Dan Greenberg, co-founder and CEO of native advertising solutions provider Sharethrough, discusses how buyers’ increased desire for contextual targeting and native advertising will benefit premium publishers in 2018.

William Bao Bean, managing director of Chinaccelerator, explains how investments in artificial intelligence and machine learning are helping marketers improve user targeting and return on investment.

12 March 2015 , Volume 91, Number 2

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.

This manuscript has been withdrawn by the Author.

Gram-negative bacteria possess an asymmetric outer membrane (OM) composed primarily of lipopolysaccharides (LPS) on the outer leaflet and phospholipids (PLs) on the inner leaflet. Loss of this asymmetry due to mutations in the lipopolysaccharide (LPS) biosynthesis or transport pathways causes externalization of PLs to the outer leaflet of the OM and leads to OM permeability defects. Here, we employed metabolic labeling to detect a compromised OM in intact bacteria. Phosphatidylcholine synthase (Pcs) expression in Escherichia coli allowed for incorporation of exogenous propargylcholine (PCho) into phosphatidyl(propargyl)choline (PPC) and for incorporation of exogenous 1-azidoethyl-choline (AECho) into phosphatidyl(azidoethyl)choline (AEPC) as confirmed by LC-MS analyses. A fluorescent copper-free click reagent poorly labeled AEPC in intact wild-type cells, but readily labeled AEPC from lysed cells. Fluorescence microscopy and flow cytometry analyses confirmed the absence of significant AEPC labeling from intact wild-type E. coli strains, and revealed significant AEPC labeling in an E. coli LPS transport mutant (lptD4213) and an LPS biosynthesis mutant (E. coli lpxC101). Our results suggest that metabolic PL labeling with AECho is a promising tool to detect a compromised bacterial OM, reveal aberrant PL externalization, and identify or characterize novel cell-active inhibitors of LPS biosynthesis or transport.

Inhibition of acid sphingomyelinase (ASM), a lysosomal enzyme that catalyzes the hydrolysis of sphingomyelin into ceramide and phosphorylcholine, may serve as an investigational tool or a therapeutic intervention to control many diseases. Specific ASM inhibitors are currently not sufficiently characterized. Here, we found that 1-aminodecylidene bis-phosphonic acid (ARC39) specifically and efficiently (>90%) inhibits both lysosomal and secretory ASM in vitro. Results from investigating sphingomyelin phosphodiesterase 1 (SMPD1/Smpd1) mRNA and ASM protein levels suggested that ARC39 directly inhibits ASM’s catalytic activity in cultured cells, a mechanism which differs from that of functional inhibitors of ASM (FIASMAs). We further provide evidence that ARC39 dose- and time-dependently inhibits lysosomal ASM in intact cells, and we show that ARC39 also reduces platelet- and ASMpromoted adhesion of tumor cells. The observed toxicity of ARC39 is low at concentrations relevant for ASM inhibition in vitro, and it does not strongly alter the lysosomal compartment or induce phospholipidosis in vitro. When applied intraperitoneally in vivo, even subtoxic high doses administered short-term induced sphingomyelin accumulation only locally in the peritoneal lavage without significant accumulation in plasma, liver, spleen or brain. These findings require further investigation with other possible chemical modifications. In conclusion, our results indicate that ARC39 potently and selectively inhibits ASM in vitro and highlight the need for developing compounds that can reach tissue concentrations sufficient for ASM inhibition in vivo.

Excessive lipid deposition is a hallmark of nonalcoholic fatty liver disease (NAFLD). Although much has been learned about the enzymes and metabolites involved in NAFLD, few studies have focused on the role of long non-coding RNAs (lncRNAs) in hepatic lipid accumulation. Here, using in vitro and in vivo models of NAFLD, we found that the lncRNA Gm15622 is highly expressed in the liver of obese mice fed a high-fat diet (HFD) and in murine liver (AML-12) cells treated with free fatty acids. Investigating the molecular mechanism in the liver-enriched expression of Gm15622 and its effects on lipid accumulation in hepatocytes and on NAFLD pathogenesis, we found that Gm15622 acts as a sponge for the microRNA miR-742-3p. This sponging activity increased the expression of the transcriptional regulator sterol regulatory element–binding transcription factor 1c (SREBP-1c) and promoted lipid accumulation in the liver of the HFD mice and AML-12 cells. Moreover, further results indicated that metformin suppresses Gm15622 and alleviates NAFLD-associated lipid deposition in mice. In conclusion, we have identified an lncRNA Gm15622–miR-742-3p–SREBP-1c regulatory circuit associated with NAFLD in mice, a finding that significantly advances our insight into how lipid metabolism and accumulation are altered in this metabolic disorder. Our results also suggest that Gm15622 may be a potential therapeutic target for managing NAFLD.

