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Author(s): Huan Yun 1, Liang Sun 1, Qingqing Wu 2, Geng Zong 1, Qibin Qi 3, Huaixing Li 1, He Zheng 1, Rong Zeng 2,*, Liming Liang 4,5,*, Xu Lin [...], Background Animal studies suggest vital roles of sphingolipids, especially ceramides, in the pathogenesis of type 2 diabetes (T2D) via pathways involved in insulin resistance, [beta]-cell dysfunction, and inflammation, but human studies are limited. We aimed to evaluate the associations of circulating sphingolipids with incident T2D and to explore underlying mechanisms. Methods and findings The current study included 826 men and 1,148 women who were aged 50-70 years, from Beijing and Shanghai, and without T2D in 2005 and who were resurveyed in 2011. Cardiometabolic traits were measured at baseline and follow-up surveys. A total of 76 sphingolipids were quantified using high-coverage targeted lipidomics. Summary data for 2-sample Mendelian randomization were obtained from genome-wide association studies of circulating sphingolipids and the China Health and Nutrition Survey (n = 5,731). During the 6-year period, 529 participants developed T2D. Eleven novel and 3 reported sphingolipids, namely ceramides (d18:1/18:1, d18:1/20:0, d18:1/20:1, d18:1/22:1), saturated sphingomyelins (C34:0, C36:0, C38:0, C40:0), unsaturated sphingomyelins (C34:1, C36:1, C42:3), hydroxyl-sphingomyelins (C34:1, C38:3), and a hexosylceramide (d18:1/20:1), were positively associated with incident T2D (relative risks [RRs]: 1.14-1.21; all P < 0.001), after multivariate adjustment including lifestyle characteristics and BMI. Network analysis further identified 5 modules, and 2 modules containing saturated sphingomyelins showed the strongest associations with increased T2D risk (RR.sub.Q4 versus Q1 = 1.59 and 1.43; both P.sub.trend < 0.001). Mediation analysis suggested that the detrimental associations of 13 sphingolipids with T2D were largely mediated through [beta]-cell dysfunction, as indicated by HOMA-B (mediation proportion: 11.19%-42.42%; all P < 0.001). Moreover, Mendelian randomization evidenced a positive association between a genetically instrumented ceramide (d18:1/20:1) and T2D (odds ratio: 1.15 [95% CI 1.05-1.26]; P = 0.002). Main limitations in the current study included potential undiagnosed cases and lack of an independent population for replication. Conclusions In this study, we observed that a panel of novel sphingolipids with unique structures were positively associated with incident T2D, largely mediated through [beta]-cell dysfunction, in Chinese individuals.

Sphingosine-1-phosphate (S1P) was found previously to inhibit [Na.sup.+]-[K.sup.+] ATPase in HepG2 cells. Whether fingolimod (FTY720), a S1P receptor (S1PR) agonist, similarly inhibits the ATPase is a question that needs to [...]

Author(s): Michael Maceyka [sup.1] , Sarah Spiegel [sup.1] Author Affiliations: (1) Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, USA Main In the 1880s, the [...], Sphingolipids are ubiquitous building blocks of eukaryotic cell membranes. Progress in our understanding of sphingolipid metabolism, state-of-the-art sphingolipidomic approaches and animal models have generated a large body of evidence demonstrating that sphingolipid metabolites, particularly ceramide and sphingosine-1-phosphate, are signalling molecules that regulate a diverse range of cellular processes that are important in immunity, inflammation and inflammatory disorders. Recent insights into the molecular mechanisms of action of sphingolipid metabolites and new perspectives on their roles in regulating chronic inflammation have been reported. The knowledge gained in this emerging field will aid in the development of new therapeutic options for inflammatory disorders.

Vasoactive properties of sphingosine 1-phosphate (SIP) have been demonstrated by many investigators to vary in systemic vascular beds. These variations appear to reflect differential S IP receptor expression in the vasculature of these tissues. Although S 1P has been demonstrated to enhance endothelial barrier function, induce airway hyperresponsiveness, and modulate immune responses in the lung, the pulmonary vasomotor effects of S 1P remain poorly defined. In the present study, we sought to define the vasoregulatory effects of SIP in the pulmonary vasculature and to elucidate the underlying mechanisms operative in effecting the response in the intact lung. S1P (10 [micro]M) increased pulmonary vascular resistance (PVR) by 36% in the isolated perfused mouse lung. SIP-induced vasoconstriction was reduced by 64% by concomitant administration of the Rho-kinase inhibitor Y27632 (10 [micro]M). Similarly, the S1P response was attenuated by >50% after S1P2 receptor antagonism (JTE-013; 10 [micro]M) and in S1P2 receptor null mice. In contrast, [S1P.sub.3] receptor antagonism (VPC23019; 10 [micro]M) had no effect on the contractile response to S1P. Furthermore, we confirmed the role of Rho-kinase as an important regulator of basal vasomotor tone in the isolated perfused mouse lung. These results suggest that S1P is capable of altering pulmonary vascular tone in vivo and may play an important role in the modulation of pulmonary vascular tone both in the normal lung and under pathological conditions. lung; sphingolipid; vasoregulation; pulmonary hypertension doi: 10.1152/ajplung.00233.2009.

