Tài liệu Quantitative aspects of ruminant digestion and metabolism - Phần 13 docx

17 Metabolic Regulation R.G Vernon Hannah Research Institute, Ayr KA6 5HL, UK Introduction Ruminants, like other animals, have to meet the nutritional demands of the many organs and cell types of the body This has to be done against a background of a varying, and not always adequate, supply of nutrients Thus, once absorbed there are a number of potential fates for a given nutrient and a plethora of mechanisms and factors, which influence the probability of a given fate Such mechanisms operate within cells, between different cells and types within a tissue, and between organs Mechanisms may be brought into play to deal with acute or chronic challenges: the former are important for homoeostasis while the latter are critical for the homoeorrhetic adaptations needed for different developmental, physiological, nutritional or pathological states The nature of these mechanisms and the various types of factors involved are considered in subsequent sections It will be obvious to those familiar with the previous edition of this book that the flavour of the current chapter is very different from that written by the late Bernard Crabtree He focused on the important but still rather specialized field of mathematical modelling of metabolic pathways and their regulation; for those interested in this aspect I strongly recommend Bernard’s chapter (Crabtree, 1993) and also articles by Brown (1994), Kacser et al (1995) and Hofmeyr and Cornish-Bowden (2000) Levels of Metabolic Control Within cells Metabolic pathways Within cells the fate of a nutrient is determined not only by the activity of relevant enzymes but, in some cases at least, by: (i) translocases, reflecting the fact that the ß CAB International 2005 Quantitative Aspects of Ruminant Digestion and Metabolism, 2nd edition (eds J Dijkstra, J.M Forbes and J France) 443 444 R.G Vernon cell is highly structured and a metabolic pathway may be split between more than one compartment, necessitating translocation of metabolites between compartments; and (ii) binding proteins because some metabolites may have deleterious effects at high concentration (e.g long-chain fatty acids and their acyl-CoA esters), hence binding proteins are used to protect against this In general terms a nutrient such as glucose, a fatty acid or an amino acid, on entry into a cell, is first activated by a reaction involving ATP or another nucleoside triphosphate Subsequently the ‘activated’ nutrient will be further metabolized, often by a branching metabolic pathway Many studies have focused on identifying and characterizing key rate-limiting enzymes of these pathways However, proponents of ‘metabolic control theory’ have made the point that within a linear pathway the flux through each individual step will be the same, and any step can be involved in determining the rate (Kacser et al., 1995) Thinking has been coloured in part by studies of pathways of tryptophan synthesis and glycolysis in yeast and other microorganisms, which showed that increasing the amount in individual compartments had little effect on overall flux (Oliver, 2002) By contrast, studies with transgenic mice have shown, for example, that increasing the amount of the glucose transporter, Glut 4, in muscle and fat increased glucose uptake and subsequent rate of metabolism (Wallberg-Henriksson and Zierath, 2001) While it is true that if the activity of any component enzyme is reduced sufficiently it can constrain the overall flux through a pathway, some enzymes are clearly more important in this respect than others Such enzymes catalyse essentially irreversible reactions and are usually subject to complex control by covalent modification (e.g phosphorylation–dephosphorylation) and non-covalent control by metabolites and other small molecules Enzyme phosphorylation most commonly involves key serine residues, and can lead to activation (e.g activation of hormone-sensitive lipase by protein kinase A) (Yeaman et al., 1994) or inhibition (e.g phosphorylation of acetylCoA carboxylase (ACC) by AMP-stimulated kinase) (Barber et al., 1997) Control can be complex: protein kinase A and AMP-stimulated kinase phosphorylate different serine residues of hormone-lipase which are separated by a single amino acid; phosphorylation of one serine prevents phosphorylation of the other (Yeaman et al., 1994) There are many examples of activity being modulated by small molecules: in some cases a molecule interacts directly with the catalytic site on the enzyme, in other cases the effector molecule interacts with a distant site causing a conformational change which results in altered activity (allosteric regulation) There can be simple product inhibition (e.g inhibition of hexokinases I and II by glucose-6-phosphate); inhibition by the final product of a pathway (e.g inhibition of ACC by fatty acids); inhibition by a component of another pathway (e.g inhibition of carnitine palmitoyl-CoA transferase-1 by malonyl-CoA and methylmalonyl-CoA, intermediates of fatty acid synthesis and propionate metabolism, respectively) It is not always inhibition as glycogen synthase, for example, is activated by glucose-6-phosphate The complexity of control is illustrated by the fact that phosphofructokinase is inhibited by both citrate and ATP (substrate) and activated by fructose6-phosphate (substrate), ADP (product) and AMP In general, changes in Metabolic Regulation 445 phosphorylation are due to extracellular stimuli, whereas modulation by small molecules is a response to intracellular stimuli Effective activity can also be modulated by translocation from one part of a cell to another For example, activation of hormone-sensitive lipase by catecholamines in adipocytes results not only in increased enzyme activity, but also a movement of the enzyme from the cytosol to the surface of the fat droplet (Londos et al., 1999) Stimulation of glucose transport by insulin into adipocytes and muscle cells involve a translocation of Glut 4-containing vesicles from the interior of the cell to the plasma membrane (Mueckler, 1994) The effective activity of an enzyme is also determined by the concentration of the substrates The importance of this depends on the concentration of substrate relative to the affinity of the enzyme for the substrate Thus for both long-chain and short-chain (volatile) fatty acids, the Km of the activating enzymes is relatively high so flux varies with fatty acid concentration over the normal physiological range (Bell, 1980) Similarly the Km of hepatic hexokinase IV (glucokinase) is very high, so flux varies directly with glucose concentration (Bollen et al., 1998) By contrast, the Km of muscle hexokinase for glucose is very low; hence flux is less sensitive to glucose concentration The above mechanisms all provide for rapid changes in effective activity of an enzyme and hence are of considerable importance for homoeostatic control (see below) In addition there are changes in the amount of enzymes, translocases and binding proteins, providing further, longer-term control