Overexpression of apolipoprotein A-IV enhances lipid secretion in IPEC-1 cells by increasing chylomicron size

Intestinal apolipoprotein A-IV expression is highly regulated by dietary lipid in newborn swine, suggesting a role in lipid absorption. Constitutive overexpression of apoA-IV in newborn swine enterocytes enhances basolateral secretion of triacylglycerol (TG) in TG-rich lipoproteins 4.9-fold (Lu, S., Yao, Y., Meng, S., Cheng, X., and Black, D. D. (2002) J. Biol. Chem. 277, 31929-31937). To investigate the mechanism of this enhancement, IPEC-1 cells were transfected with a tetracycline-regulatable expression system (Tet-On). In cells incubated with oleic acid, a dose response relationship was observed between medium doxycycline concentration and basolateral apoA-IV and TG secretion. Similarly regulated expression of apoA-I did not enhance lipid secretion. The mean diameter of TG-rich lipoproteins secreted from doxycycline-treated cells was larger than from untreated cells (87.0 nm versus 53.4 nm). Basolateral apoB secretion decreased. Using the same expression system, full-length human apoA-IV (376 amino acids); a "pig-like" human apoA-IV, lacking the C-terminal EQQQ repeats (361 amino acids); and a "chicken-like" apoA-IV, further truncated to 343 amino acids, were expressed in IPEC-1 cells. With increasing protein secretion, cells expressing the full-length human apoA-IV displayed a 2-fold increase in TG secretion; in sharp contrast, cells expressing the pig-like human apoA-IV displayed a 25-fold increase in TG secretion and a 27-fold increase in lipoprotein diameter. When human apoA-IV was further truncated to yield a chicken-like protein, TG secretion was inhibited. We conclude that overexpression of swine apoA-IV enhances basolateral TG secretion in a dose-dependent manner by increasing the size of secreted lipoproteins. These data suggest that the region in the human apoA-IV protein from residues 344 to 354 is critical to its ability to enhance lipid secretion, perhaps by enabling the packaging of additional core TG into chylomicron particles. The EQQQ-rich region may play an inhibitory or modulatory role in chylomicron packaging in humans.     

Curcumin stimulates exosome/microvesicle release in an in vitro model of intracellular lipid accumulation by increasing ceramide synthesis

Curcumin, a hydrophobic polyphenol found in the rhizome of Curcuma longa, has been shown to reduce intracellular lipid accumulation in mouse models of lysosomal storage diseases such as Niemann-Pick type C. Exosomes are small extracellular vesicles secreted by cells in response to changes in intracellular ceramide composition. Curcumin can induce exosome/microvesicle release in cellular models of lipid deposition; however, the mechanism by which curcumin stimulates this release is unknown. In a model of lipid trafficking impairment in C6 glia cells, we show that curcumin stimulated ceramide synthesis by increasing the intracellular concentration of ceramide-dihydroceramide. Ceramide overload increased exosome/microvesicle secretion 10-fold, thereby reducing the concentration of lipids in the endolysosomal compartment. These effects were blocked by inhibitors of serine palmitoyltransferase (myriocin) and ceramide synthase (fumonisin B1). It is concluded that the decrease in intracellular lipid deposition induced by curcumin is mediated by increased ceramide synthesis and exosome/microvesicle release. This action may represent an additional health benefit of curcumin.     

