Metabolomics/Metabolites/Lipids/Steroids/Cholesterol

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Introduction

Cholesterol is a fat-like substance that is found in the bloodstream and cells (particularly phospholipids bilayer) of humans. It is used to produce cell membranes, to create hormones and serves several other functions. It is attained through the diet and through metabolic processes in the body. In excess, it creates a problematic condition of hypercholesterolemia (high cholesterol) – a serious risk for coronary heart disease, obesity, artherosclerosis, stroke and other medical ailments.

Good vs. Bad Cholesterol

HDL Cholesterol

High Density Lipoprotein: these are lipoproteins (combinations of fats and proteins) in the form in which they can be transported in the blood. The high-density lipoproteins transport cholesterol from the tissues of the body to the liver so it can be gotten rid of (in the bile). HDL cholesterol is therefore called the "good" cholesterol. The higher the HDL cholesterol level, the lower the risk of coronary artery disease. For each 1 mg/dl increase in HDL cholesterol there has shown to be a 2 to 4% reduction in the risk of coronary heart disease. Generally, the beginning steps to raise HDL cholesterol levels are involved in life style modification. Regular aerobic exercise, loss of excess weight (fat), and cessation of cigarette smoking cigarettes will increase HDL cholesterol levels. Moderate alcohol consumption (such as one drink a day) also raises HDL cholesterol. Some medications that are commonly used to raise HDL cholesterol include niacin, lopid, estrogen, and sometimes the statin drugs.

LDL Cholesterol

Low Density Lipoprotein - is called "bad" cholesterol, because elevated levels of LDL cholesterol are considered to be associated with an increased risk of coronary heart disease. But research shows that only oxidized LDL inflamed the epithelial cell, which makes the epithelial cell to secrete adhesive like molecule. This promotes the formation of plague, the plague not only contains LDL, but also calcium deposits and other dead cells. Therefore, only oxidized LDL lipoprotein contributes to the deposition of cholesterol on the artery walls, causing the formation of a hard, thick substance called cholesterol plaque. Over time, cholesterol plaque causes thickening of the artery walls and narrowing of the arteries, a process called atherosclerosis.

Low-density lipoprotein (LDL) receptors are typically regulated by feedback systems that depend upon intracellular cholesterol levels. Researchers have demonstrated that cytokines induce unmodified LDL accumulation in peripheral cells via disrupting cholesterol-mediated LDL receptor feedback regulation. These researchers have also investigated the regulation of cholesterol exogenous uptake via LDL receptor and its underlying mechanisms. Experiments were conducted on human hepatic cell line (HepG2) cells under physiological and inflammatory conditions.

Enzymic assay was employed to measure intracellular total cholesterol (TC), free cholesterol (FC) and cholesterol ester (CE) concentrations. Utilizing real-time quantitative PCR, LDL receptor, sterol regulatory element binding protein (SREBP)-2 and SREBP cleavage-activating protein (SCAP) mRNA levels were detected in total cellular RNA that was isolated from cells. Western blotting was used to analyze LDL receptor and SREBP-2 expression. Confocal microscopy involved with dual staining with anti-human SCAP and anti-Golgin antibodies was employed to examine the translocation of SCAP-SREBP complexes from the endoplasmic reticulum (ER) to the Golgi apparatus.

Experimental results indicated that, under physiological conditions, an increase in LDL concentration lead to an increase in intracellular cholesterol level, which in turn reduced LDL receptor mRNA presence and protein expression in HepG2 cells. By comparison, an increase of interleukin 1β (IL-1β) levels also increased intracellular levels of cholesterol, with LDL present. This was a result of increased expression of proteins and LDL receptor mRNA in HepG2. In addition, LDL was determined to reduce SREBP and SCAP mRNA levels, under these physiological conditions. Undeterred by high LDL concentration, translocation augmentation of SCAP-SREBP from the ER to the Golgi occurred via IL-1β exposure. This exposure was a result of over-expression of SREBP-2 and the disruption of the normal distribution of the SCAP-SREBP complex in HepG2.

