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Activation of sphingomyelin synthesis by oxysterol binding protein involves phosphatidylinositol...
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CPL 3739 1–20 Abstracts / Chemistry and Physics of Lipids xxx (2008) xxx–xxx 9 plasma membrane polarity, and suggest that sphingolipids, their regulated metabolism, and cholesterol are involved at multiple levels. doi:10.1016/j.chemphyslip.2008.05.023 PL 12 The phosphoinositides in membrane trafficking in health and disease Giovanni D’Angelo, Mariella Vicinanza, Maria Antonietta De Matteis Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro, Chieti, Italy The phosphoinositides (PIs) are quantitatively minor components of cell membranes that derive from the reversible phosphoryla- tion of three of the hydroxyl groups of the inositol headgroup of PtdIns. The list of cell processes regulated by the PIs is steadily increasing, with recently recognized roles in transcription and sph- ingolipid metabolism in addition to the established ones in cell growth, actin cytoskeleton remodelling, and membrane trafficking. The importance of the PIs is highlighted by the serious conse- quences for an organism with genetic defects that affect enzymes involved in their metabolism. The versatile and key role of the PIs in so many important functions is mainly due to their fast and highly regulated interconversion. It is now clear that the different PIs serve not only as intermediates in the synthesis of the higher phosphorylated species, but also as active regulators of different protein targets in their own right. This is achieved through the direct binding to specific PIs of these protein targets via specific domains, such as the PH, FYVE, PX, ENTH domains. This is the case, for instance, for PtdIns4P, which for many years was considered as just the precursor of PtdIns(4,5)P 2 . Over the last decade, however, a large body of evidence has accumulated that shows that PtdIns(4)P is indeed a direct regulator of important cell functions, including membrane trafficking at the Golgi complex and regulation of lipid- transfer proteins involved in sphingolipid synthesis, such as CERT and FAPP2. CERT and FAPP2 belong to a family of lipid-transfer pro- teins that includes OSBP (the ‘COFs’): the members of this family share a common domain organization, with a closely homologous PH domain at their N-terminus, and a distinct additional lipid- binding/transfer domain at their C-terminus. Their homologous PH domains interact with PtdIns(4)P and ARF, and are responsible for the association of the COFs with the Golgi complex, while their dis- tinct lipid-transfer domains bind ceramide, oxysterols/cholesterol and glucosylceramide in CERT, OSBP and FAPP2, respectively. doi:10.1016/j.chemphyslip.2008.05.024 SO 10 Activation of sphingomyelin synthesis by oxysterol binding pro- tein involves phosphatidylinositol 4-kinase activation at the Golgi Sangeeta Banerji , Neale D. Ridgway Depts. Pediatrics and Biochemistry & Molecular Biology, Atlantic Research Centre, Dalhousie University, 5849 University Ave, Halifax, Nova Scotia B3H 4H7, Canada Oxysterols are important regulators of cholesterol homeostasis and are implicated in the genesis of diseases such as arteriosclerosis. Cholesterol and its oxysterol derivatives bind to a cytosolic recep- tor termed oxysterol binding protein (OSBP), which belongs to a mammalian gene family of 12 members that regulate cell signaling, vesicle transport and lipid synthesis. OSBP is targeted to the Golgi apparatus after stimulation of cells with 25-hydroxycholesterol (25OH). OSBP translocation triggers Golgi recruitment of ceramide transfer protein (CERT) leading to increased sphingomyelin syn- thesis. The mechanism for CERT translocation is unknown. Our hypothesis is that OSBP translocation increases Golgi phos- phatidylinositol 4-phosphate (PI4P) content and this promotes translocation of CERT via its pleckstrin homology domain. To test this, CHOK1 cells were treated with 25OH or solvent and isolated Golgi were assayed for PI4 kinase activity. A transient 50% increase in Golgi-associated PI4 kinase activity was observed after 30 min stimulation with 25OH that was dependent on OSBP expression. An increase of Golgi PI4P levels can be initiated by the activation of PI4 kinase II or III. Knockdown of PI4 kinase III by RNAi did not lead to reduced basal or 25OH-stimulated SM synthesis in CHOK1 cells, indicating that the kinase was not required for OSBP-mediated CERT activation. Knockdown of PI4 kinase II on the other hand diminished 25OH-mediated activation of SM syn- thesis. Ongoing experiments suggest that OSBP