Mechanism of hormone action. Role of nucleotides in gluconeogenesis

Life Sciences Vol . 8, Part II, pp. 427-434, 1989. Printed in Great Britain.

MECHANISM OF HORMONE ACTION .

Perga .mon Press

ROLE OF NOCLEOTIDES IN GLIICONEOGENESIS* N. R. Sarkar

Animal Research Institute, Research Branch, Canada Department of Agriculture, Ottawa, Canada

(Received 10 December 1968; in final form 29 Jam~ary 1969) The glycogenolytic effect of epinephrine and glucagon is known to be medsated through their actions on adenyl cyclase, the enzyme which catalyzes the formation of cyclic 3'-5'-AMP from ATP (1, 2) .

The comrersion of phosphoryLsse

b (inactive form) to phoephorylase a (the active form) is greatly accelerated by thin cyclic nucleotide (3, 4) .

The gluconeogenic effect of glucagon is also

known to be mediated through the action of cyclic AMP (4-7) .

This nucleotide,

according to some workers (8, 9) increases the activity of pyruvate carboaylase and according to others activates lipase activity (10,

11) .

Cyclic AMP also in-

hibits fructose-1, 6-diphosphatase (FDPase) activity (12) as 5'-AMP does (12-14) . The latter nucleotide can also stimulate phosphofructokinase (PFR) (15) . Although these nucleotides influence the activities of many important enzymes in glycogenolysis, glycolyaie, and gluconeogenesis, their effects on the activities of glucose-6-phosphatase (G-6-Pase), the last enzyme operating in gluconeogenic pathway, are not known .

It is also not certain whether or not the

inhibitory effect of cyclic AMP on FDPase as observed by Mendicino et al . (12) is caused by the cyclic compound itself or by its hgdrolytic product 5'-AMP . Because of the importance of these nucleotides in contro111nA the rate of carbohydrate metabolism, the effects of various nucleotides on G-6-Pace and FDPase have been investigated .

The results are presented in this paper .

* Presented at the 5th Meeting of the Federation of European Biochemical Societies held at Prague, Czechoslovalia, July 15-20, 1968 .

427

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Materiale and Methods The cell-free extract was prepared by centrifuging a lOX rat liver hanogenate, made in 0 .25H sucrose, between 600xg and 2000xg for 10 min at OC ° in an International Refrigerated Centrifuge . and FDPase enzymes .

This was used as the source of G-6-Pase

The deoxycholate-treated rat liver microsomes, prepared

according to previously published method (16), were also used as the source of G-6-Pase .

The high speed supernatant fraction, obtained by centrifuging the

cell-free extract at 40,000 rpm for 2 hr 1n a preparative ultracentrifuge (Model L), also served as the source of FDPase in the present study .

Partially

purified FDPase was prepared according to the method described by Pontremoli et al .

(17) . The activities of G-6-Pase and FDPase were determined by the methods of

Swanson (18) and Freedlaad and Harper (19) respectively, inorganic phosphorus by the method of Fiske and Subba Row (20), and protein by the Biuret method (21) . G-6-Pase, FDPase, and cyclic 3'-5'-AMP were purchased from the Sigma Chemical Company, 5'-AMP, 5' -GTSP , 5'-GMP, d5'-AMP, ~3'-AMP, ATP, dATP, GTP, CTP and UTP from Mann Research Laboratories, and partially purified venom phosphodieaterase from Calbiochem .

Crotalus adamenteus venom was a gift from Professor Find lay E .

Russell . Results The hydrolysis of glucose-6-phosphate (G-6-P) and fructose-1, 6~iphosphate (PDP) by their respective enzymes present in cell-free extract was significantly inhibited by cyclic AMP and 5'-AMP namely cyclic AMP at 2mM concentration, in hibited the activities of G-6-Pase and FDPase by 38X and 42X, whereas inhibibona of 44X and 52X were noted in the presence of the concentration of 5'-AMP . Much reduced inhibitions were observed in the presence of lower concentrations of these nucleotides (Table I) .

3'-AMP, d5'-AMP, d5'-AMP, d5'-CMP and cyclic

2'-3'-AMP were not significantly effective .

At 2mM concentration 5'-CHP was

only partially effective, namely inhibitions of 12X and 17X were observed . Hydrolysis of FDP by high speed supernatant fraction was not inhibited by cyclic

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AMP, but in the presence of 5'-AMP an inhibition of 40X was noted . TABLE II The Effects of Triphoephates of Various Nucleotides, Pyrophosphate, Orthophosphate and Citrate on Glucose-6-phosphatase and Fructose-1, 6-diphoephatase . Nucleotide Addition

G-6-Pace (cell-free preparation) Inhibition in X

FDPase (cell-free preparation) Inhibition in X

None ATP (10)

95

97

ATP

(5)

49

51

ATP

~2)

14

16

GTP (10)

0

0

CTP (10)

7

6

UTP (10)

9

7

dATP (10)

1

2

ADP (10)

86

82

Pyrophosphate (sodium salt)

(10)

80

75

Pyrophosphate (sodium salt)

(5)

67

60

Orthophosphate (sodium salt)

(10)

4

7

Citrate

(2)

7

4

Conditions of the experiment are the same as described in Table I . The effects of ATP, ADP, pyrophosphate (PP), orthophosphate (Pi) and citrate on the hydrolysis of G-6-P and FDP by their respective enzymes present .in the cell-free extract are shown in Table II .

