Lipid Metabolism

Fat gets a bad rap because it is implicated as the major culprit behind the obesity pandemic worldwide. The so-called fast-food diet that the United States has exported worldwide is blamed for the increase in body mass index (BMI), which is associated with health risks. Recent studies have emphasized that where you carry the fat matters in terms of health risks. For example, people carrying excess fat in the abdomen have higher risks of cancer and cardiovascular disease. It is clear that the biology of fat is complex. Fat, which is synonymous with lipids, plays an important role in our cells to maintain homeostasis. Lipids generate ATP and are involved in the synthesis of vitamins, hormones, bile salts, eicosanoids, and cellular membranes, as well as the regulation of cellular signaling. Cholesterol and phospholipids are essential components of membranes within the cell. The anabolism and catabolism of lipids is compartmentalized. Anabolism primarily occurs in the cytosol and endoplasmic reticulum, whereas the catabolism primarily occurs in mitochondria.

Lipids constitute an enormous topic and have many ramifications concerning human disease. This review will cover three basic aspects of lipid biology: (1) production of lipids, (2) catabolism of lipids to generate ATP, and (3) lipids as signaling molecules.

QUICK GUIDE TO LIPIDS

• Lipids, such as triacylglycerol (TAG) and phospholipids, are generated from glucose-derived glycerol and mitochondrial-derived fatty acids (Fig. 1).

• Fatty acid synthesis takes place in the cytosol, where mitochondrial citrate serves as the precursor to eventually generate palmitate, which can be modified to other fatty acids.

• Fatty acid β-oxidation occurs in the mitochondrial matrix. Fatty acids are transported into the matrix through carnitine acyltransferase I (CPTI) located in the outer mitochondrial membrane, along with carnitine acyltransferase II (CPTII) and carnitine-acylcarnitine translocase, located in the inner mitochondrial membrane.

• Fatty acid synthesis is coupled to NADPH → NADP⁺, whereas fatty acid oxidation generates acetyl-CoA, NADH, and FADH₂ to produce ATP through oxidative phosphorylation.

• Fatty acid synthesis is regulated by acetyl-CoA carboxylase (ACC), which is activated by citrate and inhibited by the fatty acid palmitate.

• Fatty acid β-oxidation is regulated by malonyl-CoA, which inhibits carnitine acyltransferase (CPTI) activity, thereby preventing fatty acid import into the mitochondrial matrix for β-oxidation.

• Lipids can modify proteins to alter their function. Notable modifications are N-myristoylation, S- or N-palmitoylation, and S-prenylation.

• Lipids, such as eicosanoids, phosphoinositides, and sphingolipids, serve as signaling molecules.

• The cholesterol biosynthetic pathway initiates in the cytosol and is controlled by the enzyme 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase), the target of statins (class of cholesterol-lowering drugs).

Figure 1.

Overview of lipid metabolism. Lipid synthesis requires the glycolytic intermediate dihydroxyacetone phosphate and TCA cycle intermediate citrate to generate glycerol 3-phosphate and acetyl-CoA, respectively. Fatty acid synthase (FASN) converts acetyl-CoA to palmitate, which, together with glycerol 3-phosphate, generates lipids, such as triglycerides and phospholipids. Acetyl-CoA is also used to generate cholesterol. Lipids break down into fatty acids, which can be used by mitochondria in β-oxidation to generate ATP.

GLYCOLYSIS AND MITOCHONDRIAL METABOLISM GENERATE LIPIDS

There are multiple types of lipids, including TAGs (commonly known as triglycerides) and phospholipids. TAG is a glycerol attached to mixture of saturated and unsaturated fatty acids (Fig. 2). Phospholipids are typically composed of two fatty acids linked to a glycerol and are attached to a polar molecule, like choline, via a phosphate group (Fig. 2). A close examination of the structure of TAGs and phospholipids informs us that there is a glucose-derived product (glycerol) attached to fatty acids, a long hydrocarbon chain composed of multiple acetyl groups, indicating that the two metabolic pathways involved in generating TAGs and phospholipids are glycolysis and TCA cycle. Fatty acid synthesis occurs in the cytosol, where there is a high NADPH/NADP⁺ ratio to drive the reactions. The addition of glycerol to fatty acids to generate TAGs and phospholipids occurs in the endoplasmic reticulum.

Figure 2.

Structures of lipids. Triglycerides consist of three fatty acids linked to glycerol. Phospholipids contain two fatty acids linked to glycerol that is attached to a polar molecule like choline via phosphate. Saturated fatty acids have no double bonds. Monounsaturated fatty acids have one carbon double bond, whereas polyunsaturated fatty acids have two or more carbon double bonds.

Fatty acids are classified according to the number of carbon double bonds. Saturated fatty acids have no double bonds. Monounsaturated fatty acids have one double bond and polyunsaturated fatty acids have two or more double bonds. Some common saturated fatty acids are palmitic, butyric, and stearic acids; oleic and linoleic acids are mono- and polyunsaturated fatty acids, respectively (Fig. 2). Fatty acid synthesis combines eight two-carbon acetyl groups derived from mitochondrial citrate-generated acetyl-CoA to form a palmitate, a 16-carbon saturated fatty acid. Palmitate can be modified into other fatty acids by undergoing desaturation to generate unsaturated fatty acids or further chain elongation to produce longer fatty acids.