Amide analogues of CD1d agonists modulate iNKT-cell-mediated cytokine production

Invariant natural killer T (iNKT) cells are restricted by the non-polymorphic MHC class I-like protein, CD1d, and activated following presentation of lipid antigens bound to CD1d molecules. The prototypical iNKT cell agonist is α-galactosyl ceramide (α-GalCer). CD1d-mediated activation of iNKT cells by this molecule results in the rapid secretion of a range of pro-inflammatory (Th1) and regulatory (Th2) cytokines. Polarization of the cytokine response can be achieved by modifying the structure of the glycolipid, which opens up the possibility of using CD1d agonists as therapeutic agents for a range of diseases. Analysis of crystal structures of the T-cell receptor-α-GalCer-CD1d complex led us to postulate that amide isosteres of known CD1d agonists should modulate the cytokine response profile upon iNKT-cell activation. To this end, we describe the synthesis and biological activity of amide analogues of α-GalCer and its non-glycosidic analogue threitol ceramide (ThrCer). All of the analogues were found to stimulate murine and human iNKT cells by CD1d-mediated presentation to varying degrees; however, the thioamide and carbamate analogues of ThrCer were of particular interest in that they elicited a strongly polarized cytokine response (more interferon-gamma (IFN-γ), no interleukin-4 (IL-4)) in mice. While the ThrCer-carbamate analogue was shown to transactivate natural killer (NK) cells, a mechanism that has been used to account for the preferential production of IFN-γ by other CD1d agonists, this pathway does not account for the polarized cytokine response observed for the thioamide analogue.     

Hyperresponsiveness of mice deficient in plasma-secreted sphingomyelinase reveals its pivotal role in early phase of host response

Plasma secretion of acid sphingomyelinase is a hallmark of cellular stress response resulting in the formation of membrane embedded ceramide-enriched lipid rafts and the reorganization of receptor complexes. Consistently, decompartmentalization of ceramide formation from inert sphingomyelin has been associated with signaling events and regulation of the cellular phenotype. Herein, we addressed the question of whether the secretion of acid sphingomyelinase is involved in host response during sepsis. We found an exaggerated clinical course in mice genetically deficient in acid sphingomyelinase characterized by an increased bacterial burden, an increased phagocytotic activity, and a more pronounced cytokine storm. Moreover, on a functional level, leukocyte-endothelial interaction was found diminished in sphingomyelinase-deficient animals corresponding to a distinct leukocytes’ phenotype with respect to rolling and sticking as well as expression of cellular surface proteins. We conclude that hydrolysis of membrane-embedded sphingomyelin, triggered by circulating sphingomyelinase, plays a pivotal role in the first line of defense against invading microorganisms. This function might be essential during the early phase of infection leading to an adaptive response of remote cells and tissues.     

Irreversible inactivation of magnesium-dependent neutral sphingomyelinase 1 (NSM1) by peroxynitrite,a nitric oxide-derived oxidant

Previous results have indicated that the generation of ceramide by hydrolysis of sphingomyelin by magnesium-dependent neutral sphingomyelinase 1 (NSM1) is reversibly inhibited by hydrogen peroxide (H2O2) and oxidized glutathione (GSSG). This redox-dependent reversible regulation of NSM1 activity has been shown to involve the reversible formation and breakage of disulfide bonds. In this paper, we show that peroxynitrite, a nitric oxide-derived oxidant generated by SIN1, inactivates dose-dependently the NSM1 activity in an irreversible manner. In addition, we show that, in contrast to the reversible inhibition of NSM1 by H2O2 or GSSG which involves the formation of disulfide bonds, irreversible inactivation of this enzyme by peroxynitrite generated from SIN1 is likely due to definitive oxidative thiol modification. These results suggest that depending on the nature of the oxidative stress, the enzymatic activity of NSM1 could be reversibly or irreversibly inactivated.     

New CD1d agonists: synthesis and biological activity of 6″-triazole-substituted α-galactosyl ceramides

Huisgen [3+2] dipolar cycloaddition of 6″-azido-6″-deoxy-α-galactosyl ceramide 11 with a range of alkynes (or a benzyne precursor) yielded a series of triazole-containing α-galactosyl ceramide (α-GalCer) analogues in high yield. These α-GalCer analogues and the precursor azide 11 were tested for their ability to activate iNKT cells and stimulate IL-2 cytokine secretion in vitro, and IFN-γ and IL-4 cytokine secretion in vivo. Some of these analogues, specifically 11, 12b, 12f and 13, were more potent IL-2 stimulators than the prototypical CD1d agonist, α-GalCer 1. In terms of any cytokine bias, most of the triazole-containing analogues exhibited a small Th2 cytokine-biasing response relative to that shown by α-GalCer 1. In contrast, the cycloaddition precursor, namely azide 11, provided a small Th1 cytokine-biasing response.     

