Disorders of Lipid Metabolism

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1 CHAPTER 37 Disorders of Lipid Metabolism CLAY F. SEMENKOVICH ANNE C. GOLDBERG IRA J. GOLDBERG Lipid Biochemistry and Metabolism, 1660 Nuclear Receptors and Lipid Metabolism, 1665 Plasma Lipoproteins, Apolipoproteins, Receptors, and Other Proteins, 1666 Integrative Physiology of Lipid Metabolism, 1674 Overview of Hyperlipidemia and Dyslipidemia, 1675 Hypertriglyceridemia, 1676 Hypercholesterolemia Without Hypertriglyceridemia, 1680 Elevated Triglycerides and Cholesterol, 1682 Hypocholesterolemia, 1684 Overview of Atherogenesis, 1685 Evidence Supporting Treatment of Lipid Disorders, 1685 Treatment of Lipid Disorders, 1687 KEY POINTS Abnormalities of lipid metabolism cause heart disease, pancreatitis, vitamin deficiencies, and gallstones. Endocrine disorders such as diabetes and obesity have protean effects on lipid metabolism. Lowering low-density lipoprotein (LDL) with statins decreases vascular disease and prolongs life. Extreme elevations of triglycerides should be treated to avoid pancreatitis. Moderate elevations of triglycerides may be associated with vascular disease, but optimal treatment for this condition is unknown. Pharmacologic elevation of high-density lipoprotein (HDL) is not clearly beneficial. Substantial additional lowering of LDL beyond that seen with statins can be achieved by inhibiting proprotein convertase subtilisin/kexin type 9 (PCSK9). LIPID BIOCHEMISTRY AND METABOLISM Endocrine disorders have important effects on serum and tissue lipids, making the mechanisms underlying primary and secondary disorders of lipid metabolism relevant to clinicians as well as basic scientists. Some primary disorders of lipid metabolism, such as familial hypercholesterolemia (FH), are uncommon but important to understand. The LDL receptor pathway altered in FH helps explain genetic predispositions to heart disease; the mechanism of action of statin drugs, which decrease the risk of vascular events and prolong life; 1,2 and the mechanism of action of PCSK9 inhibitors, which strikingly lower lipid levels even in the setting of statin treatment. 3 The quintessential secondary disorder of lipid metabolism is that seen in diabetes, a disease so frequently characterized by abnormalities of fat that lipids have been implicated in its pathogenesis. Lipids are ubiquitous. They constitute the physical bilayer that allows the formation of cell membranes, which are required for specialized organelles inside the cell and for regulating transport between the extracellular and intracellular environments. They circulate in the blood, with fatty acids and triglycerides providing an energy source to tissues such as heart and skeletal muscle and non-nutritive sterols providing substrates for hormone production by gonads and adrenals. Their specialized functions include the development of surfactant in lung to maintain patency of alveoli, formation of bile to facilitate excretion of a variety of metabolites, and constitution of myelin throughout the nervous system to ensure the fidelity of nerve transmission. Lipids are also signaling molecules, serving as targets of lipid kinases that perpetuate signaling cascades, substrates for cyclooxygenases and related enzymes that generate prostaglandins, and ligands for nuclear receptors such as the peroxisome proliferator-activated receptors (PPARs). The broad spectrum of lipid functions results in part from their biophysical characteristics. Simple and Complex Lipids Lipids owe their functional versatility to their hydrophobic structure. Because of the presence of fairly long carbon chains, lipids tend to associate with each other and have limited or no solubility in water. Fatty acids and cholesterol are simple lipids, whereas triglycerides and phospholipids are complex lipids (Fig. 37-1). Fatty Acids Chemical structures for the fatty acids are determined by the number of carbon atoms and the number of double bonds (see Fig. 37-1A). For example, stearic acid has 18 carbon atoms and is saturated, meaning that it has no double bonds; this is designated by the abbreviation C18:0. The 18-carbon monounsaturated fatty acid oleic acid (C18:1) has one double bond, and the polyunsaturated fatty acid linoleic acid (C18:2) has two double bonds. Linoleic acid and arachidonic acid (C20:4) are ω-6 fatty acids, meaning that a double bond is present at the sixth carbon from the end of the molecule farthest from the carboxy (COOH)-terminal. Fish oils, which lower lipids, are ω-3 fatty acids, with a double bond present at the third carbon from the end opposite the COOH-terminal. Saturated fatty acids and some unsaturated fatty acids such as oleic acid are nonessential (i.e., they can be synthesized). Most ω-6 and ω-3 fatty acids are essential; they cannot be 1660

2 CHAPTER 37 Disorders of Lipid Metabolism 1661 A Stearic acid: Oleic acid: Linoleic acid: Fatty acids CH 3 (CH 2 ) 16 COOH CH 3 (CH 2 ) 7 CH = CH (CH 2 ) 7 COOH CH 3 (CH 2 ) 4 CH = CH CH 2 CH = CH (CH 2 ) 7 COOH Triglycerides O H 2 C O C (CH 2 ) 16 CH 3 O HC O C (CH 2 ) 16 CH 3 O Phospholipids O H 2 C O C Fatty acid O HC O C Fatty acid O CH 3 H 2 C O C (CH 2 ) 16 CH 3 H 2 C O P O CH 2 CH 2 N + CH 3 Glycerol Fatty acid O CH 3 Choline Phosphatidylcholine B Tristearin C Cholesterol CH 3 CH 3 D OH 3 CH CH 2 CH 2 CH 2 CH CH 3 Figure 37-1 Structures of common lipids, exemplified by the stearic, oleic, and linoleic fatty acids (A), the triglyceride tristearin (B), the phospholipid phosphatidylcholine (C), and cholesterol (D). TABLE 37-1 Major Fatty Acids Chemical Designation Common Name Common Food Saturated Fatty Acids (No Double Bonds) C12:0 Lauric Coconut oil C14:0 Myristic Coconut oil, butter fat C16:0 Palmitic Butter, cheese, meat C18:0 Stearic Beef, chocolate Monounsaturated Fatty Acids (One Double Bond) C18:1 Oleic Olive and canola oils Polyunsaturated Fatty Acids (Two or More Double Bonds) Omega-6 Fatty Acids C18:2 Linoleic Sunflower, corn, soybean, and safflower oils C20:4 Arachidonic Omega-3 Fatty Acids C18:3 α-linolenic Canola, flaxseed, and soybean oils C20:5 Eicosapentaenoic (EPA) Salmon, cod, mackerel, tuna C22:6 Docosahexaenoic (DHA) Salmon, cod, mackerel, tuna synthesized and are usually required for health, especially during development and in times of physiologic stress. Table 37-1 shows major food sources of fatty acids. Triglycerides The structure of tristearin, a triglyceride with three molecules of stearic acid connected to a glycerol molecule by means of ester linkages, is shown in Figure 37-1B. Other triglycerides have a similar structure with alternative fatty acids esterified to the glycerol backbone. Most of the mass of adipose tissue in the body is composed of triglycerides; triglycerides that circulate in the blood mostly reflect the fatty acid composition of adipose tissue triglycerides, and both sources reflect dietary fatty acid composition. Butter in Western diets consists of similar amounts of palmitate and oleate with a lesser amount of stearate; adipose tissue and circulating triglycerides in persons eating Western diets contain mostly palmitate and oleate. Olive oil in Mediterranean diets is predominantly oleate with much less palmitate, so fat and circulating triglycerides in people eating a Mediterranean diet are enriched in oleic acid. Extremely high levels of triglycerides in the blood predispose to pancreatitis. Phospholipids The chemical structure for a generic phosphatidylcholine, a type of phospholipid, is shown in Figure 37-1C. As with triglycerides, phospholipids have a glycerol backbone to which fatty acids are esterified at the first two alcohols. The characteristics of these fatty acids are important for determining cell membrane shape and function. 4 The third alcohol is esterified to a phosphate moiety linked to another molecule, such as choline, ethanolamine, or serine. The presence of long-chain fatty acids comprising hydrophobic regions and the charged species at the end of the molecule make phospholipids perfect for generating cell membranes and lipoprotein surface components: the bilayer is oriented so that the hydrophobic regions point toward each other, and the hydrophilic regions interact with the aqueous environment. Phospholipids are distributed asymmetrically in cell membranes, with cholinecontaining lipids directed toward the outer surface and amine-containing lipids directed toward the cytoplasmic surface. Appearance of the aminophospholipid phosphatidylserine on the cell surface initiates blood clotting and marks apoptotic cells for phagocytosis.

3 1662 SECTION VIII Disorders of Carbohydrate and Fat Metabolism Cholesterol The structure of cholesterol is shown in Figure 37-1D. The presence of cholesterol in the plasma membrane is critical for maintaining membrane fluidity, probably by disrupting the interactions between phosphatidylcholine and other molecules. The concentration of cholesterol is enriched in the plasma membrane, with much lower levels detected in the membranes of most intracellular organelles. Cholesterol is necessary for the synthesis of estrogen, progestins, androgens, aldosterone, vitamin D, glucocorticoids, and bile acids. Cholesterol deficiency is associated with severe developmental defects, as manifested in the rare Smith- Lemli-Optiz syndrome, which is likely caused by disruption of the Hedgehog signal transduction pathway. 5 Cholesterol excess is associated with gallstones and vascular disease. Fatty Acid Metabolism Fatty Acid Biosynthesis In humans eating a typical Western diet, the overall contribution of de novo lipogenesis to lipid metabolism is small because the ingestion of exogenous fat is sufficient to suppress the energy-requiring process of synthesizing fats from carbohydrates. However, high-carbohydrate diets, especially those containing fructose, 6 substantially increase lipogenesis in liver and adipose tissue of humans. Most tissues carry out fatty acid biosynthesis to at least a small degree regardless of nutritional status. Several of the key steps in fatty acid biosynthesis, presented in Figure 37-2, also have major effects on systemic metabolism. Citrate derived from the tricarboxylic acid (TCA) cycle is converted to acetyl coenzyme A (acetyl CoA) in the cytoplasm by the action of adenosine triphosphate (ATP) citrate lyase. Acetyl CoA is then converted to malonyl CoA by acetyl CoA carboxylase (ACC), which exists in two iso- forms: ACC1 (encoded by the gene ACACA) is cytosolic and important in liver and fat for de novo lipogenesis, and ACC2 (ACACB) is associated with mitochondria, also plays a role in liver metabolism, and is expressed at highest levels in muscle and heart. Antisense targeting of ACC isoforms has been shown to improve lipid metabolism and insulin sensitivity. 7 Malonyl CoA inhibits carnitine palmitoyltransferase 1 (CPT1), which transports fatty acids into mitochondria, thereby preventing the catabolism of fats under physiologic conditions in which energy is being stored as fat through fatty acid biosynthesis. Malonyl CoA also serves as substrate for fatty acid synthase, which sequentially connects two carbon fragments to generate saturated fatty acids such as palmitate. Inhibition of fatty acid synthase in the hypothalamus suppresses appetite, inducing weight loss and improving insulin sensitivity. 8 Pharmacologic inhibition of fatty acid synthase improves glucose metabolism and fatty liver in mice. 9 Palmitate is converted to stearate through the action of a long-chain fatty acid elongase, which, when inactivated, promotes obesity but prevents insulin resistance. 10 Stearate is subsequently converted to oleate by stearoyl-coa desaturase 1, which, when inactivated, increases fatty acid oxidation and protects against diet-induced obesity and insulin resistance. 11 Fatty Acid Oxidation Metabolism of fatty acids provides more energy than metabolism of carbohydrates or proteins. Fatty acids undergo the process of β-oxidation in mitochondria (see Fig. 37-2). They are transported across (or diffuse across) the plasma membrane, converted to acyl-coa species by acyl-coa synthase, and then translocated to the mitochondrial matrix by CPT1 and CPT2. β-oxidation removes two carbon fragments through the sequential actions of Fatty acids ACS Acyl CoA Mitochondria Citrate ATP citrate lyase Acetyl CoA CPT1 β-oxidation TCA cycle Acetyl CoA carboxylase Malonyl CoA Acetyl CoA Fatty acid synthase Palmitate Glucose Fructose Glycolysis Pyruvate Ketones ELOVL6 Stearate SCD-1 Oleate Figure 37-2 Fatty acid metabolism. Fatty acids are substrates for acyl-coa synthase (ACS), which generates CoA moieties that are transported into mitochondria by carnitine palmitoyltransferase 1 (CPT1). Here, β-oxidation generates acetyl CoA, which can also be generated from glycolysis (bottom left). Acetyl CoA can be used to produce ketones, or it may enter the TCA cycle, leading to production of citrate; in the cytoplasm, citrate is a substrate for ATP citrate lyase, which produces acetyl CoA. The acetyl CoA serves as a substrate for de novo synthesis of fatty acids, as depicted on the right side of the figure. ATP, adenosine triphosphate; CoA, coenzyme A; ELOVL6, elongation of very long chain fatty acid protein 6; SCD-1, stearoyl-coa desaturase 1; TCA, tricarboxylic acid.

