Lipid trafficking and cholesterol (slide deck 3)

dietary fats are abdorbed in

the small intestine

bile salts role in digestion

• Bile salts emulsify dietary fats in the small intestine, forming mixed micelles

  • Bile acids synthesized in the liver and stored in the gall bladder

  • Triggered release when you eat fats

  • Cholesterol cannot emulsify fats since it is not amphipathic enough ie. not polar enough

Exogenous lipid trafficking steps 

1. Fats ingested in diet

2. Bile salts emulsify dietary fats in the small intestine, forming mixed micelles

3. Intestinal lipases degrade triacylglycerols

4. Fatty acids and other breakdown products are taken up by the intestinal mucosa and converted into triacylglycerols

5. Triacylglycerols are incorporated, with cholesterol and apolipoproteins, into chylomicrons

   1. Chylomicrons are synthesized from dietary fats in the ER of enterocytes (enterocytes in the small intestine assemble dietary triglycerides, cholesterol, and fat-soluble vitamins into chylomicrons. ApoB-48 is added in the ER)

6. Chylomicrons move through the lymphatic system and bloodstream to tissues

7. Lipoprotein lipase, activated by apoC-II (transferred to chylomicrons from HDL) in the capillary, converts triacylglycerols to free fatty acids and monoacylglycerols

8. Fatty acids enter cells

9. Fatty acids are oxidized as fuel (CO₂ or ATP) or reesterified for storage (myocyte or adipocyte)

10. Chylomicron remnants move through the bloodstream to the liver

11. Receptors in the liver recognize and bind to the apoE in the remnants and mediate uptake of these remnants by endocytosis

chylomicrons

• Chylomicrons are particles consisting of traicylglycerols, cholesterol, and apolipoproteins → the largest, and least dense of the lipoproteins contain a high percentage of TAGs. 

• Become larger and more abundant in response to increased fat intake because they package more dietary triglycerides into a single particle

• Deliver fat to where it is needed

• Apolipoproteins allow for traffic back to liver once completed

True or False: If you prevent the synthesis of bile acids, this will reduce the amount of dietary fat absorbed in the intestine + explain

• True since dietary fat absorption requires bile acid

• Why wouldn’t we want to make bile acid inhibitors though?

  • Bile acids play a crucial role in fat digestion, and by inhibiting them, you could cause malabsorption of not only fats but also fat-soluble vitamins (A, D, E, K)

• Only way to naturally get rid of high cholesterol → ex. Oat fibre since there are no degradation pathways for cholesterol in humans

lipoproteins

• Spherical aggregates of apoliproteins and lipids

• Arranged with hydrophobic lipids at the core and hydrophilic protein side chains and lipid head groups at the surface

• Various in densities depending on combinations of lipid and protein

  • Range from chylomicrons and very-low-density lipoproteins (VLDL) to very-high-density lipoproteins (VHDL)

apolipoprotein B-48 (apoB-48)

Primary protein component of chylomicrons

apolipoprotein C-II (apoC-II)

• Protein picked up in the blood by chylomicrons from high-density lipoprotein (HDL) particles

• It allows lipases to bind to extract fat

lipoprotein lipase

• Extracellular enzyme in the capillaries of muscle and adipose tissue that hydrolyzes triacylglycerols to free fatty acids and monoacylglycerols

• Activated by apoC-II

ultracentrifugation

• Separates by size and density

• Things will sediment quicker the more dense they are

Four major classes of human plasma lipoproteins

• Different combinations of lipids and proteins produce particles of different densities

  • Size and density are inversely proportional

• Can be separated by ultracentrifugation 

• Very-low-density lipoprotein (VLDL)

  • Lipoproteins that carry cholesteryl esters or triacylglycerols from the liver to muscle and adipose tissue

  • apoC-II activates lipoprotein lipase to release FFAs from traicylglycerols

• Intermediate-density lipoprotein (IDL)

  • Lipoproteins that have donated some of their fatty acids to other cells for energy

• Low-density lipoprotein (LDL) 

  • “Bad” cholesterol involved in returning cholesterol to the liver which prevents cholesterol build up 

  • Associated with risk of heart disease

• High-density lipoprotein (HDL)

  • “Good” cholesterol are lipoprotein particles that are comprised predominantly of cholesterol

lipid transport is made possible by…

• packing large numbers of fats into micelles that are complex with specialized proteins known as lipoproteins. The amount of fat stored in these particles and their associated lipoproteins defines what type of particle they are -- the less concentrated they are the denser they will be

• Fatty acids can be extracted from these lipoprotein particles by lipases that bind to the lipoprotein apoC-II and cleave off free fatty acids for use in specific cells

the endogenous pathway

The pathway from VLDL formation in the liver to LDL return to the liver

• Steps:

1. The liver synthesizes triglycerides and cholesterol. These lipids are packaged into VLDL (very-low-density lipoprotein) particles. apoB-100 is loaded onto VLDL as its structural protein.