Acetyl-CoA is the precursor for fatty acid synthesis in the cytoplasm. Acetyl-CoA is generated in the mitochondria from pyruvate (see Chandel 2020a), which is derived from glucose by glycolysis (see Chandel 2020b) or amino acid metabolism (see Chandel 2020c). Acetyl-CoA combines with oxaloacetate and serves as a substrate for citrate synthesis. Acetyl-CoA cannot be transported across mitochondrial membranes; however, the tricarboxylate transporter can transport citrate out of the mitochondria to the cytoplasm. Subsequently, the ATP citrate lyase (ACL) splits citrate into cytoplasmic acetyl-CoA for fatty acid and cholesterol synthesis and oxaloacetate. Malate dehydrogenase reduces oxaloacetate to malate by coupling NADH oxidation to NAD⁺ (Fig. 3). Malate can be transported back into the mitochondrial matrix or undergo oxidation to pyruvate in the cytosol by malic enzyme. The latter generates cytosolic NADPH that can be used to drive fatty acid synthesis. The pyruvate produced returns to the mitochondrial matrix. The pentose phosphate pathway, one-carbon metabolism, and isocitrate dehydrogenase 1 also generate NADPH for fatty acid synthesis (see Chandel 2020d).

Figure 3.

Mitochondrial citrate generates acetyl-CoA for fatty acid synthesis. Citrate synthase produces citrates from mitochondrial acetyl-CoA and oxaloacetate. Citrate is transported into the cytosol where the ACL splits citrate into cytoplasmic acetyl-CoA and oxaloacetate. Acetyl-CoA is used for fatty acid and cholesterol synthesis. Malate dehydrogenase 1 converts oxaloacetate to malate by coupling NADH oxidation to NAD⁺. Malate can be transported back into the mitochondrial matrix for regeneration of oxaloacetate or is converted into pyruvate in the cytosol by malic enzyme 1. (Modified, with permission, from Nelson and Cox 2013, p. 841, © W.H. Freeman.)

Fatty acid synthesis starts with the irreversible reaction catalyzed by ACC, which causes carboxylation of acetyl-CoA to malonyl-CoA (Fig. 4). The Gibbs free energy to drive this reaction comes from ATP → ADP + P_i. Subsequently, fatty acid synthase, an enzyme encoded by the FASN gene, generates palmitate. FASN is a multifunctional protein consisting of two identical multifunctional polypeptides. This complex contains seven different catalytic sites: acetyl transferase, malonyl transferase, β-ketoacyl synthase, β-ketoacyl-acyl carrier protein (ACP) reductase, 3-hydroxyacyl-ACP dehydratase, enoyl-ACP reductase, and thioesterase. These different enzymes are linked covalently in this complex, thus allowing intermediates to be handed efficiently from one active site to another without leaving the assembly.

Figure 4.

Fatty acid synthesis pathway. Fatty acid synthesis starts with the irreversible reaction catalyzed by ACC, which carboxylates acetyl-CoA to malonyl-CoA. The elongation phase of fatty acid synthesis starts with the formation of acetyl-ACP and malonyl-ACP from acetyl-CoA and malonyl-CoA by acetyl transacylase and malonyl transacylase, respectively. Fatty acid synthase uses acetyl- and malonyl-ACP to synthesize the 16-carbon fatty acid palmitate by the repetition of the reaction sequence condensation → reduction → dehydration → reduction.

The intermediates in fatty acid synthesis are linked to the sulfhydryl terminus of a phosphopantetheine group, which is, in turn, attached to a serine residue of the ACP. CoA also contains a phosphopantetheine group. The elongation phase of fatty acid synthesis starts with the formation of acetyl-ACP and malonyl-ACP by acetyl transacylase and malonyl transacylase catalyzing the reactions:

$$\begin{array}{rl}& \begin{array}{c}\mathrm{acetyl}\underset{\_}{\mathrm{CoA}}+\underset{\_}{\mathrm{ACP}}\rightleftharpoons \mathrm{acetyl}\underset{\_}{\mathrm{ACP}}+\underset{\_}{\mathrm{CoA},}\\ \mathrm{acetyl}\mathrm{transacylase},\\ \mathrm{malonyl}\underset{\_}{\mathrm{CoA}}+\underset{\_}{\mathrm{ACP}}\rightleftharpoons \mathrm{malonyl}\underset{\_}{\mathrm{ACP}}+\underset{\_}{\mathrm{CoA},}\\ \mathrm{malonyl}\mathrm{transacylase}.\end{array}\end{array}$$

Once acetyl-ACP and malonyl-ACP are formed, fatty acids are synthesized by the repetition of the following reaction sequence: condensation → reduction → dehydration → reduction (Fig. 4). The acetyl-ACP and malonyl-ACP react to form the four-carbon acetoacetyl-ACP with CO₂ as a product by β-ketoacyl-ACP synthase (also referred to as acyl-malonyl-ACP condensing enzyme). It is important to note that the synthesis of four-carbon acetoacetyl-ACP from two-carbon acetyl-ACP and three-carbon malonyl-ACP reactions is a more favorable reaction than two molecules of acetyl-ACP. ATP is used to carboxylate acetyl-CoA to malonyl-CoA, and the free energy stored in malonyl-CoA is released in the decarboxylation during the generation of acetoacetyl-ACP.

These next three reactions—a reduction, dehydration, and second reduction—convert acetoacetyl-ACP into butyryl-ACP to complete the first elongation cycle. Two NADPH molecules drive these reactions. In cycle 2 of fatty acid synthesis, four-carbon butyryl-ACP condenses with malonyl-ACP to form a C₆-β-ketoacyl-ACP, with CO₂ being released. This reaction is similar to the first-round reaction, in which acetyl-ACP condenses with malonyl-ACP to form a C₄-β-ketoacyl-ACP. Reduction, dehydration, and a second reduction convert the C₆-β-ketoacyl-ACP into a C₆-ACP, which is ready for a third round of elongation. The elongation cycles continue until C₁₆-acyl-ACP is formed. Subsequently, a thioesterase hydrolyzes C₁₆-ACP to yield palmitate and ACP. The stoichiometry of the synthesis of palmitate is shown in Figure 4.

