The lipolytic action of fructose-1-6-diphosphate

The Lipolytic Action of Fructose-16Diphosphate ByC.

CHLOUVERAKIS markedly stimulated lipolysis in both intact tissue and homogenates. This effect of F-l-6-DiP is not due to its generating L-cY-glycerophosphate, and is probably a specific activating effect of F-l-6-DIP on the lipolytic (enzyme) system of adipose tissue. A comparison between the lipolytic action of adrenaline and of FI-6-DiP has suggested that the mechanism of action of these two agents on lipolysis is not identical. (Metabolism 17: No. 8, August, 708-716, 1968)

In an attempt to elucidate the mechanism of the lipolytic action of glucose, the effect on lipolysis of (a) inhibitors of aerobic processes, (b) stimulants of the shunt pathway, and (c) glycolytic intermediates was studied in an in vitro system using intact rat adipose tissue and homogenates. (a) completely abolished the lipolytic effect of glucose and (b) had no effect. Most of (c) had a small lipolytic effect comparable to that of glucose with the exception of fructose-l-Sdiphosphate (F-l-6-DIP) which

MECHANISM whereby glucose enhances the rate of adrenalinestimulated lipolysisl and of basal lipolysis2 is not known. Previous evidence obtained in this laboratory2 suggested that the lipolytic effect of glucose was caused by one or more of its intermediary metabolites rather than being due to the steric conf?guration of its molecule. Jungas and Ball1 have speculated that this glucose-metabolite could be L-a-glycerophosphate, whose action on lipolysis would be the result of its promoting esterification of FFA within adipose tissue. The present work was undertaken in an attempt to elucidate the mechanism of the lipolytic action of glucose.

T

HE

MATERIALS Crystallised

bovine

plasma

albumin

was

AND METHODS obtained

from

Armour

Pharmaceutical

Co.,

Eastbourne, England; Glucose, adrenaline, sodium azide, potassium cyanide, 2’, 4’-dinitrophnicotinic acid, sodium salicylate, enol (2’, 4’-DNP), phenasine methosulphate, ouabain, iodoacetic acid and the disodium salt of ethylene diamine tetra-acetic acid (EDTA) were obtained from B.D.H., Poole, Dorset; glucose-g-phosphate (sodium salt), glucose-l-phosphate (crystalline disodium salt), fructose-g-phosphate (barium salt) and fructose-1-6-diphosphate (sodium salt) were obtained from the Boehringer Corp., London. The barium salt of fructose-6-phosphate was converted to the sodium salt by running it through an ion exchange column in the sodium form. The latter was prepared by washing the resin in the hydrogen form (AGSOW-X8, 200400 mesh, Bio-Rad Laboratories, Richmond, Calif.) with a volume

(2.5 times the volume of the resin bed) of 10 per cent NaCl,

the excess of

From the Medical Research Council Metabolic Reactions Unit, Department of Biochemisty, Imperial College of Science, London, England. Receiaed for publication December 7, 1967. Presented in part at the meeting of the Medical and Scientific section of the British Diabetic Association, Leicester, England, in April 1967. C. CHLOUVERAKIS, M.D.: Medical Research Council Metabolic Reactions Research Unit, Department of Biochemistry, Imperial College of Science, London, England. 708

LIPOLYTIC

709

ACTION

Table L-Effect Methosulphafe _____

of Glucose With and Without Phenazine and Sodium Azide on Rate of Lipolysis in Rat Adipose Tissue Glycerol Release (pM/Gm./3 hrs. 1

Glucose (100 mg.%:) Glucose+Phen. Slethosulph. (10 mM)

Concentration of glucose, Each value is mean of 6 rats.

1-“C-glucose Oxidation (dpm/Gm./3 brs. ) ___.

4.72

7.51 x 10::

-

1.50 -7.08

SOW?

(100 uM) Glucose+Na-Azide

~_.

Lactate ( pM/Gm./B hrs. ) .___ --.-_

when

present,

2.01

-

0.90

7.95

in incubation

medium

-

79.86

x 10:: -

was

100

mg./lOO

ml.

NaCl having been removed with water. The eluate containing the fructose-6-phosphate was mixed with activated charcoal and after centrifugation the clear supernatant was Iyophilized. D-L-u-glycerophosphate

(disodium

salt)

was

obtained

from

Sigma

Chemical

Co.

(St.

