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Regulation of intermediary metabolism by nucleotides: The mechanism of action of insulin
THE
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OF INSULIN
Lardy, 1954). Other enzymes require specific energy donors: e.g. with fructokinase, which converts fructose to fructose-l-phosphate, ATP cannot be replaced by UTP or ITP (Parks, Ben-Gershom & Lardy, 1957). In particular, many biosynthetic pathways require specific energy donors: glycogen synthesis requires UTP (Munch-Petersen, Kalckar, Cutolo & Smith, 1953), phospholipid synthesis requires CTP (Kennedy, 1956) and incorporation of amino-acids into protein requires the presence of GTP (Keller & Zamecnik, 1956). It is therefore evident that the rate of synthesis in particular anabolic pathways may well depend on the availablity of specific nucleoside triphosphates, provided, of course, that the usually adequate amounts of substrate and intact enzyme chains are present. Production of Nucleoside Triphosphates In order to postulate an exact site for insulin action, it is necessary to review briefly the biosynthesis of the nucleoside triphosphates. Certain nucleotides are phosphorylated during the course of glycolysis and substrate oxidation in the Krebs cycle (Table 1). However, the most important product quantitatively is the ATP produced by oxidative phosphorylation. TABLE
1
Direct production of nucleosidetriphosphates
_---
Reaction
-__--
Nucleotide Produced
P-enol-pyruvate to pyruvate P-enol-pyruvate to oxalacetate
ATP, UTP, ITP, GTP, CTP GTP, ITP
Succinyl-CoA to succinate
GTP, ITP
---
Reference Strominger (1955) Nordlie & Lardy (1963) Sanadi, Gibson & Ayengar (1954)
A number of enzymes catalyzing transphosphorylation of nucleotides have been found in yeast, muscle and liver cells. 1. Nucleoside monophosphate-ATP transphosphorylases(Fig. 1: la, lb, Ic) (Strominger, Heppel & Maxwell, 1959). This group of enzymes catalyzes the phosphorylation of any nucleoside monophosphate except IMP. IDP is formed instead by the deamination of ADP (Fig. 1: Id) (Deutsch & Nilsson, 1954). Certain specific members of this group have been separated or isolated :
268
M.
D. Purine
ATP
KLACHKO
nucieotides
Pyrimidine
c
ADP
AMP\ lo
20
ADP x
nucleotldes
1 ADP”
ATP ADP
r
ADP’
L
ATP
FIG. 1. Nucleotide transphosphorylases. See text for names of enzymes. All reactions are reversible.
la, lb, lc, NMP-ATP transphosphorylases (Strominger, Heppel & Maxwell, 1959); la, Adenylate kinase; (Colowick & Kalckar, i943); Id, ADP deaminase (Deutsch & Nilsson, 1954); 2a, 2b, 2c, NTP-AMP transphosphorylases (Heppel, Strominger & Maxwell, 1959): 2a, ATP-AMP transphosphorylase (Chiga & Plaut, 1960); 2b, 6-oxypurine NTP-AMP transphosphorylase (Chiga, Rogers & Plaut, 1961) ; 3, ATP-NDP transphosphorylase (NDP Kinase) (Berg & Joklik, 1954; Krebs & Hems, 1953; Ratliffe, Weaver, Lardy & Kuby, 1964). NMP, NDP, NTP; nucleoside mono-, di-, triphosphate.
(A) ATP+AMP
F? 2 ADP.
This enzyme, adenylate kinase, was the first nucleotide transphosphorylase discovered (Colowick & Kalckar, 1943). The fractionation procedure used by Strominger, Heppel & Maxwell (1959) completely removed the activity of the enzyme responsiblefor the reaction : (B) ATP+GMP
ti ADP+GDP,
whereas the majority of the remaining activity catalyzed the reaction: (C) ATP+UMP
(CMP) F? ADP+UDP
(CDP).
2. Nucleoside triphosphate-AMP transphosphoryluses(Fig. 1: 2a, 2b, 2c) (Heppel, Strominger & Maxwell, 1959). (A) 2 ADP P AMP+ATP. This specific ATP-AMP transphosphorylase, similar to the adenylate kinase of muscle described above, has been isolated from mammalian liver (Chiga & Plaut, 1960). (B) ADP+GDP (IDP) P AMP+GTP (ITP). This separate fraction, 6-oxypurine nucleoside triphosphate-AMP transphosphorylase, has been partially purified (Chiga, Rogers & Plaut. 1961).