IL-1β evidently disrupted cholesterol-mediated LDL receptor feedback regulation via augmenting SCAP-SREBP complex translocation from the ER to the Golgi. This resulted increased expression of SREBP-2 mediated LDL receptors, regardless of high LDL concentration. As a result, LDL receptor pathway produced an LDL cholesterol accumulation, in hepatic cells, under inflammatory stress.

References:

http://www.cmj.org/Periodical/paperlist.asp?id=LW20071217574113104650&linkintype=pubmed

Dietary vs. Endogenous Cholesterol

While it is easy to assume that cholesterol comes only from what you eat, surprisingly, dietary cholesterol makes up only 15% of the cholesterol in your body – the rest comes as a product of liver and cellular function (endogenous cholesterol). Although the level of cholesterol in your blood comes primarily from your body – it can be increased by high consumption of cholesterol and saturated fats in your diet

Dietary Cholesterol

Cholesterol from food is absorbed by the intestines and sent into the blood circulation. There, cholesterol is packaged in a protein coat. This cholesterol-protein coat complex is called a chylomicron. After a meal, the liver removes chylomicrons from the blood circulation. In between meals, the liver manufactures and secretes cholesterol back into the blood circulation.

•Sources include: Saturated fats, meat, poultry, fish, seafood and dairy products

Endogenous Cholesterol

Cholesterol Transport

Cholesterol is synthesized in the endoplasmic reticulum (ER), Accordingly, newly formed cholesterol goes first into the nonraft membrane but seems to become incorporated into rafts in the Golgi. From the Golgi, rafts that are formed are sent to distributed into the cell surface. Because of the high mole fraction of raft lipids in the apical membrane of epithelial cells, it has been predicted that these membranes rafts constitute the connected phase and allow for free diffusion of raft proteins over the entire membrane. Another mechanism for cholesterol transport exists in adrenal and gonadal cells utilizing cholesterol as a precursor for steroid hormone synthesis. Cholesterol is transported to the mitochondria through the cytosol (unlikely that there is a vesicular pathway connected to the mitochondria). An alternative mechanism that has been suggested involves membrane-membrane contact between the ER and mitochondria. This type of interorganelle transport has been seen in phospholipids transport and may also apply for cholesterol transfer. The key protein involved in shuttling cholesterol across the outer and inner mitochondrial membranes is the steroidogenic acute regulatory protein (StAR). Its structure has recently been determined and it has a binding pocket for cholesterol.

This figure is a schematic of cellular cholesterol distribution, processing, and trafficking. Cholesterol is synthesized in the endoplasmic reticulum (ER). Part of it is transported via the Golgi complex (1) and the trans-Golgi network (TGN) to the plasma membrane, where it is distributed either to raft (2, red) or nonraft (3, blue) microdomains. Most of the cholesterol, however, bypasses the Golgi and goes directly (4) to the cell surface. Cholesterol can be taken into the cell from the plasma membrane by endocytosis using clathrin-coated vesicles (5) or other pathways, including caveolae (small vesicles, or recesses, that have the ability to communicate with the outside of a cell and extend inward, indenting the cytoplasm and the cell membrane) (6). Rafts that have been brought into the cytosol via endocytosis can be located in endosomes specialized for sorting and recycling. From these endocytic circuits, cholesterol may be recycled to the surface (7) or transported back to the ER (8). In addition, certain routes from the Golgi complex (9) recycle cholesterol to the ER. Cholesterol is endocytosed in LDL via clathrin-coated pits (10) and transported to sorting endosomes (SE; 11). From there, it can be recycled to the surface either via a rapid route (12) or through slower routes utilizing recycling endosomes (RE; 13, 14). Cholesterol is also transported to late endocytic structures [15, late endosomes (LE) and lysosomes (LY)] that can fuse with each other (16). Sorting, recycling, and late endosomes communicate with the exocytic pathway (17 through 19), thus exchanging cholesterol between the endocytic and exocytic routes. Cholesterol esters in LDL are hydrolyzed before being released from the endocytic organelles, but cholesterol returning to the ER may become re-esterified. Cholesterol esters (CE) are deposited in cytosolic lipid droplets (20). Cholesterol can be mobilized upon ester hydrolysis from these cytosolic lipid droplets (21). Cholesterol and cholesterol esters can also be exchanged directly between circulating lipoproteins and the plasma membrane. Cholesterol can be released from cells - both from nonraft (24) and raft domains (25), the release of the raft domains may be involving caveolae (26).

