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Sympathetic nervous control of adipose tissue lipolysis
ht. J. Neuropharmacol.,
1965,7,393-403 Pergamon Press.Printed inGt.Britain.
SYMPATHETIC
Laboratory
NERVOUS CONTROL OF ADIPOSE TISSUE LIPOLYSIS
BENJAMINWEISS*, and RCGER P. MAIcIcELt of Chemical Pharmacology, National Heart Institute, National Institutes of Health, Bethesda, Maryland (Accepted 9 January 1968)
Summary-The in vitro electrical stimulation of nerves innervating rat epididymal fat pads produces a consistent and measurable biochemical change; i.e. the hydrolysis of triglycerides to FFA. The optimum electrical conditions for the nervous stimulation in eliciting a lipolytic response were 5 V square wave impulses delivered at a frequency of 50 per set and of 1 msec duration. Pretreatment of the animals with dibenamine, ergotamine, syrosingopine, bretylium or the bretylium-like agent, BW 392C60, antagonized the usual lipolytic response elicited by nervous stimulation. The in vitro addition of the beta adrenergic blocking agent, DCR, completely blocked lipolysis caused by electrical stimulation. Pargyline, atropine and theophylline all enhanced the electrically-induced increase in the rate of hydrolysis of triglycerides. Adrenalectomy decreased the response while cortisone treatment enhanced the response to nervous stimulation of fat pads. These results indicate that the process of a physiological event inducing a biochemical response can be demonstrated. The data show further that the lipolytic response is under the direct control of nerve fibers which are functionally sympathetic, since this response can be modified by a wide variety of compounds which are known to influence sympathetic activity both at the neuro-effector junction and at the postjunctional sites of hormonal action. INTRODUCTION
lipolytic effect of catecholamines in vivo (ABBOUD et al., 1963; HAMBURGER et al., 1963; and GRAHAM et al., 1964) can be blocked by pretreatment of the animals with suitable blocking agents (GOODMAN and KNOBIL, 1959; HAVEL and GOLDFEIN, 1959; FROBERGand ORO, 1963; BURNS et al., 1964; KVAM et al., 1965). Similarly, adipose tissue in vitro produces increased quantities of free fatty acids (FFA) when epinephrine (E) or norepinephrine (NE) is added to the medium (BUCKLE and BECK, 1962; RUDMAN et al., 1964; WENKE et al., 1964); this response is also blocked by adrenergic blocking agents (SCHOTZ and PAGE, 1960; LOVE et al., 1963a, LOVE et al., 1963b; WENKE et al., 1964). These observations clearly demonstrate the ability of exogenous catecholamines to stimulate lipolysis in adipose tissue. The role of endogenous catecholamines in regulation of lipolytic activity is less clear. HAUSBURGER (1934) presented anatomical evidence for sympathetic nervous innervation of adipose tissue, a finding subsequently confirmed by SIDMAN and FAWCETT (1954). However, in a more recent study utilizing fluorescent microscopy, WIRSEN (1964) could not demonstrate a sympathetic plexus around fat cells. On the other hand, NE has been identified as a constituent of adipose tissue in concentrations exceeding those required THE WELL-KNOWN
*Present address: Department of Pharmacology, College of Physicians and Surgeons of Columbia University, New York, New York. IPresent address: Departments of Pharmacology and Psychology, Indiana University, Bloomington, Indiana. 395
396
B. WEISSand R. P. MAICKEL
for circulatory control (PAOLETTIet al., 1961), and the catecholamine content of adipose tissue has been shown to decrease following denervation (SIDMAN et al., 1962). Since exogenous catecholamines stimulate lipolysis in adipose tissue, it seemed reasonable to assume that increased sympathetic tone, through a release of NE at nerve endings in adipose tissue, would also stimulate lipolytic activity. Preliminary work from this laboratory (NASH et al., 1961) gave evidence to support this supposition. Using an in situ preparation of dog omental fat, it was shown that electrical stimulation of the sympathetic fibers evoked an increased output of FFA into the venous drainage of the tissue. This preparation, however, proved to be rather cumbersome and inconvenient, and yielded erratic results. A better preparation, yielding similiar results, has been described by ORO et al. (1965). CORRELL(1963) described a preparation of the rat epididymal fat pad in vitro which permitted electrical stimulation of the nerve plexus leading to the fat pad. In this preparation, stimulation increased the output of FFA into the medium, an increase partially blocked by dibenamine. The present paper describes a detailed study of factors influencing the lipolytic response of rat epididymal fat pads electrically stimulated in vitro, including parameters of stimulation and the effects of various drugs. The results are further evidence that the lipolytic activity of mammalian adipose tissue is under the control of the autonomic nervous system. A preliminary report has been presented (WEISS and MAICKEL, 1964).
