The composition-function relationship of HDL particles and its effects on the mechanisms driving coronary heart disease (CHD) is poorly understood. We tested the hypothesis that the functionality of HDL particles is significantly influenced by their lipid composition. Using a novel 3D-separation method, we isolated five different-sized HDL subpopulations from CHD patients who had low preβ-1 functionality (low-F) (ABCA1-dependent cholesterol-efflux normalized for preβ-1 concentration) and controls who had either low-F or high preβ-1 functionality (high-F). Molecular numbers of apoA-I, apoA-II, and eight major lipid classes were determined in each subpopulation by LC-MS. The average number of lipid molecules decreased from 422 in the large spherical α-1 particles to 57 in the small discoid preβ-1 particles. With decreasing particle size, the relative concentration of free cholesterol (FC) decreased in α-mobility but not in preβ-1 particles. Preβ-1 particles contained more lipids than predicted; 30% of which were neutral lipids (cholesteryl ester and triglyceride), indicating that these particles were mainly remodeled from larger particles not newly synthesized. There were significant correlations between HDL-particle functionality and the concentrations of several lipids. Unexpectedly, the phospholipid:FC ratio was significantly correlated with large-HDL-particle functionality but not with preβ-1 functionality. There was significant positive correlation between particle functionality and total lipids in high-F controls, indicating that the lipid-binding capacity of apoA-I plays a major role in the cholesterol efflux capacity of HDL particles. Functionality and lipid composition of HDL particles are significantly correlated and probably both are influenced by the lipid-binding capacity of apoA-I.

Bile acids (BAs) facilitate intestinal absorption of lipid-soluble nutrients and modulate various metabolic pathways through the farnesoid X receptor (FXR) and Takeda G-protein-coupled receptor 5. These receptors are targets for therapy in cholestatic and metabolic diseases. However, dissimilarities in BA metabolism between humans and mice complicate translation of preclinical data. Cytochrome P450 family 2 subfamily c polypeptide 70 (CYP2C70) was recently proposed to catalyze the formation of rodent-specific muricholic acids (MCAs). With CRISPR/Cas9-mediated somatic genome editing, we generated an acute hepatic Cyp2c70 knockout mouse model (Cyp2c70^ako) to clarify the role of CYP2C70 in BA metabolism in vivo and evaluate whether its activity modulates effects of pharmacologic FXR activation on cholesterol homeostasis. In Cyp2c70^ako mice, chenodeoxycholic acid (CDCA) increased at the expense of βMCA, resulting in a more hydrophobic human-like BA pool. Tracer studies demonstrated that, in vivo, CYP2C70 catalyzes the formation of βMCA primarily by sequential 6β-hydroxylation and C7-epimerization of CDCA, generating αMCA as an intermediate metabolite. Physiologically, the humanized BA composition in Cyp2c70^ako mice blunted the stimulation of fecal cholesterol disposal in response to FXR activation compared with WT mice, predominantly due to reduced stimulation of transintestinal cholesterol excretion. Thus, deletion of hepatic Cyp2c70 in adult mice translates into a human-like BA pool composition and impacts the response to pharmacologic FXR activation. This Cyp2c70^ako mouse model may be a useful tool for future studies of BA signaling and metabolism that informs human disease development and treatment.

Diets high in calories can be used to model metabolic diseases, including obesity and its associated comorbidities, in animals. Drosophila melanogaster fed high-sugar diets (HSDs) exhibit complications of human obesity including hyperglycemia, hyperlipidemia, insulin resistance, cardiomyopathy, increased susceptibility to infection, and reduced longevity. We hypothesize that lipid storage in the high-sugar-fed fly’s fat body (FB) reaches a maximum capacity, resulting in the accumulation of toxic lipids in other tissues or lipotoxicity. We took two approaches to characterize tissue-specific lipotoxicity. Ultra-HPLC-MS/MS and MALDI-MS imaging enabled spatial and temporal localization of lipid species in the FB, heart, and hemolymph. Substituent chain length was diet dependent, with fewer odd chain esterified FAs on HSDs in all sample types. By contrast, dietary effects on double bond content differed among organs, consistent with a model where some substituent pools are shared and others are spatially restricted. Both di- and triglycerides increased on HSDs in all sample types, similar to observations in obese humans. Interestingly, there were dramatic effects of sugar feeding on lipid ethers, which have not been previously associated with lipotoxicity. Taken together, we have identified candidate endocrine mechanisms and molecular targets that may be involved in metabolic disease and lipotoxicity.

