**2. De novo lipogenesis**

In general, the fatty acid profiles of swine carcass lipids are reflective of dietary fatty acid composition and *de novo* lipogenesis. The level of unsaturation in dietary fat sources is mimicked in the carcass fatty acid profile, altering the lipid firmness by increasing the degree of unsaturation. Stress has also been shown to impact growth performance, and can have an impact on the swine industry both by altering growth performance and carcass lipid firmness. Fatty acids synthesized *de novo* are products of pathways tightly regulated by rate-limiting enzymes. Nutritional and hormonal regulators of the enzymes which regulate these pathways can alter rates in lipid synthesis, oxidation, and desaturation.

The first step in *de novo* lipogenesis is the generation of the main fatty acid subunit, malonyl-CoA. The production of malonyl-CoA from acetyl-CoA is catalyzed by acetyl-CoA carboxylase (ACC; EC 6.4.1.2) [1]. Acetyl-CoA is a single polypeptide chain which contains a biotin carboxyl carrier protein, biotin carboxylase, and carboxyl transferase domains [2, 3]. Acetyl-CoA is present as ACCα (~265 kDa) in liver and adipose tissue and catalyzes fatty

© 2013 Latour et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

acid synthesis [2, 3, 4]. In liver, heart, and muscle tissues ACCβ (~280 kDa) controls fatty acid oxidation [2, 3, 4]. The ACC reaction is a two-step reaction in which the biotin molecule, covalently attached by holo-carboxylase synthetase to the ε-amino group of a lysine residue, acts as the carboxyl carrier [5]. The first step results in the formation of carboxy-biotinyl-ACC at the biotin carboxylase active site and is ATP-dependent. During the second step, the carboxyl group is transferred from biotin to acetyl-CoA forming the malonyl-CoA product [5].

Impacts of Nutrition and Environmental Stressors on Lipid Metabolism 213

Acetyl-CoA carboxylase is also allosterically regulated, resulting in active and inactive protein conformations [1,5]. Phosphorylation of four or more serine residues on ACC results in inactivation [5, 8]. Phosphorylation of ACCα is by AMP-activated protein kinase (AMPK) while phosphorylation of ACCβ is by protein kinase A (PKA) [5, 8]. In liver and heart cells, insulin activates ACCα by dephosphorylating the AMPK site although this mechanism has

Transcriptional regulation is the primary means of controlling FAS [1, 7]. The FAS promoter has been studied in the rat, human, and chicken and the sequence is highly conserved among species [7]. The 5' flanking region of the promoter is 2.1 kb long and has transcription factor binding sites which determine tissue specificity of expression [7]. Promoter activity and FAS expression have been shown to increase in transgenic mice when high carbohydrate diets are fed, after fasting, and with increased insulin and glucocorticoid levels. Dietary polyunsaturated fatty acids (PUFA) decrease hepatic and adipose FAS mRNA levels and is a part of the mechanism of dietary fats to reduce *de novo* fatty acid synthesis [7]. Another mode of FAS regulation is stability of FAS mRNA [7]. In diabetic rats, thyroid hormone regulates FAS mRNA stability and in fetal rat lung, glucocorticoids

Stearoyl-CoA desaturase (SCD; EC 1.14.19.1), also known as ∆9 desaturase, is an endoplasmic reticulum associated enzyme that catalyzes the conversion of saturated fatty acids to monounsaturated fatty acids (MUFA) [9, 10]. Palmitoyl-CoA and stearoyl-CoA are the primary substrates of the desaturation reaction and are converted to palmitoleoyl-CoA and oleoyl-CoA, respectively [10, 11]. In liver, SCD is also required for synthesis of cholesteryl esters [9]. There are four isomers of SCD: SCD-1, found in adipose and liver tissue; SCD-2 and -3, found in the brain and harderian gland; and SCD-4, found in the heart [9]. The action of SCD to add a double bond to the ∆9 position of a saturated fatty acid starts the desaturation process. More double bonds can then be added by the elongation pathways discussed below [11]. Desaturation of 12 to 19 carbon fatty acyl-CoAs catalyzed by SCD-1, -2, -3, and -4 results in the addition of a *cis*-double bond between carbons nine and 10 and this reaction requires

Control of SCD-1, -2, -3, and -4 is mainly by transcriptional regulation [9]. Dietary omega-3 and -6 PUFAs, thyroid hormone, glucagon, thiazolidinediones, and leptin suppress SCD-1 expression, while cholesterol, vitamin A, PPARα, SREBP-1c, and high carbohydrate feeding induce expression [9, 12]. Increased SCD-1 activity thus increases the conversion of saturated fatty acids to unsaturated fatty acids and changes the ratio of carcass fatty acids.