Ether lipids (ELs) are lipids characterized by the presence of either an ether linkage (alkyl lipids) or a vinyl ether linkage (i.e. plasmalogens [Pls]) at the sn1 position of the glycerol backbone and they are enriched in PUFAs at the sn2 position. In this review, we highlight that ELs have various biological functions, act as a reservoir for second messengers (such as PUFAs), and have roles in many diseases. Some of the biological effects of ELs may be associated with their ability to regulate ion channels that control excitation-contraction/secretion/mobility coupling and therefore cell physiology. These channels are embedded in lipid membranes, and lipids can regulate their activities directly or indirectly as second messengers or by incorporating into membranes. Interestingly, ELs and EL-derived PUFAs have been reported to play a key role in several pathologies, including neurological disorders, cardiovascular diseases, and cancers. Investigations leading to a better understanding of their mechanisms of action in pathologies have opened a new field in cancer research. In summary, newly identified lipid regulators of ion channels, such as ELs and PUFAs, may represent valuable targets to improve disease diagnosis and advance the development of new therapeutic strategies for managing a range of diseases and conditions.

Ceramides are the predominant lipids in the stratum corneum (SC) and are crucial components for normal skin barrier function. Although the composition of various ceramide classes in the human SC has been reported, that in mice is still unknown, despite mice being widely used as animal models of skin barrier function. Here, we performed LC–MS/MS analyses using recently available ceramide class standards to measure 25 classes of free ceramides and 5 classes of protein-bound ceramides from the human and mouse SC. Phytosphingosine-type ceramides (P-ceramides) and 6-hydroxy sphingosine-type ceramides (H-ceramides), which both contain an additional hydroxyl group, were abundant in human SC (35% and 45% of total ceramides, respectively). In contrast, in mice, P-ceramides and H-ceramides were present at ~1% and undetectable levels, respectively, and sphingosine-type ceramides accounted for ~90%. In humans, ceramides containing α-hydroxy FA were abundant, whereas ceramides containing β-hydroxy FA (B-ceramides) or -hydroxy FA were abundant in mice. The hydroxylated β-carbon in B-ceramides was in the (R)-configuration. Genetic knockout of β-hydroxy acyl-CoA dehydratases in HAP1 cells increased B-ceramide levels, suggesting that β-hydroxy acyl-CoA, an FA-elongation cycle intermediate in the endoplasmic reticulum, is a substrate for B-ceramide synthesis. We anticipate that our methods and findings will help to elucidate the role of each ceramide class in skin barrier formation and in the pathogenesis of skin disorders.

Individuals with Netherton syndrome (NTS) have increased serine protease activity, which strongly impacts the barrier function of the skin epidermis and leads to skin inflammation. Here, we investigated how serine protease activity in NTS correlates with changes in the stratum corneum ceramides, which are crucial components of the skin barrier. We examined two key enzymes involved in epidermal ceramide biosynthesis, glucocerebrosidase (GBA) and acid-sphingomyelinase (ASM). We compared in situ expression levels and activities of GBA and ASM between NTS patients and controls and correlated the expression and activities with i) stratum corneum ceramide profiles, ii) in situ serine protease activity, and iii) clinical presentation of patients. Using activity-based probe labeling, we visualized and localized active, epidermal GBA, and a newly developed in situ zymography method enabled us to visualize and localize active ASM. Reduction in active GBA in NTS patients coincided with increased ASM activity, particularly in areas with increased serine protease activity. NTS patients with scaly erythroderma exhibited more pronounced anomalies in GBA and ASM activities than patients with ichthyosis linearis circumflexa. They also displayed a stronger increase in stratum corneum ceramides processed via ASM. We conclude that changes in the localization of active GBA and ASM correlate with i) altered stratum corneum ceramide composition in NTS patients, ii) local serine protease activity, and iii) the clinical manifestation of NTS. 