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(C) ADP+ UDP (CDP) it AMP+UTP (CTP). Although this reaction has been found to occur, the enzyme responsible has not yet been isolated. 3. ATP-nucleoside diphosphate transphosphorylase (Nucleoside diphosphokinase) (Fig. 1 : 3) (Berg & Joklik, 1954; Krebs & Hems, 1953; Ratliff, Weaver, Lardy & Kuby, 1964). This is a single enzyme which has been isolated in crystalline form (Ratliff et al., 1964). It catalyzes the phosphorylation, by ATP, of a number of purine and pyrimidine ribo- or desoxyribonucleoside diphosphates to the respective triphosphates. The above enzymes provide a network of transphosphorylations of nucleotides and thereby regulate the balance between the various nucleoside triphosphates. It can be seen that factors affecting one or more of these enzymes can influence the concentration of particular nucleoside triphosphates, and so affect the rate of biosynthesis in the pathways controlled by those nucleoside triphosphates. The mitochondria contain nucleoside diphosphokinase but lack nucleoside monophosphate kinases except for adenylate kinase. The adenosine nucleotides are preferentially phosphorylated by mitochondria (Herbert & Potter, 1956) and, with the nucleoside monophosphate kinases, no transphosphorylation has yet been discovered in which neither reactant contains adenosine (Strominger, Heppel & Maxwell, 1959). It thus appears that ATP formation is the primary result of oxidative phosphorylation and the high energy phosphate is then transferred to other nucleotides by the reactions described above. The probable course of “energy flow” is depicted in Fig. 2. Monosaccharides Amino acids
co2 “20
Creatjne
P
FIG. 2. “Energy flow”. 1. Oxidative phosphorylation; 2. Nucleoside diphosphokinase. NMP, NDP, NTP: mono-, di-, and triphosphates of nucleosides other than adenosine. T.B. 18
270
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M.
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The Mechanism of Action of Insulin The effects of insulin include: (a) increased synthesis of glycogen, proteins, fatty acids (Krahl, 1961) and RNA (Wool, 1963); (b) suppression of biosynthesis in liver of the key gluconeogenetic enzymes induced by glucocorticoids (Weber, Singhal & Srivastava, 1965); (c) induction of biosynthesis in liver of the key glycolytic enzymes (Weber & Singhal, 1965), glycogen synthetase (Steiner & King, 1964), and enzymes for synthesis of fatty acids (Gellhorn & Benjamin, 1964); (d) activation of glycogen synthetase from the glucose-6-phosphate dependent (D) to the glucose-6-phosphate independent (I) form (Villar-Palasi & Larner, 1960); (e) increased transport of sugars and some amino-acids into cells of muscle and adipose tissue; (f) increased glycolysis in muscle and adipose tissue; (g) increased incorporation of inorganic phosphate into nucleotide phosphate in rat diaphragm muscle (Clauser, Volfin & Eboue-Bonis, 1962). Recently there has been great interest in the apparent independence of many of these effects from each other. In diabetic rats, actinomycin D blocks the action of insulin on accumulation of hepatic glycogen and repair of the enzymatic defects in synthesis of fatty acids by adipose tissue and mono-unsaturated fatty acids by liver microsomes. However, it does not block the hypoglycemic action of insulin (Gellhorn & Benjamin, 1964). Inhibitors of protein synthesis, such as puromycin and ethionine, also prevent new enzyme synthesis without preventing the hypoglycemic effect of insulin (Steiner & King, 1964). In vitro, despite inhibition of RNA synthesis by actinomycin, insulin stimulates protein synthesis, uptake of glucose and aminoisobutyric acid, and incorporation of inorganic phosphate into nucleotide phosphate in rat diaphragm muscle (Wool & Moyer, 1964; Eboue-Bonis, Chambaut, Volfin & Clauser, 1963), and fatty acid synthesis by mammary gland slices (Mayne & Barry, 1965). In the presence of puromycin inhibition of protein synthesis, insulin continues to stimulate the uptake of glucose, galactose, d-xylose and aminoisobutyric acid, RNA synthesis, and incorporation of inorganic phosphate into nucleotide phosphate in rat diaphragm muscle (Carlin & Hechter, 1964; Burrow & Bondy, 1964; Eboue-Bonis et al., 1963) as well as oxidation of glucose and leucine, and lipid synthesis in rat epididymal fat pads (Dawson & Beck, 1965). These findings suggest that the increased protein and RNA synthesis may be secondary to a more fundamental effect of insulin: that of stimulating anabolic processes. However, the induction or repression of enzyme formation more probably is due to the stimulation of new RNA and protein synthesis in