β-oxidation is the catabolic process, occurring primarily in the mitochondria of the cell, that breaks down fatty acids into acyl-CoA molecules. These two carbon molecules can then enter the tricarboxylic acid cycle for energy production. β-oxidation involves three key

NADH, oxygen, NADH-cytochrome *b*5 reductase and cytochrome *b*5 [9].

not been observed in fat or liver cells [5].

stabilize FAS mRNA [7].

**2.3. β-oxidation** 

**2.2. Desaturation of fatty acids** 

The fatty acid synthase (FAS; EC 2.3.1.85) pathway is responsible for *de novo* lipogenesis which stores excess energy as fatty acids in liver and adipose tissue [1,6]. This pathway occurs within the cytosol and is a sequence of seven steps which are NADPH-dependent and utilize one acetyl-CoA and seven malonyl-CoAs as the base molecules to produce palmitate [1]. The NADPH required for each reaction is derived from activity of malic enzyme and the pentose phosphate shunt [7]. Though palmitate is the main product, stearic, mysristic and shorter fatty acids may also be produced [7]. Fatty acids produced from *de novo* lipogenesis are primarily saturated or monounsaturated and may be used in phospholipid and triacylglycerol synthesis [7].

Fatty acid synthase is a multifunctional enzyme composed of two identical monomers, each ~270 kDa [3]. Each monomer contains six functional domains which are β-ketoacyl synthase (KS), acetyl/malonyl transacylase (AT/MT), β-hydroxyacyl dehydratase (DH), enoyl reductase (ER), β-ketoacyl reductase (KR), acyl carrier protein (ACP) and thioesterase (TE) in order from the N-terminus [3]. The condensation of seven C2 moieties to the acetyl unit involves specific functions of the monomer components [3]. The reaction begins when the two substrates, acetyl-CoA and malonyl-CoA, are transferred to the KS and ACP, respectively, which is catalyzed by acetyl and malonyl transacylases. The condensation of these two substrates is catalyzed by KS and thus acetoacetyl-ACP is formed and CO2 is released. Acetoacetyl is reduced to a β-hydroxyacyl chain by KR and the product is then dehydrated and reduced a second time by DH and ER, respectively. The resulting product is a four-carbon fatty acid which is attached to ACP and transferred, by KS, from the ACP to a Cys-SH group on the KS. The ACP is then free to accept another malonyl unit. The addition of two carbon units from malonyl-CoA to the growing acyl chain leads to the synthesis of palmitate which is released after being hydrolyzed by TE [3].

#### **2.1. Regulation of de novo lipogenesis**

Regulation of ACC and FAS are important as they are the rate limiting steps of lipogenesis. Transcriptional regulation of ACC-α and -β is controlled by three promoters, PI, II, and III [5]. These promoters respond to glucose, insulin, thyroid hormone, catabolic hormones, and leptin [5]. Additional regulation occurs by sterol-regulatory-element-binding protein 1c (SREBP1c) and peroxisome-proliferator-activated receptors (PPAR) [5]. Fasting inhibits ACC expression though re-feeding returns expression to normal levels. Insulin exposure activates ACC, while catecholamines or glucagon exposure will inhibit ACC [5]. Activation and inhibition by insulin, catecholamines and glucagon, respectively, occur within minutes of exposure [5].

Acetyl-CoA carboxylase is also allosterically regulated, resulting in active and inactive protein conformations [1,5]. Phosphorylation of four or more serine residues on ACC results in inactivation [5, 8]. Phosphorylation of ACCα is by AMP-activated protein kinase (AMPK) while phosphorylation of ACCβ is by protein kinase A (PKA) [5, 8]. In liver and heart cells, insulin activates ACCα by dephosphorylating the AMPK site although this mechanism has not been observed in fat or liver cells [5].

Transcriptional regulation is the primary means of controlling FAS [1, 7]. The FAS promoter has been studied in the rat, human, and chicken and the sequence is highly conserved among species [7]. The 5' flanking region of the promoter is 2.1 kb long and has transcription factor binding sites which determine tissue specificity of expression [7]. Promoter activity and FAS expression have been shown to increase in transgenic mice when high carbohydrate diets are fed, after fasting, and with increased insulin and glucocorticoid levels. Dietary polyunsaturated fatty acids (PUFA) decrease hepatic and adipose FAS mRNA levels and is a part of the mechanism of dietary fats to reduce *de novo* fatty acid synthesis [7]. Another mode of FAS regulation is stability of FAS mRNA [7]. In diabetic rats, thyroid hormone regulates FAS mRNA stability and in fetal rat lung, glucocorticoids stabilize FAS mRNA [7].