However, the seven malonyl-CoA molecules were originally derived from seven acetyl-CoA molecules using seven ATP molecules. Hence, the overall stoichiometry for the synthesis of palmitate is

$$\begin{array}{rl}& 8\text{acetyl CoA}+7\text{ATP}+14\text{NADPH}+6{\text{H}}^{+}\\ & -\to \text{palmitate}+14\text{NAD}{\text{P}}^{+}\\ & +8\text{CoA}+6{\text{H}}_2\text{O}+7\text{ADP}+7{\text{P}}_{\mathrm{i}}.\end{array}$$

Palmitate produced by fatty acid synthase can generate other longer fatty acids by elongases, which lengthen palmitate or undergo desaturation by desaturases to generate unsaturated fatty acids.

To activate palmitate for elongation, acyl-CoA synthetase adds a CoA thioester by an ATP-dependent reaction. The elongation occurs by adding malonyl-CoA to palmitate or other saturated or unsaturated fatty acyl-CoA substrates by fatty acyl synthase enzyme on the cytosolic face of the endoplasmic reticulum (Fig. 5). This condensation reaction is driven by the decarboxylation of malonyl-CoA. Note there is no ACP involved and no multifunctional enzyme.

Figure 5.

Synthesis of lipids. Fatty acids combine with glycerol 3-phosphate to generate TAGs and phospholipids through reactions that, in part, take place in the cytosol and endoplasmic reticulum.

Once the fatty acids are generated, they can combine with glycerol 3-phosphate to generate TAG. Glycerol 3-phosphate dehydrogenase converts the glycolytic intermediate dihydroxyacetone phosphate into glycerol 3-phosphate (Fig. 5), which is primed for sequential addition of three fatty acids to make a TAG (Fig. 5). The first fatty acid is added by glycerol 3-phosphate acyltransferase (GPAT) to generate lysophosphatidic acid, and this is, in turn, acylated by an acylglycerophosphate acyltransferase (AGPAT) to generate phosphatidic acid, a key intermediate in the biosynthesis of all glycerol-derived lipids. The phosphate group is removed by lipins, which act as phosphatidic acid phosphohydrolases (PAPs) to produce diacylglycerols (DAGs). Finally, the resulting DAG is converted to TAG through the action of diacylglycerol acyltransferase (DGAT) enzymes. GPAT, AGPAT, PAPs, and DGATs are localized to the endoplasmic reticulum. The intermediates, phosphatidic acid and DAG, can produce the phospholipids involved in generating membranes, such as cardiolipin and phoshatidyl serine, respectively.

LIPID CATABOLISM GENERATES ATP

TAGs undergo hydrolysis to fatty acid and glycerol by lipases. The glycerol is converted into the glycolysis intermediate dihydroxyacetone phosphate by glycerol kinase and glycerol phosphate dehydrogenase. Fatty acids undergo β-oxidation to generate ATP in the mitochondrial matrix. Fatty acid oxidation is the primary source of ATP when glucose levels are low in cells. If glucose cannot provide pyruvate to generate acetyl-CoA, then fatty acid oxidation provides acetyl-CoA to initiate the TCA cycle. Fatty acids have to be transported from the cytosol to mitochondrial matrix. The first activation step uses fatty acyl-CoA synthetase in the cytosol to generate fatty acyl-CoA in a two-step reaction. In the first step, fatty acid uses ATP to form an acyl-adenylate intermediate (Fig. 6). This generates an inorganic pyrophosphate that is rapidly converted to inorganic phosphate. This removal keeps the pyrophosphate concentration low to preserve the favorable reaction. In the second step, the fatty acyl-adenylate intermediate attacked by the thiol group of Co-A generates AMP and fatty acyl-CoA, which is imported into mitochondrial matrix. The AMP is converted by adenylate kinase to ADP by using ATP (AMP + ATP → 2ADP). Overall, this reaction uses two ATP molecules.

Figure 6.

Carnitine shuttle to transport fatty acids into mitochondria. The import of fatty acyl-CoA into the inner mitochondrial membrane is accomplished by tagging carnitine to fatty acyl-CoA by the carnitine acyltransferase I (CPT1), located in the outer mitochondrial membrane. The carnitine-translocase protein on the inner membrane is an antiporter, which exchanges fatty acyl carnitine for a carnitine. Subsequently, the fatty acyl carnitine in the matrix is converted into fatty acyl-CoA by carnitine acetyltransferase II (CPTII), thereby releasing carnitine, which is shuttled back across the inner membrane to continue the cycle. The number of FADH₂ and NADH are shown for palmitate-driven fatty acid oxidation. (Adapted, with permission, from Mehta 2013.)

The import of fatty acyl-CoA into the mitochondrial matrix is accomplished by the carnitine transport cycle (Fig. 6). In the first step, carnitine acetyl transferase (CPT1), located in the outer mitochondrial membrane, replaces the CoA moiety with carnitine to form a fatty acyl carnitine molecule, which translocates to the inner mitochondrial membrane. The carnitine-acylcartinine translocase protein, located in the inner mitochondrial membrane, is an antiporter that exchanges fatty acyl carnitine for a carnitine. The fatty acyl carnitine in the matrix is converted into fatty acyl-CoA by carnitine acetyltransferase II, thereby releasing carnitine, which is shuttled back across the inner membrane. Thus, carnitine serves as a tag to get fatty acyl-CoA into the mitochondrial matrix. Subsequently, fatty acyl-CoA enters the β-oxidation pathway, where long-chain fatty acyl-CoA is sequentially degraded into one two-carbon acetyl-CoA, accompanied by the generation of one NADH and one FADH₂ by four reactions.

