Lipids

Hypercholesterolemia

Cholesterol , a critical structural element, comprises about 25% of the cell membrane and occurs in an equal proportion to phospholipid constituents (Fig. 2.7). It provides some rigidity to an otherwise completely flexible membranous structure. Its presence in the membrane facilitates the diffusion of nonpolar molecules. Cholesterol is essential to life. However, disease situations occur when blood cholesterol cannot be handled properly, that is, when the transporters of cholesterol, the low-density lipoproteins ( LDL s), cannot effectively move cholesterol into the peripheral tissues. Movement of cholesterol from LDLs into peripheral tissues occurs through the binding of LDL to the LDL receptor (LDLR) site on the tissue cell membrane. A model of the LDLR structure is shown in Fig. 9.1 .

The ligand-binding domain of the LDLR recognizes a lipoprotein on the surface of the LDL. A drawing of an LDL particle encircled by the ApoB-100 protein is shown in Fig. 9.2A and B . The figure shows the positively charged region of the molecule that constitutes the receptor-binding domain.

There is a negative cholesterol feedback mechanism which, when cholesterol is high in liver cell membranes, on the one hand, leads to the reduction of the number of LDLRs in cell membranes and, on the other, leads to the repression of the synthesis of cholesterol in the liver. This results from the failure of a fragment of the SREBP (sterol regulatory element-binding protein) to enter the nucleus, activate the LDLR gene , and induce the enzymes of cholesterol synthesis. This system will be discussed in connection with the biosynthesis of cholesterol.

Familial hypercholesterolemia ( FH ) is the inherited form of hypercholesterolemia and is semidominant. There are two alleles for the LDLR in FH. In the heterozygous condition (1 in 500 individuals), there is one defective gene and one normal gene. This condition would result in a lower number of LDLRs on the membranes of tissue cells resulting in higher levels of circulating cholesterol. Table 9.1 lists the levels of circulating cholesterol and LDL cholesterol that are either normal or characteristic of disease.

The worst case is the homozygous disease that can occur when both parents are heterozygous and the offspring is homozygous (two defective genes). In this case (1 in 1 million individuals), virtually no (or few) LDLRs are produced in the membranes of tissue cells. These patients rarely live beyond 20 years and can experience heart attacks as early as age 2 years. There are many sites on the LDLR gene that can be mutated ( Fig. 9.3 ) leading to the possibility for many forms of the disease with varying degrees of severity. There can be more than 300 gene defects in the LDLR gene and an allele can carry more than one mutation. Not every mutation is pathogenic. In FH, patients can have cholesterol levels that are six times the normal.

These elevated levels can lead to the formation of plaques in arterial walls that, in turn, can lead to myocardial infarcts.

In terms of function of the LDLR , there are five classes of mutations ( Fig. 9.3 ): class 1: mutation in this region affects the synthesis of the LDLR in the endoplasmic reticulum; class 2: prevention of proper transport of the LDLR to the Golgi apparatus for modification of the receptor [truncation, missing domains of the epidermal growth factor precursor domain ( Fig. 9.1 ); domains 3, 4, and 5]; class 3: mutation in repeat six in the ligand-binding domain (N-terminal in the extracellular fluid) is deleted; class 4: inhibition of internalization of the LDLR complex (residue C, 807). This domain is responsible for recruitment of clathrin and other proteins involved in endocytosis of LDL and, therefore, the mutation lowers LDL internalization into the tissue cells; and class 5: this mutation causes improper receptor recycling. A deletion mutation indicates that part of the DNA or chromosome is missing; a missense mutation is a change of a single nucleotide; an insertion mutation is the addition of one or more nucleotide base pairs into a DNA sequence and a nonsense mutation is a change in a base to a stop codon so that the product translated from an mRNA is shorter than the normal product. Other genetic factors in hypercholesterolemia include a deficiency in lecithin–cholesterol acyltransferase ( LCAT ), apolipoprotein that has been altered in some way so as to decrease its ability to bind to the LDLR as well as the point mutations in the LDLR mentioned earlier. As a consequence of mutations of the LDLR, LDLs in the blood increase and they are not taken up in the liver (or other tissues) as shown in Fig. 9.4 .