Hydrolysis of G-6-P and FDD were

inhibited by 95X and 97X in the presence of ATP,

86X and 82X in the presence of

ADP, and 67X and 60X in the presence of PP respectively, at 10 mM concentration . ATP at 2mM concentration, inhibited their hydrolysis by only 14X and 16X . Ortho-

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phosphate at lOmli was not active, nor citrate at 2mM was effective . Hydrolysis of G-6-P by deoxycholate-treated microeomea fractionated between 30 to 40X (NH4)2 50 4 was also inhibited by all the nucleotides, nucleotide triphoaphates and PP to the same extents as noted when cell-free extract was used (not ahawa) . TABLE IÎI The Effect of Venom Phosphodiesterase on Cyclic 3'-5'-AMP and Venom 5'Jnucleotidase on 5'-AMP in flelation to Their Actions on Purified Fructose-1, 6-diphosphatase Activity . Nucleotide Addition

FDPase activity (umolea of P1 liberated)

Inhibition in X

None

4 .60

ATP (ADP or PP)

4 .54

Cyclic 3'-5'-AMP

4 .56

Cyclic 3'-5'-AMP + venom phoaphodieaterase

3,.21

30

5'-AMP

2.55

44

5'-AMP + venom 5'-nucleotidase

4 .52

2

Cyclic 3'-5'-AMP (20 umolea) was incubated with partially purified venom phosphodieateraee (50 unite) in the presence of 25 umolea of tris-HC1 buffer pH 8.6 and 2 .5 ymolea of MgCl p , in a total volume of 1 ml at 37 °C for 30 min . 0 .1 ml of this mixture was added to the assay system whic~contained partially purified FDPase (or soluble fraction) . Addition of Mg was omitted . 5'-AMP (20 ymolea) was incubated with 100 yg of snake (Crotalus adamenteus) venom in the presence of 25 umolea of tris buffer pA 8.4 and 2 .5 ymolea of MgC1 2 for 30 min . 0.1 ml of thin mixture was added ~ the assay system which contained partially purified FDPase . Addition of MR was omitted . The results of Table III show the inability of cyclic AMP, ATP, ADP and PP to inhibit the hydrolysis of FDP by partially purified FDPase .

A 30X inhibition

was observed in the presence of venom phoaphodieaterase treated cyclic AMP, 5'-AMP inhibited the hydrolysis of FDP by the purified FDPase by 38X .

This

latter nucleotide was not effective as an inhibitor after treatment with venom 5'-nucleotidase (Table III) .

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Discussion The cyclic AMP and 5'-AMP were both found to be capable in inhibiting significantly the hydrolysis of FDP by FDPase and G-6-P by G-6-Pass when cell-free extract was used as the source of the enzymes .

The hydrolysis of FDP was not

affected by cyclic AMP when the high speed supernatant fraction as the source of FDPase or partially purified FDPase was used . hydrolysis of FDP under these conditions .

Aowever, 5`-AMP inhibited the

The cyclic AMP also did, but only

after treatment with venom phosphodiesterase .

Since venom phodieaterase hydro-

lyses cyclic AMP to 5'-AMP and this enzyme occurs in the liver, it is reasonable to assume that cyclic AMP'exerta its inhibitory effect through the formation of 5'-AMP .

The inhibition of G-6-Pass activity by cyclic AMP may have also been

caused by 5'-AMP although the failure of the cyclic nucleotide to inhibit G-6Pass activity could not be demonstrated because G-6-Pace free from phosphodieaterase activity could not be prepared .

It is to be noted that deoxycholate-

treated rat liver microsomea, fractionated between 30 to 40% saturation of (NA4) 2 504 , showed the presence of both the enzymes . The inhibitory effects of ATP, ADP and PP on FDPase noted when cell-free extract or high speed supernatant fraction was used as the source of the enzyme, could not be detected when the hydrolysis of FDP was catalyzed by partially pur ified FDPase .

Mendicino et al .

(12) did not âlso notice any inhibition of puri-

fled FDPase activity by ATP, but did notice an inhibition of FDPase activity when swine kidney extract was also present in the assay system .

They suggested

that the cause of inhibition was due to some structural changes in the enzyme molecule induced by ATP, making the enzyme unable to form enzyme-substrate complex .

However, the observed inhibition can also be attributed to the action of

5-AMP which can be formed under the conditions of experiments employed by Mendicino et al . (12) .

Which one of the two above suggestions can best explain

the observed facts is now under investigation .

The inhibition of G-6-Pass and

FDPase activities by ATF, ADP and PP does not appear to be significantly important in regulating glycolyais and gluconeogeneais since inhibitions are observed

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only at high concentrations of these substances .

