Effect of insulin on the phosphorus-32 mononucleotide and phosphocreatine labelling in the isolated diaphragm of the normal, the hypophysectomized, and the hypophysectomized growth-hormone treated rat

GENERAL

.4ND

COMPARATIVE

Effect

of Insulin

creatine

on the

Labelling

the

Phosphorus-32 in

the

de Chimie

biologirlue

Mononucleotide

Isolated

Hypophysectomized, Growth-Hormone H. CLAUSER,

Laboratoire

2, 369-384 (1962)

ENDOCRINOLOGY

and

P. VOLFIN,

de la FacultS

Diaphragm

and of

the

PhosphoNormal,

the Hypophysectomized Treated Rat’ AND D. EBOUI&BONIS

des Sciences,

Received February

96 Boulevard

Rnspail,

Paris

VI,

Francr

26, 1962

The rate of penetration of inorganic phosphate (NaZH3*P0,) and the turnover of the acid-soluble organic phosphates, including free nucleotides, has been determined on the isolated diaphragm of the normal, the hypophysectomized and the hypophysectomized growth-hormone treated rat. The influence of insulin on this turnover has been studied in the presence and in the absence of glucose. Insulin has no effect on the rate of penetration of inorganic phosphate into the intracellular space, but promotes, in every case, a striking enhancement of the labelling of organic phosphates, both in the presence and in the absence of extracellular glucose. Insulin specifically increases the labelling of the second phosphate of ATP both in the normal diaphragm and in the diaphragm of hypophysectomized rats, when no substrate is added to the incubation medium. With the diaphragm of the hypophysectomized rat, in the presence of glucose, and with the diaphragm of the hypophysectomized growth-hormone treated rat, the labelling of the terminal phosphate of ATP is specifically increased by insulin. Significant effects of insulin and growth hormone on uridineand guanosine-nucleotide labelling are also observed. The significance of these results is discussed with respect to the metabolic patterns, which are obtained in the isolated diaphragm under various physiological conditions. INTRODUCTION

Since the demonstration by Levine and Goldstein (1955) of a direct effect of insulin on the transport and the distribution space of metabolizable and nonmetabolizable sugars in the peripheral tissues of the eviscerated dog, recent work on the mode of action of this hormone has been mainly focused on the question of how this action could be obtained on either muscle or adipose tissue, t.he two main tissues concerned. ‘Abbreviations used are as follows: TCA, trichloroacetic acid; I.P., inorganic phosphate ; P.C., phosphocreatine ; AMP, ADP, ATP, adenosine mono-, di- and triphosphate; similarly for UMP, UDP, UTP (uridine), GMP, GDP, GTP, (guanosine) ; UDPG, uridine diphosphate glucose ; IJDPAG, uridine diphosphoacetylglurosamine. 369

The main work on muscle has been carried out on the isolated “caged” rat diaphragm (Kipnis, 1959; Kipnis and Cori, 195’7, 1959) and on the isolated perfused rat heart (Morgan et al., 1961a,b). Conclusive evidence that insulin directly affects the transport of metabolizable and nonmetabolizable sugars into the cell or from the cell to the perfusion medium has been obtained. Investigations on the adipose tissue have been mainly concentrated on t,he isolated epididymal fat-pad of the rat (Winegrad and Renold, 1958; Ball and Cooper, 1960; Ball et al., 1959) : increase of the R.Q. to values superior to 1, indicating biosynthesis of long-chain fatty acids has been observed. This effect exhibited a complete

glucose-dependence, strongly suggesting that the stimulation of fatty-acid synthesis could be mediated by an increased penetration of glucose into the cell of adipose tissue. Within the last few years, however, several groups of investigators have insisted on the fact that insulin exerts clear-cut effects on these tissues in the absence of added glucose : thus, Manchester and Young (1958, 1960)) Wool and Krahl (1959) and Manchester and Krahl (1959) reported an increased labelling of proteins from C14-labelled precursors in the isolated rat-diaphragm, and the same effect was observed by Krahl (1959) on the isolated adipose tissue. Simultaneously, Barrnett and Ball (1960) observed ultrastruct,ural changes in adipose tissue under the influence of insulin. Their conclusions were that insulin initiates pinocytosis in adipose tissue, the presence of glucose having no on this process. influence whatsoever Finally it must be noted that an insulin effect, which could not be explained by increased penetration of glucose into the cell, has been observed on glucose metabolism by Beloff-Chain et al. (1959). In view of these results, an investigation of the effect of insulin on the surviving tissue has been undertaken from a different point of view. Most metabolic changes in the tissue are bound t,o be reflected in the turnover of the nucleotide coenzymes, including the adenosine-nucleot.ides, the role of which in the act.ivation of sugars, fatty acids (Berg, 1956) and amino acids (Hoagland, 1955) is known. The study of the turnover of labile phosphat,es, such as phosphocreatine and ATP, was undertaken several years ago on the isolated rat diaphragm by Sacks (1952)) who observed a stimulat,ion in the presence of insulin and glucose; no separate determination of phosphate penetration and turnover of the labile fract,ions within t.he cell was carried out in this work and the different phosphates of ATP, uridineand guanosine-nucleotides were not separated. The same author later (Sacks, 1959), studied the labelling of phosphorylated intermediates in the cat gastrocnemius muscle, stimulated in situ.