Influx of Exogenous Cholesterol

Cholesterol can be taken up from lipoproteins in the circulation by mechanisms involving desorption (transfer of cholesterol from the lipoprotein to the exoplasmic leaflet of the plasma membrane bilayer) or by receptor-mediated uptake. The best understood and quantitatively the most important process is the one involving low-density lipoprotein (LDL), and its LDL-receptor. LDL is released from its receptor in the sorting endosomes and the LDL-receptor gets recycled to the cell surface. Cholesterol esters are hydrolyzed from LDL, and free cholesterol is continuously cycled to the plasma membrane. The main mechanism responsible for exit of LDL-cholesterol from late endosomes or lysosomes (membrane-bound organelles in the cytoplasm of most cells; they contain various hydrolytic enzymes that function in intracellular digestion) involves a certain protein - the NPC1 protein. This protein is defective in Niemann-Pick type C disease, a disease in which cholesterol and other lipids accumulate in vesicles derived from lysosomes. The NPC1 protein has been found in only late endocytic structures; its primary function and routes, however, have not yet been distinguished. The NPC1 protein is a multispanning membrane protein that contains a sterol-sensing domain. Mutations in this domain can result in the inactivation of the protein. Similar domains can be found in membrane proteins that regulate cholesterol synthesis. Cholesterol binding is a possible activity of this sterol-sensing domain. The NPC1 protein may also act in the removal of cholesterol from degradative endosomes by facilitating sterol transport to the Golgi or other destinations.

Cholesterol Efflux from Cells

Cellular cholesterol is continuously being lost by release of cholesterol to circulating lipoproteins. This loss can be quite rapid, up to 0.1% of total cholesterol per minute. The release from the plasma membrane can take place by desorption of cell surface cholesterol into lipoproteins. The liver and the intestine release cholesterol to the circulation mostly as esters by synthesizing and secreting lipoproteins. Another mechanism of cholesterol removal is by membrane shedding, a process that releases vesicles of membrane that may be enriched in raft lipids. Studies have shown that raft cholesterol is more slowly extracted by cyclodextrin and by HDL. Non-raft cholesterol, therefore, is the most likely source for efflux. A possible protein involved in cholesterol efflux has been identified through studies of Tangier's disease. Tangier’s disease is a genetic disease caused by the loss of function of an ATP-binding cassette transporter ABCA1 (formerly ABC1). This defect results in increased catabolism of HDL caused by a lowered efflux of cellular cholesterol. The increase in cellular cholesterol levels results in increased deposits of cholesterol esters in lipid droplets of the cytoplasm. One speculated function of ABCA1 could be to promote translocation of cholesterol from the cytoplasmic to the exoplasmic bilayer – facilitating efflux. It may also be involved in the transport of cholesterol from the Golgi to the cell surface (possibly involving rafts). This transporter may aid the formation of transport carriers by regulating lipid asymmetry. The ABCA1 protein has been determined to exist both in the plasma membrane and in the Golgi complex.