regulates PI4 kinase II by controlling Golgi cholesterol levels. In conclusion, SM syn- thesis is regulated by sterol-signaling through OSBP leading to PI4 kinase activation at the Golgi. doi:10.1016/j.chemphyslip.2008.05.025 PL 13 Session 4: Fatty acids, lipids and the metabolic syndrome Lipid metabolism and mitochondrial function in the metabolic syndrome A. Bonen 1 , G.P. Holloway 1 , L.L. Spriet 1 , J.F.C. Glatz 2 , J.J.F.P. Luiken 2 1 Department of Human Biology and Health, University of Guelph, Guelph, Canada 2 Department of Molecular Genetics, Maastricht University, Maas- tricht, The Netherlands Insulin resistance in heart and muscle is linked to the dysregulation of fatty acid metabolism, as intramuscular lipid metabolites inter- fere with the activation of the insulin-signalling pathway. Excess lipid accumulation is thought to be due to a reduced fatty acid oxidation and/or an altered rate of fatty acid uptake. Fatty acid entry into many tissues occurs via a protein-mediated mechanism (for review see Bonen et al., 2007) involving fatty acid translocase/CD36 (FAT/CD36) and plasma membrane associated fatty acid binding protein (FABPpm). These fatty acid transport pro- teins can be induced to translocate by contraction and insulin in muscle and the heart via activation of phosphoinositol 3-kinase (PI3-kinase) (for review see Bonen et al., 2007). In animal models of insulin resistance (Coort et al., 2004; Han et al., 2007; Luiken et al., 2001), as well as in human obesity and type 2 diabetes (Bonen et al., 2004), fatty acid transport is increased, due to a permanent relocation of FAT/CD36 to the plasma membrane, without altering its protein expression. FAT/CD36 is also present in mitochondria in rat (Campbell et al., 2004) and human muscle (Holloway et al., 2006), where this protein contributes to the regulation of fatty acid oxidation. In insulin resistant obese muscles FAT/CD36 but not FABPpm traf- ficking is impaired (Han et al., 2007), and mitochondrial FAT/CD36 is increased, concomitantly with the upregulation of mitochon- drial fatty acid oxidation (Holloway et al., 2007). Improvements in skeletal muscle insulin sensitivity have been linked to a PGC-
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Transcript of Activation of sphingomyelin synthesis by oxysterol binding protein involves phosphatidylinositol...
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Abstracts / Chemistry and Phplasma membrane polarity, and suggest that sphingolipids, theirregulated metabolism, and cholesterol are involved at multiplelevels.
doi:10.1016/j.chemphyslip.2008.05.023
PL 12
The phosphoinositides in membrane trafficking in health anddisease
Giovanni D’Angelo, Mariella Vicinanza,Maria Antonietta De Matteis
Department of Cell Biology and Oncology, Consorzio Mario Negri Sud,66030 Santa Maria Imbaro, Chieti, Italy
The phosphoinositides (PIs) are quantitatively minor componentsof cell membranes that derive from the reversible phosphoryla-tion of three of the hydroxyl groups of the inositol headgroup ofPtdIns. The list of cell processes regulated by the PIs is steadilyincreasing, with recently recognized roles in transcription and sph-ingolipid metabolism in addition to the established ones in cellgrowth, actin cytoskeleton remodelling, and membrane trafficking.The importance of the PIs is highlighted by the serious conse-quences for an organism with genetic defects that affect enzymesinvolved in their metabolism. The versatile and key role of the PIsin so many important functions is mainly due to their fast andhighly regulated interconversion. It is now clear that the differentPIs serve not only as intermediates in the synthesis of the higherphosphorylated species, but also as active regulators of differentprotein targets in their own right. This is achieved through thedirect binding to specific PIs of these protein targets via specificdomains, such as the PH, FYVE, PX, ENTH domains. This is the case,for instance, for PtdIns4P, which for many years was considered asjust the precursor of PtdIns(4,5)P2. Over the last decade, however, alarge body of evidence has accumulated that shows that PtdIns(4)Pis indeed a direct regulator of important cell functions, includingmembrane trafficking at the Golgi complex and regulation of lipid-transfer proteins involved in sphingolipid synthesis, such as CERTand FAPP2. CERT and FAPP2 belong to a family of lipid-transfer pro-teins that includes OSBP (the ‘COFs’): the members of this familyshare a common domain organization, with a closely homologousPH domain at their N-terminus, and a distinct additional lipid-binding/transfer domain at their C-terminus. Their homologous PHdomains interact with PtdIns(4)P and ARF, and are responsible for
