Dietary Origins of Lipids

The predominant form of dietary lipid in the human diet is triglyceride (TG or TAG). Gastrointestinal lipid digestion and absorption is discussed in Chapter 43. Briefly, the digestion of dietary triglyceride begins in the stomach with the action of gastric lipase and continues in the duodenum via the concerted actions of gastric lipase and pancreatic lipase. Digestion of dietary phospholipids takes place in the duodenum as well via the action of pancreatic phospholipase A₂ (PLA₂), yielding free fatty acids and lysophospholipids. Digestion of dietary cholesterol esters results via the action of carboxyl ester lipase (CEL). Intestinal absorption of diacyl- and monoacylglycerides, lysophospholipids, free fatty acids, and cholesterol occurs at the interface between the brush border membranes of intestinal enterocytes and the lipid micelles and involves both passive diffusion and transport protein–mediated uptake mechanisms.

Discussion of the various lipoproteins present in human circulation can be found in Chapter 28. As chylomicrons circulate in the vasculature, fatty acids are removed from the TG fraction through the action of endothelial cell–associated lipoprotein lipase (LPL). The free fatty acids are then absorbed by the cells and the glycerol is returned via the blood to the liver where it is utilized as a carbon skeleton for glucose synthesis via gluconeogenesis (Chapter 13).

Mobilization of Fat Stores

The primary sources of fatty acids for oxidation are dietary and mobilization from cellular stores. Fatty acids from the diet are absorbed from the gut, packaged into lipoprotein particles called chylomicrons within intestinal enterocytes and then delivered to cells of the body via transport in the blood. Fatty acids are stored in the form of triglycerides (TAGs or TGs) within all cells but predominantly within adipose tissue. In response to energy demands, the fatty acids of stored TGs can be mobilized for use by peripheral tissues. The release of these stored fatty acids is controlled by a complex series of interrelated cascades that result in the activation of triglyceride hydrolysis. The primary intracellular lipases involved in the mobilization of stored fatty acids are adipose triglyceride lipase (ATGL, also called desnutrin), hormone-sensitive lipase (HSL), and monoacylglyceride lipase (MGL).

Adipose Triglyceride Lipase

Adipose triglyceride lipase (ATGL)/desnutrin belongs to the family of patatin domain-containing proteins that consists of 9 human members. Because some members of the family act as phospholipases, the proteins were originally called patatin-like phospholipase domain-containing proteins A1 to A9 (PNPLA1–PNPLA9). ATGL (designated PNPLA2 in the patatin domain nomenclature) preferentially hydrolyzes TGs. ATGL expression and enzyme activity are both under complex regulation. Expression of ATGL is induced by peroxisome proliferator–activated receptor (PPAR) agonists, glucocorticoids, and fasting. The level of lipase activity of ATGL (as well as HSL) does not always correlate to the level of expression of the gene. The discrepancy between ATGL and HSL mRNA level and the level of enzyme activity is the result of extensive posttranslational regulation of both of these lipases.

There are 2 known serine residues in ATGL that are subject to phosphorylation. Unlike phosphorylation of HSL (described below), ATGL phosphorylation is not PKA-dependent. Some lines of evidence suggest that AMPK phosphorylates ATGL resulting in increased lipase activity. In addition to phosphorylation regulating ATGL activity, the enzyme requires a coactivator protein for full activity. This coactivator is known as comparative gene identification-58 (CGI-58). The official nomenclature for CGI-58 is α/β-hydrolase domain-containing protein-5 (ABHD5), owing to the presence of an α/β-hydrolase domain commonly found in esterases, thioesterases, and lipases. Additional proteins that are associated with lipid droplets (LD) in adipocytes participate in the ABHD5-mediated regulation of ATGL such as perilipin-1, Plin1.

In nonadipose tissues with high rates of TG hydrolysis, such as skeletal muscle and liver, regulation of ATGL activity occurs via a mechanism distinct from that in adipose tissues. In these tissues, perilipin-1 is replaced by perilipin-5 which recruits both ATGL and ABHD5 to LD by direct binding of the enzyme and its coactivator. Other perilipins exist in cells including perilipin-2, -3, and -4 but it is unclear if these proteins are also involved in regulating the association of ATGL with LD.

