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Cholesterol Metabolism- Subjective Questions (Set-2)
Give the reaction catalyzed by HMG Co A reductase and elaborate on the significance of this reaction.
Answer- Cholesterol can be obtained from the diet or it can be synthesized de novo. An adult on a low-cholesterol diet typically synthesizes about 800 mg of cholesterol per day. The liver is the major site of cholesterol synthesis in mammals, although the intestine also forms significant amounts. Regulation of cholesterol synthesis is exerted near the beginning of the pathway, at the HMG-CoA reductase step. this enzyme catalyzes the formation of mevalonate, the committed step in cholesterol biosynthesis.
HMG CoA reductase is controlled in multiple ways:
A) Fine control (Effect on catalytic activity)-
a) Feedback inhibition– HMG-CoA reductase in liver is inhibited by Mevalonate, the immediate product of the pathway, and by cholesterol, the main product.
b) Covalent Modification– Phosphorylation decreases the activity of the reductase. The enzyme exists in 2 forms- phosphorylated (Inactive) and dephosphorylated (active) form. The phosphorylation is brought about by reductase kinase which itself is phosphorylated by c-AMP dependent protein kinase ,through phosphorylation of another intermediary enzyme reductase kinase kinase (Figure-1)
Figure-1-Mechanism of regulation of HMG Co A reductase by covalent modification
The enzyme is converted back to its active form by dephosphorylation mediated by the action of protein phosphatase . Dephosphorylation is promoted by Insulin through stimulation of phosphatase , while phosphorylation is promoted by Glucagon through stimulation of c-AMP dependent phosphorylation cascade.
Insulin or thyroid hormone increases HMG-CoA reductase activity, whereas glucagon or glucocorticoids decrease it. Activity is reversibly modified by phosphorylation-dephosphorylation mechanisms, and therefore cholesterol synthesis ceases when the ATP level is low. The reduced synthesis of cholesterol in starving animals is accompanied by a decrease in the activity of the enzyme.
c) Competitive inhibition– A family of drugs known as statins, have proved highly efficacious in lowering plasma cholesterol and preventing heart disease.The statins act as competitive inhibitors of the enzyme HMG-CoA reductase. Lovastatin (Figure-2) is a member of a class of drugs (Atorvastatin, fluvastatin, pravastatin and Simvastatin are others in this class) called statins that are used to treat hypercholesterolemia. These molecules mimic the structure of the normal substrate of the enzyme (HMG-CoA) and act as transition state analogues. While the statins are bound to the enzyme, HMG-CoA cannot be converted to mevalonic acid, thus inhibiting the whole cholesterol biosynthetic process.
Figure-2- Lovastatin, a Competitive Inhibitor of HMG-CoA Reductase. The part of the structure that resembles the 3-hydroxy-3-methylglutaryl moiety is shown in red.
d) A diurnal variation occurs in both cholesterol synthesis and reductase activity.
B) Coarse control- changes in the concentration of enzyme
a) Feedback regulation- The rate of cholesterol formation is highly responsive to the cellular level of cholesterol. This feedback regulation is mediated primarily by changes in the amount of 3-hydroxy-3-methylglutaryl CoA reductase. As the intracellular cholesterol concentration rises(dietary cholesterol brought by LDL) the gene for HMG Co A reductase is repressed, However, it is only hepatic synthesis that is inhibited by dietary cholesterol. Reverse occurs when the intracellular cholesterol concentration goes down, the gene for HMG Co A reductase is induced. The rate of translation of reductase mRNA is also inhibited by nonsterol metabolites derived from mevalonate as well as by dietary cholesterol.
b) Sterol regulatory element binding protein (SREBP) dependent regulation- The rate of synthesis of reductase mRNA is controlled by the sterol regulatory element binding protein (SREBP). This transcription factor binds to a short DNA sequence called the sterol regulatory element (SRE) on the 5 side of the reductase gene. In its inactive state, the SREBP is anchored to the endoplasmic reticulum or nuclear membrane. When cholesterol levels fall, the amino-terminal domain is released from its association with the membrane by two specific proteolytic cleavages. The released protein migrates to the nucleus and binds the SRE of the HMG-CoA reductase gene, as well as several other genes in the cholesterol biosynthetic pathway, to enhance transcription. When cholesterol levels rise, the proteolytic release of the SREBP is blocked, and the SREBP in the nucleus is rapidly degraded. These two events halt the transcription of the genes of the cholesterol biosynthetic pathways.