Overexpression of apolipoprotein A-IV enhances lipid transport in newborn swine intestinal epithelial cells

Apolipoprotein A-IV (apoA-IV) has myriad functions, including roles as a post-prandial satiety factor and lipid antioxidant. ApoA-IV is expressed in mammalian small intestine and is up-regulated in response to lipid absorption. In newborn swine jejunum, a high fat diet acutely induces a 7-fold increase in apoA-IV expression. To determine whether apoA-IV plays a role in the transport of absorbed lipid, swine apoA-IV was overexpressed in a newborn swine enterocyte cell line, IPEC-1, followed by analysis of the expression of genes related to lipoprotein assembly and lipid transport, as well as quantitation of lipid synthesis and secretion. A full-length swine apoA-IV cDNA was cloned, sequenced, and inserted into a Vp and Rep gene-deficient adeno-associated viral vector, containing the cytomegalovirus immediate early promoter/enhancer and neomycin resistance gene, and was used to transfect IPEC-1 cells. Control cells were transfected with the same vector minus the apoA-IV insert. Using neomycin selection, apoA-IV-overexpressing (+AIV) and control (-AIV) clones were isolated for further study. Both undifferentiated (-D) and differentiated (+D) +AIV cells expressed 40- to 50-fold higher levels of apoA-IV mRNA and both intracellular and secreted apoA-IV protein compared with -AIV cells. Expression of other genes was not affected by apoA-IV overexpression in a manner that would contribute to enhanced lipid secretion. +D +AIV cells secreted 4.9-fold more labeled triacylglycerol (TG), 4.6-fold more labeled cholesteryl ester (CE), and 2-fold more labeled phospholipid (PL) as lipoproteins, mostly in the chylomicron/very low density lipoprotein (VLDL) density range. ApoA-IV overexpression in IPEC-1 cells enhances basolateral TG, CE, and PL secretion in chylomicron/VLDL particles. This enhancement is not associated with up-regulation of other genes involved in lipid transport. ApoA-IV may play a role in facilitating enterocyte lipid transport, particularly in the neonate receiving a diet of high fat breast milk.     

Growth hormone-induced diacylglycerol and ceramide formation via Galpha i3 and Gbeta gamma in GH4 pituitary cells. Potentiation by dopamine-D2 receptor activation

Growth hormone (GH) secretion is regulated by indirect negative feedback mechanisms. To address whether GH has direct actions on pituitary cells, lipid signaling in GH(4)ZR(7) somatomammotroph cells was examined. GH (EC(50) = 5 nm) stimulated diacylglycerol (DAG) and ceramide formation in parallel by over 10-fold within 15 min and persisting for >3 h. GH-induced DAG/ceramide formation was blocked by pertussis toxin (PTX) implicating G(i)/G(o) proteins and was potentiated 1.5-fold by activation of G(i)/G(o)-coupled dopamine-D2S receptors, which had no effect alone. Following PTX pretreatment, only PTX-resistant Galpha(i)3, not Galpha(o) or Galpha(i)2, rescued GH-induced DAG/ceramide signaling. GH-induced DAG/ceramide formation was also blocked in cells expressing Gbetagamma blocker GRK-ct. In GH(4)ZR(7) cells, GH induced phosphorylation of JAK2 and STAT5, which was blocked by PTX and mimicked by ceramide analogue C2-ceramide or sphingomyelinase treatment to increase endogenous ceramide. We conclude that in GH(4) pituitary cells, GH induces formation of DAG/ceramide via a novel Galpha(i)3/Gbetagamma-dependent pathway. This novel pathway suggests a mechanism for autocrine feedback regulation by GH of pituitary function.     

