Cholesteryl Ester Content Research Articles

From LAL-D to MASLD: Insights into the role of LAL and Kupffer cells in liver inflammation and lipid metabolism

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a prevalent liver pathology worldwide, closely associated with obesity and metabolic disorders. Increasing evidence suggests that macrophages play a crucial role in the development of MASLD. Several human studies have shown an inverse correlation between circulating lysosomal acid lipase (LAL) activity and MASLD. LAL is the sole enzyme known to degrade cholesteryl esters (CE) and triacylglycerols in lysosomes. Consequently, these substrates accumulate when their enzymatic degradation is impaired due to LAL deficiency (LALD). This study aimed to investigate the role of hepatic LAL activity and liver-resident macrophages (i.e., Kupffer cells (KC)) in MASLD. To this end, we analyzed lipid metabolism in hepatocyte-specific (hep)Lal−/− mice and depleted KC with clodronate treatment. When fed a high-fat/high-cholesterol diet (HF/HCD), hepLal−/− mice exhibited CE accumulation and an increased number of macrophages in the liver and significant hepatic inflammation. KC were depleted upon clodronate administration, whereas infiltrating/proliferating CD68-expressing macrophages were less affected. This led to exacerbated hepatic CE accumulation and dyslipidemia, as evidenced by increased LDL-cholesterol concentrations. Along with proteomic analysis of liver tissue, these findings indicate that hepatic LAL-D in HF/HCD-fed mice leads to macrophage infiltration into the liver and that KC depletion further exacerbates hepatic CE concentrations and dyslipidemia.

Sensing Biomolecules Associated with Cells' Radiosusceptibility by Advanced Micro- and Nanospectroscopy Techniques.

Radiotherapy is one of the most common approaches for cancer treatment, especially in the case of peripheral nervous system tumors. As it requires exposure to high doses of ionizing radiation, it is important to look for substances that support efficient reduction of the tumor volume with simultaneous prevention of the surrounding noncancerous cells. Cannabidiol (CBD), which exhibits both anticancer and neuroprotective properties, was applied as a potential modulator of radiological response; however, its influence on cells undergoing irradiation remains elusive. Here, we have applied high-resolution optical spectroscopy techniques to capture biomolecules associated with CBD shielding of normal and damaging cancerous cells upon X-ray exposure. Conventional Raman (RS) and Fourier transformed infrared (FT-IR) spectroscopies provided semiquantitative information mainly about changes in the concentration of total lipids, DNA, cholesteryl esters, and phospholipids in cells. A through assessment of the single cells by atomic force microscopy coupled with infrared spectroscopy (AFM-IR) allowed us to determine not only the alterations in DNA content but also in its conformation due to cell treatment. Pronounced nanoscale changes in cholesteryl ester metabolites, associated with CBD treatment and radiation, were also observed. AFM-IR chemoselective maps of the single cells indicate the modified distribution of cholesteryl esters with 40 nm spatial resolution. Based on the obtained results, we propose a label-free and fast analytical method engaging optical spectroscopy to assess the mechanism of normal and cancerous cell susceptibility to ionizing radiation when pretreated with CBD.

A Facile LC-MS Method for Profiling Cholesterol and Cholesteryl Esters in Mammalian Cells and Tissues.

Cholesterol is central to mammalian lipid metabolism and serves many critical functions in the regulation of diverse physiological processes. Dysregulation in cholesterol metabolism is causally linked to numerous human diseases, and therefore, in vivo, the concentrations and flux of cholesterol and cholesteryl esters (fatty acid esters of cholesterol) are tightly regulated. While mass spectrometry has been an analytical method of choice for detecting cholesterol and cholesteryl esters in biological samples, the hydrophobicity, chemically inert nature, and poor ionization of these neutral lipids have often proved a challenge in developing lipidomics compatible liquid chromatography-mass spectrometry (LC-MS) methods to study them. To overcome this problem, here, we report a reverse-phase LC-MS method that is compatible with existing high-throughput lipidomics strategies and capable of identifying and quantifying cholesterol and cholesteryl esters from mammalian cells and tissues. Using this sensitive yet robust LC-MS method, we profiled different mammalian cell lines and tissues and provide a comprehensive picture of cholesterol and cholesteryl esters content in them. Specifically, among cholesteryl esters, we find that mammalian cells and tissues largely possess monounsaturated and polyunsaturated variants. Taken together, our lipidomics compatible LC-MS method to study this lipid class opens new avenues in understanding systemic and tissue-level cholesterol metabolism under various physiological conditions.

Effects of Recombinant Human Lecithin Cholesterol Acyltransferase on Lipoprotein Metabolism in Humans.