Hormone-sensitive Lipase

The expression profile of hormone-sensitive lipase (HSL) essentially mirrors that of ATGL. Highest mRNA and protein concentrations are found in WAT and brown adipose tissue (BAT) with low levels of expression found in muscle, testis, steroidogenic tissues, and pancreatic islets as well as several other tissues. HSL was originally thought to be rate limiting for the catabolism of fat stores in adipose tissue and many nonadipose tissues. However, HSL actually has a higher level of activity as a diglyceride (DG) hydrolase than as a TG hydrolase. This became evident when HSL-deficient mice were produced and shown to efficiently hydrolyze TG and they do not accumulate TG in either adipose or nonadipose tissues, but they do accumulate large amounts of DGs in many tissues. It is now accepted that ATGL is responsible for the initial step of lipolysis in human adipocytes, and that HSL is rate-limiting for the catabolism of DG. HSL not only hydrolyzes DG but is also active at hydrolyzing ester bonds of many other lipids including TG, MG, cholesteryl esters, retinyl esters, and short-chain carbonic acid esters.

In adipose tissue, HSL activity is strongly induced by β-adrenergic stimulation; conversely insulin has a strong inhibitory effect. While β-adrenergic stimulation regulates ATGL primarily via recruitment of the coactivator ABHD5, HSL is a major target for PKA-mediated phosphorylation. Additional kinases, including AMPK, extracellular signal-regulated kinase (ERK), glycogen synthase kinase-4 (GSK-4), and Ca²⁺/calmodulin-dependent kinase 1 (CAMK1), also phosphorylate HSL to modulate the activity of the enzyme. Although phosphorylation affects HSL activity, full activation requires access to LD, which in adipose tissue is mediated by Plin1 (Figure 25-1).

In nonadipose tissues, such as skeletal muscle, HSL is activated by phosphorylation in response to epinephrine (β-adrenergic receptor–mediated activation of PKA) and muscle contraction (calcium release from sarcoplasmic reticulum).

Insulin-mediated deactivation of lipolysis is associated with transcriptional downregulation of ATGL and HSL expression. Insulin signaling also results in the activation of various phosphodiesterase (PDE) isoforms leading to PDE-catalyzed hydrolysis of cAMP which in turn results in reduced activation of PKA. These actions turn off lipolysis by preventing phosphorylation and activation of HSL. In addition to its peripheral action, insulin also functions within the sympathetic nervous system to inhibit lipolysis in WAT. Increased insulin levels in the brain inhibit both HSL and Plin1 phosphorylation which results in reduced HSL and ATGL activities.

Monoacylglyceride Lipase

Monoacylglyceride lipase (MGL) is considered to be the rate-limiting enzyme for the breakdown of MG that are the result of both extracellular and intracellular lipolysis pathways. The extracellular generation of MG is the result of the action of endothelial cell lipoprotein lipase (LPL) on lipoprotein particle–associated TG. Intracellular hydrolysis of TG by ATGL and HSL as well as intracellular phospholipid hydrolysis by phospholipase C (PLC) and membrane-associated DG lipase α and β results in the generation of MGL substrates.

MGL is ubiquitously expressed with highest levels of expression in adipose tissue. MGL shares homology with esterases, lysophospholipases, and haloperoxidases. MGL is critically important for efficient degradation of MG since it has been shown in mouse models that lack of MGL impairs lipolysis and is associated with increased MG levels in adipose and nonadipose tissues alike. MGL has received particular attention in recent years due to the discovery that the enzyme is responsible for the inactivation of 2-arachidonoylglycerol (2-AG) which is an endogenous cannabinoid (endocannabinoid).

Lipid Transporters and Cellular Uptake of Fats

When fatty acids are released from adipose tissue stores, they enter the circulation as free fatty acids (FFA) and are bound to albumin for transport to peripheral tissues. When the fatty acid-albumin complexes interact with cell surfaces, the dissociation of the fatty acid from albumin represents the first step of the cellular uptake process. Cellular uptake of fatty acids involves several members of the fatty acid receptor family including fatty acid translocase (FAT/CD36), plasma membrane-associated fatty acid-binding protein (FABP_pm), and at least 6 fatty acid transport proteins (FATP1-FATP6). The FATP facilitate the uptake of very-long-chain (VLCFA) and long-chain fatty acids (LCFA) (Table 25-1).