Involvement of de novo ceramide synthesis in pro-inflammatory adipokine secretion and adipocyte–macrophage interaction

Interaction between adipocytes and macrophages has been suggested to play a central role in the pathogenesis of obesity. Ceramide, a sphingolipid de novo synthesized from palmitate, is known to stimulate pro-inflammatory cytokine secretion from multiple types of cells. To clarify whether de novo synthesized ceramide contributes to cytokine dysregulation in adipocytes and macrophages, we observed cytokine secretion in mature 3T3-L1 adipocytes (L1) and RAW264.7 macrophages (RAW) cultured alone or co-cultured under the suppression of de novo ceramide synthesis.Palmitate enhanced ceramide accumulation and stimulated the expression and secretion of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) in L1. The suppression of serine-palmitoyl transferase, a rate-limiting enzyme of de novo ceramide synthesis, by myriocin or siRNA attenuated those palmitate-induced alterations, and a ceramide synthase inhibitor fumonisin B1 showed similar results. In contrast, the inhibitor of sphingosine kinase or a membrane-permeable ceramide analogue augmented the cytokine secretion. Myriocin effects on the palmitate-induced changes were not abrogated by toll-like receptor-4 blockade. Although palmitate stimulated RAW to secrete tumor necrosis factor-α (TNF-α), it did not significantly increase ceramide content, and neither myriocin nor fumonisin B1 attenuated the TNF-α hypersecretion. The co-culture of L1 with RAW markedly augmented IL-6 and MCP-1 levels in media. Myriocin or fumonisin B1 significantly lowered these cytokine levels and suppressed the gene expression of TNF-α and MCP-1 in RAW and of IL-6 and MCP-1 in L1.In conclusion, de novo synthesized ceramide partially mediates the palmitate effects on pro-inflammatory adipokines and is possibly involved in the interaction with macrophages.     

Ceramide kinase regulates TNFα-stimulated NADPH oxidase activity and eicosanoid biosynthesis in neuroblastoma cells

A persistent inflammatory reaction is a hallmark of chronic and acute pathologies in the central nervous system (CNS) and greatly exacerbates neuronal degeneration. The proinflammatory cytokine tumor necrosis factor alpha (TNFα) plays a pivotal role in the initiation and progression of inflammatory processes provoking oxidative stress, eicosanoid biosynthesis, and the production of bioactive lipids. We established in neuronal cells that TNFα exposure dramatically increased Mg(2+)-dependent neutral sphingomyelinase (nSMase) activity thus generating the bioactive lipid mediator ceramide essential for subsequent NADPH oxidase (NOX) activation and oxidative stress. Since many of the pleiotropic effects of ceramide are attributable to its metabolites, we examined whether ceramide kinase (CerK), converting ceramide to ceramide-1-phosphate, is implicated both in NOX activation and enhanced eicosanoid production in neuronal cells. In the present study, we demonstrated that TNFα exposure of human SH-SY5Y neuroblastoma caused a profound increase in CerK activity. Depleting CerK activity using either siRNA or pharmacology completely negated NOX activation and eicosanoid biosynthesis yet, more importantly, rescued neuronal viability in the presence of TNFα. These findings provided evidence for a critical function of ceramide-1-phospate and thus CerK activity in directly linking sphingolipid metabolism to oxidative stress. This vital role of CerK in CNS inflammation could provide a novel therapeutic approach to intervene with the adverse consequences of a progressive CNS inflammation.     