#### **2.2. Desaturation of fatty acids**

212 Lipid Metabolism

[5].

phospholipid and triacylglycerol synthesis [7].

palmitate which is released after being hydrolyzed by TE [3].

**2.1. Regulation of de novo lipogenesis** 

acid synthesis [2, 3, 4]. In liver, heart, and muscle tissues ACCβ (~280 kDa) controls fatty acid oxidation [2, 3, 4]. The ACC reaction is a two-step reaction in which the biotin molecule, covalently attached by holo-carboxylase synthetase to the ε-amino group of a lysine residue, acts as the carboxyl carrier [5]. The first step results in the formation of carboxy-biotinyl-ACC at the biotin carboxylase active site and is ATP-dependent. During the second step, the carboxyl group is transferred from biotin to acetyl-CoA forming the malonyl-CoA product

The fatty acid synthase (FAS; EC 2.3.1.85) pathway is responsible for *de novo* lipogenesis which stores excess energy as fatty acids in liver and adipose tissue [1,6]. This pathway occurs within the cytosol and is a sequence of seven steps which are NADPH-dependent and utilize one acetyl-CoA and seven malonyl-CoAs as the base molecules to produce palmitate [1]. The NADPH required for each reaction is derived from activity of malic enzyme and the pentose phosphate shunt [7]. Though palmitate is the main product, stearic, mysristic and shorter fatty acids may also be produced [7]. Fatty acids produced from *de novo* lipogenesis are primarily saturated or monounsaturated and may be used in

Fatty acid synthase is a multifunctional enzyme composed of two identical monomers, each ~270 kDa [3]. Each monomer contains six functional domains which are β-ketoacyl synthase (KS), acetyl/malonyl transacylase (AT/MT), β-hydroxyacyl dehydratase (DH), enoyl reductase (ER), β-ketoacyl reductase (KR), acyl carrier protein (ACP) and thioesterase (TE) in order from the N-terminus [3]. The condensation of seven C2 moieties to the acetyl unit involves specific functions of the monomer components [3]. The reaction begins when the two substrates, acetyl-CoA and malonyl-CoA, are transferred to the KS and ACP, respectively, which is catalyzed by acetyl and malonyl transacylases. The condensation of these two substrates is catalyzed by KS and thus acetoacetyl-ACP is formed and CO2 is released. Acetoacetyl is reduced to a β-hydroxyacyl chain by KR and the product is then dehydrated and reduced a second time by DH and ER, respectively. The resulting product is a four-carbon fatty acid which is attached to ACP and transferred, by KS, from the ACP to a Cys-SH group on the KS. The ACP is then free to accept another malonyl unit. The addition of two carbon units from malonyl-CoA to the growing acyl chain leads to the synthesis of

Regulation of ACC and FAS are important as they are the rate limiting steps of lipogenesis. Transcriptional regulation of ACC-α and -β is controlled by three promoters, PI, II, and III [5]. These promoters respond to glucose, insulin, thyroid hormone, catabolic hormones, and leptin [5]. Additional regulation occurs by sterol-regulatory-element-binding protein 1c (SREBP1c) and peroxisome-proliferator-activated receptors (PPAR) [5]. Fasting inhibits ACC expression though re-feeding returns expression to normal levels. Insulin exposure activates ACC, while catecholamines or glucagon exposure will inhibit ACC [5]. Activation and inhibition by insulin, catecholamines and glucagon, respectively, occur within minutes of exposure [5].

Stearoyl-CoA desaturase (SCD; EC 1.14.19.1), also known as ∆9 desaturase, is an endoplasmic reticulum associated enzyme that catalyzes the conversion of saturated fatty acids to monounsaturated fatty acids (MUFA) [9, 10]. Palmitoyl-CoA and stearoyl-CoA are the primary substrates of the desaturation reaction and are converted to palmitoleoyl-CoA and oleoyl-CoA, respectively [10, 11]. In liver, SCD is also required for synthesis of cholesteryl esters [9]. There are four isomers of SCD: SCD-1, found in adipose and liver tissue; SCD-2 and -3, found in the brain and harderian gland; and SCD-4, found in the heart [9]. The action of SCD to add a double bond to the ∆9 position of a saturated fatty acid starts the desaturation process. More double bonds can then be added by the elongation pathways discussed below [11]. Desaturation of 12 to 19 carbon fatty acyl-CoAs catalyzed by SCD-1, -2, -3, and -4 results in the addition of a *cis*-double bond between carbons nine and 10 and this reaction requires NADH, oxygen, NADH-cytochrome *b*5 reductase and cytochrome *b*5 [9].