Myelin is a unique lipid-rich membrane structure that accelerates neurotransmission and supports neuronal function. Sphingolipids are critical myelin components. Yet sphingolipid content and synthesis have not been well characterized in oligodendrocytes, the myelin-producing cells of the CNS. Here, using quantitative real-time PCR, LC-MS/MS-based lipid analysis, and biochemical assays, we examined sphingolipid synthesis during the peak period of myelination in the postnatal rat brain. Importantly, we characterized sphingolipid production in isolated oligodendrocytes. We analyzed sphingolipid distribution and levels of critical enzymes and regulators in the sphingolipid biosynthetic pathway, with focus on the serine palmitoyltransferase (SPT) complex, the rate-limiting step in this pathway. During myelination, levels of the major SPT subunits increased and oligodendrocyte maturation was accompanied by extensive alterations in the composition of the SPT complex. These included changes in the relative levels of two alternative catalytic subunits, SPTLC2 and -3, in the relative levels of isoforms of the small subunits, ssSPTa and -b, and in the isoform distribution of the SPT regulators, the ORMDLs. Myelination progression was accompanied by distinct changes in both the nature of the sphingoid backbone and the N-acyl chains incorporated into sphingolipids. We conclude that the distribution of these changes among sphingolipid family members is indicative of a selective channeling of the ceramide backbone toward specific downstream metabolic pathways during myelination. Our findings provide insights into myelin production in oligodendrocytes and suggest how dysregulation of the biosynthesis of this highly specialized membrane could contribute to demyelinating diseases.

Loss of pancreatic β-cell mass and function as a result of sustained ER stress is a core step in the pathogenesis of diabetes mellitus type 2. The complex control of β-cells and insulin production involves hedgehog (Hh) signaling pathways as well as cholesterol-mediated effects. In fact, data from studies in humans and animal models suggest that HDL protects against the development of diabetes through inhibition of ER stress and β-cell apoptosis. We investigated the mechanism by which HDL inhibits ER stress and apoptosis induced by thapsigargin, a sarco/ER Ca²⁺-ATPase inhibitor, in β-cells of a rat insulinoma cell line, INS1e. We further explored effects on the Hh signaling receptor Smoothened (SMO) with pharmacologic agonists and inhibitors. Interference with sterol synthesis or efflux enhanced β-cell apoptosis and abrogated the anti-apoptotic activity of HDL. During ER stress, HDL facilitated the efflux of specific oxysterols, including 24-hydroxycholesterol (OHC). Supplementation of reconstituted HDL with 24-OHC enhanced and, in cells lacking ABCG1 or the 24-OHC synthesizing enzyme CYP46A1, restored the protective activity of HDL. Inhibition of SMO countered the beneficial effects of HDL and also LDL, and SMO agonists decreased β-cell apoptosis in the absence of ABCG1 or CYP46A1. The translocation of the SMO-activated transcription factor glioma-associated oncogene GLI-1 was inhibited by ER stress but restored by both HDL and 24-OHC. In conclusion, the protective effect of HDL to counter ER stress and β-cell death involves the transport, generation, and mobilization of oxysterols for activation of the Hh signaling receptor SMO