Ceramide-dependent caspase 3 activation is prevented by coenzyme Q from plasma membrane in serum-deprived cells

Coenzyme Q (CoQ) is the key factor for the activity of the eukaryotic plasma membrane electron transport chain. Consequently, CoQ is essential in the cellular response against redox changes affecting this membrane. Serum withdrawal induces a mild oxidative stress, which produces lipid peroxidation in membranes. In fact, apoptosis induced by serum withdrawal can be prevented by several antioxidants including CoQ. Also, CoQ can maintain cell growth in serum-limiting conditions, whereas plasma membrane redox system (PMRS) inhibitors such as capsaicin, which compete with CoQ, inhibit cell growth and induce apoptosis. To understand how plasma membrane CoQ prevents oxidative stress-induced apoptosis we have studied the induction of apoptosis by serum withdrawal in CEM cells and its modulation by CoQ. Serum-withdrawal activates neutral sphingomyelinase (N-SMase), ceramide release and caspase-3-related proteases. CoQ addition to serum-free cultures inhibited a 60% N-SMase activation, an 80% ceramide release, and a 50% caspase-3 activity induced by serum deprivation. Caspase activation dependent on ceramide release since C 2 -ceramide was only able to mimic this effect in 10% foetal calf serum cultured cells but not in serum-free cultures. Also, in vitro experiments demonstrated that C 2 -ceramide and ceramide-rich lipid extracts directly activated caspase-3. Taken together, our results indicate that CoQ protects plasma membrane components and controls stress-mediated lipid signals by its participation in the PMRS.     

Neutral Sphingomyelinase in Physiological and Measles Virus Induced T Cell Suppression

T cell paralysis is a main feature of measles virus (MV) induced immunosuppression. MV contact mediated activation of sphingomyelinases was found to contribute to MV interference with T cell actin reorganization. The role of these enzymes in MV-induced inhibition of T cell activation remained equally undefined as their general role in regulating immune synapse (IS) activity which relies on spatiotemporal membrane patterning. Our study for the first time reveals that transient activation of the neutral sphingomyelinase 2 (NSM2) occurs in physiological co-stimulation of primary T cells where ceramide accumulation is confined to the lamellum (where also NSM2 can be detected) and excluded from IS areas of high actin turnover. Genetic ablation of the enzyme is associated with T cell hyper-responsiveness as revealed by actin dynamics, tyrosine phosphorylation, Ca²⁺-mobilization and expansion indicating that NSM2 acts to suppress overshooting T cell responses. In line with its suppressive activity, exaggerated, prolonged NSM2 activation as occurring in co-stimulated T cells following MV exposure was associated with aberrant compartmentalization of ceramides, loss of spreading responses, interference with accumulation of tyrosine phosphorylated protein species and expansion. Altogether, this study for the first time reveals a role of NSM2 in physiological T cell stimulation which is dampening and can be abused by a virus, which promotes enhanced and prolonged NSM2 activation to cause pathological T cell suppression.     

Ceramide 1-phosphate, a mediator of phagocytosis

The agonist-stimulated metabolism of membrane lipids produces potent second messengers that regulate phagocytosis. We studied whether human ceramide kinase (hCERK) activity and ceramide 1-phosphate formation could lead to enhanced phagocytosis through a mechanism involving modulation of the membrane-structural order parameter. hCERK was stably transfected into COS-1 cells that were stably transfected with the FcgammaRIIA receptor. hCERK-transfected cells displayed a significant increase in phagocytic index in association with increased ceramide kinase activation and translocation to lipid rafts after activation with opsonized erythrocytes. When challenged with opsonized erythrocytes, hCERK-transfected cells increased phagocytosis by 1.5-fold compared with vector control and simultaneously increased ceramide 1-phosphate levels 2-fold compared with vector and unstimulated control cells. Control and hCERK-transfected cells were subjected to cellular fractionation. Utilizing an antibody against hCERK, we observed that CERK translocates during activation from the cytosol to a lipid raft fraction. The plasma membrane-structural order parameter of the transfectants was measured by labeling cells with Laurdan. Cells transfected with hCERK showed a higher liquid crystalline order than control cells with stimulation, conditions that are favorable for the promotion of membrane fusion at the sites of phagocytosis. The change in the structural order parameter of the lipid rafts probably contributes to phagocytosis by promoting phagosome formation.     

