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Control of adipose tissue lipase activity by the sympathetic nervous system
Liie Sciences No . 3, pp . 210-214, 1963 . Pergamon Press, Inc . Printed in the United States .
OONTROL OF ADIPOSE TISSUE LIPASE ACTIVITY BY THE SYMPATHETIC NERVOUS SYSTEM R . Maickel, H . Suasman, K . Yamada*
and B . Brodie
Laboratory of Chemical Pharmacology National Heart Institute, Bethesda, Maryland (Received 4 March 1963) A number of studies have shown that exogenous epinephrine or norepinephrine elicits a pronounced mobilization of free fatty acids (FFA) in the intact rat and from adipose tissue in vitro (1) .
Recent papers have reported that the
activity of adipose tissue lipase is stimulated by the incubation of epididymal fat pads with small quantities of catecholamines
(2-4) .
These observations
along with the discovery that adipose tissue contains considerable amounts of norepinephrine suggested that mobilization of FFA is regulated by the sympathetic nervous system through activation of adipose tissue lipase (5,6) . A previous paper from this laboratory reported that an intact sympathetic nervous system is required for an increased output of FFA and glucose in rats exposed to a low temperature (4'C) (7) .
After complete depletion of
peripheral catecholamines, animals exposed to cold were unable to mobilize sufficient quantities of these metabolic substrates to maintain a normal body temperature . The present paper explores the possibility that the sympathetic nervous system controls the output of FFA by regulating the activity of adipose tissue lipase .
The results indicate that adipose tissue lipase is stimulated and
that plasma FFA are elevated in rata by the administration of catecholamines *Present address :
Faculty of Medicine, University of Tokyo, Japan
No . 3
ADIPO.SS TZ38US LIPA3$ AIm 3Y1~ATHETIC IO~t9pU8 SYS1'B~
or a ganglionic stimulant, or by exposure to Cold .
211
Ia contrast, adipose tiaaue
lipase and plasma FFA are unaffected by cold-exposure or by ganglionic atimilation in the absence of a functional sympathetic nervous system . Materials and Methods 8xperimeats were carried out in adult, male, Sprague-Dawley rata (180200 grams) .
Adrenal-demedullated rata (Hormone Assay Laboratories, Chicago,
Illinois) were maintained oa 1X saline-5X glucose for seven days and then given tap water .
They were used for ezperimeats 12 to 20 days after surgery when
their adrenal glands weighed 80 to 90X of normal .
Chemical sympathectomy was
performed by administration of nonsedative doses of reaerpine to adrenaldemedullated rats as described in a previous paper (7) .
The rata were placed
in the cold room (4° C) in individual cages . To assay plasma FFA, animals were stunned and immediately decapitated ; blood was collected into heparinized beakers, transferred to tubes sad i~ediately centrifuged .
For FFA assay, aliquots of plasma were removed and trans
ferred to the solvent mixture of Dole (8) .
To measure the activity of adipose
tiaaue lipase, epididymal fat pads were removed from the animals, quickly chilbed in ice, weighed sad immediately homogenis ed in 0 .1 M phosphate buffer, pH 7 .0 ; adipose tiaaue lipase was assayed by a modification of the method of Rizack (2), measuring the glycerol formed from tributyrin (9) . Results One hour after exposure of normal or adrenal demedullated rata to a temperature of 4 ° C, there was a pronounced rise in adipose tiaaue lipase activity as well as in plasma FFA levels .
In contrast, the FFA and lipase were not
increased if animals were pretreated aith a ganglionic blocking agent (chlorieondamine) or subjected to cheaical sympathectomy .
(Table 1)
One hour after the administration of the catecholamines, increases in adipose tissue lipase and in plasma FFA were observed both in normal rata and in chemically sympathectomized or chlorieoadamine-treated animals .
(Table 2)
212
ADIPOSE TISSUS LIPASE AND SYI~ATHETIC NERVOUS SYSTEM
No . 3
TABLE 1 Effects of Adrenal Demedullation, Ganglionic Blockade or Chemical Sympathectomy on Plasma FFA and Adipose Tissue Lipase in Rata Exposed to Cold (4 ° C) .