Oxidized low-density lipoprotein (oxLDL) is a known risk factor for atherogenesis. This study aimed to reveal structural features of oxLDL present in human circulation related to atherosclerosis. When LDL was fractionated on an anion-exchange column, in vivo-oxLDL, detected by the anti-oxidized phosphatidylcholine (oxPC) monoclonal antibody, was recovered in flow-through and electronegative LDL (LDL(-)) fractions. The amount of the electronegative in vivo-oxLDL, namely oxLDL in LDL(-) fraction, present in patients with acute myocardial infarction (AMI) was three-fold higher than that observed in healthy subjects. Surprisingly, LDL(-) fraction contained apoA1 in addition to apoB, and HDL-sized particles were observed with transmission electron microscopy. In LDL(-) fractions, acrolein adducts were identified at all lysine residues in apoA1, with only a small number of acrolein-modified residues were identified in apoB. The amount of oxPC adducts of apoB was higher in LDL(-) than in L1 fraction as determined using western blotting. The electronegative in vivo-oxLDL was immunologically purified from the LDL(-) fraction with an anti-oxPC monoclonal antibody. Majority of PC species was not oxidized, whereas oxPC and lysoPC did not accumulate. Here, we propose that there are two types of in vivo-oxLDL in human circulating plasma and the electronegative in vivo-oxLDL accompanies oxidized HDL.

Niemann–Pick type C1 (NPC1) disease is a rare genetic condition in which the function of the lysosomal cholesterol transporter NPC1 protein is impaired. Consequently, sphingolipids and cholesterol accumulate in lysosomes of all tissues, triggering a cascade of pathological events that culminate in severe systemic and neurological symptoms. Lysosomal cholesterol accumulation is also a key-factor in the development of atherosclerosis and non-alcoholic steatohepatitis (NASH). In these two metabolic diseases, the administration of plant stanol esters has been shown to ameliorate cellular cholesterol accumulation and inflammation. Given the overlap of pathological mechanisms among atherosclerosis, NASH and NPC1 disease, we sought to investigate whether dietary supplementation with plant stanol esters improves the peripheral features of NPC1 disease. To this end, we used an NPC1 murine model featuring an Npc1 null allele (Npc1^nih), creating a dysfunctional NPC1 protein. Npc1^nih mice were fed a two or six percent plant stanol esters–enriched diet over the course of 5 weeks. During this period, hepatic and blood lipid and inflammatory profiles were assessed. Npc1^nih mice fed the plant stanol–enriched diet exhibited lower hepatic cholesterol accumulation, damage and inflammation than regular chow–fed Npc1^nih mice. Moreover, plant stanol consumption shifted circulating T-cells and monocytes in particular towards an anti-inflammatory profile. Overall, these effects were stronger following dietary supplementation with 6% stanols, suggesting a dose-dependent effect. The findings of our study highlight the potential use of plant stanols as an affordable complementary means to ameliorate disorders in hepatic and blood lipid metabolism and reduce inflammation in NPC1 disease.

Bis(monoacylglycero)phosphate (BMP), also known as lysobisphosphatidic acid (LBPA), is a phospholipid that promotes lipid sorting in late endosomes/lysosomes by activating lipid hydrolases and lipid transfer proteins. Changes in the cellular BMP content therefore reflect an altered metabolic activity of the endo-lysosomal system. Surprisingly, little is known about the physiological regulation of BMP. In this study, we investigated the effects of nutritional and metabolic factors on BMP profiles of whole tissues and  parenchymal and non-parenchymal cells. Tissue samples were obtained from fed, fasted, two-hours refed, and insulin-treated mice, as well as from mice housed at  5°C, 22°C, or 30°C. These tissues exhibited distinct BMP profiles, which were regulated by the nutritional state in a tissue-specific manner. Insulin treatment was not sufficient to mimic refeeding-induced changes in tissue BMP levels indicating that BMP metabolism is regulated by other hormonal or nutritional factors. Tissue fractionation experiments revealed that fasting drastically elevates BMP levels in hepatocytes and pancreatic cells. Furthermore, we observed that the BMP content in brown adipose tissue strongly depends on housing temperatures. In conclusion, our observations suggest that BMP concentrations adapt to the metabolic state in a tissue-and cell type-specific manner in mice. Drastic changes observed in hepatocytes, pancreatic cells, and brown adipocytes suggest that BMP possesses a role in the functional adaption to nutrient starvation and ambient temperature.