C9-methylated glucosylceramide is a fungus-specific sphingolipid. This lipid is a major membrane component in the cell and is thought to play important roles in the growth and virulence of several fungal species. To investigate the importance of the methyl branch of the long-chain base in glucosylceramides in pathogenic fungi, we identified and characterized a sphingolipid C9-methyltransferase gene (MTS1, C9-MethylTransferase for Sphingolipid 1) in the pathogenic yeast Candida albicans. The mts1 disruptant lacked (E,E)-9-methylsphinga-4,8-dienine in its glucosylceramides and contained (E)-sphing-4-enine and (E,E)-sphinga-4,8-dienine. Reintroducing the MTS1 gene into the mts1 disruptant restored the synthesis of (E,E)-9-methylsphinga-4,8-dienine in the glucosylceramides. We also created a disruptant of the HSX11 gene, encoding glucosylceramide synthase, which catalyses the final step of glucosylceramide synthesis, in C. albicans and compared this mutant with the mts1 disruptant. The C. albicans mts1 and hsx11 disruptants both had a decreased hyphal growth rate compared to the wild-type strain. The hsx11 disruptant showed increased susceptibility to SDS and fluconazole, similar to a previously reported sld1 disruptant that contained only (E)-sphing-4-enine in its glucosylceramides, suggesting that these strains have defects in their cell membrane structures. In contrast, the mts1 disruptant grew similarly to wild-type in medium containing SDS or fluconazole. These results suggest that the C9-methyl group of a long-chain base in glucosylceramides plays an important role in the hyphal elongation of C. albicans independent of lipid membrane disruption. DOI 10.1099/mic.0.033985-0

The present study determined whether activation of acid sphingomyelinase (ASM) drives membrane proximal lysosomes to fuse to the cell surface, facilitating membrane lipid rafts (LRs) clustering in coronary arterial endothelial cells (CAECs) and leading to endothelial dysfunction. By confocal microscopy, the activators of ASM, phosphatidylinositol (PI), and his (monoacylglyceryl) phosphate (Bis), and an inducer of ASM, butyrate, were found to increase LRs clustering in bovine CAECs, which was blocked by lysosome fusion inhibitor vacuolin-1. However, arsenic trioxide (Ars), an inducer of de novo synthesis of ceramide, had no such effect. Similarly, vacuolin-l-blockable effects were observed using fluorescence resonance energy transfer detection. Liquid chromatography-electrospray ionization-tandem mass spectrometry analysis demonstrated that all of these treatments, even Ars, increased ceramide production in CAECs. When ASM gene was silenced, all treatments except Ars no longer increased ceramide levels. Furthermore, dynamic fluorescence monitoring in live cells showed that PI and Bis stimulated lysosome-membrane fusion in CAECs. Functionally, PI and Bis impaired endothelium-dependent vasodilation in perfused coronary arteries, which was blocked by vacuolin- 1 and a lysosome function inhibitor, bafilomycine. FasL (Fas ligand), a previously confirmed lysosome fusion stimulator as a comparison, also produced a similar effect. It is concluded that ASM activation serves as a triggering mechanism and driving force, leading to fusion of membrane proximal lysosomes into LR clusters on the cell membrane of CAECs, which represents a novel mechanism mediating endothelial dysfunction during death receptor activation or other pathological situation. membrane microdomains; sphingolipids; acid vesicles; coronary circulation; vascular endothelium doi:10.1152/ajpheart.00958.2009