As shown in Figure 7, the 16-carbon palmitate fatty acid is converted into palmitoyl-CoA and transferred into the mitochondrial matrix to become the 14-carbon myristoyl-CoA, which is a substrate for another round of β-oxidation. Thus, the complete oxidation of palmitoyl-CoA requires seven rounds of β-oxidation to generate eight molecules of acetyl-CoA, plus seven NADH and FADH₂ molecules. The net reaction is

$$\begin{array}{rl}& \text{palmitoyl-CoA}+7\text{CoA}+7\text{FAD}\\ & +7{\text{NAD}}^{+}+7{\text{H}}_2\text{O}\\ & \to 8\text{acetyl-CoA}+7{\text{FADH}}_2\\ & +7\text{NADH}+7{\text{H}}^{+}.\end{array}$$

Figure 7.

Mitochondrial β-oxidation. Fatty acids, such as the 16-carbon palmitate fatty acid, are converted into palmitoyl-CoA and transferred into the mitochondrial matrix by the carnitine shuttle. Palmitoyl-CoA enters the β-oxidation pathway and is sequentially degraded into one two-carbon acetyl-CoA accompanied by generation of one NADH and one FADH₂ by four reactions to become the 14-carbon myristoyl-CoA, which is a substrate for another round of β-oxidation. The complete oxidation of palmitoyl-CoA requires seven rounds of β-oxidation to generate eight molecules of acetyl-CoA plus seven NADH and FAHD₂ molecules.

Each acetyl-CoA generates 3 NADH, 1 FADH₂, and 1 GTP, bringing the total to 24 NADH, 8 FADH₂ molecules, and 8 GTP from 8 acetyl-CoA. The total NADH and FADH₂ are 31 NADH and 15 FADH₂. Remember that each NADH and FADH₂ generates ∼2.5 ATP and 1.5 ATP through oxidative phosphorylation, respectively (see Chandel 2020a). This yields 77.5 ATP (31 NADH × 2.5 ATP) plus 22.5 ATP (15 FADH2 × 1.5 ATP) for a total of 100 ATP. The eight GTP can be converted to eight ATP, resulting in 108 ATP. The activation step used two ATP to generate palmitoyl-CoA from palmitate. Thus, after subtracting these two ATP molecules, the final ATP generated by oxidizing palmitate is 106 ATP.

BOX 1.

FASTING PRODUCES KETONE BODIES

The nonviolent hunger strike campaigns of Mahatma Gandhi against the British Empire in the 20th century are well documented. During these protests, the very lean Gandhi, in 1924 and 1933, survived for as long as 21 days only on sips of water. The Mexican–American activist Cesar Chavez survived fasting for 25 days in 1968 and 36 days in 1988. A key response to fasting is the maintenance of blood glucose levels that is vital for brain to function. Gluconeogenesis in the liver as a mechanism to maintain blood glucose levels was discussed in Chandel (2020e). During starvation, skeletal muscles undergo protein catabolism, resulting in the release of alanine into the blood. The liver absorbs alanine, and it is converted into pyruvate, which feeds into gluconeogenesis to maintain blood glucose levels. This is referred to as the glucose–alanine cycle. Fasting also decreases insulin levels and increases glucagon levels, resulting in depletion of glycogen stores in the liver to maintain blood glucose levels. Furthermore, TAGs stored in adipose tissues are broken down into free fatty acids and glycerol. The latter is converted into glucose by the liver. Free fatty acids can be used by mitochondria to generate ATP through β-oxidation in a variety of energy demanding tissues, such as the heart. The liver uses acetyl-CoA generated from β-oxidation to produce ketone bodies in the form of three molecules: acetoacetate, β-hydroxybutyrate, and acetone (Box 1, Fig. 1). The brain does not use free fatty acids, but does use acetoacetate and β-hydroxybutyrate to generate acetyl-CoA for ATP generation. The brain sustains its metabolic rate by using glucose and ketone bodies. The biosynthesis and use of ketone bodies share enzymes with the exception of one enzyme in the ketone body biosynthetic pathway, β-ketoacyl-CoA transferase (Box 1, Fig. 1). The liver lacks this enzyme to prevent the futile cycle of synthesis and use of acetoacetate. During starvation, concentrations of ketone bodies increase to millimolar range in blood. People undergoing ketosis (elevated levels of ketone bodies) can be easily detected by the volatile odor of acetone, which is not metabolized for ATP generation. A fascinating new development in the field of ketone bodies is the recognition that ketone bodies are regulators of signaling and dictating biological outcomes. Specifically, β-hydroxybutyrate is an endogenous inhibitor of histone deacetylases.

BOX 1, Figure 1.

Ketone bodies production and use. Enzymatic reactions that generate the ketone bodies acetoacetate, β-hydroxybutyrate, and acetone. (B) Enzymatic reactions that convert ketone bodies into acetyl-CoA.