It seems, howcvcr, that. the study of the turnover of labile phosphates in the muscle of the living animal is not an easy task, randomizat,ion of the labelling being cxtremely rapid under these conditions. This has been established by Fleckenst,ein et al. (1959a, also Fleckenstein and .Janke, 1959) and unpublished experiments on the rat diaphragm (Volfin et nl., 1960) confirm these views. Conversely it was found (Volfin et al., 1961b,c) that in the surviving rat diaphragm, equilibration of the different organic phosphates with their common precursor, intracellular inorganic phosphate, is much slower: hence an insulin effect on this labelling could be demonstrated. The present work reports the results of an invest.igation of the in vitro effect of insulin on the penetration of inorganic phosphate and on t,he labelling of organic phosphates in the excised diaphragm of normal, hypophysectomized, and hypophysectomized growth-hormone inject,ed rats. MATERIALS

AND

METHODS

REAGENTS Analytical grade reagents and glass redistilled water were used. Isohutanol was distilled twice (on CaO and H,PO,) from a commercial grade reagent. The charcoal used for the nucleotide adsorption was Norit SX 11 (N. V. Norit, Amsterdam), similar to the SX 30 used previousl? (Volfin et ol., 1961a). The substrates used were n-glucose (Hoffmann La Roche, Paris) and DLP-hydroxybutyrate (Sodium Salt, B.D.H., London). Insulin (N. V. Org:non, Oss, Holland and Laboratoires de 1’Endopancrine Gisors, France) consisted of two samples: a crystallized sample (25.9 I.U. per mg), containing 0.2% of zinc and an amorphous one (22.5 I.U. per mg) containing no zinc. No differences were found between the actions of the two samples. The bovine growth-hormone was an electrophoretically-homogeneous preparation, kindly provided by Professor C. H. Li (1954). Radioactive phosphate (NaHz3*P04) from the C.E.A. (Saclay) was used at the concentration of 100-200 &! per ml in 0.9% NaCl after previous hydrolysis and neutralization, in order to eliminate any pyrophosphate present. Radioactivity was counted with a TGC2 Tracerlab Geiger counter, after drying of the sample on a small glass counting dish.

INSULIN

AND PHOSPHATE

ANIMALS Female W&tar Rats in a weight range of 199 to 150 gm were used. The weights of the animals were matched in order to avoid extensive variations of the weight of the diaphragms. Hypophysectomy was performed from 2 to 6 weeks heforc the rats were used and was checked by inspection of the sella turcicn; animals were discarded when completeness of removal of the pituitary gland was uncertain. Two injections of growth-hormone (250 fig each) were administered subcutaneously in 0.5 ml of 1.3% NaHCO,, 30 and 20 hours before the animals were killed. The animals were fasted for 18 hours before the csperiment was carried out. INCUBATION

PROCEDURE

The animals were killed by decapitation and the diaphragm dissected rapidly on an ice-chilled plate: only the la.teral portions of the muscle were used for the experiment. Washing of the diaphragm took place for 5 minutes at 37” in a glucose-free Krebs-Ringer bicarbonate medium (Umbreit et al., 1951) in an atmosphere of 95% 02 plus 5% co,.z The diaphragms were then carefully blotted and reincubated separately in 2.5 ml of Krebs-Ringer bicarbonate medium (containing Mg+- at the concentration of 5.9 X 1O-3 or 11.2 X 10.-“M) at 37”, in 95% 02 plus 5% CO?, with or without substrates and 200 mU (10 pg)3 insulin per ml, as specified below. Usually four experiments with four diaphragms were performed simultaneously. Gassing (5 minutes) and preincubation (10 minutes) took place under constant shaking. After this period, NabH3’P0, (19-20 pC in 0.1 ml isotonic NaCl/NaHC03 was tipped into the medium and the incubation continued for 10 or 30 minutes. The diaphragms were then rapidly transferred to a chilled plate, and were blotted, weighed and homogenized in 10 ml of 10% TCA, in a chilled “Virtis 45” homogenizer. The TCA extract was centrifuged at 0” for 20 ‘It is well known (Brown et al., 1952) that washing of the diaphragm promotes an increase of the glucose uptake: the procedure outlined limits this increase, glucose uptake in the presence of insulin being on the average 70-1007~ higher than in the controls under the experimental conditions used. ‘Note added in proof: Similar effects have been observed with 20 mp (1 pg) and 2 mp (0.1 ,sg) per ml, in presence of bovine serum albumin (1 mg/ml). A detailed report on the correlation between dose and effect will be given in a forthcoming paper.

