Lipid Droplets Flashcards
Name storage forms of lipids
Cholesterol esters
Triglycerides
Describe cholesterol esters - gen
Synthesized by acat in er membranes
Polar
To make hydrophobic =attach fatty acid
Describe triglycerides - gen
Fatty acid storage
Synthesized by dgat1, dgat2 in er membranes
Describe dgat - gen
diacylglycerol acyltransferase
= enzyme catalyzing the last step in The production of triglycerides
Triglycerides = highly insoluble, like Cholesterol esters, and must be sequestered into lipoprotein particles or Lipid droplets.
Dgat1 and 2 = do same thing but diff structures - dgat1= passes through membrane multiple time
Dgat2 = has hair pin loop= halfway through membrane
Describe acat - Gen
Found in diff cells
1 and 2 - found in intestines
Acyl-coenzyme a - cholesterol acid acyltransferase
Found in er memebranes - have to be in lipid belayer to make cholesterol esters
Makes cholesterol esters from cholesterol
Cholesterol —> cholesteryl oleate = remove h, storage form
Describe where Dgats found
In er membrane
Describe lipid droplets
Can be very large
Lipid monolahyer = stable in cytoplasm
Store them = formed outside lumen er in cytoplasm
Describe lipoprotein particles
Some cells
Found in er lumen
What are lipid droplets
Cytoplasmic organelles with phospholipid mono layer and a core consisting of triglycerides and cholesterol esters
Role = lipid storage
Interior hydrophobic
Could have integral membrane proteins but can’t have one that goes through bilayer - dgat2 can only
What cells can make lipid droplets
Almost all cells
Some specialized cells have more than others
Hepatocytes (temp storage then secreted as lipoprotein particles), epithelial cells in mammary glands = 2 other cell types that can contain numerous lipid droplets
Describe cytoplasm of adipocytes
Consists of single large lipid droplet - very big
Lipid droplets size
Much bigger than lipoprotein particles
Sizes range from 100nm —> several microns
Typically larger can see in lm
Describe structure and function of lipid droplet
Often in contact with er membrane - lipid flow can flow between both ways
Also with mito - also makes membrane lipids, mito needs fatty acids for beta oxidation = mito produces atp
Mono layer, tiny layer
Ratios of cholesterol esters and triglycerides diff - many cholesterol esters in hepatocytes, mostly all triglycerides in adipocytes
Describe labelling mammary gland with radioactive oleic acid
Incorporated and brought into lipid droplet quickly
Makes fat globule in milk = pushes through memervane
Does not have happen anywhere else
Describe ldl - process generally
Lipoprotein particle secreted form liver
Rich in cholesterol esters and have triglycerides
Circulate in blood stream - if low ldl =cells short on cholesterol and synthesize ldl receptor to bring it in
Describe ldl - pathologcally
Form where do not want = if cell has too much cholesterol or fatty acids = to avoid stress in er
Eat too much ice cream = all cells have enough cholesterol, so ldl particles circulate - stop producing ldl r
Bad = bc can oxidize - if circulate too long and damage ldl particle = macrophage scavenger receptors - take them up = eat ldl = damage then turn into foam cells - many lipid droplets - can be reversible but at a point it is not anymore = can hurt artery walls = bad, cholesterol builds up = atherosclerosis
Where does acat have to be
In lipid bilayer
Where do dgats have to be
Dgat1 = spans membrane multiple times
Dgat2 = only in one layer = can fit surface of lipid droplet
Where are acat and dgat found
In er membranes
What are lipid droplets produced from
Likely produced from er membranes -
Often show tight associations with er - so may grow by acquiring new triglycerides/cholesterol ester from er (can have membrane bridge between er and lipid droplet, dgat2 can move in = so can create more triglycerides even if not connected to er)
What can lipid droplets do with each other
Fuse with each other
What can lipids be connected to
Can still be connnected to er
Describe addition of new lipid to lipid droplets
Triglycerides = dgat1, dgat2
Cholesterol = acat1, acat2
Where does dgat 1 go
When cell loaded with triglycerides = dgat1 remains in er membranes
Where does dgat2 go
Moves to newly formed lipid droplet - diffuse while lipid still connected
Then stable on lipid proteins
New triglycerides made by dgat2 = will be incorporated preferentially into existing lipid droplets = reduce er stress
Describe dgat2 mapple in hela cells
Red fluorescent protein
Add oleic acid = induce lipid droplets = after 16 hrs = see droplets
2-3 microns across
Easy to see in lm
TISSUE CULTURE CELL
Describe visualizing lipid droplets - fluorescent dyes
Variety of fluorescent dyes that partition into hydrophobic environments
= Nile red and bodipy
