4 and PC 38:7 as well as acylcarnitines (CARs), CAR 16:0, CAR 16:1, CAR 18:1 and CAR 18:2 (Fig.\u0026nbsp;1B). In blood cells volcano plot analysis showed a decrease of TG species in VCDD compared to VCDD\u0026thinsp;+\u0026thinsp;Hcy as well as increased TG species in HCD compared to VCD/HCD group (Fig.\u0026nbsp;1A). Volcano plot analysis in blood cells also showed decreased ceramides (Cer) as well as increased CAR in VCDD compared to SD, decreased Cer, PC, PE and CAR in VCDD\u0026thinsp;+\u0026thinsp;Hcy compared to VCDD and increased CAR, Cer and PE in VCD/HCD compared to HCD (Fig.\u0026nbsp;1A). In the heart, volcano plot analysis revealed a decrease of CAR and increase of TG in VCDD compared to SD. Furthermore, in heart VCDD\u0026thinsp;+\u0026thinsp;Hcy led to dysregulation of CAR and PC species as well as decreased TG and elevated PE compared to VCDD, while VCD/HCD exhibited decreased TG and SM in addition to increased PC in hearts compared to HCD (Fig.\u0026nbsp;1B).\u003c/p\u003e \u003cp\u003e \u003cb\u003e2. Hcy injections into VCDD-fed rabbits lead to decreased TG levels in blood cells, but not in the heart\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWhile TG levels in blood cells from VCDD-fed rabbits exhibited a significant increase compared to SD, elevation of plasma Hcy by intravenous injections of Hcy into VCDD-fed rabbits resulted in a significant decrease of total TG levels in blood cells compared to rabbits fed VCDD only (Fig.\u0026nbsp;2A). In blood cells rabbits from the VCDD group accumulated primarily TG species with 2\u0026ndash;7 double bonds and containing fatty acids (FAs) with a total of 50\u0026ndash;56 carbons compared to SD (Fig.\u0026nbsp;2A). Similarly, rabbits from the VCDD\u0026thinsp;+\u0026thinsp;Hcy group, while exhibiting decrease over all analyzed TG species compared to VCDD alone, the decrease of TG species containing 2\u0026ndash;7 double bounds and total fatty acid chain lengths of 50\u0026ndash;56 carbons was less pronounced compared to other species (Fig.\u0026nbsp;2A and S1A). Feeding rabbits VCD/HCD, similarly to VCDD, led to accumulation of total TG levels in blood cells, albeit tendentially (Fig.\u0026nbsp;2A). However, in contrast to rabbits from the VCDD and VCDD\u0026thinsp;+\u0026thinsp;Hcy groups, blood cells from rabbits fed VCD/HCD accumulated TG species containing polyunsaturated FAs with 6 or more double bonds compared to either SD, HCD and VCDD, and similarly to the VCDD\u0026thinsp;+\u0026thinsp;Hcy group exhibited decreased levels of saturated TG species (TG X:0) (Fig.\u0026nbsp;2A and S1A). In contrast to blood cells, hearts from VCDD-fed rabbits showed only a tendential increase of total TG levels compared to SD, however similarly to blood cells intravenous Hcy injections into VCDD-fed rabbits also resulted in tendential decrease of total TG levels in hearts compared to VCDD alone (Fig.\u0026nbsp;2B). Furthermore, similarly to blood cells VCD/HCD resulted in a drop of total TG levels in the heart compared to VCDD group, but in contrast to blood cells it led to their decrease also compared to HCD (Fig.\u0026nbsp;2B). Additionally, in contrast to blood cells, both the VCDD\u0026thinsp;+\u0026thinsp;Hcy and VCD/HCD groups exhibited a decrease of all TG species in the heart regardless of saturation or chain lengths (Fig.\u0026nbsp;2B and S1B).\u003c/p\u003e \u003cp\u003e \u003cb\u003e3. Hcy injections into VCDD-fed rabbits lead to phospholipid remodeling both in blood cells and heart\u003c/b\u003e \u003c/p\u003e \u003cp\u003eVCDD feeding also resulted in dysregulation of phospholipid (PL) metabolism in blood cells. VCDD both in combination with either Hcy injections or high cholesterol led to increased total PC levels, in particular an increase in PC 34:2, PC 36:3 and PC 36:4 species compared to either VCDD alone or HCD alone respectively (Fig.\u0026nbsp;2A and S1A). Furthermore, rabbits from the VCD/HCD group also displayed an increase in PC 32:1, PC 32:2, PC 38:5, PC 38:6, PC 40:4, PC 40:5 and PC 40:6 species compared to HCD, which were not affected in blood cells from rabbits in the VCDD\u0026thinsp;+\u0026thinsp;Hcy group. Moreover, rabbits from the VCDD\u0026thinsp;+\u0026thinsp;Hcy group showed increased levels of saturated PC species and species with lower number of double bonds, namely PC 32:0, PC 34:0, PC 36:1 and PC 36:2 compared to rabbits fed VCDD alone (Fig.\u0026nbsp;2A and S1A). Interestingly, HCD with or without VCDD led to a decrease in these species compared to SD (Fig.\u0026nbsp;2A and S1A). While in blood cells VCDD in combination with Hcy injections resulted in an accumulation of PC species containing lower number of double bonds and shorter fatty acid chain lengths compared to VCDD alone, VCDD in combination with HCD led to an accumulation of PC species containing more unsaturated and longer fatty acids compared to HCD alone (Fig.\u0026nbsp;2A).\u003c/p\u003e \u003cp\u003eIn contrast to PC, elevation of total PE levels in blood cells was detected only in response to VCDD\u0026thinsp;+\u0026thinsp;Hcy, but not in response to VCDD, HCD or VCD/HCD (Fig.\u0026nbsp;2A). In the VCDD\u0026thinsp;+\u0026thinsp;Hcy group all analyzed PE species with total FA chain lengths of 32\u0026ndash;36 carbons as well as PE 38:6 were increased compared to VCDD alone (Fig.\u0026nbsp;2A and S1A). Noteworthy, longer and more unsaturated PE species, namely PE 40:4 and PE 40:5, were increased in blood cells from rabbits in the VCD/HCD group compared to HCD alone similarly to PC, but due to low abundance of these species their increase did not affect overall total PE levels in this group (Fig.\u0026nbsp;2A and S1A). Noteworthy, while levels of total PC and PE were significantly elevated in blood cells, but not in hearts of rabbits from the VCDD\u0026thinsp;+\u0026thinsp;Hcy group compared to rabbits fed VCDD alone, PC/PE ratios were unchanged both in blood cells and hearts of rabbits in response to both VCDD\u0026thinsp;+\u0026thinsp;Hcy and VCDD groups compared to SD (Fig.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eWhile HCD led to an increase of total lyso-PC (LPC) levels in blood cells compared to SD as well as in VCD/HCD compared to HCD, total LPC levels were unchanged in response to VCDD compared to SD or VCDD\u0026thinsp;+\u0026thinsp;Hcy compared to VCDD (Fig.\u0026nbsp;2A). Furthermore, both rabbits from HCD and VCD/HCD groups exhibited an accumulation of all analyzed LPC species with exception of LPC 14:1 in blood cells compared to either SD or HCD respectively (Fig.\u0026nbsp;2A and S1A). Interestingly, while VCDD alone did not lead to any changes in blood cell LPC levels compared to SD, combination of HCD and VCDD led to even stronger accumulation of LPCs rather than HCD alone (Fig.\u0026nbsp;2A).\u003c/p\u003e \u003cp\u003eIn contrast, in heart none of the groups exhibited changes in total PC or PE levels (Fig.\u0026nbsp;2B). Regardless of unchanged total PC levels, both HCD and VCD/HCD resulted in elevation of PC species containing longer fatty acids or a higher number of double bonds and in the heart, specifically PC species containing fatty acids with a total of 5\u0026ndash;8 double bonds or total chain lengths of 37\u0026ndash;40 carbons compared to SD (Fig.