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J Clin Pathol: first published as 10.1136/jcp.s1-2.1.66 on 1 January 1969. Downloaded from

J. clin. Path., 22, suppl. (Ass. clin. Path.), 2, 66-71

Intermediary

P. J. RANDLE From the Department ofBiochemistry, University of Bristol

Current interest in mammalian carbohydrate meta- may act by raising Km or lowering Vmax bolism is largely centred on the operation of or both. They may conform to classical inhibitor metabolic pathways in tissues and whole . kinetics (see Dixon and Webb. 1958) or the rela- Recent studies have indicated that the various tionship between degree of inhibition and inhibitor pathways may be controlled by one or two key concentration may be sigmoid (ie, show a threshold). which as pacemakers. Trans- Similar considerations may be applied to activators. porting systems in the plasma membrane and also In general it is important to distinguish between in intracellular membranes, such as the mitochon- those inhibitors which influence Vmax alone, Km drial membrane, are of increasing importance in alone, and both Vmax and Km. Where only K. is control and hence in the operation of metabolic altered inhibition may be overcome by compensatory pathways. changes in substrate concentration; this will not be the case when only Vmax is altered. CONTROL IN TISSUES The identification of pacemaker reactions in metabolic pathways has depended upon simul- The majority of enzymes and transporting systems taneous measure-ments of rates of flow and con- in probably conform to centrations of substrates and products of individual

Michaelis-Menten kinetics. The relationship be- reactions in tissues. The concepts of mass action copyright. tween substrate concentration (or gradient) and ratios and of crossover have been particularly reaction (or transport) velocity is hyperbolic, and valuable.3 A pacemaker reaction is rate-limiting in velocity is also directly proportional to its pathway and an increase in flow is due in concentration. In terms of pathway operation re- particular to an increase in the activity of a pace- action velocity (flow) is most sensitive to substrate maker en7yme. It follows that a pacemaker reaction concentration at values below the Kin,' and capacity, must be displaced from equilibrium, under condi- ie, Vmax2 or maximum flow, is mainly dependent tions where it limits flow in the whole pathway, ie, upon enzyme or carrier concentration. Some en- the mass action ratio of products and reactant will http://jcp.bmj.com/ zymes do not conform to Michaelis-Menten kinetics differ markedly from the equilibrium constant for the and of particular interest from the point of view of reaction. It is important to know whether the equili- control are those enzymes which exhibit sigmoid brium constant is dependent on factors such as pH rate curves, ie, show a sigmoid relationship between (as in the case of ), or divalent ions (as in the reaction velocity and substrate concentration. From case of aconitase), in interpreting mass action ratios. the point ofview ofpathway operation these enzymes In the case of transport processes the mass action have a threshold, and reaction velocity (or flow) is ratio is given by the ratio of concentrations on the on September 29, 2021 by guest. Protected most sensitive to substrate concentration at values two sides of the membrane; and the equilibrium above the threshold and below the Km. Enzymes constant is given by the ratio of concentrations at which show sigmoid rate curves appear invariably to equilibrium. In general when flow through a path- be regulatory enzymes, though there are some regu- way is increased by activation of a pacemaker latory enzymes which show hyperbolic rate curves. reaction, the mass action ratio is changed in the Although substrate concentration may be an direction of the equilibrium constant. The identi- important determinant of rate for some regulatory fication of a pacemaker reaction may be most enzymes the concentration of effectors is of more clearly made when a crossover occurs, ie when for a general importance. Such effectors are particular reaction in a an which activate or inhibit enzymes for which they increased rate of flow is accompanied by a decrease may or may not be substrates. Inhibitory effectors in substrate concentration(s) and an increase in concentration(s) or vice versa. Unfor- 'Michaclis constant, ie, substrate concentration at which reaction velocity = half maximum velocity. ED. tunately there are instances where no clear crossover 'Vm.. =maximum velocity of an enzyme reaction. 'See Editors' Appendix. 66 J Clin Pathol: first published as 10.1136/jcp.s1-2.1.66 on 1 January 1969. Downloaded from