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conjunction with the mechanisms of substrate induction and end-product inhibition. The basic effect of insulin as a potent stimulator of all anabolic processes had long been considered to be related to an increased provision of substrates in the cell. Initially this was thought to be specifically due to the increased transport into cells of glucose (Levine & Goldstein, 1955). Later experiments have shown that insulin stimulates glycogen formation to a greater extent than can be accounted for by the increase in glucose uptake (Norman, Menozzi, Reid, Lester & Hechter, 1959; Larner, Villar-Palasi & Richman, 1959). Even in the absence of extracellular glucose, insulin stimulates the biosynthesis of proteins from labelled precursors (Manchester, 1961), and increases labelling of RNA from [i4C]adenine (Wool, 1963). It is also accepted that the stimulation of protein synthesis is independent of any other effect of insulin on aminoacid transport into the ceil (Lukens, 1964). The possibility that insulin acts by increasing the amount of ATP in the cell has been investigated. Conflicting results have been reported. ATP formation was normal in liver mitochondria of alloxan-diabetic rats (Parks, Adler & Copenhaver, 1955), but was decreased in those from depancreatized cats (Vester & Stadie, 1957). In 1960, Blaylock, Rothrock & Sacks found decreased levels of ATP in the livers of fed, alloxan-diabetic rats, but insulin injection caused increased glycogen synthesis despite a continued low concentration of ATP. Although ATP is the prime source of energy for muscular work, ion transport and many other cellular functions, it is not the final “energy donor” for many anabolic reactions. As described earlier, other specific nucleoside triphosphates are required. Clauser, Volfin & Eboue-Bonis (1962) showed that insulin stimulates the incorporation of labelled inorgamc phosphate into nucleotide phosphate in rat diaphragm muscle, and causes an increase in the ratio of phosphate incorporated into guanosine and uridine nucleotides, relative to that incorporated into adenosine nucleotides, both in the presence and absence of glucose. This effect of insulin cannot be inhibited by either puromycin or actinomycin (Eboue-Bonis et al., 1963). It therefore appears probable that insulin stimulates anabolic processes by increasing the rate of transfer of high energy phosphate from ATP to the other nucleotides. The effects of insulin on glucose metabolism also require consideration. Both glucose transport and glycolysis in muscle and adipose tissue are increased by anoxia and agents which uncouple oxidative phosphorylation. This is presumed to be a mechanism for the maintenance of a relatively constant intracellular concentration of ATP and it has been suggested that the membrane barrier to the uptake of sugars is maintained by a “high
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energy” phosphate compound which may be ATP (Randle & Smith, 1960). The rate-limiting step in glycolysis is the enzyme phosphofructokinase (Newsholme & Randle, 1962). Isolation of the enzyme has shown that it contains a nucleic acid, or possibly a mixture of nucleotides, and that the net forward rate of the phosphorylation of fructose-6-phosphate to fructose1,6-diphosphate depends on the amount of nucleotide present (Hofer & Pette, 1965). Ling & Lardy (1954) reported that phosphofructokinase can utilize UTP and ITP as efficiently as ATP. ATP can also be replaced by CTP or GTP (Utter, 1960). High concentrations of ATP inhibit the phosphorylation of fructose-6-phosphate (Mansour, 1963 ; Passonneau & Lowry, 1962) but, at least with yeast phosphofructokinase, GTP does not (Vinuela, Salas & Sols, 1963). The above data are consistent with the hypothesis that insulin acts by accelerating the transfer of “high energy” phosphate from ATP to the non-adenosine nucleoside diphosphates thus causing an initial fall in the peripheral concentration of ATP in the cell and consequent stimulation of glucose uptake and glycolysis. The same mechanism may explain the conversion of glycogen synthetase from the D form to the I form as the D enzyme is a phosphorylated form of the I enzyme and is maintained in this state by ATP and Mg2+ (Friedman & Larner, 1962). Within the cell, numerous enzymes compete for available ATP. The “flow of energy” within the cell would be regulated by the location of the ATP as well as by the relative affinity of the enzymes for ATP. That changes in enzyme affinity for ATP is a control mechanism in the cell is suggested by the fact that the stimulation of acetyl-coenzyme A carboxylase by isocitrate, in vitro, is associated with a decrease in the Km for ATP by a factor of 5 (Waite & Wakil, 1962). The Site of Insulin Action