4 CHAPTER 37 Disorders of Lipid Metabolism 1663 acyl-coa dehydrogenases (e.g., medium-chain acyl-coa dehydrogenase [MCAD] and very long chain acyl-coa dehydrogenase [VLCAD]), enoyl-coa hydratase, hydroxy- CoA dehydrogenase, and thiolase. This process generates reduced nicotinamide adenine dinucleotide (NADH) and reduced flavin adenine dinucleotide (FADH), which participate in electron transport to yield ATP. After multiple cycles, acetyl CoA is produced, which is a substrate for the TCA cycle and for ketogenesis. Ketogenesis is necessary for life during times of nutritional deprivation. Extreme production of ketones occurs in the setting of insulin deficiency and represents a threat to life. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG- CoA) synthase (rate-limiting in mitochondria) converts acetyl CoA to hydroxymethylglutaryl CoA, which is converted to acetoacetate by HMG-CoA lyase. Acetoacetate is either reduced to β-hydroxybutyrate or converted to acetone. Defects in fatty acid oxidation are among the most common inborn errors of metabolism. Presentations include nonketotic hypoglycemia, liver dysfunction, and cardiomyopathy. 12 Triglyceride and Phospholipid Metabolism Dietary fat consists of triglycerides and phospholipids, which are digested in the stomach and proximal small intestine. Triglycerides are broken down into component fatty acids in part through the action of pancreatic lipase, which is activated by bile acids. Bile salts form micelles that acquire fatty acids and interact with the unstirred water layer of the intestine, where fatty acids are absorbed. Longchain fatty acids are taken up by enterocytes, re-esterified into triglycerides, and exported into the lymph as lipoproteins. Medium-chain ( C10) fatty acids directly enter the portal vein to access the liver. Lipolysis of Triglyceride Stores in Adipose Tissue The greatest triglyceride mass resides in adipose tissue, and turnover of energy stores at this site has important effects on lipid metabolism, normal physiology, and human health. Increased lipolysis in adipose tissue of the obese results in elevated circulating levels of free fatty acids, which may cause dysfunction in pancreatic beta cells, liver, skeletal muscle, and heart. Healthy subjects whose parents have type 2 diabetes mellitus have impaired insulinmediated suppression of circulating fatty acids, 13 suggesting that an early defect in adipose tissue fatty acid metabolism contributes to the evolution of diabetes. Release of free fatty acids and glycerol from adipose tissue is controlled by a variety of hormones, many of which act through G protein coupled receptors. The most robust mediators of fatty acid release are catecholamines, which bind to β-adrenergic receptors, activating stimulatory G proteins (G s ) that prompt an increase in the activity of cyclic adenosine monophosphate and protein kinase A. Glucagon, adrenocorticotropic hormone, α-melanocytestimulating hormone, and thyroid-stimulating hormone also induce lipolysis through activity of G s proteins. Adenosine suppresses lipolysis by binding to receptors that activate inhibitory G proteins (G i ). Niacin, which suppresses lipolysis, binds to the G protein receptor GPR109A, but this interaction does not mediate the effects of this vitamin on lipid metabolism. 14 A major mediator of lipolytic inhibition is insulin, which activates the insulin receptorsignaling cascade and suppresses lipolysis at many steps, one of which includes a decrease in protein kinase A activity. Adipocyte Triglycerides ATGL/ CGI-58 Diglycerides Hormonesensitive lipase Monoglyceride lipase Monoglycerides Glycerol Fatty acids Figure 37-3 Lipolysis in adipocytes. Stored triglycerides are metabolized to yield the fatty acids that circulate in plasma through the action of three distinct lipases with separate substrate specificities. Triglycerides are acted on by adipose triglyceride lipase (ATGL) in complex with the coactivator protein CGI-58 to yield diglycerides, which are acted on by hormone-sensitive lipase to yield monoglycerides. The monoglycerides, in turn, are acted on by monoglyceride lipase to yield glycerol. Lipid droplet proteins modulate this lipolytic process. At least three enzymes and two accessory proteins are required for the normal process of hormone-induced lipolysis in adipose tissue. 15 Stored triglycerides are acted on by the enzyme, adipose triglyceride lipase (encoded by PNPLA2), which requires the coactivator protein CGI-58. Diglycerides are hydrolyzed by hormone-sensitive lipase, yielding monoglycerides that are metabolized by monoglyceride lipase. This process cannot occur unless perilipin, a protein that coats small lipid droplets, is phosphorylated by protein kinase A. This process is depicted schematically in Figure Human mutations in adipose triglyceride lipase or CGI-58 are responsible for two variants of neutral lipid storage disease that are characterized by hepatic steatosis, lipid accumulation in muscle tissues, neurologic problems, and, in one variant, skin defects. Triglyceride and Phospholipid Synthesis and Tissue Delivery of Lipids Triglyceride Synthesis. Key steps in triglyceride synthesis have major effects on systemic metabolism. Most triglycerides are synthesized through the glycerol phosphate pathway (Fig. 37-4, top portion) by a sequence of acylations. Another pathway, the monoacylglycerol pathway, is believed to be active only in the small intestine. Glycerol- 3-phosphate is acted on by one of the glycerol-3-phosphate acyltransferases (GPATs) to generate lysophosphatidic acid. An important isoform is GPAT1, which is thought to compete with CPT1 for fatty acyl CoA molecules inside the cell, with GPAT1 prevailing when energy is to be stored and CPT1 dominant when energy is required. The next acylation is mediated by acylglycerol-3-phosphate acyltransferases (AGPATs) and generates phosphatidic acid. Human mutations in AGPAT2 are responsible for the disease known as congenital generalized lipodystrophy. Phosphatidic acid represents an important branch point in lipid metabolism. It serves as substrate for synthesis of either cytidine diphosphate diacylglycerol (CDP-DAG, the precursor for molecules such as phosphatidylinositol) or diacylglycerol (DAG). Synthesis of DAG requires a phosphatase activity provided by lipins. 16 These proteins have complicated effects on insulin sensitivity and metabolism.

5 1664 SECTION VIII Disorders of Carbohydrate and Fat Metabolism Glycerol-3-phosphate GPATs Lysophosphatidic acid AGPATs Triglycerides Phosphatidic acid DGATs Lipins DAG CDP-DAG Phosphatidylcholine Phosphatidylethanolamine Phosphatidylinositol Phosphatidylglycerol Cardiolipin Phosphatidylserine Figure 37-4 Phospholipid and triglyceride synthesis. Glycerol-3-phosphate is converted by glycerol-3-phosphate acyltransferases (GPATs) to lysophosphatidic acid, which is converted to phosphatidic acid by acylglycerol-phosphate acyltransferases (AGPATs). Phosphatidic acid can be converted to cytidine diphosphate diacylglycerol (CDP-DAG), to fuel one arm of phospholipid synthesis, or to diacylglycerol (DAG), which is a substrate for another arm of phospholipid synthesis and for acyl-coa:diacylglycerol acyltransferases (DGATs), which generate triglycerides. DAG can serve as a signaling molecule and as substrate for synthesis of either triglycerides or common phospholipids. The acylation of DAG to form triglycerides is catalyzed by acyl-coa:diacylglycerol acyltransferases (DGATs). Inactivation of DGAT1 renders mice resistant to diet-induced obesity. 17 Phospholipid Synthesis. As shown in the bottom portion of Figure 37-4, phospholipid synthesis is intimately related to triglyceride synthesis. Generation of one of the most important phospholipids, phosphatidylcholine, occurs mostly through the Kennedy pathway, which utilizes choline as an initial substrate and DAG at the final step. Mammalian liver is also able to generate phosphatidylcholine from phosphatidylethanolamine through successive methylations. Both phosphatidylcholine and phosphatidylethanolamine can be converted to phosphatidylserine. Lipoprotein Lipase. Most lipids are delivered to peripheral tissues such as muscle and fat through the activity of lipoprotein lipase (LPL). LPL, which is rate-limiting for clearance of plasma triglycerides and essential for generation of HDL particles, 18 hydrolyzes triglycerides (and, to a lesser extent, phospholipids) in circulating triglyceride-rich lipoproteins to allow peripheral sites such as adipose tissue and muscle access to preformed fatty acids. Much of this lipid flux is controlled by insulin, which increases LPL in fat and decreases LPL in muscle. Exercise tends to have the opposite effect, 19 ensuring appropriate energy supplies are available to meet metabolic demands. Free fatty acids released from lipoproteins by the action of LPL presumably diffuse into resident cells in the local tissues, where they are converted to acyl CoA species and either stored as triglycerides or subjected to fatty acid oxidation. This hydrolytic release reaction occurs at the capillary endothelium. LPL is not synthesized in endothelial cells but is produced in adipocytes, cardiac myocytes, and skeletal myocytes and then secreted and targeted to the luminal surface of the endothelium through mechanisms that are poorly understood. At the endothelium, LPL and triglyceride-rich lipoproteins bind to glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1). GPIHBP1 is believed to serve as a platform for lipolysis in the plasma. 20 Cholesterol Metabolism In adults, dietary cholesterol is not required because many tissues are capable of cholesterol synthesis. However, most diets include animal products, the source of cholesterol. Plants do not have cholesterol, but their membranes contain phytosterols, which are structurally similar to cholesterol and are useful in the dietary treatment of hypercholesterolemia because they compete with cholesterol for absorption. The liver and intestine are quantitatively the most important sites for cholesterol metabolism in humans, although a very small amount of cholesterol is also lost through the normal turnover of skin. Cholesterol Absorption, Synthesis, and Excretion Cholesterol is absorbed through a process that requires the formation of bile salt micelles. The efficiency of absorption varies widely in humans. There is a gradient of absorption through the intestine that is greatest in the proximal small intestine and least in the ileum. This gradient parallels the expression of Niemann-Pick C1 like 1 (NPC1L1), a transmembrane protein with a sterol-sensing domain that is involved in cholesterol absorption. 21 NPC1L1 is the target of ezetimibe, a drug that lowers cholesterol and has been shown to decrease heart disease (see later discussion). NPC1L1 also absorbs phytosterols such as sitosterol. Sterols are pumped out of the enterocyte and into the intestinal lumen by two ATP-binding cassette (ABC) transporters, ABCG5 and ABCG8. Human mutations in these transporters cause the rare disorder sitosterolemia, 22 characterized by increased absorption and circulating levels of sitosterol and cholesterol, xanthomas, and heart disease (see later discussion). Figure 37-5A illustrates cholesterol synthesis. Acetate is converted to HMG-CoA. The latter is a substrate for HMG-CoA reductase, the enzyme that is rate-limiting for cholesterol biosynthesis and is inhibited by statin drugs. Cells exquisitely regulate cholesterol acquisition. 23 When levels are low, mechanisms are activated to increase cholesterol biosynthesis and import cholesterol from the extracellular environment. Statins, by lowering cholesterol and