Amounts of such proteins are determined both by synthesis and degradation, but in most cases it is the former that is the key determinant Protein synthesis is regulated at the level of gene transcription and, in some cases, translation of the corresponding mRNA Gene expression is regulated by promoters usually located upstream of the 5’ end of the coding region The key lipogenic enzyme ACC illustrates the complexity of control This enzyme occurs as two distinct isoforms, coded by different genes (Travers and Barber, 2001) ACC-a is the major isoform of the liver (in non-ruminants), adipocyte and lactating mammary gland – all tissues with very high rates of lipogenesis ACC-b is found in a wider variety of tissues including heart and skeletal muscle; ACC-b activity is lower than that of ACC-a, and is thought to have an important regulatory, rather than synthetic, role as its product, malonyl-CoA, is a key modulator of fatty acid oxidation (Zammit, 1999) ACC-a gene expression is regulated by at least three promoters (Fig 17.1), with expression by these different promoters showing tissue specificity; PI is the major promoter of adipocytes whereas PIII is important in lactating mammary tissue (Travers and Barber, 2001) Most studies of this type have focused on non-ruminant species, but in the case of ACC-a much of the data comes from work on sheep tissue Expression via the different promoters is under distinct physiological and hormonal control The decrease in ACC-a expression in sheep adipose tissue during lactation, for example, is due mostly to a fall in expression via the PI promoter with only a small decrease in expression via the PII promoter (Travers and Barber, 2001) Regulation of gene expression via hormones and nutrients is mediated by transcription factors, which bind to response elements in the promoter regions of the gene 446 R.G Vernon 10 20 30 EXON EXON 242 bp 96 bp EXON 47 bp 40 50 EXON 250 bp EXON 5A 422 bp 60 70 kbp EXON 137 bp (EXON 3) PI P II P III 5Ј untranslated region Tissue distribution Class transcripts Adipose tissue Class transcripts Liver, mammary gland, all other tissues 1,5 AUG Class transcripts Exons represented in 5Ј untranslated region 1,4,5 AUG Mammary gland 2,4,5 2,5 5A Fig 17.1 Structure of the regulatory region of the ovine acetyl-CoA carboxylase-a gene (adapted from Travers and Barber, 2001) Molecular biological approaches have not only revealed the complexity of promoter systems, they have also shown that many proteins exist in more isoforms than previously thought For example, a novel form of ACC-a was found in sheep mammary gland, which has a missing sequence of eight amino acids prior to a key serine that is thought to be important for control of ACC-a activity by phosphorylation–dephosphorylation (Travers and Barber, 2001) Whether the altered amino acid sequence influences the phosphorylation of this serine is not known, but interestingly expression of this isoform of the enzyme in the mammary gland is increased markedly by lactation (Travers and Barber, 2001) Signal transduction pathways As many hormones and growth factors have receptors in the plasma membrane, signals have to be transmitted to sites within the cell via signalling pathways For some, e.g catecholamine activation of lipolysis in adipocytes and its antagonism by adenosine and prostaglandin E, the signalling pathway appears to be well defined (Fig 17.2) However, for many hormones the pathways are only partly resolved Thus we know that insulin activates a series of branching pathways which mediate effects on metabolism, protein synthesis, mitogenesis, etc (Fig 17.3), but while early steps transmitting metabolic signals appear to be known, downstream effectors are still unresolved (Pessin and Saltiel, 2000; Litherland et al., 2001) Furthermore, novel pathways continue to be identified For example, insulin stimulation of glucose transport in adipocyte and muscle cells is thought to be mediated, in part at least, via the phosphoinositide-3 kinase/protein Metabolic Regulation 447 Gs ATP β-receptor Adenylate cyclase Catecholamines A-kinase (inactive) α2-receptor PGE Adenosine Insulin Hormone sensitive lipase (inactive, cytosol) Gi PGE receptor Cyclic AMP Adenosine receptor Triacylglycerol Insulin receptor A-kinase (active) Cyclic AMP phosphodiesterase Hormone sensitive lipase (active, fat droplet) AMP Fatty acids, glycerol Fig 17.2 Lipolytic signalling cascade of adipocytes PGE, prostaglandin E; Gs , stimulatory GTPbinding protein; Gi , inhibitory GTP-binding protein kinase B pathway (Fig 17.3), but recently a new pathway involving the proteins TC10 and flotillin, which binds to lipid rafts in the plasma membrane, has been implicated as well (Litherland et al., 2001) For some important metabolic hormones, e.g growth hormone, even less is known Frustratingly for this key hormone with its important chronic homoeorrhetic metabolic effects (Bauman and Vernon, 1993; Etherton and Insulin Insulin receptor Insulin receptor substrate 1,2 shc Phosphoinositide-3 kinase MAP kinase Protein kinase B Protein kinase C zeta Metabolic effects Mitogenic effects Fig 17.3 Some of the insulin signal transduction system MAP kinase, mitogen-activated protein kinase; shc, src homology collagen-related protein 448 R.G Vernon Bauman, 1998) most research has focused on systems of questionable physiological significance (a transient insulin-like effect seen in rodent tissue after a period of abstinence from growth hormone, and a ‘commitment to differentiation’ effect observed in a preadipocyte cell line) (Herrington and Carter-Su, 2001) This reflects a tendency to study what is easy rather than what is important! To add to the complexity, we now know that many signal transduction components exist in several isoforms; for example there are at least three isoforms of the b-adrenergic receptor (Carpene et al., 1998), two of the GTPbinding protein Gs , at least three isoforms of Gi (Manning and Woolkalis, 1994) and nine of adenylate cyclase (Simonds, 1999) The proportion of the different isoforms varies with cell type and implies that the functions of the signal systems will show subtle variations depending on the isoforms involved A confusing feature of signalling is that many hormones and related factors appear to use the same intracellular signalling components, raising questions as to how specificity of effect is achieved (Dumont et al., 2002) This could arise from use of different isoforms or activation of components in different parts of the cell It may be that while a number of hormones may activate a similar network of signalling pathways, the individual receptors may interact slightly differently with the various components, thus achieving distinct, specific outcomes (Dumont et al., 2002) As the various signalling pathways are resolved, this problem of specificity should provide an interesting challenge for modellers! Within tissues Tissues are composed of multiple cell types, which communicate with each other via autocrine and paracrine signals that can influence the fate of nutrients within a tissue In addition, different cell