Intermediary carbohydrate metabolism 67 occurs although an increase in mass action ratio Metabolic integration may involve both receptor may accompany an increase in flow. This may be and effector systems and is mediated in part by the seen, for example, when two or more pacemaker endocrine system and in part by the nervous system. reactions are in close apposition. The secretion of is regulated in the ,- by An additional and necessary approach involves the blood concentration. In this instance screening individual enzymes for activation or the receptor system lies within the cell secreting the inhibition by a wide variety of metabolites. This effector (insulin). The secretion of pituitary growth type of study can indicate possible regulatory may also depend upon blood glucose enzymes and suggest whole experiments to concentration but in this instance the receptor cells establish their regulatory role. It has been parti- may be located in the hypothalamus. Some con- cularly valuable in situations where two or more sideration is given in a later section to the gluco- regulatory enzymes are closely apposed in metabolic receptor mechanism in the . pathways. The elucidation of mechanisms of regu- lation is similarly dependent upon the detection of CONTROL OF IN MUSCLE effectors for individual enzymes and the demon- stration of effector mechanisms in the cell. Table I shows the enzymes and transport systems Studies along these lines have indicated probable concerned with glucose uptake, glycolysis, control mechanisms for a number of pathways of synthesis, , and pyruvate oxidation in carbohydrate metabolism and have provided a muscle. Glucose uptake appears to be controlled by qualitative basis for understanding their operation. the two apposed steps of plasma membrane trans- Fuller understanding can come only from quan- port of glucose and its intracellular titative studies in which the operation of the pathway by . The glycolytic pathway between may be described in the form of mathematical glucose 6-phosphate and pyruvate appears to be models based on the kinetic properties and con- controlled only by . Glycogen centrations of enzymes together with the concen- synthesis may be regulated by glycogen synthetase

trations of their substrates and effectors. Some and glycogen breakdown by phosphorylase. The copyright. progress along these lines is now being made in entry of glucose into the citrate cycle may be regu- restricted segments of pathways but this approach lated by . raises special problems. Enzyme kinetics have mostly The control systems appear to subserve three been derived from studies with enzymes in dilute major physiological roles. One set of signals may solution where substrate or effector concentrations vary rates of glucose uptake, glycogen breakdown, are in vast excess. In cells the concentrations of some and glycolysis according to the state of ATP syn- enzymes may be comparable to that of their sub- thesis and breakdown. Thus glucose transport, strates or effectors and it is not known whether this phosphorylase b, and phosphofructokinase may be http://jcp.bmj.com/ will appreciably modify their kinetic properties. activated by breakdown products of ATP (ADP, The control of glycolysis in muscle is discussed in AMP, and phosphate) and inhibited by ATP. a later section as an illustration of these techniques Another set of signals may regulate rates of glucose and problems. uptake, glycogen synthesis and breakdown, gly- colysis and pyruvate oxidation according to the CONTROL IN ANIMALS: METABOLIC INTEGRATION availability of alternative respiratory fuels such as

fatty or bodies. The major signals on September 29, 2021 by guest. Protected The control mechanisms which have been outlined appear to be acetyl CoA, CoA, NADH2, and NAD for tissues are mechanisms common to both uni- which may regulate pyruvate dehydrogenase, and cellular and to individual cells of animals. citrate formed from acetyl CoA which may regulate Additional control mechanisms are seen in animals phosphofructokinase. A third mechanism is con- which lead to integration of the varied metabolic cerned with hormone action. Insulin may stimulate pathways of individual tissues. These control glucose uptake by activating glucose transport and mechanisms are partly a consequence of metabolic stimulate glycogen formation by causing the con- specialization (for example in version of glycogen synthetase from D to I form. In and synthesis and breakdown in adipose its D form the activity of glycogen synthetase is tissue); and partly a consequence of the relatively critically dependent upon glucose 6-phosphate small circulating pools of metabolites. The plasma concentration. The I form is independent of glucose free fatty pool, for example, may turn over in 6-phosphate. In this regard the action of insulin three to five minutes; that of glucose in 30 to 60 may be visualized as causing an enzyme conversion minutes. The pools are thus small in relation to which enables glycogen synthesis to be independent feeding habits. of intracellular signals (in this case glucose 6- J Clin Pathol: first published as 10.1136/jcp.s1-2.1.66 on 1 January 1969. Downloaded from

