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Metabolism of Macro- and Micronutrients Topic 1 Module 1.3 Metabolism of Lipids: New Insight Regina Komsa-Penkova, Department of Biochemistry, Medical Faculty, Medial University –Pleven, Bulgaria Lubos Sobotka, Department of Metabolic Care and Gerontology, Medical Faculty, Charles University, Hradec Kralove, Czech Republic . Learning objectives To To To To To learn the important dietary lipids; learn the role of free fatty acids; understand the main metabolic lipids’ pathways ; learn the functions of lipoproteins; learn the main steps of lipid metabolism. Contents: 1. Introduction: dietary lipids 2. Main classes of fatty acids (FA) 2.1. Saturated FA 2.2. Cis MUFAs 2.3. PUFAs 2.3.1. - 6 PUFAs 2.3.2. -3 PUFAs 2.4. Trans FA 2.5. Conjugated FA 3. Metabolism of lipoproteins 3.1. Chylomicron production 3.2. VLDL and LDL 3.3. HDL 4. Lipid metabolism 4.1. Lipolysis 4.2. FFA in circulation 4.3. Fatty acid oxidation 4.4. Reesterification of FA into TAGs 4.5. Lipid droplets 4.6. Adipokines 5. References Copyright © by ESPEN LLL Programme 2014 1. Introduction: Dietary Lipid Lipids are important source of energy but possess many other metabolic functions as the structural components of cellular and organelle membranes and essential precursors for hormones, local mediators and regulatory molecules. The accumulation of energy as fat was very important for survival of our ancestors in the past. However, at present this accumulation leads to the development of obesity. The increase in obesity incidence led to extensive research in the area of lipid metabolism, food intake, appetite control as well as its contribution to the metabolic changes in dyslipidemias, cardiovascular disease, endocrine disorders and cancer. Recent investigations on metabolism highlighted the role of lipids as inflammatory and allergic components by variety of pro and anti-inflammatory eicosanoids, specific cell signalling molecules for PPAR, GP 120, Nf-kB, toll like receptors (TLR), influencing cell’s receptiveness, involved in growth and development processes etc. New investigated molecules of lipid mediators like resolvins, protectins, sirtulins, and maresins provided new insights into the inflammation process. SREBP (sterol regulatory element binding protein), grelin, leptin, fatty acid transporters have changed the understanding of lipid regulatory mechanisms. According to general recommendations lipids should provide around 20-35 % of energy intake in healthy individual. Moreover, lipids are necessary for absorption and transport of lipid-soluble vitamins. Ingested lipids are either oxidised or used as building material in the body (cell membranes, neural tissue, etc.) Excess of fat is accumulated in fat stores as the body energy reserve. The energy yield of lipid is approximately 9.4 Kcal/g (39.3 kJ/g), compared to 4.2 Kcal/g (17.6 kJ/g) for carbohydrates. In fact, the energy storage capacity for fat is almost unlimited in human body. Usually the total amount of energy stored in adipose tissue as triacylglycerol (TAG) (80,000-140,000 kcal) is 40-70 times higher than that stored under the form of glycogen (1,700-2,000 kcal)(1, 2) and in obese subjects is even higher. Fig. 1 Energy reserves of human body: TAG- 80000-140000 kcal, usable protein 25-30000 kcal, liver glycogen 400-600 kcal, blood glucose 40 kcal and daily needs 2500-2800 kcal. Blood glucose at 5mM is sufficient for a few minutes. However, the excessive accumulation of TAG in human adipose tissue leads to obesity development. Moreover, the accumulation of lipids in other tissues like skeletal muscle and the liver, can be associated with insulin resistance and organ dysfunction. For example excessive TAG deposition in the liver is associated with nonalcoholic steatohepatitis, in skeletal muscles with insulin resistance and in the heart with cardiomyopathy (3, 4). Copyright © by ESPEN LLL Programme 2014 2. Main Classes of Fatty Acids (FA) Dietary lipids consist primarily of TAG - (97%), phospholipids (PL) and sterols. They are also transporters for fat soluble vitamins. Quantitatively the major component of TAG and PL are fatty acids (FA). FA with the chain length of 14 to 22 carbons, with minor quantity of shorter and longer chain FA (see Table 1). FAs are classified according to their main characteristics: chain length as short-chain (2-4 carbons), medium chain (6-12 carbons) and long chain FA (14 to 22 