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

pose tissue, for example, comprises about 85% triacylglycerol by weight, butadipocytes, while being very large cells, comprise only about 10% of the totalcell number of the tissue in the

Levels of Metabolic Control

Within cells

Metabolic pathways

Within cells the fate of a nutrient is determined not only by the activity of relevantenzymes 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

cell is highly structured and a metabolic pathway may be split between more thanone compartment, necessitating translocation of metabolites between compart-ments; and (ii) binding proteins because some metabolites may have deleteriouseffects at high concentration (e.g long-chain fatty acids and their acyl-CoAesters), 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 anothernucleoside triphosphate Subsequently the ‘activated’ nutrient will be furthermetabolized, often by a branching metabolic pathway Many studies havefocused on identifying and characterizing key rate-limiting enzymes of thesepathways However, proponents of ‘metabolic control theory’ have made thepoint that within a linear pathway the flux through each individual step will bethe 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 trypto-phan synthesis and glycolysis in yeast and other microorganisms, whichshowed that increasing the amount in individual compartments had little effect

on overall flux (Oliver, 2002) By contrast, studies with transgenic mice haveshown, for example, that increasing the amount of the glucose transporter,Glut 4, in muscle and fat increased glucose uptake and subsequent rate ofmetabolism (Wallberg-Henriksson and Zierath, 2001) While it is true that ifthe activity of any component enzyme is reduced sufficiently it can constrainthe overall flux through a pathway, some enzymes are clearly more important

in this respect than others Such enzymes catalyse essentially irreversible tions and are usually subject to complex control by covalent modification (e.g.phosphorylation–dephosphorylation) and non-covalent control by metabolitesand other small molecules

reac-Enzyme phosphorylation most commonly involves key serine residues, andcan lead to activation (e.g activation of hormone-sensitive lipase by proteinkinase A) (Yeaman et al., 1994) or inhibition (e.g phosphorylation of acetyl-CoA carboxylase (ACC) by AMP-stimulated kinase) (Barber et al., 1997).Control can be complex: protein kinase A and AMP-stimulated kinase phos-phorylate different serine residues of hormone-lipase which are separated by asingle amino acid; phosphorylation of one serine prevents phosphorylation ofthe other (Yeaman et al., 1994) There are many examples of activity beingmodulated by small molecules: in some cases a molecule interacts directly withthe catalytic site on the enzyme, in other cases the effector molecule interactswith a distant site causing a conformational change which results in alteredactivity (allosteric regulation) There can be simple product inhibition (e.g.inhibition of hexokinases I and II by glucose-6-phosphate); inhibition by thefinal product of a pathway (e.g inhibition of ACC by fatty acids); inhibition by acomponent of another pathway (e.g inhibition of carnitine palmitoyl-CoAtransferase-1 by malonyl-CoA and methylmalonyl-CoA, intermediates of fattyacid synthesis and propionate metabolism, respectively) It is not always inhib-ition 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 6-phosphate (substrate), ADP (product) and AMP In general, changes in

fructose-phosphorylation are due to extracellular stimuli, whereas modulation by smallmolecules is a response to intracellular stimuli.

Effective activity can also be modulated by translocation from one part of acell to another For example, activation of hormone-sensitive lipase by cat-echolamines 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 adipo-cytes and muscle cells involve a translocation of Glut 4-containing vesicles fromthe 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 ofsubstrate relative to the affinity of the enzyme for the substrate Thus for bothlong-chain and short-chain (volatile) fatty acids, the Km of the activating en-zymes is relatively high so flux varies with fatty acid concentration over thenormal physiological range (Bell, 1980) Similarly the Km of hepatic hexoki-nase IV (glucokinase) is very high, so flux varies directly with glucose concen-tration (Bollen et al., 1998) By contrast, the Km of muscle hexokinase forglucose 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, translo-cases and binding proteins, providing further, longer-term control Amounts ofsuch proteins are determined both by synthesis and degradation, but in mostcases it is the former that is the key determinant

Protein synthesis is regulated at the level of gene transcription and, in somecases, translation of the corresponding mRNA Gene expression is regulated bypromoters usually located upstream of the 5’ end of the coding region The keylipogenic enzyme ACC illustrates the complexity of control This enzymeoccurs as two distinct isoforms, coded by different genes (Travers and Barber,2001) ACC-a is the major isoform of the liver (in non-ruminants), adipocyteand 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 import-ant regulatory, rather than synthetic, role as its product, malonyl-CoA, is a key

regulated by at least three promoters (Fig 17.1), with expression by thesedifferent promoters showing tissue specificity; PI is the major promoter ofadipocytes whereas PIII is important in lactating mammary tissue (Traversand Barber, 2001) Most studies of this type have focused on non-ruminantspecies, but in the case of ACC-a much of the data comes from work on sheeptissue Expression via the different promoters is under distinct physiological and

during lactation, for example, is due mostly to a fall in expression via the PIpromoter with only a small decrease in expression via the PII promoter (Traversand Barber, 2001) Regulation of gene expression via hormones and nutrients

is mediated by transcription factors, which bind to response elements in thepromoter regions of the gene