371

IN RAT DIAPHRAGM minutes at 18,060 X g and processed as described below.

the

supernatant

FRACTIONATION OF THE TCA-EXTRACT The total phosphate and total radioactivity of the extract, as well as the extracellular phosphate and radioactivity in the incubation medium, were determined as desrribed in a previous work (Volfin et al., 1962). All phosphate determinations were performed according to the method of Berenblum and Chain (1938). Separation of inorganic phosphate and phosphocreatine was carried out as follows: 1.5 ml of the ice-cold extract was added to 05 ml of 2.5% ammoniummolybdate and 1.5 ml isobutanol in a chilled, glass-stoppered, graduated tube and shaken for 30 seconds. The lower phase, containing unhydrolyzed organic phosphates, was immediately transferred into another graduated tube, care being taken that no contamination with the upper phase occurred, even if this procedure involved small losses of the lower phase. The isobutanol phase was adjusted to 5 ml with isobutanol, and aliquots taken for phosphate determination (0.5 ml) and radioactivity measurement (0.1 ml) of the inorganic phosphate. The lower phase was allowed to stand at room temperature for 1 hour, after which it was adjusted to 2.5 ml with isobutanol-saturated water and extracted with 2.5 ml of water-saturated isobutanol. The upper phase contained phosphate arising from the hydrolysis of phosphocreatine. Aliquots were taken for the determination of phosphate (0.5 ml) and radioactivity (0.2 ml). Preliminary experiments (not reported in the present work) using mixtures of radioactive inorganic phosphate and nonradioactive phosphocreatine, indicated that no contamination of phosphocreatine with radioactive material occurred under these circumstances, and that the overall hydrolysis of phosphocreatine in the TCA extract, during homogenization, centrifugation and extraction of I.P., averaged 5% and never exceeded 10% of the phosphocreatine present. Thus, only a small amount of contamination of I.P. with phosphocreatine-phosphate is possible. This may lower the specific radioactivity of I.P. by a value of only 2-3%, and no corrections have been made to allow for this. The adsorption of nucleotides on Norit SX 11 and their separation by paper chromatography according to an adaptation of the method of Threlfall (1957) has been described in a previous paper (Volfin et al., 1961a). Nucleotide chromatographies were carried out in duplicate, one of the spots being used for phosphate determination and the other for determination of radioactivity after

372

('LAIXEI1,

VOLFIS

clution with 0.1 11: NH,OH as stated preliously (Volfin et al., 1961a, 1962). The “residual phosphate,” which in the diaphragm seems to mainly comprise phosphorylated intermediates of glycolysis. has been determined on the filtrate of the charcoal columns, which remove the nucleotides. This filtrate was transferred to a graduated tube, 1 ml of concentrated HCl and 0.5 ml of 2.5% ammonium molybdate were added, and the total volume adjusted to 10 ml. Hydrolysis was allowed to proceed at room temperature for 2-3 hours, after which extractions with equal volumes of isobutanol were performed four times, in order to remove inorganic phosphate and hydrolyzed phosphate from phosphocreatine. The lower phase was readjusted to 10 ml and aliquots of 1 ml were taken for the determination of radioactivity and phosphate, which was assayed after wet ashing in the usual way. A 90 to 100% recovery of phosphate and radioactivity by this procedure was always observed.

DETERMINATION

4R'I) EBOUI?-BOXIS sntisfurtory. So influence on the rxtrac*rllular space

Incubation

OF GLUCOSE IN THE TISSUE

In order to avoid contamination by other reducing substances, glucose was determined in the tissue extract of the diaphragm by a chromatographic procedure. This method includes removal of the TCA from the extract by ether, deionisation with Amberlite MB3 and quantitative chromatography of the neutral substances on paper, according to a procedure described in n previous work (Harbon-Chabbat et al., 1961). The spots were localized by the calorimetric method of Trevelyan et nl. (1950) with 2,3,5triphenyltetrazolium-chloride. Control experiments showed that no gluc.osc was lost by this procedure.

EXTRACELLULAR INCUBATED

SPACE OF THE D1.4pmmM

This value was established by extrapolating to 0 time the linear portion of the curve (Fig. 1) indicating the total labelling (viz. inorganic + organic phosphates) of the incubated rat diaphragm, as has been done before with the incubated rat uterus (Volfin et al., 1962). Phosphate penetration into the muscle is slow and equilibration of the extracellular space fast, so that such a method seems reasonable. An extracellular space of 19 (-CO.65)% was found, but the linearity of phosphate penetration at 2, 5, and 10 minutes was less perfect than with the uterus. However, considering the close agreement between the value found by this method and the value established by Kipnis and Cori (1957) on the intact diaphragm (18%). thr results wcrc considered to be

of insulin was found.

and

glucose,

time (min.)

FIG. 1. Total labelling of rat diaphragm in percentage of the extracellular radioactive medium at different incubation times. The st,raight line has been plotted according to the method of least squares. By space

extrapolation to zero time of 19.0 + 0.65% is calculated.

