Biochemistry - 37 - Cholesterol Metabolism 2023.pdf

Objective B Cholesterol Synthesis: Overview • All carbons come from acetyl CoA • Squalene is built up from 5-carbon isoprene units • Cholesterol synthesis can be divided into three stages and proceeds as follows: C2 à C6 à C5 à C10 à C15 à C30 à C27 • Most of the first two stages of synthesis occ...

Objective B Cholesterol Synthesis: Overview • All carbons come from acetyl CoA • Squalene is built up from 5-carbon isoprene units • Cholesterol synthesis can be divided into three stages and proceeds as follows: C2 à C6 à C5 à C10 à C15 à C30 à C27 • Most of the first two stages of synthesis occur in the cytosol but two enzymes (HMG CoA reductase and squalene synthase) are located on the cytoplasmic face of the endoplasmic reticulum. • The final stage occurs entirely with ER bound enzymes 5 5 Cholesterol Synthesis 6 6 Objective C Cholesterol Synthesis: Cytoplasm • The acetyl CoA for cholesterol synthesis is formed in the mitochondrial matrix (following glycolysis) and is transported to the cytosol by the citrate transport system • Fed State + Insulin: excess glucose à make FAs and make cholesterol • Both involve citrate shuttle to get acetyl CoA out to cytoplasm 7 7 Objective C Regeneration of Pyruvate • In the cytosol, citrate lyase converts citrate back to acetyl CoA and OAA • OAA is reduced to malate by cytosolic malate dehydrogenase using NADH • Malate is oxidatively decarboxylated by malic enzyme forming NADPH, pyruvate and CO2. • Pyruvate reenters the mito matrix and can be reconverted to OAA by pyruvate carboxylase or to acetyl CoA by PDH as needed. • Notice the induced enzymes…patients with chronically high carbohydrate intake will need this pathway more and will have higher levels of these enzymes. 8 8 Stage 1: Condensation of Acetyl CoA to Form Mevalonate Objective D • The first two reactions of cholesterol synthesis are the same for ketone body synthesis except that they involve cytoplasmic isozymes • Two acetyl CoAs condense to form acetoacetyl CoA • A third acetyl CoA is added to form 3-hydroxy-3-methylgutaryl CoA (HMGCoA) • HMG-CoA reductase reduces HMG-CoA to mevalonate using two NADPH • Clinically this is the most focused on aspect of cholesterol synthesis – statin drugs. 9 9 Objective D HMG-CoA Reductase • The committed step and the rate-limiting step of cholesterol synthesis • the site of regulation of cholesterol synthesis • located on the cytoplasmic face of the ER, has a cytoplasmic catalytic domain and a regulatory membrane domain 10 10 Stage 3: Formation of Cholesterol from Squalene • These reactions all occur on the ER • Hydroxylation at C-3 and ring closure to form lanosterol • • • Squalene binds to a specific sterol carrier protein Squalene monooxygenase adds a hydroxyl group at C-3 Lanosterol cyclase forms the ring structure • Monooxygenases or mixed function oxygenases consume one molecule of O2 per atom of oxygen introduced in to a substrate. The other oxygen forms water. • NADPH provides reducing power for ER monooxygenases • Lanosterol binds to a second carrier protein and lanosterol undergoes 20 further reactions to form cholesterol. 13 13 Objective E HMG-CoA Reductase Regulation AMPK AMPK-P • HMG-CoA reductase activity is highly regulated • Short term regulation is by phosphorylation / dephosphorylation primarily by the same AMP-activated kinase as regulates acetyl CoA carboxylase activity. • When AMP is high (ATP low) HMG-CoA reductase is phosphorylated, decreasing its activity • Long term: modify enzyme level in cell (DNA / mRNA) 14 14 Objective E Transcriptional Control of Cholesterol Synthesis Primary means to control cholesterol synthesis is to control HMG-CoA reductase activity • Transcriptional control • SREBP: sterol regulatory element binding protein • activates over 30 genes related to fat metabolism: cholesterol, FA, TG, PL, and NADPH production proteins • short half life, must be continually produced • SCAP: SREBP cleavage-activating protein AND S2P: site 2 protease • Senses sterol level and stimulate production of SREBP • When cholesterol is high, cholesterol binds to SCAP. SREBP protein is intact and quiescent. When cholesterol is low, SCAP does not bind cholesterol, SREBP is activated, and S2P cleaves the DNA binding domain of SREBP. This signals DNA transcription. 