Effect of ascorbic acid and calcium on catecholamine-induced lipolysis in vitro

Life Sciences Vol . 6, pp . 2191-2197, 1967 . Printed in Great Britain .

Pergamon Press Ltd .

EFFECT OF ASCORBIC ACID AND CALCIUM ON CATECHOLAMINE-INDUCED LIPOLYSIS IN VITRO Giuliana Fassina Institute of Pharmacology, IInivereity of Padua, Padua (Italy) (Received 2 June 1967 ; in final form 4 August 1967) In previous studies we observed that a decreased calcium concentration in the incubation medium of adipose tissue

reduced

the action of noradrenaline on lipolysie . Ascorbic acid counter acted the effect of calcium deficiency . A possible influence of calcium ions in the noradrenalinestimulated lipolysie was also suggested by the results

obtained

with digitozin and prostaglandin E1 . The antagonistic effect

of

these drugs against noradrenaline was clearly influenced

a

by

decreased calcium concentration in the incubation medium of rat epididymal fat (1,2) . The purpose of the following work was to investigate

the

parameters of the aforesaid actions of calcium and ascorbic acid in the lipolysic process stimulated by catecholamines . Material and Methods Epididymal fat was obtained from fed iNistar rate (200-250 g) under ethereal anaesthesia . The fat pads were immediately divided in pieces of 100 + 10 mg and placed in a . solution of Srebe-Ringer bicarbonate buffer pH 7 .2 which contained 2 .5 (erection Y, Sigma) and were then incubated at

9~

bovine

37°

for

albumin 150 min in

a metabolic shaker . At the end of the incubation period, 0.1 ml of H2S0 4 2 .5 N was introduced in each assay for stopping the reaction . Free fatty acids ware determined in the medium actor ding

to Dole (3) and glycerol according to Born (4) . All the

drugs were added to the incubation medium before introducing the 2191

2192

LIPOLYSIS

Vol . 6, No . 20

fat. Calcium in the modified Srebe-Ringer bicarbonate buffer was substituted with sodium . Noradrenaline bitartrate monohydrate was from Recordati (Milano,

Italy), ascorbic acid and glutathion i~om Merck

(Darm-

stadt, Germany), rotenone sad 2,4-dinitrophenol from British Drug Houses Ltd . (Poole, Eagland) and oligomycin (a mixture

of

oligomycin A and B) from Upjohn (Salamazoo, U.S .A .) . N6 C2 - dibutyryl cyclic 3'S'-AMP was a generous gift of Prof . M. Carissimi (Maggioni, Milano, Italy) . Results Effeot of ascorbic acid on the noradrenaline-induoed lipolysis . Basal lipolyeis was not affected by the presence

of

ascorbic

aci8 (200 }~g/ml) in the incubation medium of adipose tissue . The lower concentrations of noradrenaline (0 .02 and 0 .2 x 105M) were potentiated by ascorbic acid, while the higher ones were aot influenced (FIG .

1) . Only the free Patty acid (FFA)

release is reported in figure ; the results concerning

glycerol

release are quite correspondent. Ascorbic acid does not

change

the maximum effect of noradrenaline, nor the concentration

at

which this maximum lipolytio activity appears (0 .5 z 10 5M) . Thus the effect of ascorbic acid in our ezperimental conditions apparently results in an increase of the "affinity" (according

to

Ariëne nomenclature, 5) of noradrenaline for the receptor system in adipose tissue (pD2 in the absence of ascorbic acid = 5 .96, in the presence = 6 .59) and, contemporarily, in a decrease of the slope of the log cono .-action curve of noradrenaline (FIG .

1) .

EYfect of calcium deficiency . When adipose tissue was incubated in e medium prepared with Krebs-Ringer bicarbonate buffer

con-

taining a calcium concentration reduced to one half of the normal the basal lipolysis was not affected, the activity of the concentrations of noradrenaline was decreased, the absolute