types have different types and amounts of transporters needed to move nutrients across the plasma membrane Adipose tissue, for example, comprises about 85% triacylglycerol by weight, but adipocytes, while being very large cells, comprise only about 10% of the total cell number of the tissue in the adipose tissue of adult sheep (Travers et al., 1997) Other cell types include preadipocytes, endothelial cells and macrophages The growing problem of obesity has focused much attention on adipose tissue in recent years and we now know that it secretes a whole battery of factors of various types (Table 17.1) Some substances are secreted by adipocytes (e.g leptin, adipsin), some by other cell types of the tissue (e.g interleukin-6, oestrone) and some by both (e.g adenosine, prostaglandin E) (Vernon and Houseknecht, 2000) Some (e.g leptin, adiponectin, sex steroids) are hormones and are released into the general circulation, influencing events elsewhere in the body (Vernon, 2003) Many, however, are locally active and may influence the fate of nutrients within the tissue For example, there is an apparent relationship between lipolysis in adipocytes and blood flow through the tissue (Vernon and Clegg, 1985), and several locally produced factors modulate both (Vernon and Houseknecht, 2000; Vernon, 2003) Metabolic Regulation Table 17.1 449 Some substances secreted by adipose tissue Metabolic modulators Lipoprotein lipase Acylation-stimulating protein Apoprotein E Fatty acids Prostaglandin E2 Hormones Oestrone Oestradiol Testosterone IGF-1 Complement system Factor B Factor C Factor D (adipsin) Binding proteins Adipocytokines IGF-binding proteins Vasoactive factors Leptin Retinol-binding protein Prostacyclin (prostaglandin I2 ) Tumour necrosis factor a Cholesterol ester transfer protein Monobutryin Interleukin-6 Angiotensinogen/angiotensin II Resistin Other Atrial natriuretic peptide Adiponectin Plasminogen activator inhibitor-I Fatty acids released from adipose tissue are transported in the blood bound to serum albumin Albumin has two high-affinity binding sites for fatty acids and a further five low-affinity binding sites The concentration of albumin in the blood is about 0.5 mM, so mM fatty acid will potentially saturate both highaffinity binding sites; indeed a decreased release of fatty acids has been observed when the concentration exceeded about mM (Vernon and Clegg, 1985) The blood flow through sheep adipose tissue is about 50 ml=min=g tissue before a meal (Barnes et al., 1983) and this will support a rate of fatty acid release of about 50 nmol/min/g tissue The limited amount of data available suggests a rate of lipolysis of about nmol fatty acid released per per g tissue in the fed state, rising to about 15 nmol/min/g tissue on fasting in sheep (Vernon and Clegg, 1985) A substantial proportion of the binding sites of albumin entering the tissue will already be occupied by fatty acids in the fasted state, hence only a limited number will be free to accommodate newly released fatty acids The various estimates come from a number of different studies, but the general point is that blood flow, or to be precise freebinding sites, has the potential to limit lipolysis Catecholamines both stimulate lipolysis and are vasoactive (Vernon and Clegg, 1985) In addition, stimulation of lipolysis in sheep adipose tissue in vivo by catecholamines resulted in a concomitant rise in prostaglandin E2 (Doris et al., 1996) which is vasodilatory and which also acts to attenuate lipolysis (Crandall et al., 1997) (Fig 17.4) The rise in prostaglandin E2 production was associated with a fall in glycerol output, due either to decreased lipolysis, increased blood flow or both Adenosine could have a similar role (Vernon, 1996a) It is also noteworthy that prostaglandin E2 and adenosine are produced by the stromal-vascular cells of adipose tissue as well as adipocytes (Vernon and Houseknecht, 2000) Indeed it has been suggested that prostaglandin production requires both adipocytes and stromal–vascular cells, arachidonic acid released from adipocytes being metabolized to prostaglandin by the stromal–vascular cells (Richelsen, 1992) 450 R.G Vernon Nerve endings Adipocyte Noradrenaline (acute) (+) (−) Lipolysis 20:4 20:4 Angiotensinogen Stromal vascular cells (−) PGE2 PGI2 PGE2 Adenosine Angiotensin II Vasodilation Vasoconstriction Noradrenaline (chronic) Blood flow Fig 17.4 Modulation of lipolysis and blood flow by local factors in adipose tissue PGE2 , prostaglandin E2 ; PGI2 , prostaglandin I2 (prostacyclin); 20:4, arachidonic acid Between organs and tissues Nutrients need to be apportioned appropriately between the various organs and tissues of the body Key factors are blood flow, metabolic capacity of cells and hormonal and nervous signals Blood flow varies considerably from tissue to tissue (Table 17.2) and there is even marked variation within some tissues such as skin (Bell et al., 1983; Gregory and Christopherson, 1986) Differences in blood flow between organs in general reflect the differences in metabolic activity (Table 17.3) (Rolfe and Brown, 1997) A relationship between blood flow and metabolic activity within an organ has been demonstrated for the mammary gland in lactating goats (Linzell, 1974) and portal-drained viscera in sheep and cattle (see Chapter 12) Blood flow, and hence nutrient supply, to a tissue varies with physiological and nutritional state For example, on feeding in sheep, blood flow increased to the rumen epithelium and salivary glands, decreased to abdominal adipose tissue, but did not change to heart, kidney and subcutaneous adipose tissue (Barnes et al., 1983) The onset of lactation in goats results in a fivefold increase compared to pregnancy in blood flow to the mammary gland (Linzell, 1974) Exercise or stress induces marked changes in blood flow with a much greater proportion of cardiac output going to skeletal muscle (Bell et al., 1983) Blood flow is under complex control, involving paracrine and autocrine factors (e.g Fig 17.4), hormones and the nervous system Catecholamines are vasoactive and can both accentuate and attenuate blood flow, depending on which receptors are activated Increased sympathetic activity during exercise, for example, causes increased release of adrenalin from the adrenal medulla, which increases blood flow through skeletal muscle In adipose tissue increased sympathetic activity can lead to initial vasoconstriction due to activation of Metabolic Regulation 451 Table 17.2 Blood flow of various tissues in sheep (data from Barnes et al., 1983; Bell et al., 1983; Gregory and Christopherson, 1986; Weaver et al., 1990) Tissue Blood flow (ml/min/100 g) Brain Heart Kidney Lactating mammary gland Gastrointestinal tract Rumen Abomasum Small intestine Large intestine Liver – hepatic artery Liver – hepatic portal vein Skeletal muscle Adipose tissue Skin 69, 70 62, 95, 110, 154 460, 550, 650 50 24, 112 67, 105, 204 60, 62, 130 48, 64, 80, 105 4, 8, 13 285 2, 9, 10–65 0.3, 4, 8–23 1–13, 3, 2–20 a-adrenergic receptors, followed by vasodilatation due to activation of badrenergic receptors (Vernon and Clegg, 1985) Access by nutrients to most cells requires their passage from the blood to the extracellular space Endothelial cell permeability thus provides another means of manipulating nutrient fate (Vernon and Peaker, 1983) The liver in particular has a very ‘leaky’ endothelium, reflecting the important role of the liver in the uptake and degradation of proteins and even larger structures such Table 17.3 Tissue oxygen use as percentage of whole body oxygen use and blood flow as percentage of cardiac output in sheep (data also from A.W Bell, unpublished observations) Blood flow (per cent cardiac output) Tissue Gastrointestinal tract Liver Brain Heart Kidney Skeletal muscle Bone Skin Adipose tissue a Oxygen use (per cent use by whole body) 20 20 10 10 20 (skin and adipose tissue combined) Hales (1973) Weaver et al (1990) 33 – 2.3 6.9 16.5 12 – 13 28 5.8a 2.6 4.8 19 15.6 3.9 7.5 – 1.5 Via hepatic vein; liver also receives blood from gastrointestinal tract via hepatic portal vein 452 R.G Vernon as lipoprotein remnants By contrast, the brain has a very tight endothelium, creating the so-called ‘blood–brain barrier’ The cellular distribution of translocases and the nature of the isoforms have important roles in determining the partitioning of nutrient between organs/ tissues For example, there are at least six well-characterized glucose transporters involved in transport across the plasma membrane (Mueckler, 1994; Hocquette et al., 1996) and new ones continue to be discovered The Glut-4 transporter is insulin-sensitive and is found in adipocytes and myocytes – cells with a high capacity for glucose metabolism (Mueckler, 1994; Hocquette et al., 1996) Thus, if plasma glucose is increased, for example after a meal, the concomitant rise in serum insulin will cause a preferential uptake of glucose by cell types expressing Glut-4 Even in ruminants, which are thought to be less responsive to insulin than most non-ruminants, insulin-infusion induced a sixfold increase in glucose uptake across the hind limb of mature sheep (Fig 17.5) The corollary, of course, is that when serum insulin and glucose concentrations are low as during fasting, utilization of glucose by other tissues (e.g brain) will be favoured Fatty acids are mostly supplied to tissues either as non-esterified fatty acids (NEFA) bound to albumin or as a part of triacylglycerols, which are transported as part of very low-density lipoproteins (VLDL) secreted by the liver, and chylomicrons secreted by the gastrointestinal cells VLDL and chylomicrons are too large to cross the endothelial cell barrier, so triacylglycerols are hydrolysed by the action of lipoprotein lipase, an enzyme secreted by a variety of cells including adipocytes, myocytes and mammary epithelial cells (Barber et al., 1997) Following secretion it is transported to the luminal surface of the Glucose uptake (mM) 1.2 0.8 0.4 0 10 20 30 40 Blood insulin (µg/I) Fig 17.5 Effect of insulin on glucose arteriovenous difference across the hind limb of lactating (*) and non-lactating (*) sheep (data from Vernon et al., 1990) 454 R.G Vernon depots, and so may act to influence fatty acid partitioning amongst them (Cianflone, 1997) Little is known about the role of acylation-stimulating protein in ruminants (normally ruminant diets have a relatively low fat content), but a recent paper shows that acylation-stimulating protein caused a small increase in fatty acid esterification in bovine adipose tissue in vitro (Jacobi and Miner, 2002) Co-ordinating these various mechanisms are hormones and the nervous system Hormones and neurohormonal transmitters such as catecholamines can alter the amount and activation status of enzymes and translocases in a tissue-specific manner, reflecting tissue-specific differences in the numbers and sometimes isoforms of their receptors Some, such as the insulin receptor, are almost ubiquitous but others are much more restricted Glucagon, for example, targets the liver but in ruminants it has no effect on other major metabolic tissues such as myocytes, adipocytes or mammary epithelial cells (She et al., 1999) With respect to isoforms, the b1 and b2 adrenergic receptors, for example, are widespread, whereas the b3 adrenergic receptor is confined to adipocytes (Carpene et al., 1998) Leptin has at least six receptors; the socalled long-form of the receptor, Ob-Rb, which has full signalling capacity, is localized primarily in the hypothalamus where it has an important role in appetite regulation and energy balance (Ahima and Flier, 2000; Vernon et al., 2001) Other isoforms of the leptin receptor are more widespread in their distribution (Ahima and Flier, 2000) Acutely acting hormones such as insulin and glucagon and also catecholamines achieve their effects primarily by changing the activities of key enzymes and translocases (e.g by changes in phosphorylation status) Such hormones often have mutually antagonistic effects: e.g insulin and glucagon stimulate synthesis and degradation of glycogen in the liver, while insulin and catecholamines stimulate synthesis and degradation of triacylglycerol in adipocytes Chronically acting hormones can modulate function by changing the amount of key metabolic enzymes and translocases, but in addition such hormones may alter the ability of specific cell types to respond to acutely activating hormones Growth hormone, for example, antagonizes the ability of adipocytes to respond to insulin and accentuates response to catecholamines (Bauman and Vernon, 1993; Etherton and Bauman, 1998) The mechanism whereby growth hormones antagonize the response to insulin is still unresolved, but effects of growth hormone on the lipolytic-signalling pathway have been studied in some detail in ruminants and are complex In sheep, but not cattle, growth hormone causes a small increase in response and sensitivity to b-adrenergic agonists, at least partly due to an increase in the number of badrenergic receptors of adipocytes (Vernon, 1996a; Etherton and Bauman, 1998) By contrast, in sheep and cattle growth hormone attenuates response to the antilipolytic effect of adenosine and also prostaglandin E2 in sheep (Doris et al., 1996; Etherton and Bauman, 1998) Furthermore, growth hormone decreases the catecholamine-induced increase in prostaglandin E2 production in sheep adipose tissue in vivo (Doris et al., 1996) Thus in sheep, growth hormone facilitates lipolysis by at least three mechanisms (Fig 17.7) All these effects of growth hormone are chronic, taking a number of hours to become manifest Metabolic Regulation 455 Catecholamines Prostaglandin E Adenosine β-Adrenergic receptor PGE receptor Adenosine receptor GH Gi Gs − + Adenylate cyclase Lipolysis Fig 17.7 Modulation of lipolytic regulatory systems by growth hormone GH, growth hormone; PGE, prostaglandin E; Gs , stimulatory GTP-binding protein; Gi , inhibitory GTP-binding protein Homoeostasis and Homoeorrhesis Homoeostasis The top priority of the various mechanisms described in the preceding sections is to allow the animal to achieve homoeostasis throughout the body At its simplest, all cells need to