Compared with humans, rodents have higher synthesis of cholesterol and bile acids (BAs) and faster clearance and lower levels of serum LDL-cholesterol. Paradoxically, they increase BA synthesis in response to bile duct ligation (BDL). Another difference is the production of hydrophilic 6-hydroxylated muricholic acids (MCAs), which may antagonize the activation of FXRs, in rodents versus humans. We hypothesized that the presence of MCAs is key for many of these metabolic differences between mice and humans. We thus studied the effects of genetic deletion of the Cyp2c70 gene, previously proposed to control MCA formation. Compared with WT animals, KO mice created using the CRISPR/Cas9 system completely lacked MCAs, and displayed >50% reductions in BA and cholesterol synthesis and hepatic LDL receptors, leading to a marked increase in serum LDL-cholesterol. The doubling of BA synthesis following BDL in WT animals was abolished in KO mice, despite extinguished intestinal fibroblast growth factor (Fgf)15 expression in both groups. Accumulation of cholesterol-enriched particles ("Lp-X") in serum was almost eliminated in KO mice. Livers of KO mice were increased 18% in weight, and serum markers of liver function indicated liver damage. The human-like phenotype of BA metabolism in KO mice could not be fully explained by the activation of FXR-mediated changes. In conclusion, the presence of MCAs is critical for many of the known metabolic differences between mice and humans. The Cyp2c70-KO mouse should be useful in studies exploring potential therapeutic targets for human disease.

Membrane-bound proteins have been proposed to mediate the transport of long-chain FA (LCFA) transport through the plasma membrane (PM). These proposals are based largely on reports that PM transport of LCFAs can be blocked by a number of enzymes and purported inhibitors of LCFA transport. Here, using the ratiometric pH indicator (2',7'-bis-(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein and acrylodated intestinal FA-binding protein-based dual fluorescence assays, we investigated the effects of nine inhibitors of the putative FA transporter protein CD36 on the binding and transmembrane movement of LCFAs. We particularly focused on sulfosuccinimidyl oleate (SSO), reported to be a competitive inhibitor of CD36-mediated LCFA transport. Using these assays in adipocytes and inhibitor-treated protein-free lipid vesicles, we demonstrate that rapid LCFA transport across model and biological membranes remains unchanged in the presence of these purported inhibitors. We have previously shown in live cells that CD36 does not accelerate the transport of unesterified LCFAs across the PM. Our present experiments indicated disruption of LCFA metabolism inside the cell within minutes upon treatment with many of the "inhibitors" previously assumed to inhibit LCFA transport across the PM. Furthermore, using confocal microscopy and a specific anti-SSO antibody, we found that numerous intracellular and PM-bound proteins are SSO-modified in addition to CD36. Our results support the hypothesis that LCFAs diffuse rapidly across biological membranes and do not require an active protein transporter for their transmembrane movement.

Geranylgeranoic acid (GGA) originally was identified in some animals and has been developed as an agent for preventing second primary hepatoma. We previously have also identified GGA as an acyclic diterpenoid in some medicinal herbs. Recently, we reported that in human hepatoma-derived HuH-7 cells, GGA is metabolically labeled from ¹³C-mevalonate. Several cell-free experiments have demonstrated that GGA is synthesized through geranylgeranial by oxygen-dependent oxidation of geranylgeraniol (GGOH), but the exact biochemical events giving rise to GGA in hepatoma cells remain unclear. Monoamine oxidase B (MOAB) has been suggested to be involved in GGOH oxidation. Here, using two human hepatoma cell lines, we investigated whether MAOB contributes to GGA biosynthesis. Using either HuH-7 cell lysates or recombinant human MAOB, we found that: 1) the MAO inhibitor tranylcypromine dose-dependently downregulates endogenous GGA levels in HuH-7 cells; and 2) siRNA-mediated MAOB silencing reduces intracellular GGA levels in HuH-7 and Hep3B cells. Unexpectedly, however, CRISPR/Cas9-generated MAOB-KO human hepatoma Hep3B cells had GGA levels similar to those in MAOB-WT cells. A sensitivity of GGA levels to siRNA-mediated MAOB downregulation was recovered when the MAOB-KO cells were transfected with a MAOB-expression plasmid, suggesting that MAOB is the enzyme primarily responsible for GGOH oxidation and that some other latent metabolic pathways may maintain endogenous GGA levels in the MAOB-KO hepatoma cells. Along with the previous findings, these results provide critical insights into the biological roles of human MAOB and provide evidence that hepatic MAOB is involved in endogenous GGA biosynthesis via GGOH oxidation.