METHODS Male Sprague-Dawley rats weighing 180-200 g were given free access to food and water. Adrenalectomized and adrenal demedullated rats were purchased from Hormone Assay Laboratories, Chicago, Illinois. They were given 5 % glucose and 1% saline in their drinking water for 4 days. Adrenalectomized rats were given tap water for 1 day and sacrificed the following day; demedullated animals were maintained on tap water until used (34 weeks). Animals were killed by cervical dislocation, and the epididymal fat pads with attached pedicle, containing the internal spermatic artery and its accompanying nerve, were isolated and placed in a 25-ml Erlenmeyer flask containing 4 ml Krebs-Ringer phosphate buffer (pH 7.4) with 5 % bovine serum albumin. The preparation is basically the same as that described by CORRELL(1963). The fat pad was immersed in the buffer with its neurovascular pedicle draped over platinum electrodes above the incubation medium. The pedicle was held in place by attaching it to a metal rod embedded in a rubber stopper. The electrodes were encased in glass tubing for support and insulation. In practice, several flasks were arranged in parallel so that a constant voltage was maintained across each pair of electrodes. Square wave pulses were provided with a laboratory stimulator (American Electronic Laboratories). FFA in the medium were determined as described by DOLE and MEINERTZ (1960) and glycerol released into the incubation medium was assayed in deproteinized (5 % tricholoracetic acid) filtrates by the periodate oxidation procedure of LAMBERTand NEISH (1950). Whenever possible, a non-stimulated fat pad from the same rat served as a control. MATERIALS Bovine serum albumin fraction V (fatty acid poor) was purchased from Nutritional Biochemical Corp. DCB, [1-(2’,4’-dichlorophenyl)-l-hydroxy-2-(t-butylamino) ethane
Sympathetic nervous control of adipose tissue lipolysis
397
HCl] was supplied by Eli Lilly & Co. BW-392C60 (N-orthochlorobenzyl-N’,N”-dimethylguanidine sulfate) was provided by Burroughs Wellcome 8z Co., and pargyline (MO-911) (N-benzyl-N-methyl-2-propylamine HCl) by the courtesy of Abbott Laboratories. RESULTS
The effects of varying the frequency of stimulation, while maintaining a constant voltage and duration, on the lipolytic response are shown in Table 1. At a frequency of l/set, no measurable lipolytic response was evident, whereas frequencies of 10, 30, 50 and lOO/sec produced approximately the same response. The most reproducible effects were elicited with impulses of 50/set. At a constant frequency of SO/set, and a duration of 1 msec, the optimum voltage for release of FFA from epididymal fat pads was 5 V. TABLET. STIMULATIONPARAMETERS--EFFECTOFVOLTAGEANDFREQUEN~YOFELECTRICAL STIhWLATIONONTHERELEASEOFFREEFAlTYACIDSFROMRATEPILMDYhfALFATPADS
No. of fat pads
Stimulus frequency (impulses I=)
6 12 12 47 11
1: 30 50 100
5’ 5 5 5
-0*02&0.03 0.42&0*10 0*42z!zO.O8 044&0*05 0.43 50.07
8
50
1
0.21 &O-O6
Stimulus intensity 0
Increase due to electrical stimulation &Eq FFA released/g/hr)fS.E.
Rat epididymal fat pads were incubated for 1 hr in Rrebs-Ringer phosphate buffer (pH 7.4) containing 5% bovine serum albumin. Fat pad nerves were stimulated with square wave impulses of 1 m set duration. FFA released into the medium were determined as described in Methods.
Utilizing the conditions of 50 pulses/set, 1-msec duration and an intensity of 5 V, the time relationships for the release of FFA and glycerol were studied. As seen from Fig. 1, a measurable lipolytic response was produced within 30 min. Lipolysis was linear for at least 1 hr and continued to increase for the 2-hr period of stimulation. Little or no additional lipolysis occurred from 2 to 4 hr in fat pads stimulated for a 4-hr period. In the studies reported below, a 1-hr period of incubation was employed. To relate the lipolytic response to the activity of the sympathetic nervous system, various drugs were employed which affect sympathetic function. The data in Table 2 show the effects of adrenergic blocking agents and indicate that fat pads taken from animals treated with dibenamine or ergotamine were less responsive to the lipolysis induced by electrical stimulation. Addition of DCB, an analogue of the beta adrenergic blocking agent dichloroisoproterenol (DCI), to the incubation medium produced some stimulation of lipolysis, but completely prevented any additional lipolytic response to subsequent electrical stimulation. Indeed, the slight lipolytic effect produced by DCB tended to decrease in the fat pad which was stimulated. Table 3 shows the effects of compounds which are known to influence sympathetic function without having typical adrenergic blocking activity. Pretreatment of the animals with syrosingopine or BW 392C60 effectively blocked (P
398
B.
