Very-low-density lipoprotein receptor-enhanced lipid metabolism in pancreatic stellate cells promotes pancreatic fibrosis.

Lipoprotein disorder is a common feature of chronic pancreatitis (CP); however, the relationship between lipoprotein disorder and pancreatic fibrotic environment is unclear. Here, we investigated the occurrence and mechanism of pancreatic stellate cell (PSC) activation by lipoprotein metabolites and the subsequent regulation of type 2 immune responses, as well as the driving force of fibrotic aggressiveness in CP. Single-cell RNA sequencing revealed the heterogeneity of PSCs and identified very-low-density lipoprotein receptor (VLDLR)+ PSCs that were characterized by a higher lipid metabolism. VLDLR promoted intracellular lipid accumulation, followed by interleukin-33 (IL-33) expression and release in PSCs. PSC-derived IL-33 strongly induced pancreatic group 2 innate lymphoid cells (ILC2s) to trigger a type 2 immune response accompanied by the activation of PSCs, eventually leading to fibrosis during pancreatitis. Our findings indicate that VLDLR-enhanced lipoprotein metabolism in PSCs promotes pancreatic fibrosis and highlight a dominant role of IL-33 in this pro-fibrotic cascade.

The emerging role of ethanolamine phosphate phospholyase in regulating hepatic phosphatidylethanolamine and plasma lipoprotein metabolism in mice.

Ethanolamine phosphate phospholyase (ETNPPL) is an enzyme that irreversibly degrades phospho-ethanolamine (p-ETN), an intermediate in the Kennedy pathway of phosphatidylethanolamine (PE) biosynthesis. PE is the second most abundant phospholipid in mammalian membranes. Disturbance of hepatic phospholipid homeostasis has been linked to the development of metabolic dysfunction-associated steatotic liver disease (MASLD). We generated whole-body Etnppl knockout mice to investigate the impact of genetic deletion of Etnppl on hepatic lipid metabolism. Primary hepatocytes isolated from Etnppl-/- mice showed increased conversion of [3H]ethanolamine to [3H]p-ETN and [3H]PE compared to Etnppl+/+ mice. Male and female Etnppl+/+ and Etnppl-/- mice were fed either a chow or a western-type diet (WTD). Irrespective of diet, Etnppl-/- mice had elevated fasting levels of total plasma cholesterol, triglyceride (TG) and apolipoprotein B100 (VLDL particles). Interestingly, hepatic TG secretion was unchanged between groups. Although hepatic lipids (phosphatidylcholine (PC), PE, TG, and cholesterol) were not different between mice, RNA sequencing analysis showed downregulation in genes related to cholesterol biosynthesis in Etnppl-/- mice. Furthermore, hepatic low-density lipoprotein receptor-related protein1 (LRP1) protein level was lower in female Etnppl-/- mice, which may indicate reduced uptake of remnant VLDL particles from circulation. Hepatic PE levels were only increased in WTD-fed female Etnppl-/- mice, not chow diet-fed mice. However, hepatic lipid accumulation and metabolic dysfunction-associated steatohepatitis (MASH) development were unchanged between Etnppl+/+ and Etnppl-/- mice. To conclude, ETNPPL has a role in regulating plasma lipoprotein metabolism independent of hepatic TG levels.

Unlocking the mysteries of VLDL: exploring its production, intracellular trafficking, and metabolism as therapeutic targets.

Reducing circulating lipid levels is the centerpiece of strategies for preventing and treating atherosclerotic cardiovascular disease (ASCVD). Despite many available lipid-lowering medications, a substantial residual cardiovascular risk remains. Current clinical guidelines focus on plasma levels of low-density lipoprotein (LDL). Recent attention has been given to very low-density lipoprotein (VLDL), the precursor to LDL, and its role in the development of coronary atherosclerosis. Preclinical investigations have revealed that interventions targeting VLDL production or promoting VLDL metabolism, independent of the LDL receptor, can potentially decrease cholesterol levels and provide therapeutic benefits. Currently, methods, such as mipomersen, lomitapide, and ANGPTL3 inhibitors, are used to reduce plasma cholesterol and triglyceride levels by regulating the lipidation, secretion, and metabolism of VLDL. Targeting VLDL represents an avenue for new lipid-lowering strategies. Interventions aimed at reducing VLDL production or enhancing VLDL metabolism, independent of the LDL receptor, hold promise for lowering cholesterol levels and providing therapeutic benefits beyond LDL in the management of ASCVD.