LCAT (lecithin cholesterol acyl transferase) catalyzes the conversion of unesterified, or free cholesterol, to cholesteryl ester, which moves from the surface of HDL (high-density lipoprotein) into the neutral lipid core. As this iterative process continues, nascent lipid-poor HDL is converted to a series of larger, spherical cholesteryl ester-enriched HDL particles that can be cleared by the liver in a process that has been termed reverse cholesterol transport. We conducted a randomized, placebocontrolled, crossover study in 5 volunteers with atherosclerotic cardiovascular disease, to examine the effects of an acute increase of recombinant human (rh) LCAT via intravenous administration (300-mg loading dose followed by 150 mg at 48 hours) on the in vivo metabolism of HDL APO (apolipoprotein)A1 and APOA2, and the APOB100-lipoproteins, very low density, intermediate density, and low-density lipoproteins. As expected, recombinant human LCAT treatment significantly increased HDL-cholesterol (34.9 mg/dL; P≤0.001), and this was mostly due to the increase in cholesteryl ester content (33.0 mg/dL; P=0.014). This change did not affect the fractional clearance or production rates of HDL-APOA1 and HDL-APOA2. There were also no significant changes in the metabolism of APOB100-lipoproteins. Our results suggest that an acute increase in LCAT activity drives greater flux of cholesteryl ester through the reverse cholesterol transport pathway without significantly altering the clearance and production of the main HDL proteins and without affecting the metabolism of APOB100-lipoproteins. Long-term elevations of LCAT might, therefore, have beneficial effects on total body cholesterol balance and atherogenesis.

Abstract PO5-24-05: The Metabolic and Epigenetic Contributions of Fibroblast Growth Factor Receptor-Mediated Breast Cancer Progression

Abstract Despite recent improvements in treatment options, breast cancer remains the second leading cause of cancer-related deaths among women, with metastasis of the primary tumor accounting for most of these deaths. Signaling by fibroblast growth factor receptors (FGFRs) is active in 85% of breast cancers and results in enhanced proliferation, migration, invasion, and therapeutic resistance of tumor cells. A common feature of breast cancer is altered cholesterol metabolism including an increase in cholesterol stored intracellularly as cholesteryl esters (CEs). In breast cancer cells, high levels of CEs are associated with aggressive disease and CEs are essential for growth and migration of cancer cells. Furthermore, there is mounting evidence that modifications to metabolic pathways impact epigenetic processes including DNA methylation and histone post-translational modifications. These epigenetic changes support tumor progression and metastasis through oncogene activation and silencing of tumor suppressors. While there have been numerous studies focused on the role of metabolic and epigenetic changes in breast cancer, none have attempted to connect these altered pathways to upstream growth factor receptor pathways, such as the FGFR pathway, in cancer cells. We hypothesize that FGFR signaling promotes changes to cholesterol metabolism and epigenetic regulation which lead to breast cancer progression. To study the proposed roles of FGFR signaling, we utilized murine mammary cells of differing metastatic abilities with inducible, oncogenic FGFR1 signaling. We also included the FGFR-dependent murine mammary tumor lines 4T1 and 4T07 which demonstrate differing metastatic capabilities. Through gene expression studies, we found an FGFR-mediated increase in cholesterol metabolic processes including cholesterol storage, uptake, and synthesis. Along with this, we found an FGFR-mediated increase in CE content in our models. Interestingly, these changes were exclusive to our highly metastatic models. Additionally, we have identified FGFR-mediated changes in the expression of histone modifying enzymes in our cell lines. Many of the enzymes modified by FGFR have been previously reported to play a role in breast cancer metastasis. To further elucidate the role of enhanced cholesterol storage in breast cancer progression, we treated cells with pharmacological inhibitors of cholesterol storage. Using in vitro assays, we demonstrate that cell survival, migration, and invasion all decrease when cholesterol storage is inhibited. Again, we observe that our highly metastatic cell line models are more sensitive to this inhibition. Taken together, these data suggest a role for FGFR signaling in promoting breast cancer progression and metastasis. With further studies, this connection may inform the development of novel therapeutic strategies to combat therapeutic resistance in breast cancer. Citation Format: Jennifer Tuokkola, Lyndsay Reese, Kaylee Schwertfeger. The Metabolic and Epigenetic Contributions of Fibroblast Growth Factor Receptor-Mediated Breast Cancer Progression [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-24-05.