Y '
WEISS and
R. P. MAICKEL
I
IO
20
30
40
50
60
70
80
90
100
110
120
DURATION OF STIMULATION(mm) FIG. 1. Electrical stimulation of rat epididymal fat pad: Time-Response. Rat epididymal fat pads were incubated in Krebs-Ringer phosphate buffer (PH 7.4) containing 5% bovine serum albumin. Nerves supplying the fat pads were stimulated with square wave impulses (1 msec; 5 V; 5O/sec). FFA and glycerol released into the medium were determined as described in Methods.
n - - - - - n glycerol; 0-0
TABLE 2.
No. of fat pads
FFA.
EFFECT OF ADRENERGICBLOCKING AGENTS ON RESPONSEOF EPIDIDYMALFAT PAD TO ELECTRICAL STIMULATION
Pretreatment
Added to medium
FFA
released @Eq/g/hr)
Non-stimulated
f S.E.)
Stimulated
Change due to electrical stimulation
6 6
Solvent Dibenamine
-
0.19&0.07 0*12*oGI
0~41*0~11 0.21 *o*os
0.22*0.12 0~09f0~02
6 6
Saline Ergotamine
-
0*07*0.04 0.41 kO.04
0.67f0.19 0*74&0*12
0.6OrtO.20 0*33&0*11
-
Saline DCB
0.07*0.04 0.73*0*11
0.49&0*09 055*0*17
0*42&-0.12 -0*18&0*21*
Rat epididymal fat pads were incubated for 1 hr in Krebs-Ringer phosphate buffer (pH 7.4) containing 5 % bovine serum albumin. Nerves supplying fat pads were stimulated with square wave impulses (1 msec; 5 V; SO/se@. FFA released in the medium were determined as described in Methods. Dibenamine HCl: 25 mg/kg i.p.; 44 hr and 20 hr prior to sacrifice. Ergotamine tartrate: 5 mg/kg Lp.; 1 hr prior to sacrifice. DCB: 1-(2’,4’-dichlorophenyl)-l-hydroxy-2-(t-butylamino) ethane HCl (lO-SM). *P < 0.05 compared with changes caused by electrical stimulation in control tissues.
399
Sympathetic nervous control of adipose tissue lipolysis
TABLE 3. EFFJXTOF SYMPATHOLYTICS ON RESPONSE OF EPIDIJXMALFAT PAD TO ELECTRICALSTBKJLATION Change
No. of
FFA released (rEq/g/hr)&S.E.
fat pads
Pretreatment
10
Non-stimulated
Stimulated
due to electrical stimulation
10
Solvent Syrosingopine
0.20f0.03 0.14f0.03
O-48*0.07 0*22&0.03
0.28f0.06 0.08 *0.04*
:
Saline BW 392C60
0.16f0.05 0.11+0.02
0.45 jzo.07 0.12*0.03
0.29f0.10 O-01&0.02*
4 4
Saline Bretylium
0.17f0.08 O.l7=tO*O6
0.48kO.13 0.39*0.07
0.31&0*13 0*22+oGI
Experimental
conditions and statistical notations were the same as those outlined in Table 2.
Syrosingopine: 3 mg/kg i.p. ; 22 hr and 5 hr prior to sacrifice (all rats were demedullated 3 weeks prior to use). BW 392C60: N-orthochlorobenzyl-N’,N”-dimethylguanidine 20 mg/kg i.p.; 4 hr prior to sacrifice. Bretylium tosylate: 40 mg/kg i.p.; 24 hr and 2.5 hr prior to sacrifice. *P < 0.05 compared with change caused by electrical stimulation in control tissues.
the fat pads to electrical stimulation. Bretylium was considerably less potent in this regard. Increased responsiveness of fat pads to electrical stimulation was produced by the addition of three structurally dissimilar compounds (Table 4). Pretreatment of rats with pargyline, a non-hydrazine monoamine oxidase inhibitor (TAYLOR et al., 1960), increased the lipolytic response to electrical stimulation by about 45%. Preincubation of the fat pads with either theophylline, a compound which inhibits phosphodiesterase, or the cholinergic blocking agent, atropine, also caused an enhanced release of FFA induced by nervous stimulation. TABLE 4.
EFPECTOPVAR~OUSDRUGSONRESPONSEOFEPIDIDYMALFATPADTOELECTRICALSTIMULA~ON
No. of fat pads
Pretreatment
6 6
Saline MO 911
Stimulated
Change due to electrical stimulation
0.08 f 0.03 0.25 kO.08
064&0.17 1*22+0*21
0*56+0.16 0.97f0.21
FFA released bEq/g/hr) Added to medium -
Non-stimulated
f S.E.