Molecular biological approaches have not only revealed the complexity ofpromoter systems, they have also shown that many proteins exist in more iso-forms than previously thought For example, a novel form of ACC-a was found insheep 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 byphosphorylation–dephosphorylation (Travers and Barber, 2001) Whether thealtered amino acid sequence influences the phosphorylation of this serine is notknown, but interestingly expression of this isoform of the enzyme in the mam-mary gland is increased markedly by lactation (Travers and Barber, 2001).Signal transduction pathways

As many hormones and growth factors have receptors in the plasma brane, signals have to be transmitted to sites within the cell via signallingpathways For some, e.g catecholamine activation of lipolysis in adipocytesand its antagonism by adenosine and prostaglandin E, the signalling pathwayappears to be well defined (Fig 17.2)

mem-However, for many hormones the pathways are only partly resolved Thus

we know that insulin activates a series of branching pathways which mediateeffects on metabolism, protein synthesis, mitogenesis, etc (Fig 17.3), butwhile early steps transmitting metabolic signals appear to be known, down-stream 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

1,4,5 1,5

2,4,5 2,5

EXON 4

47 bp EXON 5

kinase B pathway (Fig 17.3), but recently a new pathway involving the proteinsTC10 and flotillin, which binds to lipid rafts in the plasma membrane, has beenimplicated as well (Litherland et al., 2001).

For some important metabolic hormones, e.g growth hormone, evenless is known Frustratingly for this key hormone with its important chronichomoeorrhetic metabolic effects (Bauman and Vernon, 1993; Etherton and

Adenylate cyclase

Cyclic AMP ATP

AMP

A-kinase (active)

Hormone sensitive lipase (active, fat droplet)

Fatty acids, glycerol

α 2 -receptor

β-receptor

A-kinase (inactive)

Hormone sensitive lipase (inactive, cytosol)

Fig 17.3 Some of the insulin signal transduction system MAP kinase, mitogen-activatedprotein kinase; shc, src homology collagen-related protein

Bauman, 1998) most research has focused on systems of questionablephysiological significance (a transient insulin-like effect seen in rodent tissueafter a period of abstinence from growth hormone, and a ‘commitment todifferentiation’ effect observed in a preadipocyte cell line) (Herrington andCarter-Su, 2001) This reflects a tendency to study what is easy rather thanwhat is important!

To add to the complexity, we now know that many signal transductioncomponents exist in several isoforms; for example there are at least three iso-

GTP-binding protein Gs, at least three isoforms of Gi(Manning and Woolkalis, 1994)and nine of adenylate cyclase (Simonds, 1999) The proportion of the differentisoforms varies with cell type and implies that the functions of the signal systemswill show subtle variations depending on the isoforms involved

A confusing feature of signalling is that many hormones and related factorsappear to use the same intracellular signalling components, raising questions as

to how specificity of effect is achieved (Dumont et al., 2002) This could arisefrom use of different isoforms or activation of components in different parts ofthe cell It may be that while a number of hormones may activate a similarnetwork of signalling pathways, the individual receptors may interactslightly differently with the various components, thus achieving distinct, specificoutcomes (Dumont et al., 2002) As the various signalling pathways areresolved, this problem of specificity should provide an interesting challengefor modellers!

Within tissues

Tissues are composed of multiple cell types, which communicate with eachother via autocrine and paracrine signals that can influence the fate of nutrientswithin a tissue In addition, different cell types have different types and amounts

of transporters needed to move nutrients across the plasma membrane pose tissue, for example, comprises about 85% triacylglycerol by weight, butadipocytes, while being very large cells, comprise only about 10% of the totalcell number of the tissue in the adipose tissue of adult sheep (Travers et al.,1997) Other cell types include preadipocytes, endothelial cells and macro-phages The growing problem of obesity has focused much attention onadipose tissue in recent years and we now know that it secretes a whole battery

Adi-of factors Adi-of various types (Table 17.1) Some substances are secreted byadipocytes (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 eventselsewhere in the body (Vernon, 2003) Many, however, are locally active andmay influence the fate of nutrients within the tissue For example, there is anapparent relationship between lipolysis in adipocytes and blood flow throughthe tissue (Vernon and Clegg, 1985), and several locally produced factorsmodulate both (Vernon and Houseknecht, 2000; Vernon, 2003)