The yeast protein Mpo1 belongs to a protein family that is widely conserved in bacteria, fungi, protozoa, and plants, and is the only protein of this family whose function has so far been elucidated. Mpo1 is an Fe²⁺-dependent dioxygenase that catalyzes the α-oxidation reaction of 2-hydroxy (2-OH) long-chain FAs produced in the degradation pathway of the long-chain base phytosphingosine. However, several biochemical characteristics of Mpo1, such as its catalytic residues, membrane topology, and substrate specificity, remain unclear. Here, we report that yeast Mpo1 contains two transmembrane domains and that both its N- and C-terminal regions are exposed to the cytosol. Mutational analyses revealed that three histidine residues conserved in the Mpo1 family are especially important for Mpo1 activity, suggesting that they may be responsible for the formation of coordinate bonds with Fe²⁺. We found that, in addition to activity toward 2-OH long-chain FAs, Mpo1 also exhibits activity toward 2-OH very-long-chain FAs derived from the FA moiety of sphingolipids. These results indicate that Mpo1 is involved in the metabolism of long-chain to very-long-chain 2-OH FAs produced in different pathways. We noted that the growth of mpo1 cells is delayed upon carbon deprivation, suggesting that the Mpo1-mediated conversion of 2-OH FAs to non-hydroxy FAs is important for utilizing 2-OH FAs as a carbon source under carbon starvation. Our findings help to elucidate the as-yet-unknown functions and activities of other Mpo1 family members.

Multi-component lipid emulsions, rather than soy-oil emulsions, prevent cholestasis by an unknown mechanism. Here, we quantified liver function, bile acid pools, and gut microbial and metabolite profiles in premature, parenterally fed pigs given a soy-oil lipid emulsion, Intralipid (IL); a multi component lipid emulsion, SMOFlipid (SMOF); a novel emulsion with a modified fatty-acid composition (EXP); or a control enteral diet (ENT) for 22 days. We assayed serum cholestasis markers; measured total bile acid levels in plasma, liver, and gut contents; and analyzed colonic bacterial 16S rRNA gene sequences and metabolomic profiles. Serum cholestasis markers (i.e. bilirubin, bile acids, and g-glutamyl transferase) were highest in IL-fed pigs and normalized in those given SMOF, EXP, or ENT. Gut bile acid pools were lowest in the IL treatment and were increased in the SMOF and EXP treatments and comparable to ENT. Multiple bile acids, especially their conjugated forms, were higher in the colon contents of SMOF and EXP than in IL pigs. Colonic microbial communities of SMOF and EXP pigs had lower relative abundance of several Gram-positive anaerobes, including Clostridrium XIVa, and higher abundance of Enterobacteriaceae than those of IL and ENT pigs. Differences in lipid and microbial-derived compounds were also observed in colon metabolite profiles. These results indicate that multi-component lipid emulsions prevent cholestasis and restore enterohepatic bile flow in association with gut microbial and metabolomic changes. We conclude that sustained bile flow induced by multi-component lipid emulsions likely exerts a dominant effect in reducing bile acid–sensitive, Gram-positive bacteria.

The levels and composition of sphingolipids and related metabolites are altered in aging and common disorders such as diabetes and cancers, as well as in neurodegenerative, cardiovascular, and respiratory diseases. Changes in sphingolipids have been implicated as being an essential step in mitochondria-driven cell death. However, little is known about the precise sphingolipid composition and modulation in mitochondria or related organelles. Here, we used LC–MS/MS to analyze the presence of key components of the ceramide metabolic pathway in vivo and in vitro in purified endoplasmic reticulum (ER), mitochondria-associated membranes (MAM), and mitochondria. Specifically, we analyzed the sphingolipids in the three pathways that generate ceramide: sphinganine in the de novo ceramide pathway, sphingomyelin in the breakdown pathway, and sphingosine in the salvage pathway. We observed sphingolipid profiles in mouse liver, mouse brain, and a human glioma cell line (U251). We analyzed the quantitative and qualitative changes of these sphingolipids during staurosporine (STS)-induced apoptosis in U251 cells. Ceramide, especially C16-ceramide, levels increased during early apoptosis possibly through a conversion from mitochondrial sphinganine and sphingomyelin, but sphingosine and lactosyl- and glucosyl-ceramide levels were unaffected. We also found that ceramide generation is enhanced in mitochondria when sphingomyelin levels are decreased in the MAM. This decrease was associated with an increase in acid sphingomyelinase (ASM) activity in MAM. We conclude that meaningful sphingolipid modifications occur in MAM, the mitochondria, and ER during the early phases of apoptosis.