Regulation of microsomal triglyceride transfer protein by apolipoprotein A-IV in newborn swine intestinal epithelial cells

Apolipoprotein (apo) A-IV overexpression enhances chylomicron (CM) assembly and secretion in newborn swine intestinal epithelial cells by producing larger particles (Lu S, Yao Y, Cheng X, Mitchell S, Leng S, Meng S, Gallagher JW, Shelness GS, Morris GS, Mahan J, Frase S, Mansbach CM, Weinberg RB, Black DD. J Biol Chem 281: 3473-3483, 2006). To determine the impact of apo A-IV on microsomal triglyceride transfer protein (MTTP), IPEC-1 cell lines containing a tetracycline-regulatable expression system were used to overexpress native swine apo A-IV and "piglike" human apo A-IV, a mutant human apo A-IV with deletion of the EQQQ-rich COOH-terminus, previously shown to upregulate basolateral triglyceride (TG) secretion 5-fold and 25-fold, respectively. Cells were incubated 24 h with and without doxycycline and oleic acid (OA, 0.8 mM). Overexpression of the native swine apo A-IV and piglike human apo A-IV increased MTTP lipid transfer activity by 39.7% (P = 0.006) and 53.6% (P = 0.0001), respectively, compared with controls. Changes in mRNA and protein levels generally paralleled changes in activity. Interestingly, native swine apo A-IV overexpression also increased MTTP large subunit mRNA, protein levels, and lipid transfer activity in the absence of OA, suggesting a mechanism not mediated by lipid absorption. Overexpression of piglike human apo A-IV significantly increased partitioning of radiolabeled OA from endoplasmic reticulum (ER) membrane to lumen, suggesting increased net transfer of membrane TG to luminal particles. These results suggest that the increased packaging of TG into nascent CMs in the ER lumen, induced by apo A-IV, is associated with upregulation of MTTP activity at the pretranslational level. Thus MTTP is regulated by apo A-IV in a manner to promote increased packaging of TG into the CM core, which may be important in neonatal fat absorption.     

Changes induced by formalin in the hypothalamic neurosecretory system of the spotted owlet, Athene brama temminck.

Histological changes induced in the HNS of the spotted owlet, Athene brama Temminck, by injection of 1 ml 5 or 10% formalin are described. No difference could be detected in the response of the HNS to 5 or 10% formalin administration. In the HNS of birds killed within 5 min of formalin administration, there was only partial depletion of NSM from the neurons, the tract and the NL; the quantity of NSM in the AME remained more or less unchanged. In animals killed 10-90 min after formalin injection, the depletion of NSM from the neurons, the tract and the NL was more complete. The neurons of the preoptic division of the SON exhibited the maximum response; these neurons were also moderately hypertrophied. The NL also was hypertrophied in some animals; the NSM in the AME registered only a partial loss. The interval between formalin administration and killing did not influence the degree of changes in the HNS. The depletion of NSM was no greater at 90 min following formalin injection than at 10 min. Since it is well established that formalin stress causes augmented secretion of ADH and that there is a close functional relationship existing between ADH and NSM, the depletion of NSM noticed in the HNS of the spotted owlet following formalin administration is interpreted as indicating augmented secretion of ADH. Hence it seems that the response of the HNS of birds to formalin stress are comparable to those of the HNS of mammals. The results thus provide histological evidence in favour of the concept that stressful stimuli cause increased secretion of ADH.     