Following uptake into cells, fatty acids must be activated to acyl-CoA intermediates. Several members of the FATP family possess acyl-CoA synthetase (ACS) activity. The overall process of cellular fatty acid uptake and subsequent intracellular utilization represents a continuum of dissociation from albumin by interaction with the membrane-associated transport proteins, activation to acyl-CoA (in many cases via FATP action) followed by intracellular trafficking to sites of metabolic disposition.

Mitochondrial β-Oxidation Reactions

The primary sites of fatty acid β-oxidation are the mitochondria and the peroxisomes. Fatty acids of between 4 and 8 and between 6 and 12 carbon atoms in length, referred to as short- and medium-chain fatty acids (SCFA and MCFA), respectively, are oxidized exclusively in the mitochondria. Long-chain fatty acids (LCFA: 10-16 carbons long) are oxidized in both the mitochondria and the peroxisomes with the peroxisomes exhibiting preference for 14-carbon and longer LCFA. Very-long-chain fatty acids (VLCFA: 17-26 carbons long) are preferentially oxidized in the peroxisomes.

Fatty acids must be activated in the cytoplasm before being oxidized in the mitochondria. Activation is catalyzed by fatty acyl-CoA synthetases (also called acyl-CoA ligases or thiokinases). The net result of this activation process is the consumption of 2 molar equivalents of ATP. The process of mitochondrial fatty acid oxidation is termed β-oxidation since it occurs through the sequential removal of 2-carbon units by oxidation at the β-carbon position of the fatty acyl-CoA molecule.

The transport of fatty acyl-CoA into the mitochondria is accomplished via an acyl-carnitine intermediate, which itself is generated by the action of carnitine palmitoyltransferase 1 (CPT-1 or CPT-I) an enzyme that resides in the outer mitochondrial membrane (Figure 25-2 and Clinical Box 25-1). There are 3 CPT-1 genes in humans identified as CPT-1A, CPT-1B, and CPT-1C. Expression of CPT-1A predominates in the liver and is thus, referred to as the liver isoform. CPT-1B expression predominates in skeletal muscle and is thus, referred to as the muscle isoform. CPT-1C expression is exclusive to the brain and testes. The activity of CPT-1C is distinct from those of CPT-1A and CPT-1B in that it does not act on the same types of fatty acyl-CoAs that are substrates for the latter 2 enzymes. However, CPT-1C does exhibit high-affinity malonyl-CoA binding. Following carnitine acyl-carnitine–mediated transfer of the CPT-1-generated fatty acyl-carnitines across the inner mitochondrial membrane, the fatty acyl-carnitine molecules are acted on by the inner mitochondria membrane carnitine palmitoyltransferase 2 (CPT-2 or CPT-II) regenerating the fatty acyl-CoA molecules (Figure 25-2).

Each round of β-oxidation involves 4 steps that, in order, are oxidation, hydration, oxidation, and cleavage (Figure 25-3, steps 2-5). The first oxidation step β-oxidation involves a family of FAD-dependent acyl-CoA dehydrogenases. Each of these dehydrogenases has a range of substrate specificity determined by the length of the fatty acid. Short-chain acyl-CoA dehydrogenase (SCAD, also called butyryl-CoA dehydrogenase) prefers fats of 4 to 6 carbons in length; medium-chain acyl-CoA dehydrogenase (MCAD; see Clinical Box 25-2) prefers fats of 4 to 16 carbons in length with maximal activity for C10 acyl-CoAs; long-chain acyl-CoA dehydrogenase (LCAD) prefers fats of 6 to 16 carbons in length with maximal activity for C12 acyl-CoAs. Deficiencies in several of the acyl-CoA dehydrogenases are known to result in impaired β-oxidation with MCAD deficiency being the most common form.

The next 3 steps in mitochondrial β-oxidation involve a hydration step, another oxidation step, and finally a hydrolytic reaction that requires CoA and releases acetyl-CoA and an acyl-CoA 2 carbon atoms shorter than the initial substrate. The water addition is catalyzed by an enoyl-CoA hydratase activity, the second oxidation step is catalyzed by an NAD-dependent long-chain hydroxyacyl-CoA dehydrogenase activity (3-hydroxyacyl-CoA dehydrogenase activity), and finally the cleavage into an acyl-CoA and an acetyl-CoA is catalyzed by a thiolase activity. These 3 activities are encoded in a multifunctional enzyme called the mitochondrial trifunctional protein (MTP). MTP is composed of 8 protein subunits, 4 α-subunits encoded by the HADHA gene, and 4 β-subunits encoded by the HADHB gene. The α-subunits contain the enoyl-CoA hydratase and long-chain hydroxyacyl-CoA dehydrogenase activities, while the β-subunits possess the 3-ketoacyl-CoA thiolase (β-ketothiolase or just thiolase) activity. The mammalian genome actually encodes 5 distinct enzymes with thiolase activity (Table 25-2).