the association of the COFs with the Golgi complex, while their dis-tinct lipid-transfer domains bind ceramide, oxysterols/cholesteroland glucosylceramide in CERT, OSBP and FAPP2, respectively.doi:10.1016/j.chemphyslip.2008.05.024
SO 10
Activation of sphingomyelin synthesis by oxysterol binding pro-tein involves phosphatidylinositol 4-kinase activation at theGolgi
Sangeeta Banerji, Neale D. Ridgway
Depts. Pediatrics and Biochemistry & Molecular Biology, AtlanticResearch Centre, Dalhousie University, 5849 University Ave, Halifax,Nova Scotia B3H 4H7, Canada
Oxysterols are important regulators of cholesterol homeostasis andare implicated in the genesis of diseases such as arteriosclerosis.Cholesterol and its oxysterol derivatives bind to a cytosolic recep-tor termed oxysterol binding protein (OSBP), which belongs to amammalian gene family of 12 members that regulate cell signaling,
f Lipids xxx (2008) xxx–xxx 9
vesicle transport and lipid synthesis. OSBP is targeted to the Golgiapparatus after stimulation of cells with 25-hydroxycholesterol(25OH). OSBP translocation triggers Golgi recruitment of ceramidetransfer protein (CERT) leading to increased sphingomyelin syn-thesis. The mechanism for CERT translocation is unknown. Ourhypothesis is that OSBP translocation increases Golgi phos-phatidylinositol 4-phosphate (PI4P) content and this promotestranslocation of CERT via its pleckstrin homology domain. To testthis, CHOK1 cells were treated with 25OH or solvent and isolatedGolgi were assayed for PI4 kinase activity. A transient 50% increasein Golgi-associated PI4 kinase activity was observed after 30 minstimulation with 25OH that was dependent on OSBP expression.An increase of Golgi PI4P levels can be initiated by the activationof PI4 kinase II� or III�. Knockdown of PI4 kinase III� by RNAidid not lead to reduced basal or 25OH-stimulated SM synthesisin CHOK1 cells, indicating that the kinase was not required forOSBP-mediated CERT activation. Knockdown of PI4 kinase II� onthe other hand diminished 25OH-mediated activation of SM syn-thesis. Ongoing experiments suggest that OSBP regulates PI4 kinaseII� by controlling Golgi cholesterol levels. In conclusion, SM syn-thesis is regulated by sterol-signaling through OSBP leading to PI4kinase activation at the Golgi.
doi:10.1016/j.chemphyslip.2008.05.025
PL 13
Session 4: Fatty acids, lipids and the metabolic syndrome
Lipid metabolism and mitochondrial function in the metabolicsyndrome
A. Bonen 1, G.P. Holloway 1, L.L. Spriet 1, J.F.C. Glatz 2,J.J.F.P. Luiken 2
1 Department of Human Biology and Health, University of Guelph,Guelph, Canada2 Department of Molecular Genetics, Maastricht University, Maas-tricht, The Netherlands
Insulin resistance in heart and muscle is linked to the dysregulationof fatty acid metabolism, as intramuscular lipid metabolites inter-fere with the activation of the insulin-signalling pathway. Excesslipid accumulation is thought to be due to a reduced fatty acid
CPL 3739 1–20
oxidation and/or an altered rate of fatty acid uptake.Fatty acid entry into many tissues occurs via a protein-mediated
mechanism (for review see Bonen et al., 2007) involving fatty acidtranslocase/CD36 (FAT/CD36) and plasma membrane associatedfatty acid binding protein (FABPpm). These fatty acid transport pro-teins can be induced to translocate by contraction and insulin inmuscle and the heart via activation of phosphoinositol 3-kinase(PI3-kinase) (for review see Bonen et al., 2007). In animal modelsof insulin resistance (Coort et al., 2004; Han et al., 2007; Luiken etal., 2001), as well as in human obesity and type 2 diabetes (Bonenet al., 2004), fatty acid transport is increased, due to a permanentrelocation of FAT/CD36 to the plasma membrane, without alteringits protein expression.
FAT/CD36 is also present in mitochondria in rat (Campbell etal., 2004) and human muscle (Holloway et al., 2006), where thisprotein contributes to the regulation of fatty acid oxidation. Ininsulin resistant obese muscles FAT/CD36 but not FABPpm traf-ficking is impaired (Han et al., 2007), and mitochondrial FAT/CD36is increased, concomitantly with the upregulation of mitochon-drial fatty acid oxidation (Holloway et al., 2007). Improvementsin skeletal muscle insulin sensitivity have been linked to a PGC-
![Sphingomyelin Liposomes Containing Porphyrin phospholipid ...phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG-2K, Avanti #880120P), and Sphingomyelin (SPM, # Coatsome](https://static.fdocuments.net/doc/165x107/5f3f9b782f336f6958157d47/sphingomyelin-liposomes-containing-porphyrin-phospholipid-phosphoethanolamine-n-methoxypolyethylene.jpg)