15 15 Objective E Regulation of HMG-CoA Reductase II • Panel B: Proteolytic degradation: • removes the enzyme from the pathway • Panel C: Reversible covalent modification • Don’t want to synthesize cholesterol during fasting… • Glucagon stimulates HMG-CoA reductase phosphorylation = off • or when ATP is low… • or when sterol level is high. • High [AMP] or high [sterol] turns off HMG-CoA reductase 16 16 Objective F Cholesterol Esterification • The hydroxyl group at C-3 on cholesterol can be esterified with a fatty acid • Increases the hydrophobicity of cholesterol, which increases is solubility in lipoprotein particles an in lipid droplets in the cytosol of cells. • Two main enzymes that esterify cholesterol: • Acyl:cholesterol acyltransferase (ACAT) (left) • located in cells • concentrated in cells that need to store cholesterol for steroid synthesis. • Lecithin:cholesterol acyltransferase (LCAT) (right) • located in blood • esterifies cholesterol associated with HDL 18 18 Other Functions of Cholesterol and Degradation • Precursor for: • Bile salts • Vitamin D (from precursor) • Steroid hormones • • • • • Glucocorticoids Mineralcorticoids Androgens Estrogens Progestogens Objective G • Cholesterol Degradation? Taken up by liver and reprocessed or discarded. • Cholesterol is a precursor for so many other molecules, that it is very rare that it ever needs to be degraded and eliminated. • One of the main “losses” of cholesterol occurs in the digestive tract with bile salts that are not resorbed properly. 19 19 Objective H Bile Acid / Bile Salt Metabolism • Bile acid / bile salt metabolism has direct effect on cholesterol metabolism. • Patients who do not efficiently resorb the bile salts must constantly resynthesize them from cholesterol. • These patients often have upregulated cholesterol synthesis. 20 20 Objective H Synthesis of Bile Acids • Recall the bile acids / bile salts are more polar and hydrophilic than cholesterol. • A Cytochrome P450 monooxygenase, 7α-hydroxylase incorporates an -OH group first. • Note the use of NADPH, typical of Cytochrome P540 monooxygenases. • KEY: rate-limiting step in bile acid/salt synthesis, committed step • Strongly inhibited by bile salts / bile acids 21 21 Primary and Secondary Bile Acids / Salts • Primary bile salts are synthesized in the liver and sent to the gall bladder (bile). • Once released into digestive tract, the primary bile salts *can* be modified by gut bacteria, forming the secondary bile salts. Some remain in the primary form. • Primary bile salts possess all hydroxyl groups • 3 for cholic acid • 2 for chenodeoxycholic acid • Secondary bile salts lack a hydroxyl group at position 7 • Lithocholic acid is only hydroxylated at position 3 • It is the least soluble bile salt • Its major fate is excretion Objective I 22 22 Objective J Two Classes: Lots of Conjugation • Bile acids do not remain “acids” after synthesis • Conjugated by amino acids to becom bile “salts” • Glycine or taurine (metabolite of cysteine) • Lowers pKa of ionizable group • More ionized in lumen of gut, making better detergents 23 23 Question Statin drugs are inhibitors of the enzyme HMG-CoA reductase and are often used for patients that have chronically high cholesterol levels in the blood. Which of the following would be an immediate change in cells for a patient on statin drugs? The amount of mevalonate in the cell should decrease. The amount of acetyl CoA in the cell will decrease. More lanesterol will be converted to cholesterol to compensate. The free isoprenes will be converted to triglyceride. 26 26 Question A patient has an unusually low level of cholesterol in her cells. Which of the following enzymes, if deficient, could account for the? SREBP Cleavage Activating Protein (SCAP) Acetyl CoA Carboxylase Malic Enzyme LDL Receptor Phosphoenolpyruvate carboxykinase 27 27