REGULATION OF FATTY ACID ANABOLISM AND CATABOLISM

Metabolites, hormones, and posttranslational modifications of enzymes all regulate fatty acid synthesis and oxidation. Here, the focus will be on how key intermediates and phosphorylation regulate fatty acid oxidation and synthesis (Fig. 8). Fatty acid synthesis is regulated by ACC, citrate, and palmitoyl-CoA. ACC is active in a homopolymeric form and inactive as a monomer. Citrate and palmitoyl-CoA bind to an allosteric site on this enzyme to stimulate polymerization or depolymerization, respectively. Acetyl-CoA levels in the cytosol are dependent on how much citrate is exported from mitochondria through the tricarboxylate transporter. As citrate levels accumulate in the cytosol, they activate ACC in a feed-forward mechanism to convert acetyl-CoA to malonyl-CoA, thus, increasing fatty acid synthesis. If the cell accumulates palmitate beyond the cell's metabolic needs, then fatty acid synthesis is diminished. Palmitate can be converted into palmitoyl-CoA to synthesize other fatty acids. The high palmitoyl-CoA levels in the cytosol serve as a feedback inhibitor on ACC and tricarboxylate transporter to decrease flux through the fatty acid synthesis pathway.

Figure 8.

Metabolic regulation of fatty acid synthesis and oxidation. Acetyl-CoA carboxylase (ACC) is positively and negatively regulated by citrate and palmitoyl-CoA, respectively. An important mechanism to prevent simultaneous fatty acid oxidation and synthesis is an increase in cytosolic malonyl-CoA, which allosterically inhibits CPTI activity to prevent mitochondrial import of fatty acyl-CoA molecules for β-oxidation.

The other major regulator of fatty acid synthesis is malonyl-CoA that allosterically inhibits CPTI activity to prevent mitochondrial import and degradation of newly synthesized fatty acyl-CoA molecules by β-oxidation. This is an important mechanism to prevent simultaneous fatty acid oxidation and synthesis. An increase in malonyl-CoA serves as a signal to favor fatty acid synthesis over fatty acid oxidation. Conversely, if a cell is in need of ATP, then ACC activity is diminished by phosphorylation by AMP-activated protein kinase (AMPK), leading to a decrease in malonyl-CoA levels and thus relieving the inhibition of carnitine acyltransferase. This promotes fatty acid oxidation and subsequent generation of mitochondrial ATP.

BOX 2.

PEROXISOMES, THE FORGOTTEN ORGANELLES, ALSO CONDUCT FATTY ACID OXIDATION

Peroxisomes are single membrane-enclosed organelles that are understudied and underappreciated. Mammalian peroxisomes have multiple metabolic functions, including β-oxidation of very-long-chain fatty acids, α-oxidation of branched-chain fatty acids, and synthesis of ether lipids and bile acids, as well as detoxification of ROS. Genetic defects in genes encoding peroxisomal proteins results in a myriad of devastating pathologies. Pertinent to this review, let us just focus on oxidation of fatty acids. β-oxidation of very-long-chain fatty acids (more than 26 carbons) ensues primarily in peroxisomes, but not in mitochondria. This oxidation shortens fatty acids that can be further oxidized by mitochondria to generate ATP. Peroxisomes lack a respiratory chain, thus, oxidation of very-long-chain fatty acids to shorter fatty acids generates heat rather than ATP. The transport of fatty acids into peroxisomes also differs from transport into mitochondria. Mitochondria rely on the carnitine exchange system, whereas peroxisomes use three ATP-binding cassette transporter D subfamily proteins that are localized to the peroxisomal membrane: ABCD1, ABCD2, and ABCD3. ABCD1 is mutated in the human disease adrenoleukodystrophy (ALD). The movie Lorenzo's Oil is based on the story of a boy who suffered from a deficiency in ABCD1 protein. The inability to oxidize long-chain fatty acids in patients with ALD causes accumulation of these large fatty acids that destroy the myelin sheath “insulation” around nerve cells. The other distinct function of peroxisomes compared with mitochondria is their ability to oxidize branched-chain fatty acid through α-oxidation. Branched-chain fatty acids have a methyl group on the third carbon atom (γ position) that prevents β-oxidation. Therefore, branched-chain fatty acids undergo oxidative decarboxylation (α-oxidation) in peroxisomes to remove the terminal carboxyl group, such as CO₂, resulting in a methyl group on the second carbon, which allows for β-oxidation in peroxisomes or mitochondria. The significance of peroxisome's diverse metabolic functions is not fully understood, yet recent studies have linked peroxisome metabolic functions to human pathologies, including cancer, diabetes, and neurodegeneration. Hopefully, the next generation of scientists will embrace this neglected organelle.

LIPIDS ACTIVATE CELLULAR SIGNALING PATHWAYS

There are multiple ways that lipids can intersect with signaling, including attachment of lipids to proteins that are necessary for the activity of the protein (Fig. 9). Recent studies implicate deregulation of lipid-dependent signaling as an important mechanism for the inflammatory and metabolic diseases. Lipids act as signal transduction messengers at the cell membrane level; a specific lipid can stimulate different cellular responses, depending on cell type and signaling network. The most common function for the attachment of lipids to proteins is to allow water-soluble proteins to interact with hydrophobic membranes. Other functions of lipid modification include forming part of a protein–protein interaction or an integral part of the protein tertiary structure to stabilize the conformation of the protein. It is estimated that 1000 proteins have lipophilic groups covalently attached to them, including fatty acids, phospholipids, sterols, isoprenoids, and glycosylphosphatidyl inositol anchors. All of these modifications confer distinct properties to the modified proteins that are reversible. Notable modifications are N-myristoylation, S- or N-palmitoylation, and S-prenylation. Proteins can contain more than one type of these modifications.

Figure 9.

Overview of lipid signaling. Lipids can modulate signaling pathways by modifying proteins (N-myristoylation, S- or N-palmitoylation, and S-prenylation) or serve as signaling molecules (eicosanoids, phosphoinositides, and sphingolipids).