Inverse association between apolipoprotein C-II and cardiovascular mortality: role of lipoprotein lipase activity modulation.

AIMS: Apolipoprotein C-II (ApoC-II) is thought to activate lipoprotein lipase (LPL) and is therefore a possible target for treating hypertriglyceridemia. Its relationship with cardiovascular risk has not been investigated in large-scale epidemiologic studies, particularly allowing for apolipoprotein C-III (ApoC-III), an LPL antagonist. Furthermore, the exact mechanism of ApoC-II-mediated LPL activation is unclear. METHODS AND RESULTS: ApoC-II was measured in 3141 LURIC participants of which 590 died from cardiovascular diseases during a median (inter-quartile range) follow-up of 9.9 (8.7-10.7) years. Apolipoprotein C-II-mediated activation of the glycosylphosphatidylinositol high-density lipoprotein binding protein 1 (GPIHBP1)-LPL complex was studied using enzymatic activity assays with fluorometric lipase and very low-density lipoprotein (VLDL) substrates. The mean ApoC-II concentration was 4.5 (2.4) mg/dL. The relationship of ApoC-II quintiles with cardiovascular mortality exhibited a trend toward an inverse J-shape, with the highest risk in the first (lowest) quintile and lowest risk in the middle quintile. Compared with the first quintile, all other quintiles were associated with decreased cardiovascular mortality after multivariate adjustments including ApoC-III as a covariate (all P < 0.05). In experiments using fluorometric substrate-based lipase assays, there was a bell-shaped relationship for the effect of ApoC-II on GPIHBP1-LPL activity when exogenous ApoC-II was added. In ApoC-II-containing VLDL substrate-based lipase assays, GPIHBP1-LPL enzymatic activity was almost completely blocked by a neutralizing anti-ApoC-II antibody. CONCLUSION: The present epidemiologic data suggest that increasing low circulating ApoC-II levels may reduce cardiovascular risk. This conclusion is supported by the observation that optimal ApoC-II concentrations are required for maximal GPIHBP1-LPL enzymatic activity.

Elevated remnant cholesterol, plasma triglycerides, and cardiovascular and non-cardiovascular mortality.

AIMS: Cholesterol carried in triglyceride-rich lipoproteins, also called remnant cholesterol, is being increasingly acknowledged as an important causal risk factor for atherosclerosis. Elevated remnant cholesterol, marked by elevated plasma triglycerides, is associated causally with an increased risk of atherosclerotic cardiovascular disease. The association with cause-specific mortality is, however, unclear. The aim of this study was to test the hypothesis that elevated remnant cholesterol and plasma triglycerides are associated with increased mortality from cardiovascular disease, cancer, and other causes. METHODS AND RESULTS: Using a contemporary population-based cohort, 87 192 individuals from the Copenhagen General Population Study aged 20-69 years at baseline in 2003-2015 were included. During up to 13 years of follow-up, 687 individuals died from cardiovascular disease, 1594 from cancer, and 856 from other causes, according to the National Danish Causes of Death Registry. In individuals with remnant cholesterol ≥1.0 mmol/L (≥39 mg/dL; 22% of the population) compared with those with levels <0.5 mmol/L (<19 mg/dL), multivariable-adjusted mortality hazard ratios were 2.2 (95% confidence interval 1.3-3.5) for cardiovascular disease, 1.0 (0.7-1.3) for cancer, and 2.1 (1.4-3.3) for other causes. Exploratory analysis of the cause of death subcategories showed corresponding hazard ratios of 4.4 (1.6-11) for ischemic heart disease, 8.4 (2.0-34) for infectious diseases, and 9.1 (1.9-43) for endocrinological diseases. Results for plasma triglycerides >2 vs. <1 mmol/L (>177 vs. <89 mg/dL) were similar. CONCLUSION: Remnant cholesterol of ≥1 mmol/L (39 mg/dL), present in 22% of the population, and plasma triglycerides of ≥2 mmol/L (177 mg/dL), present in 28% of the population, were associated with two-fold mortality from cardiovascular and other causes, but not from cancer. This novel finding should be confirmed in other cohorts.