6 CHAPTER 37 Disorders of Lipid Metabolism 1665 A Acetate Cholesterol Acetate Cholesterol Bile acids HMG-CoA Mevalonic acid Cholesterol HMG-CoA reductase Cholesterol biosynthesis 50% Reabsorption Liver Cholesterol Bile Bile acids Intestine 97% Reabsorption B Enterohepatic circulation of cholesterol and bile acids Figure 37-5 A, Cholesterol biosynthesis. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is the rate-limiting enzyme regulating cholesterol biosynthesis. The enzyme is downregulated by excess cholesterol in the cell. B, Enterohepatic circulation of cholesterol and bile acids. Approximately 50% of cholesterol and 97% of bile acids are reabsorbed from the intestine and recirculated to the liver. (A, Modified from Brown MS, Goldstein JL. A receptor-mediated pathway for cholesterol homeostasis. Science. 1986;232:34-47.) preventing cholesterol biosynthesis, work predominantly by increasing liver uptake of cholesterol from the plasma through the LDL receptor (see later discussion) and promoting its excretion. Free cholesterol in cells is esterified to form cholesteryl esters for storage. This esterification reaction is carried out by acyl CoA:cholesterol acyltransferases (ACATs). These endoplasmic reticulum enzymes exist in two forms: ACAT1 is present in macrophages and has been implicated in atherosclerosis, and ACAT2 is present in liver and intestine and is implicated in cholesterol absorption. Nonspecific ACAT inhibition in humans does not affect serum lipids and does not have beneficial effects on atherosclerosis. 24 Cholesterol, which is non-nutritive and cannot be catabolized to carbon dioxide and water, is either secreted into the bile as free cholesterol (about half of which is reabsorbed) or converted to bile acids for secretion into bile. Most bile acids are reabsorbed in the terminal ileum. This enterohepatic circulation of cholesterol and bile acids is shown in Figure 37-5B. The rate-limiting enzyme for bile acid synthesis is cholesterol 7α-hydroxylase, which is under feedback regulation by bile acids. Interruption of the enterohepatic circulation of bile acids through the use of bile acid sequestrants (BAS) increases bile acid synthesis, lowers plasma cholesterol, and decreases vascular disease events. At least in mice, evidence suggests that cholesterol can be excreted directly by enterocytes (independent of the biliary system) through an active metabolic process termed transintestinal cholesterol excretion (TICE). 25 NUCLEAR RECEPTORS AND LIPID METABOLISM Feedback regulation Excretion 50% 3% Nuclear receptors, usually transcription factors with ligandbinding and DNA-binding domains, affect lipid metabo- Adipose tissue PPARs Fatty acids PPAR LXR FXR Liver Bile acids Bile acids VLDL Fatty PPARs acids LXR, FXR Muscle Intestine Chylomicrons Figure 37-6 Nuclear receptors in lipid metabolism. Peroxisome proliferatoractivated receptors (PPARs) are active in adipose tissue, which is a source of fatty acids that are transported to liver, where PPARα, the liver X receptors (LXR), and farnesoid X receptor (FXR) are active. Bile acids produced by the liver participate in an enterohepatic circulation with the intestine, another site of LXR and FXR expression. Very low density lipoprotein (VLDL), produced by liver, and chylomicrons, from intestine, are metabolized to release fatty acids that fuel muscle (another site of PPAR expression) and may be stored by adipose tissue. lism. Classic hormones that interact with nuclear receptors and have important lipid effects include thyroid hormone, glucocorticoids, estrogen, and testosterone. Thyroid hormone regulates cholesterol metabolism through direct effects on the gene for a transcription factor that controls LDL-receptor expression, sterol regulatory element binding protein 2 (SREBP2). 26 This role explains why lipid levels tend to be high in hypothyroid patients and low with hyperthyroidism. Glucocorticoids have robust effects on multiple aspects of lipid metabolism, inducing expression of HMG-CoA reductase to promote cholesterol synthesis, increasing expression of fatty acid synthase to promote fatty acid synthesis, and decreasing LPL to impair clearance of circulating lipids. Accordingly, hyperlipidemia is seen commonly in the setting of glucocorticoid treatment, and insulin resistance induced by glucocorticoids amplifies the hyperlipidemia. Estrogens and selective estrogen receptor modulators such as raloxifene lower cholesterol 27 by inducing LDL-receptor activity; they tend to increase triglyceride levels, especially when higher oral doses are administered. Derivatives of cholesterol can serve as selective estrogen-receptor modulators (SERMs) to affect the vasculature. 28 Androgens, by activating the androgen receptor, decrease levels of HDL. 29 Aside from classic hormones and their receptors, other nuclear receptors affect lipid metabolism after interacting with several types of metabolic by-products. These receptors include the PPARs, the liver X receptors (LXRs), and the farnesoid X receptor (FXR). A schematic view of the roles of these receptors in lipid metabolism is presented in Figure There are three known types of PPARs: α, γ, and δ. PPARα promotes fatty acid oxidation as well as ketogenesis and is induced by starvation. It is expressed at highest levels in tissues that are adapted to metabolize fats, such as liver and skeletal muscle, but is also present at numerous other sites. In humans, pharmacologic activation of PPARα with fibrates lowers triglycerides and increases HDL. Fatty acids interact with the receptor, but a phosphatidylcholine species was identified as an endogenous ligand for PPARα. 30 Whereas PPARα facilitates energy utilization, PPARγ activates genes that promote energy storage. It is expressed at highest levels in adipose tissue and is also found in macrophages, where it may help coordinate the complex relationship between inflammation and metabolism. Ether

7 1666 SECTION VIII Disorders of Carbohydrate and Fat Metabolism lipids, phospholipids generated in peroxisomes, appear to be endogenous ligands for PPARγ. 31,32 Pharmacologic activation of PPARγ in humans with thiazolidinediones results in insulin sensitization and weight gain. The latter effect occurs because this nuclear receptor promotes adipogenesis as well as fluid retention through effects on the kidney. Thiazolidinedione treatment in humans tends to lower triglycerides and increase HDL, probably by modulating insulin signaling, but the impact of these agents on atherosclerosis is unclear. Dual agonists for PPARα and PPARγ were shown to lower hemoglobin A 1c as well as serum lipids in humans but also increased all-cause mortality rate. PPARδ has important effects in tissues such as skeletal muscle, and its activation may mimic the effects of exercise. Agents targeting this receptor in humans appear to show benefit, in part by increasing fatty acid oxidation. 33 LXRs and FXR are also involved in lipid metabolism. LXRα and LXRβ are activated by oxysterols (modified derivatives of cholesterol) to increase the conversion of cholesterol into bile acids, increase bile acid excretion, and decrease cholesterol absorption. 34 LXR activation inhibits cholesterol uptake by inducing the degradation of the LDL receptor. 35 LXRs also induce fatty acid and triglyceride synthesis. FXR is activated by bile acids to stimulate bile acid secretion as well as reabsorption. Administration of a BAS to humans with diabetes lowers blood sugar, which may be a consequence of effects on FXR as well as activation of the G protein coupled receptor TGR5 to increase glucagonlike peptide 1 by the intestine. 36 Additional nuclear receptors also play important roles in lipogenesis the process of converting carbohydrates to triglycerides rather than glycogen. Carbohydrate response element binding protein (ChREBP) may control as much as 50% of this process. It responds to carbohydrate excess by transactivating a series of glycolytic and lipogenic genes. SREBP1 is also critical for this process. 37 PLASMA LIPOPROTEINS, APOLIPOPROTEINS, RECEPTORS, AND OTHER PROTEINS Physiologic requirements at sites remote from the source of external lipids (i.e., the gut) have resulted in selective pressure to develop a system capable of moving nutrients, vitamins, structural components, and proteins with specialized functions through the plasma compartment. The effectors of this system are lipoproteins, spherical particles that circulate in the blood. Appropriate concentrations of lipoproteins are essential for health. When certain lipoproteins are present at high levels in the circulation, cardiovascular disease and pancreatitis may result. When other lipoproteins are absent or present at very low levels, vitamin deficiency syndromes and cardiovascular disease may develop. The roles of the various lipoproteins are discussed in detail in the following sections. Major Lipoproteins A prototypical lipoprotein is shown in Figure The fundamental structure of a lipoprotein exploits the biochemical characteristics of its components. The surface consists of charged molecules that interact with the aqueous environment, such as phospholipids and free cholesterol. Amphipathic proteins (with hydrophilic as well as hydrophobic domains), known as apolipoproteins (or simply apoproteins), are also present on the surface, with their hydrophilic domains oriented toward the plasma and Apoprotein B100 Apoprotein E Apoprotein C Surface Phospholipid Core Triglyceride and cholesteryl esters Free cholesterol Figure 37-7 General structure of lipoproteins: schematic representation of a very low density lipoprotein (VLDL) particle. their hydrophobic domains toward the core of the particle. Apolipoproteins direct lipoproteins toward their appropriate sites of metabolism. The lipoprotein core consists of neutral (uncharged) lipids such as triglycerides and cholesteryl esters. Lipoprotein movement through the plasma compartment is dynamic. Humans spend most of their lives in the postprandial state. Eating is associated with generation of lipoproteins, induction of enzymes that metabolize those lipoproteins, interactions among lipoproteins in the plasma involving the exchange of both lipid and protein components, rapid alterations of lipoprotein size as large particles are metabolized to smaller ones, genesis of new lipoproteins in the circulation as excess surface components of shrinking particles are extruded, and movement of critical nutrients and vitamins into tissues. Clinical assessment of disease risk is based on fasting measurements, but most lipid metabolism that causes disease occurs in the fed state. The major classes of lipoproteins are listed in Table Their original identification was accomplished based on migration in an ultracentrifuge, and classes were defined based on density. An alternative original classification scheme, which is no longer useful, involved electro phoretic mobility in agarose gels. Chylomicrons, chylomicron remnants, and very low density lipoproteins (VLDLs) are rich in triglycerides. Intermediate-density lipoproteins (IDLs), LDLs, and lipoprotein(a), or Lp(a), are rich in cholesterol. HDLs are enriched in phospholipids. Triglyceride-rich lipoproteins such as chylomicrons are large and generally insoluble, which accounts for the cloudy appearance of plasma when it is obtained in nonfasting subjects or in fasting subjects with some types of hyperlipidemias. Table 37-2 provides general ranges for particle size, which differs substantially among lipoproteins and within each class. Certain lipoprotein subtypes, such as small dense LDL, may promote cardiovascular disease and are more likely to occur with insulin resistance. Chylomicrons originate in the gut. They are lighter than water and float to the top of a plasma sample. The particles are cleared fairly rapidly after a meal and should be absent after an overnight fast. Their distinguishing apolipoprotein is apob48, which is the only form of apolipoprotein B