8 8
-
Saline Atropine
0.12+0.03 0*11*0*03
0*47*0*09 O-72&0.06
0*35&0*08 0.61 rtO.06,
8 8
-
Saline Theophylline
O-28&0*07 0*42*0.06
0*53&0.03 0.89f0.08
0*25&0*04 0~47*0*04t
Experimental
conditions were the same as those outlined in Table 2.
MO 911: Pargyline - 50 mg/kg i.p.; 1.5 hr prior to sacrifice. Atropine sulfate: lo-’ M. Theophylline: 4 x lo-” M. *P < @05 compared
with changes caused by electrical stimulation in control tissues.
tP < 0.01 compared
with changes caused by electrical stimulation in control tissues.
B. WEISSand R. P. MAICKEL
400 Table
5 shows
that fat pads from
medium in response to electrical controls
(P
whereas
adrenalectomized
stimulation
fat
pads
as compared
from
rats release
less FFA
into
the
with fat pads from sham operated
cortisone-treated
rats
release
significantly
(P
8
Stimulated
Change due to electrical stimulation
FFA released &Eq/g/hr)&S.E. Animal condition
Treatment
8
Sham operated Adrenalectomized
8 8
Non operated Non operated
Non-stimulated
-
0~41*0*04 056&0.07
1*05*0*12 0.84f0.11
064&0*10 0.28~0*11*
Saline Cortisone
0.22f0.05 0*29&-0.11
0.54f0.11 1*22*0*17
0*32&0.11 0.93 f0.06t
Rats were adrenalectomized 5 days prior to use. For the first 4 days, they were maintained on 5 % glucose1% saline in their drinking water; on the last day they were given tap water. All rats were given free access to food. Experimental conditions were the sameras those outlined in Table 2. Cortisone acetate: 50 mg/kg s.c.; 19 hr and 3 hr prior to sacrifice. *P
DISCUSSION The early
observations
but also terminating recent
studies.
conclusive
However,
anatomical
FAWCETT, 1954). in adipose
adipose
disposed
not only along
blood
confirmed
that
they are sympathetic
recently
reviewed
and physiological
is as yet lacking
lipolysis upon the administration
is based
largely
of catecholamine
or in vitro or the increase in plasma FFA observed when animals are subjected
Thus,
it was demonstrated
the adrenergic
blocking
that epididymal
influences upon
the
either in vivo to “stressful”
situations. The purpose of this study was to provide more direct pharmacological that white adipose tissue responds functionally like one which has a sympathetic tion.
origin,
(SIDMAN and
evidence for autonomic
by BRODIE et al. (1965),
vessels by more
since these nerves have not been traced to their central
proof
of increased
being
tissue cells have been adequately
The pharmacological
tissue,
observations
of nerve fibers
among
evidence innerva-
fat pads from rats which had received
agents,
dibenamine or ergotamine, were less responsive to the Complete inhibition of the response was seen stimulation.
lipolytic effects of electrical when DCB, a structural analogue of the beta adrenergic blocking agent, DCI, was added to the incubation medium. DCB was used because it elicits relatively slight positive lipolytic
action as compared with DC1 (LOVE et al., 1963a; 1963b). Similarly, the response to nerve stimulation was inhibited by pretreatment of the animals with either syrosingopine, which decreases the catecholamine stores in white adipose tissue to undetectable levels within 4 hr (STOCK and WESTERMANN, 1963), or with BW 392C60, which prevents the release of NE induced by electrical stimulation (BOURA and GREEN, 1963). The relative lack of effectiveness of bretylium may be due to the frequency with which the nerves were stimulated. GESSA et al. (1963), in studies involving the electrical stimulation of the splanchic nerve, have shown that bretylium blocked the response elicited by low frequency stimulation (4 c/s) but not
Sympathetic nervous control of adipose tissue lipolysis
401