maintain the ratio of ATP to ADP and AMP at an appropriate level Relative concentrations of these adenosine nucleotides are linked by the adenylate kinase reaction (ATP ỵ AMP ẳ ADP) Several formulae have been proposed to describe the ‘energy state’ of a cell (Vernon and Peaker, 1983); these include the ‘energy charge’: 0:5 Â ([ADP] ỵ 2[ATP])=([AMP] ỵ [ADP] ỵ [ATP]) If all were ATP, then the ‘energy charge’ would be 1.0 In actual fact the ratio is normally about 0.85 and is remarkably constant Another concept is based on the reaction ATP ỵ H2 O ẳ ADP ỵ Pi, and is termed phosphorylation potential’: [ATP]=([ADP] Â [Pi]) That is in essence an index of how far the reaction is from equilibrium; the greater the phosphorylation potential, the more energized the cell Values vary more than the ‘energy charge’ normally ranging from about 200 to 800 when expressed in molar terms Both equations have their limitations, but the key point is that cells need to maintain most of their small, but rapidly turning over, pools of adenosine nucleotides as ATP When nutrient supply is adequate or in excess of basic needs, maintaining homoeostasis involves the appropriate distribution of nutrients to all the cells of 456 R.G Vernon the body Excess nutrients can be used for productive processes or stored in reserves (see below); some may be dissipated as heat When nutrient supply is inadequate, a coordinated series of changes then takes place to minimize energy expenditure and to release nutrients from reserves (Shetty, 1990) Key factors include a fall in serum insulin, leptin, IGF-1, thyroid hormones and a decrease in sympathetic nervous activity; the latter two result in a reduction in basal metabolic rate (Shetty, 1990; Ahima, 2000) The fall in serum insulin results in: (i) a reduction in anabolic processes (e.g protein, glycogen, triacylglycerol synthesis); (ii) increased release of reserves of glucose (from glycogen) and fatty acids (from triacylglycerol) and during starvation; and (iii) increased proteolysis in muscle (Shetty, 1990) Furthermore, the fall in insulin results in partitioning of glucose, for example, away from use by muscle and adipose tissue while allowing continuing use by tissues such as brain Interestingly, the sensitivity of these various processes to insulin varies (Fig 17.8); the fall in serum insulin found on going from the fed to the fasted state (less than 24-h food deprivation) will decrease glucose utilization by adipose tissue and muscle and increase lipolysis in adipose tissue, but lower concentrations such as found during starvation are required to induce muscle proteolysis (Parsons, 1976) Leptin is a recently discovered peptide hormone secreted primarily by adipocytes (Zhang et al., 1994) It acts on the hypothalamus to both inhibit food intake and increase energy expenditure, thus acting as a potential adipostat (Ahima and Flier, 2000; Vernon et al., 2001) It is also thought to have a key role in the adaptations to fasting (Ahima, 2000) Fasting alters the Proportion of maximal response 1.0 Muscle proteolysis Lipolysis Access of glucose to muscle and adipose tissue Glycogen formation in liver 0 0.2 0.5 Starvation Normal fasting 10 Normal fed 20 40 Fed level in portal vein Circulating concentration of insulin (µg/l) Fig 17.8 Sensitivity of different metabolic processes to insulin (adapted from Parsons, 1976) Metabolic Regulation 457 secretion of pituitary hormones, including thyrotropin, gonadotropins, adrenocorticotropins, and leptin treatment at least partly prevents these changes (Ahima, 2000) Leptin secretion is stimulated by insulin (Ahima and Flier, 2000; Vernon et al., 2001) Thus the fall in serum insulin during periods of negative energy balance, via resultant falls in leptin and thyrotropin, will lead to a decrease in basal metabolic rate, further emphasizing the critical, central role of insulin in the regulation of energy metabolism Homoeorrhesis Homoeostasis is concerned with maintaining stability, but this operates against a background of change as animals develop and move from one physiological or nutritional state, or indeed pathological state, to another Such changes in state alter the nutrient needs of different tissues and hence require changes in the way nutrients are partitioned throughout the body This led to the concept of homoeorrhesis, a term first used by Waddington in 1957, and then recoined by Bauman and Currie in 1980; the latter defined homoeorrhesis as ‘the orchestrated or coordinated change in metabolism of the body tissue necessary to support a physiological state’ (Bauman, 2000) The coordinated adaptations to inadequate nutrition and stress noted above are of course examples of homoeorrhesis The more general need is to ensure that nutrients in excess of those needed for survival are directed to appropriate tissues in appropriate amounts for that particular state This is also important from a production point of view Thus in the growing animal, for example, nutrients should be used preferentially for muscle growth rather than accrued as adipose tissue; during pregnancy and lactation, nutrients are directed to the uterus and its contents and to the mammary gland, respectively, rather than adipose tissue It is of course much more complex than this; in the growing animal, all organs and tissues will increase in size, but at different rates depending on the stage of development During lactation, the exceptional demand of the mammary gland requires not only a partitioning of energy away from adipose tissue, but also a host of other changes (e.g Table 17.4) to ensure sufficient supplies of amino acids, glucose, calcium, etc for use by the gland Lactation also supplies some excellent quantitative examples of homoeorrhesis in ruminants For example, the study by Bergman and Hogue (1967) showed that in sheep, lactation increased glucose turnover 2.4-fold (and hence production, by liver and kidney as virtually none comes from the diet) At 2.5 weeks of lactation 64% of glucose utilized was secreted in milk as lactose and 23% was oxidized to CO2 The amount of glucose unaccounted for by CO2 and lactose secretion, which is used for other purposes, including for example synthesis of the glycerol moiety of milk fat triacylglycerols, was only 0.07 g/h/kg body weight0:75 , compared to 0.15 g/h/kg body weight0:75 in non-lactating sheep That is, the additional glucose required by the mammary gland was produced by increased gluconeogenesis in liver and 458 R.G Vernon Table 17.4 Some adaptations to lactation in various tissues Tissue Weight Blood flow Activity Mammary gland Increased Increased Gastrointestinal tract Liver Increased Increased Uptake of glucose, acetate, ketones, amino acids, VLDL-TG fatty acids, Ca2ỵ and Pi, increased Synthesis and secretion of protein, lipid and lactose increased Secretion of Ca2ỵ and Pi increased Absorptive capacity increased Increased Increased Heart Adipose tissue Skeletal muscle Bone Uptake of propionate, lactate, fatty acids, glycerol, amino acids increased Synthesis and output of glucose and ketones increased; output of VLDL-TG unchanged