In vascular endothelial cells, specialized microdomains of plasma membrane termed caveolae modulate various receptor signal transduction pathways regulated by caveolin-1, a resident protein of caveolae. We examined whether transforming growth factor-[beta]1 (TGF-[beta]1), a multifunctional cytokine, alters expression levels of caveolin-1 and influences heterologous receptor signaling. Treatment of cultured bovine aortic endothelial cells (BAEC) with TGF-[beta]1 induces marked decreases in caveolin-1 expression in a time- and dose-dependent fashion at both levels of protein and mRNA. A pharmacological inhibitor of activin receptor-like kinase 5 (ALK-5) counteracts caveolin-1 downregulation by TGF-[beta]1, indicating the involvement of ALK-5 receptor subtype for TGF-[beta]1. Sphingosine 1-phosphate (SIP) is a serum-borne angiogenic lipid growth factor that exerts a wide variety of biological actions. S1P modulates G protein-coupled S1P receptors, activating downstream molecules kinases AMP-activated protein kinase (AMPK), and Akt as well as a small G protein Racl, ultimately to promote migration. Because SIP receptor signaling is associated with caveolae/caveolin-1, we examined whether pretreatment with TGF-[beta]1 enhances effects of SIP on BAEC. Whereas SIP alone evokes robust BAEC responses to S1P, pretreatment with TGF-[beta]1 leads to even higher magnitudes of SIP-elicited signaling responses and cell migration. Conversely, genetic knockdown of caveolin-1 using small interfering RNA mimics TGF-[beta]1-induced promotion of BAEC responses to SIP. Collectively, these data demonstrate that TGF-[beta]1 downregulates caveolin-1 of cultured endothelial cells, involving ALK-5 receptor subtype. Because downregulation of caveolin-1 by TGF-[beta]1 promotes subsequent heterologous receptor signaling by SIP, these results may also identify novel point of cross-talk between cytokines and sphingolipids within endothelial signal transduction machineries. cytokines; sphingolipids; G protein-coupled receptors; endothelium; signal transduction doi: 10.1152/ajpcell.00109.2009.

The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane trafficking, we devised an immunoisolation procedure for specific recovery of post-Golgi secretory vesicles transporting a transmembrane raft protein from the TGN to the cell surface in the yeast Saccharomyces cerevisiae. Using a novel quantitative shotgun lipidomics approach, we could demonstrate that TGN sorting selectively enriched ergosterol and sphingolipid species in the immunoisolated secretory vesicles. This finding, for the first time, indicates that the TGN exhibits the capacity to sort membrane lipids. Furthermore, the observation that the immunoisolated vesicles exhibited a higher membrane order than the late Golgi membrane, as measured by C-Laurdan spectrophotometry, strongly suggests that lipid rafts play a role in the TGN-sorting machinery.

Epithelial-to-mesenchymal cell transition (EMT) is a basic process in embryonic development and cancer progression. The present study demonstrates involvement of glycosphingolipids (GSLs) in the EMT process by using normal murine mammary gland NMuMG, human normal bladder HCV29, and human mammary carcinoma MCF7 cells. Treatment of these cells with D-threo-1-(3',4'-ethylenedioxy)-phenyl-2-palmitoylamino-3-pyrrolidino-1- propanol (EtDO-P4), the glucosylceramide (GlcCer) synthase inhibitor, which depletes all GSLs derived from GlcCer, (i) down-regulated expression of a major epithelial cell marker, E-cadherin; (ii) up-regulated expression of mesenchymal cell markers vimentin, fibronectin, and N-cadherin; (iii) enhanced haptotactic cell motility; and (iv) converted epithelial to fibroblastic morphology. These changes also were induced in these cell lines with TGF-[beta], which is a well-documented EMT inducer. A close association between specific GSL changes and EMT processes induced by EtDO-P4 of TGF-[beta] is indicated by the following findings: (i) The enhanced cell motility of EtDO-P4-treated cells was abrogated by exogenous addition of GM2 or Gg4, but not GM1 or GM3, in all 3 cell lines, (ii) TGF-[beta] treatment caused changes in the GSL composition of cells. Notably, Gg4 or GM2 was depleted or reduced in NMuMG, and GM2 was reduced in HCV29. (iii) Exogenous addition of Gg4 inhibited TGF-[beta]-induced changes of morphology, motility, and levels of epithelial and mesenchymal markers. These observations indicate that specific GSLs play key roles in defining phenotypes associated with EMT and its reverse process (i.e., mesenchymal-to-epithelial transition). E-cadherin | EtDO-P4 | Gg4 | motility | TGF-beta

Sphingosine-1-phosphate (SIP) is a secreted lipid mediator that functions in vascular development; however, it remains unclear how S1P secretion is regulated during embryogenesis. We identified a zebrafish mutant, ko157, that displays cardia bifida (two hearts) resembling that in the S1P receptor-2 mutant. A migration defect of myocardial precursors in the k0157 mutant is due to a mutation in a multipass transmembrane protein, Spns2, and can be rescued by S1P injection. We show that the export of SIP from cells requires Spns2. spns2 is expressed in the extraembryonic tissue yolk syncytial layer (YSL), and the introduction of spns2 mRNA in the YSL restored the cardiac defect in the k0157 mutant. Thus, Spns2 in the YSL functions as a S1P transporter in S1P secretion, thereby regulating myocardial precursor migration.

