In rodents, sphingomyelins (SMs) species with very-long-chain polyunsaturated fatty acid (VLCPUFA) are required for normal spermatogenesis. Data on the expression of enzymes with roles in their ...biosynthesis and turnover during germ cell differentiation and on possible effects on such expression of testosterone (Tes), known to promote this biological process, were lacking. Here we quantified, in isolated pachytene spermatocytes (PtS), round spermatids (RS), and later spermatids (LS), the mRNA levels from genes encoding ceramide (Cer), glucosylceramide (GlcCer), and SM synthases (Cers3, Gcs, Sms1, and Sms2) and sphingomyelinases (aSmase, nSmase) and assessed products of their activity in cells in culture using nitrobenzoxadiazole (NBD)-labeled substrates and 3Hpalmitate as precursor. Transcript levels from Cers3 and Gcs were maximal in PtS. While mRNA levels from Sms1 increased with differentiation in the direction PtS→RS→LS, those from Sms2 increased between PtS and RS but decreased in LS. In turn, the nSmase transcript increased in the PtS→RS→LS order. During incubations with NBD-Cer, spermatocytes produced more GlcCer and SM than did spermatids. In total germ cells cultured for up to 25 h with NBD-SM, not only abundant NBD-Cer but also NBD-GlcCer were formed, demonstrating SM→Cer turnover and Cer recycling. After 20 h with 3Hpalmitate, PtS produced 3HSM and RS formed 3HSM and 3HCer, all containing VLCPUFA, and Tes increased their labeling. In total germ cells, Tes augmented in 5 h the expression of genes with roles in VLCPUFA synthesis, decreased the mRNA from Sms2, and increased that from nSmase. Thus, Tes enhanced or accelerated the metabolic changes occurring to VLCPUFA-SM during germ cell differentiation.
Ceramide phosphoethanolamine (CPE) is the major sphingolipid in invertebrates and in some bacterial species. It has been also detected in mammalian cells, although only in trace amounts. Complete ...understanding of the biophysical and physiological relevance of CPE is still lacking, and its biological role is still an open question. CPE differs in its biosynthetic mechanisms from sphingomyelin, due to the specific CPE synthase in invertebrates. In contrast to well-established sphingomyelin/cholesterol interactions that result in the formation of ordered membrane domains, the formation of ordered CPE/cholesterol domains is not favored. CPE might be crucial for the early development of Drosophila melanogaster, and it might be involved in the developmental stages of Trypanosoma brucei. As a Bacteroidetes-associated sphingolipid, CPE might also be involved in maintenance of these bacteria in their ecological niches. Therefore, efficient detection of CPE in biological systems is needed to better define its distribution and biological role(s).
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•Ceramide phosphoethanolamine (CPE) is the major sphingolipid in invertebrates.•CPE and dihydroCPE are one of the most dominant sphingolipids in Bacteroidetes.•CPE differs in its biosynthetic mechanisms from sphingomyelin.•The formation of ordered CPE/cholesterol domains is not favored.•CPE might be crucial for the development of some insects and protozoa.
All-atom simulation data are presented for ternary mixtures of palmitoyl sphingomyelin (PSM), cholesterol, and either palmitoyl oleoyl phosphatidyl choline or dioleoyl phosphatidyl choline (DOPC). ...For comparison, data for a mixture of dipalmitoyl phosphatidyl choline (DPPC), cholesterol, and DOPC are also presented. Compositions corresponding to the liquid-ordered phase, the liquid-disordered phase, and coexistence of the two phases are simulated for each mixture. Within the liquid-ordered phase, cholesterol is preferentially solvated by DOPC if it is available, but if DOPC is replaced by POPC, cholesterol is preferentially solvated by PSM. In the DPPC mixtures, cholesterol interacts preferentially with the saturated chains via its smooth face, whereas in the PSM mixtures, cholesterol interacts preferentially with PSM via its rough face. Interactions between cholesterol and PSM have a very particular character: hydrogen bonding between cholesterol and the amide of PSM rotates the tilt of the amide plane, which primes it for more robust hydrogen bonding with other PSM. Cholesterol-PSM hydrogen bonding also locally modifies the hexagonal packing of hydrocarbon chains in the liquid-ordered phase of PSM mixtures.