Increased (?) Increased Cardiac output increased Decreased ? Uptake of acetate, glucose and VLDL-TG fatty acid decreased Synthesis of lipid decreased Output of fatty acids and glycerol increased Decreased Unchanged Uptake of glucose and acetate may decrease; uptake of fatty acids increased Output of lactate, amino acids increased Protein synthesis decreased; proteolysis increased Decreased ? Uptake and accretion of Ca2ỵ and Pi decreased; resorption and output of Ca2ỵ and Pi increased VLDL-TG, very low-density lipoproteins triacylglycerol; Pi, phosphate kidney and by a reduction in glucose utilization by processes other than oxidation to CO2 by various non-mammary tissues of the body Fatty acid synthesis provides another well-studied example The estimated rate of fatty acid synthesis of adipose tissue per sheep in non-lactating animals is about half that of the mammary gland in lactating sheep (Table 17.5) However, during lactation the rate falls by over 90% in adipose tissue, hence lipogenic precursors will be preferentially used by the mammary gland There are similar homoeorrhetic adaptations in cattle (Vernon, 1996b) Changes in fatty acid synthesis in the two tissues are paralleled, qualitatively, by reciprocal changes in the amount of mRNA, total enzyme activity (Table 17.5) and activation status of ACC-a in adipose tissue and mammary gland (Travers et al., 1997) The proportionately greater changes in lipogenic flux than in ACC activity are due to changes in the activation state of the latter (Travers et al., 1997) Interestingly there are differential changes in ACC-a expression via the three promoters in the two tissues with lactation In adipose tissue, expression via PI and PII is reduced to 11% and 43%, respectively, of that seen in non-lactating sheep By contrast, in the mammary gland there is a 15-fold increase in expression via the PIII promoter (yielding a different isoform of the enzyme) and only a threefold increase in expression via the ubiquitous PII promoter (Travers and Barber, 2001) A different strategy is employed for Metabolic Regulation 459 Table 17.5 Homoeorrhetic changes in fatty acid synthesis and acetyl-CoA carboxylase in sheep adipose tissue and mammary gland during lactation (data from Bauman et al., 1974; Vernon et al., 1987; Barber et al., 1997) Non-lactating Lactating Adipose tissue Tissue weight (kg) Fatty acid synthesis (mmol acetate incorporated per h) Acetyl-CoA carboxylase (mmol/h) Mammary gland Adipose tissue Mammary gland 14.0 33.0 0.05 0.01 8.4 0.25 1.5 65.0 40.0 0.03 2.0 50.0 glycerol-3-phosphate acyltransferase, a key enzyme of fatty acid esterification For this enzyme total adipose tissue activity falls from 60 to 23 mmol=min per sheep with lactation, whereas mammary activity is increased to 990 mmol=min per sheep (Vernon et al., 1987) In this case the homoeorrhetic change in adipose tissue is not so important Thus various homoeorrhetic strategies are used to ensure the preferential use of nutrients by the mammary gland Homoeorrhetic changes are not achieved just by tissue-specific changes in activities of important metabolic enzymes In addition, there are also tissuespecific changes in response and sensitivity to hormones and other regulatory factors such as insulin, catecholamines and adenosine For example, the ability of insulin to increase glucose uptake by the hind limb was decreased by lactation in sheep (Fig 17.5); this is likely to contribute to the decreased use of glucose for processes other than oxidation to CO2 and lactose found by Bergman and Hogue (1967) While the mechanism of this diminished response of muscle to insulin is not resolved, it is known from studies in non-ruminants that lipid accumulation in muscle cells leads to insulin resistance (Shulman, 2000) and increased lipid has been found in sheep muscle during lactation, possibly as a result of the hypoleptinaemia (Vernon, 2003) Adipocytes also become less responsive to insulin during lactation (Vernon, 1996b) The molecular mechanism is not known; there does not appear to be a change in amount or activity of early steps in the insulin-signalling pathway, including activation of protein kinase B, but there does appear to be a decrease in amount of protein kinase C zeta (Fig 17.3) (Vernon and Finley, 1999) Not only some tissues become less responsive to insulin during lactation but, in cattle at least, the pancreatic islets become less responsive to insulinotropic agents, contributing to the hypoinsulinaemia of early lactation (Lomax et al., 1979) In contrast to the response to insulin, the response of adipocytes to catecholamines is enhanced by lactation in cattle and sheep, in part due to an increased number of b-adrenergic receptors of adipocytes (Vernon, 1996a) Paradoxically, however, there is also an increased response of adipocytes to the antilipolytic effect of adenosine (Vernon, 1996a) The latter is unexpected as sheep and cattle are usually in negative energy balance during early lactation and so are actively mobilizing adipose tissue lipid 460 R.G Vernon The factors responsible for these chronic, homoeorrhetic adaptations to lactation have not been identified but growth hormone and glucocorticoids are probably involved, especially with respect to the increased response of adipocytes to catecholamines (Vernon, 1996a) Glucocorticoids, but not growth hormone, could also be responsible for the increased response of adipocytes to adenosine (Vernon, 1996a) Growth hormone is probably at least partly responsible for the decreased fatty acid synthesis of adipocytes during lactation but it is not clear whether growth hormone is responsible for the diminished response of adipocytes to insulin (Bauman and Vernon, 1993; Etherton and Bauman, 1998) Growth hormone prevents a sustained activation of protein kinase B by insulin, whereas activation of this kinase by insulin does not appear to be impaired by lactation (Vernon and Finley, 1999) The onset of lactation is associated with a three- to fourfold increase in blood flow per g tissue in the mammary gland in goats (Linzell, 1974) and mammary gland weight increases by 30-fold or more in ruminants (comparing peak lactation with non-lactating, non-pregnant size) (Table 17.5) The overall effect is that the proportion of cardiac output going to the gland increases from a negligible amount to 10–20% of total In addition, a highly significant correlation between mammary blood flow and milk yield has been demonstrated (Linzell, 1974) Such changes suggest that altering blood flow, and hence nutrient supply, could be an important homoeorrhetic mechanism This raises the question of whether blood flow determines or responds to metabolic activity One situation where blood flow has a critical partitioning effect is during exercise/response to stress when changes in blood flow, due primarily to release of adrenalin from the adrenal gland, results in a preferential use of nutrients by skeletal muscle (Bell et al., 1983) Competition or coordination More than 50 years ago Hammond (1944) proposed that tissues compete for nutrients and suggested that during lactation