carbons), according to the presence or absence of double bonds (saturated / unsaturated) and their number (mono-, polyunsaturated). The location of the first double bond from the methyl end is also important for FA nomenclature: (-3, -6 and -9 or n-3, n-6, or n-9 respectively). The double bonds can occur in either the cis or trans configuration. The major dietary FAs are saturated, monounsaturated and polyunsaturated. Table 1 Classification of FA and the distribution in TAG of adipose tissue in human Class FA Subclasses Individual FA Short chain 2:0 Acetic acid 3:0 Valeric acid 4:0 Butyric acid 6:0 Caproid acid 8:0 Caprylic acid 10:0 Caproic acid 12:0 Lauric acid 14:0 Myristic acid 16:0 Palmitic acid 18:0 Stearic acid 18:1n-9 Oleic acid 14:1n-7 Myristoleic acid 16:1n-7 Palmitoleic acid 18:1n-7 Vaccenic acid 20:1n-9 Eicosenoic acid 22:1n-9 Erucic acid 18:2 Linoleic acid 18:3 γ-Linolenic acid 20:3 Dihomo-γ-linolenic acid 20:4 Arachidonic acid 22:4 Adrenic acid 22:5 Docosapentaenoic acid 18:3 α-Linolenic acid 20:5 Eicosapentaenoic acid 22:5 Docosapentaenoic acid 22:6 Docosahexaenoic acid 9-trans,12-cis 18:2; 9-cis,12-trans 18:2 Saturated FAs Medium chain Long Chain Mono unsatureated Cis FA n-6 FAs Polyunsaturated Cis FA Polyunsaturated Cis FA Trans FAs n-3 FAs TAG of adipose tissue in human % 5% 24% 8% 46% 7% 7% 1% In general, animal lipids have high content of saturated and monounsaturated FAs (MUFA), and are mainly solid (lard, tallow). Plant lipids are mainly oils and have a high content of unsaturated FAs. Exceptions to this rule are coconut oil and palm kernel oil, which are high in saturated lipid or waxes. Copyright © by ESPEN LLL Programme 2014 2.1 Saturated FA Recently saturated FAs have been considered to be associated with increased atherogenic risk and adverse health outcome (5). Numerous studies have been conducted to investigate the effect of saturated FAs on serum cholesterol concentration. Meta-analysis of metabolic studies showed that the higher the intake of saturated FAs, the higher the serum total cholesterol (6). It has been shown that especially lauric, myristic, and palmitic, acids (intermediate chain lengths 12:0–16:0) may increase the synthesis and accumulation of triglyceride and cholesterol in liver (7), each resulting in the suppression of hepatic LDL receptor mRNA levels (8). Replacing saturated FA with MUFA and PUFA, can reduce plasma LDL cholesterol (LDL-C) (9, 10) via an increase in LDL receptor (LDLR)-mediated uptake of LDL-C from circulation (11). LDLR-mediated uptake, however, is impaired by obesity. The greater rate of hepatic cholesterol synthesis in obese individuals suppresses the expression of hepatic LDL receptors (LDLR), thereby reducing hepatic LDL uptake. Not all the saturated FAs exhibit this effect. Shorter chain saturated FAs (6:0–10:0) have little effect on plasma cholesterol concentrations. Stearic acid, is neutral with respect to HDL cholesterol, and can lower LDL cholesterol and the ratio of total to HDL cholesterol. Stable isotope tracer methods have shown that approximately 9 to 14 % of dietary stearic acid is converted to oleic acid (12). 2.2 Cis Monounsaturated FAs Cis monounsaturated FAs (MUFAs) contain one double bond with the hydrogen atoms positioned on the same side of the double bond. Plant lipids are rich in cis MUFA like olive oil, sunflower oil and canola oils. The double bond is localised usually in -9 position and partially in position -7 (n-7). The most frequent MUFA is oleic acid (-9), which accounts for about 92 % of dietary MUFAs. Palmitoleic acid (-7) is presented in minor amount in the diet. There is convincing evidence that replacing carbohydrates with MUFA increases HDL cholesterol concentration in plasma and improves insulin sensitivity; replacing of SFA (C12:0–C16:0) with MUFA reduces LDL cholesterol concentration and total/HDL cholesterol ratio (13). 2.3 Polyunsaturated FAs Polyunsaturated FAs (PUFAs) are essential FA as mammalian cells do not have the enzymatic system which inserts a cis double bond at the -6 or -3 positions of a FA chain. There are two important classes of PUFAs -3 and -6 (double bond located at 3 and 6 carbon atoms from the methyl end respectively). Linoleic acid (LA; 18:2n-6) is the quantitatively most important PUFA, comprising 84–89% of the total PUFA energy, whereas -linolenic acid (ALA; 18:3n-3) contributes 9–11% of the total PUFA energy. The recommended adequate intake of LA is 17 g/d for young men and 12 g/d for young women, whereas of ALA is 1.6 and 1.1 g/d for men and women, respectively. Copyright © by ESPEN LLL Programme 2014 Fig. 2 3D structure of Linoleic acid (LA;18:2 n-6), -Linolenic acid (ALA;18:3 n-3) and DHA (22: 6n- 3) Usually in western diet PUFAs contribute <7% of total energy intake and 19–22% of energy intake from lipid in the diets of adults. PUFAs serve as the precursors to eicosanoids, components of membrane phospholipids, and are also important in cell signalling pathways. 