N-myristoylation is the attachment of a 14-carbon saturated fatty acid, myristate, onto the amino-terminal glycine residue of target proteins and catalyzed by the enzyme N-myristoyltransferase. Myristoylated proteins reversibly flip between a myristoyl-accessible state, in which the myristoyl group is available for binding to membranes or other proteins, and a myristoyl-inaccessible state, in which the myristoyl group is located in a hydrophobic-binding pocket within the protein. Myristoylation itself is not strong enough for stable binding to membranes. Thus, many proteins bound to membranes undergo S-palmitoylation, which is an attachment of the C16 palmitoyl group from palmitoyl-CoA to the thiolate side chain of cysteine residues within proteins catalyzed by palmitoyl acyltransferases. Because of the longer hydrophobic group compared with the C14 myristoyl moiety, this can permanently anchor the protein to the membrane. Palmitoyl thioesterases cause depalmitoylation of proteins to release proteins from membranes, and this makes S-palmitoylation a reversible switch to regulate membrane localization. Many signaling proteins, such as the Src family of kinases, use N-myristoylation and S-palmitoylation as a mechanism to localize to the plasma membrane, which is essential for their biological activity (Fig. 9).

A second type of palmitate attachment is N-palmitoylation, which is essential for the activity of secreted proteins, such as Hedgehog (Hh) and Wnt, that are necessary for proper embryonic development. Hedgehog acyltransferase (Hhat) and porcupine (Porcn) catalyze N-palmitoylation of Hh and Wnt proteins in the endoplasmic reticulum lumen to a cysteine and serine residue, respectively. Although cholesterol attachment is not a common lipid modification on proteins, Hh is modified by cholesterol, allowing it to diffuse during development. Many cancer cells also use Hh and Wnt proteins to drive their proliferation and survival. Small-molecule inhibitors against Hhat and Porcn are being tested to block Hh- and Wnt-driven cancers.

Another important lipid modification is S-prenylation, which covalently adds a farnesyl (C15) or geranylgeranyl (C20) group to specific cysteine residues within five amino acids from the carboxyl terminus via farnesyl transferase (FT) or geranylgeranyl transferases, respectively. S-prenylation of proteins allows them to become membrane-associated because of the hydrophobic nature of farnesyl or geranylgeranyl groups. One example is the Ras GTPase family of proteins, which requires this modification for their optimal activity. Ras can undergo mutations that make it oncogenic, prompting the development of drugs that target FT to inhibit oncogenic Ras activity.

In addition to being posttranslational modifications on proteins, lipids can themselves serve as signaling molecules. Eicosanoids, which are primarily derived from the 20-carbon polyunsaturated fatty acid arachidonic acid, are one example. Eicosanoids are sometimes referred to as local hormones because they are rapidly degraded and have specific effects on targets cells close to their site of production. The three major classes of arachidonate-derived eicosanoids are prostaglandins, leukotrienes, and thromboxanes, which regulate diverse physiological effects, including induction of inflammation, regulation of blood pressure, and blood clotting. The release of eicosanoids affects neighboring cells usually by interacting with their plasma membrane G-protein-coupled receptors to activate diverse cell-signaling pathways.

Eicosanoid synthesis begins with the release of arachidonic acid from phospholipids by phospholipase A2. Arachidonic acid is converted into leukotrienes by lipoxygenase enzymes or into prostaglandin H2 (PGH2) by cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzymes. PGH2 is a precursor for prostaglandins and thromboxanes. Eicosanoids, derived from PGH2, can amplify inflammation; thus, there are drugs that target COX-1 and COX-2 to reduce inflammation, fever, and pain. These drugs are classified as nonsteroidal anti-inflammatory drugs (NSAIDs). The first NSAID found to inhibit COX-1 and COX-2 was salicylic acid. The German pharmaceutical company Bayer synthesized a pure and stable form of acetylsalicylic acid, which was less irritating than salicylic acid and is marketed as aspirin. Presently, there is host of NSAIDs, such as ibuprofen (Motrin) and naproxen (Alleve), which inhibit COX-1 and COX-2. These molecules can have harmful side effects, such as stomach bleeding. There are multiple other effects exerted by salicylates beyond COX-1 and COX-2 inhibition, including metabolic responses that, in part, are triggered by salicylate activation of AMPK.

Phosphoinositides and sphingolipids are an important class of lipids, worth highlighting, that participate in signaling. Phospholipase C hydrolyzes phosphatidylinositol-4,5-bisphosphate to DAG and inositol-1,4,5-trisphosphate, which trigger the activation of protein kinase C and the release of Ca²⁺ from internal stores, respectively. Furthermore, there are inositol and phosphoinositide kinases that generate an array of soluble inositol polyphosphates and membrane polyphosphoinositide lipids. Notably, phosphoinositide 3-kinase promotes the formation of phosphatidylinositol-3,4,5-trisphosphate, a signaling lipid that activates the kinase AKT to regulate metabolism (see Chandel 2020f). Sphingolipids are synthesized de novo from serine and palmitoyl-CoA. Sphingolipids, such as sphingosine-1-phosphate and ceramide, have been implicated in a variety of biological outcomes, including cell proliferation and death. Importantly, both phosphoinositides and sphingolipids have been implicated as drivers of multiple pathologies, including diabetes, cancer, and inflammation.