8 CHAPTER 37 Disorders of Lipid Metabolism 1667 TABLE 37-2 Major Classes of Plasma Lipoproteins Type Density (g/ml) Origin Major Lipids Major Apolipoproteins Size (nm) Chylomicrons <0.95 Intestine 85% Triglyceride B48, AI, AIV, E, CI, CII, CIII ~ Chylomicron remnants <1.006 Derived from chylomicrons 60% Triglyceride B48, E ~ % Cholesterol VLDL <1.006 Liver 55% Triglyceride B100, E, CI, CII, CIII % Cholesterol IDL Derived from VLDL 35% Cholesterol B100, E % Triglyceride LDL Derived from IDL 60% Cholesterol B % Triglyceride HDL Liver, intestine, plasma 25% Phospholipid AI, AII, CI, CII, CIII, E % Cholesterol 5% Triglyceride Lp(a) Liver 60% Cholesterol 5% Triglyceride B100, apo(a) ~30 apo, apolipoprotein; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; Lp(a), lipoprotein(a); VLDL, very low density lipoprotein. produced by intestinal cells in humans. Chylomicrons acquire apoc and apoe molecules by interacting with HDL particles, a process that promotes chylomicron metabolism and conversion to chylomicron remnants. Chylomicron remnants, also characterized by the presence of apob48, are cleared rapidly from the plasma. Remnant particles may promote ischemic heart disease, particularly in obesity. 38 VLDL particles are of hepatic origin. Smaller than chylomicrons, their distinguishing apolipoprotein is apob100, the form of apob produced by the liver. VLDLs also carry apoc molecules that modulate the conversion of VLDLs to IDLs, which are VLDL remnants and are thought to be atherogenic. IDL particles contain apob100 and apoe, and they are converted to LDL, which is characterized by carrying essentially only apob100 as an apolipoprotein. LDL, known as bad cholesterol, is the major carrier of cholesterol in most humans, and its measurement forms the basis for coronary heart disease (CHD) risk stratification and treatment goals. For most clinical laboratories, LDL results represent both IDL and LDL particles. HDL particles have a complex biology. They can be generated by liver and intestine or assembled in the plasma as a consequence of the metabolism of other lipoproteins. They are arbitrarily divided into HDL 2 (less dense, at to g/ml), which typically contains apoai as well as apocs, and HDL 3 (more dense, at to 1.21 g/ml), which typically contains apoai, apoaii, and apocs. There is also a minor subclass known as HDL 1 that carries a large percentage of plasma apoe. HDL is known as good cholesterol and high levels are associated with low cardiovascular risk, but it is not known if HDL plays a direct role in atherosclerosis. Concentrations of HDL may not be helpful in determining risk. Instead, a functional assay, cholesterol efflux capacity, may be a useful biomarker for vascular disease. This activity, reflecting the movement of labeled cholesterol from a cultured macrophage cell line to apobdepleted plasma, is inversely associated with cardiovascular events. 39 Lp(a), produced by the liver, consists of an LDL particle in which the apolipoprotein apo(a) has been covalently linked to apob100. Apo(a) has substantial protein homology to plasminogen, required for the endogenous thrombolytic response, and it exists in isoforms based on kringle repeats (named after a type of pastry). Isoforms with fewer repeats, and therefore lower mass, tend to circulate at higher concentrations. Higher levels increase the risk of myocardial infarction and aortic valve calcification. 40,41 Major Apolipoproteins The chromosomal location, size, sites of synthesis, and major functions of important apolipoproteins are summarized in Table Apolipoproteins AI, AII, AIV, and AV ApoAI is the most abundant apolipoprotein in HDL. It is synthesized by the liver and intestine and is known to activate the enzyme lecithin:cholesterol acyltransferase (LCAT), which transfers a fatty acid from lecithin to the free hydroxyl group on cholesterol to generate cholesteryl ester. This activity is involved in the maturation of HDL particles, which begin as lipid-poor discs containing apoai and then acquire free cholesterol; they convert this cholesterol to cholesteryl ester through the activity of LCAT and expand into spheres as cholesteryl ester is stuffed into the growing core. ApoAI is important for mediating the efflux of cholesterol from peripheral tissues, an important step in the process of reverse cholesterol transport. 42 Human genetic mutations in apoai cause low levels of HDL and corneal opacities. ApoAI is considered to be an antiatherogenic protein, but genetic defects in apoai are not consistently associated with coronary artery disease. Its presence may be important in the setting of an atherosclerotic environment with elevated levels of atherogenic lipoproteins. ApoAII is present with apoai in some HDL particles. Synthesized mostly in the liver, it has been implicated in the activation of hepatic lipase, an enzyme involved in lipoprotein processing, including HDL metabolism, and in the inhibition of LCAT. ApoAII may disrupt the ability of HDL to promote reverse cholesterol transport, but the genetic absence of this protein in humans does not seem to be associated with a phenotype. 43 ApoAIV originates in the gut, and its secretion is induced by the consumption of a high-fat meal. It may affect food intake in mice, but information in humans is not available. ApoAV is encoded by a locus near the apoaiv gene in the apoai/ciii/aiv/av gene cluster on chromosome 11. It is produced by liver and circulates at low concentrations in association with VLDL particles in humans. ApoAV is involved in the hydrolysis of triglyceride-rich lipoproteins by LPL, its expression in mice is inversely related to triglyceride levels, and it promotes lipoprotein clearance by hepatic proteoglycans. 44

9 1668 SECTION VIII Disorders of Carbohydrate and Fat Metabolism TABLE 37-3 Major Apolipoproteins Apolipoprotein (Chromosome No.) Molecular Weight (kda) Synthesis Functions AI (11) ~29 Liver, intestine Structural protein (HDL) Cofactor for LCAT Crucial role in reverse cholesterol transport Ligand for ABC-A1 and SR-BI AII (1) ~17 (dimer) Liver Inhibits apoe binding to receptors Activates hepatic lipase Inhibits LCAT AIV (11) ~45 Intestine Potential satiety factor Activator of LCAT Facilitates lipid secretion from intestine AV (11) 39 Liver Activator of LPL-mediated lipolysis Might inhibit hepatic VLDL synthesis B100 (2) ~500 Liver Structural protein (VLDL and LDL) Ligand for LDL receptor B48 (2) ~200 Intestine Structural protein (chylomicrons) CI (19) 6.6 Liver Modulates remnant binding to receptors Activates LCAT CII (19) 8.9 Liver Cofactor for LPL CIII (11) 8.8 Liver Modulates remnant binding to receptors Inhibitor of LPL E (19) ~34 Liver, brain, skin, testes, spleen Ligand for LDL and remnant receptors Local lipid redistribution Reverse cholesterol transport (HDL with apoe) apo(a) (6) ~ Liver Modulates thrombosis/fibrinolysis ABCA1, adenosine triphosphate binding cassette transporter A1; apo, apolipoprotein; HDL, high-density lipoprotein; LCAT, lecithin:cholesterol acyltransferase; LDL, low-density lipoprotein; LPL, lipoprotein lipase; SR-B1, scavenger receptor class B type 1; VLDL, very low density lipoprotein. 5 Gln CAA H 2 N COOH H 2 N COOH ApoB protein B100 B Figure 37-8 Synthesis of apolipoprotein B100 and apob48 by a messenger RNA (mrna) editing mechanism. In the human intestine, a specific cytosine (C) is changed to a uracil (U) in the apob mrna. This change results in a stop codon and the formation of apob48, which contains only the first 2152 amino acids of the full-length apob100 (4536 amino acids). COOH, carboxy-terminus; Gln, glutamine; H 2 N, amino-terminus. Apolipoprotein B mrna editing (Stop) UAA 3 ApoB gene ApoB mrna There are two forms of this apolipoprotein, apob100 and apob48, which are derived from a single gene by a unique mechanism that involves RNA editing (Fig. 37-8). In both liver and intestinal cells, the same messenger RNA is transcribed, but an editing protein complex interacts with the message only in the intestine (in humans) to change the cytosine at nucleotide position 6666 to a uracil. 45 This enzymatic effect converts a glutamine codon to a stop codon, resulting in an intestinal protein that is approximately 48% of the length of apob100 hence, the name apob48. ApoB48, in essence a truncated form of apob100, thus originates in the gut, where it is important for the assembly of chylomicrons. 46 There are one or two B48 molecules on each chylomicron, where they provide structural support to the particle. The COOH-terminus of apob100, missing in apob48, determines interaction with the LDL receptor, so apob48 does not appear to be involved in the clearance of gut-derived lipoproteins. ApoB100 originates in the liver, where it is cotranslationally associated with lipids to coordinate the formation of VLDL particles. VLDL assembly and export, which affect the levels of circulating atherogenic lipoproteins, are determined not by transcriptional control of the apob gene but by a unique mechanism involving stabilization of the apob protein by lipid. VLDL production is shown in Figure Assembly is thought to involve two distinct processes. First, as the apob message is translated on the rough endoplasmic reticulum, it binds to lipids that are provided by microsomal triglyceride transfer protein (MTP, the target of a new medication, see later). This protein heterodimerizes with protein disulfide isomerase, which remodels the apob protein by rearranging the positions of disulfide bonds in the molecule to accommodate incoming lipid. Most of this lipid originates in adipose tissue, where triglyceride lipolysis releases free fatty acids that are transported to the liver. Phospholipids and cholesterol also associate with apob at this step. If sufficient lipids are not available in the liver, apob (which is constitutively produced) is ubiquitinated and degraded in the proteasome. Second, maturing VLDL particles fuse with additional lipid droplets in the Golgi apparatus, a process facilitated by apoe. The triglyceriderich particles are then secreted into the space of Disse. Because these particles carry the apolipoproteins that determine VLDL binding to liver receptors, it might be expected that they would be taken up immediately and never access the circulation. This does not occur, probably because high concentrations of phosphatidylethanolamine in nascent VLDL obscure receptor binding sites. These sites are revealed in the circulation as phospholipids are removed. Transfer of apolipoproteins from other lipoproteins in the circulation also modifies VLDL structure to promote metabolism in the periphery.