lower

Vol . 6, No . 20

W

w

LIPOLYSIS

2193

100 1

z

t W

20 y 0~

0.002

.02 0

NORADRENALINE

0.2

.S 0

S

10

CDNC . x 10' s M

FIG . 1 Concentration-effect Curves for Noradrenaline-induced 1Gipolyeis in vitro under the Influence of Ascorbic Acid and Ca++ Deficiency Abscissa : molar cone . of noradrenaline in the incubation medium of rat epididymal fat (log scale) . Ordinate : free fatty acid percent relative increase in the medium . The free fatty acid increase from control induced by noradreaaline 10 -5 M in the normal incubation medium was taken as 100 96 of the effect . Experim= ental conditions as in TABLE 1 . ~---~ Normal medium . Q~---d Nfedium containing ascorbic acid 200 Kg/ml . p--~ Medium prepared with Krebs-Ringer bicarbonate buffer containing a Ca++ cone . 1~iedium containing a Ca++ cone . reduced to half the normal . reduced to half the normal and ascorbic acid 200 ~g/ml. Mean values of 4 to 11 assays . Vertical bars represents s. e. of the means .

~--r

maximum activity was the same as when calcium is normally present in the medium but it was induced by a concentration of noradrenaline about 4 times higher (FIG . 1) . The log cone .-action curve of noradrenaline is thus parallely displaced to higher conoen trations (pD2 = 5 .52) . In this case too, the curves obtained by determining the glycerol release were quite corresponding to those given by FFA release. Effect of ascorbic acid in antaQOaizin~r the calcium deficiency . In the presence of ascorbic acid, the Ca ++ deficiency is

2194

LIPOLYSIS

Vol . 6, No . 20

largely antagonized (TABLE 1) . TABLE 1 Effect of Ascorbic Acid on the Noradrenaline-induced in a Calcium deficient Incubation Medium

Noradrenaline Conc . in the Medium

FFA

Lipolysis

~zEq/g/150 min ~ Ringer without Ca++

Normal Ringer

Ascorbic acid 5 .66 ± 0 .74

5 .18 ± 0 .80

5.72 _+ O .g3

0.2 x 10 5 M 0.5 x 10 5 M

20 .14 ± 2 .10

8 .59 ± 0 .71

18 .97 _+ 0 .88

34 .23 + 2 .05

14 .92 _+ 1 .13

30 .32 _+ 2 .16

2 x 10 5 M

28 .32 ± 1 .95

17 .42 + 0 .60

30 .66 + 4 .45

-

Rat epididymal fat (100 ± 10 mg) was incubated for 150 min at 37° in 2 ml of grebe-Ringer bicarbonate buffer pH 7 .2 containing 3 gb bovine albumin. Ascorbic acid was added, where indicated, to the medium in conc . of 200 ~g/ml . Data are eapressed as mean ± s .e . of 4 to 11 assays . TABLE 2 Influence of Ascorbic Acid and of Calcium Deficiency on the Dibutyryl 3'5'-~-induced Lipolysis in vitro

Dibutyryl 3'S'-AMP M conc . in the Medium 10

4

10 3 2 x 10-3 -3 5 x 10

~

FFA }~Eq/g/150 minim

-

Ascorbic acid

Without Ca++

1 .31

0 .82

-

8 .56

6 .44

-

22 .10

19 .45

-

28 .07

24 .13

26 .10

Experimental conditions as in TABLE 1 . Ascorbic acid 200 ~g/ml . Results of two parallel experiments . FFA absolute increase from control (fat incubated without dibutyryl 3'5'-AàiP) in the incubation medium .

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LIPOLYSIS

2195

Ascorbic acid and calcium defici enc V in th e dibutyr~!1 3'S '-AMPinduced lipolYSis . The action of dibutyryl 3'5'-AMP is not affected by the presence of ascorbic acid, nor by the

calcium

deficiency in the incubation medium of adipose tissue (TABLE 2) . Ascorbic acid and oxidative phosnhorslation inhibitors .

In the

presence of ascorbic acid, the antagonistic action of oligomycin, rotenone and 2,4-dinitrophenol on the noradrenaline-induced lipolysis is much less evident than in a normally

composed

medium (TABLE 3) . TABLE 3 Effect of Ascorbic Acid on the Antagonism between Oxidative Phosphorylation Inhibitors and Noradrenaline on Lipolysis in vitro

Drugs in the incubation medium M conc .