2.3.1 -6 PUFA The most abundant -6 polyunsaturated FAs in our diet are: Linoleic acid - LA Arachidonic acid - AA Dihomolinoleic acid - DHLA Arachidonic acid and other PUFAs are involved in regulation of gene expression resulting in decreased expression of proteins that regulate the enzymes involved in FA synthesis (14). This may partly explain the ability of PUFAs to influence the hepatic synthesis of FAs. A lack of dietary -6 polyunsaturated FAs is characterized by rough and scaly skin, dermatitis, and an elevated eicosatrienoic acid to arachidonic acid ratio. AA is the substrate for the production of a wide variety of eicosanoids (20-carbon AA metabolites). Some are proinflammatory, vasoconstrictive, and/or proaggregatory, such as prostaglandin E2, thromboxane A2, and leukotriene B4. However, others are antiinflammatory/antiaggregatory, such as prostacyclin, lipoxin A4, and epoxyeicosatrienoic acids. 11, 12-Epoxyeicosatrienoic acids are FA epoxides produced from AA by a cytochrome P450 epoxygenase. Dihomo-γ-linolenic acid, formed from linoleic acid, is also an eicosanoid precursor. 2.3.2 -3 PUFA Polyunsaturated FAs in diet are: -linolenic acid - ALA Eicosapentaenoic acid - EPA Docosahexaenoic acid - DHA They play an important role as structural membrane lipids, particularly in nerve tissue and the retina, and also serve as precursors to regulatory eicosanoids. ALA (18:3n-3) is an essential FA, lack of ALA results in adverse clinical symptoms, including neurological abnormalities, scaly dermatitis and poor growth. Vegetable oils are the major source of ALA, fish oils contains EPA (20:5n-3) and DHA (22:6n-3) (15). ALA is the precursor of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are synthesized in human organism. These very long chain PUFAs (EPA, DHA) are direct precursors of eicosanoids with lower inflammatory activity than majority of pro-inflammatory eicosanoids which are metabolites of AA. Copyright © by ESPEN LLL Programme 2014 It was shown that increased consumption of -3 FAs from fish or fish-oil supplements (20 and 22 carbons), but not of ALA (18 carbons), reduces the rates of all-cause mortality, cardiac(16) and sudden death (17). This is probably due to their anti-inflammatory effects. A considerable number of observational and interventional studies, systematic reviews and meta-analyses of the relationship of dietary -3 FAs and CVD events have been published (18, 19). The inverse relationship between -3 FA intake and CVD events was found and confirmed for EPA and DHA. Moreover, EPA and DHA from fish oil have regulatory role in the expression of genes involved in lipid and energy metabolism. Two transcriptional factors, particularly, sterol regulatory element binding protein-1c (SREBP-1c) and peroxisome proliferator activated receptor (PPAR ), were investigated as the regulators of gene expression by PUFA. -3 PUFA suppress the induction of lipogenic enzymes by inhibiting the expression and processing of SREBP-1c (by antagonizing LXR-dependent activation of SREBP-1c) (20). PPAR plays a key role in metabolic adaptation to fasting by inducing the genes for mitochondrial and peroxisomal FA oxidation as well as those for ketogenesis in mitochondria. 2.4 Trans FAs Trans FAs contain at least one double bond in the trans configuration, which results in a chain straight shape more similar to saturated FAs. A major trans FA is elaidic acid (9-trans 18:1). Trans FAs are mainly produced by industrial hydrogenation of plant oils to produce margarines. Partial hydrogenation of polyunsaturated oils causes saturation and hardening of oils to margarines, however, isomerisation and migration of double bonds, can result in a production of mixed 9-trans,12-cis 18:2; 9-cis,12-trans 18:2 FA (20). There is a positive linear trend between trans FA intake in diet and plasma LDL cholesterol concentration and increased risk of CHD. An inverse association between total trans FAs and AA and DHA and concentrations in cholesteryl esters in plasma was described (22) as well as between plasma cholesteryl esters elaidic acid (18:1trans), and birth weight of premature infants (23) were also reported. 