Treatment
Number of Plasma FFA Animals ~E ml t S .D,
Intact Control Intact Control + Cold-Exposure Adrenal Demedullated Adrenal Demedullated + Cold-Exposure Gaaglionic Blockade* + Cold-Exposure Chemical Sympathectomy + Cold-Exposure
Adipose Tissue Li ass G.imol g hr t S .D,
16 9
0 .41 ± .05 1 .03 ± .12
6 .8 t 1 .0 13 .9 ± 1 .2
7 9
0 .44 t .08 0 .98 ± .10
6 .4 ± 1 .3 12 .0 ± 1 .3
11 12
0 .47 t .04 0 .40 t .05
4 .7 ± 1 .0 5 .9 ± 1 .3
*Chlorisondamine (Ecolid), 5 mg/kg i .p ., 2 hra before cold-exposure . TABLE 2 Effects of Ganglionic Blockade or Chemical Sympathectomy on Plasma FFA and Adipose Tissue Lipase in Rats Given Epinephrine or Norepinephrine .
Treatment
Number of Animals
Intact Control
Plasma FFA_ uE0 ml ± S .D .
Adipose Tissue Li ass umol A hr t S,D .
16
0 .41 t 0 .5
6 .8 ± 1 .0
Intact Control + Catecholamine* 12 Ganglionic Blockade** + Catecholamine* 11 Chemical 3ympathectomy + Catecholamine* 14
1 .20 t .11 1 .09 t .08 1 .12 ± .05
15 .3 ± 1 .9 14 .7 t 1 .2 15 .1 t 1 .9
*Epinephrine or norepinephriae, 0 .2 mg/kg, i .m . **Chlorisondamine (Ecolid), 5 mg/kg, i .p ., 2 hra before epinephrine . These results indicate that the failure of the animals to respond to coldexposure after gaaglionic blockade or chemical sympathectomy was owing to an interference with sympathetic function . The administration of the ganglionic stimulant, DMPP (1,1-dimethyl-4pheaylpiperaziaium iodide) to adrenal-demedullated animals produced an increase in plasma FFA and in adipose tissue lipase within one hour (Table 3) . responses to DMPP did not occur in chemically sympathectomised animals .
These
No . 3
ADIP03B TISSIJi LIPASE AND SYl~AT88TIC NERVOUS SYS'i~t
213
TABIE 3 Effects of D1~P on Plasma FFA Level and Adipose Tieaue Lipase Activity in Rata Number of Animals
Treatment Intact Control Intact Control + Dl~P* Adrenal Demedullated Adrenal Demedullated + DMPP* Che~ical Syu~athectomy + II~!!PP*
Plasma FFA ~EQ ml t 3,D,
Adipose Tissue Li ass 4xmo1 g hr t S,D,
16 4
0 .41 t .05 0 .99 t .10
6 .8 f 1 .0 14 .9 t 1 .6
7 5 5
0 .44 t .08 0 .88 t .08 0 .43 t .06
6 .4 t 1 .3 12 .9 t 1 .4 7 .2 ± 1 .1
*~P, 0 .5 mg/kg, i .m . This dose of drug produces signs typical of increased sympathetic tone including piloerection and exophthalmue . Larger doses of drug may cause convulsions . Mscusaion Previous publications have reported that the exposure of rata to cold (4 ° C) markedly increases the plasma levels of FFA (6,7,10) .
Since the increase
in FFA waa prevented by chemical or functional sympathectomy but was not pre vented by removal of the adrenal medulla, it was concluded that norepinephrine at peripheral nerve endings is important in mobilizing FFA (7) . The present paper suggests that the increased mobilization of FFA is mediated by the sympathetic nervous system through the activation of adipose tissue lipase .
Evidence for this is shown by a number of experimental facts .
For example, the injection of norepinephriae or epinephrine stimulates both the enzyme and the output of FFA in the intact animal . Furthermore the exposure of rata to cold (4 ° C) elevates the level of plasma FFA and the activity of adipose tissue lipase, presumably by releasing norepinephrine at adipose tissue nerve endings .
When sympathetic function is
blocked by ganglionic blockade or by chemical sympathectomy, the cold-induced mobilization of FFA and activation of adipose tissue lipase is prevented . Stimulation of sympathetic ganglia by the administration of D1~P provides additional evidence that a release of norepinephrine at adipose tissue nerve endings activates adipose tissue lipase and elicits a rise in plasma FFA levels .