Lee & Williams (1954), using [‘3’I]insulin, showed that at least 667; of insulin in liver was intracellular within five minutes after intravenous administration. In epididymal adipose tissue, in vitro, insulin causes invagination of the cell membrane with formation of pinocytotic vesicles as well as changes in the mitochondria suggestive of increased activity or growth (Barrnett & Ball, 1960). These changes occur with or without glucose in the medium, and pinocytosis may be the means by which the insulin molecules enter the fat cell. As detailed above, the action of insulin could be explained by an increased rate of transfer of “high energy” phosphate from ATP to non-adenosine nucleotides. Anabolic processes require particularly large amounts of energy. For example, the incorporation of each glucose molecule into
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glycogen or each choline molecule into lecithin is associated with the conversion of a nucleoside triphosphate to the corresponding nucleoside monophosphate. This then requires two molecules of ATP and participation of at least two enzymes for the regeneration of the one triphosphate. The widespread stimulation of anabolism produced by insulin would require an increased rate of formation of UTP for glycogen synthesis and GTP for protein synthesis, whereas RNA synthesis requires the simultaneous presence of UTP, GTP, CTP and ATP. Although it is possible that insulin could act on all the nucleotide transphosphorylases, all its effects could be explained by a stimulation of the single enzyme nucleoside diphosphokinase (Fig. I : 3) which accelerates the phosphorylation of all nucleoside diphosphates at the expense of ATP. Considered teleologically, at times of food intake and excess of available substrate, insulin switches the pattern of metabolism from catering primarily to immediate needs to one promoting a state of general synthesis and storage. Other peptide hormones may affect other nucleotide transphosphorylases, leading to a relative preponderance of single nucleoside triphosphates and stimulating particular metabolic pathways. The possible actions of peptide hormones on this network of nucleotide transphosphorylases, leading to competition between them for available ATP, could provide an explanation for the many synergistic and antagonistic effects of such hormones. Conclusion
Nucleotides not only carry the genetic code that determines the overall metabolic constitution of every cell, but also appear to direct the energy flow to specific anabolic pathways. It is postulated that certain peptide hormones act on the nucleotide transphosphorylases to regulate the flow of “high energy” phosphate from ATP, thus determining the relative quantities of nucleoside triphosphates in the cell and indirectly controlling the pattern of intermediary metabolism. The effects of insulin could be explained on the basis of an increased rate of transfer of “high energy” phosphate from ATP to all non-adenosine nucleoside diphosphates, causing an increased level of all non-adenosine nucleoside triphosphates resulting in general increases in rates of synthesis and storage within the cell. A possible single site for this major action of insulin is the enzyme nucleoside diphosphokinase. I wish to express my appreciation to my colleagues Drs Thomas W. Burns, J. Earle White and Nathaniel Winer for their encouragement and helpful comments, and to Mrs Gloria Patterson and Miss Karen Stone for typing the manuscript.
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8, 83. BERG, P. & JOKLIK, W. K. (1954). J. biol. Chem. 210, 657. BLAYLOCK, B. A., ROTHROCK, E. & SACKS, J. (1960). Am. J. Physiol. 198, 1063. BURROW, G. N. & BONDY, P. K. (1964). Endocrinology, 75,455. CARLIN, H. & HECHTER, 0. (1964). Proc. Sot. exp. Biol. Med. 115, 127. CHIGA, M. & PLAUT, G. W. E. (1960). J. biol. Chem. 235, 3260. CHIGA, M., ROGERS, A. E. & PLAUT, G. W. E. (1961). J. Biol. Chem. 236, 1800. CLAUSER, H., VOLFIN, P. & EBOIJE-BONIS, D. (1962). Gen. camp. Endocr. 2, 369. COLOWICK, S. P. & KALCKAR, H. M. (1943). J. biol. Chem. 148, 117. DAWSON, K. G. & BECK, J. C. (1965). Biochim. biophys. Acta, 107, 163. DEUTSCH, A. & NILSSON, R. (1954). Acfa them. stand. 8, 1106. EBOUE-BONIS, D., CHAMBAIJT, A. M., VOLFIN, P. & CLAUSER, H. (1963). Nature, Lond.
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1183. FRIEDMAN, D. L. & LARNER, J. (1962). Biochim. biophys. Acra, 64, 18.5. GELLHORN, A. & BENJAMIN, W. (1964). Science, N. Y. 146, 1166. HEPPEL, L. A., STROMINGER, J. L. & MAXWELL, E. S. (1959). Biochim. biophys. 422, HERBERT, E. & POTTER, V. R. (1956). J. biol. Chem. 222,453. HOFER, H. W. & PETTE, D. (1965). Life Sciences, 4, 1591, 1597. KELLER, E. B. & ZAMECNIK, P. C. (1956). J. biol. Chem. 221, 45, KENNEDY, E. P. (1956). Can. J. Biochem. Physiol. 34, 334.
