Week 5 Lecture 8 Cholesterol PDF

Week 5 Lecture 8  Cholesterol What do you know? Is it derived from diet? Do we make it? Is having too much a problem? Why? Where can you find it?  Cholesterol is the major sterol in animal tissues. Cholesterol is pr...

Week 5 Lecture 8  Cholesterol What do you know? Is it derived from diet? Do we make it? Is having too much a problem? Why? Where can you find it?  Cholesterol is the major sterol in animal tissues. Cholesterol is present in tissues and in plasma either as free cholesterol in membranes or as a storage form, combined with a long- chain fatty acid as cholesteryl ester. In plasma, both free cholesterol and cholesterol esters are transported in lipoproteins. Plasma LDL carries cholesterol into many tissues. Free cholesterol is removed from tissues by plasma high-density lipoprotein and transported to the liver, where it can be excreted as bile acids in a process known as reverse cholesterol transport. In free form, the hydroxyl group of cholesterol interacts with the phospholipid head group so that the hydrophobic side chain of cholesterol is oriented in parallel with the fatty acids of phospholipids Cholesterol constitutes nearly 25% of the lipids in plasma membranes of some nerve cells but may be absent in intracellular membranes. Cells can regulate the amount of cholesterol in membranes by esterifying “excess” cholesterol with a fatty acid and storing the cholesterol esters in vesicles within the cytosol. When unesterified (free) cholesterol is needed, the cholesterol esters are hydrolyzed and free cholesterol is transported back to the membrane. Through the interaction with the phospholipid fatty-acid chains, cholesterol increases membrane packing, which both alters membrane fluidity and maintains membrane integrity so that animal cells do not need to build cell walls (like plants and most bacteria). The membrane remains stable and durable without being rigid, allowing animal cells to change shape and animals to move.  SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver Genes regulated by SREBPs. The diagram shows the major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides. In vivo, SREBP-2 preferentially activates genes of cholesterol metabolism, whereas SREBP-1c preferentially activates genes of fatty acid and triglyceride metabolism. DHCR, 7- dehydrocholesterol reductase; FPP, farnesyl diphosphate; GPP, geranylgeranyl pyrophosphate synthase; CYP51, lanosterol 14α- demethylase; G6PD, glucose-6- phosphate dehydrogenase; PGDH, 6- phosphogluconate dehydrogenase; GPAT, glycerol-3-phosphate acyltransferase. J Clin Invest. 2002;109(9):1125-1131. https://doi.org/10.1172/JCI15593. The SREBP pathway in mammals. In the presence of cholesterol or oxysterols, SREBP-Scap is retained in the ER by binding to Insig. In the absence of sterols, Insig no longer binds SREBP-Scap and SREBP- Scap is loaded into COPII vesicles through an interaction with Sec23/24. After transport to the Golgi, the transcription factor domain of SREBP is released from the membrane by two sequential proteolytic cleavage events mediated by the Site-1 (S1P) and Site-2 (S2P) proteases. The nuclear form of SREBP activates target genes such as HMG-CoA reductase and the LDL receptor through binding to sterol regulatory element (SRE) sequences in gene promoters. Cholesterol controls transport of SREBPs from the ER to Golgi complex by regulating the binding between Insig-1 and Scap. In cells depleted of cholesterol, Insig-1 is dissociated from Scap and degraded by proteasome. This allows the Scap/SREBP complex to be incorporated into COP-II-coated vesicles and transported to the Golgi complex, in which SREBPs are proteolytically activated. The NH2-terminal domain of SREBPs enters the nucleus to activate genes required for cholesterol synthesis as well as the gene encoding Insig-1. The proteolytic processing of SREBPs will not be terminated until two SREBP-induced products converge on Scap simultaneously: (1) Newly synthesized cholesterol accumulated in the ER that induces a conformational change in Scap, resulting in its increased affinity with Insig-1; and (2) Newly synthesized Insig-1 that interacts with Scap. In these cells with cholesterol restoration, binding between Scap and Insig-1 stabilizes Insig-1 and prevents incorporation of the Scap/SREBP complex into COP-II-coated vesicles  Cholesterol biosynthesis in hepatocytes indicating the negative regulatory effect of cholesterol on the HMG-CoA reductase reaction. Not all reactions are shown. HMG-CoA Reductase HMG-CoA is reduced to mevalonate by NADPH. This is the rate limiting step in cholesterol synthesis, which is why this enzyme is a good target for pharmaceuticals (statins). The mevalonate pathway, also known as the isoprenoid pathway or HMG-CoA reductase pathway is an essential metabolic pathway present in eukaryotes, archaea, and some bacteria. The pathway produces two five- carbon building blocks called isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are used to make isoprenoids, a diverse class of over 30,000 biomolecules such as cholesterol, vitamin K, coenzyme Q10, and all steroid hormones. HMG-CoA reductase is the rate controlling step in cholesterol biosynthesis and the target of Statin drugs. HMGCR catalyzes the conversion of HMG-CoA to mevalonic acid, a necessary step in the biosynthesis of cholesterol. Normally in mammalian cells this enzyme is competitively suppressed so that its effect is controlled. HMG-CoA reductase is activated by insulin, inhibited by glucagon and oxysterols. Oxysterols are derivatives of cholesterol and accumulate when there is excess cholesterol. When oxysterol levels are high they will also block LDL-receptor mediated endocytosis. When energy levels are low in the cell and [ATP] is decreased, AMPK will be activated and will lead to inhibition of HMG-CoA reductase. Domain structure of HMG CoA reductase. (A) HMG CoA reductase consists of two distinct domains: a hydrophobic N- terminal domain with eight membrane-spanning segments that anchor the protein to ER membranes, and a hydrophilic C- terminal domain that projects into the cytosol and exhibits all of the enzyme's catalytic activity. (B) Amino acid sequence and topology of the membrane domain of hamster HMG CoA reductase. The lysine residues implicated as sites of Insig- dependent, sterol-regulated ubiquitination are highlighted in red and denoted by arrows. The YIYF sequence in the second membrane-spanning helix that mediates Insig binding is highlighted in yellow. If you had to design a sterol sensing domain on HMG CoA reductase, where would you place it? Domain structure of HMG CoA reductase. (A) HMG CoA reductase consists of two distinct domains: a hydrophobic N- terminal domain with eight membrane-spanning segments that anchor the protein to ER membranes, and a hydrophilic C- terminal domain that projects into the cytosol and exhibits all of the enzyme's catalytic activity. (B) Amino acid sequence and topology of the membrane domain of hamster HMG CoA reductase. The lysine residues implicated as sites of Insig- dependent, sterol-regulated ubiquitination are highlighted in red and denoted by arrows. The YIYF sequence in the second membrane-spanning helix that mediates Insig binding is highlighted in yellow. HMGCR is subject to negative and positive regulation. In particular, the ability of oxysterols and intermediates of the mevalonate pathway to stimulate its proteasomal degradation is an exquisite example of metabolically controlled feedback regulation. Bile acids and bile salts are critical components of bile that act as detergents in the small intestine to emulsify dietary lipids for digestion and absorption. The liver synthesizes two bile acids, cholic acid and chenodeoxycholic acid, each of which is conjugated with either glycine or taurine, resulting in four different primary bile salts. After the newly formed bile salts enter the small intestine via bile secretion, they are subject to dehydroxylation by bacteria, thus producing secondary bile salts. All of the bile salts can be reabsorbed into the enterohepatic circulation and returned to the liver. In this way, secondary bile salts, while not directly synthesized by the liver, are present in gallbladder bile. Some of the cholesterol present in food is esterified with a fatty acid. About 10% of the cholesterol in egg yolks is esterified, whereas about 50% in meat and poultry is esterified. Cholesterol esters cannot be absorbed and therefore must be hydrolyzed to free cholesterol and free fatty acid to be incorporated into micelles for delivery to intestinal cells. Hydrolysis is achieved by cholesterol esterase, made and secreted by the pancreas. Free cholesterol from the diet (and from bile) requires no digestion and can directly incorporate in micelles. Cholesterol esterase also hydrolyzes phytosterol esters consumed in the diet. As previously mentioned, free phytosterols can displace cholesterol from the micelle, resulting in less cholesterol being available for absorption. Cholesterol that enters the small intestine comes from two sources: the diet and bile. As previously mentioned, dietary intake of cholesterol is about 300 mg/day, whereas the bile contributes 800–1,400 mg/day. Because the majority of cholesterol available for absorption is of hepatic origin, the efficiency of absorption can affect how much cholesterol is retained in the body. Cholesterol not absorbed is excreted in the feces. Given that no oxidative pathway for cholesterol exists in humans, fecal excretion represents the primary catabolic route in which whole-body cholesterol homeostasis in maintained. Therefore, the efficiency of cholesterol absorption is a critical point of regulation and the target of drug and dietary therapies that block absorption and promote the removal of cholesterol from the body. Cholesterol in the intestine must incorporate into micelles for delivery to the