Aat

[Alanine ]

I Lactate I

Note that there are three places in which the pathways of glycolysis and gluconeogenesis are separate: glucokinase/glucose-6-phosphatase, phosphofructokinase/fructose-1,6 bisphosphatase; pyruvate kinase/(pyruvate carboxylase and phosphoenolpyruvate carboxykinase). Pyruvate carboxylase is present within the mitochondrion, but PEPCK in humans is equally distributed between cytosol and mitochondrion. [Since oxaloacetate cannot cross the mi-tochondiral membrane, it is converted to malate for transport to the cytosol; this is not shown.]

The three major substrates for gluconeogenesis are shown (dark boxes) together with the places at which they enter the pathway. They arise from tissues outside the liver.

Regulation

The pathways of glycolysis and gluconeogenesis catalyse opposite functions, and conditions that favour one tend to suppress the other. In general, glycolysis is favoured under 'fed' conditions, gluconeogenesis under 'starved' conditions. There are three major modes of regulation: allosteric, covalent (phosphorylation) and gene expression. The last of these is relatively long term (hours rather than minutes) and affects GK, G-6-Pase and PEPCK activities as shown. Allosteric and covalent regulation by hormones works mainly through adenylyl cyclase/cAMP (see Box 2.4 for more details). The principal features are as follows:

Glucokinase is inhibited allosterically by a specific regulatory protein that is activated by fructose 6-phosphate (i.e. tending to limit glycolysis when flux is high), but fructose 1-phosphate, a product of fructose metabolism, relieves the inhibition of glucokinase.

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphos-phatase is regulated by phosphorylation by PKA (glucagon high) and de-phosphorylation by protein phosphatase 2A (insulin high). In the phospho-rylated form it catalyses breakdown of F 2,6-P2; in the dephosphorylated form it catalyses formation of F 2,6-P2.

F 2,6-P2 is a potent activator of PFK and inhibitor of FBPase; thus, when insulin is elevated glycolysis is favoured; when glucagon is high relative to insulin, the F 2,6-P2 concentration falls and gluconeogenesis is favoured. The enzyme PK (glycolysis pathway) is inhibited by phosphorylation by PKA (glucagon high); insulin inhibits this phosphorylation (i.e. maintains the enzyme active).

In addition, PK is activated allosterically by F 1,6-P2 (thus, its activity is maintained when glycolytic flux is high).

Other compounds within the cell also play a role in allosteric regulation. Of these, the most important are probably: (i) the adenine nucleotides (ATP,

ADP, AMP) regulate several steps such that when the cellular energy state is low (low ATP/ADP ratio) glycolysis is favoured; (ii) citrate inhibits PFK. The importance of these additional controls may lie in the fact that when cellular energy is plentiful, e.g. because the cell is oxidising fatty acids (and thus citrate concentrations are also high), gluconeogenesis will be favoured. There is much experimental evidence that an increased rate of fatty acid oxidation in the liver increases the rate of gluconeogenesis.

Glucagon is an example of a hormone counteracting insulin (a counter-regulatory hormone). In certain 'stress' conditions (see Section 7.3.1) adrenaline and noradrenaline may play the equivalent role, raising cyclic AMP concentrations through P-adrenergic receptors.

Based in part on Hue & Rider (1987), Pilkis & Granner (1992) and Nordlie et al. (1999).

blood into the liver cell. The net result is again that, in conditions where glucagon predominates over insulin, the liver will produce glucose 6-phosphate that is directed, by the mechanisms discussed earlier, into export as glucose into the circulation. It will be apparent that the processes of glycogenolysis and gluconeogenesis tend to be active at the same time in normal daily life. This is not so in more prolonged starvation, a condition in which gluconeogenesis becomes particularly important but there is little glycogen in the liver to mobilise; this will be discussed fully in Chapter 8.