SEPARATE

an extracellulal

CALCULATION OF SPECIFIC RADIOACTIVITIES

The amount and specific radioactivity of intracellular phosphate was calculated from the total inorganic phosphate and the phosphate of the medium (considered to be equal to the extracellular phosphate), assuming an extracellular space of 19%, as indicated above. The specific radioactivities of phosphocreatinc and “residual phosphate” were determined hy direct measurements, as specified above. No labelling of the ribose-linked phosphates occurs within the limited incubation times used in the present work. Hence labelling of ADP refers exclusively to the labile phosphate present in this compound. The specific activity of the terminal phosphatr of ATP was calculated on the assumption that the second phosphate of this compound is in isotopic equilibrium with the labile phosphate of ADP. The correctness of this assumption is founded on the work of Fleckenstein and Janke (1957), who found no difference what,soever between the specific activity of ADP and of the

INSULIN

AlrjD PHOSPHATE

second phosphate of ATP in the surviving muscle, and on the work of Harth and Mandel (1961), who demonstrated that, in all tissues studied, ADP isolated by column chromatography was almost exclusively an artifact arising from the limited breakdown of ATP during the handling of the tissue and the isolation procedure. Endogenous concentrations of ADP are likely to be many times lower than the concentrations found in the present work. No attempt was made to separate the uridineand guanosine-phosphates during the experiments involving I’“’ turnover. The radioactivity of this fraction was calculated on the assumption that all uridine- and guanosine-phosphates were present as UTP and GTP: thus, the specific activities mentioned are merely a lower limit of the real specific activities of the compounds present. It must be pointed out that UMP and GMP, the Rf of which are different, are not included in this fraction. STATISTICAL

ANALYSIS

Although an insulin effect on the labelling of organic phosphates was found in every single experiment with four diaphragms, noticeable variations occurred between the experiments, especially as far as the labelling of organic phosphates with respect to intracellular I.P. is concerned. These variations which so far have not been completely controlled are mainly influenced concentration and the weight of the by Mg” diaphragms. For these reasons, differences between the experiments were considered as “nonrandom variations” and segregated, according to the method of “randomized groups,” as described by Bliss (1952). RESULTS ACID-SOLUBLE DIAPHRAGM

PHOSPHATES IN THE OF THE NORMAL RAT

The levels of inorganic phosphate, phosphocreatine, and free-nucleotides in t,he diaphragm of the normal rat before incubation are shou?nin Table 1. Phosphocreatine content seems higher than in cardiac muscle (2 ,LJV per gm fresh weight) (Mommaerts, 1951), but. lower than in skeletal muscle (20 PLMper gm fresh weight) ; ATP content, however, approaches the level found in skeletal muscle (Mommaerts, 1951). The free nucleotide content is consistently higher than that reported by RyeAlertsen et al. (1958). Guanosine and uridine nucleotides have been separated by

373

IN RAT DIAPHRAGM TABLE 1 ACID-S• UJBLE MONONUCLEOTIDES IN E~AT DIAPHRAGM fig P/IO0 mg F.W. I.P. P.C. ATP ADP AMP G + Ua DPN IMP

24 17 37 12 1.8 3.6 3.8 4.9

rM/smF.W.

_+ 0.9 + 1.3 f 0.9 f 0.9 f 0.35 k 0.2 k 0.2 * 0.34

7.75 5.50 3.98 1.94 0.58 0.60 0.60 1.58

a Guanosine + Ilridine. GTP 0.48 ) GDP 0.43 ) GMP 0.62 ) UTP 0.58 UDP 0.84 UMP 0.09 (0.18) (from UDP glucose) UDPAG 0.33

0.w11 1 }

0.28

J

means of the N-propanol/NH,OH/watr solvent-system of Hanes and Isherwood (1949). Extensive hydrolysis of guanosine and uridine compounds seems to occur in this solvent, as has been mentioned by Tsuboi (1959), with respect to UDPglucose. Hence, the relative proportions of mono-, di- and triphosphates do not reflect the levels of these nucleotides in the muscle. However, the molar ratio between guanosine and uridine compounds is not affected by phosphate hydrolysis and seems close to 1. FREE GLUCOSE IN THE INCUBATED DIAPHRAGM

Influence of the washing method outlined above is demonstrated in Fig. 2. Washing with glucose-containing medium yields glucose contents, which are somewhat higher (glucose space 24.6%) than would have been expected if the extracellular space alone had been saturated with glucose. Five minutes washing in glucose-free medium lowers the glucose cont.ent to less than 10% of the former value, the presence of insulin in the medium having no effect on this level. Reincubation in fresh glucose-free Krebs-Ringer bicarb0nat.e me-

374

CLALW31& VOLFIK,

clium (2.5 ml) further reduces the gluco~cl content to a value lower than 4.2 pg pel gm fresh weight. This small value may have arisen from the limited breakdown of glucose-l-phosphate or even of glycogen during extraction and chromatographical procedure. It seems therefore that when the experiment with PnZ starts, the concentration of glucose in the diaphragm has dropped to exceedingly small values.

t

500

i .P 5 5a, t p

250

P 8 8 .2 c7 I T

1234

FIG. bated

2. Glucose content of rat diaphragm incuunder various conditions. T, t,heoretical value for (1) (Estracellular space 19%): 1, washed 5 minutes, 37”, Krebs-Ringer glucose 2 mg/ml; 2, washed 5 minutes, 37”, no glucose; 3, washed 5 minutes, 37”, no glucose, insulin 10 &g/ml; 4, same as 3 plus incubation fresh medium 15 minutes.