THE SYNTHESIS AND REGULATION OF CHOLESTEROL

Cholesterol becomes an obsession for many as they age. You cannot help but notice how many people having dinner at a restaurant or party will obsess about whether their cholesterol levels are high or if they should take cholesterol-lowering drugs. Although high cholesterol levels need to be dealt with, it is important to realize that cholesterol has important biological functions. Cholesterol can incorporate into cell membranes, function as a modification on proteins, and serve as precursor for the generation of steroid hormones and vitamin D. Cholesterol is not essential in our diet because most cells can generate cholesterol. The highest producer of cholesterol is the liver, where it can be esterified into cholesterol esters, which are stored in lipid droplets, packaged into lipoprotein particles, and exported to the peripheral tissues, or converted into bile acids, which are secreted into the small intestine through the bile duct to act as emulsifying agents in the digestion of dietary fat. Some of the bile is excreted as waste, thus getting rid of excess cholesterol, but most returns to the liver and then is moved to the gall bladder for storage and use in the digestion of lipids. The most abundant bile acid is cholic acid (cholate).

The cholesterol biosynthetic pathway takes place in the cytosol and is initiated by the enzyme HMG-CoA reductase, the target of statin drugs (see Box 3), to generate the six-carbon molecule mevalonate from one molecule of acetyl-CoA and one molecule of acetoacetyl-CoA (Fig. 10). Mevalonate is then phosphorylated and decarboxylated to form the activated 5-carbon isoprenoid intermediate, isopentenyl pyrophosphate. Next, three molecules of isopentenyl pyrophosphate are combined to form farnesyl pyrophosphate (C15), which is then used to generate squalene, a C30 cholesterol precursor. C15 can be used to prenylate proteins, such as Ras. Squalene synthase catalyzes condensation of two molecules of C15 with reduction by NADPH to generate squalene, which undergoes multiple reactions to generate a 27-carbon cholesterol molecule.

BOX 3.

THE STORY OF STATINS AS CHOLESTEROL-REDUCING AGENTS

A detrimental effect of rising cholesterol levels in the blood is that they are associated with the formation of atherosclerotic plaques in the lining of blood vessels. If atherosclerotic plaques rupture, pieces of fibrous material break off and travel to smaller blood vessels, where they cause blood clots (thrombosis) that can block blood flow to vital organs, such as the brain and heart, causing stroke and heart attack. In thinking about how to lower cholesterol levels to reduce atherosclerotic plaques, scientists targeted the cholesterol biosynthesis pathway. Initial efforts targeted late steps in the pathway using the compound Triparanol. However, this drug was withdrawn from clinical use because of adverse effects, including development of cataracts. Triparanol caused the accumulation of desmosterol by inhibiting the enzyme that converts desmosterol to cholesterol. In contrast, inhibition of HMG-CoA reductase, early in the cholesterol pathway, results in accumulation of hydromethylglutrate, which is not toxic and has alternative metabolic pathways for its breakdown. In the 1970s, the Japanese microbiologist Akira Endo discovered ML236B (compactin or mevastatin) in a fermentation broth of Penicillium citrinum as an HMG-CoA reductase inhibitor. Sankyo, in Japan, developed this compound for clinical use; however, this inhibitor never made it to the market because of fears of toxicity. The inhibitor that eventually made it to the market was lovastatin in 1987. This inhibitor, initially named mevinolin, was discovered at Merck Research Laboratories in a fermentation broth of Aspergillus terreus in the late 1970s. Subsequently, the second statin approved was simvastatin (Zocor) in 1988, which differs from lovastatin by having an additional methyl group as a side chain. Pravastatin (Pravachol), derived from compactin by Sankyo, followed in 1991. The next statins, fluvastatin (Lescolin) in 1994, atorvastatin (Lipitor) in 1997, cerivastatin (Baycol) in 1998, and rosuvastatin (Crestor) in 2003, were all synthetic compounds and not derived from microorganisms. For his pioneering efforts in discovering the original statin compactin, Dr. Endo received the prestigious 2008 Lasker-DeBakey Award for Clinical Medical Research. Forty years after his initial discovery, millions have used statins to reduce the morbidity and mortality associated with cardiovascular diseases.

Figure 10.

Cholesterol biosynthesis pathway. The cholesterol biosynthetic pathway is initiated by the enzyme HMG-CoA reductase, the target of statin drugs, to generate the six-carbon molecule mevalonate from one molecule of acetyl-CoA and one molecule of acetoacetyl-CoA. Mevalonate undergoes multiple reactions to generate a 27-carbon cholesterol molecule. Important by-products derived from cholesterol are the steroid hormones, bile salts, vitamin D, plasma lipoproteins, and structural elements of membranes.

Important by-products derived from cholesterol are the steroid hormones. Most of us are familiar with the steroid hormones estrogen and testosterone because of their pivotal role in reproductive physiology in females and males, respectively. These two hormones, along with the glucocorticoid cortisol and mineralocorticoid aldosterone, are derived from progesterone (Fig. 10). Cortisol induces gluconeogenesis to increase blood sugar and is a powerful suppressor of the immune system; aldosterone regulates ion transport in the kidney. Cholesterol generates pregenolone to produce progesterone. Because of their pleiotropic roles, synthetic steroids have been generated as pharmacological agents to treat a wide range of pathologies, including asthma (glucocorticoids).

MULTIPLE MECHANISMS REGULATE CELLULAR CHOLESTEROL LEVELS

Cholesterol is essential for multiple biological processes, thus cells have mechanisms for short- and long-term regulation of cholesterol synthesis. The rate-limiting step in cholesterol synthesis is HMG-CoA reductase, which localized to the endoplasmic reticulum membrane. AMPK, which phosphorylates to inhibit the activity of this enzyme, regulates this enzyme in metabolically stressed cells to shut off cholesterol synthesis. As we have already discussed, AMPK activation also stimulates fatty acid oxidation to stimulate mitochondrial-generated ATP. Remember, metabolically stressed cells will typically shut down ATP-consuming anabolic functions and concomitantly promote ATP-generating catabolic functions to maintain a high ATP/ADP ratio.