enterocyte. Uptake by the cell is mediated by a brush border protein called Niemann-Pick C1 like 1 (NPC1L1). Once inside the cell, cholesterol is carried through the cytosol by sterol carrier proteins. Cholesterol may incorporate into enterocyte membranes, although the majority is esterified in preparation for transport out of the cell as a component of chylomicrons. Cholesterol esterification is catalyzed by acyl-CoA:cholesterol acyltransferase 2 (ACAT2), which is required for chylomicron formation to occur. Phytosterols are also transported into the intestinal cell by NPC1L1. Despite the ability of NPC1L1 to transport both cholesterol and phytosterols, essentially no phytosterols incorporate into chylomicrons or enter the circulation. This is due to the presence of two additional proteins—members of the ATP-binding cassette (ABC) transporter family called ABCG5 and ABCG8—that reside adjacent to NPC1L1 in the brush border membrane. The role of ABCG5 and ABCG8 is to redirect phytosterols back into the intestinal lumen immediately after being taken into the cell. ABCG5 and ABCG8 also redirect some cholesterol back into the intestinal lumen, so that the overall efficiency of cholesterol absorption is about 50–60%. A rare autosomal recessive disorder called sitosterolemia can occur as a result of mutations in either ABCG5 or ABCG8, causing hyperabsorption of cholesterol and phytosterols. Strategies to block cholesterol absorption date back to the 1950s when patients with elevated blood cholesterol were given a commercial preparation of phytosterols suspended in fruit-flavored syrup (marketed as Cytellin). Phytosterols are known to displace cholesterol from micelles and compete for binding to NCP1L1. The product had limited success and was largely replaced by powerful prescription drugs, including ezetimibe, which directly inhibits NPC1L1, resulting in significant reductions (about 18%) in blood cholesterol levels. For patients who cannot tolerate prescription drugs, foods and supplements enriched with phytosterols are increasingly available and effective at reducing blood cholesterol concentration by 10% or more. Ezetimibe (10 mg/day) was found to inhibit cholesterol absorption by an average of 54% in hypercholesterolemic individuals. Ezetimibe alone reduced plasma total and LDL- Cholesterol (18%) levels in patients with primary hypercholesterolemia. When ezetimibe was added to on-going statin treatment, an additional 25% reduction in LDL-C was found in patients with primary hypercholesterolemia and an additional 21% reduction in LDL-C in homozygous familial hypercholesterolemia.  A 10-year-old girl with homozygous Familial Hypercholesterolemia. Note the elevated orange-yellow xanthomas lying superficially over the knees, the wrists, and interdigital webs. These xanthomas arise from the deposition of plasma LDL-derived cholesterol into macrophages of the skin. The rate of deposition is proportional to the severity and duration of the elevation in plasma LDL. A similar deposition of LDL-derived cholesterol occurred in the coronary arteries of this girl, producing atheromas of artery wall, which led to her first myocardial infarction at age 8. Joseph L. Goldstein. Arteriosclerosis, Thrombosis, and Vascular Biology. The LDL Receptor, Volume: 29, Issue: 4, Pages: 431-438, DOI: (10.1161/ATVBAHA.108.179564) Regulation of HMG-CoA reductase activity in fibroblasts from a normal subject and from an FH homozygote. A, Monolayers of cells were grown in dishes containing 10% fetal calf serum. On day 6 of cell growth (zero time), the medium was replaced with fresh medium containing 5% human serum from which the lipoproteins had been removed. At the indicated time, extracts were prepared, and HMG-CoA reductase activity was measured. B, 24 hours after addition of 5% human lipoprotein-deficient serum, human LDL was added to give the indicated cholesterol concentration. HMG-CoA reductase activity was measured in cell free extracts at the indicated time. (Modified from Goldstein and Brown.8)  In more than 75% of cases of familial hypercholesterolemia (FH), the LDL receptor is defective, owing to mutations in the LDLR gene. Because familial hypercholesterolemia is inherited in an autosomal dominant fashion, most patients who have it are heterozygous, possessing 1 normal allele and 1 mutated allele. The prevalence of heterozygous familial hypercholesterolemia is about 1 in 220, based on large genetic studies. Homozygous familial hypercholesterolemia, in which the patient possesses 2 mutated alleles, is much less prevalent, with a frequency One of the main signs of FH is LDL cholesterol levels over 190 estimated at 1 in 300,000. Patients with mg/dL in adults (and over 160 mg/dL in children). In addition, homozygous disease face a worse most people with FH have a family health history of early heart prognosis. disease or heart attacks. In some cases, elevated LDL levels are found through routine blood cholesterol screening.