\u0026nbsp;2B and S1B). Similarly, rabbits from the VCDD\u0026thinsp;+\u0026thinsp;Hcy group exhibited significantly increased PC species containing 4 and 7 double bonds in the heart as well as tendency towards an accumulation of PC species containing longer fatty acids with total chain lengths of 34\u0026ndash;40 carbons, whereas PC species containing shorter fatty acids with total chain lengths of 24\u0026ndash;28 carbons were decreased compared to VCDD alone (Fig.\u0026nbsp;2B and S1B).\u003c/p\u003e \u003cp\u003eFurthermore, similar to blood cells HCD and VCD/HCD feeding led to significant accumulation of total LPC levels in hearts compared to non-HCD groups (Fig.\u0026nbsp;2B). However, in contrast to blood cells VCD/HCD did not lead to higher total LPC levels in heart compared to HCD alone (Fig.\u0026nbsp;2B). Increase of LPC species in HCD and VCD/HCD groups was distributed equally (Fig.\u0026nbsp;2B and S1B).\u003c/p\u003e \u003cp\u003e \u003cb\u003e4. Hcy injections into VCDD-fed rabbits lead to an accumulation of free cholesterol in blood cells and heart, as well as total fatty acids in blood cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAnalysis of free cholesterol levels in balloon-injured NZW rabbits fed VCDD in the absence or presence of HCD, or with additional intravenous injections of Hcy revealed that also cholesterol metabolism was dysregulated in response to elevated Hcy. While intravenous injections of Hcy into VCDD-fed rabbits led to significant increase of free cholesterol levels in blood cells compared to VCDD, both VCDD without Hcy injections compared to SD as well as VCD/HCD compared to HCD showed only tendential increase of free cholesterol levels in blood cells (Fig.\u0026nbsp;3A). Moreover, VCDD led to tendentially increased cholesterol ester levels in blood cells compared to SD and similarly VCD/HCD resulted in an accumulation of cholesterol esters compared to HCD (Fig.\u0026nbsp;3A). In contrast, additional injections of Hcy into rabbits fed VCDD resulted in tendentially decreased cholesterol ester levels in blood cells compared to VCDD alone (Fig.\u0026nbsp;3A).\u003c/p\u003e \u003cp\u003eNext, we analyzed total fatty acid levels in blood cells from rabbits fed VCDD in the absence or presence of HCD, or with additional intravenous injection of Hcy. While VCDD alone resulted in only minor increase of total fatty acids in blood cells compared to SD, additional injections of Hcy into rabbits fed VCDD led to twofold increase in total fatty acids compared to both SD and VCDD (Fig.\u0026nbsp;3A). Similarly, while total fatty acid levels in blood cells from rabbits fed HCD showed an increase compared to SD, combination of VCDD and HCD led to further increase in total fatty acid levels compared to HCD alone (Fig.\u0026nbsp;3A).\u003c/p\u003e \u003cp\u003eIn hearts rabbits from the HCD, VCD/HCD as well as VCDD\u0026thinsp;+\u0026thinsp;Hcy groups, but not rabbits fed VCDD alone, showed significantly increased free cholesterol levels compared to SD (Fig.\u0026nbsp;3B). Rabbits from the VCDD\u0026thinsp;+\u0026thinsp;Hcy group, similarly to rabbits from the VCDD group, also exhibited an increase of total cholesterol esters in hearts compared to SD, albeit insignificant (Fig.\u0026nbsp;3B). Furthermore, hearts from both HCD- and VCD/HCD-fed rabbits, similarly to blood cells, exhibited also a strong accumulation of total cholesterol esters compared to SD (Fig.\u0026nbsp;3B). However, in contrast to blood cells, levels of cholesterol esters in hearts from rabbits in the VCD/HCD group were not further increased compared to rabbits fed HCD alone (Fig.\u0026nbsp;3B).\u003c/p\u003e\n\u003ch3\u003e5. Hcy injections into VCDD-fed rabbits lead to ceramide and sphingomyelin accumulation in blood cells\u003c/h3\u003e\n\u003cp\u003eAnalysis of Cer and SM content revealed that elevation of Hcy levels by intravenous injections of Hcy into VCDD-fed rabbits also affected sphingolipid metabolism. While VCDD alone did not lead to an increase of total Cer levels in blood cells compared to SD, rabbits from the VCDD\u0026thinsp;+\u0026thinsp;Hcy group displayed a significant increase of total Cer levels in blood cells compared to VCDD (Fig.\u0026nbsp;4A). Furthermore, VCD/HCD also led to a significant increase of total Cer levels in blood cells compared to VCDD as well as a tendential increase of total Cer levels compared to HCD (Fig.\u0026nbsp;4A). Rabbits from both VCD/HCD group compared to HCD and VCDD\u0026thinsp;+\u0026thinsp;Hcy group compared to VCDD accumulated Cer species containing fatty acids with total chain lengths of 40\u0026ndash;44 carbons and a total of 1\u0026ndash;4 double bonds, namely Cer d40:1, Cer d40:2 and Cer d44:4 in the VCD/HCD group and Cer d42:2, Cer d42:3 and Cer d44:3 in VCDD\u0026thinsp;+\u0026thinsp;Hcy group, in addition to Cer d38:3, Cer d42:1, Cer d42:4 and Cer d44:2 accumulating in both groups (Fig.\u0026nbsp;4A and S2A). Total SM levels, similarly to Cer levels, were also significantly increased in blood cells of rabbits from the VCDD\u0026thinsp;+\u0026thinsp;Hcy group compared to VCDD and tendentially increased in rabbits from the VCD/HCD group compared to HCD (Fig.\u0026nbsp;4A). Particularly, rabbits from the VCDD\u0026thinsp;+\u0026thinsp;Hcy group exhibited an accumulation of SM species containing longer fatty acids, namely SM d40:1, SM d42:1, SM d42:2 and SM d42:3 (Fig.\u0026nbsp;4A and S2A). In contrast, SM species containing shorter fatty acids, including SM d32:1, SM d34:1, SM d34:2, SM d36:1, SM d36:2 and SM d40:1 were elevated in blood cells from rabbits in the VCD/HCD group compared to HCD, and SM d42:2 was elevated both in HCD and VCD/HCD groups compared to SD (Fig.\u0026nbsp;4A). Of note, VCDD alone did not lead to an accumulation of total SM, but displayed an elevation of SM d42:3 in comparison to SD (Fig.\u0026nbsp;4A).\u003c/p\u003e \u003cp\u003eIn heart, VCDD both with or without additional Hcy injections did not show any changes of total Cer levels (Fig.\u0026nbsp;4B). However, HCD and VCD/HCD feeding resulted in a tendential increase of total Cer levels as well as significant increase of monounsaturated Cer species such as Cer d34:1, Cer d38:1, Cer d39:1, Cer d40:1, Cer d41:1 and Cer d42:1 compared to SD (Fig.\u0026nbsp;4B and S2B), but VCD/HCD did not show further increase of Cer levels compared to HCD (Fig.\u0026nbsp;4B and S2B). Similar to Cer, HCD and VCD/HCD but not VCDD with or without Hcy injections led to an accumulation of total SM in the heart compared to SD (Fig.\u0026nbsp;4B). In particular, rabbits from the HCD and VCD/HCD groups accumulated saturated and mono-unsaturated SM species, including SM d34:0, SM d38:0, SM d40:1, SM d41:0, SM d41:1 and SM d42:1, whereas rabbits from the HCD group additionally exhibited an elevation of SM d34:1, SM d38:0, SM d38:1 and SM d42:3 in the heart compared to SD, whereas VCD/HCD did not show further elevation of SM species in hearts compared to HCD (Fig.\u0026nbsp;4B and S2B).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e6. Hcy injections into VCDD-fed rabbits lead to significantly decreased acylcarnitine levels in the heart\u003c/h2\u003e \u003cp\u003eAnalysis of CAR levels in balloon-injured NZW rabbits fed VCDD in the absence or presence of HCD, or with additional intravenous injections of Hcy revealed that elevation of Hcy levels also affected CAR metabolism. While VCDD did not change the levels of total CAR in blood cells, additional injections of Hcy into VCDD-fed rabbits resulted in significantly increased total CAR levels in blood cells compared to VCDD alone (Fig.