PHOSPHATE

TURNOVER IN THE DIAPHRAGM OF THE NORMAL RAT

The level of the various phosphate fractions, the percentage labelling of the intracellular I.P. with respect to the I.P. of the medium, the percentage labelling of organic labile phosphates wit.h respect to the intracellular I.P. or the terminal phosphate of ATP in the incubated diaphragm of the normal rat are shown in Table 2 (no substrate present) and Table 3 (in the presence of glucose) at. two different incubation times. The levels of the phosphate fractions differ markedly from the values found in diaphragms before incubation (Table l), inorganic phosphorus being on the average 35% higher and organic fractions (except phosphocreatine) 40 to 50%

.iSI)

EIWr-ti-M)S~s

loa-er. X0 differences w
of experiments

Number

organic

P (sum)

35.8 15 27.8 5.3 2.1

f 2.6 * 1.5 k 0.3 + 0.0 +0.2 4.8

2

0

3.3 1.9 2.8 0.6 0.5 1.0

38.7 14.4 23.4 5.3 1.5

33.3 13.2 28.5 6.1 2.5 4.8

2

+

19.6 9.2 11.1 5.1 9.9

6.0 2.63 2.53 1.18 1.94 1.67

IL 0.6 & 1.2 +2.7 + 1.6 + 1.7

19.1 12.0 14.9 9.8 12.6

-

-

* + * k

2

+ + * * f +

2

5.0 3.75 3.03 1.80 2.54 2.10

+

1.0 0.35 0.34 0.12 0.24 0.22

0

* + + * + f

3

+

0 3

P)

Specific activity (6% extracellular

4.5 2.0 2.0 3.5

B. Incubation

0.4 0.73 0.38 0.20 0.36 0.28

A. Incubation

I

38 f 5.3

58 IL 9.7

-

50 + 6

26 + 7.3 50 f 7.5

2

+

64 !c 4.2 80 k 8

30 minutes

47 dc 4.5 56 3~ 12.1

2

0

time:

37 zk 4.9 52 f 1.4 45k5.4

22 f 1.7 30 IL 2.8 36 f 5.7

3

+

I’)

56 k 6.8 56 + 6.0

10 minutes

Specific activity (% intracellular

DIAPHRAGJI

43 f 2.8 42 rk 4.8

3

0

time:

TABLE 2 OF PHOSPHATES IN THE ISOLATED .~BSEKE OF ADDED SUBSTRATE

minutes and 30 minutes incubation time). P values greater than group of ATP. group of ADP. The specific activity refers to the labile phosphate groups if only

0.1 1 0.4 0.7 0.1

+ 1.9 * 2.9 IL 2.9 kO.8 k 0.5 * 1.1

4

+

+ * * f k 4.6

0 P refers to the sum of the experiments (10 b The specific activity refers to the terminal C The specific activity refers to the terminal d Sum of uridine and guanosine phosphates.

Labile

Intrac. I. P. P.C. ATP* 4DPc U + Gd “Residual” phosphorus

10 .g/ml

Insulin

* * f + + +

Intrar. I. P. P.C. ATP* ADF U + Gd “Residual” phosphorus

31.7 16.1 29.1 5.9 2.6 4.6

4

of experiments

Number

0

10 pg/ml

fresh weight

/

ON THE LABELLIR.G

PI3 p/100 mg

OF INSULIX

Insulin

INFLUENCE

-

-

IJTP

and

4

0

49 lk 7.9 77 f 6.0 73 * 5.5

were

included.

-

4

+

activity P of ATP)

64 + 1.4

86 *2.5 -

2

+

61 + 6.0 93f12 63f5.9

100 k3.6

present.

46 k 7 ..I! 91 +7 -

86 + 10.8 -

2

0

IS THE

Xpecific (% terminal

RAT

104 k 3.8

GTP

0.05 are not

0.01

-

0.01

0.001

Pa

0.01 0.05 -

0.001

Pa

OF THE NORMAL

0.01 -

-

-

Pa

0.01 -

-

-

PQ

of experiments

Number

39.7 20 28.5 5.8 1.8

+o.ci k 3.6 + 0.7 f 0.8 kO.4 6.1

2

0

2.7 3.4 3.4 0.4 0.4 1.3

fresh

mg

kO.1

2.4

6.8

k 1.6 * 1.1

I

-

f0.25 zk 0.25 f 0.06 f 0.39 zk 0.26

1.3 2.9 2.8

46 k 6.2 71 If: 1.0 -.

-

.~. 63 f. i 80 k 10

2

11 k2.7 13 + 1.3

2

0

30 minutes

time:

B.

Incubation

a1 + 2.0 47 + 9.0 52 If: 10.4

7 f3.0 10 f 3.0

+

-

10 minutes

49.0 + 1.8 58 + 1.0

2

0

time:

DIAPHRAGM

P)

2

75

+!I

57 +2.0

84 83 3~ 6 t

___.~

+

39 $7 1.0 69k5.8 65f10.2

72 f. 2.5 TO * 3.0

2

+

Specific activity (To intracellular

f 0.16 & 0.02 & 0.28

f 0.15 * 0.13

+ 0.5

.4. Incubation

I

ISOLATED

(2 MC/ML)

18.3 k 4 15 * 0.3 15 k 2.7

1.8 3.2 3.0

3.6 3.4

4.6

a

+

P)

3 THE

19.3 9 f 1.7 11 f 2.0

2

0

k 0.6

3.1 3.4

2

IN

OF GLUCOSE

Specific activity (70 extracellular

PRESENCE

0

TABLE OF PHOSPHATES

5.6

LABELLINC;