Percent inhibition -

Oligomycin 107 -6 10

Rotenone

49 + 6

Ascorbic acid 9

~T7±4

32±9

0. 01

10-5 -4 10 10-7

87 ± 3

48 ± 7

0. 01

99 ± 1

71 ± 3

0. 01

26 + 9

3 _+ 3

0. 01

10

45 ± 9

20 ± 9

0.05

69 ± 2

47 ± 5

0. 01

86 ± 6

76 ± 3

40 ± 8

19 ± 9

70 ± 5

43 ± 7

-6

10-5 

P

10-4

2,4-dinitrophenol 10

4

10 -3

0. 05

Experimental conditions as in TABLE 1 . The activity of metabolic inhibitors was tested against a conc . of noradrenaline in the incubatiôn medium = 2 x 10-5 M. Ascorbic acid 200 }~g/ml . ~ Statistical significance Diean _+ s .e . of 4 to 7 assays . of the difference .

2196

Vol . 6, No . 20

LIPOLYSIS

Effect of ~lutathion . Glutathion 10

4

M and 10

-3

M was found to

be without effect on the noradrenaline-stimulated lipolytic

as

well as on the antagonism between oxidative phosphorylation inhibitors and noradrenaline in the process . Discu~sion The presence of a certain concentration of calcium

is

necessary for an optimum of activity of noradrenaline on lipolytis in vitro .

This result apparently contrasts with the findings

reported by Lopez, ~lhite and Engel (6) and by Engel and ~lhite (7) demonstrating that adipose tissue depleted of ionized calcium , diminishes or abolishes the lipolytic effect of ACTH but not that of adrenaline . The contrast may be, however, only apparent and dependent on the unique concentration of catecholamine used by those Authors . The deficiency of calcium induces a

parallel

shift of noradrenaline log dose-action curve to higher concen trations of the drug (FIG . 1) . The concentration of adrenaline (about 5 x 10 5 h) used by Lopez, VPhite and Engel (6) is thus probably largely sufficient to induce the maximum

lipolytic

effect and to antagonize calcium deficiency . The effect of calcium deficiency is counteracted by ascorbic acid . The action of both ascorbic acid and calcium seems to

be

localized before the formation of cyclic 3'5'-AMP, as indicated by the lack of influence of these two substances on the dibutyryl 3'S'-AMP-induced lipolytis . The action of ascorbic acid presumably consists not only

in

preserving catecholamines from oxidation, but it appears to be a more specific one, as suggested by the lack of effect of another reducing substance as glutathion, by the varied slope of the log cone .-action curve of noradrenaline, and by the fact that ascorbic acid counteracts the effect of oxidative phosphorylation inhibitors on the process . Rotenone, oligomycin and 2,4-dinitrophenol are non-competitive antagonists of noradrenaline

on

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LIPOLYSIS

2197

lipolysis in vitro (8) . Their antagonistic power is

therefore

independent from the concentration of the agonist (5) . Thus, a simple preservation of noradrenaline from oxidation should not have a significant

influence

oa

oxidative phosphorylation

inhibitors antagonism . Glutathion has no similar effect on the process. The results described, and particularly

the

variations

induced on the noradrenaline log cone -effect curve,

lead

to

consider the possibility that calcium and ascorbic acid somehow influence the relationship between adrenergic hormones and their receptor sites in adipose tissue . This could explain the varied responsiveness of epididymal

fat to

noradrenaline

in

the

presence of ascorbic acid or in calcium deficiency .

Aclrnowled~emente The technical assistance of bfr. G. F. Daniel is

gratefully

aclrnowledged. References 1.

G . FASSINA and A.R . CONTESSA, Abe . Third Int . Pharmacol. ConAresa , Sâo Paulo, Brazil, July 24-30 (1966) .

2.

G . FASSINA and A.R . CONTESSA, Biochem. Pharmacol., in the press.

3.

V.P . DOLE, J . Clin . Invent ., ~,

4.

E .D . iCORN, J . Biol . Chem .,

21~,

150 (1956) . 1 (1955) .

5 . E.J . ARIËNS and J .L~ . VAN ROSSUM, Arch . int . Pharmacodvn., 110, 275 (1957) . 6.

E . LOPEZ, J .E . NHITE and F .L . ENGEL, J . Biol . Chem ., 2254 (1959) "

7.

F .L . ENGEL and J . E. ,7HITE, Am . J . Clin . Nut ., ~, 691 (1960) .

8.

G. FASSINA, I . h1ARAGNO and P . DORIGO, Biochem. Pharmacol ., in the press .