that produced by high frequency stimulation (32 c/s). On the other hand, BW 392C60 blocked the response in both high and low frequency stimulatio. It has been shown (STOCK and WESTERMANN,1963) that inhibition of monoamine oxidase increases NE levels in adipose tissue. Administration of pargyline, a potent monoamine oxidase inhibitor, resulted in an increased lipolytic response to electrical stimulation, again indicating an adrenergic component to the lipolytic effects of nervous stimulation. Further evidence that the lipolytic response of rat epididymal fat pads is typically sympathetic is given from results with adrenalectomized rats. Adrenalectomy, which decreases the response to sympathetic stimulation in vivo (BRODIEet al., 1966), also decreases FFA release in response to electrical stimulation. Similarly, pretreatment of rats with corisone, which has been shown to increase the lipolytic response to catecholamines (SHAFRIRand KERPEL, 1964; DAVIESand WEISS,unpublished observations), increases the the lipolytic response to electrical stimulation of nerves supplying epididymal fat pads. In an attempt to gain further insight into the more discrete mechanisms involved in the sequence of reactions between the release of NE from nerve terminals and the lipolytic response, fat pads were incubated with theophylline prior to stimulation of the nerve fibers. This compound inhibits cyclic nucleotide phosphodiesterase prepared from beef heart (BUTCHERand SUTHERLAND,1962) as well as that prepared from rat adipose tissue (WEISSet al., 1966; BRODIEet al., 1966; HYNIEet al., 1966). Cyclic 3’,5’-AMP, the substrate for this enzyme, stimulates lipolysis in isolated fat cells and has been implicated as a mediator of the hormone-induced lipolytic response (MAICICEL et al., 1965; BUTCHWet al., 1965; WEISSet al., 1966). So far, the major evidence for a role of the cyclic nucleotide in lipolysis has been provided with studies of exogenously administered agents. However, the demonstration that theophylline increases the lipolysis caused by electrical stimulation indicates that catecholamines endogenously released from nerve endings may induce an increased formation of the cyclic nucleotide, thereby causing the lipolytic response. The stimulatory effect of atropine on the response of fat pads to electrical stimulation lends support to the possibility of a cholinergic influence on adipose tissue lipolysis. If there are cholinergic as well as adrenergic fibers innervating adipose tissue, then one would expect that the two systems would be antagonistic. Electrical stimulation of the entire neurovascular pedicle would then necessarily excite both types of fibers. By selectively inhibiting the cholinergic response with atropine, the restraining influence would be removed, and a greater lipolytic action would become manifest. The apparent reversal of the effect seen with DCB may also be explained by the existence of both types of fibers. DCB, by blocking only the stimulatory fibers, would thus unmask the effect of the inhibitory ones. The observation of SIDMANand FAWCETT(1954) that nerves entering fat bodies are mixed nerves carrying more than one type of fiber is in accord with this hypothesis. An alternate explanation for the decreased lipolytic response to electrical stimulation in the presence of DCB should be considered in light of the recent results of TURTLEand KIPNIS (1967), which indicate that alpha receptor stimulation decreases the concentration of cyclic 3’,5’-AMP in adipose tissue, whereas stimulation of beta receptors increases cyclic 3’,5’-AMP levels. Thus, the beta blocking drug, DCB, may have unmasked the alpha stimulating effect of sympathetic nervous stimulation, resulting in an inhibition of lipolysis.
402
B. WEISSand R. P. MAICKEL
REFERENCES ABBOUD,F. M., WENDLING,M. G. and ECKSTE~N,J. W. (1963). Effect of norepinephrine on plasma free fatty acids in dogs treated with reseroine. Am. J. Phvsiol. 205: 57-59. BOURA;A. L. A. and-GREEN, A. F. (1963): Adrenergic neurone blockade and other acute effects caused by Nbenzyl-N’N”-dimethylguanidine and its ortho-chloro derivative. Br. J. Pharmac. Chemother. 20: 36-55. BRODIE,B. B., DAVIES,J. I., Hm, S., KRISHNA.G. and WEISS,B.