5.4 2.1 1.1 1.0 0.4 1.6

+ 3.8 + 5.1

2

+

f * k + + +

3

+

THE

35.4 17.9 27.3 5.5

38.4 14.0 20.6 5.6 1.8 i.3

weight

P&! p/100

ON

0.01 --

0.001

PQ

0.01 -

0.001

P

OF THE

I

NORMAL

f

8.0

59 & 14.i 90 * 10

Labile

organic

P (sum)

104

*

1.0

:3

+

activity P of ATI’)

il i 5.3 !E * 8

1012

2

+

54 k2.1 95 If: 2 .!I 8Ykli

THE

present

is * 1.:3

2

0

39 + 2.8 75 * 9 83 * 11

93

3

0

Is

Specific (% terminal

RAT

I 53 + 2. 1 70 f 2.9 0.01 I-I-(1 P refers t,o the sum of the experiments (10 minutes and 30 minutes inrubat.ion time). I’ values superior to 0.05 are not included. * The specific activity refers to the terminal group of ATP. c The specific activity refers to the terminal group of ADP. d Sum of uridine and guanosine phosphates. The specific activity refers to the labile phosphate groups if only 1JT1’ and GTP were

Intrac. I. P. P.C. ATPb ADF U + Gd “Residual” phosphorus

10 rg/ml

Insulin

f * + + * +

Intrac. I. P. P.C. ATP* ADP U + Gd “Residual” phosphorus

30.3 15.5 27.9 5.6 1.9 6.4

3

of experiments

Number

0

10 pg/ml

I

OF INSULIN

Insulin

INFLUENCE

0.05 -

Pa

--

0.05

PI

INSULIN

ANU PHOSPHATE

ever, that insulin enhances the labelling of this fraction, not only with respect, to I.P., but also to the terminal phosphate of ATP. No specific action of glucose on this labelling seems to take place, which disagrees with what had been stated in a preliminary report (Volfin et al., 1961c). Large scattering also occurred on the labellings obtained with “residual phosphorus.” Here the glucose effect seems predominant in promoting t,he equilibration of this fraction with ATP. Figure 3 summarizes the results on the diaphragm of the normal rat as far as the labellings of phosphocreatine, the two labile phosphates of ATP, and the sum of guanosine and uridine-phosphates are concerned.

377

IN RAT I)IAPHRAGM

reaches isotopic equilibrium with the terminal phosphate. Stimulation of the turnover of all the other phosphates is limited (terminal P of ATP + 150/o), but it must be kept in mind that the extra labelling of the second group of ATP compulsorily derives from the terminal ATP phosphate, so that any increased labelling of the second group is bound to reflect an increased labelling of the terminal phosphate. The addition of substrates (Table 4 B) to these diaphragms almost completely reverses the insulin effect. Specific labelling of the second group of ATP is no longer found, whereas a highly significant effect appears on the terminal phosphate of this nucleotide (+54%). Insulin action on all other organic labile phosphates closely

loo5

I) PC

2)ATP(terminal

4) Uridine+

phosphate)

guanasine

C-P)

t

30

IO Incubation

FIG. 3. The action of insulin on the labelling no insulin; of means.

---,

insulin

present

(10 pg/ml)

time

10

Table 4 A shows the results of similar experiments performed on the isolated diaphragm of hypophysectomized rats in the absence of glucose. The levels of organic phosphates are slightly lower than in the normal rat at the same incubation time. Insulin stimulation of phosphate turnover involves almost exclusively the second phosphate of ATP (+1300/o), which nearly

3(

(min.)

of phosphates in ; G, glucose present:

PHOSPHATE TURNOVER IN THE DIAPHRAGM OF THE HYPOPHYSECTOMIZED RAT

-1

the isolated rat diaphragm. vertical lines, t standard

- - - -, errors

matches this increase and hence seems merely to be a consequenceof the increased labelling of the terminal ATP phosphate. PHOSPHATE TURNOVER IN THE DIAPHRAGM OF THE HYPOPHYSECTOMIZED GROWTHHORMONE TREATED RAT

Diaphragms from hypophysectomized growth-hormone treated rats no longer exhibit sensitivity to the presence of substrates (Table 5). The main insulin effect

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is observed on the terminal *ATP phosphat,e; the other phosphates, including ADP, seem to lag behind ATP labelling in the presence of insulin. The effect of insulin on ATP and the overall effect on the sum of labile organic phosphates arc statist,ically significant,. INFLUENCE OF R/IAC;NESIUM IONS ON THE LABELLING OF PHOSPHATES IN THE ISOLATED DIAPHRAGM

As stated above, two concentrat,ions of Mg++ were used in the present work. Differences were noticed between t,he two concentrations, although no systematic examination of this phenomenon has been carried out so far. At the higher Mg++ concentrat,ion, phosphat~e penetration into the diaphragm is reduced and percentage eouilibrium of organic phosphates with respect to intracellular I.P. is increased. The action of insulin on the labelling of the various groups, however, is identical at both Mg++ levels. DISCUSSION