10 CHAPTER 37 Disorders of Lipid Metabolism 1669 Endothelial cells VLDL Golgi SER Sinusoid Space of Disse Secretory vesicle VLDL Hepatocyte Nascent ApoB-containing lipoproteins RER Nucleus Apolipoproteins CI, CII, and CIII These small apolipoproteins are encoded by loci residing at two different locations in the genome. ApoCI and apocii are transcribed from a site on chromosome 19 near the apoe gene. The apociii gene is a component of the apoai/ CIII/AIV/AV cluster on chromosome 11. ApoCs, which can be exchanged freely among lipoprotein particles, are important for triglyceride metabolism because their presence either interferes with the recognition of apoe by lipoprotein receptors or displaces apoe from lipoproteins (both of which would increase triglycerides by impairing their clearance). The function of apocii is more complex than that of CI and CIII. High levels in mice cause elevated triglycerides by displacing apoe, but normal levels of apocii are required for normal lipid clearance because this apolipoprotein is a cofactor for the enzyme LPL. Mutations of apocii in humans cause severe hypertriglyceridemia, mimicking LPL deficiency. ApoCIII may be particularly relevant to human health. Its levels are increased in the setting of many dyslipidemias, and most lipid-lowering medications lower apociii levels. A mutation in the apociii gene causing lower apociii levels is associated with an improved lipid profile and less atherosclerosis, 49 suggesting that therapies targeted at apociii might provide clinical benefit. In patients with extremely high triglycerides due to the familial chylomicronemia syndrome (see later discussion), inhibiting the apociii mrna results in substantial triglyceride lowering. 50 Figure 37-9 Very low density lipoprotein (VLDL) biosynthesis by hepatocytes. The nascent apolipoprotein B (apob)-containing apolipoproteins synthesized by the rough endoplasmic reticulum (RER) are thought to combine with lipids in the smooth endoplasmic reticulum (SER). The VLDLs are processed in the Golgi apparatus and accumulate in large secretory vesicles. They are then released into the space of Disse and enter the plasma. (Modified from Alexander CA, Hamilton RL, Havel RJ. Subcellular localization of B apoprotein of plasma lipoproteins in rat liver. J Cell Biol. 1976;69: ; by copyright permission of the Rockefeller University Press.) Increased VLDL production, fueled by the increased availability of lipid, is predominantly responsible for the dyslipidemia seen with obesity and diabetes. Hepatitis C, a major cause of human liver disease, circulates in VLDL particles, and this virus assimilates the VLDL assembly machinery. There is one copy of apob100 on each VLDL particle, and this relationship is retained as these lipoproteins are metabolized to IDL and then to LDL. Therefore, measurements of apob100 in the plasma reflect particle number, and higher levels of apob are associated with cardiovascular disease. The complete absence of apob, which occurs in the rare human disorder abetalipoproteinemia, is not caused by mutations in apob but by defects in MTP. 47 Patients with this disease have severe neurologic deficits, probably reflecting vitamin E deficiency, because triglyceride-rich lipoproteins transport this lipid-soluble vitamin. Very low, but not absent, apob, which occurs in the human disorder hypobetalipoproteinemia, is caused by mutations in apob. These individuals present with low levels of cholesterol and triglycerides and appear to be healthy. A mutation at amino acid residue 3500 of the apob100 protein, within the COOH-terminal region of the molecule that mediates binding to the LDL receptor, causes familial defective apob100. These individuals have high levels of LDL cholesterol, mimicking the presentation of FH. 48 Apolipoprotein E ApoE biology is more complex than that of other apolipoproteins. The highest level of apoe expression is found in liver, with the second highest in brain. Many other cell types synthesize the protein, including macrophages. In brain, astrocytes and microglial cells make apoe, but it can also be produced by injured neurons. ApoE circulates in plasma as a part of every lipoprotein with the probable exception of LDL. Its principal function involves interactions with the two major receptors mediating the clearance of plasma lipoproteins, the LDL receptor and the LDL receptor related protein (i.e., LRP1, also known as the chylomicron remnant receptor). Therefore, it is apoe that is primarily responsible for the clearance of intestinal-derived lipoproteins after a meal and for the clearance of VLDL and IDL particles before they are converted to LDL. There are three major apoe isoforms: E2, E3, and E4. They are encoded, respectively, by alleles referred to as ε2, ε3, and ε4, which exist because of charge differences caused by variations in amino acids at residues 112 and 158 in the protein. ApoE3 is considered to be the normal isoform; it has a cysteine at residue 112 and an arginine at 158. ApoE2 has cysteines at both 112 and 158, and apoe4 has an arginine at both 112 and 158. These variations have structural and functional consequences (Fig ). The protein has two domains: an amino (NH 2 )-terminus interacts with lipoprotein receptors, and a COOH-terminus interacts with lipids (Fig A). In apoe4, the isoform associated with disease, these domains interact; this does not occur with apoe3 (Fig B). Comprehensive data (>86,000 individuals for lipids, >37,000 for coronary events) link apoe allele and genotype frequencies, lipid levels, and coronary risk. 51 Allele frequencies in healthy adults are 7% for ε2, 82% for ε3, and 11% for ε4. Genotype frequencies are 0.7% for ε2/ε2, 11.6% for ε2/ε3, 2.2% for ε2/ε4, 62.3% for ε3/ε3 (the most abundant genotype), 21.3% for ε3/ε4, and 1.9% for ε4/ε4. There is a

11 1670 SECTION VIII Disorders of Carbohydrate and Fat Metabolism Arg 158 Cys 150 Receptor binding region 136 A NH 2 -terminal domain 112 Arg Cys 244 Major lipid-binding region 272 CO 2 H-terminal domain ApoE4 ApoE3 Figure A, The amino (NH 2 )-terminal domain of apoprotein E is composed of a four-helix bundle. A region of random structure encompassing residues 165 to 200 forms a connector or hinge region linked to the carboxy (CO 2 H-terminal domain. There are two major functional regions. Residues 136 to 150 (yellow helix) encompass the receptor-binding region; residues 240 to 260 in the carboxyterminal domain encompass the lipidbinding region. B, ApoE4 displays the unique property of domain interaction that distinguishes it from apoe3 (Arg61 in the amino-terminal domain interacts with Glu255 in the carboxy-terminal domain). Arg, arginine; Cys, cysteine; Glu, glutamic acid. B Glu-255 NH 2 NH 2 Arg-61 NH 2 -terminal domain Arg-112 CO 2 H-terminal domain NH 2 -terminal domain Arg-61 Cys-112 Glu-255 CO 2 H-terminal domain linear relationship between the genotype and both LDL cholesterol level and coronary risk, from least to most, as follows: ε2/ε2 < ε2/ε3 < ε2/ε4 < ε3/ε3 < ε3/ε4 < ε4/ε4. Compared to the reference group (ε3/ε3), the presence of the ε2 allele decreases coronary risk by about 20%, and the presence of the ε4 allele slightly increases risk. These observations are interesting for two reasons. First, ε2/ε2 individuals, although they are protected from CHD on a population basis, are at risk for dysbetalipoproteinemia. In the setting of appropriate additional conditions, about 5% of ε2/ε2 individuals will develop this disorder, which is associated with aggressive vascular disease. Second, the E2 protein binds less well to the LDL receptor than E3 and E4 do. This suggests that LDL cholesterol in patients with the E2 protein should be higher (because it is less likely to be cleared by this receptor), yet the opposite is observed. These data suggest that other receptor-mediated processes, such as those mediated by heparan sulfate proteoglycans (HSPG), may be critical for clearance of apoe-containing lipoproteins. 52 ApoE is involved in Alzheimer disease. Risk of this neurodegenerative disease increases approximately threefold in those with one ε4 allele and 12-fold in those with two ε4 alleles. 53 The presence of an ε2 allele is protective. These relationships hold for both early- and late-onset Alzheimer disease. There are HDL-like lipoproteins in the central nervous system (CNS), and apoe-mediated delivery of cholesterol is important for normal synaptic function. The relation of lipid metabolism to Alzheimer disease is incompletely understood, but some evidence suggests that deposition of amyloid-β (the major constituent of the plaques that characterize the disease) begins sooner in the brains of those with the E4 protein. Because people with the ε4 allele are also more likely to have atherosclerosis, CNS vascular disease may help explain why this apoe variant is involved in neurodegeneration. Major Receptors Involved in Lipid Metabolism Low-Density Lipoprotein Receptor Gene Family Including distant relatives, the LDL receptor family contains at least 10 members. The two most important ones for systemic lipid metabolism are the LDL receptor and LRP1. The LDL receptor recognizes apob100 as well as apoe, whereas LRP1 recognizes apoe but not apob100. Other core family members (those that share considerable structural homology) include the VLDL receptor (VLDLR), the apolipoprotein E receptor 2 (apoer2 or LRP8), LRP4, LRP1B, and megalin (LRP2, also known as gp330 and as the major Heymann nephritis antigen).

12 CHAPTER 37 Disorders of Lipid Metabolism 1671 Three family members lack some of the structural features of the others. They are sortilin-related receptor L1 (LR11/SORL1), LRP5, and LRP6. Aside from the LDL receptor and LRP1, these receptors appear to be most important for brain development, synaptic function, and neuroprotection, making them relevant to Alzheimer disease. 54 LRP5 and LRP6 are involved in endocrine disease. Both are coreceptors for a family of G protein coupled receptors known as frizzled receptors. Frizzled receptors bind the Wnt molecule to induce an important signaling cascade upstream of the transcription factor β-catenin. Genetic variants in LRP5 are associated with obesity, 55 and a human mutation in LRP6 results in the metabolic syndrome and coronary artery disease. 56 Loss-of-function mutations in LRP5 or LRP6 cause osteoporosis in humans. These observations suggest that insulin resistance, coronary disease, and osteoporosis, common comorbid conditions in patients, may be related to abnormal Wnt signaling. Low-Density Lipoprotein Receptor. The LDL receptor is a large (160 kda) glycoprotein expressed on most cells. Because it recognizes apob100 as well as apoe, it is involved in the uptake of LDL, chylomicron remnants, VLDL, and IDL. Most HDL particles do not have apoe and therefore do not interact with this receptor or the LRP. The discovery of this receptor in the 1970s by Brown and Goldstein was important because its deficiency explained a human disease (FH), its physiologic regulation explained the mechanism of action of drugs that lower cholesterol, and its biology defined receptor-mediated endocytosis as a paradigm for providing cells with critical components from the external environment. 57 Domains shared with other members of this receptor family comprise the LDL receptor. These include the ligandbinding domain, the epidermal growth factor (EGF) precursor domain, the O-linked sugar domain at the cell surface, the membrane-spanning domain, and the cytoplasmic domain at the COOH-terminus (Fig ). In the ligandbinding domain, there are seven repeats of approximately 40 amino acids, each containing six cysteines that form three disulfide bonds within the repeat to stabilize the structure. Each repeat also includes negatively charged amino acids that interact with positive charged residues on apob and apoe and with calcium ions that allow the repeat to bind to the ligand. The EGF precursor domain consists of three EGF-like repeats (see Fig ) with a structure known as a β-propeller located between repeats B and C. The O-linked sugar domain is the site at which carbohydrate moieties attach to the molecule, and this is followed by a short sequence that traverses the membrane. The cytoplasmic domain consists of 50 residues that include an NPXY (asparagine, proline, any amino acid, tyrosine) targeting sequence where adapter proteins dock, which leads to receptor clustering in coated pits. Coated pits are specialized regions of the cell surface that are characterized by the presence of the protein complex clathrin. When LDL receptors bind lipoproteins, they migrate to coated pits, and clathrin directs the receptors to a cell membrane region that folds inward, creating an intracellular vesicle or endosome (Fig ). Endosomes become acidic, which prompts the lipoprotein to be displaced from the LDL receptor by the β-propeller of the EGF precursor domain. 58 The unoccupied receptor recycles back to the cell surface. In the presence of PCSK9 (see later discussion), the LDL receptor conformation is altered, promoting its degradation and preventing its recycling to the cell surface. 59 Lipoproteins are degraded in lysosomes. Lysosomal lipids appear to affect longevity in model organisms. 60 Cholesterol is transported out of the lysosomes through 1 NH 2 Cysteine COOH C Figure Functional domains of the low-density lipoprotein receptor. Numbers 1 through 7 indicate repeats in the ligand-binding domain. A, B, and C are epidermal growth factor (EGF)-like repeats in the EGF precursor domain. See text for complete description. Rough endoplasmic reticulum LDL receptors Clathrin Golgi apparatus Lysosome B A Lipoprotein Coated vesicle Domain Coated pit Endosome Ligand binding EGF precursor O-Linked sugar Membrane spanning Cytoplasmic Receptor recycling Figure Low-density lipoprotein (LDL) receptor pathway. LDL interacts with receptors on the cell surface. The complex enters the coated pit and is internalized. The coated vesicle loses its clathrin coat and becomes an endosome, the site of lipoprotein and receptor dissociation. The receptors recycle to the cell surface, and the lipoproteins are degraded. Alternatively, new receptors are synthesized in the rough endoplasmic reticulum and transported to the cell surface. (Modified from Brown MS, Goldstein JL. A receptor-mediated pathway for cholesterol homeostasis. Science. 1986;232:34-47; and Myant NB. Cholesterol Metabolism, LDL, and the LDL Receptor. San Diego: Academic Press; 1990.) the action of two proteins, Niemann-Pick C1 and C2 (NPC1 and NPC2), which are mutated in the human disease Niemann-Pick type C. Accumulation of cholesterol and other lipids characterizes this disorder. It is believed that NPC2, which is soluble, binds cholesterol after lipoprotein hydrolysis in the lysosome and moves this sterol to the membrane-associated NPC1 for subsequent release to the cell, where it serves structural and regulatory functions.