(1965). Interrelationships of catecholamine with other endocrine systems. Pharmac. Rev. 18: 273-289. BRODIE,B. B., MAICKEL,R. P. and STERN,D. N. (1965). Autonomic nervous system and adipose tissue. In Handbook of Physiology-Adipose Tissue, pp. 583-600. Am. Physiol. Sot., Washington, D.C. BUCKLE, R. M. and BECK, J. C. (1962). Modifying influence of glucose on fatty acid-mobilizing activities of epinephrine, ACTH and growth hormone in vitro. Metabolism 11: 235-243. BURNS, J. J., COLVILLE,K. I., LINDSAY,L. A. and SALVADOR,R. A. (1964). Blockade of some metabolic effects of catecholamines by N-isopropyl methoxamine(B.W. 61-43). J. Pharmac. exp. Ther. 144: 163-171. BUTCHER,R. W., Ho, R. J., MENG, H. C. and SUTHERLAND, E. W. (1965). 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: 4515-4523. BUTCHER,R. W. and SUTHERLAND,E. W. (1962). Adenosine 3’,5’-phosphate in biological materials I. Purification and properties of cyclic 3’,5’-nucleotide phosphodiesterase and use of this enzyme to characterize adenosine 3’,5’-phosphate in human urine. J. biol. Chem. 237: 1244-1250. CORRELL,J. W. (1963). Adipose tissue: Ability to respond to nerve stimulation in vitro. Science 140: 387-388. DOLE, V. P. and MEINERTZ,H. (1960). Microdetermination of long-chain fatty acids in plasma and tissues. J. biol. Chem. 235: 2595-2599. FROBERG,S. and ORO, L. (1963). The effects of nicotinic acid, phentolamine and nethalide on the plasma free fatty acids and the blood pressure in the dog. Acta med. stand. 174: 635-641. GESSA,G. L., CUENCA, E. and COSTA,E. (1963). On the mechanism of hypotensive effects of MAO inhibitors. Ann. N.Y. Acad. Sci. 107: 935-941. GOODMAN,H. M. and KNOBIL, E. (1959). Effect of adrenergic blocking agents on fatty acid metabolism during fasting. Proc. Sot. exp. Biol. Med. 102: 493-495. GRAHAM,M. H., ABBOUD,F. M. and ECKSTEIN,J. W. (1964). Effect ofnorepinephrine on plasma nonesterified fatty acid concentrations after administration of cocaine. J. Pharmac. exp. Ther. 143: 340-343. HAMBURGER,J., Slwnl, R. W., JR. and MILLER, J. M. (1963). Effects of epinephrine on free fatty acid mobilization in hyperthyroid and hypothyroid subjects. Metabolism 12: 821-828. HAUSBURGER,F. X. (1934). Uber die Innervation der Fettorgane. Z. Mikroscop. Anat. Forsch. 26: 231-266. HAVEL, R. J. and GOLDFIEN,A. (1959). The role of the sympathetic nervous system in the metabolism of free fatty acids. J. Lipid Res. 1: 102-108. HYNIE, S., KRISHNA, G. and BRODIE,B. B. (1966). Theophylline as a tool in studies of the role of cyclic adenosine 3’,5’-monophosphate in hormone-induced lipolysis. J. Pharmac. exp. Ther. 153: 90-96. KVAM, D. C., RIGGILO, D. A. and LISH, P. M. (1965). Effect of some new Badrenergic blocking agents on certain metabolic responses to catecholamines. J. Pharmac. exp. Ther. 149: 183-192. LAMBERT,M. and NEISH, A. C. (1950). Rapid method for estimation of glycerol in fermentation solutions. Can. J. Res. 28: 83-89. LOVE, W. C., CARR, L. and ASHMORE,J. (1963a). Lipolysis in adipose tissue: Effects of dl-3,4-di chloroisoproterenol and related compounds. J. Pharmac. exp. Ther. 140: 287-294. LOM, W. C., CARR, L. and ASHMORE,J. (1963b). Influence of isoproterenol and l-(2/,4’, dichlorophenyl)2-t-butylaminoethanol (DCB) and on free fatty acid release and glucose metabolism in adipose tissue. J. Pharmac. exp. Ther. 142: 137-140. MAICKEL,R. P., DAVIES,J. I. and WEISS,B. (1965). Cylic 3’,5’-AMP-An intermediate in the activation of adipose tissue lipolytic activity. Fedn Proc. 24: 299. NASH, C. W., Sm, R. L., MAICKEL,R. P. and PAOLE’ITI,R. (1961). In situ study of fatty acid release from adipose tissue. Pharmacologist 3: 55. ORO, L., ROSELL, S. and WALLENBERG,L. (1965). Circulatory and metabolic processes in adipose tissue in vivo. Nature, Lond. 205: 178-179. PAOLETTI,R., Shrrr~, R. L., MAICKEL,R. P. and BRODLE,B. B. (1961). Identification and physiological role of norepinephrine in adipose tissue. Biochem. biophys. Res. Commun. 5: 424429. RUDMAN,D., GARCIA,L. A., BROWN,S. J., MALKIN, M. F. and PERL, W. (1964). Dose-response curves for the adipokinetic action of aromatic amines and adrenocorticotropin upon the isolated adipose tissue of the hamster. J. Lipid Res. 5: 28-37. SCHOTZ, M. C. and PAGE, I. H. (1960). Effect of adrenergic blocking agents on the release of free fatty acids from rat adipose tissue. J. Lipid Res. 1: 466-468.