The main conclusions which may be drawn from these experimental results concern phosphate penetration into the cell, overall labelling of organic phosphates, and specific labelling of various labile groups. Phosphate penetration into the surviving Gssue can be demonstrated by the percentage labelling of intracellular I.P. versus I.P. of the medium. Insulin or substrate dependence of this penetration could in no case be observed, which seemsto be contradictory to the results reported by Park et aZ. (1961)) who found an increase of phosphate uptake in the isolated perfused rat-heart under the influence of insulin. This lack of agreement may, however, be explained by the differences cxisting between the intact muscle fibers of the heart and t.he cut muscle fibers of the excised diaphragm. It may be relevant to mention that the specific activity of intracellular I.P. is consistently higher than the specific activity of any organic fraction t.ested. It has already been noted that, when experiments were performed in vim, isotopic equilibrium was almost im-

Ah-11

E:HOI!lhBOSIS

mediately attained between intracellular I.P., phoxphocreatine, and both labile groups of ATP. Thus, no evidence was ohtained for a direct, labelling of organic compounds, as ATP, from the extracellulai I.P., nor was there any anomalous labelling of organic phosphates, which could have been ascribed to nonhomogeneities of the various pools. On the contrary, all the rcsults were consistent with the hypothesis that labelling of int,racellular I.P. preceded Iabelling of the terminal phosphate of ATP which, in turn, seemsto act, as a precursor of all other organic phosphates. This may be correlated wit.h recent data of Fleckenstein et al. (1959133,who, by studying 0’” incorporation into single P-O bonds, found a much higher labelling of intracellular I.P. than of ATP; as no exchange of 0” between phosphate and H,O takes place in the absence of Gssue, these results indicate t,hat phosphate cmers the cell by an active transport process of some kind, which, however, does not. seem to involve stoichiometric participat,ion of ATP. As far as the sum of labile organic phosphates is concerned, the results demonstrate that. insulin always promotes a significant increase of the overall labelling of this fraction. The fact, that, t,lie action of insulin is glucose-independent agrees with numerous other glucose-independent effects of insulin on isolated tissues mentioned above (Manchester and Young, 1958, 1960; Wool and Krahl, 1959; Manchester and Krahl, 1959; Krahl, 1959; Barrnett and Ball, 1960). This does not signify, of course, that, the act.ion of insulin on glucose transport’ and dist,ribution space is not a direct effect., but clearly means that insulin may also act, directly on some ot,her mctabolic events in the muscle without any mediation of t,his effect through an increased glucose penetration. Information on the precise point where such insulin action could intervene is provided by the specific labelling of various phosphate groups. The way, however, in which insulin affects these turnovers may be t.wofold: either changes of the relative labelling of specific phosphate groups reflect metabolic changes induced by insulin

INSULIN

AND

PHOSPHATE

in the tissue, or insulin does not modify metabolism in the absence of glucose but promotes increased phosphate penetration into some restricted compartment of the muscle cell, where most phosphorylations take place, such as the sarcosomes. In the latter case, changes in the labelling of specific phosphates would merely reflect more closely the kind of metabolism already established in the tissue before addition of insulin. A definite solution of this important problem is at present impossible, but it seems reasonable to preclude the second hypothesis on the ground that, if it was correct, prolonged incubation (Table 2)) which results in closer equilibrium between intracellular I.P. and organic phosphates, should also, as does insulin, promote an increased equilibrium between the different organic phosphates which clearly is not the case. Hence the first hypothesis concerning insulin-induced metabolic changes must be carefully considered. In the normal diaphragm (Tables 2 and 3), where insulin increases the labelling of all organic phosphates, a preferential action on ADP resuIts in a cIoser equilibrium between both labile groups of ATP. This could be explained by an increase of “pyrephosphate splits” or an increased dephosphorylation of ADP through the action of myokinase and subsequent rephosphorylation of AMP by an adenylate-kinase system. Increased action of myokinase is unlikely to occur for the reason that the remarkable constancy of the level of energy rich-phosphates, especially phosphocreatine and ATP, within the incubation times used in the present work, indicates that the muscle is not in a “state of emergency.” Moreover, the close isotopic equilibrium between phosphocreatine and the terminal ATP phosphate demonstrates that rephosphorylation of creat,ine from ATP proceeds normally, which is a further indication of steady state equilibrium condit.ions in the muscle. Pyrophosphate split is known to take place in amino acid (Hoagland, 1955) and fatty acid (Berg, 1956) activation. Increase of protein synthesis has been reported, as mentioned above (Manchester and Young,