Elevated remnant cholesterol and atherosclerotic cardiovascular disease in diabetes: a population-based prospective cohort study.

AIMS/HYPOTHESIS: Elevated remnant cholesterol is observationally and causally associated with increased risk of atherosclerotic cardiovascular disease (ASCVD) in the general population. This association is not well studied in individuals with diabetes, who are often included in clinical trials of remnant cholesterol-lowering therapy. We tested the hypothesis that elevated remnant cholesterol is associated with increased risk of ASCVD in individuals with diabetes. We also explored the fraction of excess risk conferred by diabetes which can be explained by elevated remnant cholesterol. METHODS: We included 4569 white Danish individuals with diabetes (58% statin users) nested within the Copenhagen General Population Study (2003-2015). The ASCVDs peripheral artery disease, myocardial infarction and ischaemic stroke were extracted from national Danish health registries without losses to follow-up. Remnant cholesterol was calculated from a standard lipid profile. RESULTS: During up to 15 years of follow-up, 236 individuals were diagnosed with peripheral artery disease, 234 with myocardial infarction, 226 with ischaemic stroke and 498 with any ASCVD. Multivariable adjusted HR (95% CI) per doubling of remnant cholesterol was 1.6 (1.1, 2.3; p=0.01) for peripheral artery disease, 1.8 (1.2, 2.5; p=0.002) for myocardial infarction, 1.5 (1.0, 2.1; p=0.04) for ischaemic stroke, and 1.6 (1.2, 2.0; p=0.0003) for any ASCVD. Excess risk conferred by diabetes was 2.5-fold for peripheral artery disease, 1.6-fold for myocardial infarction, 1.4-fold for ischaemic stroke and 1.6-fold for any ASCVD. Excess risk explained by elevated remnant cholesterol and low-grade inflammation was 14% and 8% for peripheral artery disease, 26% and 16% for myocardial infarction, 34% and 34% for ischaemic stroke, and 24% and 18% for any ASCVD, respectively. LDL-cholesterol did not explain excess risk, as it was not higher in individuals with diabetes. We also explored the fraction of excess risk conferred by diabetes which can be explained by elevated remnant cholesterol. CONCLUSIONS/INTERPRETATION: Elevated remnant cholesterol was associated with increased risk of ASCVD in individuals with diabetes. Remnant cholesterol and low-grade inflammation explained substantial excess risk of ASCVD conferred by diabetes. Whether remnant cholesterol should be used as a treatment target remains to be determined in randomised controlled trials.

Fenofibrate Mitigates Hypertriglyceridemia in Nonalcoholic Steatohepatitis Patients Treated With Cilofexor/Firsocostat.

BACKGROUND & AIMS: Patients with advanced fibrosis due to nonalcoholic steatohepatitis (NASH) are at high risk of morbidity and mortality. We previously found that a combination of the farnesoid X receptor agonist cilofexor (CILO) and the acetyl-CoA carboxylase inhibitor firsocostat (FIR) improved liver histology and biomarkers in NASH with advanced fibrosis but was associated with hypertriglyceridemia. We evaluated the safety and efficacy of icosapent ethyl (Vascepa) and fenofibrate to mitigate triglyceride elevations in patients with NASH treated with CILO and FIR. METHODS: Patients with NASH with elevated triglycerides (≥150 and <500 mg/dL) were randomized to Vascepa 2 g twice daily (n = 33) or fenofibrate 145 mg daily (n = 33) for 2 weeks, followed by the addition of CILO 30 mg and FIR 20 mg daily for 6 weeks. Safety, lipids, and liver biochemistry were monitored. RESULTS: All treatments were well-tolerated; most treatment-emergent adverse events were Grade 1 to 2 severity, and there were no discontinuations due to adverse events. At baseline, median (interquartile range [IQR]) triglycerides were similar in the Vascepa and fenofibrate groups (median, 177 [IQR, 154-205] vs 190 [IQR, 144-258] mg/dL, respectively). Median changes from baseline in triglycerides for Vascepa vs fenofibrate after 2 weeks of pretreatment were -12 mg/dL (IQR, -33 to 7 mg/dL; P = .09) vs -32 mg/dL (IQR, -76 to 6 mg/dL; P = .012) and at 6 weeks were +41 mg/dL (IQR, 16-103 mg/dL; P < .001) vs -2 mg/dL (IQR, -42 to 54 mg/dL; P = .92). In patients with baseline triglycerides <250 mg/dL, fenofibrate was more effective vs Vascepa in mitigating triglyceride increases after 6 weeks of combination treatment (+6 vs +39 mg/dL); similar trends were observed in patients with baseline triglycerides ≥250 mg/d (-61 vs +99 mg/dL). CONCLUSIONS: In patients with NASH with hypertriglyceridemia treated with CILO and FIR, fenofibrate was safe and effectively mitigated increases in triglycerides associated with acetyl-CoA carboxylase inhibition. CLINICALTRIALS: gov, Number: NCT02781584.