the high metabolic activity of the mammary gland allows it to compete very successfully The concept of homoeorrhesis, on the other hand, emphasizes coordination, with a change in physiological state resulting in a series of changes, sometimes reciprocal, in functions in a number of organs to meet the needs of the new state (Bauman, 2000) It is arguable that both are right For example, all cells of the body have access to the blood glucose pool and during the course of the day all cells will remove some molecules of glucose from the pool The cellular fate of a particular molecule of glucose is not predetermined – it could be used by any cell, and in this respect, cells are all in competition with each other, both within a tissue and between tissues What homoeorrhetic mechanisms is alter the probability of a specific molecule being used by a particular cell Thus, for example, from the data of Table 17.5 it can be calculated that in the nonlactating sheep the chance of a molecule of acetate being used for fatty acid synthesis by adipose tissue rather than the mammary gland is about 3000:1 However, at peak lactation this has reversed to about 250:1 in favour of the Metabolic Regulation 461 mammary gland, due to reciprocal changes in the rate of fatty acid synthesis in the two tissues and an increase in size of the mammary gland The greater blood flow to the mammary gland (Table 17.2) should increase even further the chance of a molecule of acetate being used by the mammary gland rather than adipose tissue Thus, it is not really a case of ‘competition’ or ‘coordination’, but that animals, by their homoeorrhetic adaptations, manipulate the probability of a nutrient molecule being used by a particular cell to meet the needs of the current physiological state Supply and Demand Studies with microorganisms have shown that demand for product is a key determinant of metabolic flux (Hofmeyr and Cornish-Bowden, 2000; Oliver, 2002) For example, increasing the expression of specific glycolytic enzyme genes had little impact on the rate of glycolysis in microorganisms, but when a constitutively active ATPase was overexpressed, the glycolytic rate was markedly increased, suggesting that it is demand for ATP which determines glycolytic flux, rather than the ability to synthesize ATP (Oliver, 2002) Mammals have more complex requirements than microorganisms, but again demand for the product is a critical determinant of flux through a metabolic pathway This of course is the reason for feedback inhibition, often ignored when considering how a pathway is regulated The importance of demand is diminished if there is a sink for the product Cells can secrete a product, but this merely moves the problem elsewhere – a substance secreted into the blood is a stage in a pathway and not an end-product, unless it is then excreted Milk production provides a considerable sink; however, this is normally constrained by the demands of the young This is easily shown for species with multiple young, when varying the number of young alters the rate of milk production (Vernon et al., 2002) Again there is an apparent feedback mechanism via the production of an inhibitor of milk secretion and hence production on milk accumulation in the gland (Peaker and Wilde, 1996) Machine milking creates, potentially, an insatiable demand Increasing milking frequency increases milk production but eventually nutrient supply or mammary capacity becomes limiting Thus a study with high-yielding cows, in which milk production was increased to very high levels by four times daily milking plus treatment with bST, showed that when milking was reduced to once daily for one-half of the udder, milk production was increased in the other half of the udder, which continued to be milked four times daily (Sorensen and Knight, 1999) In this case nutrient supply rather than mammary capacity was the constraint on yield A sink can also be created by storage of product Some glucose can be stored as glycogen, especially in liver and skeletal muscle, but the capacity for glycogen storage is quite limited (about 5% of liver weight and less in muscle) The constraint on amount may well be physical as stored glycogen is hydrated and comprises about 75% water; hence 5% glycogen would in fact represent about 20% of the weight of an hepatocyte 462 R.G Vernon By contrast to glycogen, animals appear to have an almost unlimited capacity to store triacylglycerol in adipose tissue – at least this appears to be the case in humans, where in some individuals it can exceed 80% of body weight Triacylglycerol is hydrophobic, hence contains virtually no water when stored All vertebrates have to meet their needs (maintaining homoeostasis, etc.) against a background of varying food availability, hence the requirement to store some nutrients during periods of surplus for use when supply is inadequate (Pond, 1992) The energy requirements of poikilotherms are relatively low compared to homoeotherms, especially when ambient temperatures are low (Sheridan, 1994) Fish store lipid in liver and muscle, which is sufficient to meet their needs (Sheridan, 1994) However, reptiles have mesenteric adipose tissue (Pond, 1992), which is arguably an extension of the liver for fatty acids released pass into the portal blood and hence go through the liver before entering the general circulation Homoeotherms require mechanisms for the active generation of heat, thus markedly increasing the energy requirements of mammals and birds compared to poikilotherms; a consequence of this is a need for greater stores of energy (Pond, 1992) Thus mammals have multiple adipose tissue depots distributed throughout the body, some in the abdominal cavity, some under the skin and within the musculature (both inter- and intramuscular) (Pond, 1986) This distribution is present in marsupials and has been retained, with occasional modification, in eutherian mammals (Pond, 1986) While adipocytes are large cells, and vary markedly in size as fat is accreted or mobilized, they have a maximum size (about nl in cattle) (Vernon and Houseknecht, 2000) As adipocytes get large, this appears to induce the production of new adipocytes from precursor cells within the tissue (Faust et al., 1978) Thus the capacity for storing fat is considerable, but in reality there are important constraints While a large amount of adipose tissue provides a buffer against starvation, it can render an animal much more susceptible to predation Thus, animals normally adjust the size of their adipose tissue reserves depending on whether starvation or predation is the greater threat to survival (Vernon and Houseknecht, 2000) So, while adipose tissue is potentially a vast sink for excess nutrients, the size of this reserve has to be carefully controlled, at least in the wild Hence antelope on the plains of Africa, where predation is a greater threat than starvation, have very limited reserves of adipose tissue, but, by contrast, prior to an Arctic winter, reindeer accumulate substantial amounts of adipose tissue (Vernon and Houseknecht, 2000) as for them starvation is the