Lipidomics and plasma hormone analysis differentiate reproductive and pregnancy statuses in Florida manatees (Trichechus manatus latirostris)

Florida manatees (Trichechus manatus latirostris) are protected as a threatened species, and data are lacking regarding their reproductive physiology. This study aimed to (1) quantify plasma steroid hormones in Florida manatees from two field sites, Crystal River and Indian River Lagoon, at different gestational stages and to (2) identify individual lipids associated with pregnancy status. Ultra-high performance liquid chromatography-tandem mass spectrometric analysis was used to measure plasma steroid hormones and lipids. Pregnant female manatees were morphometrically distinct from male and non-pregnant female manatees, characterized by larger body weight and maximal girth. Progesterone concentrations in manatees were also elevated during early gestation versus late gestation. Cholesterol, an important metabolic lipid, and precursor for reproductive steroids, was not different between groups. Mass spectrometry quantified 949 lipids. Plasma concentrations of glycerophospholipids, glycerolipids, sphingolipids, acylcarnitines, and cholesteryl esters were associated with pregnancy status in the Florida manatee. Most of the lipid species associated with pregnancy were triacylglycerides, phosphatidylethanolamines, and ether-linked phosphatidylethanolamines, which may serve as energy sources for fetal development. This research contributes to improving knowledge of manatee reproductive physiology by providing data on plasma steroid hormones relative to reproductive status and by identifying plasma lipids that may be important for pregnancy. Elucidation of lipid species directly associated with pregnancy has the potential to serve as a diagnostic approach to identify pregnant individuals in fresh and archived samples. These biochemical and morphometric indicators of reproductive status advance the understanding of manatee physiology.

Evidence for alterations in lipid profiles and biophysical properties of lipid rafts from spinal cord in sporadic amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an age-dependent neurodegenerative disease affecting motor neurons in the spinal cord and brainstem whose etiopathogenesis remains unclear. Recent studies have linked major neurodegenerative diseases with altered function of multimolecular lipid-protein complexes named lipid rafts. In the present study, we have isolated lipid rafts from the anterior horn of the spinal cords of controls and ALS individuals and analysed their lipid composition. We found that ALS affects levels of different fatty acids, lipid classes and related ratios and indexes. The most significant changes affected the contents of n-9/n-7 monounsaturated fatty acids and arachidonic acid, the main n-6 long-chain polyunsaturated fatty acid (LCPUFA), which were higher in ALS lipid rafts. Paralleling these findings, ALS lipid rafts lower saturates-to-unsaturates ratio compared to controls. Further, levels of cholesteryl ester (SE) and anionic-to-zwitterionic phospholipids ratio were augmented in ALS lipid rafts, while sulfatide contents were reduced. Further, regression analyses revealed augmented SE esterification to (mono)unsaturated fatty acids in ALS, but to saturates in controls. Overall, these changes indicate that lipid rafts from ALS spinal cord undergo destabilization of the lipid structure, which might impact their biophysical properties, likely leading to more fluid membranes. Indeed, estimations of membrane microviscosity confirmed less viscous membranes in ALS, as well as more mobile yet smaller lipid rafts compared to surrounding membranes. Overall, these results demonstrate that the changes in ALS lipid rafts are unrelated to oxidative stress, but to anomalies in lipid metabolism and/or lipid raft membrane biogenesis in motor neurons.Key messagesThe lipid matrix of multimolecular membrane complexes named lipid rafts are altered in human spinal cord in sporadic amyotrophic lateral sclerosis (ALS).Lipid rafts from ALS spinal cord contain higher levels of n-6 LCPUFA (but not n-3 LCPUFA), n-7/n-9 monounsaturates and lower saturates-to-unsaturates ratio.ALS lipid rafts display increased contents of cholesteryl esters, anomalous anionic-to-zwitterionic phospholipids and phospholipid remodelling and reduced sulphated and total sphingolipid levels, compared to control lipid rafts.Destabilization of the lipid structure of lipid raft affects their biophysical properties and leads to more fluid, less viscous membrane microdomains.The changes in ALS lipid rafts are unlikely related to increased oxidative stress, but to anomalies in lipid metabolism and/or raft membrane biogenesis in motor neurons.

Scavenger Receptor BI Deficiency in Mice Is Associated With Plasma Ceramide and Sphingomyelin Accumulation and a Reduced Cholesteryl Ester Fatty Acid Length and Unsaturation Degree.