The LDLR gene is located on human chromosome 19 and the inheritance is autosomal dominant. If one parent has FH and the other does not, half of the offspring will be affected. Death from heart failure in the 1940s is not unusual and death can occur in the 1950s, 1960s, and 1970s depending on the severity of FH. Today, regulation of diet coupled with exercise and the use of new drugs ( statins ) to inhibit cholesterol synthesis in the liver form a treatment for this disease. Also, surgical intervention using the bypass technique is needed in some cases.

ApoB-100 Protein Mutations

ApoB-100 is embedded in the phospholipid outer layer of LDL particles ( Fig. 9.2 ) and is recognized by the LDLR. LDLR also recognizes the apoE protein , as a ligand, in chylomicron remnants and in very LDL ( VLDL s). The normal LDLR binding to apoB-100 involves an interaction between arginine 3500 (R3500) and tryptophan 4369 (W4369) spanning the region between R3500 and W4369 as shown in Fig. 9.5 .

LDLR binds to the apoB-100 protein (ApoB-100) in the phospholipid outer layer of LDL particles. There are two forms of apoB protein, a 48-kDa form synthesized exclusively in the intestinal tract and a 100-kDa form made in the liver. Both ApoB-48 and ApoB-100 are encoded within a single gene that transcribes a long mRNA and both proteins have an identical N-terminal sequence. ApoB-100 is a component of VLDL s, intermediate-density lipoproteins (IDLs), and LDLs all of which are transporters of fats and cholesterol in the blood. ApoB-100 facilitates the attachment of these particles to specific liver cell membrane receptors. These receptors transport LDLs into the cell where they are degraded to release free cholesterol that is either stored in a lipid droplet (LD) or removed. There are more than 90 mutations possible in the APOB gene and these mutations cause hypobetalipoproteinemia that impedes the bodily absorption and transport of fat. This inherited disease is familial hypobetalipoproteinemia ( FHBL ). Many of the mutations of the APOB gene that cause FHBL generate an abnormally shortened form of the ApoB protein. Inherited hypercholesterolemia can result from any of about five point mutations (changes in an individual amino acid in the resulting protein) in the APOB gene and it is called familial defective apolipoprotein B-100 ( FDB ). This produces severely elevated blood cholesterol and greatly increases the risk of heart disease. In FDB, LDLs are unable to bind effectively to cell membrane receptors, resulting in the excess of circulating cholesterol that is abnormally deposited in the skin, the walls of arteries (especially those supplying blood to the heart), and tendons.

Normal LDL Function at Tissue Cells

LDLs transfer their cholesterol content as cholesteryl esters ( CE s) to the interior of tissue cells. First, the LDL binds to the LDLRs on the surface of the tissue cell. This is accomplished by the recognition of the LDL through the ligand-binding domain of the apoprotein on the surface of the LDL particle ( Fig. 9.2 ). The process of endocytosis begins when the LDL is encapsulated by the invaginated membrane covered on the interior with clathrin proteins and other proteins involved in the inward transport. This forms an endosome inside the cell and the LDL dissociates from the receptor in the endosome caused by its acidic environment. Eventually, the LDL is moved into the lysosome where it is degraded and releases cholesterol or CE. The residual LDLR is returned to the cell surface by the recycling vesicle in preparation for the next event of LDL binding. The subsequent regulation of cholesterol synthesis involves a fragment from the steroid regulatory element-binding protein cleavage-activating protein-SREBP2 complex that is the nuclear SREBP2. These events are summarized in Fig. 9.5A . SREBP2 regulates cholesterol synthesis by activating genes that express the messengers for HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis, HMG-CoA synthase, and mevalonate kinase (see Fig. 9.7 ). The forward and return passage of the endosome between the cell membrane at the beginning and its return takes about 10 minutes and it lasts for about 20 hours before it is degraded. The CE in the lysosome can be released as unesterified cholesterol (eventually located mainly in the membrane) by the action of CE hydrolase after it has become activated [phosphorylation by protein kinase A (PKA)]. The unesterified free cholesterol in membranes in part can be returned as the CE to the liver by way of high-density lipoprotein (HDL) ( Fig. 9.27 ) (with a protein on its surface) and acyl-coenzyme A (CoA) cholesterol acyltransferase ( ACAT ) attached in the reverse cholesterol transport system that will be shown later. Cholesterol synthesis in the liver will contribute to the unesterified cholesterol pool. As the cholesterol pool in the cell increases, there will be a negative feedback on cholesterol synthesis and a positive effect on ACAT that will catalyze the conversion of the free form of cholesterol to the CE. In specific types of cells, CE can be stored into a fat droplet ( Fig. 9.40A and B ) and CE can be made available as free cholesterol to the mitochondria in cells that synthesize steroid hormones, for example, by the action of CE hydrolase. The normal functioning of LDLs is capitulated in Fig. 9.6 .