Cholesterol Homeostasis

Recent, scientific advances have shown that cholesterol completes many of its functions primarily by maintaining sphingolipid rafts in a functional state. However, how rafts contribute to cholesterol metabolism and transport is still debatable. Cellular cholesterol levels are precisely controlled by biosynthesis, efflux from cells and influx of lipoprotein cholesterol. The regulation of cholesterol homeostasis has changed perspectives and may open up more understandings of diseases caused by excess cholesterol, atherosclerosis for instance. Cells also continuously lose cholesterol to the outside circulation. Regulation of synthesis, influx and efflux keeps overall cholesterol levels controlled. The necessity of cholesterol homeostasis (and its maintenance), however, is not completely clear. Cholesterol has been thought to simply add rigidity to the plasma membrane – reducing permeability and increasing the durability of the lipid bilayer. Cholesterol was also thought to regulate the thickness of the bilayer – facilitating post-Golgi protein sorting. Cholesterol’s activities have now been expanded with the introduction of its role in lipid rafts.

Lipid Rafts of Cellular Membranes

Lipid rafts are physical bodies made up of cholesterol and sphingolipids (sphingomyelin and glycosphingolipids) in the exoplasm of the lipid bilayer. Cholesterol occupies the spaces between the saturated hydrocarbon chains of sphingolipids, thus, condensing the packing of sphingolipid molecules. This association is presumably strengthened by hydrogen bonding between the 3'-OH group of the sterol and the amide function of the sphingolipid ceramide backbone.

Removal of cholesterol from rafts (by cyclodextrin treatment, for example) dissociates the raft proteins from the lipids. Average raft size has been difficult to determine, but recent studies using photonic force microscopy have demonstrated that plasma membrane rafts usually maintain a diameter of about 50 nm, meaning about 3500 sphingomyelin molecules. Because of the small size of the raft, it can be assumed that probably not more than 10 to 30 proteins are contained in each raft. Raft size depends on the concentration of sphingolipids and cholesterol in the membrane. If the concentration of the raft lipids is increased and exceeds a critical value, the rafts would disrupt the connected phase and the liquid-disordered phase of the membrane. Such changes may play a role in regulating membrane properties both under physiological conditions and in the pathogenesis of diseases such as lipid storage disorders and atherosclerosis. Lipid rafts are dynamic assemblies that have functions in sorting and distributing lipids and proteins to the cell surface. At the cell surface, they play an important role in signal transduction and the creation of cell surface polarity. For these functions, it is crucial that not only the total cellular level of cholesterol but also its distribution are regulated.

This picture shows a lipid raft model. Lipids within the liquid-ordered phase are shown as red and in the liquid-disordered phase as blue. Cholesterol molecules, indicated by orange, arrange themselves preferentially into the liquid-ordered phase.

Cholesterol and Health

Factors that Influence Cholesterol Levels

•Diet: There are two factors associated with increased blood cholesterol

1.Saturated fats (in foods containing and not containing cholesterol) – they include foods with high levels of hydrogenated vegetable oils (trans fats) (palm and coconut oils), avocados and similar high fat foods of vegetable origin

2.High Cholesterol foods – only foods of animal origin actually contain cholesterol – most common culprits include eggs, red meat, lard and shrimp

•Age: Typically as we age, blood levels of cholesterol increase due to lifestyles that have long accumulated cholesterol.

•Weight: People who are overweight are more likely to have high blood cholesterol. Also, when extra weight is centered around the abdominal region (opposed to legs and buttocks) seems to be attributable to high cholesterol

•Gender: Trends indicate that before the age of 50, men tend to have higher LDL levels and lower HDL levels than women. After the age of 50, women’s levels of LDL cholesterol usually increase (thought to be due to the post-menopausal decrease in estrogen.

•Genetics: Many people can have a predisoposal to be high in cholesterol. Also, several, minor genetic defects can result in the increased production of LDL and the decreased ability to remove them.

•Diseases: Certain diseases like diabetes can lower HDL levels , increase triglycerides and speed up the development of artherosclerosis. Hypertension (high blood pressure) can also increase blood cholesterol levels and hasten artherosclerosis

•Lifestyle: Certain lifestyle choices and environments can raise cholesterol levels including cigarette smoking, lack of exercise and stress.