\u0026nbsp;5A). Analysis of CAR species in blood cells of rabbits from the VCDD\u0026thinsp;+\u0026thinsp;Hcy group revealed that they accumulated CAR 16:0 and CAR 18:1 (Fig.S3A). Similar to VCDD\u0026thinsp;+\u0026thinsp;Hcy group, total CAR levels were also elevated in blood cells from rabbits from the HCD group, with no further increase in CAR levels in blood cells in response to VCD/HCD compared to HCD alone (Fig.\u0026nbsp;5A). However, analysis of CAR species in blood cells from rabbits from the VCD/HCD group exhibited an increase of CAR 18:2 compared to HCD (Fig.S3A).\u003c/p\u003e \u003cp\u003eIn contrast, VCDD led to significantly decreased total CAR levels in the heart compared to SD and an even stronger decrease of myocardial total CAR levels when VCDD was combined with intravenous Hcy injections compared to VCDD alone (Fig.\u0026nbsp;5A). Furthermore, VCD/HCD also resulted in decreased total CAR levels in the heart compared to HCD alone (Fig.\u0026nbsp;5B). Noteworthy, in the heart VCDD resulted in a decrease of saturated and mono-unsaturated CAR species, while rabbits from the VCDD\u0026thinsp;+\u0026thinsp;Hcy group showed a significant decrease of CAR species in the heart regardless of saturation level, namely Car 3:0, Car 6:0, Car 12:0, Car 14:0, Car 16:0, Car 18:1, Car 18:3, Car 19:1 and Car 22:5 (Fig.\u0026nbsp;5B and S3B).\u003c/p\u003e \u003cp\u003e \u003cb\u003e7. Block in Hcy degradation in VCDD-fed rabbits leads to deregulation of lipid metabolism in aorta\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNext, we analyzed lipids in the aortas from balloon-injured rabbits fed VCDD, HCD, VCD/HCD or SD. While OPLS scoring plot did not show differences between the groups, both OPLS loading plot and volcano plot indicated a dysregulation of TG and PL metabolism in aortic tissue from rabbit fed different diets, similarly to blood cells (Fig.\u0026nbsp;6A). In line with our findings in blood cells, both VCDD and VCD/HCD resulted in an accumulation of total TG in the aortic wall compared to SD or HCD, respectively (Fig.\u0026nbsp;6B). Furthermore, analysis of individual TG species showed that in the VCDD group elevation was equally distributed, while in the VCD/HCD group preferably TG species containing longer and unsaturated fatty acids accumulated, similar to species distribution found in blood cells (Fig. S4). Interestingly, despite no differences in total PC and PE levels in response to VCDD in blood cells or in heart, in aorta VCDD, HCD and VCD/HCD groups exhibited decreased levels of total PC and increased levels of total PE compared to SD, while an increase in PE levels was the highest in the VCDD group (Fig.\u0026nbsp;6B). In aortas from rabbits from the VCDD group, PC species containing longer FAs with no or low number of double bonds were decreased, while in HCD and VCD/HCD groups also species containing 4\u0026ndash;5 double bonds were decreased compared to SD (Fig. S4). Noteworthy, in blood cells an accumulation of similar PC species was found in response to VCD/HCD, but not VCDD alone. In contrast, while aortas from rabbits from the VCDD group exhibited elevation of PE species containing longer, highly unsaturated fatty acids, both HCD and VCD/HCD feeding resulted in an accumulation of PE species containing shorter fatty acids with a total of 1\u0026ndash;3 double bonds compared to SD (Fig. S4).\u003c/p\u003e \u003cp\u003eAnalysis of sphingolipids in aortas from rabbits fed different diets showed a tendency towards increased levels of total Cer in response to HCD and VCD/HCD, similar to both blood cells and heart (Fig.\u0026nbsp;6D). However, in contrast to blood cells and heart total Cer levels in aorta were also found to be slightly increased in response to VCDD alone (Fig.\u0026nbsp;6D). Furthermore, in contrast to Cer levels in aorta, total SM levels were slightly decreased in response to VCDD as well as HCD and VCD/HCD feeding in comparison to SD (Fig.\u0026nbsp;6D). Noteworthy, both in blood cells and heart total SM levels were found to be increased in response to HCD and VCD/HCD in contrast to aorta, whereas VCDD did not lead to any changes in total SM levels in blood cells and aorta compared to SD.\u003c/p\u003e \u003cp\u003eAnalysis of total CAR levels in aortas from rabbits fed different diets showed a tendential increase in response to VCDD, HCD and VCD/HCD compared to SD, while HCD and VCD/HCD showed higher elevation of total CAR than VCDD (Fig.\u0026nbsp;6E), which is in line with total CAR levels in blood cells, whereas in hearts total CAR levels were decreased in response to VCDD and VCD/HCD compared to SD or HCD, respectively (Fig.\u0026nbsp;5).\u003c/p\u003e \u003cp\u003eAnalysis of total fatty acids in aortas from rabbits fed different diets showed an elevation of fatty acid levels in aortas from rabbits fed VCDD as well as in both high cholesterol-containing diet groups, HCD and VCD/HCD, compared to SD (Fig.\u0026nbsp;6C). However, in contrast to increased total fatty acid levels in blood cells from rabbits fed VCD/HCD compared to HCD fed rabbits, in aortic tissue VCD/HCD feeding did not lead to an accumulation of total fatty acids compared to HCD (Fig.\u0026nbsp;6C).\u003c/p\u003e \u003cp\u003eScanning transmission electron microscopy (STEM) and transmission electron microscopy (TEM) revealed an accumulation of lipid droplets (LDs) in aortic media from rabbits fed VCDD compared to SD (Fig.\u0026nbsp;7A-B), similarly as in rabbits fed HCD and, however to a lower extend compared to HCD alone, in rabbits fed VCD/HCD (Fig.\u0026nbsp;7C-D), in line with an accumulation of LDs in aortic neointima from rabbits fed either VCDD, HCD or VCD/HCD (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Furthermore, in aortic media rabbits fed VCDD exhibited an accumulation of dilated endoplasmic reticulum (ER) and electron-dense mitochondria in smooth muscle cells, which were not observed in response to SD (Fig.\u0026nbsp;7A-B). Similarly, an accumulation of dilated ER in smooth muscle cells was also found in aortic media from rabbits fed VCD/HCD, but not HCD alone, whereas electron-dense mitochondria were observed in VCDD-fed rabbits as well as in response to both, VCD/HCD and HCD (Fig.\u0026nbsp;7C-D). Of note, dilated ER was also observed both in smooth muscle cells and fibroblasts in aortic intima of rabbits fed VCDD and VCD/HCD, but not HCD or SD(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e8. VCDD leads to phosphatidylcholine remodeling in serum, but not in the liver\u003c/h2\u003e \u003cp\u003eNext, to better understand PC metabolism in rabbits fed VCDD with or without HCD, or with additional intravenous injections of Hcy we analyzed metabolites of PC metabolism in the liver and serum by NMR. Choline levels were significantly increased in response to HCD in the liver and in response to VCD/HCD in the serum compared to SD (Fig.\u0026nbsp;8A). Feeding VCDD, despite decreased choline content in the diet, resulted in tendential increase of choline both in the liver and serum compared to SD (Fig.