The process of moving cholesterol from the bloodstream into the tissues reduces the circulating LDL cholesterol.

Biosynthesis of Cholesterol

Although most tissue cells have the capacity to synthesize cholesterol, the main organs that do so are the liver, the adrenal cortex, and the ovaries and testes. The latter three involve cholesterol as the starting molecule for the synthesis of adrenal cortical hormones and the sex hormones . The 4-ring system of cholesterol cannot be broken down in the body but the side chains, double bonds, and substituted groups all can be removed or altered. About 75% of the body’s cholesterol is formed in the liver. The diet is also important and supplies the rest, although absorption into the bloodstream involves about 25% of ingested cholesterol, mostly in animal fats and sterols from plant sources. The cholesterol molecule is derived from the acetate of acetyl-CoA. The synthesis can be visualized from the point of view of the number of carbons involved in the intermediates as shown in Fig. 9.7 .

Three molecules of acetyl-CoA (three times C2 for acetyl) are converted to 3-hydroxy-3-methylglutaryl-CoA ( HMG - CoA ) in a few steps, and HMG-CoA is then converted to mevalonate (C6 compound) in the endoplasmic reticulum. This step is catalyzed by hydroxyl-methylglutaryl-CoA reductase ( HMG - CoAR ), the rate-limiting enzyme in the overall pathway for the synthesis of cholesterol. As indicated, this enzymatic activity is stimulated by insulin (stimulates phosphatases, e.g., Fig. 6.30) and thyroxine (increases HMG - CoA reductase mRNA production) and inhibited by the action of glucagon [inhibits protein phosphatases and elevates cyclic adenosine monophosphate (cAMP) leading to activation of PKA and the phosphorylation of inhibitor protein phosphatase inhibitor-1 (PPI-1) to its activated form that then inhibits the dephosphorylation of HMG-CoAR-P to its active form]. Mevalonate is converted to isopentenyl diphosphate (C5), the “active isoprene.” Six isopentenyl diphosphates are polymerized to form squalene (C30). Squalene is converted to lanosterol (C30) in two steps. Finally, the 2-methyl groups at C4 of the A ring are removed in multiple steps and cholesterol (C27) is formed. Note the numbering of the carbons and the labeling of the 4-ring system as A, B, C, and D in the cholesterol molecule. The upper limit of the normal level of plasma cholesterol is 200 mg/dL. Values of 240 mg/dL or above are considered to be elevated. When the circulating cholesterol level is above 300 mg/dL, the risk of a fatal heart attack is five times that of a person with a level of 200 mg/dL or below.

HMG-CoAR catalyzes the rate-limiting step and is the regulated enzyme in this pathway. The enzyme is inactive in the phosphorylated form and active in the nonphosphorylated form. Phosphorylation of the enzyme to the less active form is carried out by AMP kinase ( AMPK ). In turn, AMPK, itself, is active in a phosphorylated form and less active in the nonphosphorylated form. Phosphorylation is catalyzed by either of two kinases and dephosphorylation occurs through the action of protein phosphatase 2C (PP′ase 2C) . The dephosphorylation of HMG-CoAR-P occurs through the action of HMG - CoAR phosphatase that, in turn, is controlled by a PPI-1. The inhibitor is active in its phosphorylated form. These elaborate reactions involving phosphorylation–dephosphorylation to control the activity of HMG-CoAR are outlined in Fig. 9.8 . This is another facet in the regulation of the synthesis of cholesterol in the liver and, in the overall sense, may be less determinative for circulating cholesterol levels than the mutations in the LDLR gene.