Hepatic gluconeogenesis can also be stimulated by an increase in the supply of substrate from other tissues. One example is the period after physical exercise when there are elevated concentrations of lactate in the blood, some of which will be reconverted to glucose in the liver. During starvation, an increased concentration of blood glycerol arising from adipose tissue lipolysis (see Section 4.5.3.2) will have the same effect. However, there is one common situation in which hormonal factors will be tending to suppress gluconeogen-esis whilst substrate supply increases it. This is the situation after a meal. It leads to a phenomenon known as the glucose paradox. It was noted some years ago that an isolated liver, perfused with an artificial 'blood' medium, will synthesise glycogen under appropriate conditions. However, the highest rates of glycogen synthesis are observed not when glucose alone is supplied at high concentration in the perfusate, but when it is supplied together with a precursor of gluconeogenesis such as lactate. Under these conditions lactate rather than glucose appears to be the true substrate for glycogen synthesis. (Lactate must first be converted to glucose 6-phosphate by the pathway of gluconeogenesis - see Box 4.2.) Findings in an isolated tissue like the perfused liver must be interpreted with caution for the reason discussed earlier, that in the body there are special relationships between different organs and tissues which are not reproduced in this laboratory situation. However, the result has since been confirmed in humans: hepatic glycogen synthesis after a meal comes about by a combination of the 'direct pathway' (glucose uptake, glucose 6-phosphate formation, glycogen synthesis) and the 'indirect pathway' (uptake of three-carbon gluconeogenic substrates, particularly lactate, formation of glucose 6-phosphate and glycogen synthesis). The origin of the lactate in this situation is not yet completely clear. One suggestion is that the small intestine itself, during the process of glucose absorption, metabolises a proportion of the glucose to lactate, which passes to the liver through the portal vein (see Section 3.3.1). Again, the anatomical relationship of liver to intestine is important. It is also possible that some hepatocytes produce lactate whilst others use it for gluco-neogenesis. Other tissues may produce some lactate from glucose in the plasma: red blood cells, adipose tissue and muscle play some part in this, although it is not yet clear how important any particular tissue is in the postprandial period (the period after a meal). The essential point, however, is still that glycogen is synthesised by the liver after a meal, although the pathways involved are not quite so straightforward as we would have thought fifteen years ago.

The pentose phosphate pathway

Figure 4.2 shows an alternative fate for glucose 6-phosphate. It may be converted to five-carbon sugars (pentoses), particularly ribose 5-phosphate, which is required for synthesis of nucleic acids. This pathway is a relatively minor route for disposal of glucose 6-phosphate but is important because the partial oxidation that it brings about releases reducing power, in the form of NADPH (rather than NADH). NADPH is required for fatty acid synthesis. The first step in this pathway is catalysed by glucose-6-phosphate dehydrogenase, which forms 6-phosphogluconate. 6-Phosphogluconate dehydrogenase is the next step. Both these dehydrogenases produce NADPH. The activity of glucose-6-phosphate dehydrogenase is increased in conditions of carbohydrate excess, and it has been grouped with the 'lipogenic' enzymes because of its role in releasing the reducing power needed for lipogenesis. Activation of glucose-6-phosphate dehy-drogenase by carbohydrate availability involves increased gene expression but also appears to involve increased stability of its mRNA. The pentose phosphate pathway is present in most tissues, but only in the liver and adipose tissue is the activity of glucose-6-phosphate dehydrogenase regulated by carbohydrate availability, stressing the link with fat synthesis.

4.1.2.2 Fat metabolism in the liver

The main pathways of fatty acid metabolism in the liver, and their hormonal regulation, are shown in Fig. 4.3. The liver can both oxidise and synthesise fatty acids. In humans the overall rate of fatty acid synthesis from other molecules (glucose in particular) is usually small in comparison with dietary fatty

Fatty a transpc

Fatty adds

GLU1 Glucose -

Fatty a transpc

Fatty adds

GLU1 Glucose -

Fig. 4.3 Overview of fatty acid metabolism in the liver. Fatty acids cross the hepatocyte membrane mainly by a carrier-mediated process (see Table 2.4). Inside the liver cell they are transported through the cytosol by binding to specific fatty acid binding proteins, and activated by esterification to coenzyme-A (CoASH) by the enzyme acyl-CoAsynthase (ACS). In order to enter the mitochondrion (dotted box) for oxidation in the tricarboxylic acid cycle (TCA cycle), fatty acyl-CoA esters are converted to acyl-carnitine derivatives by the action of carnitine-palmitoyl transferase-1 (CPT-1) (further details on Fig. 4.4). This enzyme is inhibited by malonyl-CoA, an intermediate in the pathway of de novo lipogenesis. Insulin inhibits fatty acid oxidation by (1) increasing the concentration of malonyl-CoA via activation of acetyl-CoA carboxylase (ACC) and (2) stimulating fatty acid esterification to form triacylglycerol. Glucagon increases fatty acid oxidation, possibly by a direct effect on CPT-1. Note that acetyl-CoA formation from glucose is over-simplified: see Fig. 4.5 for further details. PDH, pyruvate dehydrogenase.