Expression of the phospholipid-dependent Escherichia coli sn-1,2-diacylglycerol kinase in COS cells perturbs cellular lipid composition

The Escherichia coli sn-1,2-diacylglycerol (DAG) kinase has been successfully expressed in COS cells. The E. coli dgkA locus which contains the coding sequences for DAG kinase was subcloned into an eukaryotic expression vector, pMT2. COS cells transfected with the vector pMT2dgk expressed the DAG kinase as shown by Western analysis. Immunofluorescence studies revealed that the E. coli DAG kinase was prominently but not exclusively located in the endoplasmic reticulum. In addition, mixed micellar assays in beta-octyl glucoside revealed that membranes prepared from pMT2dgk-transfected COS cells contained over a 1500-fold increase in DAG kinase activity: 107 nmol/min/mg compared with only 0.067 nmol/min/mg for controls. DAG kinase activity from the E. coli enzyme was distinguished from endogenous COS cell activity based on differences in thermolability and the ability of the E. coli enzyme to use ceramide as a substrate. No ceramide kinase activity was detected in control COS cells, so the activity detected in pMT2dgk transfectants must have resulted from the expressed E. coli DAG kinase. The Km values for DAG kinase derived from E. coli and COS cells were nearly identical. Finally, transfected COS cells were labeled with [32P]Pi to investigate possible perturbations in lipid composition induced by the action of the E. coli DAG kinase. Ceramide (generated by the action of sphingomyelinase) was also used to clearly implicate the E. coli enzyme. Levels of ceramide phosphate increased more than 150-fold in pMT2dgk-transfected cells relative to controls. The results of these studies show that the E. coli enzyme expressed in COS cells is active and perturbs lipid composition in the intact cell system; the absolute lipid cofactor requirement of E. coli DAG kinase can be satisfied in COS cells.     

Adaptation to short-term extreme fat consumption alters intestinal lipid handling in male and female mice

The small intestine is a highly adaptable organ serving as both a barrier to the external environment and a conduit for nutrient absorption. Enterocytes package dietary triglycerides (TG) into chylomicrons for transport into circulation; the remaining TGs are stored in cytosolic lipid droplets (CLDs). The current study aimed to characterize the impact of diet composition on intestinal lipid handling in male and female wild-type mice. Mice were continued on their grain-based diet (GBD) and switched to either a high-fat, high cholesterol Western-style diet (WD) or a ketogenic diet (KD) for 3 or 5 weeks. KD-fed mice displayed significantly higher plasma TG levels in response to an olive oil gavage than WD- and GBD-fed mice; TG levels were ~2-fold higher in male KD-fed mice than female KD-fed mice. Poloxamer-407 experiments revealed enhanced intestinal-TG secretion rates in male mice fed a KD upon olive oil gavage, whereas secretion rates were unchanged in female mice. Surprisingly, jejunal CLD size and TG mass after oil gavage were similar among the groups. At fasting, TG mass was significantly higher in the jejunum of male KD-fed mice and the duodenum of female KD-fed mice, providing increased substrate for chylomicron formation. In addition to greater fasting intestinal TG stores, KD-fed male mice displayed longer small intestinal lengths, while female mice displayed markedly longer jejunal villi lengths. After 5 weeks of diet, 12 h fasting-2 h refeeding experiments revealed jejunal TG levels were similar between diet groups in male mice; however, in female mice, jejunal TG mass was significantly higher in KD-fed mice compared to GBD- and WD-fed mice. These experiments reveal that KD feeding promotes distinct morphological and functional changes to the murine small intestine compared to the WD diet. Moreover, changes to intestinal lipid handling in response to carbohydrate and protein restriction manifest differently in male and female mice.     