High dietary fat intake can cause elevated serum and hepatic lipids, as well as contribute to gut dysbiosis, intestinal barrier dysfunction and increased circulating lipopolysaccharide (LPS). Dietary ...milk sphingomyelin (SM) has been shown to inhibit lipid absorption in rodents. We evaluated the effects of milk SM on lipid metabolism and LPS levels in C57BL/6J mice fed a high-fat diet for 4weeks and compared it with egg SM. Mice were fed a high-fat diet (45%kcal from fat) (CTL, n=10) or the same diet modified to contain 0.25% (wt/wt) milk SM (MSM, n=10) or 0.25% (wt/wt) egg SM (ESM, n=10). After 4weeks, MSM had gained significantly less weight and had reduced serum cholesterol compared to CTL. ESM had increases in serum cholesterol, triglycerides, phospholipids and SM compared to CTL. MSM significantly decreased, while ESM increased, hepatic triglycerides. This may have been related to induction of hepatic stearoyl-CoA desaturase-1 mRNA observed in ESM. MSM displayed intestinal and hepatic gene expression changes consistent with cholesterol depletion. MSM had significantly lower serum LPS compared to CTL, which may have been due to altered distal gut microbiota. Fecal Gram-negative bacteria were significantly lower, while fecal Bifidobacterium were higher, in MSM. These results suggest that milk SM is more effective than egg SM at combating the detrimental effects of a high-fat diet in mice. Additionally, distal gut microbiota is altered with milk SM and this may have contributed to the lower serum LPS observed.
Milk sphingomyelin (SM), a polar lipid (PL) component of milk fat globule membranes, is protective against dyslipidemia. However, it is unclear whether ingestion of milk PLs protect against ...atherosclerosis. To determine this, male LDLr−/− mice (age 6 weeks) were fed ad libitum either a high-fat, added-cholesterol diet (CTL; 45% kcal from fat, 0.2% cholesterol by weight; n=15) or the same diet supplemented with 1% milk PL (1% MPL; n=15) or 2% milk PL (2% MPL; n=15) added by weight from butter serum. After 14 weeks on diets, mice fed 2% MPL had significantly lower serum cholesterol (−51%) compared to CTL (P<.01), with dose-dependent effects in lowering VLDL- and LDL-cholesterol. Mice fed 2% MPL displayed lower inflammatory markers in the serum, liver, adipose and aorta. Notably, milk PLs reduced atherosclerosis development in both the thoracic aorta and the aortic root, with 2% MPL-fed mice having significantly lower neutral lipid plaque size by 59% (P<.01) and 71% (P<.02) compared to CTL, respectively. Additionally, the 2% MPL-fed mice had greater relative abundance of Bacteroidetes, Actinobacteria and Bifidobacterium, and lower Firmicutes in cecal feces compared to CTL. Milk PL feeding resulted in significantly different microbial communities as demonstrated by altered beta diversity indices. In summary, 2% MPL strongly reduced atherogenic lipoprotein cholesterol, modulated gut microbiota, lowered inflammation and attenuated atherosclerosis development. Thus, milk PL content may be important to consider when choosing dairy products as foods for cardiovascular disease prevention.