Inhibition of Liver HMG-CoA Reductase by Drugs (Statins)

This enzyme, catalyzing the rate-limiting reaction of cholesterol synthesis in the liver, is a prime chemotherapeutic target. A group of drugs called statins mimics the structure of 3-hydroxy-3-methylglutaryl-CoA (HMG - CoA ), the substrate of the enzyme. These drugs are competitive inhibitors (Figs. 5.5 and 5.7) of HMG-CoAR. Some of the structures of the statin drugs are shown in Fig. 9.9 .

The main feature of a competitive inhibitor is that it should resemble the natural substrate enough to compete with it for binding to the active site but not so alike that it will form a product in the reaction. To ascertain that this is the case with a statin, Fig. 9.10 shows the comparison of the structures of lovastatin to that of the natural substrate HMG - CoA in the active site of HMG - CoAR . As a result of the action of statin inhibition, the synthesis of cholesterol in the liver is reduced (to as low as 50%). When this occurs, as elaborated by the work of Michael S. Brown and Joseph L. Goldstein, the transcription factor SREBP is transported to the Golgi apparatus for proteolytic processing that yields an active fragment bHLH (basic helix-loop-helix) that enters the nucleus and activates the LDLR gene and the genes for enzymes of cholesterol synthesis. This results in the production of more LDLRs that clear the blood of LDL cholesterol and replaces the deficit in cellular cholesterol. The action of statins counteracts the situation occurring when high-fat diets are ingested and the influx of cholesterol causes elevated cholesterol levels in liver cell membranes. When an excess of cholesterol develops in cell membranes, SREBP remains in the endoplasmic reticulum and it is not processed to the fragment that activates the LDLR gene . Consequently, the production of LDLR is reduced and LDL accumulates in the bloodstream. The mechanism, elaborated by Brown and Goldstein for the action of SREBP, is shown in Fig. 9.11 .

The ARH Protein

The ARH protein is involved with the clathrin coat apparatus. It is required for the activity of the LDLR in the endocytic process and directly binds soluble clathrin trimers and to clathrin adapter proteins . The N-terminal region of ARH contains a phosphotyrosine domain . This domain interacts with the internalization sequence , NPVY (Asn-Pro-Val-Tyr), in the cytoplasmic tail of LDLR . Mutations in the internalization sequence motif of LDLR abolish its binding to ARH. The sequence in ARH that binds to clathrin is LLDLE . ARH also binds to phosphoinositides that regulate clathrin bud assembly at the cell surface. In the C-terminal domain of ARH, there is a highly conserved sequence of 20 amino acids that binds to the β-2-adaptin subunit of the adapter protein , AP-2 . Patients with ARH mutations have hepatocytes with a defective adapter function leading to aberrant LDLR trafficking in the cell, specifically in the formation of endosomes . Mutations in the phosphotyrosine-binding domain cause recessive hypercholesterolemia .

Bile Acids

Cholesterol forms bile acids in the liver ( Fig. 9.12 ).

They are transported to the intestine where they solubilize ingested fats through micelle formation ( Fig. 9.22 ). In the circulation of bile acids, about 10% is lost through the feces. Increasing the loss of bile acids from the body is one measure to reduce cholesterol levels because more liver cholesterol would be diverted to the synthesis of bile acids. The beneficial effects of fiber in the diet, in part, accomplish this in that bile acids combine with fiber (they also bind to certain resins, e.g., cholestyramine ) and are excreted through the intestine. The circulation of bile acids is shown in Fig. 9.13 .