2.5 Conjugated Linoleic Acids (CLAs) CLAs are geometric and positional isomers of linoleic acid containing conjugated trans and cis double bonds. Nine different isomers of CLA as minor constituents of food were reported, but only two of the isomers, cis-9, trans-11 and trans-10, cis-12, possess biological activity including anticancer, anti-atherosclerosis and prevention of obesity development (24). CLAs are naturally present in dairy products and ruminant meats as a consequence of biohydrogenation in the rumen. 3. Metabolism of Lipoproteins 3.1 Chylomicron Production The main pathway for dietary fats to enter the bloodstream is through chylomicron (CM) formation in the intestine. Once released by pancreatic lipase into the intestine, FA and monoacylglycerol (MAG) molecules are absorbed into enterocytes, where they are reesterified into TAGs and incorporated into TAG-rich particles covered by phospholipids. After acquiring of Apo B-48 (the main apoprotein of chylomicron) by the action of microsomal transfer protein they are called chylomicrons. The production of apo B-48 (1.3 mgkg-1d-1) varies on the basis of dietary fat intake. The average residence time of apo B-48 in plasma is 4.8 h (25). Copyright © by ESPEN LLL Programme 2014 CMs enter the lymphatic system and the circulation through thoracic duct. In peripheral tissues (manly adipose and muscle tissue) chylomicrones gain apoprotein C and then they are hydrolysed by lipoprotein lipase (LPL), which degrades their core of TAGs; this lead to the release of Fas and surface phospholipids. Released Fas are taken up by various tissues. However some FA are not rapt at the place of hydrolysis but rather leak out into the plasma as FFA pool were they are bound on albumin. Then these FFA are taken up by the liver or other tissues. LPL is activated by an apoprotein CII, transferred to CM from other lipoproteins (mainly HDL). After CMs lose much of their TAG, they acquire cholesterol ester from other lipoproteins via the action of cholesterol ester transfer protein (CETP). During this process CM also acquire apo E from HDL and they turn into chylomicron remnants which are taken up by the liver. TAG originating from CM-remnant as well these synthesized de novo in liver are repacked into very low-density lipoproteins (VLDLs), thereby recycling the dietary Fas. However, the rate of incorporation de novo synthesized Fas into VLDL-TAG is much slower in contrast to the incorporation of Fas from the plasma FFA pool. Impaired postprandial plasma TAG clearance by adipose tissue was reported in obese subjects after ingestion of a single mixed meal(26). This is partly explained by a lower functional LPL activity per unit fat mass in combination with the absence of postprandial up regulation of adipose tissue LPL in obesity. 3.2 VLDL and LDL In the endogenous pathway the hepatic TAGs synthesized from glucose, Fas and other lipids, are packaged into VLDL – very low density lipoproteins which are rich in triacylglycerol and contain mainly protein Apo B-100. Apo B-100 is the major apoprotein of VLDL and the sole protein of LDL. About 20.4 mgkg-1d-1 of apo B is packed into VLDL along with cholesterol and phospholipids (23, 27). VLDLs transport TAGs from the liver to peripheral tissues, such as muscle and adipose tissue, where in capillaries on the endothelial cell surface undergo intravascular lipolysis by LPL. In circulation VLDL acquire another apoproteins (apo C-I and – III and apo E) and phospholipids from HDL particles. ApoC-I and ApoC-III are small proteins that modulate lipolysis (activation of LPL) and interaction of TAG rich particles with receptors. After losing core triacylglyceroles approximately half of the VLDL remnants (intermediate density lipoproteins – IDL) are cleared from the circulation by LDL receptor (LDL-R) mediated endocytosis in the liver, and the residue undergoes further lipolysis to produce LDL (28). VLDL apo B-100 has a residence time of 3.6h. Most of LDL is removed from the circulation after binding to the hepatic LDL-R via apoB-100 (29). An estimated 70% of circulating LDL is cleared by LDL-R in the liver. During postprandial lipolysis TAG lipoprotein derived Fas are stored in the adipose tissue, but also contribute significantly