Cell membrane stiffness is critical for cellular function, with cholesterol and sphingomyelin as pivot contributors. Current methods for measuring membrane stiffness are often invasive, ex situ, and ...slow in process, prompting the need for innovative techniques. Here, we present a fluorescence resonance energy transfer (FRET)-based protein sensor designed to address these challenges. The sensor consists of two fluorescent units targeting sphingomyelin and cholesterol, connected by a linker that responds to the proximity of these lipids. In rigid membranes, cholesterol and sphingomyelin are in close proximity, leading to an increased FRET signal. We utilized this sensor in combination with confocal microscopy to explore changes in plasma membrane stiffness under various conditions, including differences in osmotic pressure, the presence of reactive oxygen species (ROS) and variations in substrate stiffness. Furthermore, we explored the impact of SARS-CoV-2 on membrane stiffness and the distribution of ACE2 after attachment to the cell membrane. This tool offers substantial potential for future investigations in the field of mechanobiology.
Maintenance of lipid asymmetry across the two leaflets of the plasma membrane (PM) bilayer is a ubiquitous feature of eukaryotic cells. Loss of this asymmetry has been widely associated with cell ...death. However, increasing evidence points to the physiological importance of non-apoptotic, transient changes in PM asymmetry. Such transient scrambling events are associated with a range of biological functions, including intercellular communication and intracellular signaling. Thus, regulation of interleaflet lipid distribution in the PM is a broadly important but underappreciated cellular process with key physiological and structural consequences. Here, we compile the mounting evidence revealing multifaceted, functional roles of PM asymmetry and transient loss thereof. We discuss the consequences of reversible asymmetry on PM structure, biophysical properties and interleaflet coupling. We argue that despite widespread recognition of broad aspects of membrane asymmetry, its importance in cell biology demands more in-depth investigation of its features, regulation, and physiological and pathological implications.
Pathogenic bacteria enter the cytosol of host cells through uptake into bacteria-containing vacuoles (BCVs) and subsequent rupture of the vacuolar membrane 1. Bacterial invaders are sensed either ...directly, through cytosolic pattern-recognition receptors specific for bacterial ligands, or indirectly, through danger receptors that bind host molecules displayed in an abnormal context, for example, glycans on damaged BCVs 2–4. In contrast to damage caused by Listeria monocytogenes, a Gram-positive bacterium, BCV rupture by Gram-negative pathogens such as Shigella flexneri or Salmonella Typhimurium remains incompletely understood 5, 6. The latter may cause membrane damage directly, when inserting their Type Three Secretion needles into host membranes, or indirectly through translocated bacterial effector proteins 7–9. Here, we report that sphingomyelin, an abundant lipid of the luminal leaflet of BCV membranes, and normally absent from the cytosol, becomes exposed to the cytosol as an early predictive marker of BCV rupture by Gram-negative bacteria. To monitor subcellular sphingomyelin distribution, we generated a live sphingomyelin reporter from Lysenin, a sphingomyelin-specific toxin from the earthworm Eisenia fetida 10, 11. Using super resolution live imaging and correlative light and electron microscopy (CLEM), we discovered that BCV rupture proceeds through two distinct successive stages: first, sphingomyelin is gradually translocated into the cytosolic leaflet of the BCV, invariably followed by cytosolic exposure of glycans, which recruit galectin-8, indicating bacterial entry into the cytosol. Exposure of sphingomyelin on BCVs may therefore act as an early danger signal alerting the cell to imminent bacterial invasion.
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•Lysenin serves as a reporter of sphingomyelin exposure in the mammalian cytosol•Chemical-, toxin-, or pathogen-induced membrane damage exposes sphingomyelin•Sphingomyelin exposure precedes catastrophic breakage of bacteria-containing vacuoles•Cytosolic sphingomyelin is indicative of membrane stress and imminent pathogen entry
Ellison et al. show that the pore-forming toxin Lysenin can be used as a reporter for detecting sphingomyelin in the cytosolic leaflet of cellular membranes. The breakout of Gram-negative bacteria from vacuoles into the host cytosol follows a precisely choreographed process in which sphingomyelin exposure precedes catastrophic membrane damage.
Although dietary DHA alleviates Toll-like receptor (TLR)–associated chronic inflammation in fish, the underlying mechanism is not well understood.