IN

RAT

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381

1958, 1960; Manchester and Krahl, 1959; Wool and Krahl, 1959; Krahl, 1959)) and an increased level of peptidic materia1 linked t.o glucose has equally been observed under the influence of insulin by Walaas et a2. (1960). On the other hand, increase of fatty acid activation under the influence of insulin may occur, for it is known from the work of Neptune et al. (1959a,b) that fatty acids seem to be the main subst.rate of the oxidative metabolism of the excised resting rat diaphragm. It seems unlikely that protein biosynthesis in the surviving tissue could be sufficient,ly active to account for an almost 100% increase of ADP-labelling. Thus it may be concluded that this increase reflects predominantly fatty acid breakdown. This interpretation is st,rongly supported from the results with hypophysectomized rats (Table 4), the muscles of which are known to contain smaller levels of glycogen, especially after fasting (Russell, 1957). Hence it may be supposed that, in the absence of added substrates, fatty acids are, even more than in the diaphragm of normal rats, the main substrat.e available to oxidative metabolism: in agreement with this view, insulin in this case promotes an 130% increase of the labelling of ADP, which almost reaches isotopic equilibrium with the terminal ATP phosphate. If, however, glucose is added, insulin action mainly affects the terminal phosphate of ATP, as if the metabolism had switched from fatty acid to glucose degradation. This would not be surprising, for the diaphragm of hypophysectomized rats may preferentially degrade glucose if this substrate is available to the muscle. High glucose uptakes have been frequently reported with the excised diaphragm of the hypophysectomixed rat (Krahl and Park, 1948; Villee and Hast,ings, 1949; Manchester et al., 1959). This increase does not seem to take place in the absence of cut muscle fibers (Henderson et al., 1961), but hypophysectomy definitely counteracts the decrease of glucose phosphorylation in diabetic animals (Morgan et al., 1961c). Hence the results observed in the present work on the diaphragm of the hypophysectomized rat are

by no means in contradiction to the known effects of hypophysectomy on muscle metabolism. With regard to the hypoltl~yscctomized growt,h-hormone injected rat (Table 51, action of insulin on phosphate labelling can st.ill be demonstrated. This action however is no longer influenced by the presence 01 absence of substrates, which may bc correlated with the glycostatic effect. of growth-hormone (Russell and Wilhelmi, 1950). ADP labelling in the absence of insulin is consistently higher than with the noninjected hypophysectomized animal under the same conditions (compare Table 4). The addition of insulin mainly promotes increase of the labelling of the terminal ATP-phosphate. This effect, suggests a swikh from lipid t.o glucose or to glycogen degradation under the influence of insulin in the hypophysectomized growthhormone treated rat. A more detailed interpretation awaits closer examination of the effect of growth-hormone in t.he hypophysectomized or hypophyscctomized alloxandiabetic rat. Similarly, explanation of uridine- and guanosine-phosphate labelling needs a detailed investigation and separation of the relevant fractions. It should be mentioned that, in the normal rat, insulin seems t,o increase isotopic equilibrium between this fraction and the terminal ATP phosphate (Tables 2 and 3). Growth-hormone in the absence of insulin has the same effect on the hypophysectomized rat (compare Tables 4 and 5). To what extent this may be due to an increase of UTP-turnover [through synthesis of glycogen (Larner et nl., 1959; Villar-Palasi and Larner, 1960)] or on GTP turnover [through prot.ein synthesis (Hoagland et al., 1957) ] remains to be seen. The glucose dependence of the amount and labelling of “residual phosphate” has already been mentioned. This result is not unexpected but a detailed investigation of this fraction remains to be carried out. It appears that systematic investigation on the labelling of specific phosphorylated coenzymes and metabolites may give valuable information on the metabolic patterns

prevailing in the surviving diaphragm, ant1 on the influence of various physiological condit,ions on these pat,terns. “Metabolic fingerprints” of this kind have been already obtained on the uterus (Volfin et al., 1962) and the principle of this method may be applicable to various other surviving Cssues. ACKNOWLEDGMENTS It is :I pleasure to express our gratitude to Prof. C. H. Li, Berkeley, California, for his generous gift of pure bovine hypophyseal growthhormone, to the Labora.toires Choay, Paris for the gift of hypophysectomized rats and to the Laboratoires de l’Endopancrine, Gisors, France, for the two samples of insulin used throughout this work. The technical assistance of Miss M. Bourdain has been of great value and is gratefully acknowledged. REFERENCES BALL, E. G., AND COOPER,0. (1960). Studies on the metabolism of adipose tissue. III. The response to insulin by different types of adipose tissue and in the presence of various metabolites. J. Biol.

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BALL, E. G., MARTIN, D. B., AND COOPER, 0 (1959). Studies on the metabolism of adiposr tissue. I. The effect of insulin on glucose utilizntion as measured by the manometric determination of carbon dioxide output,. J. Biol. Chem. 234,

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BARRNETT, R. J., AND BALL, E .G. (1960). Metaho!ic and ultrastructural changes induced in adipose tissue by insulin. J. Biophys. B&hem. Cytol. 8, 83-101. BELOFF-CHAIN, A., CATANZARO, R., CHAIN, E. B., LONGINOTTI, L., MASI, I., AND POCCHIARI, F. (1959). Influence of anaerobiosis on glucose metabolism in the isolated rat diaphragm muscle. Selected Sci. Papers Inst. super. Sanit6 2, 13%149. BERENBLUM, I., AND CHAIN, E. (1938). An improved method for the calorimetric determination of phosphate. Biochem. J. 32, 295-298. BERG, P. (1956). Acyl adenylates: an enzymatic mechanism of acetate activation. J. Biol. Chem. 222,

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BLISS, C. I. (1952). “The Statistics of Bioassay.” pp. 474-482. Academic Press, New York. BROWN, D. H., PARK, C. R., DAUCHADAY, W. H., AND CORNBLATH, M. (1952). The influence of preliminary soaking on glucose utilization by diaphragm. J. Biol. Chem. 197, 167-174.

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