1.3.3.2 Fat deposition and mobilisation
Most dietary fat is in the form of triacylglycerol (Table 4.1). Within the small intestine, dietary triacylglycerol molecules are hydrolysed by intestinal lipases and the products are absorbed into the cells lining the intestines (mucosal or epithelial cells, collectively known as enterocytes). The products of lipolysis are recombined with the enterocytes to form new triacylglycerol. These triacylglycerols, composed of dietary fat, are liberated into the circulation as lipoprotein particles: in fact, the largest and most fat-enriched of the lipoprotein particles, known as chylomicrons (more detail in Chapters 4 and 10). At target tissues, the triacylglycerol in the lipoprotein particles is hydrolysed by a lipase bound to the endothelial cells lining the capillaries, known as lipoprotein lipase. The resulting fatty acids are taken up by cells, and have two potential fates: (i) re-esterification with glycerol 3-phosphate to make new triacylglycerol (and other lipids) – the pathway of fat deposition; or (ii) oxidation. The former is the major route by which dietary fat is laid down for storage in adipose tissue (Figure 1.17; Section 5.2.2.1).
When the stored fat is required as a source of energy, for instance during physical activity when muscles will oxidise fatty acids, or during periods between meals, then the stored triacylglycerol is hydrolysed by a series of intracellular lipases to liberate fatty acids and glycerol, which can be released into the plasma. This is the process known as fat mobilisation. As noted earlier, these non-esterified (‘free’) fatty acids are transported bound to albumin. Glycerol, which is freely soluble, will travel mainly to the liver where it is a substrate for gluconeogenesis as described above (Section 1.3.2.1.5). On average, in a mature adult who is weight-stable, the amount of fat stored in a typical day will equal the amount mobilised. Most tissues can utilise NEFAs, but importantly fatty acids cannot cross the blood-brain barrier and therefore cannot be used as an energy source by the central nervous system. Also, their oxidation requires mitochondria, meaning that red blood cells (erythrocytes), which lack mitochondria, are unable to use them. However, fatty acids are a major fuel source for muscle and kidney, and for the heart and liver under certain conditions.
1.3.3.3 Fatty acid oxidation
Following uptake into cells, fatty acids are rapidly ‘activated’ by esterification to CoA, forming fatty acyl-CoA; this esterification (known as thio- esterification because of the –SH thio group of the CoA molecule) also removes the amphipathic, detergent-like character of the fatty acid, making it less toxic in the membrane-rich cytosol. This reaction requires ATP and releases inorganic pyrophosphate, PPi. PPi is rapidly broken down to Pi, meaning that this step is essentially irreversible. It therefore achieves the same end as glucose phosphorylation to glucose 6-phosphate on entering a cell: it both traps the fatty acid within the cell, and creates a concentration gradient to draw more fatty acids into the cell. The enzymes concerned are known as acyl-CoA synthases (ACSs): again there is a family of these, suited for fatty acids of different carbon chain lengths. The action of the ACSs may be intimately linked to the process of fatty acid transport into the cell, discussed further in Chapter 2.
The fatty acyl moiety may then undergo β-oxidation to yield acetyl-CoA (together with NADH and FADH2) for further oxidation and ATP formation (Figure 1.16 and Box 1.7). This process occurs in mitochondria (hence cells lacking this organelle, such as red blood cells, are unable to derive energy from fatty acids because they cannot oxidise substrates). However, in order to get inside the mitochondria, the fatty acyl-CoA must cross the IMM on the carnitine shuttle (Box 1.7), and it is the activity of the carnitine shuttle that regulates the rate of supply of fatty acids to the mitochondria, and hence of the rate of β-oxidation.
Box 1.7 Fatty acid oxidation
Once a fatty acid enters the cell it is rapidly joined to Coenzyme A (CoASH) to form fatty acyl-CoA, by the enzyme ACS – it has been suggested that it may be linked with fatty acid transport into the cell so that the intracellular concentration of free fatty acids is kept very low. The fatty acyl-CoA may undergo esterification to triacylglycerol (for example, in adipose tissue) or it may be oxidised for energy release, by β-oxidation in the mitochondrion. However, long chain fatty acyl-CoA cannot cross the highly selective inner mitochondrial membrane (IMM), therefore the fatty acid is transported across on the carnitine shuttle. Carnitine is a highly charged molecule ((CH3)3N+CH2CH(OH)CH2COO−) and there is a specific translocase for it to move (with and without esterified acyl group) across the mitochondrial membranes. The carnitine shuttle is initiated by carnitine palmitoyl transferase-1 (CPT-1) on the outer mitochondrial membrane (OMM), which transfers the fatty acyl group from CoA to carnitine. This compound can cross the IMM in association with a translocase before being reconverted to fatty acyl-CoA by carnitine palmitoyl transferase-2 (CPT-2). CPT-1 is strongly inhibited by malonyl-CoA, the first intermediate of the ‘opposite’ pathway – lipogenesis – hence a reciprocal regulatory mechanism prevents fatty acid degradation (oxidation) and synthesis from occurring simultaneously, which would represent an inefficient futile cycle.
The fatty acyl-CoA that results in the mitochondrial matrix now undergoes β-oxidation. β-oxidation is so called because the β-carbon (second methyl carbon) of the FA chain is attacked, in a reaction sequence involving oxidation, hydration and thiolysis, releasing the 2-carbon acetyl group, again attached to CoA, all within mitochondria. The process is cyclically repeated until the entire FA chain has been broken down to acetyl-CoA 2 carbon units. β-oxidation occurs by a multienzyme trifunctional protein complex which catalyses an oxidative cycle generating acetyl-CoA, NADH and FADH2. The acetyl-CoA undergoes further oxidation to CO2 in the TCA cycle (also within the mitochondrial matrix), whilst the NADH and FADH2 (that derived both from oxidation of the acetyl-CoA by the TCA cycle and also that derived from β-oxidation itself) are then re-oxidised by the electron transport chain, yielding ATP. Each cycle of β-oxidation produces a theoretical maximum of 17 ATPs (though due to some inherent inefficiencies including some proton ‘leak’ across the IMM, in practice ∼14 ATP) – hence palmitate (16 carbons → 8 acetyl-CoA) yields a theoretical maximum of 106 ATP. Odd number carbon fatty acids (which are relatively rare) produce the 3-carbon propionyl-CoA in their final β-oxidation cycle; this can go on to produce succinyl-CoA, an intermediate of the TCA cycle, and hence an anaplerotic substrate – an example of lipids potentially producing carbohydrates, though limited by the relative rarity of these fatty acids.
Although most β-oxidation occurs in mitochondria, some fatty acid oxidation also takes place in organelles called peroxisomes. Peroxisomes seem to be particularly responsible for oxidation of fatty acids of relatively unusual (or at least, relatively rare) structure, especially very long chain fatty acids (22 or more carbons) and branched chain fatty acids, such as phytanic acid. Peroxisomal β-oxidation of very long chain fatty acids produces medium chain fatty acids which can then be further oxidised in mitochondria (at least in humans), but also produces hydrogen peroxide (H2O2), a reactive oxygen species which must be reduced. Very long-