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SHAFRIR, E. and KERPEL, S. (1964). Fatty acid esterification and release as related to the carbohydrate metabolism of adipose tissue: Effect of epinephrine, cortisol and adrenalectomy. Archs biochem. Biophys. 105:231-246. SIDMAN,R. L. and FAWCE~~, D. W. (1954). The effect of peripheral nerve section on some metabolic responses of brown adipose tissue in mice. Anat. Rec. 118:487-507. SDMAN,R. L., PERKMS, M. and WEINER, N. (1962). Noradrenaline and adrenaline content of adipose tissue. Nature, Lond. 193: 36. STOCK, K. and WESTERMANN,E. 0. (1963). Concentration of norepinephrine, serotonin, and histamine, and of amine-metabolizing enzymes in mammalian adipose tissue. J. Lipid Res. 4: 297-304. TAYLOR,J. D., WYKES, A. A., GLADISH,Y. C. and MARTIN, W. B. (1960). New inhibitor of monoamine oxidase. Nature, Lond. 187: 941-942. TURTLE,J. R. and Kmms, D. M. (1967). An adrenergic receptor mechanism for the control of cyclic 3’,5’adenosine monophosphate synthesis in tissues. Biochem. biophys. Res. Commun. 28: 797-802. WEISS,B., DAMES, J. I. and BRODIE,B. B. (1966). Evidence for a role of adenosine 3’,5’-monophosphate in adipose tissue lipolysis. Biochem. Pharmac. 15:1553-1561. WEISS, B. and MAICKEL, R. P. (1964). Pharmacological demonstration of sympathetic innervation of adipose tissue. Pharmacologist 6: 172. WENKE, M., MUHLBACHOVA,E., SCHUSTEROVA, D., ELISOVA,K. and HYNIE, S. (1964). Effect of directly acting sympathomimetic drugs on lipid metabolism in vitro. Znt. J. Neuropharmac. 3: 283-292. WIRSEN,C. (1964). Adrenergic innervation of adipose tissue examined by fluorescence microscopy. Nature, Land. 202: 913.
1965,7,393-403 Pergamon Press.Printed inGt.Britain.
SYMPATHETIC
Laboratory
NERVOUS CONTROL OF ADIPOSE TISSUE LIPOLYSIS
BENJAMINWEISS*, and RCGER P. MAIcIcELt of Chemical Pharmacology, National Heart Institute, National Institutes of Health, Bethesda, Maryland (Accepted 9 January 1968)
Summary-The in vitro electrical stimulation of nerves innervating rat epididymal fat pads produces a consistent and measurable biochemical change; i.e. the hydrolysis of triglycerides to FFA. The optimum electrical conditions for the nervous stimulation in eliciting a lipolytic response were 5 V square wave impulses delivered at a frequency of 50 per set and of 1 msec duration. Pretreatment of the animals with dibenamine, ergotamine, syrosingopine, bretylium or the bretylium-like agent, BW 392C60, antagonized the usual lipolytic response elicited by nervous stimulation. The in vitro addition of the beta adrenergic blocking agent, DCR, completely blocked lipolysis caused by electrical stimulation. Pargyline, atropine and theophylline all enhanced the electrically-induced increase in the rate of hydrolysis of triglycerides. Adrenalectomy decreased the response while cortisone treatment enhanced the response to nervous stimulation of fat pads. These results indicate that the process of a physiological event inducing a biochemical response can be demonstrated. The data show further that the lipolytic response is under the direct control of nerve fibers which are functionally sympathetic, since this response can be modified by a wide variety of compounds which are known to influence sympathetic activity both at the neuro-effector junction and at the postjunctional sites of hormonal action. INTRODUCTION
lipolytic effect of catecholamines in vivo (ABBOUD et al., 1963; HAMBURGER et al., 1963; and GRAHAM et al., 1964) can be blocked by pretreatment of the animals with suitable blocking agents (GOODMAN and KNOBIL, 1959; HAVEL and GOLDFEIN, 1959; FROBERGand ORO, 1963; BURNS et al., 1964; KVAM et al., 1965). Similarly, adipose tissue in vitro produces increased quantities of free fatty acids (FFA) when epinephrine (E) or norepinephrine (NE) is added to the medium (BUCKLE and BECK, 1962; RUDMAN et al., 1964; WENKE et al., 1964); this response is also blocked by adrenergic blocking agents (SCHOTZ and PAGE, 1960; LOVE et al., 1963a, LOVE et al., 1963b; WENKE et al., 1964). These observations clearly demonstrate the ability of exogenous catecholamines to stimulate lipolysis in adipose tissue. The role of endogenous catecholamines in regulation of lipolytic activity is less clear. HAUSBURGER (1934) presented anatomical evidence for sympathetic nervous innervation of adipose tissue, a finding subsequently confirmed by SIDMAN and FAWCETT (1954). However, in a more recent study utilizing fluorescent microscopy, WIRSEN (1964) could not demonstrate a sympathetic plexus around fat cells. On the other hand, NE has been identified as a constituent of adipose tissue in concentrations exceeding those required THE WELL-KNOWN
*Present address: Department of Pharmacology, College of Physicians and Surgeons of Columbia University, New York, New York. IPresent address: Departments of Pharmacology and Psychology, Indiana University, Bloomington, Indiana. 395
396
B. WEISSand R. P. MAICKEL
for circulatory control (PAOLETTIet al., 1961), and the catecholamine content of adipose tissue has been shown to decrease following denervation (SIDMAN et al., 1962). Since exogenous catecholamines stimulate lipolysis in adipose tissue, it seemed reasonable to assume that increased sympathetic tone, through a release of NE at nerve endings in adipose tissue, would also stimulate lipolytic activity. Preliminary work from this laboratory (NASH et al., 1961) gave evidence to support this supposition. Using an in situ preparation of dog omental fat, it was shown that electrical stimulation of the sympathetic fibers evoked an increased output of FFA into the venous drainage of the tissue. This preparation, however, proved to be rather cumbersome and inconvenient, and yielded erratic results. A better preparation, yielding similiar results, has been described by ORO et al. (1965). CORRELL(1963) described a preparation of the rat epididymal fat pad in vitro which permitted electrical stimulation of the nerve plexus leading to the fat pad. In this preparation, stimulation increased the output of FFA into the medium, an increase partially blocked by dibenamine. The present paper describes a detailed study of factors influencing the lipolytic response of rat epididymal fat pads electrically stimulated in vitro, including parameters of stimulation and the effects of various drugs. The results are further evidence that the lipolytic activity of mammalian adipose tissue is under the control of the autonomic nervous system. A preliminary report has been presented (WEISS and MAICKEL, 1964).