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 theblood is about 0.5 mM, so 1 mM fatty acid will potentially saturate both high-affinity binding sites; indeed a decreased release of fatty acids has been ob-served when the concentration exceeded about 1 mM (Vernon and Clegg,

tissue before a meal (Barnes et al., 1983) and this will support a rate of fattyacid release of about 50 nmol/min/g tissue The limited amount of dataavailable suggests a rate of lipolysis of about 5 nmol fatty acid released permin per g tissue in the fed state, rising to about 15 nmol/min/g tissue onfasting in sheep (Vernon and Clegg, 1985) A substantial proportion of thebinding sites of albumin entering the tissue will already be occupied by fattyacids in the fasted state, hence only a limited number will be free to accommo-date newly released fatty acids The various estimates come from a number ofdifferent studies, but the general point is that blood flow, or to be precise free-binding sites, has the potential to limit lipolysis

Catecholamines both stimulate lipolysis and are vasoactive (Vernon andClegg, 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 E2production wasassociated with a fall in glycerol output, due either to decreased lipolysis,increased blood flow or both Adenosine could have a similar role (Vernon,

pro-duced by the stromal-vascular cells of adipose tissue as well as adipocytes(Vernon and Houseknecht, 2000) Indeed it has been suggested that prosta-glandin production requires both adipocytes and stromal–vascular cells, arachi-donic acid released from adipocytes being metabolized to prostaglandin by thestromal–vascular cells (Richelsen, 1992)

Table 17.1 Some substances secreted by adipose tissue

Adipocytokines IGF-binding proteins

Prostacyclin (prostaglandin I2) Tumour necrosis factora Cholesterol ester transfer protein

Atrial natriuretic peptide Adiponectin Plasminogen activator inhibitor-I

Between organs and tissues

Nutrients need to be apportioned appropriately between the various organsand tissues of the body Key factors are blood flow, metabolic capacity of cellsand 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 andBrown, 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 andnutritional state For example, on feeding in sheep, blood flow increased to therumen 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 increasecompared to pregnancy in blood flow to the mammary gland (Linzell, 1974).Exercise or stress induces marked changes in blood flow with a much greaterproportion of cardiac output going to skeletal muscle (Bell et al., 1983).Blood flow is under complex control, involving paracrine and autocrinefactors (e.g Fig 17.4), hormones and the nervous system Catecholamines arevasoactive and can both accentuate and attenuate blood flow, depending onwhich 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 increasedsympathetic activity can lead to initial vasoconstriction due to activation of

Nerve endings

Noradrenaline

(acute)

Noradrenaline (chronic)

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

a-adrenergic receptors, followed by vasodilatation due to activation of adrenergic receptors (Vernon and Clegg, 1985).

b-Access by nutrients to most cells requires their passage from the blood tothe extracellular space Endothelial cell permeability thus provides anothermeans of manipulating nutrient fate (Vernon and Peaker, 1983) The liver inparticular has a very ‘leaky’ endothelium, reflecting the important role of theliver in the uptake and degradation of proteins and even larger structures such

Table 17.2 Blood flow of various tissues in sheep (data from Barnes

et al., 1983; Bell et al., 1983; Gregory and Christopherson, 1986;

Blood flow(per cent cardiac output)Hales

(1973)

Weaveret al.(1990)

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 haveimportant roles in determining the partitioning of nutrient between organs/tissues For example, there are at least six well-characterized glucose trans-porters involved in transport across the plasma membrane (Mueckler, 1994;Hocquette et al., 1996) and new ones continue to be discovered The Glut-4transporter is insulin-sensitive and is found in adipocytes and myocytes – cellswith a high capacity for glucose metabolism (Mueckler, 1994; Hocquette et al.,1996) Thus, if plasma glucose is increased, for example after a meal, theconcomitant rise in serum insulin will cause a preferential uptake of glucose bycell types expressing Glut-4 Even in ruminants, which are thought to be lessresponsive to insulin than most non-ruminants, insulin-infusion induced a six-fold 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 concentrationsare low as during fasting, utilization of glucose by other tissues (e.g brain) will

endothelium within the tissue where it can then hydrolyse lipoprotein glycerols Fatty acids thus released are mostly taken up by cells of the tissue,although some escape into the general circulation The proportion escapingimmediate uptake probably varies from tissue to tissue and may be influenced

triacyl-by blood flow Studies with lactating mammary gland, for example, which has ahigh rate of blood flow, suggest about 30% of fatty acids released escape fromthe tissue (Mendelson and Scow, 1972) Uptake may be more efficient inadipose tissue and skeletal muscle, which have much lower rate of blood flowthan the lactating mammary gland (Table 17.2) The amount of effective (i.e.located on the endothelium) lipoprotein lipase of a tissue will thus be a majordeterminant of fatty acid availability for uptake by the tissue Lipoprotein lipase

is under tissue-specific control; fasting, for example, decreases activity in pose tissue but increases activity in muscle, while lactation increases activity inmammary tissue while decreasing it in adipose tissue (Vernon and Clegg, 1985;Barber et al., 1997) By contrast, NEFA are available to all tissues and their use