\u0026nbsp;8A). Similarly, combination of VCDD and HCD increased serum choline levels compared to HCD group (Fig.\u0026nbsp;8A). While, additional Hcy injections into rabbits fed VCDD did not change liver choline levels, they resulted in decreased serum choline levels compared to VCDD alone (Fig.\u0026nbsp;8A).\u003c/p\u003e \u003cp\u003ePhosphorylcholine was similarly affected as choline in the liver, but was oppositely affected in the serum of rabbits from different groups (Fig.\u0026nbsp;8B). In particular, phosphorylcholine in serum showed a tendency towards lower levels in response to VCDD, HCD and VCD/HCD groups compared to SD, whereas additional intravenous injections of Hcy into VCDD fed rabbits did not change serum phosphorylcholine levels compared to SD and increased them compared to VCDD (Fig.\u0026nbsp;8B). Glycerophosphocholine levels in liver, similar to phosphorylcholine levels, were not affected in response to VCD/HCD or VCDD compared to SD, but in contrast to phosphorylcholine they were tendentially decreased in livers of rabbits fed HCD (Fig.\u0026nbsp;8C). Noteworthy, combination of VCDD and HCD tendentially increased liver glycerophosphocholine levels compared to HCD alone and elevation of plasma Hcy levels by intravenous Hcy injections into VCDD-fed rabbits tendentially increased liver glycerophosphocholine levels compared to VCDD (Fig.\u0026nbsp;8C). In contrast, glycerophosphocholine levels in serum showed tendential increase in response to VCDD compared to SD as well as in response to VCD/HCD compared to HCD (Fig.\u0026nbsp;8C).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eElevated Hcy is linked to many human diseases including CVD (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Although the underlying mechanisms leading to human pathology are insufficiently understood, downstream pathways triggered by elevated Hcy are likely to involve inhibition of SAM-dependent methyltransferases due to an accumulation of their product inhibitor SAH (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), interference with protein structure and function by homocysteinylation of critical protein lysines (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e), induction of oxidative stress (\u003cspan additionalcitationids=\"CR46 CR47\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e), deregulation of lipid metabolism (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) and activation of UPR (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHHcy is classified into severe, moderate and mild forms based on the levels of fasting plasma Hcy. Severe HHcy characterized by plasma Hcy levels above 100 \u0026micro;M is due to a block in cystathionine-\u0026szlig;-synthase (CBS) that hinders Hcy degradation by transsulfuration and is associated with fatty liver, lean phenotype and CVD (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Moderate HHcy characterized by plasma Hcy levels above 25 \u0026micro;M is in most cases due to a block in methyltetrahydrofolate reductase (MTHFR) that hinders folate-dependent remethylation of Hcy to methionine and is associated with fatty liver, CVD, aortic lipid deposition (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) and abdominal fat accumulation (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Mild HHcy characterized by plasma Hcy levels above 15 \u0026micro;M is due to deficiency of vitamins required for Hcy degradation (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), represents two thirds of all HHcy cases (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e) and is associated with CVD (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) and obesity (\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur previous work in yeast showed that elevated Hcy leads to accumulation of TG and total FAs (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). In accordance, it was reported that elevated Hcy via activation of sterol regulatory element-binding proteins (SREBPs) upregulates genes responsible for cholesterol/triglyceride biosynthesis and uptake in human hepatocytes, vascular endothelial and aortic smooth muscle cells (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Moreover, dietary induction of HHcy in mice leads to an elevation of cholesterol and TG levels in the liver and plasma, and increased secretion of VLDL-TG and VLDL-cholesterol (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn accordance with hepatic lipid accumulation in response to elevated Hcy, both CBS and MTHFR knockout mice develop fatty liver (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). However, in contrast to CBS knockout mice, which exhibit loss of fatty tissue (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), MTHFR knockout mice had significantly increased abdominal fat mass (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e) suggesting a dose-dependent effect of Hcy on lipid metabolism. In accordance, mouse models of CBS deficiency were reported to reveal significant threshold effects of elevated Hcy, however, its effects on lipid metabolism were not addressed in these mice (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Threshold effects of elevated Hcy observed in CBS knockout mice are similar to graded CVD risk with no threshold conferred by elevated Hcy (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Here we aimed to analyze and compare dysregulation of lipid metabolism in various tissues including blood cells, heart and aorta from rabbits fed VCDD in the absence or presence of additional intravenous Hcy injections as well as hypercholesterolemia.\u003c/p\u003e \u003cp\u003eFeeding rabbits VCDD in the absence of hypercholesterolemia led to a significant elevation of total TG levels in blood cells and tendential TG elevation in aorta compared to SD. In line with elevation of aortic TG levels, electron microscopy of aortic media from rabbits fed VCDD, but not SD, showed an accumulation of LDs, similarly to an accumulation of LDs in aortic intima from rabbits fed VCDD (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). However, TG accumulation was not observed in blood cells of rabbits from the VCDD\u0026thinsp;+\u0026thinsp;Hcy group. Similarly, total TG levels in the hearts of rabbits from the VCDD group but not from the VCDD\u0026thinsp;+\u0026thinsp;Hcy group were elevated, albeit non-significantly. In accordance we and others have shown that elevated Hcy leads to an accumulation of total TG in yeast (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), human hepatocytes, vascular endothelial and aortic smooth muscle cells as well as in livers and plasma of mice in response to dietary caused HHcy (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). Decrease of TG levels in blood cells and hearts from rabbits from the VCDD\u0026thinsp;+\u0026thinsp;Hcy group compared to VCDD alone suggest an activation of lipolysis by elevated Hcy. Indeed, HHcy induced by 2% high methionine diet over 8 week or administration of 1.8 g/L in drinking water for 2 and 4 weeks in mice demonstrated that Hcy activates adipocyte lipolysis and increases release of free FAs and glycerol (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). In accordance, free FAs and glycerol were elevated in blood cells from rabbits