greater threat to survival Sheep also show seasonal cycles of adipose tissue accretion and loss if fed ad libitum (Vernon et al., 1986) Thus, while adipose tissue can respond to ‘supply’ by depositing excess nutrient as fat, there are clearly signals which put constraints on this In essence adipose tissue is under a form of autonomic control, secreting peptide hormones (adipocytokines), at a rate varying with the degree of adiposity, which modulate both nutrient supply and adipose tissue metabolism (Vernon, 2003) As mentioned above, leptin acts on hypothalamic neurones to limit appetite and increase energy expenditure, and so is, in theory at least, a feedback inhibitor of adipocyte size (Ahima and Flier, 2000; Vernon, 2003) Other factors such as tumour necrosis factor a and resistin act locally as insulin Metabolic Regulation 463 antagonists and also promote lipolysis (Vernon, 2003) Such factors should limit the rate of lipid accretion in adipocytes, but while these, and probably other mechanisms, are effective in the wild, it is painfully apparent in humans at least that such mechanisms not necessarily protect from the accumulation of excess adipose tissue lipid The liver is also an important tissue from the point of view of supply and demand due to its role as a modulator of blood composition Thus the liver responds to nutrient supply and may take up nutrients in excess of the actual needs, processing and eventually secreting the surplus nutrients For example, in species in which blood glucose increases substantially following a meal, glucose is accumulated in the liver as glycogen for release as glucose in the subsequent postprandial period To facilitate this, the liver has a bidirectional glucose transporter (Glut-2) and a hexokinase (glucokinase) with a Km for glucose of about 20 mM, so flux through this reaction varies with plasma concentration as it changes following a meal (Bollen et al., 1998) Interestingly, hepatic glucose metabolism is modulated by adipose tissue as it secretes adiponectin, a peptide hormone, which modulates hepatic sensitivity to insulin (Vernon, 2003) The liver also takes up fatty acids and again uptake and metabolism vary with plasma concentration (Zammit, 1990; Drackley et al., 2001) The liver either oxidizes these fatty acids or uses them for the synthesis of lipid Some fatty acids will be oxidized to provide ATP for use in the liver, but some oxidation products are released into the circulation as ketones (acetoacetate and b-hydroxybutyrate) to be used by other tissues (Zammit, 1990; Drackley et al., 2001) The liver (e.g for membrane turnover) uses some lipids produced by esterification but most are secreted as lipoproteins, transporting lipids, including triacylglycerols, to other tissues Problems with the functioning of this system can occur during periods of sustained high rates of lipolysis (for example during early lactation) when the supply of fatty acids to the liver exceeds the capacity of the tissue to process and release them (Zammit, 1990; Drackley et al., 2001) Curiously, in ruminants the capacity of the liver to secrete triacylglycerols as very low-density lipoproteins is quite limited; a consequence of this is that when supply of fatty acids is substantial, some triacylglycerols are retained within the hepatocyte (Zammit, 1990; Drackley et al., 2001) If the high rate of fatty acid supply is not sustained these stored triacylglycerols will be eventually secreted, but when high rates of fatty acid supply are sustained, as can happen during early lactation, excess triacylglycerols can accumulate in the liver leading to ‘fatty liver disease’ and subsequently ketosis Conclusions Ruminants are no different from other mammals in having to rise to the challenge of meeting the nutrient demands of tissues against a background of variable supply For much of the time this problem is tempered in ruminants by their eating behaviour, the nature of the diet and the digestive processes, which ensure a more continuous absorption of nutrients than in meal-eating species 464 R.G Vernon Nevertheless, supply does not always equal demand, even in domestic species (e.g during early lactation) Under such circumstances demand is diminished where possible by decreasing basal metabolic rate and anabolic processes and, in addition, animals draw on reserves of nutrients When nutrient supply exceeds needs some of the excess is stored in reserves but some is also dissipated as heat For some nutrients (e.g calcium, protein) there are no specialized reserves but animals can avail themselves to some extent of such nutrients contained in structural tissues (bone, muscle) For energy there is a specialized reserve, adipose tissue, the size of which has to be carefully regulated depending on the needs of the animal, including whether starvation or predation is a greater threat Adjusting supply and demand does not just concern quantities of nutrient, it is also concerned with quality, the liver having a key role in this respect Whereas most tissues are concerned with meeting their own demands, the liver is equally concerned with supply; this can in certain circumstances lead to a failure of control when supply exceeds the ability of the tissue to handle the nutrient influx The objectives of metabolic control are thus meeting these multiple challenges A plethora of mechanisms and signals operate within the cell, within the tissue and within the body which coordinate the fate of nutrients, both in the short-term, meeting homoeostatic demands, and in the longer term via homoeorrhetic mechanisms While the basic metabolic pathways and their key regulatory steps are known, more is still to be learnt about the intracellular organization of such pathways By contrast to the metabolic pathways, new regulatory factors continue to be discovered and the signal transduction pathways, and their intracellular organization, which transmit signals to the metabolic pathways, are still largely unresolved even for very important hormones like insulin and growth hormone Furthermore, it appears that hormones not activate simple linear signalling pathways, rather many hormones and regulatory factors activate signalling networks, often with common components, raising questions of how specificity of signalling is achieved As these various networks and control processes are unravelled, describing them in quantitative terms will present a major challenge for future modellers! 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J.M and France, J (eds) Quantitative Aspects of Ruminant Digestion and Metabolism CAB International, Wallingford, UK, pp 222–248 Crandall, D.L., Hausman, G.J and Kral, J.G (1997) A review of the... complexity of control This enzyme occurs as two distinct isoforms, coded by different genes (Travers and Barber, 2001) ACC-a is the major isoform of the liver (in non-ruminants), adipocyte and lactating... mammary gland – all tissues with very high rates of lipogenesis ACC-b is found in a wider variety of tissues including heart and skeletal muscle; ACC-b activity is lower than that of ACC-a, and is