Must work in both fixed and live cells
Describe visualizing lipid droplets - transmitted light
Oil red o - Sudan red
Describe visualizing lipid droplets - ‘em
Lipid droplets can be recognized by em with normal prep/staining
Describe visualizing lipid droplets - fluorescently tagged substrates
For dgat and acat = can also be sued - like bodipy cholesterol
Fluorescent tag = big as tagged molecule so category a bit risky
Describe visualizing lipid droplets - gfp tagged
Gfp tagged proteins that associate with lipid droplets = frequently used
Describe lipid droplet structure
Core of cholesterol ester and triglcuyerides
Probably phosphpylid monolaer on surface
What is found on surface lipid droplets
Family of proteins called pat proteins = attached to mono layer
Ex = perilipin, adrp, tip47
Rab18
On lipid droplets
Recruit tethering proteins to attach to er
Describe biochemical studies of lipid droplets = purifying
Purifying lipid droplets = easy
Break cells, pellet debris then float lipid droplets on sucrose gradient (density gradient centrifugation)
But lipid droplets often attach to er and mitochondria - so there will be significant contamination from these organelles
Describe biochemical studies of lipid droplets = if dissolve lipid droplets
If lipid droplets dissolved - with organic solvents = proteins can be extracted for western blot, proteomics analysis or other techniques
Describe biochemical studies of lipid droplets = tlc
Thin layer chromatography can be used to analyze lipid content of purified droplets
Describe pat proteins
Adrp and perilipin can be phosphrylated by protein kinase a (pka)
Gets lipids out of lipid droplets
Name pat protein mechanisms
Hormone sensitive lipase
Also autopahgy can work
Describe hormone sensitive lipase - pat protein mechanism
Hsl can bind to phoshorylated pat proteins where it can release free fatty acids from triglycerides
- brought to emmervane by phosphryaltion
what are other roles pat proteins
May also play structural role = stabilizing surface of lipid droplet
Retrieval of lipids from lipid droplets
- Triglycerides – HSL, ATGL
- Cholesterol – neutral cholesterol lipases? HSL? (Autophagy more effective)
- Both – autophagy, lysosomal acid lipase (LAL) (release free fatty acids from both triglycerides and cholesterols)
Retrieval of fatty acids - Hsl
Triggered by epinephrine/adrenaline
Take lipids out for energy boost
Hsl binds to phosphorylated pat proteins on lipid droplets and can release free fatty acids from triglycerides
Retrieval of fatty acids -Atgl
Another lipolytic enzyme that can bind to pat proteins on lipid droplets
Regulation unclear
Retrieval of fatty acids - associate with
Lipids often associate with mito, peroxiosmes or er
Retrieval of fatty acids - To mito
Fatty acids transferred to mito or peroxsiomes can undergo beta oxidation
Retrieval of fatty acids - To er
Fatty acids transferred to er can be incorporated into phospholipids - to make more membrane
Name the 2 enzymes that are ale to release free fatty acids from triglycerides
Hsl
Atgl
Describe whole process of hormone sensitive lipase
Beta adrenergic receptor activated - by addition of adrneline - epinephrine
A coupled G protein activated and camp produced
Camp activated pka
Pka phoshporylates Hsl in cytoplasm and some pat proteins - adrp or perilipin on surface of lipid droplets
Phosphorylated Hsl binds to pat proteins on surface of lipid droplet and hydrolzyes triglycerides = release free fatty acids
Targets of pka
Many
Describe retrieval of cholesterol
Hsl has some cholesterol esterase activity
But may be other cholesterol esterases in cell
Enzymes appear to be largely absent from macropahesg (but these cells have lots of cholesterol/lipid droplets) - macrophages are capable of exporting cholesterol via Hdl
Autophagy of lipid droplets = major mechanism for cholesterol esterase hydrolysis in these cells
Describe lipophagy
Possible to transfer lipid droplets to lysosomes by variant of autophagy = lipophagy
Lysosomes have abundant lapses and can liberate both free fatty acids and cholesterol - rab18
Describe lipophagy - exp
Test bu blocking lysosomal degradation (with weak bases) or by blocking autophagy (inhibitors like 3-methyladenine or Sirna ok of proteins essential for autophagy)
Cholesterol stuck in lipid droplets
Describe secretion of lipi droplet by mammary gland
How fat globules in milk produced
One of few sreetory processes that does not involve secretory pathway
Lipid droplet - little membrane around = pushed through apical surface of epithelial cell in mammary gland
Describe lipoproteins - image
Diff
Much smaller - produced form er membrane, goes to lumen er and traverse secretory pathway
Apolipoproetin b
Specialized cells - mostly in enterocytes (intestines) and hepatocytes