No . 3
ADIPO~ TISSUE LIPASE AND SYI~ATHE1'IC NERVOUS SY~EM
214
These responses depend upon a functional sympathetic system ;
DMPP fails to
affect the plasma FFA or the adipose tissue lipase after chemical sympathectomy . Adrenal demedullation has little or no effect on the mobilization of FFA induced by cold-eapoaure or by DMPP .
This suggests that the mobilization a~f
FFA is regulated by the sympathetic nerve endings in adipose tissue itself ; adrenal medullary amines appear to be of little importance .
In contrast, the
adrenal medullary amines appear to be of prime importance in regulating the blood levels of glucose (7) . In conclusion, the activation of the enzyme adipose tissue lipase by the sympathetic nervous system is a unique confluence of biochemistry and physiology in which a neurohormone released at nerve endings stimulates a biochemical mechanism . References 1.
D . STEINBERG and M, VAUGHAN, Proc . 5th Int . Congr . Biochem . Moscou, in press, (1961) .
2.
M,A . RI2ACR, J . Biol . Chem . 2~, 657 (1961)
3.
H .H . SUSSMAN, R . P, MAICREL, R . YAMALIA, D . N . STERN and B .B . BRODIE, The Pharmacologiat , 4,
173 (1962)
4.
P . BJORNTORP and R . H . FUlalAN, Amer . J . Phyaiol . 203, 316 (1962)
5.
R . PAOLETTI, R .L . SMITH, R . P, MAICREL and B .B .BRODIE, Biochem . Biophys . Rea . Comm . 5, 424 (1961) .
6.
R . PAOLETTI, R .P . MAICREL, R .L . SMITH and B .B . BRODIE, Proc .
lat Int .
Pha :macol . Meeting , Stockholm, in press (1961) . 7.
A, GILGEN, R,P . MAICREL, 0 . NIRODLTEVIC and B .B . BRODIE, Life Sci . 1, 709 (1962)
S.
V,P . DOLE, J . Clin . Invest ., 35, 150 (1956)
9.
W .M . BUTLER, JR ., H .M . MALING, M .G . HORNING and B,B, BRODIE, J . Lipid Rea . 2, 95 (1960)
10 . R,P . MAICREL, E,O . WESTERMANN and B .B . BRODIE, J . Pharm . Exptl . Therap . 167
(1961) .
1~34~
OONTROL OF ADIPOSE TISSUE LIPASE ACTIVITY BY THE SYMPATHETIC NERVOUS SYSTEM R . Maickel, H . Suasman, K . Yamada*
and B . Brodie
Laboratory of Chemical Pharmacology National Heart Institute, Bethesda, Maryland (Received 4 March 1963) A number of studies have shown that exogenous epinephrine or norepinephrine elicits a pronounced mobilization of free fatty acids (FFA) in the intact rat and from adipose tissue in vitro (1) .
Recent papers have reported that the
activity of adipose tissue lipase is stimulated by the incubation of epididymal fat pads with small quantities of catecholamines
(2-4) .
These observations
along with the discovery that adipose tissue contains considerable amounts of norepinephrine suggested that mobilization of FFA is regulated by the sympathetic nervous system through activation of adipose tissue lipase (5,6) . A previous paper from this laboratory reported that an intact sympathetic nervous system is required for an increased output of FFA and glucose in rats exposed to a low temperature (4'C) (7) .
After complete depletion of
peripheral catecholamines, animals exposed to cold were unable to mobilize sufficient quantities of these metabolic substrates to maintain a normal body temperature . The present paper explores the possibility that the sympathetic nervous system controls the output of FFA by regulating the activity of adipose tissue lipase .
The results indicate that adipose tissue lipase is stimulated and
that plasma FFA are elevated in rata by the administration of catecholamines *Present address :
Faculty of Medicine, University of Tokyo, Japan
No . 3
ADIPO.SS TZ38US LIPA3$ AIm 3Y1~ATHETIC IO~t9pU8 SYS1'B~
or a ganglionic stimulant, or by exposure to Cold .
211
Ia contrast, adipose tiaaue
lipase and plasma FFA are unaffected by cold-exposure or by ganglionic atimilation in the absence of a functional sympathetic nervous system . Materials and Methods 8xperimeats were carried out in adult, male, Sprague-Dawley rata (180200 grams) .
Adrenal-demedullated rata (Hormone Assay Laboratories, Chicago,
Illinois) were maintained oa 1X saline-5X glucose for seven days and then given tap water .