68 P. J. Randle TABLE I ENZYME ACTIVITIES IN MOUSE ISLETS WHICH MAY REGULATE RATES OF GLUCOSE PHOSPHORYLATION Pathway Segment Enzyme (or Carrier) Activator Inhibitor Glucose uptake Glucose transport Insulin ?ATP ?AMP Hexokinase Glucose 6-phosphate AMP, ADP. Glycolysis Phosphoglucoisomerase Phosphofructokinase AMP, ADP ATP Phosphate Citrate 6-phosphate Fructose 1 :6-diphosphate Sulphate Cyclic 3'5'-AMP Aldolase phosphate Triose phosphate dehydrogenase Phosphoglyceromutase Pyruvate Glycerolphosphate dehydrogenase Glycogen UDP glucose pyrophosphorylase synthesis Glycogen synthetase (D form) Glucose 6-phosphate Glycogen Glycogen synthetase (I forn) Branching enzyme Glycogenolysis Phosphorylase b AMP ATP Glucose 6-phosphate

Malate copyright. Phosphorylase a --1 :6-Amyloglycosidase Aerobic Pyruvate dehydrogenase CoA Acetyl CoA oxidations NAD NADH2 Regulation of Citrate transport Malate phosphofructokinase (niitochondrial membrane by citrate http://jcp.bmj.com/ phosphate). Similarly the action of in activate glycogen synthetase (D form); and fructose causing glycogen breakdown appears to involve the 6-phosphate may activate phosphofructokinase. The conversion of phosphorylase from the b form, re- effects of glucose 6-phosphate are on the Vmax of quiring activation by AMP and inhibited by ATP the three enzymes concerned. This may be especially and glucose 6-phosphate, to the a form which is not important with hexokinase where inhibition by dependent upon AMP for activity and which is glucose 6-phosphate may increase the intracellular largely insensitive to effectors (AMP, ATP or glu- glucose concentration. Because the inhibition by on September 29, 2021 by guest. Protected cose 6-phosphate). glucose 6-phosphate affects Vmax and not Km there In terms of operation glucose uptake, glycogen may be little or no compensatory increase in the rate synthesis and breakdown and glycolysis may func- of glucose phosphorylation when glucose accumu- tion as a single complex unit. For example, glycolytic lates. The control of phosphofructokinase, on the is dependent upon glucose uptake or glycogen other hand, appears to be through effects on the Km breakdown or a combination ofthe two. More detail- for fructose 6-phosphate. The enzyme is inhibited by edexamination ofthecontrol ofhexokinase, glycogen ATP which raises the Km for fructose 6-phosphate synthetase (D form), phosphorylase b, and phos- and this inhibition is overcome by fructose 6- phofructokinase suggests that they may act as a phosphate. Rate curves for fructose 6-phosphate are single complex unit. The connecting link appears to thus sigmoid, because fructose 6-phosphate is both be the concentrations of glucose 6-phosphate and substrate and activator, and the Km is dependent on fructose 6-phosphate which are held in equilibrium ATP concentration. Citrate increases the ATP by phosphoglucoisomerase. Glucose 6-phosphate inhibition and further raises the Km. Phosphate, may inhibit hexokinase and phosphorylase b and ADP and AMP, and fructose 1 :6-diphosphate J Clin Pathol: first published as 10.1136/jcp.s1-2.1.66 on 1 January 1969. Downloaded from Intermediary carbohydrate metabolism 69