Control of SCD-1, -2, -3, and -4 is mainly by transcriptional regulation [9]. Dietary omega-3 and -6 PUFAs, thyroid hormone, glucagon, thiazolidinediones, and leptin suppress SCD-1 expression, while cholesterol, vitamin A, PPARα, SREBP-1c, and high carbohydrate feeding induce expression [9, 12]. Increased SCD-1 activity thus increases the conversion of saturated fatty acids to unsaturated fatty acids and changes the ratio of carcass fatty acids.

#### **2.3. β-oxidation**

β-oxidation is the catabolic process, occurring primarily in the mitochondria of the cell, that breaks down fatty acids into acyl-CoA molecules. These two carbon molecules can then enter the tricarboxylic acid cycle for energy production. β-oxidation involves three key

#### 214 Lipid Metabolism

components: activation of fatty acids in the cytosol of the cell, transport of activated fatty acids into the cell mitochondria, and oxidation.

Impacts of Nutrition and Environmental Stressors on Lipid Metabolism 215

Long-chain fatty acids are oxidized in the peroxisome by catalase, producing acetyl-CoA and hydrogen peroxide [15]. The catalase enzyme is induced by high-fat diets and proliferation of the peroxisomes is controlled by the peroxisome proliferator activated receptor (PPAR), which is part of the nuclear receptor family [9, 15]. The PPARα form is involved in regulation of β-oxidation and lipolysis in hepatocytes while PPARγ is involved in regulation of fatty acid synthesis in adipocytes [17, 18, 19]. The PPAR binding site contains both a hydrophobic ligand-binding pocket and a DNA-binding domain [9]. Stimulation of fatty acid oxidation by PPARα is by induction of CPT-I. Peroxisome

Intracellular fatty acids contribute to the overall regulation of synthesis and oxidative pathways. Fatty acids enter cells through diffusion or transporters, specifically fatty acid transport protein (FATP) or fatty acid transporter CD36 (FAT). Fatty acyl CoA synthetases or FATP then convert fatty acids into fatty acyl CoA (FACoA). Fatty acid binding proteins (FABP) then bind to and transport FACoA into intracellular compartments where they influence transcription through regulation of PPARα, γ and SREBP-1 [17, 20]. Intracellular PUFA inhibit SREBP-1 by downregulating enzymes involved in fatty acid synthesis [17, 18]. Intracellular PUFA activate

Dietary triacylglycerol composition plays a major role in determining adipose tissue composition. Monogastric animals incorporate dietary fatty acids directly into tissue lipid deposits [21, 22] and, therefore, to manipulate carcass lipid quality, it is important to understand the interactions of dietary lipids with carcass lipid. Carcass fatty acid profiles closely mimic dietary fatty acid profile [21, 23], and therefore, potential exists to modify carcass lipid properties (i.e., firmness, fatty acid profile, etc.) by altering dietary lipid composition.

One of the strongest determinants of carcass fat quality in pigs is the level and composition of lipids in the diet [24]. Because the utilization efficiency of dietary fat is 90% in pigs fed above maintenance [24] and the transfer coefficient of dietary fat to carcass lipid is as high as 31-40% [25] the carcass lipid composition is a reflection of dietary fat. The impact of dietary lipids on carcass lipid may differ depending on the timing of feeding relative to growth and

Dietary triacylglycerols alter carcass lipid composition at the level of the fatty acid profile [21]. Saturated fatty acids lack double bonds and have melting temperatures above 40ºC. Mono-, di- and poly-unsaturated fatty acids have one, two, or many double bonds, respectively and as the level of unsaturation increases, the melting point decreases [21]. The ratio of saturated to unsaturated fatty acids is a way of describing the relative saturation of a fatty acid profile [21]. Iodine value, a measure of double bonds in a lipid, is a method used to composite characteristics of lipids in regard to fluidity [21, 26]. Saturated to unsaturated ratios and iodine values can be utilized to describe the composition of lipids in both

proliferators also stimulate SCD-1 transcription levels [9].

**3. Dietary lipid composition** 

feedstuffs, total rations, and animal tissue.

**3.1. Dietary fat** 

PPAR to upregulate the transcription of the corresponding enzymes [17].

finishing, levels included in the diet, and interactions with other stressors.