Author(s): Vijay R. Varma 1, Anup M. Oommen 2, Sudhir Varma 3, Ramon Casanova 4, Yang An 1, Ryan M. Andrews 5, Richard O'Brien 6, Olga Pletnikova 7, Juan C. [...], Background The metabolic basis of Alzheimer disease (AD) is poorly understood, and the relationships between systemic abnormalities in metabolism and AD pathogenesis are unclear. Understanding how global perturbations in metabolism are related to severity of AD neuropathology and the eventual expression of AD symptoms in at-risk individuals is critical to developing effective disease-modifying treatments. In this study, we undertook parallel metabolomics analyses in both the brain and blood to identify systemic correlates of neuropathology and their associations with prodromal and preclinical measures of AD progression. Methods and findings Quantitative and targeted metabolomics (Biocrates AbsoluteIDQ [identification and quantification] p180) assays were performed on brain tissue samples from the autopsy cohort of the Baltimore Longitudinal Study of Aging (BLSA) (N = 44, mean age = 81.33, % female = 36.36) from AD (N = 15), control (CN; N = 14), and "asymptomatic Alzheimer&apos;s disease" (ASYMAD, i.e., individuals with significant AD pathology but no cognitive impairment during life; N = 15) participants. Using machine-learning methods, we identified a panel of 26 metabolites from two main classes-sphingolipids and glycerophospholipids-that discriminated AD and CN samples with accuracy, sensitivity, and specificity of 83.33%, 86.67%, and 80%, respectively. We then assayed these 26 metabolites in serum samples from two well-characterized longitudinal cohorts representing prodromal (Alzheimer&apos;s Disease Neuroimaging Initiative [ADNI], N = 767, mean age = 75.19, % female = 42.63) and preclinical (BLSA) (N = 207, mean age = 78.68, % female = 42.63) AD, in which we tested their associations with magnetic resonance imaging (MRI) measures of AD-related brain atrophy, cerebrospinal fluid (CSF) biomarkers of AD pathology, risk of conversion to incident AD, and trajectories of cognitive performance. We developed an integrated blood and brain endophenotype score that summarized the relative importance of each metabolite to severity of AD pathology and disease progression (Endophenotype Association Score in Early Alzheimer&apos;s Disease [EASE-AD]). Finally, we mapped the main metabolite classes emerging from our analyses to key biological pathways implicated in AD pathogenesis. We found that distinct sphingolipid species including sphingomyelin (SM) with acyl residue sums C16:0, C18:1, and C16:1 (SM C16:0, SM C18:1, SM C16:1) and hydroxysphingomyelin with acyl residue sum C14:1 (SM (OH) C14:1) were consistently associated with severity of AD pathology at autopsy and AD progression across prodromal and preclinical stages. Higher log-transformed blood concentrations of all four sphingolipids in cognitively normal individuals were significantly associated with increased risk of future conversion to incident AD: SM C16:0 (hazard ratio [HR] = 4.430, 95% confidence interval [CI] = 1.703-11.520, p = 0.002), SM C16:1 (HR = 3.455, 95% CI = 1.516-7.873, p = 0.003), SM (OH) C14:1 (HR = 3.539, 95% CI = 1.373-9.122, p = 0.009), and SM C18:1 (HR = 2.255, 95% CI = 1.047-4.855, p = 0.038). The sphingolipid species identified map to several biologically relevant pathways implicated in AD, including tau phosphorylation, amyloid-[beta] (A[beta]) metabolism, calcium homeostasis, acetylcholine biosynthesis, and apoptosis. Our study has limitations: the relatively small number of brain tissue samples may have limited our power to detect significant associations, control for heterogeneity between groups, and replicate our findings in independent, autopsy-derived brain samples. Conclusions We present a novel framework to identify biologically relevant brain and blood metabolites associated with disease pathology and progression during the prodromal and preclinical stages of AD. Our results show that perturbations in sphingolipid metabolism are consistently associated with endophenotypes across preclinical and prodromal AD, as well as with AD pathology at autopsy. Sphingolipids may be biologically relevant biomarkers for the early detection of AD, and correcting perturbations in sphingolipid metabolism may be a plausible and novel therapeutic strategy in AD.