Louis, Missouri) and L-u-glycerophosphate (dicyclohexylammonium salt) from Calbiochem, Los Angeles, California. The dicyclohexylammonium salt of the L-a-glycerophosphate was converted to the sodium salt by dissolving it in a solution containing a slight stoichiometric excess of NaOH and extracting the cyclohexalamine in diethylether. The aqueous phase containing the L-u-glycerophosphate was lyophilized. Samples of all the above mentioned phosphate esters prior to their being added to the incubation medium were chromatographed on thin layer, using the following system of solvents: tertiary amyl-alcohol : water : p-toluene-sulfonic acid (60:30:2). All compounds gave one spot except Fructose-I-6-diphosphate which contained a small admixture of fructose-6-phosphate. The experimental

methods

concerning

animals,

tissue

incubation,

glycerol

estimation have been described elsewhere.aJ Homogenates of rat epididymal were prepared using a Lab. Mixer-Emulsifier (Vortex-Mixers, Hampton, Middlesex,

and

FFA

fat tissue England).

Lactate in the medium was estimated enzymically using a modification (Boehringer) of the method described by Scholz et al .4 Carbon dioxide (CO?) was collected in 0.2 ml of a solution consisting of equal part of ethanolamine (B.D.H.) and methanol, using the device described by Keen et al.5 The CO, so collected was dissolved in 10 ml. of Bray’s liquid scintillanta and its radioactivity was determined in a Packard liquid scintillation spectrometer using the channel ratio method to correct for quenching. KESULTS

To investigate the possibility that the stimulation of lipolysis by glucose was due to a product of the shunt pathway, the release of glycerol into a medium containing phenazine methosulphate was measured (Table 1). Phenazine methosulphate did not influence the rate of glycerol release in spite of its stimulation of the shunt pathway, as indicated by a ten-fold increase in the rate of oxidation of the first carbon atom of glucose. Sodium azide, an inhibitor of aerobic processes stimulated lactate production hut inhibited glycerol release (Table 1). This ruled out the possibility that the lipolytic activity of glucose was due to a glucose metabolite produced at faster rates when aerobic processes were inhibited. The effect on lipolysis of various phosphate esters of carbohydrates at a concentration lo-” M is shown in Fig. 1. Glucose-l-phosphate (G-l-P) was without effect, glucose-6-phosphate (G-6-P), fructose-6-phosphate (F-6-P)

710

c.

cH.LouvERAKIs

Fig. L-Effect of Glucose-6-Phosphate (G-6-P), Glucose-l-Phosphate (G-lP), Fructose-6-Phosphate (F-6-P)) Fructose-6-Phosphate (F&P), Fructose-l6-Diphosphate (F-l-6-DiP) and D-L-a-Glycerophosphate (DL-CY-GP), all at concentration 10-3M on rate of lipolysis in rat adipose tissue. Height of each indicates mean column percentage change above or below basal lipolysis and vertical lines show . standard error of this mean. Numbers in columns indicate number of rats

used.

and D-L-a-glycerophosphate ( DL-a-GP) showed a small but significant stimulation and fructose-1-6-diphosphate (F-l-6-DIP) produced a three-fold increase in the rate of lipolysis. This effect of F-l-6-DiP was shown also in homogenates prepared from fresh adipose tissue and in addition to the activation which occurred during homogenisation of the tissue (Table 2). In homogenates prepared from tissue preincubated for 2% hours, F-l-6-DiP (2 x 1O-5 M) slightly stimulated lipolysis and at a concentration of 2 x 10S2 M the lipolytic activity exceeded that of the fresh tissue (Fig. 2). The lipolytic response of the intact fresh tissue was linearly related to the concentration of F-l-SDiP in the incubation medium (Fig. 3). The possibility that the action of F-l-6-DiP on lipolysis, was not specik, Table 2.-

Separate Efects of Homogenization, Fructose-I-6-diphosphate, (F-I-B-Dip), and (DL-glycerophosphute (DL-CY-GP) on Rate of Lipolysis in Rat Adipose Tissue Additions

Intact tissue Homogenate Homogenate Homogenate

None None F-l-&Dip (10-s M) DL-a-GP (2 x 10-3 M)

Numberof Rats

Gl cenolProduction ( &!fGm./BO min.)

eanf s.e.m.

14 14 14

0.65 & 0.09 4.98 f 0.30 16.47 + 1.42

4

5.99 f 47

Pieces of intact tissues and homogenates from same animal were incubated for 30 minutes. During this period, rate of lipolysis was found fo be constant.

LIPOLYTIC

711

ACTION

Fig. B.-Rate of lipolysis in homogenates of rat adipose tissue as influenced by previous infor cubation of tissue 21/2 hours and by varying concentrations of fructose1-6-diphosphate. F-l-6-DiP at concentration of 2 x 10-2M not only restores activity of lipolysis to levels of unincubated tissue but further enhances it. Number of rats is printed in columns and standard errors are shown by vertical lines.