reverse ATP inhibition and lower the Km for fructose PANCREATIC f-CELL GLUCORECEPTOR 6-phosphate. The operation of this complex has been deduced The secretion of insulin by pancreatic fl-cells is from studies of the effects of anoxia, of fatty acids stimulated by glucose. The rate curve for secretion and ketone bodies, and of insulin in ratheart muscle. at varying glucose concentration measured with rat Anoxia accelerates glycolysis, glucose phosphoryl- pancreas in vitro is sigmoid with a threshold at ation, and glycogen breakdown and leads to a approximately 90 mg/100 ml and with a Km of crossover between substrates and products of the approximately 180 mg/100 ml (Malaisse, Malaisse- phosphofructokinase reaction (ATP and fructose Lagae, and Wright, 1967). Several lines of evidence 6-phosphate decrease; ADP and fructose 1:6- have indicated that the effect of glucose on insulin diphosphate increase). Lack of is believed to secretion is transmitted through metabolism of the lower ATP and raise ADP, AMP, and phosphate in the fl-cell. Secretion is stimulated only by and thus activate phosphofructokinase and phos- those which can be readily metabolized phorylase b. Activation of phosphofructokinase (glucose and ). There is a close correlation raises fructose 1 :6-diphosphate (producing a further between rates of glucose oxidation and effects on activation) and lowers fructose 6-phosphate and secretion. Rate curves for glucose oxidation by mouse glucose 6-phosphate. The change in glucose 6- islets at'varying glucose concentration are also sig- phosphate may then activate hexokinase and also moid with a threshold at 90 mg/100 ml, and with a phosphorylase b. Fatty acids or ketone bodies Km of approximately 130 mg/100 ml. Glucose oxi- inhibit glycolysis and glucose phosphorylation and dation is suppressed by which also stimulate glycogen synthesis and lead to a crossover suppresses the glucose effect on secretion, whereas between substrates and products for phospho- phloridzin has no effect either on secretion or on fructokinase (fructose 6-phosphate increases and glucose oxidation. As a working hypothesis it has fructose 1 :6-diphosphate decreases; ATP and ADP been suggested that the metabolism of glucose in are little changed). They act by increasing citrate the islet cell alters the concentration of some meta- concentration thereby inhibiting phosphofructo- bolite of glucose which activates the secretory pro-

kinase and leading to accumulation of glucose 6- cess. The glucoreceptor has thus been visualized as copyright. phosphate, inhibition ofhexokinase and phosphoryl- an enzyme or transport system involved in the ase b, and possibly activation of glycogen synthetase initiation of glucose metabolism. Glucose transport (D form). Insulin accelerates glucose uptake, systems in other cells and tissues are inhibited by glycolysis, and glycogen synthesis and leads to a phloridzin. Mannoheptulose on the other hand crossover at the glucose transport step (intracellular appears to be an inhibitor of and glucose concentration is increased from an extremely and moreover it can penetrate only low Insulin is thus to accelerate those cells, eg, liver, which are freely permeable to value). thought http://jcp.bmj.com/ glucose transport and as a consequence to increase glucose, ie, cells in which phosphorylation of glucose intracellular glucose concentration and thus pI-o- is the first step in its metabolism. Since in the islet mote glucose phosphorylation. The increased rate mannoheptulose is inhibitory whereas phloridzin is of glycolysis may be due to an increase in fructose not, it has been suggested that the glucoreceptor is 6-phosphate concentration activating phospho- an enzyme or enzymes regulating glucose phos- fructokinase. phorylation. The properties of the glucoreceptor This system has yet to be evaluated quantitatively must be capable of explaining the sigmoid rate curve as a whole. However, a quantitative evaluation of for insulin secretion and for glucose oxidation with on September 29, 2021 by guest. Protected the control of hexokinase predicted from the kinetic a threshold of approximately 90 mg/100 ml and a properties of the enzyme has been shown to be valid Km of approximately 130 to 180 mg/100 ml; and of in the perfused rat heart (England and Randle, explaining the powerful inhibitory effect of manno- 1967). heptulose. One of the problems to be surmounted in an Three enzyme activities have been detected in analysis of the whole system is the effect of com- homogenates of mouse islets which may contribute partmentation. In heart muscle citrate synthesis may to the overall rate of glucose phosphorylation in be confined to the mitochondrion whereas citrate this tissue by Matschinsky and Ellerman (islets of inhibition of phosphofructokinase is cytoplasmic. obese, hyperglycaemic mice) and Ashcroft and The regulation may thus involve export of mito- Randle (islets of normal mice). Table II summarizes chondrial citrate for which there appears to be a kinetic data published by Randle and Ashcroft citrate transporting system requiring activation by (1969). Two enzymes phosphorylating glucose have malate (Chappell, Henderson, McGivan, and been detected. One is a hexokinase with a low Km Robinson, 1968; England and Robinson, 1969). for glucose, 1-3 mg/100 ml, and inhibited non- J Clin Pathol: first published as 10.1136/jcp.s1-2.1.66 on 1 January 1969. Downloaded from 70 P. J. Randle TABLE II ENZYMES OF GLYCOLYSIS AND REGULATION Km (mM) 3.70 Enzyme Activity max: (units/g) Inhibitors Ki (mM)5