Q.- Discuss the steps of formation of bile salts from cholesterol. How is this process regulated? What are the functions of bile salts?
Answer- Cholesterol is a precursor for other important steroid molecules: the bile salts, steroid hormones, and vitamin D. About 1 g of cholesterol is eliminated from the body per day. Approximately half is excreted in the feces after conversion to bile acids. The remainder is excreted as cholesterol.
As polar derivatives of cholesterol, bile salts are highly effective detergents because they contain both polar and nonpolar regions. Bile salts are synthesized in the liver, stored and concentrated in the gall bladder, and then released into the small intestine. Bile salts, the major constituent of bile, solubilize dietary lipid . Solubilization increases in the effective surface area of lipids with two consequences: more surface area is exposed to the digestive action of lipases and lipids are more readily absorbed by the intestine. Bile salts are also the major breakdown products of cholesterol.
Steps of synthesis-
A) Synthesis of Primary bile acids
The primary bile acids are synthesized in the liver from cholesterol (Figure-3 ). These are Cholic acid (found in the largest amount) and Chenodeoxycholic acid.
Step -1- The 7-α–hydroxylation of cholesterol is the first and principal regulatory step in the biosynthesis of bile acids and is catalyzed by cholesterol 7–α-hydroxylase, a microsomal enzyme. A typical monooxygenase, it requires oxygen, NADPH, and cytochrome P450. (Figure-3)
Step -2-Subsequent hydroxylation steps are also catalyzed by monooxygenases. The pathway of bile acid biosynthesis divides early into one subpathway leading to cholyl-CoA, characterized by an extra -OH group on position 12, and another pathway leading to chenodeoxycholyl-CoA (Figure-3).
A second pathway in mitochondria involving the 27-hydroxylation of cholesterol by sterol 27-hydroxylase as the first step is responsible for a significant proportion of the primary bile acids synthesized.
Figure-3- Showing the formation of primary bile acids. Secondary bile acids are formed from primary bile acids by deconjugation and dehydroxylation of primary bile acids.
Step-3-The primary bile acids (Figure 26–7) enter the bile as glycine or taurine conjugates. Conjugation takes place in peroxisomes. In humans, the ratio of the glycine to the taurine conjugates is normally 3:1. In the alkaline bile, the bile acids and their conjugates are assumed to be in a salt form—hence the term “bile salts.”
B) Secondary Bile acids-A portion of the primary bile acids in the intestine is subjected to further changes by the activity of the intestinal bacteria. These include deconjugation and 7-dehydroxylation, which produce the secondary bile acids, deoxycholic acid and lithocholic acid (Figure-3)
Enterohepatic Circulation of Bile salts-The primary and secondary bile acids are absorbed almost exclusively in the ileum, and 98–99% are returned to the liver via the portal circulation. This is known as the enterohepatic circulation (Figure-4). However, lithocholic acid, because of its insolubility, is not reabsorbed to any significant extent. Only a small fraction of the bile salts escapes absorption and is therefore eliminated in the feces. Nonetheless, this represents a major pathway for the elimination of cholesterol. Each day the small pool of bile acids (about 3–5 g) is cycled through the intestine six to ten times and an amount of bile acid equivalent to that lost in the feces is synthesized from cholesterol, so that a pool of bile acids of constant size is maintained. This is accomplished by a system of feedback controls.
Figure-4- showing the formation of primary and secondary bile acids; Enterohepatic circulation of bile salts.
Regulation of bile acid synthesis-The principal rate-limiting step in the biosynthesis of bile acids is at the cholesterol 7-α-hydroxylase reaction (Figure-3).
1) The activity of the enzyme is feedback-regulated .When the size of the bile acid pool in the enterohepatic circulation increases, transcription of the cholesterol 7-α–hydroxylase gene is suppressed. Chenodeoxycholic acid is particularly important in causing repression.
2) 7-α–hydroxylase activity is also enhanced by cholesterol of endogenous and dietary origin and regulated by insulin, glucagon, glucocorticoids, and thyroid hormone.
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