Acta,
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KRAHL, M. E. (1961). “The Action of Insulin on Cells.” Academic Press, New York. KREBS, H. A. & HEMS, R. (1953). Biochim. biophys. Acta, 12, 172. LARMR, J., VILLAR-PALASI, C. & RICHMAN, D. J. (1959). Ann. N. Y. Acad. Sci. 82, 345. LEE, N. D. & WILLIAMS, R. H. (1954). Endocrinology, 54, 5. LEVINE, R. & GOLDSTEIN, M. S. (1955). Recentprog. Horm. Res. 11, 343. LING, K-H. & LARDY, H. A. (1954). J. Am. them. Sot. 76, 2842. LUKENS, F. D. W. (1964). Diabetes, 13, 451. MANCHESTER, K. L. (1961). Biochem. J. 81, 135. MANSOUR, T. E. (1963). J. biol. Chem. 238, 2285. MAYNE, R. & BARRY, J. M. (1965). Biochim. biophys. Acta, 107, 160. MUNCH-PETERSEN, A., KALCKAR, H. M., CUTOLO, E. & SMITH, E. E. B. (1953). Nature, Land. 172, 1036. NEWSHOLME, E. A. & RANDLE, P. J. (1962). Biochrm. J. 83, 387. NORDLIE. R. C. & LARDY. H. A. (1963). J. biol. Chem. 238, 2259. NORMAN; D., MENOZZI, P., REID,‘D., LESTER, G. & HECH~ER, 0. (1959). J. gen. Physiol. 42, 1277. PARKS, R. E., JR., ADLER, J. & COPENHAVER, J. H., JR. (1955). J. biol. Chem. 214, 693. PARKS, R. E., JR., BEN-GERSHOM, E. & LARDY, H. A. (1957). J. biol. Chem. 227,231. PASSONNEAU, J. V. & LOWRY, 0. H. (1962). Biochem. biophys. Rex Commun. 7, 10. RANDLE, P. J. & SMITH, G. H. (1960). In “The Mechanism of Action of Insulin”, ed.
F. G. Young. Oxford: Blackwell. RATLIFF,
R. L., WEAVER,
R. H., LARDY,
H. A. & KUBY,
S. A. (1964).
301. ROSS, C. R. & GIBSON, D. M. (1964).
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J. biol. Chem. 239, 1694. SANADI, D. R., GIBSON, D. M. & AYENGAR, P. (1954). Biochim. biophys. Acta, 14, 434. STEINER, D. F. & KING, J. (1964). J. biol. Chem. 239, 1292. STROMINGER, J. L. (1955). Biochim. biophys. Acfa, 16, 616. STROMINGER, J. L., HEPPEL, L. A. & MAXWELL, E. S. (1959). Biochim. biophys. Acfa, 32,412. UTTER, M. F. (1960). In “The Enzymes”, ed. P. D. Boyer, H. Lardy & K. Myrback,
vol. 2, p. 87. New York: Academic Press. VESTER, J. W. & STADIE, W. C. (1957).
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227, 669.
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VILLAR-PALASI, C. & LARNER, J. (1960). Biochim. biophys. Acta, 39, 171. VINUELA, E., SALAS, M. L. & SOLS, A. (1963). Biochem. biophys. Res. Commun. 12, 140. WAITE, M. & WAKIL, S. J. (1962). J. biol. Chem. 237, 2750. WEBER, G. & SINGHAL, R. L. (1965). Life Sciences, 4, 1993. WEBER, G., SINGHAL, R. L. & SRIVASTAVA, S. K. (1965). Proc. nafn. Acad. Sci. U.S.A. 53, 96. WOOL, I. G. (1963). Biochim. biophys. Acta, 68, 28. WOOL, I. G. & MOYER, A. N. (1964). Eiochim. biophys. Actq, 91, 248.
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Lardy, 1954). Other enzymes require specific energy donors: e.g. with fructokinase, which converts fructose to fructose-l-phosphate, ATP cannot be replaced by UTP or ITP (Parks, Ben-Gershom & Lardy, 1957). In particular, many biosynthetic pathways require specific energy donors: glycogen synthesis requires UTP (Munch-Petersen, Kalckar, Cutolo & Smith, 1953), phospholipid synthesis requires CTP (Kennedy, 1956) and incorporation of amino-acids into protein requires the presence of GTP (Keller & Zamecnik, 1956). It is therefore evident that the rate of synthesis in particular anabolic pathways may well depend on the availablity of specific nucleoside triphosphates, provided, of course, that the usually adequate amounts of substrate and intact enzyme chains are present. Production of Nucleoside Triphosphates In order to postulate an exact site for insulin action, it is necessary to review briefly the biosynthesis of the nucleoside triphosphates. Certain nucleotides are phosphorylated during the course of glycolysis and substrate oxidation in the Krebs cycle (Table 1). However, the most important product quantitatively is the ATP produced by oxidative phosphorylation. TABLE
1
Direct production of nucleosidetriphosphates
_---
Reaction
-__--
Nucleotide Produced
P-enol-pyruvate to pyruvate P-enol-pyruvate to oxalacetate
ATP, UTP, ITP, GTP, CTP GTP, ITP
Succinyl-CoA to succinate
GTP, ITP
---
Reference Strominger (1955) Nordlie & Lardy (1963) Sanadi, Gibson & Ayengar (1954)
A number of enzymes catalyzing transphosphorylation of nucleotides have been found in yeast, muscle and liver cells. 1. Nucleoside monophosphate-ATP transphosphorylases(Fig. 1: la, lb, Ic) (Strominger, Heppel & Maxwell, 1959). This group of enzymes catalyzes the phosphorylation of any nucleoside monophosphate except IMP. IDP is formed instead by the deamination of ADP (Fig. 1: Id) (Deutsch & Nilsson, 1954). Certain specific members of this group have been separated or isolated :
268
M.