Induction of Membrane Ceramides: A Novel Strategy to Interfere with T Lymphocyte Cytoskeletal Reorganisation in Viral Immunosuppression

Silencing of T cell activation and function is a highly efficient strategy of immunosuppression induced by pathogens. By promoting formation of membrane microdomains essential for clustering of receptors and signalling platforms in the plasma membrane, ceramides accumulating as a result of membrane sphingomyelin breakdown are not only essential for assembly of signalling complexes and pathogen entry, but also act as signalling modulators, e. g. by regulating relay of phosphatidyl-inositol-3-kinase (PI3K) signalling. Their role in T lymphocyte functions has not been addressed as yet. We now show that measles virus (MV), which interacts with the surface of T cells and thereby efficiently interferes with stimulated dynamic reorganisation of their actin cytoskeleton, causes ceramide accumulation in human T cells in a neutral (NSM) and acid (ASM) sphingomyelinase–dependent manner. Ceramides induced by MV, but also bacterial sphingomyelinase, efficiently interfered with formation of membrane protrusions and T cell spreading and front/rear polarisation in response to β1 integrin ligation or αCD3/CD28 activation, and this was rescued upon pharmacological or genetic ablation of ASM/NSM activity. Moreover, membrane ceramide accumulation downmodulated chemokine-induced T cell motility on fibronectin. Altogether, these findings highlight an as yet unrecognised concept of pathogens able to cause membrane ceramide accumulation to target essential processes in T cell activation and function by preventing stimulated actin cytoskeletal dynamics.     

Disruption of lipid rafts stimulates phospholipase d activity in human lymphocytes: implication in the regulation of immune function

Recent evidence suggests that phospholipase D (PLD) can be regulated through its association/dissociation to lipid rafts. We show here that modifying lipid rafts either by cholesterol depletion using methyl-beta-cyclodextrin and filipin or by conversion of sphingomyelin to ceramide with exogenous bacterial sphingomyelinase (bSMase) markedly activated the PLD of human PBMC. bSMase was the most potent PLD activator, giving maximal 6- to 7-fold increase in PLD activity. Triton X-100-treated lysates prepared from control PBMC and from bSMase-treated cells were fractionated by centrifugation on sucrose density gradient. We observed that bSMase treatment of the cells induced a larger ceramide increase in raft than in nonraft membranes and displaced both the Src kinase Lck and PLD1 out of the raft fractions. In addition, the three raft-modifying agents markedly inhibited the lymphoproliferative response to mitogenic lectin. To examine further the potential role of PLD activation in the control of lymphocyte responses, we transiently overexpressed either of the PLD1 and PLD2 isoforms in Jurkat cells and analyzed the phorbol ester plus ionomycin-induced expression of IL-2 mRNA, which is one of the early responses of lymphocyte to activation. We observed a 43% decrease of IL-2 mRNA level in Jurkat cells overexpressing PLD1 as compared with mock- or PLD2-transfected cells, which indicates that elevated PLD1, but not PLD2, activity impairs lymphocyte activation. Altogether, the present results support the hypothesis that PLD1 is activated by exclusion from lipid rafts and that this activation conveys antiproliferative signals in lymphoid cells.     

Influences of hypothyroidism on lipid composition and inositol lipid-linked receptors responsiveness and protein kinase C (PKC) activity in the cerebral cortex of lewis rats

The influence of hypothyroidism (HO) induced by treatment with propylthiouracil on lipid composition, receptor responsiveness of MI-muscarinic receptors (M₁AChRs) and metabotropic glutamate receptors (mGluRs) as well as on protein kinase C (PKC) activity was investigated in the cerebral cortex of Lewis rats. HO did not influence the lipid composition. There was a significant 2-fold increase of efficacy and 6-fold decrease of potency of carbachol-induced inositol phosphate (IP) accumulation in HO, with respect to control rats. The efficacy of trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD)-induced IP accumulation was also higher in HO (by 50%), without differences in EC50 values. The activities of soluble calcium-dependent and calcium-independent PKC were higher in HO than in control rats (both roughly 30%); membrane-associated PKCs were not modified. The data indicate that HO induces an increased responsiveness of M₁AChRs and mGluRs and a rise in the soluble PKC activity that could be available and ready for translocation. Department of Metabolism and Pathological Biochemistry.