Fig. 4.3 Overview of fatty acid metabolism in the liver. Fatty acids cross the hepatocyte membrane mainly by a carrier-mediated process (see Table 2.4). Inside the liver cell they are transported through the cytosol by binding to specific fatty acid binding proteins, and activated by esterification to coenzyme-A (CoASH) by the enzyme acyl-CoAsynthase (ACS). In order to enter the mitochondrion (dotted box) for oxidation in the tricarboxylic acid cycle (TCA cycle), fatty acyl-CoA esters are converted to acyl-carnitine derivatives by the action of carnitine-palmitoyl transferase-1 (CPT-1) (further details on Fig. 4.4). This enzyme is inhibited by malonyl-CoA, an intermediate in the pathway of de novo lipogenesis. Insulin inhibits fatty acid oxidation by (1) increasing the concentration of malonyl-CoA via activation of acetyl-CoA carboxylase (ACC) and (2) stimulating fatty acid esterification to form triacylglycerol. Glucagon increases fatty acid oxidation, possibly by a direct effect on CPT-1. Note that acetyl-CoA formation from glucose is over-simplified: see Fig. 4.5 for further details. PDH, pyruvate dehydrogenase.

acid intake, but this pathway has a special significance in coordinating glucose and fat metabolism as discussed below.

Like other tissues, the liver may take up non-esterified fatty acids from the plasma. These fatty acids have two major fates within the liver: oxidation or triacylglycerol formation.

Fatty acid oxidation

The liver may oxidise fatty acids by the mitochondrial P-oxidation pathway to produce energy for its many metabolic activities. An alternative P-oxida-tion pathway in peroxisomes operates mainly to shorten very-long-chain fatty acids. It uses different enzymes, but the same metabolic steps. It has been estimated to contribute from 5% to 30% of the total rate of hepatic fatty acid oxidation, and will not be considered here except to note that, in rodents although probably not in humans, it is up-regulated by ligands of the nuclear receptor PPARa (see Table 2.7). In humans, PPARa activation increases mitochondrial fatty acid oxidation. Given that the natural ligand for PPARa is thought to be a fatty acid derivative, this may be seen as a way of increasing the oxidation of fatty acids when fatty acid supply is high.

In particular, gluconeogenesis, a pathway that requires energy and reducing equivalents (NADH), appears to be 'fuelled' by oxidation of fatty acids. If fatty acid oxidation is prevented by using a specific inhibitor, then gluconeogen-esis is suppressed: if fatty acid supply to the liver is increased experimentally, gluconeogenesis always increases.

The pathway of fatty acid oxidation diverges from that of glycerolipid synthesis when acyl-CoA enters the mitochondrion for oxidation. This step is closely regulated. The mitochondrial membrane is not permeable to acyl-CoA and the acyl group is transferred to the small molecule carnitine (Fig. 4.4). This transfer is catalysed by the enzyme carnitine-palmitoyl transferase-1 (CPT-1). The activity of this enzyme is controlled by the cellular level of the compound malonyl-CoA (Fig. 4.3), which is a potent inhibitor. The significance of this will become clear soon. This role of malonyl-CoA provides a vital link between carbohydrate and fat metabolism. It was discovered in 1977 by the British-born biochemist, J. Denis McGarry, working at the University of Dallas, Texas with Daniel W. Foster (McGarry et al. 1977).

During the oxidation of fatty acids in the liver, the ketone bodies aceto-acetate and 3-hydroxybutyrate are produced (Fig. 4.5) and exported into the circulation. The regulation of ketogenesis occurs at several steps (see Fig. 4.5) although it is clear that to a major extent ketone body production in the liver is determined by the rate of fatty acid oxidation (i.e. acetyl-CoA generation, determined in turn by the activity of CPT-1).