Bile acids are secreted from the liver into the gallbladder. Following a meal, bile acids enter the intestine through the bile duct . When bile acid formation is high, there is a negative feedback on further bile acid formation (elevating the level of cholesterol in the liver). The negative feedback occurs by suppression of cholesterol 7 α-hydroxylase activity ( Fig. 9.12 ). This may occur by bile acid repression of the transcription of human cholesterol 7 α-hydroxylase gene, CYP7A1 , resulting from effects on certain transcription factors regulating the expression of this gene. There are two lipid-activated nuclear receptors involved in the regulation of bile acid synthesis in the liver: the liver X receptor ( LXR ) and the farnesoid X receptor ( FXR ). The LXR binds oxysterols to activate it and the FXR binds bile acids. Both receptors form dimers with the retinoid X receptor and these activated receptors bind to gene promoters to generate mRNAs that are translated into proteins that prevent bile salt toxicity (the function of FXR) and the overproduction of cholesterol (LXR). When bile acid formation is low, more cholesterol would be diverted to the formation of bile acids. The mechanisms involved in the synthesis and removal of cholesterol and its products theoretically should maintain cholesterol at a homeostatic level. Possibly the maneuvers to cause higher excretion of cholesterol from the large intestine might result in enhanced cholesterol synthesis in the liver, the limiting factor in the synthesis still being HMG-CoA reductase.

Fatty Acids and Fat

Fatty acids are hydrocarbon chains with a carboxyl group at the end of the molecule. The hydrocarbon chain is a saturated fatty acid unless it contains one or more double bonds, in which case, it is an unsaturated fatty acid. Fig. 9.14 shows two fatty acids, stearic acid and oleic acid . Both contain eighteen carbons but oleic acid has one double bond in the 9–10 positions. The double bond creates a bend in the chain of about 120°.

The bend in the chain around a double bond occurs when the double bond is in cis , that is, when the hydrogen atoms on either side of the double bond are on the same side. When the hydrogen atoms on either side of the double bond are in trans , that is, on opposite sides of the double bond, there is no bend and the chain appears straight ( Fig. 9.15 ).

Oleic acid can be converted to stearic acid by hydrogenation (saturating the double bond) as shown in Fig. 9.16 .

Thus in linoleic acid , which has 18 carbons and two double bonds, the chain appears straight when both double bonds are in trans (bottom of Fig. 9.17 ), with a single bend when one double bond is in cis and the other is in trans (middle) or with two bends in the chain when both double bonds are in cis (top).

Consequently, the more cis double bonds in a hydrocarbon chain, the more bends appear in the structure. If we examine fatty acids with no double bonds (e.g., arachidic, stearic, and palmitic) compared with chains that have one cis double bond (e.g., erucic or oleic) compared to a fatty acid having two cis double bonds (e.g., linoleic) or a fatty acid having three cis double bonds (e.g., linolenic) or a fatty acid having four cis double bonds (e.g., arachidonic), we see that there is comparable bending, as shown in Fig. 9.18 .

Multiple double bonds in fatty acids often occur at three-carbon intervals (–CC–C–CC–). The double bond is usually in the cis configuration but partial hydrogenation can yield double bonds in the trans configuration. The latter is harmful in that ingestion of fats that have trans fatty acids (the so-called transfats ) increase LDLs and decrease HDLs. When fatty acids are left at room temperature, they can become rancid by oxidative breakdown into hydrocarbons, aldehydes, and ketones. Shorter chain fatty acids, like formic acid or acetic acid , are soluble in water and can produce acidity:

As the chain length of fatty acids increases, they become insoluble in water and exert little or no effect on the pH of a solution. Chemically, fatty acids behave like other carboxylated compounds; they can undergo esterification and can be reduced to fatty alcohols. When fatty acids are esterified with glycerol, they form either fats or oils depending on the types of fatty acids. Substitution with saturated fatty acids produces solid fats. Substitution with unsaturated fatty acids produces oils. Liquid fats are obtained mostly from plants. Unsaturated fatty acids such as oleic or linoleic acids are essential fatty acids and must be obtained in the diet.