The mechanism of the carnitine pathway is an ordered reaction where the binding of acyl-CoA begins the transport action [13]. Long chain fatty acids are converted to acyl-CoAs by acyl-CoA synthetase [14]. Acyl-CoAs are converted to acyl-carnitine molecules and transferred across the outer mitochondrial membrane by carnitine palmitoyltransferase-I (CPT; EC 2.3.1.21) [14]. Carnitine palmitoyltransferase-II is located on the inner mitochondrial membrane and liberates the carnitine from the acylcarnitine after transfer across the inner mitochondrial membrane [15]. After liberation, the acyl-CoA units are available for β-oxidation within the mitochondrial matrix [14]. Because CPT-II is not regulated [1] it is not pertinent to this discussion.

Carnitine palmitoyltransferase-I is located on the outer mitochondrial membrane and limits the rate of fatty acid oxidation by controlling the transportion of fatty acyl-CoA to the mitochondrial matrix where β-oxidation occurs [13, 1]. Two transmembrane domains anchor CPT-I to the outer mitochondrial membrane [13]. There are three isoforms of CPT-I [1]. In liver, kidney, lung, and heart tissue, CPT-Ia is present; CPT-Ib is present in skeletal muscle, heart, and adipose tissue; and CPT-Ic is brain tissue specific [1].

Regulation of β-oxidation occurs during the initial transport step. The main route of CPT-I regulation is by malonyl-CoA, the first product of lipogenesis, which inhibits CPT-I and aids to prevent simultaneous oxidation and synthesis [1, 16]. Regulation of CPT-I allows βoxidation to be regulated by controlling the availability of acyl-CoA in the mitochondrial matrix [1, 14]. Though the sensivitiy of the CTP-Ia and CPT-Ib to malonyl-CoA are different, they both contain binding sites on the same side of the membrane as the active site [13]. The N-terminus of the enzyme, which is not required for catalytic activity, controls the response to malonyl-CoA [13]. The kinetics of inhibition by malonyl-CoA are responsive to temperature, pH, and lipids [13, 14].

Fasting and glucagon increases CPT-I gene expression while hypothyroidism decreases expression by regulating the transcription level [13]. The insulin growth factor I receptor also controls CPT-I expression by mediating the inhibitory effects of insulin [13, 14]. Expression of CPT-I is also transcriptionally upregulated by PPARα [13]. Long chain fatty acids increase CPT-Ia mRNA expression in liver tissue by both increasing transcription levels as well as improving CPT-I mRNA stability [14].

#### **2.4. Regulation of lipid metabolism by transcription factors**

Sterol regulatory element binding proteins (SREBP) are helix loop helix proteins that are within the leucine zipper family of transcription factors [9]. The SREBPs are present as two isoforms, SREBP-1 (a and c subforms) and SREBP-2 [9]. While SREBP-2 is primarily involved in activation of cholesterol synthesis and metabolism, SREBP-1c is involved solely in regulation of fatty acid synthesis and SREBP-1a is capable of inducing both synthesis of cholesterol and fatty acids [9]. In the liver, SREBP-1c increases expression of SCD, ACC, FAS and acetyl CoA synthase [9].

Long-chain fatty acids are oxidized in the peroxisome by catalase, producing acetyl-CoA and hydrogen peroxide [15]. The catalase enzyme is induced by high-fat diets and proliferation of the peroxisomes is controlled by the peroxisome proliferator activated receptor (PPAR), which is part of the nuclear receptor family [9, 15]. The PPARα form is involved in regulation of β-oxidation and lipolysis in hepatocytes while PPARγ is involved in regulation of fatty acid synthesis in adipocytes [17, 18, 19]. The PPAR binding site contains both a hydrophobic ligand-binding pocket and a DNA-binding domain [9]. Stimulation of fatty acid oxidation by PPARα is by induction of CPT-I. Peroxisome proliferators also stimulate SCD-1 transcription levels [9].

Intracellular fatty acids contribute to the overall regulation of synthesis and oxidative pathways. Fatty acids enter cells through diffusion or transporters, specifically fatty acid transport protein (FATP) or fatty acid transporter CD36 (FAT). Fatty acyl CoA synthetases or FATP then convert fatty acids into fatty acyl CoA (FACoA). Fatty acid binding proteins (FABP) then bind to and transport FACoA into intracellular compartments where they influence transcription through regulation of PPARα, γ and SREBP-1 [17, 20]. Intracellular PUFA inhibit SREBP-1 by downregulating enzymes involved in fatty acid synthesis [17, 18]. Intracellular PUFA activate PPAR to upregulate the transcription of the corresponding enzymes [17].
