Regulation of Lipid Homeostasis: Lipid Droplets Bernd Helms Article Brand Recter Hendrik Mertens 1
The Basics I: FAs The Basics II: FA Activation 2
Basics III: TG-FA Interplay. Why? Adipocytes 3
Foam cells Pathologies associated with lipid droplets Obesity - mice with k.o. of structural protein, perilipin, or signaling molecule caveolin-1, are resistant to high fat induced obesitas Atherosclerosis accumulation of cholesterol loaded droplets in foam cells. Cells transform from lipid-storing into synthetic form during liver injury and repair Hepatitis/Fatty liver - hepatitis c virus core protein is associated with lipid droplets Mutation in lipid droplet-associated protein CGI-58 causes lipid storage disease Dorfman-Chanarin syndrome 4
What are Lipid Droplets? Lipid droplets, general principles Excess of lipids uptake, synthesis, breakdown membranes Lipid modifying enzymes Phospholipid monolayer Free fatty acid Free cholesterol Retinol TAG Cholesterol-ester Retinyl-ester Decreased usage or export 5
Lipid droplets, general principles Structural proteins Phospholipid monolayer Free fatty acid Free cholesterol Retinoic acid TAG Cholesterol-ester Retinyl-ester Lipid modifying enzymes Structural lipid droplet proteins: PAT domain proteins in mammalian cells: -Perilipin (adipocytes) -Adipophilin / ADRP -TIP47 -S3-12 -Oleosins(plant) - Hepatitis C core protein (oleosin-like domain) 6
Structural lipid droplet proteins adipocyt Wolins et al (2006) FEBS Lett 7
Lipid Droplets and Lipid Metabolism D.A. Brown, Current Biol. 2001 Structural lipid droplet proteins are also involved in the degradation of lipid droplets. AR(1) AR(3) TNF AR(2) TNF i q i AC PLC IP3 ERK 1,2 AR(1,2) DAG s PKC TNF Ca ++ ER Ca ++ camp 5 AMP TNF TNF PDE3b mrna Nucleus ERK 1,2 PKA PDE-3B Ca 2+ 5 AMPK ALBP HSL HSL ATGL Perilipins PKB P PDK1,2 PI3K p110 p85 IRS1 Insulin NEFA + Glycerol FABP + NEFA Perilipins TAG ERK 1,2 TNF 8
Release of Free Fatty Acids (FFA) from adipocyte lipid droplets Perilipin KO mice show 10x elevated lipolysis reaction and are resistant to high fat diet Londos et al, 2005 Biochemie Many cells have lipid droplets Lipid droplets as dynamic organelles? 9
Proteomics Analysis of Lipid Droplets LDs: more than just storage organelles Liu et al (2004) JBC 10
Lipid droplets, more than just fat storage organelles. Structural proteins Phospholipid monolayer Free fatty acid Free cholesterol Retinoic acid Lipid modifying enzymes TAG Cholesterol-ester Retinyl-ester Signaling molecules Trafficking - distribution or uptake of lipids Regulation of lipid modifying enzymes/ structural proteins Formation/budding lipid droplets How do Lipid Droplets form? 11
Overexpression of Rab18 induces lipid droplet - ER contact sites Control Rab18 S. Ozeki et al (2005) JCS "Classical" model Martin & PArton (2006) Nat. Rev Mol Cell Biol 12
Brown (2001)Cur biol Lipid Droplet Formation: 'Budding model' Wolins et al (2006) FEBS Lett 13
"Classical" model Lens-like structure Martin & PArton (2006) Nat. Rev Mol Cell Biol Alternative model: Adipophilin-enriched ER domains 14
Alternative model: Adipophilin-enriched ER domains Methods to study lipid droplets in vitro 15
A Lipid Droplet Visualization B CHO-wt CHO-mutant Nile Red Bodipy LDs Rab18 Actin DAPI - slow quenching Live cell imaging - no spectral overlap Multicolor imaging Spandl et al (2009) Traffic 16
Induction of LD formation Oleic Acid (18:1) CHO-K1 Bodipy α-adrp CHO-K1 24 hr oleate Bodipy α-adrp Lipidomics 4000QTRAP API-3000 2000QTRAP 17
Extracted 339 = MAG-18:1 TAG-18:1,18:1,18:1 WT CHO-K1 + Oleate DAG-18:1,18:1 TAG-16:1,18:1,18:1 TAG-18:0,18:1,22:5 TAG-18:1,18:1,18:2 TAG-18:1,18:1,22:5 TAG-18:1,18:1,22:6 TAG-16:0,18:1,18:1 TAG- 18:1,18:1,20:1 TAG- 18:0,18:1,18:1 TAG 16:0, 18:0,18:1 WT CHO-K1 + Oleate contains 18-1 Cholesterol -esters 18
Lipid droplets, more than just fat storage organelles. Structural proteins Phospholipid monolayer Free fatty acid Free cholesterol Retinoic acid Lipid modifying enzymes TAG Cholesterol-ester Retinyl-ester Signaling molecules Trafficking - distribution or uptake of lipids Regulation of lipid modifying enzymes/ structural proteins Formation/budding lipid droplets For example... Maternal Histones in Drosophila Eggs Cermelli et al (2006) Curr. Biol. 19
Refugee Proteins on Lipid Droplets Welte et al. (2007) Trends Cell Biol Lipid Droplets and Protein Availability Sequestration Garbage Dump Chaperone Vehicle Welte et al (2007) TICB 20
Function Lipid Droplets Storage FAs (prevention lipotoxicity) Energy Source ( -oxidation Fatty Acids) --------------------------------- Cholesterol and Lipid Homeostasis Cellular Signaling Storage Place for Refugee Proteins Size and Distribution are now recognized as important factors 21