13 1672 SECTION VIII Disorders of Carbohydrate and Fat Metabolism Endoplasmic reticulum Sterols + Sterols Golgi SCAP cycling S1P compartment Sp1 SCAP cycling SREBP transport S2P Release of active SREBP SREBP NH 2 SRE Sp1 TATA Nucleus SREBP COOH COOH S1P COOH COOH SCAP NH 2 NH 2 NH 2 LDL Receptor gene SCAP NH 2 SCAP Golgi cycling No SCAP cycling No SREBP transport No release of active SREBP SRE Sp1 TATA Nucleus LDL Receptor gene LDL Receptors LDL Receptors Figure Low-density lipoprotein (LDL) receptor gene regulation. S1P, site-1 protease; S2P, site-2 protease; SCAP, SREBP cleavage activating protein; SRE, sterol regulatory element; SREBP, sterol regulatory element binding protein; TATA, indicates the TATA box or core promoter sequence. One of the key regulatory functions of cholesterol is control of LDL receptor expression. Intracellular sterol concentrations are sensed by SCAP (SREBP cleavage activating protein), which binds to SREBPs in the endoplasmic reticulum. SREBPs are transcription factors that control LDL receptor expression as well as the expression of other genes important for lipid metabolism. SREBP2 is most important for LDL receptor transcription; its NH 2 -terminus contains a leucine zipper type transcription factor structure that binds to a sterol regulatory element in the promoter of the LDL receptor gene. When cells are sterol-depleted (Fig , left side), SCAP migrates to the Golgi apparatus, where sugar moieties attached to the protein are modified. This allows SCAP to transport SREBPs to the S1P compartment. There, two proteases, site-1 protease (S1P) and site-2 protease (S2P), sequentially act on SREBPs to release their NH 2 -terminus, which migrates to the nucleus and binds to the sterol regulatory element in the promoter region of lipid genes such as the LDL receptor, increasing transcription and subsequent levels of functional proteins. In the presence of sterols (see Fig , right side), SCAP does not cycle to the Golgi structure, it cannot move SREBPs to the S1P compartment, and SREBPs are not cleaved to allow their transcription factor to migrate to the nucleus. LDL Receptor Related Protein 1. LRP1 is also known as the apoe receptor or the chylomicron remnant receptor. LRP1 Sp1 roughly consists of the equivalent of four LDL receptors with a multiplicity of ligand-binding domains. It is critical for normal development, because inactivation of LRP1 (but not of the LDL receptor) is lethal in mice. The major cell types in which LRP1 is expressed are hepatocytes, neurons (where it participates in critical functions), and syncytiotrophoblasts in the placenta. Multiple different ligands bind to LRP1 and participate in nutrient flow as well as signaling. These ligands include amyloid precursor protein (relevant in Alzheimer disease because it is processed to form the amyloid-β of plaques), bacterial by-products, tissue plasminogen activator (which interacts with LRP1 to modulate physiology in the setting of brain ischemia), plasminogen activator inhibitors, and α 2 -macroglobulin (which plays multiple roles in inflammation, in part by inactivating matrix metalloproteinases). Given the promiscuity of LRP1 binding, it is not surprising it is linked with receptor-associated protein, a small protein that is involved in intracellular LRP1 processing and occupies the LRP1 binding sites during transport to the cell surface. LRP1 binds apoe but not apob100. Therefore, it mediates the metabolism of the major apoe-containing lipoproteins, including chylomicron remnants and IDL (VLDL remnants), but is not involved in LDL metabolism. The interaction between LRP1 and lipoproteins is more complex than that between LDL and the LDL receptor. Multiple apoe molecules are required for LRP1 binding, and this interaction requires an initial binding of the lipoprotein to HSPG on the cell surface. Other moieties on apoecontaining lipoproteins also are believed to facilitate the binding process. LPL, which metabolizes chylomicrons and VLDL particles, adheres to particles after mediating the release of fatty acids and other substitutents at the endothelium. Lipoprotein-bound LPL molecules (as well as hepatic lipase) are thought to interact with LRP1 and to facilitate the uptake of remnants by the liver. Pattern Recognition Receptors One of the most serious consequences of abnormal lipid metabolism is atherosclerosis, which requires the delivery of excess lipids to blood vessels. This process involves the innate immune system and at least two broad types of receptors scavenger receptors and toll-like receptors (TLRs) that preferentially recognize patterns instead of discrete species. Scavenger Receptors. The discovery of scavenger receptors was prompted by the observation that macrophages can bind and internalize modified forms of LDL but not native LDL. There are now thought to be 10 classes of these receptors 61 that are generally characterized by the ability to bind altered (e.g., oxidized, acetylated) LDL or other polyanionic ligands. Class A and class B receptors may be particularly important. Class A receptors include scavenger receptor A (SR-A types 1 and 2, which consists of alternative splice variants), macrophage receptor with collagenous structure (MARCO), scavenger receptor A 5 (SCARA5), and scavenger receptor with C-type lectin domain (SRCL-I/II, also referred to as CL-P1). SR-A, the first to be discovered, binds a wide variety of ligands (including bacterial by-products), activates stress signaling pathways including mitogen-activated protein kinases, and is believed to be involved in atherosclerosis, the clearance of apoptotic cells, and Alzheimer disease. Class B receptors include CD36 and scavenger receptor class B (SR-BI [called CLA-1 in humans]). These receptors bind modified LDL, but unlike the other classes of scavenger receptors, they also bind VLDL, native LDL, and HDL. CD36 is expressed on a wide variety of cell types, including

14 CHAPTER 37 Disorders of Lipid Metabolism 1673 monocytes, macrophages, adipocytes, platelets, endothelial cells, hepatocytes, microglial cells, and the tongue, where it detects dietary fat. In addition to lipoproteins, long-chain fatty acids are ligands for CD The tissue distribution of SR-BI is more limited, with expression on hepatocytes, monocyte/macrophages, and steroidogenic tissues. Given the panoply of ligands that bind to scavenger receptors and their effects on innate immunity, their roles in lipid metabolism and atherosclerosis may be complex. Toll-like Receptors. The TLR family comprises key effectors of the innate immune system that are required for host defense mechanisms against simple pathogens. Their activation has been implicated in many chronic inflammatory diseases, including atherosclerosis. Some scavenger receptors such as CD36 may be coreceptors for TLRs. TLRs are found on myeloid cells such as monocyte/macrophages but also on the gut epithelium; they bind ligands such as lipopolysaccharide (TLR4) and glycolipids found in bacteria (TLR2). TLR4 is also known to bind saturated fatty acids, an interaction that is believed to be involved in insulin resistance in mammals. 63 TLR2 mediates monocyte activation by apociii on triglyceride-rich lipoproteins. 64 Other Enzymes and Transfer Proteins Mediating Lipid Metabolism Hepatic Lipase Primarily a phospholipase with some triglyceride lipase activity, hepatic lipase is made in hepatocytes and is found mostly on endothelial cells in the liver and on HSPG in the space of Disse. It is also found in steroidogenic tissues but is not synthesized at those sites. Unlike LPL, which is mostly present at tissues remote from the liver to ensure the peripheral delivery of lipids and vitamins, hepatic lipase coordinates lipoprotein metabolism centrally. Its functions include the conversion of IDL to LDL, the conversion of HDL 2 to HDL 3, and probably the final metabolism of chylomicron remnants to facilitate their uptake by LRP1. Unlike LPL, hepatic lipase does not require a cofactor such as apocii, but both enzymes are displaced from their endothelial sites of activity by injection of heparin (postheparin lipase activity). High levels of hepatic lipase decrease HDL concentrations, whereas high levels of LPL increase HDL. Endothelial Lipase Evolutionarily related to hepatic lipase and LPL, endothelial lipase is a phospholipase with almost no triglyceride lipase activity. It is expressed at high levels in embryonic endothelial cells, with expression declining during maturation. Considerable levels are found in adult tissues that include the thyroid, lung, liver, placenta, and gonads (with expression in those tissues reflecting the endothelium and not parenchymal cells). In mice, overexpression decreases HDL and inactivation increases HDL. Endothelial lipase is expressed in aorta, where it may increase with atherosclerosis. Human loss-of-function mutations are associated with increased HDL cholesterol levels. 65 Proprotein Convertase Subtilisin/Kexin Type 9 PCSK9 is a secreted protease that promotes the degradation of the LDL receptor, but its catalytic activity is not required for receptor degradation. Mostly expressed in liver, intestine, and kidney, PCSK9 was found to be important in lipid metabolism when missense mutations (subsequently determined to be gain-of-function mutations) in its gene were determined to be associated with hypercholesterolemia and coronary artery disease. 66 Overexpression of PCSK9 in mice decreases LDL-receptor protein. Human loss-offunction mutations in PCSK9 are associated with low levels of LDL and decreased risk of vascular disease. 67,68 Antibodies to PCSK9 may be useful for treatment in humans (see discussion later). Lipoprotein-Associated Phospholipase A 2 Phospholipases hydrolyze the ester bond at the sn2 position of phospholipids, usually resulting in the release of a fatty acid and lysophosphatidylcholine, which can induce inflammation. This type of enzyme was originally identified as a component of snake venom, and many distinct classes of phospholipases have subsequently been characterized. For most, membrane phospholipids are the substrate. Lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ) is an exception because it can hydrolyze substrate in the aqueous phase. Lp-PLA 2 binds to LDL as well as HDL lipoproteins and is a biomarker for coronary artery disease. 69 Inhibition of this enzyme decreases the expansion of the lipid core of atherosclerotic plaques in humans. 70 However, administration of an oral inhibitor of Lp-PLA2 did not decrease cardiovascular end points in large clinical trials. 71,72 Cholesteryl Ester Transfer Protein Cholesteryl ester transfer protein (CETP) promotes the exchange between lipoproteins of two classes of neutral lipids: cholesteryl esters and triglycerides. HDL cholesteryl esters are transferred to VLDL, IDL, and chylomicron remnants; in return, triglycerides from VLDL, IDL, and remnants are transferred to HDL. Humans and other primates have CETP activity; the transfer of cholesteryl ester from HDL to apob-containing lipoproteins ultimately leads to most of their cholesterol burden being carried by LDL, and this is thought to result in atherosclerosis. Rodents and dogs do not have CETP. Most of their cholesterol is carried in HDL; levels of LDL are low, and these animals are resistant to atherosclerosis. Such observations have led to the notion of inhibiting CETP activity as a treatment for atherosclerosis in humans. One CETP inhibitor was shown to increase HDL cholesterol and lower LDL cholesterol in humans, but it also increased mortality rate, 73 perhaps due to off-target effects including increased levels of aldosterone. Another CETP inhibitor selectively increased HDL but had no impact on cardiovascular events. 74 Lecithin:Cholesterol Acyltransferase LCAT is an enzyme synthesized mostly in the liver; it circulates in the plasma associated with HDL particles and, to a lesser extent, with LDL particles. LCAT is activated by several apolipoproteins (apoai and others) and uses the phospholipid lecithin (phosphatidylcholine) and free cholesterol as substrates to generate lysolecithin (lysophosphatidylcholine) and cholesteryl ester. Most of the cholesteryl esters in lipoproteins are derived from LCAT activity. Rare human mutations in LCAT result in low HDL levels in the setting of a range of disorders, including fish-eye disease (in which activity is deficient on HDL particles but continues on LDL particles) and a more severe presentation with corneal clouding (resulting from free cholesterol in the cornea), hemolytic anemia, and renal failure. The role of LCAT in atherosclerosis is uncertain. Although LCAT deficiency might be expected to promote atherosclerosis because of low HDL levels, studies of humans with