A study was conducted to explore the structural and functional properties of carboxyl-terminus (amino acids 462-629) from the human acid sphingomyelinase (ASM), protein coded by the sphingomyelin phosphodiesterase 1 (SMPD-1). The results suggest that the carboxyl-terminus of the ASM is crucial for its protein structure which dictates the enzymatic function and secretion.

The brain is enriched with sphingolipids, which are important membrane constituents and major lipid signaling molecules that have a role in motor and cognitive behavior. Vitamin K has been implicated in brain sphingolipid metabolism for more than 30 years. The in vitro and in vivo studies to date suggest a role of vitamin K in the regulation of multiple enzymes involved in sphingolipid metabolism within the myelin-rich regions in the brain. However, the precise mechanisms of action are not well understood. Further, the physiological consequences of the observed effects of vitamin K on sphingolipid metabolism have not been systematically studied. doi: 10.1301/nr.2005.apr.111-121 Key words: vitamin K, sphingolipids, lipid signaling

The genus Sphingobacterium, whose members are Gram-negative non-fermentative rods, possesses ceramides and related sphingophospholipids (SPLs) with isoheptadecasphinganine and 2-hydroxy or non-hydroxy isopentadecanoic acid. This paper reports evidence that ceramides isolated from Sphingobacterium ATCC 33861 induce endonucleolytic DNA cleavage in human myeloid leukaemia HL-60 cells in vitro, which is the primary characteristic biochemical marker for apoptosis or programmed cell death. Ceramides and SPLs also induced DNA fragmentation and caspase-3 activation, followed by changes in morphology, such as alterations in the size of nuclei and cells, and cell cycle shortening. Apoptotic activity correlated with the ceramide structure. Ceramide with a 2-hydroxy fatty acid showed stronger apoptotic activity than ceramide with a non-hydroxy fatty acid. Furthermore, the major five SPLs (ceramide phosphorylethanolamine-1 and -2, ceramide phosphorylinositol-1 and -2, and ceramide phosphorylmannose-1) showed apoptosis-inducing activity in HL-60 cells, indicating that the ceramide moiety of the SPLs plays a crucial role as the intracellular second messenger but that their hydrophilicity is less important in this regard. The hydrophilic part of SPLs may play a role in other cellular response systems. The involvement of Fas antigen was implicated in the apoptotic event since Fas antigen expression was observed after 3 or 4 h stimulation of HL-60 cells with bacterial ceramides. However, a time-course study for caspase-3 activation indicated maximal activity at 1 h after stimulation with bacterial ceramides, suggesting that two (or possibly more) mechanisms of signal transduction, Fas-dependent and Fas-independent, may be involved. Fas antigen expression and caspase-3 activation by five kinds of SPLs were observed after 3 or 4 h. These results indicate that there is a difference in the response of HL-60 cells to bacterial ceramides and SPLs.

Ceramide, the basic structural unit of sphingolipids, controls the balance between cell growth and death by inducing apoptosis. We have previously shown that accumulation of ceramide, triggered by hydrogen peroxide ([H.sub.2][O.sub.2]) or by short-chain ceramide analogs, induces apoptosis of lung epithelial cells. Here we elucidate the link between caspase-3 activation, at the execution phase, and ceramide accumulation, at the commitment phase of apoptosis in A549 human lung adenocarcinoma cells. The induction of ceramide accumulation by various triggers of ceramide generation, such as [H.sub.2][O.sub.2], [C.sub.6]-ceramide, or UDP-glucose-ceramide glucosyltransferase inhibitor DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, triggered the activation of caspase-3. This ceramide elevation also induced the cleavage of the death substrate poly(ADP-ribose) polymerase and was followed by apoptotic cell death. Ceramide-mediated apoptosis was blocked by a general caspase inhibitor, Boc-D-fluoromethylketone, and by overexpression of the antiapoptotic protein Bcl-2. Notably, overexpression of Bcl-2 reduced the basal cellular levels of ceramide and prevented the induction of ceramide generation by [C.sub.6]-ceramide, which implies ceramide generation as a possible target for the antiapoptotic effects of Bcl-2. Bcl-2 overexpression; hydrogen peroxide; sphingolipid signaling