to the plasma FA pool (30). In patients with elevated TAGs (hyperlipidemia) VLDL are increased in number and size and contain more TAGs. These larger TAG-rich VLDL have delayed lipolysis in insulin-resistant patients, which may be due to a lower affinity for LPL, lower LPL activity or lower affinity to tissue and hepatic receptors that promote the degradation and clearance of VLDL. VLDLs with increased plasma residence time transfer some of their core TAG to HDL and LDL via CETP, in exchange for cholesterol ester (CE). Copyright © by ESPEN LLL Programme 2014 Fig. 3 Lipid metabolism and transport. Liver produces VLDL from Cholesterol and TAGs synthesized de novo and from CM-remnants (exogenous). Lecithincholesterol acyltransferase (LCAT) esterifies free cholesterol I, forming the core of newly synthesized HDL molecules. LPL hydrolyzes TG in VLDL LPL (releasing glycerol and FFA)which result in LDL molecules taken up by extrahepatic tissues and/or liver. HDL take back cholesterol to the liver in a process known as reverse cholesterol transport. (ACAT, acyl-CoA:cholesterol acyltransferase; HL, hepatic lipase). HDL take back cholesterol to the liver in a process known as reverse cholesterol transport. (ACAT, acyl-CoA:cholesterol acyltransferase; HL, hepatic lipase). Lecithin-cholesterol acyltransferase (LCAT) esterifies free cholesterol I, forming the core of newly synthesized HDL molecules. Increased numbers of apo B or LDL particles not cleared by hepatic LDL receptors have increased plasma residence time and may enter the arterial intima. Persistence of LDL in circulation leads to production of atherogenic small dense LDLs. In individuals with dyslipidaemia, LDLs and other atherogenic lipoproteins enter the arterial wall where they undergo chemical modification, including oxidation (31). These modified lipoproteins initiate the inflammatory process that culminates in atherosclerosis lesion development and coronary heart disease (CHD) (32). Patients with marked elevations of LDL cholesterol and tendinous xanthomas generally have familial hypercholesterolemia resulting from a delayed catabolism of LDL apo B-100 (33) associated with various defects in the LDL receptor (see Module 22.1). 3.3 HDL The first step in cholesterol reverse transport is the production of Apo A-I and A-II by the liver and Apo A-I by the intestine and their combination with the phospholipids and cholesterol with the subsequent formation of discoid aggregates which are HDL precursors (34). Cholesterol is taken from the cells of peripheral tissues by HDL either by passive diffusion or through the action of ATP-dependent transmembrane transporter: ATP-binding cassette transporter-1 (ABCA-1) (35). In HDL particle cholesterol then undergoes esterification by the LCAT enzyme, forming cholesterol esters. This esterification prevents Copyright © by ESPEN LLL Programme 2014 re-diffusion of cholesterol from HDL back to the membrane and amplifies cholesterol efflux, forming “mature spherical” HDL. HDL particles are captured by liver via scavenger receptor B, class 1 (SRB1) and apoE receptors (adrenal glands, ovaries). In individuals with dyslipidaemia, when LDLs and other atherogenic lipoproteins chemically modified, in the arterial wall initiate the inflammatory process and atherosclerosis lesion development, the inflammation can be reversed by HDLs via promotion of cholesterol efflux and/or inhibition of LDL oxidation and reduction of adhesion molecule expression (28). 4. Lipid Metabolism Lipid stores of white adipose tissue represent the major energy reserves in humans. After food intake, most of the FAs released by LPL during postprandial lipolysis are taken up by adipose tissue (AT) and esterified into TAG, which are subsequently stored in cytosolic lipid droplets (LDs) of adipocytes. On energy demand, TAGs are mobilized from their stores by hydrolytic cleavage and the resulting FFAs are delivered via the circulation to peripheral tissues for β-oxidation and ATP production. Lipid droplet-associated TAGs are also found in most nonadipose tissues, including liver, cardiac muscle, and skeletal muscle (36). However, whereas adipocytes are able to release FAs and provide them as systemic energy substrate, non-adipose cells do not secrete FFAs but utilize them locally for energy production or lipid synthesis. Excessive ectopic lipid deposition in non-adipose tissues leads to lipotoxicity. 