D. Purine
ATP
KLACHKO
nucieotides
Pyrimidine
c
ADP
AMP\ lo
20
ADP x
nucleotldes
1 ADP”
ATP ADP
r
ADP’
L
ATP
FIG. 1. Nucleotide transphosphorylases. See text for names of enzymes. All reactions are reversible.
la, lb, lc, NMP-ATP transphosphorylases (Strominger, Heppel & Maxwell, 1959); la, Adenylate kinase; (Colowick & Kalckar, i943); Id, ADP deaminase (Deutsch & Nilsson, 1954); 2a, 2b, 2c, NTP-AMP transphosphorylases (Heppel, Strominger & Maxwell, 1959): 2a, ATP-AMP transphosphorylase (Chiga & Plaut, 1960); 2b, 6-oxypurine NTP-AMP transphosphorylase (Chiga, Rogers & Plaut, 1961) ; 3, ATP-NDP transphosphorylase (NDP Kinase) (Berg & Joklik, 1954; Krebs & Hems, 1953; Ratliffe, Weaver, Lardy & Kuby, 1964). NMP, NDP, NTP; nucleoside mono-, di-, triphosphate.
(A) ATP+AMP
F? 2 ADP.
This enzyme, adenylate kinase, was the first nucleotide transphosphorylase discovered (Colowick & Kalckar, 1943). The fractionation procedure used by Strominger, Heppel & Maxwell (1959) completely removed the activity of the enzyme responsiblefor the reaction : (B) ATP+GMP
ti ADP+GDP,
whereas the majority of the remaining activity catalyzed the reaction: (C) ATP+UMP
(CMP) F? ADP+UDP
(CDP).
2. Nucleoside triphosphate-AMP transphosphoryluses(Fig. 1: 2a, 2b, 2c) (Heppel, Strominger & Maxwell, 1959). (A) 2 ADP P AMP+ATP. This specific ATP-AMP transphosphorylase, similar to the adenylate kinase of muscle described above, has been isolated from mammalian liver (Chiga & Plaut, 1960). (B) ADP+GDP (IDP) P AMP+GTP (ITP). This separate fraction, 6-oxypurine nucleoside triphosphate-AMP transphosphorylase, has been partially purified (Chiga, Rogers & Plaut. 1961).
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(C) ADP+ UDP (CDP) it AMP+UTP (CTP). Although this reaction has been found to occur, the enzyme responsible has not yet been isolated. 3. ATP-nucleoside diphosphate transphosphorylase (Nucleoside diphosphokinase) (Fig. 1 : 3) (Berg & Joklik, 1954; Krebs & Hems, 1953; Ratliff, Weaver, Lardy & Kuby, 1964). This is a single enzyme which has been isolated in crystalline form (Ratliff et al., 1964). It catalyzes the phosphorylation, by ATP, of a number of purine and pyrimidine ribo- or desoxyribonucleoside diphosphates to the respective triphosphates. The above enzymes provide a network of transphosphorylations of nucleotides and thereby regulate the balance between the various nucleoside triphosphates. It can be seen that factors affecting one or more of these enzymes can influence the concentration of particular nucleoside triphosphates, and so affect the rate of biosynthesis in the pathways controlled by those nucleoside triphosphates. The mitochondria contain nucleoside diphosphokinase but lack nucleoside monophosphate kinases except for adenylate kinase. The adenosine nucleotides are preferentially phosphorylated by mitochondria (Herbert & Potter, 1956) and, with the nucleoside monophosphate kinases, no transphosphorylation has yet been discovered in which neither reactant contains adenosine (Strominger, Heppel & Maxwell, 1959). It thus appears that ATP formation is the primary result of oxidative phosphorylation and the high energy phosphate is then transferred to other nucleotides by the reactions described above. The probable course of “energy flow” is depicted in Fig. 2. Monosaccharides Amino acids
co2 “20
Creatjne
P
FIG. 2. “Energy flow”. 1. Oxidative phosphorylation; 2. Nucleoside diphosphokinase. NMP, NDP, NTP: mono-, di-, and triphosphates of nucleosides other than adenosine. T.B. 18
270
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The Mechanism of Action of Insulin The effects of insulin include: (a) increased synthesis of glycogen, proteins, fatty acids (Krahl, 1961) and RNA (Wool, 1963); (b) suppression of biosynthesis in liver of the key gluconeogenetic enzymes induced by glucocorticoids (Weber, Singhal & Srivastava, 1965); (c) induction of biosynthesis in liver of the key glycolytic enzymes (Weber & Singhal, 1965), glycogen synthetase (Steiner & King, 1964), and enzymes for synthesis of fatty acids (Gellhorn & Benjamin, 1964); (d) activation of glycogen synthetase from the glucose-6-phosphate dependent (D) to the glucose-6-phosphate independent (I) form (Villar-Palasi & Larner, 1960); (e) increased transport of sugars and some amino-acids into cells of muscle and adipose tissue; (f) increased glycolysis in muscle and adipose tissue; (g) increased incorporation of inorganic phosphate into nucleotide phosphate in rat diaphragm muscle (Clauser, Volfin & Eboue-Bonis, 1962). Recently there has been great interest in the apparent independence of many of these effects from each other. In diabetic rats, actinomycin D blocks the action of insulin on accumulation of hepatic glycogen and repair of the enzymatic defects in synthesis of fatty acids by adipose tissue and mono-unsaturated fatty acids by liver microsomes. However, it