Biochemistry, Cholesterol

Fundamentals

Humans can synthesize cholesterol de novo and can also obtain it from the diet. De Novo synthesis occurs in the liver and the intestines, each organ accounting for ~ 10% of total cholesterol in the body. Dietary triglycerides and cholesterol are packaged together with Apo proteins in the liver before being released into the circulation as very low-density lipoproteins (VLDL). Packaging of cholesterol together with Apo protein is essential as the hydrophobic nature of cholesterol makes it impossible for transportation through the blood. VLDL contains triglycerides, cholesterol, and phospholipids. Degradation of triglycerides in VLDL results in smaller low-density lipoproteins (LDL) that are rich in cholesterol.[4] Cholesterol-rich low-density lipoproteins (LDLs) travel through the blood circulation. They are delivered to the peripheral tissues, where LDL is recognized by the LDL receptors on the cell membranes and is endocytosed via receptor-mediated endocytosis.[5] Besides LDL, high-density lipoproteins (HDLs) carry cholesterol from the peripheral tissues to the liver in a reverse transport mechanism to get rid of any excess cholesterol.[6]

Cellular Level

While all cells can synthesize cholesterol to a small extent, the liver is the major site of cholesterol synthesis. The cholesterol synthesis occurs in the hepatic cytoplasm and requires enzymes n the cytoplasm and the smooth endoplasmic reticulum (SER). The first step of cholesterol biosynthesis involves condensing 2 molecules of acetyl-CoA to form acetoacetyl-CoA. Next, a cytosolic enzyme HMG-CoA synthase adds a third molecule of acetyl-CoA to acetoacetyl-CoA, making a six-carbon compound called 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA). An isozyme of HMG-CoA synthase in mitochondria catalyzes the rate-limiting reaction in ketogenesis. HMG-CoA reductase, a regulatory enzyme in the smooth ER catalyzes the next step, which reduces HMG-CoA to mevalonate. Synthesis of mevalonate is the key rate-limiting, committed step in the synthesis of cholesterol. A series of reactions convert Mevalonate to 3-isopentenyl pyrophosphate, farnesyl pyrophosphate, squalene, and lanosterol. Lanosterol then goes through another19-step ER-associated process to finally synthesize cholesterol. The terminal step is catalyzed by 7-dehydrocholesterol reductase that converts 7-dehydrocholesterol to cholesterol.[7]

Molecular Level

Cholesterol synthesis is regulated by modulating HMG-CoA reductase by different mechanisms. These include covalent modification of enzymes, allosteric feedback inhibition affecting the reaction rate, hormonal control, and transcriptional control of gene expression. 

HMG CoA reductase is regulated covalently through the actions of adenosine monophosphate (AMP)-activated protein kinase (AMPK) and a phosphoprotein phosphatase which switch the enzyme between dephosphorylated (active) and phosphorylated (inactive) state. When plenty of cholesterol is available, high levels cause feedback inhibition to decrease the HMG-CoA reductase activity. In a well-fed state, with plenty of substrate availability, insulin and thyroxine cause upregulation of the enzyme. The counterregulatory hormone for cortisol, glucocorticoids, and insulin is glucagon, which has an inhibitory effect.

Transcriptional control of gene expression is another important mechanism that regulates HMG-CoA reductase. This involves the special transcription factors, known as sterol regulatory binding proteins, to be present in the membranes of ER. In cholesterol-depleted cells, the SREBPs are transported to the Golgi complex, where they are processed to release an active fragment that enters the nucleus to bind the SRE (sterol regulatory element) and activate the transcription of the genes encoding HMG-CoA reductase and other enzymes involved in cholesterol biosynthesis. When cholesterol levels are high in the cells, the transport of SREBPs to the Golgi complex is blocked as the enzyme binds to another set of proteins that retain to the ER called ‘insigs’ (insulin signaling proteins). This prevents the proteolytic release of the active fragment of SREBPs from ER membranes. Since the fragment is not available to bind the SRE in the nucleus, the transcription of the target genes no longer occurs, and HMG-CoA reductase is not activated. [8]

Function

Cholesterol fulfills several biological functions and is necessary for successful cellular homeostasis. It acts as a precursor to bile acids, assists in steroid and vitamin D synthesis, and plays a central role in maintaining cellular membrane rigidity and fluidity.[9]

All classes of steroid hormones, glucocorticoids, mineralocorticoids, and sex hormones, are derivatives of cholesterol. Synthesis occurs in the placenta and ovaries (estrogens and progestins), testes (testosterone), and adrenal cortex (cortisol, aldosterone, and androgens). The initial rate-limiting reaction converts cholesterol to pregnenolone, which is then oxidized and isomerized to progesterone. It is further modified in the ER and mitochondria by various hydroxylation reactions to other steroid hormones (cortisol, androgens, and aldosterone). Aldosterone acts primarily on the renal tubules, stimulating potassium excretion and uptake of sodium and water. Its ultimate effect is an increase in blood pressure. Cortisol allows the body to handle and respond to stress through its effects on intermediary metabolism, in other words, increased gluconeogenesis and the inflammatory and immune responses. The androgens, specifically testosterone, estrogens, and progestins, are responsible for sexual differentiation, libido, spermatogenesis, and ovarian follicle production.