in the VCDD\u0026thinsp;+\u0026thinsp;Hcy group. On the other hand, consistent with threshold effects of Hcy, supplementation of 100 to 500 \u0026micro;M Hcy was shown to inhibit release of glycerol and free FAs in primary adipocytes and fully differentiated 3T3-L1 adipocytes in a dose-dependent manner, and in differentiated 3T3-L1 adipocytes to elevate intracellular TG content by phosphorylating AMP-activated protein kinase and its downstream enzyme acetyl-CoA carboxylase (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). Furthermore, supplementation of 0.1% Hcy in the diet for 2 weeks was shown to lowered circulating glycerol and FA levels (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). While rabbits from the VCDD\u0026thinsp;+\u0026thinsp;Hcy exhibited a nonsignificant gain in weight (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), an accumulation of abdominal fat in human patient exhibiting mild HHcy (\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e) or MTHFR knockout mice (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e), but not in CBS knockout mice (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), further demonstrates a dose-dependent effect of Hcy on lipid metabolism. Interestingly, rabbits from the VCDD\u0026thinsp;+\u0026thinsp;Hcy group exhibited massively elevated TG and also cholesterol levels particularly in VLDL, but also in LDL and HDL suggesting dysregulation of lipoprotein metabolism and increased hepatic lipid synthesis in response to elevated Hcy in our model (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Noteworthy, analysis of plasma TG levels in normal population without lipid lowering treatment showed that HHcy was independently associated with hypertriglyceridemia and MTHFR knockout mice exhibit hypertriglyceridemia further indicating the central role of Hcy in TG metabolism (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). Combination of VCDD with HCD resulted in significant decrease of total TG levels in the heart compared to HCD that were even lower than in SD but not in blood cells suggesting an activation of lipolysis in the heart and sensitization towards elevated Hcy in the presence of high cholesterol.\u003c/p\u003e \u003cp\u003eDespite VCD/HCD did not affect total TG levels in blood cells and only slight elevation of TG in aorta, it led to an increase in polyunsaturated TG species containing 6 or more double bonds compared to HCD alone. Similarly, VCDD resulted in an accumulation of polyunsaturated TG species with 2\u0026ndash;7 double bonds in blood cells and combination of VCDD with intravenous Hcy injections resulted in particularly low levels of saturated TG species compared to SD. Similarly, while HCD did not affect total TG levels in the heart, it led to an accumulation of TG species containing polyunsaturated FAs. Serum saturated, monounsaturated and polyunsaturated FA and TG levels have been shown to be deregulated in extremely obese human patients as well as in mice fed high-fat diet (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). Furthermore, high-fat diet was shown to increase plasma Hcy levels by inhibiting hepatic transsulfuration pathway (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) and elevated Hcy to be linked to a decrease of decosahexaenoic acid in serum (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e) and phospholipids (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e). In line, obesity has been shown to depend on increased methionine in humans (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e) and TG accumulation and deregulation of FA metabolism has been linked to elevated Hcy in yeast (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Moreover, deficiency in phospholipid methylation in PE-methyltransferase (PEMT)-deficient mice fed choline-deficient diet that blocks an alternative Kennedy pathway for PC synthesis leads to an accumulation of polyunsaturated FAs in TG (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eElevation of plasma Hcy levels by intravenous Hcy injections into VCDD-fed rabbits led to a significant increase of total PC in blood cells compared to both VCDD and SD as well as to a significant increase of total PE compared to VCDD alone. The increase in PC and PE levels was not observed in hearts from rabbits from the VCDD\u0026thinsp;+\u0026thinsp;Hcy group. Along with significant decrease in total TG levels, PC and PE accumulation in blood cells from rabbits from the VCDD\u0026thinsp;+\u0026thinsp;Hcy group suggests membrane proliferation in response to elevated Hcy. Of note, the ratio of PC/PE was unaltered both in blood cells and heart from rabbits from the VCDD\u0026thinsp;+\u0026thinsp;Hcy group compared to all other groups. Importantly, despite total PC elevation in blood cells, rabbits from the VCDD\u0026thinsp;+\u0026thinsp;Hcy group accumulated only short chain PC species containing low number of double bonds compared to VCDD. In line, PEMT-deficient mice also accumulate PC species containing shorter FAs with low number of double bonds in liver and plasma, while PC species containing long chain polyunsaturated FAs are depleted in these mice (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn contrast, aortic tissue from rabbits fed VCDD exhibited tendentially decreased levels of total PC, increased levels of total PE and decreased PC/PE ratio compared to SD, suggesting an inhibition of PE to PC methylation. Similarly, elevation of plasma Hcy and SAH leads to an accumulation of PE and a drop in PC levels in human erythrocytes as well as significant depletion of docosahexaenoic acid in PC, but not PE (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e). Furthermore, aortas from rabbits fed VCDD exhibited an accumulation of PE species containing long chain polyunsaturated FAs compared to SD, suggesting that an increase in polyunsaturated FAs in PE in response to elevated Hcy/SAH is due to inhibition of PE to PC methylation. Indeed, synthesis of PC by methylation-dependent pathway from PE catalyzed by PEMT produces PC species containing long chain polyunsaturated FAs, whereas PC synthesis via CDP-choline pathway mainly produces PC species containing shorter FAs with lower number of double bonds (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). Decreased PC/PE ratio as well as an accumulation of long chain polyunsaturated FAs in PE may interfere with membrane properties and lead to a lipid disbalance in the membrane. Membrane aberrancy or an accumulation of unfolded proteins (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e) may be a cause for the observation of dilated ER in smooth muscle cells and fibroblasts in the aortic wall from rabbits fed VCDD. Previously, we have shown that feeding rabbits either VCDD or VCD/HCD leads to an accumulation of dilated ER in smooth muscle cells and fibroblasts in aortic neointima (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Here we show that rabbits from the VCDD and VCD/HCD groups also exhibited an accumulation of dilated ER in smooth muscle cells in aortic media. It was shown in yeast that alterations in membrane PLs and decreased PC levels leads to induction of ER stress (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e). Furthermore, Hcy was