Cmc

d F-I-~-DIP

in hmmprete

but due to its action as a chelating

agent (owing to the two phosphate groups) fat pad in the presence of varying concentrations of the disodium salt of EDTA; this did not affect the rate of glycerol release (Table 3). Also no significant effect on lipolysis was shown by L-a-glycerophosphate (Fig. 4). The presence of cupric chloride (2 x 10C4 M) an inhibitor was tested by incubating

Fig. S.-Effect of varying concentrations of fructose-l6-diphosphate (F-I-6.Dip) on rate of lipolysis in intact adipose tissue. Each point is mean of 6 animals. Each of 4 pieces of fat from same rat were incubated without and with varying concentrations of F-l-6-Dip. Vertical lines show standard error of mean.

26 1OJt-i F-t-d-DIP

1

Fig. 4. -Effect of Fructose-1-6-Dip. L-W Glycerophosphate and of both together on the rate of glycerol and FFA production by rat adipose tissue. Pieces of fat were incubated in albumin-free medium. Difference between glycerol release into medium multiplied by 3 (shaded columns) and accumulation (net production) of FFA in tissue during course of incubation is approximate measure of rate of esterification. Standard error of means (s.e.m.) are shown by vertical lines. Number of animals is printed in columns.

Glycerol&ease x3

15

pM/g/2hrs.

10

5

CUltrOt

F-1-6QP (10-3M)

L-d-GP

F-I-B-D,P

d03M)

L-&+GP

of the aldolase reaction7 caused a small but significant potentiation of the lipolytic effect of F-l-6-DiP (Table 4). To compare the effect on lipolysis of adrenaline with that of fructose-1-6diphosphate, intact rat adipose tissue was incubated in the absence and presence of each of these two agents separately and of both combined. As expected, the addition of adrenaline (1 pg./ml.) to the incubation medium led to a marked stimulation of lipolysis, as did the addition of F-l-6-Dip. The Table 3.-Efiect of Varying Concentrations of EDTA Rate of Lipolysis in Rat Adipose Tissue

on

Glycerol Release ( sM/Gm./B hrs. )

EDTA 0

1.86in0.16 2.04k 0.26 1.80k 0.14 2.35I!I 0.28

10-5M 10-4M 10-3M

Each figure is mean k standard error of mean of 6 animals. Table 4.-Effect of Fructose-I-Sdiphosphate (F-I-6-Dip) With and Without CuCZ, (an Znhibitor of Aldolase Reaction) on Rate of Lipolysis in Homogenates of Rat Adipose Tissue NUlIlbW

Additions

a. b. c.

None F-l-6-DiP (lo-3 M) F-l-6-DiP+CuC1,(0.2

FL

mM)

6 6 6

Glycerol Pmdudicm (.uM/Gm./30 min.

4.90 9.47 10.27

)

Mean Dazgg

0.80~0.2”

(c-b 1

P

0.02

LIPOLYTIC

713

ACTION

r (3lycetwl

release

)rMlgl2hrs.

Fig. 5.--E&& of Adrenaline, Fructose-1-6-Diphosphate and both together on glycerol release of rat adipose tissue. Vertical bars indicate standard error of mean. Number of animals is printed in columns.

of these two agents showed no synergism, being (Fig. 5). There were both similarities and dissimilarities in the way in which the lipolytic effects of adrenaline and F-l-6-DiP were modified by the presence of various metabolic inhibitors. Thus, potassium cyanide and sodium azide fully inhibited the lipolytic effect of both adrenaline and F-l-6-DiP whereas 2’, 4’ dinitrophenol influenced neither; ouabam, nicotinic acid, sodium salicylate and propranolol inhibited the lipolytic action of adrenaline but less that of F-l-6-DiP. The latter was uninfluenced by insulin, puromycin and pronethalol and was stimulated by iodoacetate; all of which markedly decreased the lipolytic effect of adrenaline (Table 5). combined

strictly

effect on lipolysis

additive

DISCUSSION

It has been speculated that glucose, by providing L-a-glycerophosphate, lowers the concentration of FFA in adipose tissue to the extent that lipolysis is fav0red.l However, in the experiments presented here both D-L and L-CYglycerophosphate had only a small stimulatory effect whereas F-l-6-DiP greatly enhanced the rate of lipolysis. This stimulatory effect occurred both in intact rat epididymal adipose tissue and in homogenates prepared from either fresh or from preincubated rat epididymal fat tissue. None of the other intermediates of glucose metabolism tested influenced lipolysis to the extent that F-l-6-DiP did. Furthermore, stimulation of the shunt pathway had no effect and stimulation of anaerobic glycolysis had an inhibitory action. Thus, the data obtained indicate a marked in vitro effect of F-l&Dip on the hydrolysis of glycerides in rat adipose tissue. That this effect is not likely to be due to chelation is supported by the failure of EDTA to influence

714

C.