4.1 Lipolysis Consistent with its essential importance in energy homeostasis, lipolysis occurs in essentially all tissues and cell types, however, it is most abundant in white and brown adipose tissues (37). Triacylglycerol of adipose tissue can be rapidly mobilized by the hydrolytic action of the three main lipases of the adipocyte (38). The complete hydrolysis of TAG depends on the activity of three enzymes, adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and monoacylglycerol lipase (MGL). Until recently HSL was considered to be the key ratelimiting enzyme responsible for regulating TAG mobilization. In addition to its activity towards triacylglycerols, HSL hydrolyses diacylglycerols (10 times more effectively than TAG), monoacylglycerols, retinyl esters and cholesterol esters. Recently identified enzyme - ATGL has been discussed as playing an important role in the control of fat cell lipolysis. The role of ATGL in lipolysis became evident from the observations of a severe “lipid” phenotype in ATGL-deficient mice (39). Absence of ATGL causes a reduction of FA release from white adipose tissue by more than 75%, leading to increased total fat mass, increased ectopic fat mass, and increased body weight. Fig. 4 The hydrolytic cleavage of TAG by consequent action of the enzymes Copyright © by ESPEN LLL Programme 2014 The full hydrolysis of TAG is dependent on the activity of three lipases ATGL, HSL and MGL, each of which possesses a distinct specificity and regulatory mechanism. Considerable progress has been made in understanding the mechanisms of activation of the various lipases, mostly HSL. The best understood hormonal effects on AT lipolysis concern the opposing regulation by insulin and catecholamines/glucagon, natriuretic peptides and numerous autocrine/paracrine factors originating from adipocytes. Glucagon is secreted during low glucose fasting state, and epinephrine is associated with increased metabolic demands. In these conditions the energy need is covered by oxidation of FA. Glucagon, norepinephrine, and epinephrine bind to G protein-coupled receptors that activate production of cyclic AMP, leading to activation of protein kinase A, which as a consequence activates (phosphorylates) HSL, thus stimulating lipolysis. Insulin stimulates the opposite (inhibitory) effect when blood glucose is high. Insulin activates protein phosphatase 2A, which dephosphorylates HSL, thereby inhibiting its activity. Insulin also activates the enzyme phosphodiesterase, which hydrolysis cAMP and stops the effects of protein kinase A. Although more is known about HSL, it has been shown that HSL and ATGL can be activated simultaneously, that enables HSL to access the surface of lipid droplets and stimulates ATGL. Expression of ATGL is under the influence of dietary status. In fasting animals the level of ATGL increases and then declines following re-feeding. It has recently been reported that PKA-mediated phosphorylation of perilipin A is important for ATGL-dependent lipolysis The classical pathway of lipolysis activation in adipocytes is cAMP-dependent. Several agents contribute to the control of lipolysis in adipocytes by modulating the activity of HSL and ATGL. In addition, CGI-58 has also been shown to stimulate ATGL activity. 4.2 FFA in Circulation Subcutaneous and abdominal adipose tissues are the largest fat depots and contributes the major proportion of circulating nonesterified fatty acids (NEFS a synonym of FFA), considerably less FFA comes from intraabdominal adipose tissue) (40). Circulating FFAs in blood are bound to albumin. During prolonged fasting or during longer aerobic physical activity lipids are utilised as major energy source. In the fasting state, plasma FFA arise (double at night fast) almost entirely from hydrolysis of TAG within the adipocyte. Prolonged fasting concentrations of FFA have been related to adipose tissue mass (41) and also to the eventual presence of type 2 diabetes. After a meal that contains fat, LPL in the capillaries of adipose tissue hydrolyses circulating TAG mainly in the chylomicrons. FFA released are taken up into the adipocytes for storage. However, a part of FFA always escapes and joins the plasma FFA pool (in a process called “spillover”) reaching 40–50% of the total plasma FFA pool in the postprandial period (42). The postprandial concentrations of FFA tend to remain somewhat higher in obese compared with lean people. 