What does transport of lipids between cells
Primarily carried out by liprotein particle s
= cholesterol/triglycerides between cells in body
What can produce lipoprotein particles
Only enterocytes - intestines and hepatocytes - liver can produce lipoprotein particles - excluding hdl
Lipid from these particles can be transferred to many cell types
Where do lipids come from
Outside body - diet
Or be synthesized within body
Describe lipid stores under normal conditions
Major stores of excess lipids are adipocytes - triglycerides only
And liver - triglycerides and cholesterol
Describe lipid stores pathologically
Lipid can be deposited into tissues - artery walls = leading cause of death in western counters - major medical problem - atherosclerosis + diseases
Describe ex of lipoprotein particle
Vldl - produced in hepatocytes
Apolipoprotein b - translcoated into er large hydrophobic areas, transfer lipids
Same true for chylomriocns
Describe density gradient centrifugation of lipoprotein particles
Lightest = vldl —> idl, ldl (same lipoprotein particle at diff stages of life)
Hdl = most dense
Describe size chromatography lipoprotein particles
Hdl = smallest
Vldl = largest
Name and describe all lipoprotein particle s
- Chylomicrons – secreted by enterocytes, small intestine
- HDL – APOA secreted by liver, converted to HDL in periphery (circulates and graves Choelsterol and brings to liver)
- VLDL – secreted by liver
- IDL, LDL – produced by stripping triglycerides from VLDL particles
(Others - besides hdl = deliver lipids to diff parts of body)
Describe whole process - transfer of lipids and stuff between liver and lipoprotein particles
Eat ice cream - intestine —> lipids initially packaged into chylomcirons —> circulate and gradually becomes smaller —> chylomicron remnant —> then endocytosied by hepatocyte, then lover repackages into —> vldl —>idl —> ldl
Hdl = comes from liver —> mature hdl —> liver
What is a lipoprotein particle
Composed of one or more lipoprotein molecule associated with cholesterol esters - hdl only associated with cholesterol esters
And sometimes triglycerides - usually both
Produced by cells in intestines and liver
ApoA
Found in hdl
ApoB48
Chylomicrons
Same as apob100 but spliced different = missing domain = lacks region for binding ldl receptor - cannot bind
Can be reorganized by receptor on hepatocytes
ApoB100
Vldl, idl,ldl
Can bind sequence on ldl r
Size of apolipoproteins
- ApoA–<400AA
- ApoB48 – 2153 AA BIG!!!
- ApoB100 – 4485 AA BIG!!!
- ApoC – 57 AA
- ApoE – 299 AA
Sizes of lipoprotein particles
Chylomicrons = up to one micron (scecerted by enterocytes )
Chylomicron remnants - 30-50nm
Vldl - 30-8nm secreted by hepatocytes
Idl - 20-35nm
Ldl - 20nm - varies between ppl
Hdl - 7-12nm smallest
Describe chylomicrons - first step
Transport of apoB48 into er of enterocytes - absorb lipids from diet and repackage them into chylomicrons
Then lipidated in er and golgi with triglycerides and cholesterol obtained from diet
Secreted into lymph by enterocytes in small intestines
Describe chylomicrons - remnants
Some of triglycerides are converted to free fatt acids and removed by lipoprotein lipase and attached to endothelial cells —> chylomicron remnant
Endocsytsoed in liver and are enriched in cholesterol compared to go chylomicron
Describe chylomicrons - contain
ApOB48
Which cannot bind to ldl r
But also counting apoe= can bind to receptor in liver - endocytosus by hepatocytes
Describe chylomicrons - secreted from
Basal domain of inestimable epithelium
Continue to grow as moves through secretory pathway then secreted into lymph
Role of liver - acccumlate
Accumulate choelsterol and triglycerides from endocytosed chylomicron remnants from hdl
Can also make cholesterol if none in diet
Role of liver - fatty acids
Fatty acids in triglycerides can be liberated and transferred to mito for energy in any tissue
Role of liver - cholesterol
Once acccumualted = choelsterol can be re secreted in vldl particles, stored in lipid droplets or hepatcoytes or converted to bile salts = get rid
Choelsterol cannot be destroyed - only efficient way to get rid = bile slats, only happens in liver,excrete it
Small amounts of choelsterol also converted to steroid hormones - can also lose choelsterol in liver, hair, skin etc but small amount
Role of hepatocytes - endocytose
Endocytose chylomicron remnants and hdl
Choelsterol esters and triglycerides hydorlzyed in Lysosome and free choelsterol and fatty acids released
Role of hepatocytes - make new vldl
ApoB100
cholesterol must be re converted to choelsterol Estrs in er
Fatty acids must be added to glycerol to make new triglycerides - reform them
Move to golgi then secreted to cell surface
EASIER TO MOEV CHOLESTEROLAND FATTY ACIDS THAN CHOELSTEROL ESTER AND TRIGLYCERIDES