Long-term regulation of cholesterol synthesis is accomplished by sensing fluctuations in levels of cholesterol and other sterols in the pathway, triggering changes in multiple genes in the cholesterol pathway, including HMG-CoA reductase. Cholesterol is embedded in membranes, and, therefore, sensing changes in sterol levels occurs in membranes by two proteins embedded in endoplasmic reticulum membranes that contain sterol-sensing domains, HMG-CoA and SREBP cleavage-activating protein (SCAP). HMG-CoA detects increases in sterols through its sterol-sensing domain, resulting in binding to two other ER membrane proteins called INSIG-1 and INSIG-2 (insulin-induced gene 1 and 2), which are bound to ubiquitin ligases. This triggers proteasomal degradation of HMG-CoA protein.

SCAP is essential for the activation of a family of transcription factors SREBP (sterol regulatory element-binding protein) that control genes involved in cholesterol and fatty acid synthesis. There are three closely related isoforms of SREBP in mammalian cells, referred to as SREBP1a, SREBP1c, and SREBP2. SREBP1a and SREBP1c are generated from the same gene through alternative splicing and use of different promoters and are involved in fatty acid synthesis. SREBP1c and SREBP2 control genes involved in fatty acid and cholesterol synthesis, respectively. SREBP1a can control genes in both pathways. SREBPs belong to the basic helix-loop-helix–leucine zipper (bHLH-Zip) family of transcription factors. However, they are different from other bHLH-Zip proteins in that they are synthesized as inactive precursors bound to the endoplasmic reticulum; to be transcriptionally active, they have to reach the nucleus. When cholesterol levels become low, SCAP binds to SREBPs and escorts them from the endoplasmic reticulum to the Golgi apparatus. Next, SREBPs undergo sequential proteolytic processing by Site-1 and Site-2 protease to release the amino-terminal bHLH-Zip domain from the Golgi membrane. The bHLH-Zip domain enters the nucleus and binds to a sterol response element in the enhancer/promoter region of target genes to activate their transcription. When cholesterol increases, SCAP binds to INSIGs, trapping the SREBP complex in the endoplasmic reticulum membrane. SREBPs are not able to reach the Golgi apparatus and the bHLH-Zip domain is not released to activate gene transcription.

It is important to note that two mechanisms of cholesterol pathway regulation, HMG-CoA degradation and SCAP regulation of SREBP, respond to different sterols. Lanosterol primarily triggers degradation of HMG-CoA and cholesterol inhibits SREBP activation. Lanosterol, the cholesterol precursor, is a more potent inducer of HMG-CoA degradation compared with cholesterol. Lanosterol is known to be toxic to cells; it makes sense to have it degrade HMG-CoA protein as its levels increase to shut off the pathway. If lanosterol accumulation were to shut off SREBP processing, rather than degrading HMG-CoA, then it would decrease the enzymes required for lanosterol conversion to cholesterol and this could result in transient increase in lanosterol. Thus, having lanosterol degrade HMG-CoA reductase, and not SREBP, shuts off further synthesis of lanosterol while allowing for the enzymes to convert lanosterol to cholesterol. As cholesterol increases, the SREBP genes are turned off to shut down this pathway.

The elucidation of the cholesterol pathway led to the mechanism by which statins decrease cholesterol levels in the blood, and, at first glance, this mechanism would reduce cholesterol synthesis by HMG-CoA reductase inhibition. However, the mechanism is a bit more complicated and involves induction of the low-density lipoprotein (LDL) receptor, discovered by Michael Brown and Joseph Goldstein in the 1970s. They set out to elucidate the underlying mechanism of a human disease called familial hypercholesterolemia (FH). The concentration of cholesterol in blood of patients with FH is abnormally high, and these individuals are at high risk of a heart attack early in life. By examining fibroblasts isolated from FH homozygous patients, they were able to reveal that these cells lack their ability to take up LDL. Subsequently, experiments showed that LDL binding to the LDL receptor at specific sites on the membranes, called clathrin-coated pits, triggered internalization of the receptor, followed by lysosomal hydrolysis to free the cholesterol from the LDL particle. The increase in cholesterol inhibited HMG-CoA reductase activity by degradation of the protein and decrease in transcription through inhibiting SREBP, as discussed earlier. SREBP also induces the LDL receptor. If SREBP is inhibited by the increase in cholesterol because of internalization of the LDL receptor, transcription of the LDL receptors decreases. An increase in cholesterol levels also activates the cholesterol esterfying enzyme, cholesterol acyl-transferase, which allows cholesterol to be stored in cells as ester droplets. Thus, when statins directly inhibit HMG-CoA reductase, this triggers a decrease in cholesterol, which activates SREBP-mediated induction of the LDL receptors on the surface of cells and subsequent endocytosis of LDL receptor particles from the blood. The particles taken up by the liver are shunted off to bile acids for storage and excretion from the body. Statins reduce LDL levels in serum through this mechanism by 20%–40%. This discovery of Brown and Goldstein resulted in their being awarded the1985 Nobel Prize in Physiology or Medicine “for their discoveries concerning the regulation of cholesterol metabolism.” Beyond cholesterol metabolism, the importance of their discovery lies in concept of receptor-mediated endocytosis and receptor recycling, which provided a conceptual framework by which cells can internalize hormones, growth factors, and viruses. The story of cholesterol biology and statins is a good example of how a mechanistic reductionist approach can elucidate fundamental biology and provide benefit to patients.