shown to lead to ER stress induction and dilated ER accumulation in human cardiac cells (\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e) as well as in human endothelial cells in atherosclerotic plaques (\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e). Interestingly, it was shown that supplementation of vitamin B12, required for Hcy re-methylation, leads to an amelioration of UV-radiation induced dilatation of ER in rat hepatocytes (\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn contrast to PC, total LPC levels in blood cells and hearts from rabbits from the VCDD\u0026thinsp;+\u0026thinsp;Hcy group were unaltered compared to both SD and VCDD, and increased significantly only in response to high cholesterol containing diets, HCD and VCD/HCD. Lecithin cholesterol acyl transferase (LCAT) plays a major role in reverse cholesterol transport and cholesterol metabolism, catalyzing FA transfer from PC to free cholesterol forming LPC and cholesterol esters (\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e). In line, feeding rabbits high cholesterol containing diets resulted not only in strongly elevated levels of LPC, but also proportionally increased total cholesterol ester levels in blood cells, hearts and aortas. Interestingly, hearts from rabbits fed VCD/HCD exhibited tendentially lower levels of LPC compared to HCD alone. In accordance, it has been reported that CBS-deficient mice, exhibiting HHcy, show decreased expression of LCAT in liver and decreased LCAT activity in serum (\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eElevation of plasma Hcy levels by intravenous injections of Hcy into VCDD-fed rabbits lead to significant increase of free cholesterol levels in the heart and blood cells compared to both SD and VCDD. Free cholesterol levels were also significantly elevated in rabbits fed combined VCD/HCD compared to HCD, however, only in blood cells, but not in hearts. Similarly, also only in blood cells, but not in the heart, cholesterol ester levels were non-significantly elevated in rabbits fed VCD/HCD compared to rabbits fed HCD alone. Interestingly, VCDD resulted in significant elevation of cholesterol ester levels in the heart, but not in blood cells compared to rabbits fed SD. In accordance with our data, homozygous CBS knockout mice exhibit significant elevation of free cholesterol levels in serum in comparison to CBS(+/+) or heterozygous CBS knockout mice suggesting interference between Hcy and cholesterol metabolism (\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e). Further suggesting a crosstalk between Hcy and cholesterol metabolism our previous experiments showed increased atherogenicity and elevation of LDL- and VLDL-cholesterol levels in rabbits fed VCDD, VCDD\u0026thinsp;+\u0026thinsp;Hcy and VCD/HCD compared to respective controls (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Accumulation of free cholesterol in blood cells as well as elevation of cholesterol in circulating lipoproteins is in accordance with passive equilibrium of free cholesterol pool between lipoproteins and cell membranes (\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e). Furthermore, Hcy was shown to stimulate production as well as secretion of cholesterol in human hepatic cells, vascular endothelial and aortic smooth muscle cells, and monocyte-derived macrophages (\u003cspan additionalcitationids=\"CR75\" citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e), the latter being also associated with deficient DNA methylation linked to increased expression of fatty acid-binding protein 4. In accordance, plasma Hcy was shown to be independently associated with conventional atherogenic lipid profile and remnant cholesterol in adults suggesting that Hcy-related dyslipidemia risk is clinically relevant (\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e). Moreover, increased cholesterol ester and LPC levels as well as elevated PC and free cholesterol levels in blood cells from rabbits fed VCD/HCD compared to HCD alone along with significant accumulation of free cholesterol and, significant only for blood cells, PC in blood cells and heart in rabbits from the VCDD\u0026thinsp;+\u0026thinsp;Hcy group suggest that elevated Hcy may contribute to increased atherogenicity by impacting reverse cholesterol transport and cholesterol as well as phospholipid metabolism.\u003c/p\u003e \u003cp\u003eCeramides are cholesterol-independent biomarkers causatively linked to CVD (\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e). Ceramide levels are increased significantly and sphingomyelin levels non-significantly in blood cells from rabbits from the VCDD\u0026thinsp;+\u0026thinsp;Hcy group compared to VCDD alone. Similarly, VCDD increases ceramide and sphingomyelin levels in blood cells from rabbits fed combined VCD/HCD compared to HCD alone. HCD also increases ceremide and sphingomyelin levels in blood cells compared to SD and VCDD. Elevation of ceremide and sphingomyelin levels in response to HCD or VCDD compared to SD can also be observed in the heart. Noteworthy, while in aorta ceramide levels are increased in VCDD compared to SD as well as in VCD/HCD compared to HCD, aorta sphingomyelin levels are decreased in VCDD and VCD/HCD compared to respective controls. Cholesterol interacts with sphingolipids in plasma membranes (\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e). Moreover, their synthesis and regulation are tightly linked (\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e). Particularly it has been shown, that clinical interventions to decrease elevated LDL cholesterol leads to decreased levels of ceramides (\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e) suggesting a connection between cholesterol and sphingolipid metabolism. In line, our results show that free cholesterol levels in all analyzed rabbit groups strongly correlate with total levels of Cer in blood cells, hearts and aortas as well as SM levels in blood cells and hearts.\u003c/p\u003e \u003cp\u003eElevation of plasma Hcy levels by intravenous Hcy injections into VCDD-fed rabbits leads to significant increase of total SM levels in blood cells compared to SD. In particular, rabbits from the VCDD\u0026thinsp;+\u0026thinsp;Hcy group accumulated SM d40:1, d42:1, d42:2 and d42:3 and exhibited decreased levels of SM 34:1, 36:1 and 36:2 in blood cells compared to SD. In contrast, combination of VCDD and HCD resulted in accumulation of SM d32:1, d34:1, d34:2 in addition to d40:1, but not SM d42:1, d42:2 and d42:3 compared to HCD in blood cells. In heart, HCD similar to VCD/HCD led to an accumulation of most SM molecular lipid species with exception of SM d33:1, d36:2, d40:2, d41:2, d44:1 and significantly decreased SM d38:2 compared to SD. In accordance with critical role of SM in atherosclerosis development, total SM were shown to be increased in coronary artery disease (CAD) patients and to have a predictive value for CAD after adjusting for other risk factors, including remnants (\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e). Moreover, circulating SM 34:1, which accumulated both in blood cells and in the heart in rabbits fed VCD/HCD, was shown to predict CVD mortality risk and be associated with higher risk for sudden cardiac death in humans (\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e) suggesting that dysregulated sphingolipid metabolism may contribute to CVD development in response to combination of HHcy and hypercholesterolemia.