Hexokinase Glucose 0 075 054 Glucose 6-phosphate 0-22 ATP 0 77 Mannoheptulose 0-25 Glucokinase Glucose 22 0-17 Mannoheptulose (K1 not known but inhibition weak) Glucose 6- Glucose 6- 0 70 Glucose mixed inhibition K, 9 K, 38. Not reversed phosphate 1-0 by mannoheptulose 'Kt = concsntration of inhibitor produ:ing 50 % inhibition. ED. competitively by glucose 6-phosphate and com- they have shown that enzymes have regulatory as petitively by mannoheptulose. The other, well as catalytic activity. It is not unreasonable to provisionally designated as glucokinase, has a high expect that future investigation will reveal inborn Km for glucose, 396 mg/100 ml; it is not inhibited by errors which affect the regulatory activities of glucose 6-phosphate and may be activated by it; it enzymes and not their catalytic activities. Similarly is weakly inhibited by mannoheptulose. Glucose the realization of the role of plasma membrane and 6-phosphatase activity is also present and it is intracellular membrane transporting systems in inhibited by glucose, but not by mannose. Glucose metabolic pathways and metabolic regulation sug- inhibits by raising Km as well as lowering Vmax, and gests the basis for new investigation of metabolic 50 per cent inhibition is seen at approximately disease. The specificity of some enzyme regulations 180 mg/100 ml. and some transporting systems in the operation of Although the properties of these enzymes may metabolic pathways indicates the possibility of new ultimately account for the glucoreceptor mechanism drugs. For example, in due to insulin deficiency many of the changes in the carbohydrate there are a number of unexplained facts which make copyright. it difficult to compound any satisfactory model. metabolism of muscle, which may lead to insulin Rate curves with each of the enzymes are hyperbolic unresponsiveness, are similar to the changes pro- whereas rate curves for insulin secretion and glucose duced by oxidation of fatty acids. Some of these oxidation in the islet are sigmoid. Moreover the changes have been referred to in the section on kinetics of mannoheptulose inhibition of hexokinase glycolysis. It has been suggested that these changes are such as to be incapable of explaining the inhibi- in diabetic muscle are in fact due to accelerated tory effect of mannoheptulose on glucose oxidation breakdown and oxidation. If in the islet, with the glucose Km value obtained. The this is so then inhibitors of fatty acid oxidation http://jcp.bmj.com/ inhibition of glucokinase by mannoheptulose is too should reverse the abnormalities of carbohydrate weak to explain its effect in the islet. A sigmoid rate metabolism in diabetic muscle. Some evidence for curve may be achieved by a combination of glucose this possibility has been achieved in studies with 6-phosphatase and hexokinase and taking into 2-bromo fatty acids (palmitate or stearate) which account inhibitory effects of glucose and glucose can inhibit specifically acyl in 6-phosphate. However such a combination may not the pathway of fatty acid oxidation. Experiments explain the effects of mannose on insulin release in vitro with rat heart muscle have indicated that on September 29, 2021 by guest. Protected and mannose oxidation, because mannose is not an 2-bromostearate can reverse abnormalities ofglucose inhibitor of the glucose 6-phosphatase. A more uptake, glucose phosphorylation, glycolysis, and complete evaluation taking into account, for exam- glucose oxidation due to inhibition of hexokinase, ple, the possibility of activation of glucokinase by phosphofructokinase, and pyruvate dehydrogenase glucose 6-phosphate is required. in alloxan diabetes (Burges, Butt, and Baggaley, 1968; Randle, 1969). APPLICATIONS OF CONTROL CLINICAL MECHANISMS REFERENCES Burges, R. A., Butt, W. D., and Baggaley, A. (1968). Biochem. J., The importance of inherited defects in the catalytic 109, 38P. activities of enzymes in the aetiology of some di- Chappell, J. B., Henderson, P. J. F., McGivan, J. D., and Robinson, B. H. (1968). In The interaction of Drugs and Subcellular Com- seases has been recognized for nearly half a century ponents, Edited by P. N. Campbell, p. 71. Churchill, London. following Garrod's description of inborn errors of Dixon, M., and Webb, E. C. (1958). Enzymes. Longmans Green, metabolism. Studies of control mechanisms have in London. England, P. J., and Randle, P. J. (1967). Biochem. J., 105, 907. effect added a new dimension to enzymology since -, and Robinson, B. H. (1969). Ibid., 112, 8P. J Clin Pathol: first published as 10.1136/jcp.s1-2.1.66 on 1 January 1969. Downloaded from