Scavenger receptor class B type I (SR-BI) is primarily known for its role in the selective uptake of cholesteryl esters (CEs) from high-density lipoproteins (HDLs). Here we investigated whether SR-BI deficiency is associated with other potentially relevant changes in the plasma lipidome than the established effect of HDL-cholesterol elevation. Targeted ultra-high-performance liquid chromatography-tandem mass spectrometry was utilized to measure lipid species in plasma from female wild-type and SR-BI knockout mice. SR-BI deficiency was associated with a reduction in the average CE fatty acid length (-2%; p<0.001) and degree of CE fatty acid unsaturation (-18%; p<0.001) due to a relative shift from longer, polyunsaturated CE species CE (20:4), CE (20:5), and CE (22:6) towards the mono-unsaturated CE (18:1) species. Sphingomyelin (SM) levels were 64% higher (p<0.001) in SR-BI knockout mice without a parallel change in (lyso)phosphatidylcholine (LPC) concentrations, resulting in an increase in the SM/LPC ratio from 0.102±0.005 to 0.163±0.003 (p<0.001). In addition, lower LPC lengths (-5%; p<0.05) and fatty acid unsaturation degrees (-20%; p<0.01) were detected in SR-BI knockout mice. Furthermore, SR-BI deficiency was associated with a 4.7-fold increase (p<0.001) in total plasma ceramide (Cer) levels, with a marked >9-fold rise (p<0.001) in Cer (d18:1/24:1) concentrations. We have shown that SR-BI deficiency in mice not only impacts the CE concentrations, length, and saturation index within the plasma compartment, but is also associated with plasma accumulation of several Cer and SM species that may contribute to the development of specific hematological and metabolic (disease) phenotypes previously detected in SR-BI knockout mice.

Loss of lysosomal acid lipase results in mitochondrial dysfunction and fiber switch in skeletal muscles of mice

ObjectiveLysosomal acid lipase (LAL) is the only enzyme known to hydrolyze cholesteryl esters (CE) and triacylglycerols in lysosomes at an acidic pH. Despite the importance of lysosomal hydrolysis in skeletal muscle (SM), research in this area is limited. We hypothesized that LAL may play an important role in SM development, function, and metabolism as a result of lipid and/or carbohydrate metabolism disruptions. ResultsMice with systemic LAL deficiency (Lal−/−) had markedly lower SM mass, cross-sectional area, and Feret diameter despite unchanged proteolysis or protein synthesis markers in all SM examined. In addition, Lal−/− SM showed increased total cholesterol and CE concentrations, especially during fasting and maturation. Regardless of increased glucose uptake, expression of the slow oxidative fiber marker MYH7 was markedly increased in Lal−/−SM, indicating a fiber switch from glycolytic, fast-twitch fibers to oxidative, slow-twitch fibers. Proteomic analysis of the oxidative and glycolytic parts of the SM confirmed the transition between fast- and slow-twitch fibers, consistent with the decreased Lal−/− muscle size due to the “fiber paradox”. Decreased oxidative capacity and ATP concentration were associated with reduced mitochondrial function of Lal−/− SM, particularly affecting oxidative phosphorylation, despite unchanged structure and number of mitochondria. Impairment in muscle function was reflected by increased exhaustion in the treadmill peak effort test in vivo. ConclusionWe conclude that whole-body loss of LAL is associated with a profound remodeling of the muscular phenotype, manifested by fiber type switch and a decline in muscle mass, most likely due to dysfunctional mitochondria and impaired energy metabolism, at least in mice.

Bile acids regulate SF-1 to alter cholesterol balance in adrenocortical cells via S1PR2

Glucocorticoid synthesis typically occurs in adrenal cortex and is influenced by cholesterol balance, since cholesterol is the sole precursor of steroids. Bile acids as the signaling molecules, have been shown to promote steroidogenesis in steroidogenic cells. However, whether bile acids directly regulate cholesterol balance remains elusive. In this study, we prepared cholestatic mouse models and cultured human adrenocortical cells (H295R) treated with taurochenodeoxycholic acid (TCDCA) to determine transcription levels of cholesterol metabolism associated genes and cholesterol concentrations in adrenocortical cells. Results showed that common bile duct ligation (CBDL) and chenodeoxycholic acid (CDCA) feeding elevated the mRNA levels of Abca1, Cyp51, Hmgcr, Srb1, and Mc2r in adrenals of mice. Meanwhile, the concentrations of total cholesterol and cholesteryl ester in adrenals of CBDL and CDCA-fed mice were dramatically lowered. The total and phosphorylation levels of HSL in adrenal glands of CBDL mice were also enhanced. Similarly, TCDCA treatment in H295R cells decreased intracellular concentrations of total cholesterol and cholesteryl ester and increased transcription levels of SRB1, MC2R, and HSL as well. Inhibition of bile acids’ receptor sphingosine 1‐phosphate receptor 2 (S1PR2), extracellular signal‐regulated kinase (ERK) phosphorylation, and steroidogenic factor 1 (SF-1) respectively successfully abolished effect of TCDCA on H295R cells. SF-1s was found to be phosphorylated at Thr75 in TCDCA-treated H295R cells. While a mild increase of intracellular cAMP concentration was detected upon TCDCA treatment, inhibition of PKA activity with Rp-Isomer in H295R cells failed to decrease the expression of SF-1 and its target genes. Our findings suggest that conjugated bile acids affect cholesterol balance through regulation of SF-1 in adrenocortical cells so as to provide an adequate cholesterol supply for glucocorticoid synthesis, which improves and enriches our understanding of the mechanism whereby bile acids regulate cholesterol balance to affect adrenal function.