2. VLDL enters the bloodstream. It acquires additional apolipoproteins (apoC-II, apoE) from HDL.

3. apoC-II activates lipoprotein lipase (LPL). LPL hydrolyzes triglycerides → free fatty acids (taken up for storage or energy) + glycerol (returned to liver).

4. After losing triglycerides, VLDL becomes IDL (intermediate-density lipoprotein). IDL can take two routes:

• Cleared by the liver via apoE recognition.

• Further metabolized by hepatic lipase to form LDL.

5. Formation of LDL: IDL loses more triglycerides and apolipoproteins → becomes LDL (low-density lipoprotein). LDL is cholesterol-rich, carrying mostly cholesteryl esters.

6. LDL delivers cholesterol to peripheral tissues for membrane synthesis, steroid hormone production, etc. Uptake occurs by LDL receptor (recognizes apoB-100) via receptor-mediated endocytosis.

7. Most LDL eventually returns to the liver, where it is taken up by LDL receptors. The excess cholesterol in these particles is either converted into bile acid for digestion or repackaged into a VLDL for circulation

consequence of variant that impairs function of the protein that loads Apo-B48 onto the surface of a chylomicron

messes up exogenous trafficking; micelle loses identity since you cannot load apo-B48 (involved in receptor mediated retrieval system), doesn’t know where to go and doesn’t know how to get back to liver

consequence of variant that impairs function of apo-CII

Activates lipoprotein lipase (LPL), which hydrolyzes triglycerides in chylomicrons and VLDL so that fatty acids can be taken up by muscle/adipose tissue. So this would cause Chylomicrons and VLDL to not be efficiently hydrolyzed.

consequence of variant that impairs function of phospholipid-particle associated lipases

similar to apo-CII, cannot breakdown TAGs

storage of excess fatty acids

• Fatty acids are converted to TAGs in the liver

  • Fatty acid synthesis occurs in the cytoplasm of the liver after an excess of energy is consumed

• TAGs are packaged with specific apolipoproteins into VLDLs

• VLDLs are secreted and transported in the blood to adipose tissue

• TAGs are removed and stored in lipid droplets within adipocytes in the adipose tissue

mobilization of triacylglycerols stored in adipose tissue

• Mobilization occurs when hormones (glucagon and epinephrine) signal the need for metabolic energy

  • Glucagon is released when in fasting state

• PKA (protein kinase A) triggers changes that open the lipid droplet to the action of three cytosolic lipases

  • Now have access to cleave TAGs

• Lipid droplets = organelles stored in adipocytes and steroid-synthesizing cells that contain neutral lipids

  • Contain a core of triacylglycerols and sterol esters surrounded by a monolayer of phospholipids

• Perilipins = family of proteins that coats the surface of lipid droplets to restrict access to lipid droplets

  • Prevent untimely lipid mobilization

• Release of fats from adipocytes is tightly controlled and hormones stimulate lipases to release free fatty acids. FFAs are transported by albumin to target tissues for energy derived beta-oxidation

human serum albumin 

• Free fatty acids, FFAs = fatty acids released by lipases

  • Not happy free floating in aqueous solutions 

• Serum albumin = blood protein that noncovalently binds and transports FFAs to target tissues

  • Makes up about ½ of the total serum protein

  • Have 7 hydrophobic pockets

LNPs summary

• Lipid nanoparticles allow for the transport of negatively charged nucleic acid polymers through the body to the target tissue (easiest ones currently are blood cells and liver)

• Different structural lipids have distinct properties and propensities for forming lipid structures, these can be leveraged to carry different types of cargo 

• pH dependent ionizarion of structural lipid head groups enables the uptake of the nucleic acid, as well as release during endocytosis

• We are using these particles in lots of interesting ways to deliver drugs and therapeutics

SiRNA therapies

• siRNA targets PCSK9 mRNA in the nucleus/cytoplasm.

• This reduces PCSK9 protein production.

• Less PCSK9 → more LDL receptors recycled → lower LDL levels in blood.

which proteins would you target with siRNA to limit/restrict cholesterol synthesis? (SiRNA knocks down protein levels)

• HMG-CoA reductase 

• PCSK9

• Inhibit acetyl-CoA by inhibiting citrate lyase