Research has been conducted on glycosylsphingolipids. The transbilayer movement of these glycosylsphingolipids has been characterized via the use of plasma, Golgi, ER and model membrane by means of monohexosylsphingolipids glucosylceramide and galactosylceramide and of the dihexosylsphingolipid caltosylcerabide and the details are reported.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that regulates diverse biological processes by binding to a family of G protein--coupled receptors or as an intracellular second messenger. Mammalian S1P phosphatase (SPP-1), which degrades S1P to terminate its actions, was recently cloned based on homology to a lipid phosphohydrolase that regulates the levels of phosphorylated sphingoid bases in yeast. Confocal microscopy surprisingly revealed that epitope-tagged SPP-1 is intracellular and colocalized with the ER marker calnexin. Moreover, SPP-1 activity and protein appeared to be mainly enriched in the intracellular membranes with lower expression in the plasma membrane. Treatment of SPP-1 transfectants with S1P markedly increased ceramide levels, predominantly in the intracellular membranes, diminished survival, and enhanced apoptosis. Remarkably, dihydro-S1P, although a good substrate for SPP-1 in situ, did not cause significant ceramide accumulation or increase apoptosis. Ceramide accumulation induced by S1P was completely blocked by fumonisin B1, an inhibitor of ceramide synthase, but only partially reduced by myriocin, an inhibitor of serine palmitoyltransferase, the first committed step in de novo synthesis of ceramide. Furthermore, S1P, but not dihydro-S1P, stimulated incorporation of [[sup.3]H]palmitate, a substrate for both serine palmitoyltransferase and ceramide synthase, into C16-ceramide. Collectively, our results suggest that SPP-1 functions in an unprecedented manner to regulate sphingolipid biosynthesis and is poised to influence cell fate.

In this study isolated perfused working rat hearts were used to investigate the role of palmitate-regulated protein kinase B (PKB) phosphorylation on glucose metabolism. Rat hearts were perfused aerobically in working mode with 11 mM glucose and either 100 [micro]U/ml insulin or 100 [micro]U/ml insulin and 1.2 mM palmitate. PKB activity and phosphorylation state were reduced in the presence of 1.2 mM palmitate, which correlates with a decrease in glycolysis (47%), glucose oxidation (84%), and glucose uptake (43%). In contrast to skeletal muscle, neither p38 nor ERK underwent changes in their phosphorylation states in response to insulin or insulin and palmitate. Moreover, pharmacological restoration of glucose oxidation rates in hearts perfused with 1.2 mM palmitate demonstrated no increase in PKB phosphorylation state. In cultured mouse cardiac muscle HL-1 cells, insulin markedly increased PKB phosphorylation, which was blunted by pre- and cotreatment with 1.2 mM palmitate. However, neither palmitate nor [C.sub.2]-ceramide treatment of insulin-stimulated cells was able to accelerate PKB dephosphorylation beyond that observed following the removal of insulin alone. Taken together, these experiments show the control of PKB phosphorylation by palmitate is independent of ceramide and suggest that this signaling event may be an important regulator of myocardial glucose uptake and oxidation. insulin; glucose; ceramide; Akt, metabolism

Research has been conducted on the alpha-galactosyl ceramide. The synthesis of this stimulatory agent for the immunological process which is mediated by CD1 molecules is described.

Research has been conducted on ceramide, a long-chain aliphatic 2-amido-1,3-diol with double bond. The synthesis of the unnatural ceramide analogues with incorporated diene and a double bond has been carried out and the results are described.

Results reveal that the group V secretory phospholipases A(sub)2 hydrolyze phosphatidylcholine 30 times more efficient than the group IIa enzymes, while both enzymes being active on high density lipoprotein than low density lipoprotein. Data further show that sphingomyelin inhibits the hydrolysis of phospholipids, as shown by a reversal of the inhibition by sphingomyelinase.

Research has been conducted on the D-erythro-homoceramide analogues. The synthesis of these analogues has been investigated and the results indicate that due to the lack of metabolism to glucosylhomoceramide none of the tested compounds have exhibited any biological activity.