Intermediary carbohydrate metabolism 71 Malaisse, W., Malaisse-Lagae, F., and Wright, P. H. (1967). Endo- from the equilibrium constant, often by several crinology, 80, 99. Randle, P. J. (1969). Nature (Lond.), 221, 777. orders of magnitude. and Ashcroft, S. J. H. (1969). Biochem. J., 12, IP. Acceleration of flow along the pathway is achieved by an increase in the capacity of the , and the resulting increase in activity causes EDITORS' APPENDIX a fall in the enzyme's substrate concentration and a rise in its product concentration so that the mass The mass action ratio is the concentration of the action ratio rises towards the equilibrium constant. product of an enzyme reaction divided by the con- The preceding reaction in the pathway is also accel- centration of the substrate. The velocity of an enzyme erated because the concentration of this enzyme's reaction increases with substrate concentration and product (which is the substrate of the regulatory decreases with product concentration, so that the enzyme) is now lower, resulting in a fall of the mass reaction velocity is inversely proportional to the mass action ratio away from the equilibrium constant. action ratio. When the reaction is at equilibrium, The reaction which follows the pacemaker reaction ie, when the reaction velocity is zero, the mass action is accelerated too because its substrate (which is the ratio will be at its maximum and equal to the product of the regulatory enzyme) is now higher, equilibrium constant of the enzyme. again resulting in a fall of the mass action ratio away When the rate of flow along a metabolic pathway from the equilibrium constant. Hence activation of is below its maximum because it is restricted by a the pathway is associated with a rise, usually large, rate-limiting 'pacemaker' reaction controlled by a in the mass action ratio of the regulatory enzyme regulatory enzyme, all the other enzymes of the whereas there is a fall, usually small, in the mass pathway will be running well below their capacity action ratios of the other enzymes. This 'cross-over' and hence their mass action ratios will be relatively of mass action ratios is one method of identifying high and close to the equilibrium constant. The the regulatory enzyme, another being the large rate-limiting reaction, however, will be running at displacement of its mass action ratio from the equi- its full (but limited) capacity, so that its mass librium constant and the extent of its change when action ratio will be low and considerably displaced the pathway is activated. copyright. http://jcp.bmj.com/ on September 29, 2021 by guest. Protected