Role of hepatocytes - sequester
CHOELSTERol esters and triglycerides may also be sequestered in lipid droplets within hepatocyte - stored, fatty liver disease = bad
Vldl - describe
Repackaging choelsterol and triglycerides in liver - delivered by chylomicrons or hdl
Can be secreted as very low density lipoprotein - vldl
Vldl Contains
ApoB100 = full lengths
Can bind to ldl r
Initially ldl r binding domain masked - not exposed until sufficient lipid is removed
Vldl What happens to these particles
Particles will shrink as lipoprotein lipase in endothelial cells hydrolysis and removed triglycerides - more effective for triglycerides and not choelsterol
As it shrinks = becomes more enriched in cholesterol
Idl - describe
Vldl particles become more dense and smaller as triglycerides removed
Smaller particles = intermediate density lipoproteins then low density lipoproteins - apob100 unmasked once idl is converted to ldl
Most of original choelsterol still in ldl particle but most of triglycerides have been removed
Describe ldl particles - taken up
Can be taken up into cells by binding ldl receptor
Major mechanism by which choelsterol delivered to cells in body
Describe ldl particles - too much ldl
Too much circulating ldl in blood too long = bad = eventually degrades - forms oxidized ldl
No endocytosis bc stop producing ldl receptor
Describe ldl particles - degraded ldl
Degraded LDL can be accumulated by binding to “scavenger receptors” on macrophages. However,
macrophages in artery walls can be overloaded and transformed into pathogenic “foam cells” or even killed. This is believed to be one of the initial steps in forming atheroschlerotic lesions if goes on for years
Describe genetic diseases involving excess ldl
Category 1 – No LDL receptors synthesized, Category 2 – Receptor stuck in ER (never reaches cell surface), Category 3 – Receptor reaches cell surface but fails to bind LDL
Category 4= Receptor binds LDL but fails to enter clathrin coated pits. Three mutations led to truncation of the tail, but one mutation substituted a cysteine for a
tyrosine. This led eventually to the identification of NPVY as an internalization signal for LDL receptor (tyrosine converted to cysteine) - binds arh, endocytosis not efficient - bc will not be brought to clathrin coated pit
Describe Hdl - what
ApoA secreted by liver - no lipid in it
ApoA = major apoprotein in hdl
Addition of cboelsterol in peripheral tissues is sufficient to convert ApoA to hdl - circulates
ApoA/hDL = can extract cholesterol from cells, chylomicrons remnants or lipoprotein particles
Describe Hdl -binds
Abca1 choelsterol transporter on cell surface in periphery - transfers cholesterol to hdl and once hdl full = circulates
Hdl particles then endocytosed by liver
(High ldl = bad, high hdl = associated with lower heart disease)- transfers choelsterol from peripheral cells in body to liver
Describe Hdl - abca1 transporter
Multiple Tm protein
cell disposes of unwanted choelsterol
Describe tangier disease
If no hdl =
* Results from mutation in ABCA1
* Fat accumulation in tissues
* Atherosclerosis
* Very low plasma HDL (since ApoA1 cannot be lipidated)
Describe bile acids
Cholesterol in liver can be converted to bile acids and secreted
Bile acid synthesis in liver
Bile acids - once secreted can efficiently re absorbed in intestines and recyled back to liver = reduce net loss of choelsterol from body - primary way
(Also pb = blocks resabsorption bile acids - implies eating pb = leads to more loss of choelsterol from body)
Describe transport of cholesterol inside cells
Choelsterol can be brought in from outside by endocytosed of ldl particles
Srebp = acts as cholesterol sensor, triggering production of ldl receptors when choelsterol levels are low
Choelsterol can also be synthesized de novo when cellular levels low - process much more efficient in hepatocyte compared to other cell
Describe niemann pick transporter
Endocytosis ldl - choelsterol arrives in lysosome in forms of cholesterol ester - then brown down but hydrophobic do
Niemann pick transporter takes choelsterol through membrane
= exports cholesterol from late endosome/lysosome to cytoplasm
Choelsterol not water soluble but can bind to cytosolic lipid transfer proteins such as stard4
Cytosolic cholesterol can be transferred directly to er or other membranes
Describe Niemann-Pick disease type C
- Lysosomal storage disease
- Types A and B result in mutations in sphingomyelinase
- Type C results from inability to transport cholesterol out of lysosomes
(Swollen lysosomes with much choelsterol in them - developmental issues, similar to icd)
Apoc
Small lipoprotein found in hdl
Sometimes other lipoprotein particles
Apoe
Chylomicrons, binds to receptor in liver