Many clinical studies and epidemiological investigations have clearly demonstrated that women are twice as likely to develop cholesterol gallstones as men, and oral contraceptives and other estrogen therapies dramatically increase that risk. Further, animal studies have revealed that estrogen promotes cholesterol gallstone formation through the estrogen receptor (ER) α, but not ERβ, pathway. More importantly, some genetic and pathophysiological studies have found that G protein-coupled estrogen receptor (GPER) 1 is a new gallstone gene, Lith18, on chromosome 5 in mice and produces additional lithogenic actions, working independently of ERα, to markedly increase cholelithogenesis in female mice. Based on computational modeling of GPER, a novel series of GPER-selective antagonists were designed, synthesized, and subsequently assessed for their therapeutic effects via calcium mobilization, cAMP, and ERα and ERβ fluorescence polarization binding assays. From this series of compounds, one new compound, 2-cyclohexyl-4-isopropyl-N-(4-methoxybenzyl)aniline (CIMBA), exhibits superior antagonism and selectivity exclusively for GPER. Furthermore, CIMBA reduces the formation of 17β-estradiol-induced gallstones in a dose-dependent manner in ovariectomized mice fed a lithogenic diet for 8 weeks. At 32 μg/day/kg CIMBA, no gallstones are found, even in ovariectomized ERα (^–/–) mice treated with 6 μg/day 17β-estradiol and fed the lithogenic diet for 8 weeks. In conclusion, CIMBA treatment protects against the formation of estrogen-induced cholesterol gallstones by inhibiting the GPER signaling pathway in female mice. CIMBA may thus be a new agent for effectively treating cholesterol gallstone disease in women.­

The formation and properties of liquid-ordered (Lo) lipid domains (rafts) in the plasma membrane are still poorly understood. This limits our ability to manipulate ordered lipid domain-dependent biological functions. Giant plasma membrane vesicles (GPMVs) undergo large-scale phase separations into coexisting Lo and liquid-disordered lipid domains. However, large-scale phase separation in GPMVs detected by light microscopy is observed only at low temperatures. Comparing Förster resonance energy transfer-detected versus light microscopy-detected domain formation, we found that nanodomains, domains of nanometer size, persist at temperatures up to 20°C higher than large-scale phases, up to physiologic temperature. The persistence of nanodomains at higher temperatures is consistent with previously reported theoretical calculations. To investigate the sensitivity of nanodomains to lipid composition, GPMVs were prepared from mammalian cells in which sterol, phospholipid, or sphingolipid composition in the plasma membrane outer leaflet had been altered by cyclodextrin-catalyzed lipid exchange. Lipid substitutions that stabilize or destabilize ordered domain formation in artificial lipid vesicles had a similar effect on the thermal stability of nanodomains and large-scale phase separation in GPMVs, with nanodomains persisting at higher temperatures than large-scale phases for a wide range of lipid compositions. This indicates that it is likely that plasma membrane nanodomains can form under physiologic conditions more readily than large-scale phase separation. We also conclude that membrane lipid substitutions carried out in intact cells are able to modulate the propensity of plasma membranes to form ordered domains. This implies lipid substitutions can be used to alter biological processes dependent upon ordered domains.