Fatty acids are referred to by name and information following the name in parentheses. For example, arachidonic acid is referred to as arachidonic acid (20:4) where the number 20 refers to its number of carbon atoms and the number 4 refers to its number of double bonds. Sometimes the double bonds are numbered from the carboxyl (as number 1) and indicated by “Δ” followed by the numbers of the double bonds starting at the carboxyl side of the double bond. Thus arachidonic acid is 20:4 (Δ5, 8, 11, 14). Some of the common fatty acids are shown in Fig. 9.19 .

Arachidonic acid is an important fatty acid located in the plasma membrane. It is a precursor of prostaglandins, leukotrienes, lipoxins, and anandamide as well as many other important active biological compounds.

Glycerol and Its Substituents; Triglycerides

Glycerol is a trialcohol, the structure of which is shown in Fig. 9.20 .

Glycerol is 1,2,3-propanetriol, also referred to as glycerin . As a 70% solution of glycerol, it freezes at −37.8°C and therefore can act as an antifreeze compound , like ethylene glycol. When glycerol is substituted with one fatty acid, it is a monoglyceride , with two fatty acids, a diglyceride and with three fatty acids, a triglyceride. A triglyceride is shown in Fig. 9.21 .

Fat (Triglyceride) Digestion and Uptake

Ingested lipids are emulsified by bile acids . Bile acids are synthesized in the hepatocyte from cholesterol and transported through an adenosine triphosphate (ATP) requiring transport system to the biliary canaliculus . They flow down through the biliary tract and about half of the bile acids reach the gallbladder . The bile acids, released from the gallbladder (bile) and the liver, emulsify the fat in the form of micelles that are units containing triglycerides (triacylglycerols) in the center surrounded by bile acids. Fats can be broken down by pancreatic lipase that gains access to the triglyceride through gaps between the bile salts. The formation of a micelle is shown in Fig. 9.22 .

The fats are degraded by pancreatic lipase and phospholipase A2 (secreted by the pancreas and activated by trypsin) in the intestine to a mixture of monoglycerides and diglycerides. The action of pancreatic lipase is shown in Fig. 9.23 . The action of pancreatic lipase releases two free fatty acids (FFAs) and one 2-monacyl- sn -glycerol that are rapidly absorbed through the intestinal wall. The transporter for long-chain fatty acids in the apical side of the mature enterocyte is fatty acid transporter protein 4 ( FATP4 ). In the human, there is a family of six homologous transporters (FATP1 through FATP6) and they occur variously in all the fatty acid utilizing tissues in the body. FATP4, the only fatty acid transporter in the small intestine, is located in the apical brush border and is encoded on the human chromosome 9q34. It is a protein of 71,000 Da and contains 641 amino acids. Unsaturated fatty acids are more readily transported than saturated fatty acids. FATP4, in addition to its location in the plasma and internal membranes of the small intestine, is also found in adipocytes, brain, kidney, liver, skin, and heart. The isolated FATP4 also has the enzymatic activity of (long chain) Acyl-CoA synthase and the two activities of transporter and enzyme may work in concert to facilitate the influx of fatty acids across biomembranes. Although the brain contains FATP4, it does not use fatty acids as a source of energy and this requirement, when necessary, can be fulfilled by the action of glucagon on hormone-sensitive lipase (HSL) (discussed later) to release FFAs and glycerol from stored triglycerides and glycerol can be metabolized to glucose via gluconeogenesis to feed the brain (see Fig. 6.14).

As seen from Fig. 9.23 , lipase hydrolyzes at the 1 and 3 positions of glycerol. Phospholipids are degraded by phospholipase A2 at position 2 of glycerol to release FFA and lysophospholipid as shown in Fig. 9.24 . Complete digestion of a phospholipid is accomplished by phospholipase A ₁ , phospholipase D, and phospholipase C in addition to phospholipase A ₂ . These phospholipases all originate from the pancreas. The digestion products are absorbed by the intestinal mucosal cells (apical side of mature enterocytes) where resynthesis of triacylglycerides occurs.