Regulation of low-density lipoprotein on lipid metabolism in macrophages of large yellow croaker (Larimichthys crocea)

Low-density lipoprotein (LDL) is the main carrier of cholesterol transport in plasma, which participates in regulating lipid homeostasis. Studies in mammals have shown that high levels of LDL in plasma absorbed by macrophages trigger the formation of lipid-rich foam cells, leading to the development of atherosclerotic plaques. Although lipid-rich atherosclerosis-like lesions have been discovered in the aorta of several fish species, the physiological function of LDL in fish macrophages remains poorly understood. In the present study, LDL was isolated from the plasma of large yellow croaker (Larimichthys crocea), and mass spectrometry analysis identified two truncated forms of apolipoprotein B100 in the LDL protein profile. Transcriptomic analysis of LDL-stimulated macrophages revealed that differentially expressed genes (DEGs) were enriched in various pathways related to lipid metabolism, as confirmed by the fact that LDL increased total cholesterol and cholesteryl esters content. Meanwhile, the gene and protein expression levels of perilipin2 (PLIN2), a DEG enriched in the PPAR signaling pathway, were upregulated in response to LDL stimulation. Importantly, knocking down plin2 significantly attenuates LDL-induced cholesterol accumulation and promotes cholesterol efflux. Furthermore, the transcription factor PPARγ, which is upregulated in response to LDL stimulation, can enhance the promoter activity of plin2. In conclusion, this study suggests that LDL may upregulate plin2 expression through PPARγ, resulting in cholesterol accumulation in fish macrophages. This study will facilitate the investigation of the function of LDL in regulating lipid homeostasis in macrophages and shed light on the evolutionary origin of LDL metabolism in vertebrates.

Structural insights on the deformations induced by various mutations on cholesteryl ester transfer protein

Cholesteryl Ester Transfer Protein (CETP) is a plasma glycoprotein that intervenes the reverse cholesterol transport (RCT) by equimolar exchange of Cholesteryl esters (CE) and Triglycerides (TGs) between anti-atherogenic High-Density Lipoproteins (HDLs) and pro-atherogenic Low-Density Lipoproteins (LDLs) resulting in the increased concentration of CEs in LDL. This is a potential cause for the formation of atherosclerotic plaques in blood vessels leading to fatality. Therefore, blocking the function of CETP has emerged as a novel strategy for suppressing atherosclerotic plaques. The crystal structure of CETP revealed two Cholesteryl esters (CEs) in the hydrophobic tunnel and two phospholipids (PLs) plugged on the concave surface. Previous lipid transfer assay experimental studies have shown a substantial reduction in the neutral lipid transfer in [R201S] and [I443W, V198W] mutants. However, the protein conformational arrangements due to the mutations present in the CETP system leading to a decrease in the transfer rate of neutral lipids is not explored. Thus, I explored the reason behind the decreased transfer rate in mutants using molecular dynamics (MD) simulations and free energy calculations. Resulting evidences show that R201S mutant induces unfavorable bending angle to CETP with a decreased binding efficiency between N-terminal phospholipid of CETP with S201. Also, an unfavorable conformation state of TGs is formed which makes them difficult to transfer across CETP. Likewise, [I443W, V198W] mutant induces unfavorable CE, TG, and bending angle conformation to CETP impeding neutral lipid transfer. Thus, my results provide sufficient insights on the causation for a decreased transfer rate as reported earlier. The detailed understanding obtained here could help in developing a new strategy in preventing the function of CETP by blocking the role of potential hot spot residues.

Developmental brain lipidomics is influenced by postnatal chlorpyrifos exposure and APOE genetic background in mice