They were used for ezperimeats 12 to 20 days after surgery when
their adrenal glands weighed 80 to 90X of normal .
Chemical sympathectomy was
performed by administration of nonsedative doses of reaerpine to adrenaldemedullated rats as described in a previous paper (7) .
The rata were placed
in the cold room (4° C) in individual cages . To assay plasma FFA, animals were stunned and immediately decapitated ; blood was collected into heparinized beakers, transferred to tubes sad i~ediately centrifuged .
For FFA assay, aliquots of plasma were removed and trans
ferred to the solvent mixture of Dole (8) .
To measure the activity of adipose
tiaaue lipase, epididymal fat pads were removed from the animals, quickly chilbed in ice, weighed sad immediately homogenis ed in 0 .1 M phosphate buffer, pH 7 .0 ; adipose tiaaue lipase was assayed by a modification of the method of Rizack (2), measuring the glycerol formed from tributyrin (9) . Results One hour after exposure of normal or adrenal demedullated rata to a temperature of 4 ° C, there was a pronounced rise in adipose tiaaue lipase activity as well as in plasma FFA levels .
In contrast, the FFA and lipase were not
increased if animals were pretreated aith a ganglionic blocking agent (chlorieondamine) or subjected to cheaical sympathectomy .
(Table 1)
One hour after the administration of the catecholamines, increases in adipose tissue lipase and in plasma FFA were observed both in normal rata and in chemically sympathectomized or chlorieoadamine-treated animals .
(Table 2)
212
ADIPOSE TISSUS LIPASE AND SYI~ATHETIC NERVOUS SYSTEM
No . 3
TABLE 1 Effects of Adrenal Demedullation, Ganglionic Blockade or Chemical Sympathectomy on Plasma FFA and Adipose Tissue Lipase in Rata Exposed to Cold (4 ° C) .
Treatment
Number of Plasma FFA Animals ~E ml t S .D,
Intact Control Intact Control + Cold-Exposure Adrenal Demedullated Adrenal Demedullated + Cold-Exposure Gaaglionic Blockade* + Cold-Exposure Chemical Sympathectomy + Cold-Exposure
Adipose Tissue Li ass G.imol g hr t S .D,
16 9
0 .41 ± .05 1 .03 ± .12
6 .8 t 1 .0 13 .9 ± 1 .2
7 9
0 .44 t .08 0 .98 ± .10
6 .4 ± 1 .3 12 .0 ± 1 .3
11 12
0 .47 t .04 0 .40 t .05
4 .7 ± 1 .0 5 .9 ± 1 .3
*Chlorisondamine (Ecolid), 5 mg/kg i .p ., 2 hra before cold-exposure . TABLE 2 Effects of Ganglionic Blockade or Chemical Sympathectomy on Plasma FFA and Adipose Tissue Lipase in Rats Given Epinephrine or Norepinephrine .
Treatment
Number of Animals
Intact Control
Plasma FFA_ uE0 ml ± S .D .
Adipose Tissue Li ass umol A hr t S,D .
16
0 .41 t 0 .5
6 .8 ± 1 .0
Intact Control + Catecholamine* 12 Ganglionic Blockade** + Catecholamine* 11 Chemical 3ympathectomy + Catecholamine* 14
1 .20 t .11 1 .09 t .08 1 .12 ± .05
15 .3 ± 1 .9 14 .7 t 1 .2 15 .1 t 1 .9
*Epinephrine or norepinephriae, 0 .2 mg/kg, i .m . **Chlorisondamine (Ecolid), 5 mg/kg, i .p ., 2 hra before epinephrine . These results indicate that the failure of the animals to respond to coldexposure after gaaglionic blockade or chemical sympathectomy was owing to an interference with sympathetic function . The administration of the ganglionic stimulant, DMPP (1,1-dimethyl-4pheaylpiperaziaium iodide) to adrenal-demedullated animals produced an increase in plasma FFA and in adipose tissue lipase within one hour (Table 3) . responses to DMPP did not occur in chemically sympathectomised animals .