•Central Obesity: Studies shows that obesity at the center is one major risk factor for the development of high blood cholesterol. People with central obesity is due to two much of fat deposites, only due to too much of calory intake than actual requirment.

Diseases and Conditions Related with high levels of Cholesterol

• Artherosclerosis

• Chronic Heart Disease

• Diabetes

• Hypercholesteremia

• Obesity

• Stroke

Sources

1. Gordon, Dr. Jerry. “How cholesterol Works.” How Stuff Works: It’s good to know. 21 Feb 2008.<http://health.howstuffworks. com/cholesterol2.htm>.

Summary: This site gives a general overview of cholesterol. It categorizes the specific types of cholesterol: blood vs. dietary, good vs. bad and provides explanations of how each type may be affected. Links from the page also give general information and even animations on the mechanisms by which cholesterol affects the body. Testing and related conditions of excess cholesterol are covered and it also discusses topics such as treatments, diagnoses and contributing factors in simple terms so that it is understandable to non-scientific people.

Terms: • Statins - HMG-CoA reductase inhibitors • Fibrates – a category of pharmaceuticals that lower triglycerides and can increase HDL levels. • Artherosclerosis – a disease in which fatty material are deposited on the wall of an artery

Connection: This site is still relatable because it looks at the larger scale picture of cholesterol. Rather than focusing on the specific molecule and how it behaves and how it is regulated – the site addresses its overall affect on humans and gives the need-to-know information.

2. Griffin, R. Morgan. “High Cholesterol Treatment – What Works.” WebMD: Better information. Better health. 25 Jan 2007. 21 Feb 2008. <http://www.webmd.com/cholesterol-management /features/high-cholesterol-treatment-what-works?page=4>.

Summary: This is more of a general information site that discusses in lamens terms treatments available and life style change possibilities for people diagnosed with high cholesterol. While the explanations are fairly simplified compared to what we have learned in class, it does provide a few good treatments and gives a brief description of their physiological activities.

Terms: • Bile acid resins – medications that adhere to ingested cholesterol molecules in the intestines and prevent absorption • Policosanol – is the generic term for a natural extract of plant waxes. It is used as a nutritional supplement to lower LDL cholesterol and increase HDL cholesterol and to help prevent atherosclerosis.

Connection: It relates to the class, again, in the way that it provides descriptions of mechanistic functions – however simplified. It takes more of the wholistic, health sort of approach, rather than the molecular level of biochemistry, yet successfully expresses the importance of cholesterol to well-being.

3. “High Blood Cholesterol.” U.S. Department of Health and Human Services: National Heart Lung and Blood Institute: Diseases and Conditions Index. Feb 2006. 23 Feb 2008. < http://www.nhlbi.nih.gov/health/dci/Diseases/Hbc/ HBC_WhatIs.html>.

Summary: This site gives a general overview of Cholesterol. Links from the page also discuss LDL and HDL testing and normal amounts – it discusses topics such as treatments, diagnoses and contributing factors in simple terms so that it is understandable to non-scientific people.

Terms: • Coronary Artery Disease – a disease resultant of the accumulation of fatty plaques within the walls of the arteries that supply the heart with oxygen and nutrients. • Agina - refers to a painful constriction or tightness somewhere in the body • Hyperlipidemia - the presence of raised or abnormal levels of lipids and/or lipoproteins in the blood

Connection: Cholesterol is a lipid; a subject of biochemistry. While the site does not go in depth about biochemical processes or functions involving cholesterol (other than the negative health affects related to excessive cholesterol), it is still relatable because it looks at the larger scale picture of cholesterol. Rather than focusing on the specific molecule and how it behaves and how it is regulated – the site addresses its overall affect on humans.