On the validity of fluorimetric intracellular calcium detection: Impact of lipid components.

We investigated the effects of different lipids on the activity of the angiotensin II type 1 receptor (AT1R). As calcium plays a key role in the signaling of the AT1R, we used the calcium-sensitive fluorescence indicators fura-2 to detect intracellular calcium release upon stimulation with the agonist angiotensin II. At first sight, cells preincubated with Very low-density lipoprotein (VLDL) showed a reduced calcium release triggered by angiontensin II compared to untreated control. However, on closer examination, this result seemed to be an artifact. Incubation with VLDL reduced also the amount of intracellular fura-2, as measured by fluorescence in the isosbestic point. Additionally, the maximal obtainable ratio, obtained after complete saturation with calcium ions, was reduced in cells preincubated with VLDL. These findings rendered our initial results questionable. We report the results of our work and our suggestions regarding the experimental setup to contribute to the understanding of the interpretation of fura-2 measurements and to avoid erroneous conclusions.

Up-regulated small-conductance calcium-activated potassium currents contribute to atrial arrhythmogenesis in high-fat feeding mice.

AIMS: Metabolic syndrome (MetS) is associated with arrhythmias and cardiovascular mortality. Arrhythmogenesis in MetS results from atrial structural and electrical remodelling. The small-conductance Ca2+-activated K+ (SK) currents modulate atrial repolarization and may influence atrial arrhythmogenicity. This study investigated the regulation of SK current perturbed by a high-fat diet (HFD) to mimic MetS. METHODS AND RESULTS: Thirty mice were divided into two groups that were fed with normal chow (CTL) and HFD for 4 months. Electrocardiography and echocardiography were used to detect cardiac electrical and structure remodelling. Atrial action potential duration (APD) and calcium transient duration (CaTD) were measured by optical mapping of Langendorff-perfused mice hearts. Atrial fibrillation (AF) inducibility and duration were assessed by burst pacing. Whole-cell patch clamp was performed in primarily isolated atrial myocytes for SK current density. The SK current density is higher in atrial myocytes from HFD than in CTL mice (P ≤ 0.037). The RNA and protein expression of SK channels are increased in HFD mice (P ≤ 0.041 and P ≤ 0.011, respectively). Action potential duration is shortened in HFD compared with CTL (P ≤ 0.015). The shortening of the atrial APD in HFD is reversed by the application of 100 nM apamin (P ≤ 0.043). Compared with CTL, CaTD is greater in HFD atria (P ≤ 0.029). Calcium transient decay (Tau) is significantly higher in HFD than in CTL (P = 0.001). Both APD and CaTD alternans thresholds were higher in HFD (P ≤ 0.043), along with higher inducibility and longer duration of AF in HFD (P ≤ 0.023). CONCLUSION: Up-regulation of apamin-sensitive SK currents plays a partial role in the atrial arrhythmogenicity of HFD mice.

α-Tocopherol reduces VLDL secretion through modulation of intracellular ER-to-Golgi transport of VLDL.