\u003c/p\u003e \u003cp\u003eSimilarly to SM, total Cer levels were significantly increased in blood cells but not in hearts from rabbits from the VCDD\u0026thinsp;+\u0026thinsp;Hcy group compared to both SD and VCDD. Highest Cer levels in blood cells were observed for Cer species containing longer fatty acids such as Cer d42:2, d42:3, d42:4, d44:2 and d44:3. While HCD also led to an increase in total Cer levels in blood cells, combination of VCDD and HCD resulted even in further elevation of total Cer levels in blood cells compared to HCD alone, with the highest accumulation observed for Cer d34:1, d40:1, d42:1 and d44:4. In accordance, circulating Cer 34:1 was shown to be associated with higher risk for sudden cardiac death in humans (\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e). In hearts, however, elevated total Cer levels were observed only in response to HCD alone, but not in combination with VCDD. In aorta, in contrast to total SM levels, which were decreased in HCD-fed rabbits compared to SD and even more pronounced decreased in VCD/HCD compared to HCD, total Cer levels were tendentially increased in response to VCDD compared to SD as well as VCD/HCD compared to HCD alone.\u003c/p\u003e \u003cp\u003eIn line with our results, elevated Hcy was shown to be linked to activation of ceramide de novo synthesis in rat mesangial cells (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e) as well as in liver and brain of HHcy mice (\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e). Furthermore, folate deficient rats were shown to exhibit an accumulation of ceramides in the renal cortex (\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e). Cer accumulation was shown to promote inflammation and cell death, and to be associated with adverse cardiovascular outcomes (\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e) as well as mortality in patients with chronic heart failure (\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e). In particular, patients who died due to cardiovascular causes, had significantly higher levels of plasma Cer(d18:1/16:0) and Cer(d18:1/24:1) specially when used in ratios with Cer(d18:1/24:0) (\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e). In accordance, our data showed that Cer d42:2 was dramatically increased in blood cells from rabbits from the VCDD\u0026thinsp;+\u0026thinsp;Hcy group even if used in ratio with Cer d42:1, suggesting the role of elevated Hcy in dysregulation of ceramide species linked to cardiovascular disease.\u003c/p\u003e \u003cp\u003eHigher plasma levels of Cer and SM containing palmitic acid were also associated with increased risk of heart failure, while on contrary higher levels of Cer and SM containing very long chain and/or unsaturated FAs, Cer-22, SM-20, SM-22, and SM-24, were associated with decreased risk of heart failure (\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e). Our data show that both SM d34:1 and Cer d34:1 were elevated in blood cells from rabbits fed VCD/HCD compared to HCD, but VCD/HCD feeding also led to an accumulation of Cer species containing very long fatty acids with a total of 40\u0026ndash;44 carbons compared to HCD. However, it has to be noted that very long chain FA-containing ceramides were also reported to induce mitochondrial dysfunction leading to oxidative stress and cell death in cardiomyocytes in mouse models of diabetic cardiomyopathy (\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e). Of note, electron microscopy showed that rabbits fed VCDD, HCD or VCD/HCD showed darker, electron-dense mitochondria in the aortic media in contrast to SD, suggesting potential effects of elevated Hcy on mitochondrial function likely due to changes in membrane ultrastructure and/or paracrystalline protein accumulations (\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e). Importantly, structurally abnormal mitochondria, next to dilated ER, have also been found in endothelial in human atherosclerotic plaques (\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIncreased CVD risk, cardiac hypertrophy and heart failure are also reported in association with dysregulated CAR metabolism (\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e, \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e). Suggesting that elevated Hcy may contribute to CVD via impaired CAR metabolism, our data show that feeding rabbits VCDD with or without intravenous Hcy injections both leads to significant decrease of total CAR levels in the heart compared to SD. Moreover, total heart CAR levels in rabbits from the VCDD\u0026thinsp;+\u0026thinsp;Hcy group were significantly lower than in rabbits fed VCDD alone. Combination of VCDD and HCD also led to a significant decrease of total CAR levels compared to HCD alone. Depletion of CAR suggest impaired FA transport into mitochondria, mitochondrial dysfunction and disruption of energy metabolism in the heart (\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e) in response to elevated Hcy.\u003c/p\u003e \u003cp\u003eIn contrast to heart, blood cells from rabbits from the VCDD\u0026thinsp;+\u0026thinsp;Hcy group exhibited significantly higher CAR levels compared to VCDD alone. Similarly, rabbits from the VCDD group exhibited tendential increase of total CAR levels compared to SD in both blood cells and aortic tissue. In line, medium- and long-chain acylcarnitines, preferably used for fatty acid oxidation, are markedly lower in myocardium, but exhibit no difference or were shown to be increased in plasma of human patients suffering from heart failure (\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e). Altered blood CAR levels in heart failure patients are linked to mitochondrial dysfunction and are independently associated with disease severity (\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e). Furthermore, in humans elevated serum Hcy levels, in response to deficiency of folate and vitamin B\u003csub\u003e12\u003c/sub\u003e, are positively correlated to increased CAR levels in plasma and are linked to myocardium hypertrophy, impaired mitochondrial FA oxidation and increased brain natriuretic peptide (\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e). FAs are preferred substrate in adult myocardium supplying about 70% of total ATP and are derived from circulating TG-rich lipoproteins and albumin-bound non-esterified FAs (\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e). While we have not analyzed serum free FAs in our model, we observed dramatically increased TG-VLDL levels in serum from rabbits fed VCDD in the presence of additional intravenous injections (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e) in line with limited up-take of FA by cardiomyocytes suggesting TGs accumulation in circulating lipoproteins in particular VLDL.