Lecture 11: Triglyceride and Cholesterol Biosynthesis

Recall: a 70kg human stores about 15kg of TAGs (Triacylglycerols) yielding 37 kJ/g and 555 000 kJ of total energy

Origins of Glycerol
- Usually, limiting substrate in TAG synthesis = Glycerol. It starts w/ a molecule of glycerol-3-phosphate which comes from...
  1. Recycling of glycerol produced during beta-oxidation of FAs
  2. Reduction of Dihydroxyacetone phosphate (DHAP) produced during glycolysis
  3. Reduction of DHAP produced during glyceroneogenesis (using gluconeogenesis to produce glycerol) 
In both 2 and 3, use glycerol 3 P dehydrogenase using 1 NADH

Liver: Glycerol is acquired using ALL three mechanisms 
Adipose Tissue: Adipocytes lack glycerol kinase so can only do 2 or 3; glycerol produced by beta-oxidation returned to the liver through blood stream to be reused (using glycerol kinase)

Time for TAG Synthesis!
Happens in the liver (main place), adipose tissue (to replenish stock), and intestine
Part 1. DAG Formation

1) GPAT (glycerol-3-phosphate acyltransferase) converts glycerol-3-phosphate into a glycerol fatty acid at carbon 1 
2) AGPAT converts the product into phosphatidic acid (PA) 
3) PAP1 (hydrolase) converts the PA into DAG; removes a phosphate

INTESTINE: Monacylglycerol pathway (MAG)
- Glycerol with an FA already on it (on carbon 2)
1) MGAT (monoglycerol Acyltransferase) converts MAG to DAG
- Happens on Carbon 1

Part 2. TAG Formation

TAG is formed using Diacylglycerol Acyltransferase (DGAT

ER Localization of TAG biosynthesis machinery
- Most machinery of TAG biosynthesis is found in the ER
- LOTS OF EXCHANGE between mitochondria and ER

A key intermediate for TAG synthesis is Phosphatidic acid (PA) which is important in biosynthesis of glycerophospholipids

Fate of TAGs?

1. Storage in lipid droplets (LDs)
- Highly hydrophobic so they must remain in the cells (leads to obesity); FA can be released from LDs and access bloodstream or travel to mitochondria to be used as energy source doing beta-oxidation

2. Redistribution throughout the body 
- Chylomicrons (intestine); have 86% TAGs
- Very low density lipoprotein VLDL (liver)

Source of energy, but if you don't use it you'll store it

Summary of TAGs:
- Synthesized from G3P (mainly derived from glycolysis) and fatty acyl-CoA
- Mechs responsible for TAG synthesis between liver and intestine are different (intestine only one that can use MAG)
- TAGs are distributed by chylomicrons and VLDL to various tissues where FAs can be stored or used as energy source
Now let's look at that Cholesterol Biosynthesis.
Biological functions:
  1. Structural component of cell membranes, particularly the plasma membranes
  2. Constituent of Lipoproteins
  3. Precursor for bile acids and steroid hormones
Skipping major steps

All are made from ACETATE
- WHERE FROM: Acetyl-CoA
- i.e. Building block = Acetyl-CoA

STEP 1 = Condensation of acetate to form mevalonate intermediates (C5 unit)
STEP 2 = Polymerization of mevalonate to form squalene (C30 unit)
STEP 3 = Cyclization of squalene and further modifications to form cholesterol (C27 unit)

STAGE 1: Condensation

1) From acetyl-CoA to mevalonate via 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA)
- HMG-CoA is a precursor for cholesterol and ketone bodies
- Enzymes involved in the formation of HMG-CoA as precursor for ketone bodies are found in the mitochondria
- Enzymes catalyzing formation of HMG-CoA that is utilized for synthesis of cholesterol are found in cytosol and peroxisomes

2) HMG-CoA Reductase: catalyzes controlling step of HMG-CoA -> mevalonate
- Integral membrane protein found in ER and peroxisomes
- Consumes 2 NADPH

3) mevalonate (C6) to Dimethylallyl pyrophosphate (C5) via ...
i - Mevalonate 5 phosphotransferase
ii - Phosphomevalonate kinase
iii - Pyrophosphomevalonate kinase
- Spontaneous decarboxylation into IPP
iv - Isopentenyl pyrophosphate (IPP) isomerase; transfer double bond from C1-2 to C2-3

SO FAR: used 3 ATP and 2 NADPH for just one 5C molecule

STAGE 2: Polymerization

Polymerize isoprenoids (5C) to form squalene (30C)
- Isoprenoid: building block into bigger molecule

1) ENZYME = prenyltransferase
Take one of IPP and one of DMPP
MECHANISM = Nucleophilic substitutions on C1
PRODUCT = C10 molecule Geranyl pyrophosphate

2) ENZYME = Same as 1)
Product = Farnesyl pyrophosphate (C15)

3) Enzyme = Squalene synthase (found in ER membrane)
- Joins 2 molecules of farnesyl pyrophosphate head-to-head
Uses: 2 NADPH
PRODUCT = Squalene (30C)

(Isoprene); if you cut it you have two of whatever: defn = 5C molecule with 2 DBs

STAGE 3: Cyclization

From squalene (C30) to cholesterol (C27):

1) Cyclization of squalene to form Lanosterol
- ENZYME: Cyclase
- 3-step process, one of them makes a squalene epoxide which is starting point of cyclization

2) Synthesis of cholesterol from lanosterol
- 19 steps, all really small changes 

Cholesterol and Isoprenoids are precursors of other compounds.
- TAKE-HOME message: cholesterol is costly to make so we'll do everything possible to recycle it and use it in the diet
- Biosynthesis is important for things like vitamin D and hormones
- Farnesyl-PP creates CoQ

- In the HMG-CoA pathway, the building block for de novo cholesterol biosynthesis is Acetyl-CoA
- HMG-CoA reductase catalyzes the Rate-Limiting Step
- Important intermediates are HMG-CoA, mevalonate, activated isoprenes (IPP and DMAPP)
- Enzymes are located in the peroxisome (pre-squalene) and in the ER.