This study aimed to explore the role of Tlr22 in the ...innate immunity of large yellow croaker and investigate the anti-inflammatory effects of DHA on Tlr22-triggered inflammation.
Head kidney–derived macrophages of croaker and HEK293T cells were or were not pretreated with 100 μM DHA for 10 h prior to polyinosinic-polycytidylic acid (poly I:C) stimulation. We executed qRT-PCR, immunoblotting, and lipidomic analysis to examine the impact of DHA on Tlr22-triggered inflammation and membrane lipid composition. In vivo, croakers (12.03 ± 0.05 g) were fed diets containing 0.2% control (Ctrl), 0.8%, and 1.6% DHA for 8 wk before injection with poly I:C. Inflammatory genes expression and rafts-related lipids and protein expression were measured in the head kidney. Data were analyzed by ANOVA or Student t test.
The activation of Tlr22 by poly I:C induced inflammation, and DHA diminished Tlr22-targeted inflammatory gene expression by 56–73% (P ≤ 0.05). DHA reduced membrane sphingomyelin (SM) and SFA-containing phosphatidylcholine (SFA-PC) contents, as well as lipid raft marker caveolin 1 amounts. Furthermore, lipid raft disruption suppressed Tlr22-induced Nf-κb and interferon h activation and p65 nuclear translocation. In vivo, expression of Tlr22 target inflammatory genes was 32–64% lower in the 1.6% DHA group than in the Ctrl group upon poly I:C injection (P ≤ 0.05). Also, the 1.6% DHA group showed a reduction in membrane SM and SFA-PC contents, accompanied by a decrease in caveolin 1 amounts, compared with the Ctrl group.
The activation of Tlr22 signaling depends on lipid rafts, and DHA ameliorates the Tlr22-triggered inflammation in both head kidney and head kidney–derived macrophages of croaker partially by altering membrane SMs and SFA-PCs that are required for lipid raft organization.
Colorectal cancer is the second most common cause of cancer‐related death globally, with marked differences in prognosis by disease stage at diagnosis. We studied circulating metabolites in relation ...to disease stage to improve the understanding of metabolic pathways related to colorectal cancer progression. We investigated plasma concentrations of 130 metabolites among 744 Stages I–IV colorectal cancer patients from ongoing cohort studies. Plasma samples, collected at diagnosis, were analyzed with liquid chromatography‐mass spectrometry using the Biocrates AbsoluteIDQ™ p180 kit. We assessed associations between metabolite concentrations and stage using multinomial and multivariable logistic regression models. Analyses were adjusted for potential confounders as well as multiple testing using false discovery rate (FDR) correction. Patients presented with 23, 28, 39 and 10% of Stages I–IV disease, respectively. Concentrations of sphingomyelin C26:0 were lower in Stage III patients compared to Stage I patients (pFDR < 0.05). Concentrations of sphingomyelin C18:0 and phosphatidylcholine (diacyl) C32:0 were statistically significantly higher, while citrulline, histidine, phosphatidylcholine (diacyl) C34:4, phosphatidylcholine (acyl‐alkyl) C40:1 and lysophosphatidylcholines (acyl) C16:0 and C17:0 concentrations were lower in Stage IV compared to Stage I patients (pFDR < 0.05). Our results suggest that metabolic pathways involving among others citrulline and histidine, implicated previously in colorectal cancer development, may also be linked to colorectal cancer progression.
What's new?
Metabolomics is a sophisticated method for investigating whether the metabolite profile of a patient's blood, etc., may reflect the pathophysiological state of cancers and other diseases. In the present study, the authors analyzed circulating metabolites, seeking biomarkers related to colorectal cancer progression. Their results at various stages of colorectal cancer suggest that metabolic pathways involving citrulline, histidine, and other molecules that have been previously implicated in colorectal cancer development may also be linked to progression.
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