Ceramides and sphingomyelins in skeletal muscles of the rat: content and composition. Effect of prolonged exercise. Am J Physiol Endocrinol Metab 282: E277-E285, 2002; 10.1152/ajpendo. 00151.2001.--The sphingomyelin-signaling pathway has been described in many tissues. Ceramide is the main second messenger in this pathway. Ceramide has also been shown to be present in skeletal muscles; however, there are few data on the regulation of the content of ceramide in muscle tissue. Moreover, there are no data on the content of particular ceramides or their composition in the muscles. The aim of the present study was to examine the content and composition of fatty acids (FA) in ceramide and sphingomyelin moieties and the activity of neutral [Mg.sup.2+]-dependent sphingomyelinase in different skeletal muscle types of the rat at rest and after exhausting exercise of moderate intensity. The experiments were carried out on male Wistar rats, divided into two groups: 1) control and 2) exercised until exhaustion on a treadmill. Soleus and red and white gastrocnemius muscles were taken. Ceramide and sphingomyelin were separated by TLC. The content of individual FA in the two compounds was determined by gas-liquid chromatography. Twelve different ceramides and sphingomyelins were identified and quantified in each muscle type according to the FA residues. Saturated FA consisted of 68, 67, and 66% of total ceramide-FA and 75, 77, and 78% of total sphingomyelin-FA in soleus and red and white gastrocnemius, respectively. The total content of ceramide- and sphingomyelin-FA and the activity of sphingomyelinase were highest in the soleus and lowest in the white gastrocnemius. Exercise resulted in a reduction in the total content of ceramide- and sphingomyelin-FA in each muscle. This was accounted for mostly by a reduction in content in the amount of saturated FA. Exercise reduced the activity of neutral [Mg.sup.2+]-dependent sphingomyelinase in the soleus and red gastrocnemius and did not affect its activity in the white gastrocnemius. We conclude that the sphingomyelin-signaling pathway is present in skeletal muscles and that it is influenced by prolonged exercise. sphingomyelin-signaling pathway

Research has been conducted on the glycolipid transfer from donor to acceptor vesicles. The monitoring of this transfer has been carried out via the fluorescence resonance energy transfer assay which involves anthrylvinyl-labeled galactosylceramide and perylenoyl-labeled triglyceride and the results arevreported.

Bradykinin stimulates ceramide production by activating specific BK-[B.sub.1] receptor in rat small artery. Am J Physiol Heart Circ Physiol 282: H175-H183, 2002.. First published September 27, 2001; 10.1152/ ajpheart.00379.2001.--Bradykinin (BK), a proinflammatory factor and vasodilator, causes functional change of the small artery. However, it is not clear whether any of these changes induced by BK are mediated by N-acetyl-D-sphingosine (ceramide). Therefore, we investigated whether BK affects the hydrolysis of sphingomyelin and generation of ceramide in the intact rat small artery. Our results suggest that BK induces sphingomyelin hydrolysis and increases ceramide production in a time- and dose-dependent manner. Relative to controls, BK causes a 50% decrease in sphingomyelin levels. Ceramide levels increase in response to BK with the highest level being obtained with [10.sup.-8] M BK as well as similar amounts of ceramide are generated when exogenous sphingomyelinase (SMase):is added. We then determined which of the two BK receptors (BK-[B.sub.1] antagonist Lys-Des-[Arg.sup.9]-[Leu.sup.8]-BK or the BK-[B.sub.2] antagonist HOE-140) are implicated in the BK-induced generation of ceramide. The BK-[B.sub.2] antagonist did not alter the effect of BK on ceramide generation, whereas the BK-[B.sub.1] antagonist blocked the BK-induced production of ceramide. Although ceramide had no effect on KCl-induced constrictions, ceramide dilated preconstricted (phenylephrine) small pressurized rat mesenteric arteries by ~40%. These results suggest that the activation of the BK-[B.sub.1] receptor mediates the BK-induced activation of SMase and of the production of ceramide. In conclusion, BK-mediated effects on vascular tone may be due, at least in part, to the increased production of ceramide. phenylephrine; receptor antagonists; sphingomyelin; vascular tone

Research has been conducted on the membrane microdomains enriched in cholesterol and characterized by their resistance to the detergents and on the sphingolipids modulating the signal transduction pathways. The effect of these membrane domains on the signaling through the heterotrimeric protein G (sub)b has been investigated via the cholesterol and sphingolipid cell depletion and the results are reported.