Avoiding hepatic steatosis is crucial for preventing liver dysfunction, and one mechanism by which this is accomplished is through synchronization of the rate of very low density lipoprotein (VLDL) synthesis with its secretion. Endoplasmic reticulum (ER)-to-Golgi transport of nascent VLDL is the rate-limiting step in its secretion and is mediated by the VLDL transport vesicle (VTV). Recent in vivo studies have indicated that α-tocopherol (α-T) supplementation can reverse steatosis in nonalcoholic fatty liver disease, but its effects on hepatic lipoprotein metabolism are poorly understood. Here, we investigated the impact of α-T on hepatic VLDL synthesis, secretion, and intracellular ER-to-Golgi VLDL trafficking using an in vitro model. Pulse-chase assays using [3H]-oleic acid and 100 µmol/L α-T demonstrated a disruption of early VLDL synthesis, resulting in enhanced apolipoprotein B-100 expression, decreased expression in markers for VTV budding, ER-to-Golgi VLDL transport, and reduced VLDL secretion. Additionally, an in vitro VTV budding assay indicated a significant decrease in VTV production and VTV-Golgi fusion. Confocal imaging of lipid droplet (LD) localization revealed a decrease in overall LD retention, diminished presence of ER-associated LDs, and an increase in Golgi-level LD retention. We conclude that α-T disrupts ER-to-Golgi VLDL transport by modulating the expression of specific proteins and thus reduces VLDL secretion.

Elevated remnant cholesterol increases the risk of peripheral artery disease, myocardial infarction, and ischaemic stroke: a cohort-based study.

AIMS: The atherogenic potential of cholesterol in triglyceride-rich lipoproteins, also called remnant cholesterol, is being increasingly acknowledged. Elevated remnant cholesterol is associated with increased risk of myocardial infarction and ischaemic stroke. We tested the hypothesis that elevated remnant cholesterol is also associated with increased risk of peripheral artery disease (PAD). METHODS AND RESULTS: We studied 106 937 individuals from the Copenhagen General Population Study recruited in 2003-15. During up to 15 years of follow-up, 1586 were diagnosed with PAD, 2570 with myocardial infarction, and 2762 with ischaemic stroke. We also studied 13 974 individuals from the Copenhagen City Heart Study recruited in 1976-78. During up to 43 years of follow-up, 1033 were diagnosed with PAD, 2236 with myocardial infarction, and 1976 with ischaemic stroke. Remnant cholesterol was calculated from a standard lipid profile. Diagnoses were from Danish nationwide health registries. In the Copenhagen General Population Study, elevated remnant cholesterol levels were associated with higher risk of PAD, up to a multivariable adjusted hazard ratio (HR) of 4.8 (95% confidence interval 3.1-7.5) for individuals with levels ≥1.5 mmol/L (58 mg/dL) vs. <0.5 mmol/L (19 mg/dL). Corresponding results were 4.2 (2.9-6.1) for myocardial infarction and 1.8 (1.4-2.5) for ischaemic stroke. In the Copenhagen City Heart Study, corresponding HRs were 4.9 (2.9-8.5) for PAD, 2.6 (1.8-3.8) for myocardial infarction, and 2.1 (1.5-3.1) for ischaemic stroke. CONCLUSION: Elevated remnant cholesterol is associated with a five-fold increased risk of PAD in the general population, higher than for myocardial infarction and ischaemic stroke.

Decreased Serum Stromal Cell-Derived Factor-1 in Patients with Familial Hypercholesterolemia and Its Strong Correlation with Lipoprotein Subfractions.

Stromal cell-derived factor-1 (SDF-1) is a chemokine that exerts multifaceted roles in atherosclerosis. However, its association with hyperlipidemia is contradictory. To date, serum SDF-1 and its correlations with lipid fractions and subfractions in heterozygous familial hypercholesterolemia (HeFH) have not been investigated. Eighty-one untreated patients with HeFH and 32 healthy control subjects were enrolled in the study. Serum SDF-1, oxidized LDL (oxLDL) and myeloperoxidase (MPO) were determined by ELISA. Lipoprotein subfractions were detected by Lipoprint. We diagnosed FH using the Dutch Lipid Clinic Network criteria. Significantly lower serum SDF-1 was found in HeFH patients compared to healthy controls. Significant negative correlations were detected between serum total cholesterol, triglycerides, LDL-cholesterol (LDL-C), apolipoprotein B100 (ApoB100) and SDF-1. Furthermore, serum SDF-1 negatively correlated with VLDL and IDL, as well as large LDL and large and intermediate HDL subfractions, while there was a positive correlation between mean LDL-size, small HDL and SDF-1. SDF-1 negatively correlated with oxLDL and MPO. A backward stepwise multiple regression analysis showed that the best predictors of serum SDF-1 were VLDL and oxLDL. The strong correlation of SDF-1 with lipid fractions and subfractions highlights the potential common pathways of SDF-1 and lipoprotein metabolism, which supports the role of SDF-1 in atherogenesis.