Mitochondrial membranes Outer Inner

Mitochondrial membranes Outer Inner

Fig. 4.4 Further detail of the transport of fatty acids into mitochondria. 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. ACS, acyl-Co synthase (this is membrane-associated: 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 very low); CPT, carnitine-palmitoyl transferase: CPT-1, so-called 'overt' CPT (outer mitochondrial membrane); CPT-2, inner mitochondrial membrane (not regulated); BP, binding proteins for fatty acids (FA) and acyl-CoA.

Fig. 4.4 Further detail of the transport of fatty acids into mitochondria. 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. ACS, acyl-Co synthase (this is membrane-associated: 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 very low); CPT, carnitine-palmitoyl transferase: CPT-1, so-called 'overt' CPT (outer mitochondrial membrane); CPT-2, inner mitochondrial membrane (not regulated); BP, binding proteins for fatty acids (FA) and acyl-CoA.

Oxaloacetate 2 Acetyl-CoA

Acetyl-CoA | ^^Citrate synthase acetyltransferase iyl-C°A |

Acetoacetyl-CoA Citrate ^ ^

HMG-CoA synthase CO

Acetyl-CoA V CO2

HMG-CoA

HMG-CoA lyase

Acetone ^^— Acetoacetate^^ -.^3-Hydroxybutyrate CO2 NADH NAD+

Fig. 4.5 The pathway of ketone body formation from acetyl-CoA (ketogenesis). This is all located within the mitochondrion. Acetyl-CoA is produced from P-oxidation of fatty acids. It may enter the tricarboxylic acid cycle (TCA cycle) or it may enter the ketogenesis pathway. For the latter, two molecules of acetyl-CoA condense to form acetoacetyl-CoA. A third is added to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) in a reaction catalysed by HMG-CoA synthase. This is split to release acetoacetate (a ketone body) and acetyl-CoA. The other major ketone body, 3-hydroxybutyrate, is formed by reduction of acetoacetate. A minor one, acetone, is formed by non-enzymatic breakdown of acetoacetate. The ketone bodies cannot be re-utilised in the liver and are exported into the bloodstream. The major regulation appears to be the delivery of fatty acids to the mitochondrion for oxidation. Beyond that, the availability of oxaloacetate may limit entry of acetyl-CoA into the TCA cycle. HMG-CoA synthase is also regulated by covalent modification (succinylation) by suc-cinyl-CoA, a TCA cycle intermediate. Succinyl-CoA competes with acetyl-CoA and can be displaced when acetyl-CoA concentration is high. Glucagon lowers succinyl-CoA concentration and so stimulates ketogenesis. See Further Reading for more details.

Lipid synthesis

The alternative fate for fatty acids taken up by the liver is esterification to form triacylglycerol, which is stored within hepatocytes. In addition, lipids - glycero-lipids (phospholipids and triacylglycerol) and cholesterol - may be synthesised from non-lipid precursors such as glucose and amino acids, via acetyl-CoA (Box 4.3). In fact, much like the situation of the 'glucose paradox' described earlier (Section 4.1.2.1), in isolated hepatocytes the three-carbon compounds lactate and pyruvate are better substrates for this pathway than is glucose: it is not known which are the preferred substrates in vivo. These pathways are stimulated by insulin in the short term, and in the longer term by increased expression of key enzymes (see Box 4.3). Therefore, under conditions when glucose is in excess, it is converted to lipids (maybe via three-carbon intermediates), and in addition fatty acids taken up by the liver are used for glycerolipid synthesis rather than oxidation. Malonyl-CoA, the first committed intermediate in fatty acid synthesis, is a key coordinator of glucose and fat oxidation through its ability to inhibit fatty acid entry into the mitochondrion for oxidation.