433

The inhibitions of G-6-Pace

and FDPase by 5'-AMP and the stimulation of PFK by the same nucleotide are, on the other hand, observed at much lower concentrations . The stimulating effect of cyclic AMP on many enzyme catalyzed reactions has now been well established (4)

Pasaonnesu and Lowry (15) noted increased PFR

activity in the presence of cyclic AMP .

Activation of vyruvate carboxylase,

adipose tissue lipase, and hepatic lipase by cyclic AMP has also been reported . The metabolic effects of epinephrine and glucagon in the liver are also mediated through the action of this cyclic nucleotide .

Although cyclic AMP has been im-

plicated in a wide variety of enzyme catalyzed reactions, it has not been clearly demonstrated whether or not cyclic AMP exerts its stimulating or inhibitory effect through itself or through the formation of 5'-AMP .

In many cases, where

cyclic AMP has been found to be effective, the conditions for 5'-AMP formation cannot be ruled out .

It is also interesting to note that highly purified PFR

activity can be stimulated by 5'-AMP but not by cyclic AMP (16) .

Furthermore,

the latter compound unlike 5'-AMP cannot inhibit purified FDPase activity .

Un-

til the stimulating action of cyclic AMP can be demonstrated with purified enzymes, the importance of 5'-AMP in regulating glycolysis and gluconeogenesis should not be overlooked . Summary The hydrolysis of G-6-P and FDP by their respective enzymes present in the cell-free preparation was significantly inhibited by cyclic 3'-5'-AMP and 5'-AMP . Cyclic 2'-3'-AMP, 3'-AMP, d5'-AMP, 5'-UMP or 5'-GMP could not prevent the hydro lysis of G-6-P and FDP, but ATP, ADP and PP did when used at 10 mM concentration . dA~P, GTP, CTP, UTP, orthophosphate and citrate were all inactive .

Among all

the nucleotides tested, only 5'-AMP was capable of inhibiting the hydrolysis of FDP by purified FDPase .

The hydrolysis of FDP was inhibited by ATP, ADP and PP

when cell-free extract or high speed supernatant fraction was used as the source of FDPase but not when purified FDPase was used instead .

Cyclic A~iP was active

in inhibiting the activity purified FDPase only after treatment with venom nhos-

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phodiesterase .

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The physiological significance of 5'-AMP and cyclic 3'-5'-AMP

se metabolic regulators was discussed . References 1.

E . W . SUTHERLAND and T. W. RALL, J . Biol . Chem . 232,

2.

E . W. SUTHERLAND, I . OYE and R . W . BUTCHER, Recent Prog . in Horm . Res . 21, 632 (1965) . -

3.

E . G. KREBS, C . GONZALES, J . B . POSNFR, D . S. LORE, G. E . BRATVOLD and E . H. Fischer, in Control of Glycogen Metabolism , p. 200 J. and A. Churchill, London 1964 .

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A . Robinson, R . W. Butcher, and E . W. Sutherland, Ann. Rev . Biochem. 37, 149 (1968) . -

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J . ASAMORE and G . WEBER, in Carbohydrate Metabolism , Vol . I, 333 (1968) .

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J . LARNER, Ann . N .Y . Acad . Sci . 29 (2) , 192 (1967) .

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L. MENAHAN and 0 . WIELAND, Biochem .- Bi ophys . Rea. Commu ., 29, 880,

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B. D . ROSS, R. HII~SS, and H. A. KREBS, Bioc hem . J., 102,

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J. H. SRTON, and C . R . PARK, Pharmacol . Rev .,

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10 . E. STRUCK, J . ASAMORE, and 0. WIELAND, Adv . Enzyme Regulation . 4,

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11 . J. R. WILLIAMSON, ibid . 5 , 229 1967 12 . J. MENDICINO, C . BEAUDREAU and R . N. BHATTACHARYA, Arch . Biochem. Biophys . 116, 436 (1966) . 13 . M. SALAS, E . VINUELA, J. SALAS and A . Sols, Biochem . Biophys . Res . Commu . 17, 150 (1964) . 14 . J. MENDICINO and F . VASARHELY, J. Biol . Chem . 238, 2259, (1963) . 15 . J. V . PASSONNEAU and 0 . H . LOWRY, Advances in Enzyme Regulations , Vol . 2, 265 (1964) . 16 . R. C . NORDLIE and W . J . ARION, in Methods in Enzymology , Vol . IX,Carbohydrate Metabolism, ed . Willis A. Wood, Acad . Press N.Y ., p . 619 (1966) . 17 . S . PONTREMOLI, S . TRANIELLO, B . LUPPIS and W. A . WOOD, J . Biol . Chem ., 240, 3459 (1965) . 18 . M. A . SWANSON, Methods in Enzymology , Vol . II, p. 541 (1955) . 19 . R . A. FREEDLAND and A . G . HARPER, J. Biol . Chem . 234,

1350 (1959) .

20 . C . H. FISRE and Y. SUBBA ROW, J . Biol . Chem . 66, 375 (1925) . 21 . R. W. CLELAND and E. C . SLATER, Biochem. J . 53, 548 (1953) .