Comparison of angiopoietin-like protein 3 and 4 reveals structural and mechanistic similarities.

Elevated plasma triglycerides are a risk factor for coronary artery disease, which is the leading cause of death worldwide. Lipoprotein lipase (LPL) reduces triglycerides in the blood by hydrolyzing them from triglyceride-rich lipoproteins to release free fatty acids. LPL activity is regulated in a nutritionally responsive manner by macromolecular inhibitors including angiopoietin-like proteins 3 and 4 (ANGPTL3 and ANGPTL4). However, the mechanism by which ANGPTL3 inhibits LPL is unclear, in part due to challenges in obtaining pure protein for study. We used a new purification protocol for the N-terminal domain of ANGPTL3, removing a DNA contaminant, and found DNA-free ANGPTL3 showed enhanced inhibition of LPL. Structural analysis showed that ANGPTL3 formed elongated, flexible trimers and hexamers that did not interconvert. ANGPTL4 formed only elongated flexible trimers. We compared the inhibition of ANGPTL3 and ANGPTL4 using human very-low-density lipoproteins as a substrate and found both were noncompetitive inhibitors. The inhibition constants for the trimeric ANGPTL3 (7.5 ± 0.7 nM) and ANGPTL4 (3.6 ± 1.0 nM) were only 2-fold different. Heparin has previously been reported to interfere with ANGPTL3 binding to LPL, so we questioned if the negatively charged heparin was acting in a similar fashion to the DNA contaminant. We found that ANGPTL3 inhibition is abolished by binding to low-molecular-weight heparin, whereas ANGPTL4 inhibition is not. Our data show new similarities and differences in how ANGPTL3 and ANGPTL4 regulate LPL and opens new avenues of investigating the effect of heparin on LPL inhibition by ANGPTL3.

Soluble very low-density lipoprotein receptor (sVLDLR) inhibits fibrosis in neovascular age-related macular degeneration.

Subretinal fibrosis is a key pathological feature in neovascular age-related macular degeneration (nAMD). Previously, we identified soluble very low-density lipoprotein receptor (sVLDLR) as an endogenous Wnt signaling inhibitor. This study investigates whether sVLDLR plays an anti-fibrogenic role in nAMD models, including Vldlr-/- mice and laser-induced choroidal neovascularization (CNV). We found that fibrosis factors including P-Smad2/3, α-SMA, and CTGF were upregulated in the subretinal area of Vldlr-/- mice and the laser-induced CNV model. The antibody blocking Wnt co-receptor LRP6 significantly attenuated the overexpression of fibrotic factors in these two models. Moreover, there was a significant reduction of sVLDLR in the interphotoreceptor matrix (IPM) in the laser-induced CNV model. A transgenic strain (sVLDLR-Tg) with sVLDLR overexpression in the IPM was generated. Overexpression of sVLDLR ameliorated the profibrotic changes in the subretinal area of the laser-induced CNV model. In addition, Wnt and TGF-ß signaling synergistically promoted fibrogenesis in human primary retinal pigment epithelium (RPE) cells. CRISPR/Cas9-mediated LRP6 gene knockout (KO) attenuated this synergistic effect. The disruption of VLDLR expression promoted, while the overexpression of sVLDLR inhibited TGF-ß-induced fibrosis. These findings suggest that overactivated Wnt signaling enhances the TGF-ß pathway in subretinal fibrosis. sVLDLR confers an antifibrotic effect, at least partially, through the inhibition of Wnt signaling and thus, has therapeutic potential for fibrosis.

Phosphatidylcholine (18:0/20:4), a potential biomarker to predict ethionamide-induced hepatic steatosis in rats.

