Lipid Second Messengers: Lessons from SphingolipidsIan Mitchelle De VeraLipids OverviewLipids Overview•Functional classes of lipids:Functional classes of lipids:1. Structural lipids Æ membrane lipids (5‐10% dry mass); barriers aroundcells(510% dry mass); barriers around cells and cellular compartments.2Storage lipidsÆ80% ofadipocytemass; passive2.Storage lipids Æ80% of adipocytemass; passive role in cell until enzymatically oxidized. 3Messenger lipidsÆactive role in metabolic3.Messenger lipids Æactive role in metabolic traffic; potent signals; intracellular messengers.Examples of Lipid Second MessengersExamples of Lipid Second Messengers(Prokazova, et al, 2007).Hormone‐sensitivePhospholipase C in plasma membraneFigure 1. Phosphatidylinositols in cellular regula tion (redrawn from Nelson and Cox, 2005).Figure 2. Structure and generation of lipid second messengers. a) Generation of IP3, DAG, and P tdIns(3,4,5)P3 from PtdIns(4,5)P2 (Prokazova, et al, 2007).Figure 2. Structure and generation of lipid second messengers. b) Generation of ceramide from sphingomyelin. (Prokazova, et al, 2007).Figure 3. When ceramide serves as a precursor to other sphingolipids, the major site of modification within the molecule is at the C1 hydroxyl group (Perry and Hannun, 1998)SMase:SphingomyelinaseCdase:CeramidaseCeramidaseSK:Sphingosine kinaseCAPP:Ceramide‐activatedPhosphataseIGF:Insulinlike growthInsulin‐like growthFactorIL‐1:Interleukin‐1oxLDL :Oxidized low‐Density lipoproteinPDGF:PDGF:Protein‐derived Growth factorPKC (H, B):ikiCNumbers in brackets indicate relative levelsProtein kinaseCYPK:Yeast protein kinaseFigure 4.(Hannun and Obeid, 2008)Fig 5Fig 5.(Hannun and Obeid, 2008)Fig 6MAM: mitochondria‐associatedmembranesCer : ceramideGlucer: glucosylceramideSM:sphingomyelinSM: sphingomyelinGSL: glycosphingolipidsSph: sphingosineSK: sphingosine kinaseS1Phi i1hhtS1P: sphingosine‐1‐phosphate3K: 3‐keto‐dh: dihydro‐CERT: ceramide transfer proteinpFAPP2: a type of transfer proteinDe novo synthesis of sphingolipidsoccurs in endoplasmic reticulumoccurs in endoplasmic reticulum(ER) or ER‐associated membranes such as MAMs.Cer formed in this compartmentis transported to the Golgi whereSM and GluCer are synthesized.Classes of Bioactive Lipids Based on hlBiophysical Properties 1. Carry ionic/zwitterionic charge at neutral pH and contain two hydrophobic chains (i.e. ceramide‐1‐phosphate, SM)Æ reside in compartment of generationÆunlikely to flip spontaneously acrossbilayersÆunlikely to flip spontaneously across bilayers2. Neutral, two hydrophobic chains (i.e. ceramide)Æ restricted to compartment of generationÆ readily flip‐flop across membranes3. Single‐chain lipids (i.e. sphingosine, sphingosine‐1‐phosphate,lyso‐sphingomyelin)lysosphingomyelin)Æ sufficient aqueous solubility to move between membranesÆ exert surfactant activity; therefore, lowest cellular levelsd li idamong second messenger lipids.Æ if zwitterionic (i.e S1P), unlikely to flip‐flop across membranesSphingolipids must interact with relevant direct mediators to effect functional responsesto eff ect functional responses.Example: S1P (inner) Æ tumor necrosisfactor‐alphaS1P (outer) Æ S1P receptorABC transporters regulate S1P transport such as:CFTR:cystic fibrosis transCFTR:cystic fibrosis trans‐membrane regulatorfor S1P “entrance”ABCC1: S1P “exit door”ER Golgi(CERT)Fig 7.Transport and transbilayer movement of sphingolipids in model membranes (Hannun and Obeid, 2008)Cera mide flip with ease, butCer microdomains formation results to restricted flippingPhysiological levels of lipids influence their mechanisms of results to restricted flipping.Immense eff ects to signalling.Cer (in) function not equal to Cer (out) function.action: SM (30,000); Cer(3,000); Sph (100); S1P (1)1. Trace lipids (i.e. S1P) Æ interact with high‐affinity receptors2. Intermediate concentration lipids (i.e. Cer) Æ targets with intermediate affinityintermediate affinity3. Abundant lipids (i.e. SM) Æ change overall membrane properties and substructure when their levels change. Excesspalmitateincreasesde novosynthesis ofceramide(Cer)Figure 8. Example of ceramide signaling pathways (Hannun and Obeid, 2008)Excess palmitateincreases de novo synthesis of ceramide(Cer)Higher Cer Æ activate protein phosphatase 2A (PP2A) Æ Inhibit AKTExcess Cer Æ activate PP1 phosphatase Æ cytotoxic and apoptotic responseAKT: key mediator in insulin signaling and metabolic controlAKT: key mediator in insulin signaling and metabolic controlPhysical result: deterioration in insulin responsiveness and death of islet cells.Inhibition of Cer synthesis prevented insulin resistance and obesity (in mice). Summary of Properties of Lipid Second Messengers• Lipid signaling composed of: regulated enzyme, bioactive lipid, and specific downstream targets.• Complex metabolic interconnections of bioactive lipids.• Lipids formed by different mechanisms in distinct p ycompartments, by necessity, ex ert different functions.• “The outlined biophysical and metabolic considerations introduce a level of complexity not seen in other signaling pathways”. (Hannun and Obeid, 2008)Take Home MessagesTake Home Messages• Broad spectrum of bioactive lipids reversed our assumptions about the putative functions of lipid metabolism and roles of minor lipids.“Bioactive until proven otherwise.”•Sphingolipid‐mediated pathways operate at the•Sphingolipid‐mediated pathways operate at the level of individual organelles. E i th b th d•Even in the membrane, the second messenger lipid occasionally regulate different functions ddi hth it i fdi th idepending on whether it is formed in the inner or outer leaflet.Take Home MessagesTake Home Messages• Complexity of sphingolipid metabolism provide the cell with a rich repertoire for not only generating multiple signals but also for fine‐tuning specific responses. •More work is needed to dissect each of theMore work is needed to dissect each of the regulated pathways of bioactive sphingolipids. •“The study of bioactive lipids is only now coming•The st udy of bioactive lipids is only now coming to the forefront of cell biology…” (Hannun and Obeid 2008)Obeid, 2008). ReferencesReferencesHannun, Y. A . and Obeid L.M.“Principles of bioactive lipidsignalling:Hannun, Y. A . and Obeid L.M. Principles of bioactive lipid