4.3 Fatty Acid Oxidation FFAs are taken for oxidation in many tissues; quantitatively major site is skeletal muscle (up to 80%). Fatty acids are transported into the cell by tissue specific fatty acid transport protein (FATP), fatty acid translocase (FAT/CD 36) and plasma membrane fatty acid binding protein (FABPpm) (43). Muscle uptake of FAs is dependent on plasma FFA level. Copyright © by ESPEN LLL Programme 2014 Once in the cell, FAs are activated to acyl-CoA by fatty acyl-CoA synthase (FACS). Carnitine palmitoyltransferase 1 (CPT1), carnitine translocase (CAT) and mitochondrial membrane CPT2 catalyse the consequent processes of conversion of long chain acyl-CoA into acylcarnitine, its transport across the inner mitochondrial membrane and reconversion to acyl-CoA. Medium chain fatty acids transport into mitochondria is carnitine-independent. In mitochondria acyl-CoA undergoes β-oxidation to acetyl-CoA with concomitant production of reduced NADH and FADH2. Acetyl-CoA enters the citric acid cycle to be oxidized completely to carbon dioxide. Energy production from FA in β-oxidation pathway is aerobic mitochondrial process, exceeding 100 ATP per FA molecule (around 130 for palmitic acid). Fig. 5 The availability of FA in myocyte mitochondria depends on the rate of lipolysis by lipases and rate of reesterification in adipose tissue and the rate of LPL lipolysis of triacylglycerol rich particles in postprandial conditions. FABPpm and FAT/CD36 proteins have been identified in the plasma membrane of muscle cells, which facilitate the transport of FA into the myocyte. Another pool of FA for oxidation are intramuscular triacylglycerol molecules Hydrolysis of these TAGs involves intramuscular HSL and adipose triglyceride lipase (ATGL) During fasting period acetyl CoA molecules condense to form ketone bodies in liver mitochondria. During starvation or prolonged low carbohydrate intake, ketone bodies are an important energy substrate for many tissues including the brain and skeletal muscle. Increased dietary intake of medium-chain FAs also results in the higher production of ketone bodies. This is explained by the carnitine-independent influx of medium-chain FAs into the mitochondria, thus by-passing this regulatory step of FA entry into β-oxidation. Copyright © by ESPEN LLL Programme 2014 4.4 Reesterification of Fatty Acids into TAGs FAs that do not enter into oxidative pathways can be re-esterified into TAGs or other lipids after activation into acyl-CoA derivatives. The non-oxidised fatty acids are esterified with glycerol-3 phosphate in G-3-P pathway GPAT is believed to be the rate-limiting factor in glycerophospholipid synthesis. This process starts with the acylation of glycerol-3-phosphate with a fatty acyl-CoA, production of lysophosphatidic acid (LPA), followed by further acylation by LPA acyltransferase (LPAAT) and dephosphorylation to yield diacylglycerol (DAG). DAG is then esterified with the third acyl-CoA molecule to produce TAG. DAG is also the substrate for the synthesis of phospholipids as phosphatidic choline (PC) and phosphatidic ethanolamine (PE). Fig. 6 The 2 metabolic pathways involved in the synthesis of triacylglycerol (TAG). The monoacylglycerol (MAG) pathway, also known as the remodeling pathway, begins with the acylation of MAG with fatty acyl-CoA catalyzed by monoacylglycerol acyltransferase (MGAT) to form diacylglycerol (DAG) (44). Further acylation of DAG by diacylglycerol acyltransferase (DGAT) leads to the synthesis of TAG. This pathway plays a predominant role in the enterocytes after feeding, where large amounts of 2-MAG and fatty acids are released from the digestion of dietary lipids. The MAG pathway is also active in adipose tissue, likely playing a role in a storing excess of energy in TAG. These two pathways share the final reaction, catalyzed by diacylglycerol acyltransferase (DGAT), for converting DAG to TAG. In fact there are two DGAT enzymes, which are structurally and functionally distinct. DGAT1 is expressed in skeletal muscle, skin, mammary gland and intestine, with lower levels of expression in liver and adipose tissue. DGAT2 is the Copyright © by ESPEN LLL Programme 2014 main form in hepatocytes and adipocytes (lipid droplets). Both enzymes are important modulators of energy metabolism, although DGAT2 appears to be especially important in controlling the homeostasis of triacylglycerols in vivo (45). Triacylglycerol products of the DGAT reaction may be channelled into the cores of cytosolic lipid droplets (46) or triacylglycerol-rich lipoproteins for secretion in cells such as enterocytes and hepatocytes. 