15 1674 SECTION VIII Disorders of Carbohydrate and Fat Metabolism Dietary fat and cholesterol Intestine Chylomicrons Adipose tissue LPL FFA Chylomicron remnants LPL HL Figure General scheme summarizing the major pathways involved in the metabolism of chylomicrons synthesized by the intestine and very low density lipoprotein (VLDL) synthesized by the liver. ApoB, apolipoprotein B; ApoE, apolipoprotein E; FFA, free fatty acid; HL, hepatic lipase; IDL, intermediate-density lipoprotein; LPL, lipoprotein lipase. (Modified from Mahley RW. Biochemistry and physiology of lipid and lipoprotein metabolism. In: Becker KL, ed. Principles and Practice of Endocrinology and Metabolism, 2nd ed. Philadelphia: JB Lippincott; 1995: ) Liver Remnant receptors Bile acids LDL receptors FFA FFA VLDL LPL IDL LPL HL LDL ApoE mediated ApoB mediated ApoE mediated Peripheral tissues loss-of-function LCAT mutations suggest that these individuals are not at increased risk for atherosclerosis. 75 Peripheral tissues INTEGRATIVE PHYSIOLOGY OF LIPID METABOLISM Lipid metabolism is characterized by a dynamic flux of multiple lipid species from the external environment to the liver, from the liver to peripheral tissues, from peripheral tissues back to the liver, and eventually back to the external environment through the excretion of bile acids. Integrated views of the major pathways involved are shown in Figures and Exogenous Lipid Transport Dietary fat and cholesterol (see Fig , top left) absorbed by the duodenum and proximal jejunum are used to generate chylomicrons that are secreted at the lateral borders of enterocytes and enter mesenteric lymphatics. They access the plasma via the thoracic duct and are rapidly metabolized by LPL to yield chylomicron remnants. These are taken up by remnant receptors (LRP1/HSPG) and by LDL receptors in the liver. Free fatty acids liberated by the action of LPL are available to adipose tissue for storage and to other tissues (e.g., skeletal muscle, heart) for use as energy substrates. Endogenous Lipid Transport Lipid derived from remnants and from lipolysis of adipose tissue is reassembled in the liver (see Fig , bottom left) as VLDL particles, which are secreted into the plasma. Abnormal lipid metabolism in insulin resistance is mediated in large part by overproduction of VLDL, an event that occurs through disruption of signaling downstream of the insulin receptor and the insulin receptor substrate (IRS) adapter proteins. VLDL particles are metabolized by LPL to yield IDL particles, which are metabolized by LPL and hepatic lipase to yield LDL particles. Thus, LDL is derived from VLDL, which helps explain why treatment to lower triglycerides (carried by VLDL) is frequently associated with VLDL IDL remnants Free cholesterol HDL 3 HDL 2 HDL 1 LCAT LCAT Hepatic lipase CETP Tg Figure Role of high-density lipoprotein (HDL) in the redistribution of lipids from cells with excess cholesterol to cells requiring cholesterol or to the liver for excretion. The reverse cholesterol transport pathway is indicated by arrows (net transfer of cholesterol from cells to HDL, then to LDL and liver). ApoE, apolipoprotein E; CE, cholesteryl ester; CETP, cholesteryl ester transfer protein; IDL, intermediatedensity lipoprotein; LCAT, lecithin:cholesterol acyltransferase; LDLR, low-density lipoprotein receptor; SR-BI, scavenger receptor class B, type 1; Tg, triglyceride; VLDL, very low density lipoprotein. at least transient increases in LDL. IDL can be taken up by the liver through an apoe-dependent process, and LDL is taken up by the liver through the binding of apob100 to LDL receptors. Small VLDL particles, IDL particles, and LDL particles may be taken up by peripheral tissues to deliver nutrients, cholesterol, and fat-soluble vitamins. When present in excess, each of these lipoproteins may be atherogenic. CE CE SR-BI LDLR Cholesterol delivery Liver Bile ApoE Cholesterol excretion

16 CHAPTER 37 Disorders of Lipid Metabolism 1675 Reverse Cholesterol Transport and Dysfunctional HDL Cholesterol cannot be metabolized by peripheral tissues and must be returned to the liver for excretion. This process, known as reverse cholesterol transport, is dependent on HDL and its precursors and is depicted in Figure Excess cholesterol in tissues can be effluxed either to lipidpoor apoai, mediated by the protein transporter ABCA1 (adenosine triphosphate binding cassette transporter 1), or to nascent HDL particles, mediated by ABCG1. Efflux from cultured cells to human plasma as a biomarker of cardiovascular risk 39 is thought to mostly reflect the activity of ABCA1. There is also evidence that cholesterol can be acquired by HDL without the assistance of transporters by following a concentration gradient at the cell surface. LCAT esterifies HDL-associated cholesterol to form cholesteryl ester and induce the maturation of HDL. HDL particles have three pathways for transporting sterols to the liver. First, they can directly bind to SR-BI (CLA-1) at the liver, which induces cholesteryl ester delivery through a mechanism involving lateral lipid transfer and not receptor internalization. Second, cholesteryl esters can be transferred to apob-containing lipoproteins by CETP, and these particles can deliver cholesterol to the liver through the LDL receptor. Third, a small portion of HDL can acquire apoe and bind to the liver LDL receptor. Once in the liver, cholesterol is converted to bile acids for excretion. HDL 2 particles are partially depleted of cholesteryl esters and enriched in triglycerides through the activity of CETP, which renders them suitable as substrates for hepatic lipase. Hepatic lipase hydrolyzes the triglyceride-enriched HDL 2 particles and regenerates HDL 3, yielding particles that are again suited to accept cholesterol from peripheral cells. Because cholesterol is the principal component of atherosclerotic plaque, it is reasonable to pursue the notion that atherosclerosis could be treated by promoting the efflux of cholesterol from lesions. HDL participates in this process, but static levels of HDL cholesterol are poor predictors of reverse cholesterol transport. Measurements of the rate of flux of cholesterol from the periphery to the liver, which may be possible in humans, would represent a better predictor of beneficial therapies. In vitro HDL efflux capacity represents an initial step toward assessing reverse cholesterol transport. 39 The presence of high levels of HDL particles that have been modified to prevent their capacity to promote cholesterol efflux would not be expected to decrease vascular risk. Such dysfunctional particles may explain why some HDL-elevating interventions have not been associated with decreased cardiovascular disease. In addition to participating in reverse cholesterol transport, HDL has other properties that could be impaired by a variety of processes leading to a dysfunctional particle. They include the induction of endothelial nitric oxide synthase, the transport of proteins involved in the acute phase response and inflammation, and the suppression of thrombosis through induction of prostacyclin (which decreases thrombin production via the protein C pathway and decreases platelet activation). OVERVIEW OF HYPERLIPIDEMIA AND DYSLIPIDEMIA Two major clinical disorders are associated with common lipoprotein disorders. Very elevated triglyceride levels are a risk factor for development of pancreatitis. Elevated cholesterol due to greater concentrations of LDL and remnant lipoproteins and reduced levels of HDL promote atherosclerosis. Clinicians are often faced with evaluation and treatment of patients who have hypertriglyceridemia, hypercholesterolemia, combined hyperlipidemia due to elevated cholesterol and triglycerides, and low HDL syndromes. A summary of the primary and secondary causes of each condition is presented in Table Plasma lipid levels are highly dependent on lifestyle; for example, the high-fat, high-cholesterol diets eaten in Western societies raise plasma cholesterol, and vigorous exercise lowers both atherogenic particles and triglycerides. TABLE 37-4 Differential Diagnosis of Hyperlipidemia and Dyslipidemia Hypertriglyceridemia Hypercholesterolemia Increased Cholesterol and Triglycerides Low HDL Primary Disorders LPL deficiency Familial hypercholesterolemia Familial combined hyperlipidemia Familial hypoalphalipoproteinemia ApoCII deficiency Familial defective apob100 Dysbetalipoproteinemia ApoAI mutations Familial hypertriglyceridemia Polygenic hypercholesterolemia LCAT deficiency Dysbetalipoproteinemia Sitosterolemia ABCA1 deficiency Secondary Disorders Diabetes mellitus Hypothyroidism Diabetes mellitus Anabolic steroids Hypothyroidism Obstructive liver disease Hypothyroidism Retinoids High-carbohydrate diets Nephrotic syndrome Glucocorticoids HIV infection Renal failure Thiazides Immunosuppressives Hepatitis C infection Obesity/insulin resistance Protease inhibitors Estrogens Nephrotic syndrome Ethanol Lipodystrophies Beta blockers Protease inhibitors Glucocorticoids Retinoids Bile acid binding resins Antipsychotics Lipodystrophies Thiazides ABCA1, adenosine triphosphate binding cassette transporter 1; apo, apolipoprotein; HDL, high-density lipoprotein; LCAT, lecithin:cholesterol acyltransferase; LPL, lipoprotein lipase.