Table 5.-Modification F-I-6-DiP

of Eflect of Adrenaline (lO-SM) by Various Metabolic

(l~g./ml.) Inhibitors

Per cent Ghan e of Inhibitor

-104*

signs

indicate

-8 -2o*

-18’ -36’

Na-Salicyclate (5 X 10-s M) Insulin (0.5 mu/ml.) Puromycin (lo-4 M) Iodoacetate (lo-3 M) Pronethalol (100 ug./ml.) Propranolol (100vg./ml.) Positive

-91* -105*

-105” 0

2’4’-DNP (lo-4 M) Ouabain (lo-4 M) Nicotinic Acid (lo-4 M)

and

Per cent Ch e of F-l-B-Dip 7E ect

Adrenaline Ezect

KCN (10-s M) NaN, (10-s M)

CHLOUVERAKIS

further

-2o*

-69’ -49* -23’

-37’

-75’ -53”

+31*

-13 f2 $8 -57*

-131* stimulation

indicate inhibition. Asterisks show statistically is mean of eight rats. Formula used was (A),

- (A:,

of

lipolysis

significant

x

by

changes

inhibitor; (p
negative Each

ones

number

100

(Ak - (B) where

and

(A& =

rate of lipolysis in presence of activator but absence of inhibitor.

(A),

=

rate of lipolysis in presence of both activator and inhibitor.

(B)

=

rate of basal lipolysis in absence of both activator and inhibitor.

lipolysis. A similar lack of effect was observed with 1-3-a-glycerophosphoric acid (unpublished observations). The possibility that this lipolytic action of F-l-6-DiP is due to the generation of glycerophosphate has been ruled out by the failure of D-L-cr-glycerophosphate and of L-cr-glycerophosphate to stimulate lipolysis to any sign&ant extent and by the failure of copper, an inhibitor of the aldolase reaction, to abolish the lipolytic action of F-l-6-DiP. It is not possible to say with certainty whether this in vitro effect of F-1-6DiP has any physiological significance. The concentrations which elicited a lipolytic response in vitro were much higher than those found in rat muscular tissue.8 However, only a very small proportion of the amounts added to the incubation medium would be expected to penetrate into the cells. In this case, the effective concentrations of F-l-SDiP would be much lower than those used. Although the prevailing view is that hexose-phosphate esters do not penetrate cell membranes, the similarity of the results in experiments using intact tissue or a cell-free system suggests that the cell membranes of the adipose tissue are not completely impermeable to F-l-6-DiP and possibly to other phosphate esters. Even in a cell-free system in which possible membrane barriers to phosphate ester penetration are absent physiological concentrations of F-l-SDiP failed to elicit a lipolytic response. This could, however, be due to the lipolytic enzyme having been already activated by the homogenisation of the tissue.” Regarding this lipolytic action of F-l-6-DiP in a cell-free system prepared