\u003c/p\u003e \u003cp\u003eIncreased levels of choline and glycerophosphocholine in serum in VCDD fed rabbits compared to SD, similar to rabbits from VCD/HCD group compared to HCD, suggest choline synthesis via catabolism of PC in line with dietary choline deficiency in VCDD. Furthermore, VCDD fed rabbits show a tendential decrease of phosphorylcholine in serum compared to SD in accordance with decreased PC \u003cem\u003ede novo\u003c/em\u003e synthesis. Interestingly, further elevation of plasma Hcy by intravenous injections into VCDD fed rabbits resulted in a decrease of choline and to elevation of phosphorylcholine levels in serum compared to VCDD alone, suggesting inhibition of PC catabolism in response to elevated Hcy. In accordance we have observed a significant accumulation of total PC in blood cells as well as a tendential increase of total PC in hearts compared to VCDD. In line with an adaptive response of the liver to keep choline homeostasis during choline depletion (\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e), rabbits fed VCDD compared to SD, similar to VCD/HCD compared to HCD alone, did not show alterations of choline, phosphorylcholine or glycerophosphocholine in the livers. However, altered levels of choline in serum have been associated with the development of hepatic steatosis, atherosclerosis, cardiovascular disease and increased cardiovascular mortality(\u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e, \u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eStudy limitation\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eIt was not possible to analyze lipidome in the aortic specimens from rabbits fed VCDD in combination with intravenous injections of Hcy.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eDuring preparation of the aorta it was not possible to quantitatively remove the surrounding fat layer from the aorta.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eIn conclusion, here we show that Hcy leads to dysregulation of lipid metabolism in blood cells, heart and aorta in rabbits having a graded effect on glycerolipid, cholesterol, ceramide, acylcarnitine and fatty acid metabolism in different tissues. Accumulation of triglycerides in response to VCDD and their decrease in response to VCDD in combination with Hcy injections in blood cells and heart, accumulation of cholesterol in blood cells, heart and aorta, accumulation of ceramides and fatty acids in blood cells and a drastic drop in myocardial acylcarnitines suggest mechanisms how elevated Hcy may contribute to development of CVD.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eHcy \u0026ndash; homocysteine; HHcy \u0026ndash; hyperhomocysteinemia; CVD \u0026ndash; cardiovascular disease; SD \u0026ndash; standard diet; VCDD \u0026ndash; vitamin and choline deficient diet; HCD \u0026ndash; high cholesterol diet; VCD/HCD \u0026ndash; vitamin and choline deficient high cholesterol diet; VCDD+Hcy \u0026ndash; vitamin and choline deficient diet with intravenous Hcy injections; TG \u0026ndash; triglyceride; PL \u0026ndash; phospholipid; PC \u0026ndash; phosphatidylcholine; PE \u0026ndash; phosphatidylethanolamine; LPE \u0026ndash; lysophosphatidylethanolamine, LPC \u0026ndash; lysophosphatidylcholine; FA \u0026ndash; fatty acid; Cer \u0026ndash; ceramide; SM \u0026ndash; sphingomyelin; CAR \u0026ndash; acylcarnitine; SREBP \u0026ndash; sterol regulatory element binding protein; CBS \u0026ndash; cystathionine-\u0026beta;-synthase; MTHFR \u0026ndash; methyl-tetrahydrofolate reductase; PEMT \u0026ndash; phosphatidylethanolamine methyl transferase; LCAT \u0026ndash; lecithine-cholesterol acyl transferase\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest statement\u003c/h2\u003e \u003cp\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was funded by the Austrian Science Fund (FWF) (P31105 and P33672) and BioTechMed-Graz.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eMSB, TZ, ET, GNR - lipidomics; HH, TM \u0026ndash; NMR; GA, GH, OT, GS - animal study; DK, GL - EM; OT - conceiving the study; OT, GNR, TZ, MSB, GA, GH, GS - interpretation and discussion of the results, writing the paper; MSB, ET, TZ - statistical analysis.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe would like to especially thank Brigitte Spreitzer for helping with animal experiments and technical assistance in sample preparation. 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Nutr Rev 67:615\u0026ndash;623\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"2275d99a-25ae-49b1-9271-f73b5076b985","identifier":"10.13039/501100002428","name":"Austrian Science Fund","awardNumber":"P31105 and P33672","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of Graz","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"cardiovascular disease, atherosclerosis, rabbits, homocysteine, lipids","lastPublishedDoi":"10.21203/rs.3.rs-6717713/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6717713/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCardiovascular disease, the leading cause of death worldwide, is mainly a result of atherosclerosis. However, 50% of all cases of atherosclerosis still cannot be explained by known risk factors including hypercholesterolemia. Hyperhomocysteinemia, an elevation of homocysteine (Hcy) levels in the blood, is an independent risk factor for atherosclerosis, aggravates atherosclerosis in the presence of hypercholesterolemia and strongly correlates with cardiovascular mortality. We showed that a diet deficient in vitamins and choline required for Hcy degradation (VCDD) leads to cholesterol-independent atherogenic transformation of the aorta and aortic lipid accumulation in balloon-injured rabbit model of atherosclerosis (Almer \u003cem\u003eet al\u003c/em\u003e, 2022, Biomed Pharmacother). Elevation of plasma Hcy by intravenous injections of Hcy into VCDD-fed rabbits results in further atherogenic changes, degradation of aortic lipid droplets, decreased total protein methylated arginine and altered metabolomic profiles compared to rabbits fed VCDD only (Tehlivets \u003cem\u003eet al\u003c/em\u003e, 2024, Biomed Pharmacother). Here we show that feeding VCDD with or without intravenous injections of Hcy leads to dysregulation of lipid metabolism in blood cells, heart and aorta in rabbits. Hcy has a graded effect on lipid metabolism deregulating glycerolipid, cholesterol, ceramide, acylcarnitine and fatty acid metabolism in different tissues. Accumulation of triglycerides in response to VCDD and their decrease in response to VCDD in combination with Hcy injections in blood cells and heart, accumulation of cholesterol in blood cells, heart and aorta, accumulation of ceramides and fatty acids in blood cells and drastic drop in myocardial acylcarnitines suggest mechanisms how elevated Hcy may contribute to development of CVD.\u003c/p\u003e","manuscriptTitle":"Homocysteine Leads to Decreased Acylcarnitine Levels in the Heart in a Rabbit Model of Atherosclerosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-22 14:39:31","doi":"10.21203/rs.3.rs-6717713/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"759c9f94-64f1-4b08-ab7e-b06913fdf820","owner":[],"postedDate":"May 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":48863332,"name":"General Biochemistry"}],"tags":[],"updatedAt":"2025-05-22T14:39:31+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-22 14:39:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6717713","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6717713","identity":"rs-6717713","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}