17 1676 SECTION VIII Disorders of Carbohydrate and Fat Metabolism CHD Mortality Rate per Plasma Cholesterol (mg/dl) Figure Relation between plasma cholesterol levels and coronary heart disease (CHD) mortality rate in the Multiple Risk Factor Intervention Trial. (Modified from Stamler J, Wentworth D, Neaton JD. Is relationship between serum cholesterol and risk of premature death from coronary heart disease continuous and graded? Findings in 356,222 primary screenees of the Multiple Risk Factor Intervention Trial [MRFIT]. JAMA. 1986;256: ; Copyright 1986, by the American Medical Association.) For this reason, normal blood concentrations those that are within 2 standard deviations of the mean vary among countries and over time. For Western adults, cholesterol concentrations higher than 240 mg/dl (6.2 mmol/l) or triglyceride concentrations higher than 150 mg/dl (1.7 mmol/l) constitute high-risk hyperlipidemia. The overriding influences of diet and lifestyle on plasma cholesterol were illustrated by studies of ethnic Japanese populations. Plasma cholesterol was markedly increased in Japanese-Americans and was associated with a more westernized food intake. 76 Because serum total cholesterol levels correlate with the risk for CHD over a broad range (Fig ), normal levels are often defined as those associated with minimal cardiovascular risk rather than population averages, suggesting that most of those in the developed world have lipid levels that put them at risk for heart disease. The National Cholesterol Education Program (NCEP) created a standard for cholesterol levels and pioneered a practical approach to treatment by dividing the population according to cardiac risk, based on the presence of vascular disease or other cardiac risk factors. In 2001, the NCEP classified plasma cholesterol levels lower than 200 mg/dl as desirable, those between 200 and 240 mg/dl as borderline high, and levels greater than 240 mg/dl as high. 77 (Because plasma lipid levels increase with age, values in children are lower. 78 ) Total cholesterol concentrations between 170 and 200 mg/dl are considered borderline high, and levels greater than 200 mg/dl are high. Triglyceride levels greater than 500 mg/dl carry a risk of pancreatitis, and those greater than 150 mg/dl are considered elevated. The Endocrine Society used different definitions in 2012, 79 in part influenced by clinical data that severe hypertriglyceridemia is associated with pancreatitis with triglyceride levels of greater than 2000 mg/dl. Hyperlipidemias are caused by increased concentrations of plasma lipoproteins. Although the clinical diagnosis is made solely on the basis of circulating lipids, the diseases are classified by abnormalities of lipoproteins (see Table 37-2) and are often referred to as hyperlipoproteinemias. As noted earlier, lipoproteins differ in their physiologic functions, metabolic pathways, and pathologic significance. TABLE 37-5 Criteria for Diagnosis of the Metabolic Syndrome Measure* Waist circumference Elevated triglycerides Reduced HDL-C Elevated blood pressure Elevated fasting glucose Categorical Threshold The NCEP also recognized the existence of the metabolic syndrome, a common condition linked to insulin resistance but without a unifying mechanistic cause. The presence of at least three of the five features described in Table 37-5 is sufficient to make the diagnosis. This condition is clearly associated with increased risk of vascular disease and development of type 2 diabetes and is equivalent to what was previously referred to as prediabetes. 80 Evaluation and therapy for the metabolic syndrome should be directed at individual components including hypertriglyceridemia and low HDL cholesterol (HDL-C). HYPERTRIGLYCERIDEMIA Whites, African Americans, Latin Americans: Men, 40 in; women, 35 in Asians: Men, 35 in; women, 32 in 150 mg/dl or On drug treatment for elevated triglycerides Men, <40 mg/dl; women, <50 mg/dl or On drug treatment for reduced HDL-C 130 mm Hg systolic or 85 mm Hg diastolic or On antihypertensive drug treatment 100 mg/dl or On drug treatment for elevated glucose *Three of the five measures are required for diagnosis. HDL-C, high-density lipoprotein cholesterol. Adapted from Grundy SM, Cleeman JI, Daniels SR, et al. Diagnosis and management of the metabolic syndrome: an American Heart Association/ National Heart, Lung, and Blood Institute scientific statement. Circulation. 2005;112: Fasting Hyperchylomicronemia The most dramatic example of severe hypertriglyceridemia is that of fasting hyperchylomicronemia. This can result from a primary defect in chylomicron metabolism, or it can occur secondary to increased VLDL and saturation of LPL actions to metabolize triglycerides into free fatty acids and generate remnant lipoproteins that are amenable to uptake by the liver through pathways mediated by the LDL receptor, LRP1, and proteoglycans. LPL saturation occurs when triglyceride levels exceed about 500 mg/dl. Therefore, familial hypertriglyceridemia, familial combined hyperlipidemia, and dysbetalipoproteinemia can manifest with fasting hyperchylomicronemia. One common cause of such exacerbations is out-of-control diabetes leading to increased adipose intracellular lipolysis, return of fatty acids to the liver, greater secretion of VLDL triglyceride, and saturation of LPL. Several dietary and environmental factors also modulate triglyceride production. The most dramatic is alcohol, a major substrate for triglyceride production. In addition, diets that are rich in free carbohydrates, and especially simple sugars, induce triglyceride production. Fructose also increases de novo production of lipids in the liver but has less effect on circulating triglycerides. Defective clearance of plasma lipids is a major cause of fasting hyperchylomicronemia. Patients with genetic defects leading to this condition are often referred to as having familial chylomicronemia syndrome (FCS). Genetic defects in LPL commonly cause FCS; the lack of normal

18 CHAPTER 37 Disorders of Lipid Metabolism 1677 LPL prevents chylomicron clearance. LPL deficiency usually, but not always, manifests in childhood. The symptoms vary from difficulty feeding young infants to frank pancreatitis, which is sometimes mistaken as appendicitis. The plasma is often milky, and whole blood may have a pinkish, cream of tomato soup hue. The trigger level of triglyceride elevation leading to pancreatitis is variable; some patients have triglycerides in excess of 10,000 mg/dl with no symptoms, whereas others develop pancreatitis at much lower triglyceride levels, but usually in excess of 2000 mg/ dl. However, patients presenting with pancreatitis have often avoided eating and the first measured sample may not reflect the peak triglyceride levels. Several additional mutations in proteins required for normal LPL actions also cause FCS (see later). The pathophysiology of the relationship between hyperchylomicronemia and pancreatitis is unknown. Lipid-rich blood may sludge, leading to pancreatic ischemia. The small amount of lipases that normally leak from the acinar cells may lead to exuberant local lipolysis, creation of toxic local concentrations of free fatty acids and lysolecithin, a toxic lipid produced from phosphatidylcholine, and further acinar cell damage to adjacent cells. 81 Insults to the acinar cells such as that provided by alcohol can fan this process. Although most patients with severe hyperchylomicronemia who do not develop pancreatitis are asymptomatic, a few with extreme levels exceeding 10,000 mg/dl develop the hyperchylomicronemia syndrome. These patients have dyspnea and confusion that may be indistinguishable from early dementia. Presumably this is the result of reduced blood flow or defective oxygen delivery. The marked increase in blood triglyceride concentration can lead to accumulation of triglycerides in several organs and can be observed in the blood. The latter is best appreciated by examining the blood directly, allowing the red blood cells to settle, and observing a creamy layer on the plasma, or by noting the pinkish discoloration of the blood on funduscopic examination, known as lipemia retinalis (Fig B). Eruptive xanthomas, as shown in Figure 37-17G, are 2- to 5-mm papules with a yellow center surrounded by erythema. They are caused by triglycerideenriched skin macrophages. These lesions are sometimes confused with acne or folliculitis. For unclear reasons, eruptive xanthomas are most commonly found on the buttocks, extensor surfaces of the arms, and the back. Enlargement of the liver and spleen is not uncommon and is thought to be caused by triglyceride accumulation in these organs. Aside from the severe hypertriglyceridemia, other laboratory indices are sometimes abnormal. Plasma sodium is reduced; liver transaminases are sometimes elevated. Despite the presence of pancreatitis, amylase may be normal due to an assay artifact; serum lipase is a more reliable indicator in this setting. Often the clinical laboratory will note the severe lipemia and fail to report measurements of routine chemistries due to the turbidity of the serum. If these other measurements are required, plasma can be centrifuged, the chylomicron layer removed, and the remaining plasma examined. Fasting hyperchylomicronemia in adults is frequently accompanied by comorbid conditions such as uncontrolled diabetes and excessive alcohol intake. Lipoprotein Lipase Deficiency Almost every racial group has been reported to have patients with genetic defects in LPL, 82 and a founder mutation makes the defect especially common among French Canadians. Approximately half of the cases of severe primary hypertriglyceridemia are the result of LPL defects. Most LPL enzyme deficiencies are caused by inactive LPL protein. However, lack of protein production has also been reported, and because LPL might have receptor functions that do not require catalytic function, patients with these defects may have a more severe phenotype. Although genetic LPL deficiency has been reported to manifest in adulthood, most cases of severe hyperchylomicronemia in adulthood are associated with partial LPL deficiency or other causes. In adults, the most important of these causes are type 2 diabetes and obesity, because insulin resistance is associated with defective clearance of lipoproteins. 83 Postprandial lipemia is a prominent feature of diabetes. 84 A thorough history of triglyceride-raising medications should be taken (see later discussion). Regulation of LPL is complicated, and defects in its actions are associated with genetic or acquired abnormalities that are exclusive of genetic defects in the LPL molecule. Defective apocii, the obligate cofactor for LPL, leads to deficient LPL activity. 85 Two molecular defects initially found in mice are the cause of occasional severe human hypertriglyceridemia. GPIHBP (discussed earlier) is a molecule expressed by endothelial cells whose deficiency leads to defective association of LPL with its binding site on the capillary lumen and defective intravascular lipolysis. 86 In one report, 20% of patients with FCS had GPIHBP mutations. 87 Lipase maturation factor 1 (LMF1) is an intracellular protein that is required for correct intracellular folding and activation of LPL. 88 Mutations in LPL, GPIHBP, and LMF1 all lead to reduced postheparin LPL activity; mutated apocii does not, but serum from these patients fails to maximally activate LPL. LPL deficiency also occurs as a secondary phenomenon. Autoimmune conditions can be associated with defective triglyceride catabolism due to inhibition of LPL, apocii, or heparin. Antibodies against heparin are thought to prevent normal LPL association with the endothelial surface. In addition, patients with vascular disease or generalized intravascular reactions to transfusions or chemotherapy can occasionally develop defects in LPL. Transient episodes of fasting hyperchylomicronemia have been attributed to viral infections and to excessive fat/calorie intake after fasting. Postprandial Hyperlipidemia Although plasma lipid levels are usually measured after an overnight fast, chylomicron remnants are associated with vascular disease in a number of animal models and with genetic or dietary causes of hyperlipidemia. This has led to a widely accepted hypothesis that remnant lipoproteins are an overlooked cause of human vascular disease. Postprandial lipidemia, measured as triglyceride increase, is associated with greater risk of heart disease. 89 However, postprandial triglyceride elevations are also correlated with fasting triglycerides and reduced HDL levels, so the use of postprandial measurements in clinical practice is not currently recommended. Diagnostic Evaluation of Severe Hypertriglyceridemia Assessment of underlying medical conditions, consideration of age at onset, and, in some cases, biochemical evaluation of LPL are required. Conditions that cause fasting hypertriglyceridemia (discussed later) can lead to severe hypertriglyceridemia when exacerbated by diet, drugs, or other conditions such as diabetes or pregnancy.

19 1678 SECTION VIII Disorders of Carbohydrate and Fat Metabolism A B C D E F G Figure Physical examination findings associated with hyperlipidemia. A, Xanthelasma. B, Lipemia retinalis. C, Achilles tendon xanthomas. Notice the marked thickening of the tendons. D, Tendon xanthomas. E, Tuberous xanthomas. F, Palmar xanthomas. G, Eruptive xanthomas. (A and B, Courtesy of Dr. Mark Dresner and Hospital Practice [May 1990, p 15]; C through F, courtesy of Dr. Tom Bersot; G, Courtesy of Dr. Alan Chait.)