Lipids are a major component of the brain, and are involved in structural and neurodevelopmental processes such as neurogenesis, synaptogenesis and signaling. Apolipoprotein E (apoE) is the main lipoprotein involved in lipid transport in the brain. The apoE isoforms can determine vulnerability to the toxic effects of the pesticide chlorpyrifos (CPF), which can interfere with normal neurodevelopment. We aimed to study the effects of postnatal exposure to CPF and of the APOE genotype on the lipid composition of the brain at early ages. For it, we used apoE3 and apoE4 targeted-replacement (TR) male mice, as well as wild-type C57BL/6. The mice were orally exposed to 1 mg/kg/day of CPF on postnatal days 10–15 and, four hours after the treatment, we obtained samples to assess the cerebral lipid composition. Differences between APOE genotypes were found in the cerebral lipid profile in the postnatal period. ApoE4-TR mice exhibited higher lipid concentrations compared to the other groups in most of the cases. CPF exposure led to a decrease in cholesteryl ester and triglyceride concentrations, while modulating the levels of phosphatidylcholine species based on the apoE isoform. Specifically, CPF treatment decreased the concentration of some species of this lipid (PC30:0, PC31:0, PC32:2, PC36:5, PC40:4 and PC40:5) in C57BL/6 mice exposed to CPF, increased (PC31:0 and PC37:6) in apoE3-TR exposed mice while exposed apoE4-TR mice remained unaltered. These results provide further insights into the lipid composition of the brain at early ages, and how it can be modulated by environmental and genetic factors.

Regulation of cholesterol metabolism during high fatty acid–induced lipid deposition in calf hepatocytes

Cholesterol in the circulation is partly driven by changes in feed intake, but aspects of cholesterol metabolism during development of fatty liver are not well known. The objective of this study was to investigate mechanisms of cholesterol metabolism in calf hepatocytes challenged with high concentrations of fatty acids (FA). To address mechanistic insights regarding cholesterol metabolism, liver samples were collected from healthy control dairy cows (n = 6; 7-13 d in milk) and cows with fatty liver (n = 6; 7-11 d in milk). In vitro, hepatocytes isolated from 3 healthy female calves (1 d old) were challenged with or without a mix of 1.2 mM FA to induce metabolic stress. In addition, hepatocytes were processed with 10 µmol/L of the cholesterol synthesis inhibitor simvastatin or 6 µmol/L of the cholesterol intracellular transport inhibitor U18666A with or without the 1.2 mM FA mix. To evaluate the role of cholesterol addition, hepatocytes were treated with 0.147 mg/mL methyl-β-cyclodextrin (MβCD + FA) or 0.147 mg/mL MβCD with or without 10 and 100 µmol/L cholesterol before incubation with FA (CHO10 + FA and CHO100 + FA). In vivo data from liver biopsies were analyzed by 2-tailed unpaired Student's t-test. Data from in vitro calf hepatocytes were analyzed by one-way ANOVA. Compared with healthy cows, blood plasma total cholesterol and plasma low-density lipoprotein cholesterol content in cows with fatty liver was markedly lower, whereas the hepatic total cholesterol content did not differ. In contrast, compared with healthy controls, the triacylglycerol content in the liver and the content of FA, β-hydroxybutyrate, and aspartate aminotransferase in the plasma of cows with fatty liver were greater. The results revealed that both fatty liver in vivo and challenge of calf hepatocytes with 1.2 mM FA in vitro led to greater mRNA and protein abundance of sterol regulatory element binding transcription factor 1 (SREBF1) and fatty acid synthase (FASN). In contrast, mRNA and protein abundance of sterol regulatory element binding transcription factor 2 (SREBF2), acyl coenzyme A-cholesterol acyltransferase, and ATP-binding cassette subfamily A member 1 (ABCA1) were lower. Compared with the FA group, the cholesterol synthesis inhibitor simvastatin led to greater protein abundance of microsomal triglyceride transfer protein and mRNA abundance of SREBF2, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), ACAT2, and lower ABCA1 and FASN protein abundance. In contrast, compared with the FA group, the cholesterol intracellular transport inhibitor U18666A + FA led to greater total cholesterol concentration and greater protein and mRNA abundance of FASN. Compared with the MβCD + FA group, the addition of 10 µmol/L cholesterol led to greater concentration of cholesteryl ester and excretion of apolipoprotein B100, and greater protein and mRNA abundance of ABCA1 and microsomal triglyceride transfer protein, and lower concentration of malondialdehyde. Overall, a reduction in cholesterol synthesis promoted FA metabolism in hepatocytes likely to relieve the oxidative stress caused by the high FA load. The data suggest that maintenance of normal cholesterol synthesis promotes very low-density lipoprotein excretion and can reduce lipid accumulation and oxidative stress in dairy cows that experience fatty liver.