4.5 Lipid Droplets This lipid droplets (LD) could be compared in their micellar structure to the plasma lipoproteins (47). LDs of adipocytes are enriched in triacylglycerols (energy store), while defending cells against lipotoxicity. They also contain structural components, including cholesterol and retinol, for membrane synthesis and repair. The phospholipid component of the monolayer contains significant amounts of phosphatidylcholine with a fatty acid composition distinct from that of the endoplasmic reticulum and plasma membrane. Many cell types, even ganglia in the brain, can contain small lipid droplets (of the order of 50 nm in diameter), but in adipocytes these can range to up to 200 μm in diameter. The lipid droplets (LD) like plasma lipoproteins on the surface contain a specific group of constitutively associated protein members of the PAT family: perilipin, adipophilin, TIP47 (now renamed to perilipins 1–3). Perilipins probably regulate formation, growth and lipolysis of LDs. The enzymes of lipid metabolism are also abundantly located at the LD surface . Fig. 7 Hypothetical model of triacylglycerol synthesis and the lipid droplets formation in the ER (43). The reaction of triacylglycerol synthesis catalysed by DGAT at the cytosolic surface of the ER. In the liver, TAGs can either be stored temporarily or incorporated into TAG-rich VLDL and released into the plasma. In myotubes palmitic acid is accumulated as DAG and TAG, whereas oleic acid mainly as free FA. Oleic acid, the major MUFA is oxidized, as all other FAs, by β-oxidation. Copyright © by ESPEN LLL Programme 2014 TAG synthesis is important in many of physiological processes, intestinal dietary fat absorption, intracellular storage of extra energy, lactation, attenuation of lipotoxicity, lipid transportation, and signal transduction. The importance of TAG synthesis is exemplified by severe insulin resistance in patients with lipodystrophy, a genetic condition characterized by defective TAG synthesis and storage in adipose tissues (48). Whereas excessive TAG accumulation in adipose leads to obesity, ectopic storage of TAG in nonadipose tissues such as liver and skeletal muscle is associated with insulin resistance (49). 4.6 Adipokines Lipid synthesis and storage and consequent lipolysis are the main classical functions of adipocytes. However, adipocytes also express and secrete various factors (adipokines) that exert autocrine, paracrine, and endocrine effects in the body (50). There are currently over 50 different adipokines recognized as being secreted from adipose tissue including growth factors, cytokines, chemokines, acute phase proteins, complement-like factors, and adhesion molecules like leptin, adiponectin, resistin, visfatin, apelin, vaspin and IL-1β, IL-4, IL-6, CRP et. The adipokines are involved in the modulation of several physiological responses that includes control of appetite and energy balance. Specific metabolic processes regulated by adipose tissue include lipid metabolism, glucose homeostasis, inflammatory process, angiogenesis, regulation of coagulation) and blood pressure (51). Recent evidence has demonstrated that many factors secreted from adipocytes are pro-inflammatory mediators. Table 2 Main adipokines and their functions Adipokines Function leptin Repression of food intake Stimulation of fatty acid oxidation in liver, pancreas and skeletal muscle Modulation of hepatic gluconeogenesis Modulation of pancreatic β-cell function Suppression of resistin and retinol binding protein 4 expression Stimulation of adiponectin expression) adiponectin Stimulation of fatty acid oxidation in liver and skeletal muscle Suppression of hepatic gluconeogenesis Stimulation of glucose uptake in skeletal muscle Stimulation of insulin secretion Modulation of food intake and energy expenditure resistin Stimulation of TNF-α and IL-6 expression visfatin Stimulation of TNF-α and IL-6 expression apelin apelin signaling participates in cell relaxation (smooth muscle cell) or contraction (cardiomyocyte), migration and proliferation vaspin Suppression of leptin, resistin, and TNF-α expression Of particular relevance is the ability of white adipose tissue (WAT) to increase or reduce leptin secretion under conditions of positive and negative energy balance, respectively. 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