The autosomal dominant disorder Schnyder corneal dystrophy (SCD) is caused by mutations in UbiA prenyltransferase domain-containing protein-1 (UBIAD1), which uses geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K₂ subtype menaquinone-4 (MK-4). SCD is characterized by opacification of the cornea, owing to aberrant build-up of cholesterol in the tissue. We previously discovered that sterols stimulate association of UBIAD1 with ER-localized HMG-CoA reductase, which catalyzes a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids, including GGpp. Binding to UBIAD1 inhibits sterol-accelerated ER-associated degradation (ERAD) of reductase and permits continued synthesis of GGpp in cholesterol-replete cells. GGpp disrupts UBIAD1-reductase binding and thereby allows for maximal ERAD of reductase as well as ER-to-Golgi translocation of UBIAD1. SCD-associated UBIAD1 is refractory to GGpp-mediated dissociation from reductase and remains sequestered in the ER to inhibit ERAD. Here, we report development of a biochemical assay for UBIAD1-mediated synthesis of MK-4 in isolated membranes and intact cells. Using this assay, we compared enzymatic activity of WT UBIAD1 with that of SCD-associated variants. Our studies revealed that SCD-associated UBIAD1 exhibited reduced MK-4 synthetic activity, which may result from its reduced affinity for GGpp. Sequestration in the ER protects SCD-associated UBIAD1 from autophagy and allows intracellular accumulation of the mutant protein, which amplifies the inhibitory effect on reductase ERAD. These findings have important implications not only for the understanding of SCD etiology but also for the efficacy of cholesterol-lowering statin therapy, which becomes limited, in part, because of UBIAD1-mediated inhibition of reductase ERAD.

Adipocytes take up long chain FAs through diffusion and protein-mediated transport, whereas FA efflux is considered to occur by diffusion. To identify potential membrane proteins that are involved in regulating FA flux in adipocytes, the expression levels of 55 membrane transporters without known function were screened in subcutaneous adipose samples from obese patients before and after bariatric surgery using branched DNA methodology. Among the 33 solute carrier (SLC) transporter family members screened, the expression of 14 members showed significant changes before and after bariatric surgery. One of them, Slc43a3, increased about 2.5-fold after bariatric surgery. Further investigation demonstrated that Slc43a3 is highly expressed in murine adipose tissue and induced during adipocyte differentiation in primary preadipocytes and in OP9 cells. Knockdown of Slc43a3 with siRNA in differentiated OP9 adipocytes reduced both basal and forskolin-stimulated FA efflux, while also increasing FA uptake and lipid droplet accumulation. In contrast, overexpression of Slc43a3 decreased FA uptake in differentiated OP9 cells and resulted in decreased lipid droplet accumulation. Therefore, Slc43a3 seems to regulate FA flux in adipocytes, functioning as a positive regulator of FA efflux and as a negative regulator of FA uptake.

Acne is one of the most common dermatological conditions, but the details of its pathology are unclear, and current management regimens often have adverse effects. Cutibacterium acnes is known as a major acne-associated bacterium that derives energy from lipase-mediated sebum lipid degradation. C. acnes is commensal, but lipase activity has been observed to differ among C. acnes types. For example, higher populations of the type IA strains are present in acne lesions with higher lipase activity. In the present study, we examined a conserved lipase in types IB and II that was truncated in type IA C. acnes strains. Closed, blocked, and open structures of C. acnes ATCC11828 lipases were elucidated by X-ray crystallography at 1.6–2.4 Å. The closed crystal structure, which is the most common form in aqueous solution, revealed that a hydrophobic lid domain shields the active site. By comparing closed, blocked, and open structures, we found that the lid domain-opening mechanisms of C. acnes lipases (_CAlipases) involve the lid-opening residues, Phe-179 and Phe-211. To the best of our knowledge, this is the first structure-function study of _CAlipases, which may help to shed light on the mechanisms involved in acne development and may aid in future drug design.