Inhibition of DHCR24 activates LXRα to ameliorate hepatic steatosis and inflammation

Liver X receptor (LXR) agonism has theoretical potential for treating NAFLD/NASH, but synthetic agonists induce hyperlipidemia in preclinical models. Desmosterol, which is converted by Δ24‐dehydrocholesterol reductase (DHCR24) into cholesterol, is a potent endogenous LXR agonist with anti‐inflammatory properties. We aimed to investigate the effects of DHCR24 inhibition on NAFLD/NASH development. Here, by using APOE*3‐Leiden. CETP mice, a well‐established translational model that develops diet‐induced human‐like NAFLD/NASH characteristics, we report that SH42, a published DHCR24 inhibitor, markedly increases desmosterol levels in liver and plasma, reduces hepatic lipid content and the steatosis score, and decreases plasma fatty acid and cholesteryl ester concentrations. Flow cytometry showed that SH42 decreases liver inflammation by preventing Kupffer cell activation and monocyte infiltration. LXRα deficiency completely abolishes these beneficial effects of SH42. Together, the inhibition of DHCR24 by SH42 prevents diet‐induced hepatic steatosis and inflammation in a strictly LXRα‐dependent manner without causing hyperlipidemia. Finally, we also showed that SH42 treatment decreased liver collagen content and plasma alanine transaminase levels in an established NAFLD model. In conclusion, we anticipate that pharmacological DHCR24 inhibition may represent a novel therapeutic strategy for treatment of NAFLD/NASH.

Cholesterol, Amyloid Beta, Fructose, and LPS Influence ROS and ATP Concentrations and the Phagocytic Capacity of HMC3 Human Microglia Cell Line.

Research has found that genes specific to microglia are among the strongest risk factors for Alzheimer's disease (AD) and that microglia are critically involved in the etiology of AD. Thus, microglia are an important therapeutic target for novel approaches to the treatment of AD. High-throughput in vitro models to screen molecules for their effectiveness in reversing the pathogenic, pro-inflammatory microglia phenotype are needed. In this study, we used a multi-stimulant approach to test the usefulness of the human microglia cell 3 (HMC3) cell line, immortalized from a human fetal brain-derived primary microglia culture, in duplicating critical aspects of the dysfunctional microglia phenotype. HMC3 microglia were treated with cholesterol (Chol), amyloid beta oligomers (AβO), lipopolysaccharide (LPS), and fructose individually and in combination. HMC3 microglia demonstrated changes in morphology consistent with activation when treated with the combination of Chol + AβO + fructose + LPS. Multiple treatments increased the cellular content of Chol and cholesteryl esters (CE), but only the combination treatment of Chol + AβO + fructose + LPS increased mitochondrial Chol content. Microglia treated with combinations containing Chol + AβO had lower apolipoprotein E (ApoE) secretion, with the combination of Chol + AβO + fructose + LPS having the strongest effect. Combination treatment with Chol + AβO + fructose + LPS also induced APOE and TNF-α expression, reduced ATP production, increased reactive oxygen species (ROS) concentration, and reduced phagocytosis events. These findings suggest that HMC3 microglia treated with the combination of Chol + AβO + fructose + LPS may be a useful high-throughput screening model amenable to testing on 96-well plates to test potential therapeutics to improve microglial function in the context of AD.

Abstract 2113: The age-dependent intratissue accumulation of cholesterol, resulting in reduced squalene epoxidase, is associated with a high colorectal cancer risk population

Abstract Objective: we recently reported that cholesterol accelerates CRC aggravates via the dissociation of GSK3β/p53 by the degradation of squalene epoxidase (SQLE). However, despite its clinical reports, the underlying mechanisms between age, cholesterol, and CRC aggravation have never been evaluated in human cohorts. Design: tissue lysates and human specimens collected prospectively from CRC patients and controls were grouped according to CRC grade and divided into two groups based on age 50, the average risk for CRC. The intra-tissue content of total cholesterol and cholesteryl ester were measured using CRC lysates. We then used fluorescence-based multiplex immunohistochemistry with digital image analysis and machine learning to measure the quantitative expression of SQLE, GSK3β, p53wt, and p53mt. Results: through an epidemiological study, we estimated age-dependent increases in CRC incidence and the percentage of a group with high cholesterol around age 50. Considering this age-dependency, we demonstrated age and CRC-grade-dependent manner cholesterol accumulated inside CRC tissues, SQLE, p53wt, and p53mt degraded, and GSK3β inactivated. SQLE and GSK3β were clinically associated with increased risk and showed good diagnostic and prognostic performance in CRC patients older than 50. Furthermore, the combined value for four variables exhibited superior diagnostic performance in discriminating from CRC patients after age 50 to controls. Conclusion: this study provides new biological perceptions explaining the effects of age in the progression of CRC aggravation and potential biomarkers associated with a high CRC-risk population. Citation Format: Soo Young Jun, Ji-Yong Yoon, Jeong-Ju Lee, Nam-Soon Kim. The age-dependent intratissue accumulation of cholesterol, resulting in reduced squalene epoxidase, is associated with a high colorectal cancer risk population [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2113.