Ethionamide (ETH), a second-line drug for multidrug-resistant tuberculosis, is known to cause hepatic steatosis in rats and humans. To investigate predictive biomarkers for ETH-induced steatosis, we performed lipidomics analysis using plasma and liver samples collected from rats treated orally with ETH at 30 and 100 mg/kg for 14 days. The ETH-treated rats developed hepatic steatosis with Oil Red O staining-positive vacuolation in the centrilobular hepatocytes accompanied by increased hepatic contents of triglycerides (TG) and decreased plasma TG and total cholesterol levels. A multivariate analysis for lipid profiles revealed differences in each of the 35 lipid species in the plasma and liver between the control and the ETH-treated rats. Of those lipids, phosphatidylcholine (PC) (18:0/20:4) decreased dose-dependently in both the plasma and liver. Moreover, serum TG-rich very low-density lipoprotein (VLDL) levels, especially the large particle fraction of VLDL composed of PC containing arachidonic acid (20:4) involved in hepatic secretion of TG, were decreased dose-dependently. In conclusion, the decreased PC (18:0/20:4) in the liver, possibly leading to suppression of hepatic TG secretion, was considered to be involved in the pathogenesis of the ETH-induced hepatic steatosis. Therefore, plasma PC (18:0/20:4) levels are proposed as mechanism-related biomarkers for ETH-induced hepatic steatosis.

A New Structural Model of Apolipoprotein B100 Based on Computational Modeling and Cross Linking.

ApoB-100 is a member of a large lipid transfer protein superfamily and is one of the main apolipoproteins found on low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) particles. Despite its clinical significance for the development of cardiovascular disease, there is limited information on apoB-100 structure. We have developed a novel method based on the "divide and conquer" algorithm, using PSIPRED software, by dividing apoB-100 into five subunits and 11 domains. Models of each domain were prepared using I-TASSER, DEMO, RoseTTAFold, Phyre2, and MODELLER. Subsequently, we used disuccinimidyl sulfoxide (DSSO), a new mass spectrometry cleavable cross-linker, and the known position of disulfide bonds to experimentally validate each model. We obtained 65 unique DSSO cross-links, of which 87.5% were within a 26 Å threshold in the final model. We also evaluated the positions of cysteine residues involved in the eight known disulfide bonds in apoB-100, and each pair was measured within the expected 5.6 Å constraint. Finally, multiple domains were combined by applying constraints based on detected long-range DSSO cross-links to generate five subunits, which were subsequently merged to achieve an uninterrupted architecture for apoB-100 around a lipoprotein particle. Moreover, the dynamics of apoB-100 during particle size transitions was examined by comparing VLDL and LDL computational models and using experimental cross-linking data. In addition, the proposed model of receptor ligand binding of apoB-100 provides new insights into some of its functions.

Emerging Evidence of Pathological Roles of Very-Low-Density Lipoprotein (VLDL).

Embraced with apolipoproteins (Apo) B and Apo E, triglyceride-enriched very-low-density lipoprotein (VLDL) is secreted by the liver into circulation, mainly during post-meal hours. Here, we present a brief review of the physiological role of VLDL and a systemic review of the emerging evidence supporting its pathological roles. VLDL promotes atherosclerosis in metabolic syndrome (MetS). VLDL isolated from subjects with MetS exhibits cytotoxicity to atrial myocytes, induces atrial myopathy, and promotes vulnerability to atrial fibrillation. VLDL levels are affected by a number of endocrinological disorders and can be increased by therapeutic supplementation with cortisol, growth hormone, progesterone, and estrogen. VLDL promotes aldosterone secretion, which contributes to hypertension. VLDL induces neuroinflammation, leading to cognitive dysfunction. VLDL levels are also correlated with chronic kidney disease, autoimmune disorders, and some dermatological diseases. The extra-hepatic secretion of VLDL derived from intestinal dysbiosis is suggested to be harmful. Emerging evidence suggests disturbed VLDL metabolism in sleep disorders and in cancer development and progression. In addition to VLDL, the VLDL receptor (VLDLR) may affect both VLDL metabolism and carcinogenesis. Overall, emerging evidence supports the pathological roles of VLDL in multi-organ diseases. To better understand the fundamental mechanisms of how VLDL promotes disease development, elucidation of the quality control of VLDL and of the regulation and signaling of VLDLR should be indispensable. With this, successful VLDL-targeted therapies can be discovered in the future.