adi-is not under such tadi-issue-specific regulation

Adipocytes have a curious mechanism, which facilitates uptake of fatty acidsspecifically from chylomicron triacylglycerols (Fig 17.6) Adipocytes secretesome proteins of the alternative pathway for complement production whichbind to the surface of chylomicrons where factor D (adipsin) catalyses a proteo-lytic cleavage of factor C3to C3a Factor C3a then loses its N-terminal arginine toproduce acylation-stimulating protein, which enhances fatty acid uptake andesterification by adipocytes and stimulates glucose uptake (Cianflone, 1997).Production of acylation-stimulating protein varies amongst adipose tissue

+

+ +

Chylomicron

Triacylglycerol

Fig 17.6 Production and role of acylation-stimulating protein in adipose tissue B, C3, C3a,complement factors B, C , Ca, respectively

depots, and so may act to influence fatty acid partitioning amongst them flone, 1997) Little is known about the role of acylation-stimulating protein inruminants (normally ruminant diets have a relatively low fat content), but a recentpaper shows that acylation-stimulating protein caused a small increase in fattyacid esterification in bovine adipose tissue in vitro (Jacobi and Miner, 2002).Co-ordinating these various mechanisms are hormones and the nervoussystem Hormones and neurohormonal transmitters such as catecholaminescan alter the amount and activation status of enzymes and translocases in atissue-specific manner, reflecting tissue-specific differences in the numbers andsometimes isoforms of their receptors Some, such as the insulin receptor, arealmost ubiquitous but others are much more restricted Glucagon, for example,targets the liver but in ruminants it has no effect on other major metabolictissues such as myocytes, adipocytes or mammary epithelial cells (She et al.,

adipocytes (Carpene et al., 1998) Leptin has at least six receptors; the called long-form of the receptor, Ob-Rb, which has full signalling capacity, islocalized primarily in the hypothalamus where it has an important role inappetite regulation and energy balance (Ahima and Flier, 2000; Vernon

so-et al., 2001) Other isoforms of the leptin receptor are more widespread intheir distribution (Ahima and Flier, 2000)

Acutely acting hormones such as insulin and glucagon and also amines achieve their effects primarily by changing the activities of key enzymesand translocases (e.g by changes in phosphorylation status) Such hormonesoften have mutually antagonistic effects: e.g insulin and glucagon stimulatesynthesis and degradation of glycogen in the liver, while insulin and catechol-amines stimulate synthesis and degradation of triacylglycerol in adipocytes.Chronically acting hormones can modulate function by changing the amount

catechol-of key metabolic enzymes and translocases, but in addition such hormones mayalter the ability of specific cell types to respond to acutely activating hormones.Growth hormone, for example, antagonizes the ability of adipocytes to re-spond to insulin and accentuates response to catecholamines (Bauman andVernon, 1993; Etherton and Bauman, 1998) The mechanism wherebygrowth hormones antagonize the response to insulin is still unresolved, buteffects of growth hormone on the lipolytic-signalling pathway have been stud-ied in some detail in ruminants and are complex In sheep, but not cattle,growth hormone causes a small increase in response and sensitivity tob-adrenergic agonists, at least partly due to an increase in the number of b-adrenergic 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 E2in sheep (Doris

et al., 1996; Etherton and Bauman, 1998) Furthermore, growth hormonedecreases the catecholamine-induced increase in prostaglandin E2 production

in sheep adipose tissue in vivo (Doris et al., 1996) Thus in sheep, growthhormone facilitates lipolysis by at least three mechanisms (Fig 17.7) All theseeffects of growth hormone are chronic, taking a number of hours to becomemanifest

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 itssimplest, all cells need to maintain the ratio of ATP to ADP and AMP at anappropriate level Relative concentrations of these adenosine nucleotides are

formulae have been proposed to describe the ‘energy state’ of a cell (Vernonand Peaker, 1983); these include the ‘energy charge’:

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

potential’:

[ATP]=([ADP]  [Pi])That is in essence an index of how far the reaction is from equilibrium; thegreater the phosphorylation potential, the more energized the cell Values varymore than the ‘energy charge’ normally ranging from about 200 to 800 whenexpressed in molar terms Both equations have their limitations, but the keypoint 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, maintaininghomoeostasis involves the appropriate distribution of nutrients to all the cells of

Lipolysis Adenylate cyclase

Adenosine Prostaglandin E

Catecholamines

Adenosine receptor PGE receptor

β-Adrenergic receptor

GH

− +

Fig 17.7 Modulation of lipolytic regulatory systems by growth hormone GH, growthhormone; PGE, prostaglandin E; Gs, stimulatory GTP-binding protein; Gi, inhibitory

GTP-binding protein