Berry, C., R. Touyz, A. F. Dominiczak, R. C. Webb, and D. G. Johns. Angiotensin receptors: signaling, vascular pathophysiology, and interactions with ceramide. Am J Physiol Heart Circ Physiol 281: H2337-H2365, 2001.--Angiotensin II (ANG II) is a pleiotropic vasoactive peptide that binds to two distinct receptors: the ANG II type 1 ([AT.sub.1]) and type 2 ([AT.sub.2]) receptors. Activation of the renin-angiotensin system (RAS) results in vascular hypertrophy, vasoconstriction, salt and water retention, and hypertension. These effects are mediated predominantly by [AT.sub.1] receptors. Paradoxically, other ANG II-mediated effects, including cell death, vasodilation, and natriuresis, are mediated by [AT.sub.2] receptor activation. Our understanding of ANG II signaling mechanisms remains incomplete. [AT.sub.1] receptor activation triggers a variety of intracellular systems, including tyrosine kinase-induced protein phosphorylation, production of arachidonic acid metabolites, alteration of reactive oxidant species activities, and fluxes in intracellular [Ca.sup.2+] concetrations. [AT.sub.2] receptor activation leads to stimulation of bradykinin, nitric oxide production, and prostaglandin metabolism, which are, in large part, opposite to the effects of the [AT.sub.1] receptor. The signaling pathways of ANG II receptor activation are a focus of intense investigative effort. We critically appraise the literature on the signaling mechanisms whereby [AT.sub.1] and [AT.sub.2] receptors elicit their respective actions. We also consider the recently reported interaction between ANG II and ceramide, a lipid second messenger that mediates cytokine receptor activation. Finally, we discuss the potential physiological cross talk that may be operative between the angiotensin receptor subtypes in relation to health and cardiovascular disease. This may be clinically relevant, inasmuch as inhibitors of the RAS are increasingly used in treatment of hypertension and coronary heart disease, where activation of the RAS is recognized. renin-angiotensin system; angiotensin receptor antagonist; second messenger Received 7 June 2000; accepted in final form 24 July 2001

Ceramide and sphingosine-1-phosphate (S1P) are important dietary lipids involved in cell growth, differentiation, apoptosis and cell survival. Treatment of C3H10T1/2 murine fibroblast cells (10T1/2) with ceramide did not induce apoptosis, a commonly observed effect of ceramide treatment. To determine whether the metabolism of ceramide played a role in this resistance to apoptosis, inhibitors of ceramidase and sphingosine kinase, two important enzymes in sphingolipid metabolism, were used. Treatment of 10T1/2 cells both without or with ceramide plus N-oleoyl-ethanolamine (NOE) and (1S,2R)-D-erythro-s-(N-myristoylamino)-1-phenol-1-propanol (MAPP), two ceramidase inhibitors, resulted in fourfold and eightfold increases, respectively, in apoptosis. Cells treated without or with ceramide plus N,N-dimethylsphingosine (DMS), a potent sphingosine kinase inhibitor, resulted in fourfold and sixfold increases, respectively, in apoptosis. In all treatments the induction of apoptosis was prevented by the addition of S1P. With the addition of S1P with NOE and MAPP as well as with ceramide, treatments reduced the apoptotic response by 30 and 35%, respectively; whereas the addition of S1P with the DMS only and ceramide with DMS treatments reduced the apoptotic response by 60 and 70%, respectively. Studies using labeled ceramide demonstrated ceramide was metabolized to S1P. In addition, a 14-fold increase in apoptosis occurred in cells treated with a nonhydrolyzable analog of ceramide, ceramine, compared with vehicle control. Because inhibiting the conversion of ceramide to S1P resulted in apoptosis, the lack of an apoptotic response to ceramide alone for C3H10T1/2 cells is attributable to the conversion of this pro-apoptotic sphingolipid to the anti-apoptotic metabolite S1P, which is essential for cell survival. J. Nutr. 131: 2826-2830, 2001. KEY WORDS: * C3H10T1/2 cells * ceramide * sphingosine-1-phosphate * apoptosis

Although glycosphingolipids are ubiquitously expressed and essential for multicellular organisms, surprisingly little is known about their intracellular functions. To explore the role of glycosphingolipids in membrane transport, we used the glycosphingolipid-deficient GM95 mouse melanoma cell line. We found that GM95 cells do not make melanin pigment because tyrosinase, the first and rate-limiting enzyme in melanin synthesis, was not targeted to melanosomes but accumulated in the Golgi complex. However, tyrosinase-related protein 1 still reached melanosomal structures via the plasma membrane instead of the direct pathway from the Golgi. Delivery of lysosomal enzymes from the Golgi complex to endosomes was normal, suggesting that this pathway is not affected by the absence of glycosphingolipids. Loss of pigmentation was due to tyrosinase mislocalization, since transfection of tyrosinase with an extended transmembrane domain, which bypassed the transport block, restored pigmentation. Transfection of ceramide glucosyltransferase or addition of glucosylsphingosine restored tyrosinase transport and pigmentation. We conclude that protein transport from Golgi to melanosomes via the direct pathway requires glycosphingolipids.

Research has been conducted on the ceramides which inhibit phospholipase D activity in mammalian cells. The inhibition of phospholipase D1 and D2 by these ceramides and the interaction of the ceramides with phosphatidylinositol 4,5-bisphosphate have been investigated and reported.

Research has been conducted on the rat brain mitochondrial ceramidase. The effect of the structural ceramide analogues on this ceramidase has been investigated and described.