Triglyceride-Rich Lipoproteins: Their Role in Atherosclerosis.

The term "triglyceride-rich lipoproteins" (TRLs) includes chylomicrons and their remnants, very low-density lipoproteins (VLDL), and intermediate-density lipoproteins (IDL). In this manuscript, the mechanisms by which atherogenic TRLs contribute to the formation of atheroma plaques are reviewed. Cholesterol from TRLs that can be retained in the subendothelial space (i.e., remnants, DLs, and small VLDLs) contributes to the genesis of atherosclerosis. Triglycerides of atherogenic TRLs induce inflammation of the arterial wall. Mechanisms that explain the involvement of TRLs in atherosclerosis are the generation of pro-atherogenic changes in high-density lipoproteins and low-density lipoproteins, accumulation of TRLs in plasma, and their passage to the subendothelial space where they cause endothelial dysfunction and inflammation of the vascular wall. Furthermore, plasma accumulation of TRLs causes hyperviscosity and a procoagulant state. Finally, this manuscript summarizes the controversial aspects of the clinical approach and the treatment of cases with dyslipidemia explained by atherogenic TRLs.

Wilms tumor 1 regulates lipid accumulation in human endometrial stromal cells during decidualization.

We previously reported that the transcription factor Wilms tumor 1 (WT1) regulates the expression of insulin-like growth factor-binding protein-1 (IGFBP-1) and prolactin (PRL) during decidualization of human endometrial stromal cells (ESCs). However, other roles of WT1 in decidualization remain to be fully clarified. Here, we investigated how WT1 regulates the physiological functions of human ESCs during decidualization. We incubated ESCs isolated from proliferative-phase endometrium with cAMP to induce decidualization, knocked down WT1 with siRNA, and generated three types of treatments (nontreated cells, cAMP-treated cells, and cAMP-treated + WT1-knockdown cells). To identify WT1-regulated genes, we used gene microarrays and compared the transcriptome data obtained among these three treatments. We observed that WT1 up-regulates 121 genes during decidualization, including several genes involved in lipid transport. The WT1 knockdown inhibited lipid accumulation (LA) in the cAMP-induced ESCs. To examine the mechanisms by which WT1 regulates LA, we focused on very low-density lipoprotein receptor (VLDLR), which is involved in lipoprotein uptake. We found that cAMP up-regulates VLDLR and that the WT1 knockdown inhibits it. Results of ChIP assays revealed that cAMP increases the recruitment of WT1 to the promoter region of the VLDLR gene, indicating that WT1 regulates VLDLR expression. Moreover, VLDLR knockdown inhibited cAMP-induced LA, and VLDLR overexpression reverted the suppression of LA caused by the WT1 knockdown. Taken together, our results indicate that WT1 enhances lipid storage by up-regulating VLDLR expression in human ESCs during decidualization.

The Role of Phospholipid Transfer Protein in the Development of Atherosclerosis.

PURPOSE OF REVIEW: Phospholipid transfer protein (PLTP), a member of lipid transfer protein family, is an important protein involved in lipid metabolism in the circulation. This article reviews recent PLTP research progresses, involving lipoprotein metabolism and atherogenesis. RECENT FINDINGS: PLTP activity influences atherogenic and anti-atherogenic lipoprotein levels. Human serum PLTP activity is a risk factor for human cardiovascular disease and is an independent predictor of all-cause mortality. PLTP deficiency reduces VLDL and LDL levels and attenuates atherosclerosis in mouse models, while PLTP overexpression exerts an opposite effect. Both PLTP deficiency and overexpression result in reduction of HDL which has different size, inflammatory index, and lipid composition. Moreover, although both PLTP deficiency and overexpression reduce cholesterol efflux capacity, but this effect has no impact in macrophage reverse cholesterol transport in mice. Furthermore, PLTP activity is related with metabolic syndrome, thrombosis, and inflammation. PLTP could be target for the treatment of dyslipidemia and atherosclerosis, although some potential off-target effects should be noted.