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ANAT 262 > Lipid Droplets > Flashcards

Lipid Droplets Flashcards

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1
Q

Name storage forms of lipids

A

Cholesterol esters
Triglycerides

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2
Q

Describe cholesterol esters - gen

A

Synthesized by acat in er membranes
Polar
To make hydrophobic =attach fatty acid

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3
Q

Describe triglycerides - gen

A

Fatty acid storage
Synthesized by dgat1, dgat2 in er membranes

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4
Q

Describe dgat - gen

A

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

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5
Q

Describe acat - Gen

A

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

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6
Q

Describe where Dgats found

A

In er membrane

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7
Q

Describe lipid droplets

A

Can be very large
Lipid monolahyer = stable in cytoplasm
Store them = formed outside lumen er in cytoplasm

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8
Q

Describe lipoprotein particles

A

Some cells
Found in er lumen

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9
Q

What are lipid droplets

A

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

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10
Q

What cells can make lipid droplets

A

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

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11
Q

Describe cytoplasm of adipocytes

A

Consists of single large lipid droplet - very big

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12
Q

Lipid droplets size

A

Much bigger than lipoprotein particles
Sizes range from 100nm —> several microns
Typically larger can see in lm

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13
Q

Describe structure and function of lipid droplet

A

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

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14
Q

Describe labelling mammary gland with radioactive oleic acid

A

Incorporated and brought into lipid droplet quickly
Makes fat globule in milk = pushes through memervane
Does not have happen anywhere else

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15
Q

Describe ldl - process generally

A

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

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16
Q

Describe ldl - pathologcally

A

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

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17
Q

Where does acat have to be

A

In lipid bilayer

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18
Q

Where do dgats have to be

A

Dgat1 = spans membrane multiple times
Dgat2 = only in one layer = can fit surface of lipid droplet

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19
Q

Where are acat and dgat found

A

In er membranes

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20
Q

What are lipid droplets produced from

A

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)

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21
Q

What can lipid droplets do with each other

A

Fuse with each other

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22
Q

What can lipids be connected to

A

Can still be connnected to er

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23
Q

Describe addition of new lipid to lipid droplets

A

Triglycerides = dgat1, dgat2
Cholesterol = acat1, acat2

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24
Q

Where does dgat 1 go

A

When cell loaded with triglycerides = dgat1 remains in er membranes

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25
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
26
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
27
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
28
Describe visualizing lipid droplets - transmitted light
Oil red o - Sudan red
29
Describe visualizing lipid droplets - ‘em
Lipid droplets can be recognized by em with normal prep/staining
30
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
31
Describe visualizing lipid droplets - gfp tagged
Gfp tagged proteins that associate with lipid droplets = frequently used
32
Describe lipid droplet structure
Core of cholesterol ester and triglcuyerides Probably phosphpylid monolaer on surface
33
What is found on surface lipid droplets
Family of proteins called pat proteins = attached to mono layer Ex = perilipin, adrp, tip47
34
Rab18
On lipid droplets Recruit tethering proteins to attach to er
35
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
36
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
37
Describe biochemical studies of lipid droplets = tlc
Thin layer chromatography can be used to analyze lipid content of purified droplets
38
Describe pat proteins
Adrp and perilipin can be phosphrylated by protein kinase a (pka) Gets lipids out of lipid droplets
39
Name pat protein mechanisms
Hormone sensitive lipase Also autopahgy can work
40
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
41
what are other roles pat proteins
May also play structural role = stabilizing surface of lipid droplet
42
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)
43
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
44
Retrieval of fatty acids -Atgl
Another lipolytic enzyme that can bind to pat proteins on lipid droplets Regulation unclear
45
Retrieval of fatty acids - associate with
Lipids often associate with mito, peroxiosmes or er
46
Retrieval of fatty acids - To mito
Fatty acids transferred to mito or peroxsiomes can undergo beta oxidation
47
Retrieval of fatty acids - To er
Fatty acids transferred to er can be incorporated into phospholipids - to make more membrane
48
Name the 2 enzymes that are ale to release free fatty acids from triglycerides
Hsl Atgl
49
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
50
Targets of pka
Many
51
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
52
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
53
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
54
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
55
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
56
What does transport of lipids between cells
Primarily carried out by liprotein particle s = cholesterol/triglycerides between cells in body
57
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
58
Where do lipids come from
Outside body - diet Or be synthesized within body
59
Describe lipid stores under normal conditions
Major stores of excess lipids are adipocytes - triglycerides only And liver - triglycerides and cholesterol
60
Describe lipid stores pathologically
Lipid can be deposited into tissues - artery walls = leading cause of death in western counters - major medical problem - atherosclerosis + diseases
61
Describe ex of lipoprotein particle
Vldl - produced in hepatocytes Apolipoprotein b - translcoated into er large hydrophobic areas, transfer lipids Same true for chylomriocns
62
Describe density gradient centrifugation of lipoprotein particles
Lightest = vldl —> idl, ldl (same lipoprotein particle at diff stages of life) Hdl = most dense
63
Describe size chromatography lipoprotein particles
Hdl = smallest Vldl = largest
64
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)
65
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
66
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
67
ApoA
Found in hdl
68
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
69
ApoB100
Vldl, idl,ldl Can bind sequence on ldl r
70
Size of apolipoproteins
* ApoA--<400AA * ApoB48 -- 2153 AA BIG!!! * ApoB100 -- 4485 AA BIG!!! * ApoC -- 57 AA * ApoE -- 299 AA
71
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
72
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
73
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
74
Describe chylomicrons - contain
ApOB48 Which cannot bind to ldl r But also counting apoe= can bind to receptor in liver - endocytosus by hepatocytes
75
Describe chylomicrons - secreted from
Basal domain of inestimable epithelium Continue to grow as moves through secretory pathway then secreted into lymph
76
Role of liver - acccumlate
Accumulate choelsterol and triglycerides from endocytosed chylomicron remnants from hdl Can also make cholesterol if none in diet
77
Role of liver - fatty acids
Fatty acids in triglycerides can be liberated and transferred to mito for energy in any tissue
78
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
79
Role of hepatocytes - endocytose
Endocytose chylomicron remnants and hdl Choelsterol esters and triglycerides hydorlzyed in Lysosome and free choelsterol and fatty acids released
80
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
81
Role of hepatocytes - sequester
CHOELSTERol esters and triglycerides may also be sequestered in lipid droplets within hepatocyte - stored, fatty liver disease = bad
82
Vldl - describe
Repackaging choelsterol and triglycerides in liver - delivered by chylomicrons or hdl Can be secreted as very low density lipoprotein - vldl
83
Vldl Contains
ApoB100 = full lengths Can bind to ldl r Initially ldl r binding domain masked - not exposed until sufficient lipid is removed
84
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
85
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
86
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
87
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
88
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
89
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
90
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
91
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
92
Describe Hdl - abca1 transporter
Multiple Tm protein cell disposes of unwanted choelsterol
93
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)
94
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)
95
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
96
Describe niemann pick transporter
Endocytosis ldl - choelsterol arrives in lysosome in forms of cholesterol ester - then broken down but hydrophobic so 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
97
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)
98
Apoc
Small lipoprotein found in hdl Sometimes other lipoprotein particles
99
Apoe
Chylomicrons, binds to receptor in liver

ANAT 262 (24 decks)

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