Each round of β-oxidation produces 1 mole of FADH₂, 1 mole of NADH, and 1 mole of acetyl-CoA. The acetyl-CoA, the end product of each round of β-oxidation, then enters the TCA cycle (Chapter 13), where it is further oxidized to CO₂ with the concomitant generation of 3 moles of NADH, 1 mole of FADH₂ and 1 mole of ATP. The NADH and FADH₂ generated during the fat oxidation and acetyl-CoA oxidation in the TCA cycle then can enter the respiratory pathway for the production of ATP via oxidative phosphorylation (Chapter 14).

Minor Alternative Fatty Acid Oxidation Pathway

The majority of natural lipids contain an even number of carbon atoms. A small proportion of plant-derived lipids contain odd numbers and upon complete β-oxidation these yield acetyl-CoA units plus a single mole of propionyl-CoA. The propionyl-CoA is converted in an ATP-dependent pathway to succinyl-CoA. The succinyl-CoA can then enter the TCA cycle for further oxidation (Figure 25-4).

The oxidation of unsaturated fatty acids is essentially the same process as for saturated fats, except when a double bond is encountered (Figure 25-5). In such a case, the bond is isomerized by a specific enoyl-CoA isomerase and oxidation continues. In the case of linoleate, the presence of the Δ¹² unsaturation results in the formation of a dienoyl-CoA during oxidation. This molecule is the substrate for an additional oxidizing enzyme, the NADPH requiring 2,4-dienoyl-CoA reductase.

Peroxisomal (Alpha) α-Oxidation Pathway

Phytanic acid is a fatty acid present in the tissues of ruminants and in dairy products and is, therefore, an important dietary component of fatty acid intake. Because phytanic acid is methylated, it cannot act as a substrate for the normal mitochondrial β-oxidation pathway. Phytanic acid is first converted to its CoA-ester and then phytanoyl-CoA serves as a substrate in an α-oxidation process, therefore, the process is referred to as α-oxidation (Figure 25-6). The α-oxidation reaction, as well as the remainder of the reactions of phytanic acid oxidation, occurs within the peroxisomes and requires a specific α-hydroxylase called phytanoyl-CoA hydroxylase (PhyH). The action of PhyH adds a hydroxyl group to the α-carbon of phytanic acid generating the 19-carbon homologue, pristanic acid. Pristanic acid then serves as a substrate for the remainder of the normal process of β-oxidation. Deficiency in PhyH activity results in the disorder known as Refsum disease. (see Clinical Box 25-3).

Peroxisomal β-Oxidation Reactions

In addition to mitochondrial oxidation of fatty acids, the peroxisomes play an important role in overall fatty acid metabolism. Very-long-chain fatty acids (VLCFAs: 17-26 carbons long) are preferentially oxidized in the peroxisomes with cerotic acid (a 26:0 fatty acid) being solely oxidized in this organelle. The peroxisomes also metabolize di- and trihydroxycholestanoic acids (bile acid intermediates); long-chain dicarboxylic acids that are produced by ω-oxidation of long-chain monocarboxylic acids (see below); pristanic acid via the α-oxidation pathway (see above); certain polyunsaturated fatty acids (PUFA) such as tetracosahexaenoic acid (24:6), which by β-oxidation yields the important PUFA docosahexaenoic acid (DHA); and certain prostaglandins and leukotrienes.

The enzymatic processes of peroxisomal β- oxidation are very similar to those of mitochondrial β-oxidation with 1 major difference (Figure 25-7). During mitochondrial oxidation the first oxidation step, catalyzed by various acyl-CoA dehydrogenases, results in the reduced electron carrier FADH₂ that then delivers its electrons directly to the electron transport chain for synthesis of ATP. In the peroxisome the first oxidation step is catalyzed by acyl-CoA oxidases which are coupled to the reduction of O₂ to hydrogen peroxide (H₂O₂). Thus, the reaction is not coupled to energy production but instead yields a significant reactive oxygen species (ROS). Peroxisomes contain the enzyme catalase that degrades the hydrogen peroxide back to O₂.

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