4. Lee, Dr. Dennis and Kulick, Dr. Daniel. “Lowering Your Cholesterol Heart Attack Prevention Series.” MedicineNet.com: We Bring Doctor’s Knowledge to You. 20 Feb 2008. <http://www.medicinenet.com/cholesterol/ article.htm>.

Summary: This site/article is a very good database and provides in depth overview of several of the primary topics relating to cholesterol (mainly relating to health). It addresses disorders, treatments, types of cholesterol, and uses a lot of the specific terminology – although avoiding detailed explanations of mechanisms. This would be a very good source for anyone trying to get any information – it is simple, yet detailed.

Terms: • Bile acid sequestrants - bind bile acids in the intestine and cause more of the bile acids to be excreted in the stool; they lower LDL cholesterol levels • Atorvastatin – A medication that lowers the level of cholesterol in the blood • Chylomicron – a small globule composed of protein and lipid, they are found in the blood and lymphatic fluid where they serve to transport fat from its port of entry in the intestine to the liver and to adipose tissue. • Niacin - Nicotinic acid. It is one of the B-complex B vitamins. Deficiency of niacin in the diet results in the disease pellagra, it has uses as medication for hypercholesteremia

Connections: It relates to the class in the way that it provides in depth information on this specific metabolite and goes into specific details about forms in which it is transported (chylomicrons). It also discusses the physiological functions of certain life changes and pharmaceuticals in lowering LDL cholesterol and raising HDL cholesterol.

5. Simons, Kai and Ikonen, Elina. “How Cells Handle Cholesterol.” Science Magazine. No. 5497 (1 Dec 2000). 20 Feb2008<http://www.sciencemag.org/cgi/content/full/ 290/5497/1721>.

Summary: This article explains very in-depth the mechanisms by which cholesterol is utilized, stored, endocytosed, transported, etc. This is a good review article because it has a very high level of specificity and details every important protein, pathway and cofactor of relevant functions. It gives insight specifically on topics such as how it is effluxed and influxed into the cell, how it is used in lipid rafts to maintain cellular rigidity and how it is a precursor for steroid hormones.

Terms: • Ubiquitous – Being or seeming to be everywhere at the same time • Coalescence – to unite so as to form one mass • Caveolins – a family of proteins that form oligomers and associates with cholesterol and sphingolipids in certain areas of the cell membrane, forming caveolae. • Sphingolipid - a class of lipids derived from the amino alcohol sphingosine, they are often found in neural tissue, and play an important role in both signal transmission and cell recognition. • Nascent – beginning to exist or develop

Connection: It is relatable to the class in the way that it places cholesterol into the perspective of several different functions and systems. It discusses how systems involving cholesterol are regulated and how changes in the function or homeostasis of cholesterol levels can result in toxicity and certain diseases.

6. “What is Cholesterol?” American Heart Association. 27 Aug 2007. 20 Feb 2008. <http://www.americanheart.org/presenter.jhtml?identifier=3046103>.

Summary: This site discusses cholesterol, its misconceptions relating to health, its physiological functions and the sources through which cholesterol is attained.

Connections: While cholesterol is an important biochemical molecule used as a precursor of steroid hormones and a compositional additive to cell membranes (lipid rafts) it has a much larger, application and that is typically associated with human health. The article addresses this larger issue, putting into functions of cholesterol at the cellular and molecular level into perspective of a whole being.

7. "What is high cholesterol?" Healthy Ojas. 22 June 2010 <http://healthy-ojas.com/cholesterol/cholesterol-details.html>.

Summary: This site provides high cholesterol information that helps people with high cholesterol to lower their cholesterol level and for others to avoid high cholesterol.

Connections: Cholesterol is the Greek word "chole" - means bile, "stereos" - means solid, and the chemical suffix "ol" - means alcohol. Research shows that only oxidized LDL inflamed the epithelial cell, which makes the epithelial cell to secrete adhesive like molecule. This promotes the formation of plague, the plague not only contains LDL, but also calcium deposits and other dead cells.