Vitamin D3 (cholecalciferol) from either the skin or the diet undergoes hydroxylation by 25-alpha hydroxylase to form 25-hydroxycholecalciferol (calcidiol) in the liver from lipid-soluble compounds with a 4-ringed cholesterol backbone. It is then further hydroxylated by 1-alpha hydroxylase to an active form 1,25-dihydroxycholecalciferol (calcitriol) in the kidneys. Vitamin D plays an integral role in the terminal differentiation of hypertrophic chondrocytes, subsequent calcification of the bone matrix. In addition, it plays an important role in calcium homeostasis helps by mobilizing calcium from the bones and stimulating intestinal absorption and reabsorption in the kidneys.[10]

Bile is a watery mixture of both inorganic and organic compounds, of which phosphatidylcholine and conjugated bile salts/acids are quantitatively the most important. Between 15 and 30 grams of bile salts/acids are secreted from the liver each day, but as a result of bile reabsorption, only about 0.5 grams are lost daily in the feces. As a result, to replace the amount lost, roughly 0.5 grams per day is synthesized from cholesterol in the liver. Cholesterol is incorporated as the backbone in bile acid synthesis, a complex multistep, multi-organelle process. This synthesis accomplishes 2 goals. First, it creates a way for the body to excrete cholesterol as there is no way to break it down physiologically, and it allows lipids to be digested via emulsification and subsequent break down by pancreatic enzymes.

Clinical Significance

While cholesterol is vital for the functioning of our cells, elevated levels can cause serious problems. In addition, it is implicated in many genetic diseases, such as cholelithiasis, and is also the target of many therapeutic pharmacologic drugs. 

Cholelithiasis

The formation of gallstones occurs if there is either a bile salt deficiency or excess cholesterol secreted into the bile. In other words, when the liver secretes cholesterol, there must be a proper balance of bile salts, cholesterol, and phospholipids, as an imbalance causes cholesterol to precipitate. In pathologic states of hypercholesteremia, gallstones are often formed, leading to cholecystitis or even ascending cholangitis. Understanding this precarious relationship led to the invention of two important types of antihyperlipidemic drugs: bile acid-binding resins (cholestyramine/colestipol/colesevelam) and cholesterol absorption inhibitors (ezetimibe). The former acts by blocking the reabsorption of bile acids in the small intestine, which then forces the liver to synthesize more bile acids to use up excess cholesterol in the process, thus lowering serum cholesterol levels. Ezetimibe acts similarly by blocking the absorption of cholesterol in jejunal enterocytes, allowing the body to take excess cholesterol and again secrete it into bile. It is interesting to note that Fibrates, another class of antihyperlipidemic drugs, can cause cholelithiasis. This occurs simply by causing cholesterol excretion into the bile, and as previously mentioned, if there is excess cholesterol in bile, it precipitates and forms stones.[11]

Statins

These are an important class of FDA-approved drugs used for the treatment of hyperlipidemia and hypercholesterolemia. Statins are reversible competitive inhibitors of HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis. They are structural analogs of HMG-CoA, the substrate for HMG-CoA reductase. Their action is mediated through transcriptional control of gene expression by SREBPs and by increasing the levels of LDL receptors on cells to facilitate the removal of excess LDL. When serum cholesterol levels are too high, statins may be used to stop de novo synthesis in the liver.[12]

Atherosclerosis

This is a result of an increased level of circulating LDL lipoproteins. LDLs are commonly referred to as the "bad" lipoproteins as they carry a very high concentration of cholesterol. When LDL levels are pathologically high, LDL deposits in the arterial wall and oxidize. Macrophages engulf these oxidized LDL particles, leading to their transformation that appears like foam, hence the name "foam" cells. Harvesting oxLDL by macrophages triggers the activation of cytokines, growth factors, leukocytes, and neovascularization, and smooth muscle cell proliferation. This results in fatty streak formation and eventually causes the formation of atherosclerotic plaques and consequently coronary artery disease.[13]

Familial Hypercholesterolemia 

This inherited condition is due to mutations in certain genes such as ApoB, LDLR, LDLARP1, or PCSK9 and causes familial hypercholesterolemia. The most common defect is a mutation of the LDL receptor, where LDLR can no longer clear the LDL from the blood circulation. This results in high cholesterol levels in the blood that can deposit in the blood vessels, accumulate and cause hardening of the arteries. If left untreated, the excess build-up can cause coronary heart disease.