Physical activity-induced alterations of the gut microbiota are BMI dependent.

Physical inactivity is one of the leading causes of chronic metabolic disease including obesity. Increasing physical activity (PA) has been shown to improve cardiometabolic and musculoskeletal health and to be associated with a distinct gut microbiota composition in trained athletes. However, the impact of PA on the gut microbiota is inconclusive for individuals performing PA in their day-to-day life. This study examined the role of PA and hand-grip strength on gut microbiome composition in middle-aged adults (40-65 years, n=350) with normal (18.5-24.9kg/m2 ) and overweight (25-29.9kg/m2 ) body mass index (BMI). PA was recorded using the International Physical Activity Questionnaire, and hand-grip strength was measured using a dynamometer. Serum samples were assessed for lipidomics while DNA was extracted from fecal samples for microbiome analysis. Overweight participants showed a higher concentration of triacylglycerols, and lower concentrations of cholesteryl esters, sphingomyelin, and lyso-phosphotidylcholine lipids (p < .05) compared with those with normal BMI. Additionally, overweight participants had a lower abundance of the Oscillibacter genus (p < .05). The impact of PA duration on the gut microbiome was BMI dependent. In normal but not overweight participants, high PA duration showed greater relative abundance of commensal taxa such as Actinobacteria and Proteobacteria phyla, as well as Collinsella and Prevotella genera (p < .05). Furthermore, in males with normal BMI, a stronger grip strength was associated with a higher relative abundance of Faecalibacterium and F. prausnitzii (p < .05) compared with lower grip strength. Taken together, data suggest that BMI plays a significant role in modeling PA-induced changes in gut microbiota.

Cholesterol esters form supercooled lipid droplets whose nucleation is facilitated by triacylglycerols

Cellular cholesterol can be metabolized to its fatty acid esters, cholesteryl esters (CEs), to be stored in lipid droplets (LDs). With triacylglycerols (TGs), CEs represent the main neutral lipids in LDs. However, while TG melts at ~4 °C, CE melts at ~44 °C, raising the question of how CE-rich LDs form in cells. Here, we show that CE forms supercooled droplets when the CE concentration in LDs is above 20% to TG and, in particular, liquid-crystalline phases when the fraction of CEs is above 90% at 37 °C. In model bilayers, CEs condense and nucleate droplets when the CE/phospholipid ratio reaches over 10-15%. This concentration is reduced by TG pre-clusters in the membrane that thereby facilitate CE nucleation. Accordingly, blocking TG synthesis in cells is sufficient to strongly dampen CE LD nucleation. Finally, CE LDs emerged at seipins, which cluster and nucleate TG LDs in the ER. However, when TG synthesis is inhibited, similar numbers of LDs are generated in the presence and absence of seipin, suggesting that seipin controls CE LD formation via its TG clustering capacity. Our data point to a unique model whereby TG pre-clusters, favorable at seipins, catalyze the nucleation of CE LDs.

Lower adiposity does not protect beta-2 syntrophin null mice from hepatic steatosis and inflammation in experimental non-alcoholic steatohepatitis

Visceral adiposity is strongly associated with liver steatosis, which predisposes to the development of non-alcoholic steatohepatitis (NASH). Mice with loss of the molecular adapter protein beta-2 syntrophin (SNTB2) have greatly reduced intra-abdominal fat mass. Hepatic expression of proteins with a role in fatty acid metabolism such as fatty acid synthase was nevertheless normal. This was also the case for proteins regulating cholesterol synthesis and uptake. Yet, a slight induction of hepatic cholesterol was noticed in the mutant mice. When mice were fed a methionine choline deficient (MCD) diet to induce NASH, liver cholesteryl ester content was induced in the wild type but not the mutant mice. Serum cholesterol of the mice fed a MCD diet declined and this was significant for the SNTB2 null mice. Though the mutant mice lost less fat mass than the wild type animals, hepatic triglyceride levels were similar between the groups. Proteins involved in fatty acid or cholesterol metabolism such as fatty acid synthase, apolipoprotein E and low-density lipoprotein receptor did not differ between the genotypes. Hepatic oxidative stress and liver inflammation of mutant and wild type mice were comparable. Mutant mice had lower hepatic levels of secondary bile acids and higher cholesterol storage in epididymal fat, and this may partly prevent hepatic cholesterol deposition. In summary, the current study shows that SNTB2 null mice have low intra-abdominal fat mass and do not accumulate hepatic cholesteryl esters when fed a MCD diet. Nevertheless, the SNTB2 null mice develop a similar NASH pathology as wild type mice suggesting a minor role of intra-abdominal fat and liver cholesteryl esters in this model.