These
No . 3
ADIP03B TISSIJi LIPASE AND SYl~AT88TIC NERVOUS SYS'i~t
213
TABIE 3 Effects of D1~P on Plasma FFA Level and Adipose Tieaue Lipase Activity in Rata Number of Animals
Treatment Intact Control Intact Control + Dl~P* Adrenal Demedullated Adrenal Demedullated + DMPP* Che~ical Syu~athectomy + II~!!PP*
Plasma FFA ~EQ ml t 3,D,
Adipose Tissue Li ass 4xmo1 g hr t S,D,
16 4
0 .41 t .05 0 .99 t .10
6 .8 f 1 .0 14 .9 t 1 .6
7 5 5
0 .44 t .08 0 .88 t .08 0 .43 t .06
6 .4 t 1 .3 12 .9 t 1 .4 7 .2 ± 1 .1
*~P, 0 .5 mg/kg, i .m . This dose of drug produces signs typical of increased sympathetic tone including piloerection and exophthalmue . Larger doses of drug may cause convulsions . Mscusaion Previous publications have reported that the exposure of rata to cold (4 ° C) markedly increases the plasma levels of FFA (6,7,10) .
Since the increase
in FFA waa prevented by chemical or functional sympathectomy but was not pre vented by removal of the adrenal medulla, it was concluded that norepinephrine at peripheral nerve endings is important in mobilizing FFA (7) . The present paper suggests that the increased mobilization of FFA is mediated by the sympathetic nervous system through the activation of adipose tissue lipase .
Evidence for this is shown by a number of experimental facts .
For example, the injection of norepinephriae or epinephrine stimulates both the enzyme and the output of FFA in the intact animal . Furthermore the exposure of rata to cold (4 ° C) elevates the level of plasma FFA and the activity of adipose tissue lipase, presumably by releasing norepinephrine at adipose tissue nerve endings .
When sympathetic function is
blocked by ganglionic blockade or by chemical sympathectomy, the cold-induced mobilization of FFA and activation of adipose tissue lipase is prevented . Stimulation of sympathetic ganglia by the administration of D1~P provides additional evidence that a release of norepinephrine at adipose tissue nerve endings activates adipose tissue lipase and elicits a rise in plasma FFA levels .
No . 3
ADIPO~ TISSUE LIPASE AND SYI~ATHE1'IC NERVOUS SY~EM
214
These responses depend upon a functional sympathetic system ;
DMPP fails to
affect the plasma FFA or the adipose tissue lipase after chemical sympathectomy . Adrenal demedullation has little or no effect on the mobilization of FFA induced by cold-eapoaure or by DMPP .
This suggests that the mobilization a~f
FFA is regulated by the sympathetic nerve endings in adipose tissue itself ; adrenal medullary amines appear to be of little importance .
In contrast, the
adrenal medullary amines appear to be of prime importance in regulating the blood levels of glucose (7) . In conclusion, the activation of the enzyme adipose tissue lipase by the sympathetic nervous system is a unique confluence of biochemistry and physiology in which a neurohormone released at nerve endings stimulates a biochemical mechanism . References 1.
D . STEINBERG and M, VAUGHAN, Proc . 5th Int . Congr . Biochem . Moscou, in press, (1961) .
2.
M,A . RI2ACR, J . Biol . Chem . 2~, 657 (1961)
3.
H .H . SUSSMAN, R . P, MAICREL, R . YAMALIA, D . N . STERN and B .B . BRODIE, The Pharmacologiat , 4,
173 (1962)
4.
P . BJORNTORP and R . H . FUlalAN, Amer . J . Phyaiol . 203, 316 (1962)
5.
R . PAOLETTI, R .L . SMITH, R . P, MAICREL and B .B .BRODIE, Biochem . Biophys . Rea . Comm . 5, 424 (1961) .
6.
R . PAOLETTI, R .P . MAICREL, R .L . SMITH and B .B . BRODIE, Proc .
lat Int .
Pha :macol . Meeting , Stockholm, in press (1961) . 7.
A, GILGEN, R,P . MAICREL, 0 . NIRODLTEVIC and B .B . BRODIE, Life Sci . 1, 709 (1962)
S.
V,P . DOLE, J . Clin . Invest ., 35, 150 (1956)
9.
W .M . BUTLER, JR ., H .M . MALING, M .G . HORNING and B,B, BRODIE, J . Lipid Rea . 2, 95 (1960)
10 . R,P . MAICREL, E,O . WESTERMANN and B .B . BRODIE, J . Pharm . Exptl . Therap . 167
(1961) .
1~34~