Fatty liver involves ectopic lipid accumulation and dysregulated hepatic oxidative metabolism, which can progress to a state of elevated inflammation and fibrosis referred to as nonalcoholic steatohepatitis (NASH). The factors that control progression from simple steatosis to NASH are not fully known. Here, we tested the hypothesis that dietary vitamin E (VitE) supplementation would prevent NASH progression and associated metabolic alterations induced by a Western diet (WD). Hyperphagic melanocortin-4 receptor-deficient (MC4R^–/–) mice were fed chow, chow+VitE, WD, or WD+VitE starting at 8 or 20 weeks of age. All groups exhibited extensive hepatic steatosis by the end of the study (28 weeks of age). WD feeding exacerbated liver disease severity without inducing proportional changes in liver triglycerides. Eight weeks of WD accelerated liver pyruvate cycling, and 20 weeks of WD extensively upregulated liver glucose and oxidative metabolism assessed by ²H/¹³C flux analysis. VitE supplementation failed to reduce the histological features of NASH. Rather, WD+VitE increased the abundance and saturation of liver ceramides and accelerated metabolic flux dysregulation compared with 8 weeks of WD alone. In summary, VitE did not limit NASH pathogenesis in genetically obese mice, but instead increased some indicators of metabolic dysfunction.

The pregnane X receptor (PXR) is a nuclear receptor that can be activated by numerous drugs and xenobiotic chemicals. PXR thereby functions as a xenobiotic sensor to coordinately regulate host responses to xenobiotics by transcriptionally regulating many genes involved in xenobiotic metabolism. We have previously reported that PXR has pro-atherogenic effects in animal models, but how PXR contributes to atherosclerosis development in different tissues or cell types remains elusive. In this study, we generated an LDL receptor-deficient mouse model with myeloid-specific PXR deficiency (PXR^MyeLDLR^–/–) to elucidate the role of macrophage PXR signaling in atherogenesis. The myeloid PXR deficiency did not affect metabolic phenotypes and plasma lipid profiles, but PXR^MyeLDLR^–/– mice had significantly decreased atherosclerosis at both aortic root and brachiocephalic arteries compared with control littermates. Interestingly, the PXR deletion did not affect macrophage adhesion and migration properties, but reduced lipid accumulation and foam cell formation in the macrophages. PXR deficiency also led to decreased expression of the scavenger receptor CD36 and impaired lipid uptake in macrophages of the PXR^MyeLDLR^–/– mice. Further, RNA-Seq analysis indicated that treatment with a prototypical PXR ligand affects the expression of many atherosclerosis-related genes in macrophages in vitro. These findings reveal a pivotal role of myeloid PXR signaling in atherosclerosis development and suggest that PXR may be a potential therapeutic target in atherosclerosis management.

Cellular membranes are not homogenous mixtures of proteins; rather, they are segregated into microdomains on the basis of preferential association between specific lipids and proteins. These microdomains, called lipid rafts, are well known for their role in receptor signaling on the plasma membrane (PM) and are essential to such cellular functions as signal transduction and spatial organization of the PM. A number of disease states, including atherosclerosis and other cardiovascular disorders, may be caused by dysfunctional maintenance of lipid rafts. Lipid rafts do not occur only in the PM but also have been found in intracellular membranes and extracellular vesicles (EVs). Here, we focus on discussing newly discovered functions of lipid rafts and microdomains in intracellular membranes, including lipid and protein trafficking from the ER, Golgi bodies, and endosomes to the PM, and we examine lipid raft involvement in the production and composition of EVs. Because lipid rafts are small and transient, visualization remains challenging. Future work with advanced techniques will continue to expand our knowledge about the roles of lipid rafts in cellular functioning.

When you register an Oracle table to the metadata, it might fail and generate an error similar to the following: "ERROR: An exception has been encountered...ERROR: Read Access Violation METALIB..."

When you run 32-bit SAS on Windows and disconnect from the Oracle database server, you might see the error: "ERROR: ORACLE disconnect error: ORA-03135: connection lost contact."

The problem occurs on a content release on the SAS Risk Governance Framework.

The problem occurs on a content release on the SAS Risk Governance Framework.

When you query an Oracle table and use the LIMIT clause using either SAS  Federation Server or FedSQL, a row limit is not passed to the database. In this scenario, a Full Article