715

LJS’OLY’TIC ACTION

from fresh adipose tissue, this is the only substance which has been described so far to possess such an activity. Thus, in the hands of some investigators,‘O cyclic adenosine 3’5’ monophosphate (3’,5’-AMP) stimulated an extract of the lipolytic enzyme only when the enzyme had been partially inactivated by previous incubation of the tissue and even this effect was achieved by concentrations ten thousand times larger than those normally present in adipose tissue.ll The marked stimulation of lipolysis by F-l-6-DiP together with a much smaller effect exerted by its precursors and by glucose supports the hypothesis that glucose owes its mild lipolytic properties to F-l-6-DiP which may stimulate lipolysis by acting directly on the lipolytic enzyme(s) of adipose tissue. If this hypothesis is correct, then the reaction catalysed by phosphofructokinase, already known to be a rate limiting step in glycolysis, would also be a key reaction in regulating lipolysis. The lipolytic action of F-l-6-DiP becomes of interest in view of some evidence that the activation of the hormone-sensitive lipase by adrenaline might be due to its generating cyclic adenosine 3’5’ monophosphate (3’,5’AMP),‘O-13 a known activator of the phosphofructokinase reaction.14-l6 It thus becomes conceivable that the final activator of the lipase system in adipose tissue is fructose-1-6-diphosphate, acting as a third messenger, the hormone and cyclic 3’,5’-AMP acting as a first and second messenger. This hypothesis becomes diacult to prove directly at present since the overall evidence concerning the action of cyclic 3’,5’-AMP on lipolysis is still inconclusive.17 However, on the basis of the evidence presented in this paper it would appear that the mode of activation of lipolysis by adrenaline is not identical with that due to F-l-6-Dip. Thus the use of inhibitors showed that the activation of lipolysis by both adrenaline and F-l-6-DiP is dependent on respiration (but not on oxidative phosphorylation) since this activation was abolished by inhibitors of aerobic processes but not by 2’,4’-DNP. In spite of this property, common to both systems, the way in which other metabolic inhibitors modified the lipolytic response of the tissue differed between the two systems, the most marked difference having occurred with iodoacetate which almost completely abolished the adrenaline effect whereas it further potentiated the lipolytic effect of F-l-6-DiP. The different effect of metabolic inhibitors on the two systems favours the view that the mode of activation of the lipolytic system by adrenaline differs from that of F-l-SDiP and this view is strengthened by the finding that there is no interaction between the two lipolytic agents, their combined effect being the exact sum of their separate actions. It is thus possible that the activation of lipolysis in adipose tissue is a complex phenomenon involving either one enzyme with more than one allosteric sites or a system of enzymes. ACKNOWLEDGMENT I am indebted to Dr. L. Opie for useful criticism during the preparation of this paper. The excellent technical assistance of Miss Wendy Rowland Hill and Miss L. Headley is gratefully acknowledged.

c. CHLOUVERAKIS

716 REFERENCES 1. Jungas, R. L., and Ball, E. G.: Studies on the metabolism of adipose tissue XII. The effects of insulin and epinephrine on free fatty acid and glycerol production in the presence and absence of glucose. Biochemistry (Wash.) 2:383, 1963. 2. Chlouverakis, C.: The action of glucose on lipolysis. Metabolism 16:469, 1967. 3. -: Factors affecting the inhibitory action of insulin on lipolysis in a glucosefree medium. Endocrinology 81:521, 1967. 4. Scholz, R., Schmitz, H., Bucher, T., and Lampen, J. 0.: Wber die Wirkung von Nystatin auf Backerhefe. Biochem. Z. 331: 71, 1959. 5. Keen, H., Field, J. B., and Pastan, I.: A simple method for in vitro metabolic studies using small volumes of tissue and medium. Metabolism 12:143, 1963. 6. Bray, G. A.: A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biothem. 1:279, 1960. 7. Bergmeyer, H. U.: Methods of Enzymatic Analysis, New York, Academic Press, 1963, p. 970. 8. Newsholme, E. A., and Randle, P. J.: Regulation of glucose uptake by muscle. Biochem. J. 80:655, 1961. 9. Cori, C. F.: In Gaebler, 0. H. (Ed.): Enzymes: Units of Biological Structure and Function. New York, Academic Press, 1956, p. 573. 10. Rizack, M. A.: Activation of an epinephrine-sensitive lipolytic activity from adipose tissue by adenosine 3’, 5’ monophos-

phate. J, Biol. Chem. 239:392, 1964. 11. Butcher, R. W., Ho., R. J., Meng, H. C., and Sutherland, E. W.: Adenosine 3’,5’monophosphate in biological materials. II. The measurement of adenosine 3’, 5’monophosphate in tissues and the role of the cyclic nucleotide in the lipolytic response of fat to epinephrine. J. Biol. Chem. 240: 4515, 1965. 12. Sutherland, E. W., @ye, I., and Butcher, R. W.: The action of epinephrine and the role of the adenyl cyclase system in hormone action. Rec. Progr. Hormone Res. 21:623, 1965. 13. Weiss, B., Davies, J. I., and Brodie, B. B.: Evidence for a role of adenosine 3’, 5’-monophosphate in adipose tissue lipolysis. Biochem. Pharmacol. 15:1553, 1966. 14. Mansour, T. E., and Mansour, J. M.: Effects of serotonin (5-hydroxytryptamine) on and adenosine 3’, 5’-monophosphate from the liver fluke phosphofructokinase fasciola hepatica. J. Biol. Chem. 237:629, 1962. 15. Mansour, T. E.: Studies on heart phosphofructokinase: purification, inhibition and activation. J. Biol. Chem. 238:2285, 1963. 16. Denton, R. M., and Randle, P. J.: Citrate and the regulation of adipose tissue phosphofructokinase. Biochem. J. 100:420, 1966. 17. Vaughan, M.: Effect of hormones on phosphorylase activity in adipose tissue. J. Biol. Chem. 235:3049, 1960.