does not block the hypoglycemic action of insulin (Gellhorn & Benjamin, 1964). Inhibitors of protein synthesis, such as puromycin and ethionine, also prevent new enzyme synthesis without preventing the hypoglycemic effect of insulin (Steiner & King, 1964). In vitro, despite inhibition of RNA synthesis by actinomycin, insulin stimulates protein synthesis, uptake of glucose and aminoisobutyric acid, and incorporation of inorganic phosphate into nucleotide phosphate in rat diaphragm muscle (Wool & Moyer, 1964; Eboue-Bonis, Chambaut, Volfin & Clauser, 1963), and fatty acid synthesis by mammary gland slices (Mayne & Barry, 1965). In the presence of puromycin inhibition of protein synthesis, insulin continues to stimulate the uptake of glucose, galactose, d-xylose and aminoisobutyric acid, RNA synthesis, and incorporation of inorganic phosphate into nucleotide phosphate in rat diaphragm muscle (Carlin & Hechter, 1964; Burrow & Bondy, 1964; Eboue-Bonis et al., 1963) as well as oxidation of glucose and leucine, and lipid synthesis in rat epididymal fat pads (Dawson & Beck, 1965). These findings suggest that the increased protein and RNA synthesis may be secondary to a more fundamental effect of insulin: that of stimulating anabolic processes. However, the induction or repression of enzyme formation more probably is due to the stimulation of new RNA and protein synthesis in
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conjunction with the mechanisms of substrate induction and end-product inhibition. The basic effect of insulin as a potent stimulator of all anabolic processes had long been considered to be related to an increased provision of substrates in the cell. Initially this was thought to be specifically due to the increased transport into cells of glucose (Levine & Goldstein, 1955). Later experiments have shown that insulin stimulates glycogen formation to a greater extent than can be accounted for by the increase in glucose uptake (Norman, Menozzi, Reid, Lester & Hechter, 1959; Larner, Villar-Palasi & Richman, 1959). Even in the absence of extracellular glucose, insulin stimulates the biosynthesis of proteins from labelled precursors (Manchester, 1961), and increases labelling of RNA from [i4C]adenine (Wool, 1963). It is also accepted that the stimulation of protein synthesis is independent of any other effect of insulin on aminoacid transport into the ceil (Lukens, 1964). The possibility that insulin acts by increasing the amount of ATP in the cell has been investigated. Conflicting results have been reported. ATP formation was normal in liver mitochondria of alloxan-diabetic rats (Parks, Adler & Copenhaver, 1955), but was decreased in those from depancreatized cats (Vester & Stadie, 1957). In 1960, Blaylock, Rothrock & Sacks found decreased levels of ATP in the livers of fed, alloxan-diabetic rats, but insulin injection caused increased glycogen synthesis despite a continued low concentration of ATP. Although ATP is the prime source of energy for muscular work, ion transport and many other cellular functions, it is not the final “energy donor” for many anabolic reactions. As described earlier, other specific nucleoside triphosphates are required. Clauser, Volfin & Eboue-Bonis (1962) showed that insulin stimulates the incorporation of labelled inorgamc phosphate into nucleotide phosphate in rat diaphragm muscle, and causes an increase in the ratio of phosphate incorporated into guanosine and uridine nucleotides, relative to that incorporated into adenosine nucleotides, both in the presence and absence of glucose. This effect of insulin cannot be inhibited by either puromycin or actinomycin (Eboue-Bonis et al., 1963). It therefore appears probable that insulin stimulates anabolic processes by increasing the rate of transfer of high energy phosphate from ATP to the other nucleotides. The effects of insulin on glucose metabolism also require consideration. Both glucose transport and glycolysis in muscle and adipose tissue are increased by anoxia and agents which uncouple oxidative phosphorylation. This is presumed to be a mechanism for the maintenance of a relatively constant intracellular concentration of ATP and it has been suggested that the membrane barrier to the uptake of sugars is maintained by a “high