It should be stressed again, however, that the rate of de novo fatty acid synthesis in humans is usually low (it will be considered again in Section 6.4.1.1), and the importance of this pathway under most circumstances relates to its link with fat oxidation via malonyl-CoA. But closely linked with this is a diversion of fatty acids from oxidation into glycerolipid, especially triacylglycerol, synthesis. In 'fed' conditions, when insulin is elevated, malonyl-CoA levels will be high and fatty acid oxidation will be inhibited. Fatty acids will be diverted into esterification with glycerol 3-phosphate, a process which is stimulated by insulin (although the exact locus of control is not clear). Thus, in the 'fed' state, the liver tends to store fatty acids as triacylglycerol rather than to oxidise them. Hepatic energy requirements under these circumstances will be met mainly by amino acid oxidation.

The hepatic triacylglycerol pool is not a major energy store for the rest of the body (that function is performed by the triacylglycerol stored in adipose tissue) but appears to be a local store for hepatic needs.2 The stored triacyl-glycerol acts as the substrate for hepatic secretion of fat into the bloodstream, in the form of the lipoprotein particles known as very-low-density lipoprotein (VLDL). The bulk of the lipid in VLDL is in the form of triacylglycerol, derived from the hepatic store. The details and the regulation of lipoprotein metabolism will be discussed in detail in Chapter 9.

Longer-term control of hepatic fat metabolism

Most of the description above of the regulation of fat metabolism has related to short-term control. However, many of the enzymes involved are also subject to longer-term regulation of expression by insulin and carbohydrate availability, as well as via the SREBP and PPAR systems. We can therefore imagine the metabolic pattern of the liver as shifting rapidly, on an hour-to-hour basis, as meals are taken, digested and absorbed: but these rapid fluctuations may be overlaid on a longer-term trend to increases or decreases in particular pathways. Some of the genes involved have been mentioned in passing above, and were summarised in Tables 2.4 and 2.6. In general, fatty acid synthesis and diversion of fatty acids away from oxidation is favoured by high insulin (which would usually be associated with overfeeding), but activation of PPARa, perhaps brought about by increased fatty acid availability, tends to up-regulate fatty acid oxidation.

Other roles of the liver in fat metabolism

The liver has other specialised roles in fat metabolism. These include its production of bile salts, covered in Box 3.1, and its role in uptake of circulating cholesterol (Chapter 9).

4.1.2.3 Amino acid metabolism in the liver

Under normal circumstances in adult life, our bodies do not continuously accumulate or lose protein in a net sense. The rate of amino acid oxidation in the body must therefore balance the rate of entry of dietary protein (typically 70-100 g of protein per day on a Western diet). General features of amino acid metabolism in the body will be covered later (Section 6.3). The liver plays a

Box 4.3 Synthesis of fatty acids and cholesterol from glucose

De novo lipogenesis is the term used for synthesis of fatty acids from non-lipid precursors. It is in effect a pathway for disposing of excess carbohydrate, and it is stimulated by conditions of high carbohydrate availability.

Pathways and abbreviations

Acetyl-CoA cannot cross the inner mitochondrial membrane and so is converted to citrate, for which there is a transporter. Mitochondrial citrate is regenerated by the inward transport of pyruvate, as shown. The enzyme ATP : citrate lyase is the starting point for synthesis of both fatty acids and cholesterol and has been investigated by pharmaceutical companies as a potential target for both body weight regulation and cholesterol lowering. Note that the pathway of fatty acid synthesis from acetyl-CoA (in animals) involves just two enzymes. Fatty acid synthase (FAS) is a complex enzyme with seven different functional activities in a single polypeptide chain. Fatty acid synthesis proceeds by sequential addition of two-carbon units (from malonyl-CoA, a three-carbon intermediate: one carbon is then lost from each three-carbon unit added). The combination of fatty acids with glycerol 3-phosphate, derived from glycolysis, to form triacylglycerol and phospholipids (glycerolipids) is also shown.

Cholesterol synthesis from acetyl-CoA is a more complex pathway with many enzymatic steps. 3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) is formed by reactions identical to those shown in Fig. 4.5 for ketone body synthesis, except that there are different isoforms of the enzymes expressed in the cytoplasm.

Glycerolipids Insulin -M

Fatty acids \FAS

Glycerol 3-phosphate Pyruvate

Glucose Insulin +) Glycolysis

Malate

Malonyl-CoA Insulin + | ACC'

ilin

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