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energy” phosphate compound which may be ATP (Randle & Smith, 1960). The rate-limiting step in glycolysis is the enzyme phosphofructokinase (Newsholme & Randle, 1962). Isolation of the enzyme has shown that it contains a nucleic acid, or possibly a mixture of nucleotides, and that the net forward rate of the phosphorylation of fructose-6-phosphate to fructose1,6-diphosphate depends on the amount of nucleotide present (Hofer & Pette, 1965). Ling & Lardy (1954) reported that phosphofructokinase can utilize UTP and ITP as efficiently as ATP. ATP can also be replaced by CTP or GTP (Utter, 1960). High concentrations of ATP inhibit the phosphorylation of fructose-6-phosphate (Mansour, 1963 ; Passonneau & Lowry, 1962) but, at least with yeast phosphofructokinase, GTP does not (Vinuela, Salas & Sols, 1963). The above data are consistent with the hypothesis that insulin acts by accelerating the transfer of “high energy” phosphate from ATP to the non-adenosine nucleoside diphosphates thus causing an initial fall in the peripheral concentration of ATP in the cell and consequent stimulation of glucose uptake and glycolysis. The same mechanism may explain the conversion of glycogen synthetase from the D form to the I form as the D enzyme is a phosphorylated form of the I enzyme and is maintained in this state by ATP and Mg2+ (Friedman & Larner, 1962). Within the cell, numerous enzymes compete for available ATP. The “flow of energy” within the cell would be regulated by the location of the ATP as well as by the relative affinity of the enzymes for ATP. That changes in enzyme affinity for ATP is a control mechanism in the cell is suggested by the fact that the stimulation of acetyl-coenzyme A carboxylase by isocitrate, in vitro, is associated with a decrease in the Km for ATP by a factor of 5 (Waite & Wakil, 1962). The Site of Insulin Action
Lee & Williams (1954), using [‘3’I]insulin, showed that at least 667; of insulin in liver was intracellular within five minutes after intravenous administration. In epididymal adipose tissue, in vitro, insulin causes invagination of the cell membrane with formation of pinocytotic vesicles as well as changes in the mitochondria suggestive of increased activity or growth (Barrnett & Ball, 1960). These changes occur with or without glucose in the medium, and pinocytosis may be the means by which the insulin molecules enter the fat cell. As detailed above, the action of insulin could be explained by an increased rate of transfer of “high energy” phosphate from ATP to non-adenosine nucleotides. Anabolic processes require particularly large amounts of energy. For example, the incorporation of each glucose molecule into
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glycogen or each choline molecule into lecithin is associated with the conversion of a nucleoside triphosphate to the corresponding nucleoside monophosphate. This then requires two molecules of ATP and participation of at least two enzymes for the regeneration of the one triphosphate. The widespread stimulation of anabolism produced by insulin would require an increased rate of formation of UTP for glycogen synthesis and GTP for protein synthesis, whereas RNA synthesis requires the simultaneous presence of UTP, GTP, CTP and ATP. Although it is possible that insulin could act on all the nucleotide transphosphorylases, all its effects could be explained by a stimulation of the single enzyme nucleoside diphosphokinase (Fig. I : 3) which accelerates the phosphorylation of all nucleoside diphosphates at the expense of ATP. Considered teleologically, at times of food intake and excess of available substrate, insulin switches the pattern of metabolism from catering primarily to immediate needs to one promoting a state of general synthesis and storage. Other peptide hormones may affect other nucleotide transphosphorylases, leading to a relative preponderance of single nucleoside triphosphates and stimulating particular metabolic pathways. The possible actions of peptide hormones on this network of nucleotide transphosphorylases, leading to competition between them for available ATP, could provide an explanation for the many synergistic and antagonistic effects of such hormones. Conclusion
Nucleotides not only carry the genetic code that determines the overall metabolic constitution of every cell, but also appear to direct the energy flow to specific anabolic pathways. It is postulated that certain peptide hormones act on the nucleotide transphosphorylases to regulate the flow of “high energy” phosphate from ATP, thus determining the relative quantities of nucleoside triphosphates in the cell and indirectly controlling the pattern of intermediary metabolism. The effects of insulin could be explained on the basis of an increased rate of transfer of “high energy” phosphate from ATP to all non-adenosine nucleoside diphosphates, causing an increased level of all non-adenosine nucleoside triphosphates resulting in general increases in rates of synthesis and storage within the cell. A possible single site for this major action of insulin is the enzyme nucleoside diphosphokinase. I wish to express my appreciation to my colleagues Drs Thomas W. Burns, J. Earle White and Nathaniel Winer for their encouragement and helpful comments, and to Mrs Gloria Patterson and Miss Karen Stone for typing the manuscript.
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