METHODS Male Sprague-Dawley rats weighing 180-200 g were given free access to food and water. Adrenalectomized and adrenal demedullated rats were purchased from Hormone Assay Laboratories, Chicago, Illinois. They were given 5 % glucose and 1% saline in their drinking water for 4 days. Adrenalectomized rats were given tap water for 1 day and sacrificed the following day; demedullated animals were maintained on tap water until used (34 weeks). Animals were killed by cervical dislocation, and the epididymal fat pads with attached pedicle, containing the internal spermatic artery and its accompanying nerve, were isolated and placed in a 25-ml Erlenmeyer flask containing 4 ml Krebs-Ringer phosphate buffer (pH 7.4) with 5 % bovine serum albumin. The preparation is basically the same as that described by CORRELL(1963). The fat pad was immersed in the buffer with its neurovascular pedicle draped over platinum electrodes above the incubation medium. The pedicle was held in place by attaching it to a metal rod embedded in a rubber stopper. The electrodes were encased in glass tubing for support and insulation. In practice, several flasks were arranged in parallel so that a constant voltage was maintained across each pair of electrodes. Square wave pulses were provided with a laboratory stimulator (American Electronic Laboratories). FFA in the medium were determined as described by DOLE and MEINERTZ (1960) and glycerol released into the incubation medium was assayed in deproteinized (5 % tricholoracetic acid) filtrates by the periodate oxidation procedure of LAMBERTand NEISH (1950). Whenever possible, a non-stimulated fat pad from the same rat served as a control. MATERIALS Bovine serum albumin fraction V (fatty acid poor) was purchased from Nutritional Biochemical Corp. DCB, [1-(2’,4’-dichlorophenyl)-l-hydroxy-2-(t-butylamino) ethane
Sympathetic nervous control of adipose tissue lipolysis
397
HCl] was supplied by Eli Lilly & Co. BW-392C60 (N-orthochlorobenzyl-N’,N”-dimethylguanidine sulfate) was provided by Burroughs Wellcome 8z Co., and pargyline (MO-911) (N-benzyl-N-methyl-2-propylamine HCl) by the courtesy of Abbott Laboratories. RESULTS
The effects of varying the frequency of stimulation, while maintaining a constant voltage and duration, on the lipolytic response are shown in Table 1. At a frequency of l/set, no measurable lipolytic response was evident, whereas frequencies of 10, 30, 50 and lOO/sec produced approximately the same response. The most reproducible effects were elicited with impulses of 50/set. At a constant frequency of SO/set, and a duration of 1 msec, the optimum voltage for release of FFA from epididymal fat pads was 5 V. TABLET. STIMULATIONPARAMETERS--EFFECTOFVOLTAGEANDFREQUEN~YOFELECTRICAL STIhWLATIONONTHERELEASEOFFREEFAlTYACIDSFROMRATEPILMDYhfALFATPADS
No. of fat pads
Stimulus frequency (impulses I=)
6 12 12 47 11
1: 30 50 100
5’ 5 5 5
-0*02&0.03 0.42&0*10 0*42z!zO.O8 044&0*05 0.43 50.07
8
50
1
0.21 &O-O6
Stimulus intensity 0
Increase due to electrical stimulation &Eq FFA released/g/hr)fS.E.
Rat epididymal fat pads were incubated for 1 hr in Rrebs-Ringer phosphate buffer (pH 7.4) containing 5% bovine serum albumin. Fat pad nerves were stimulated with square wave impulses of 1 m set duration. FFA released into the medium were determined as described in Methods.
Utilizing the conditions of 50 pulses/set, 1-msec duration and an intensity of 5 V, the time relationships for the release of FFA and glycerol were studied. As seen from Fig. 1, a measurable lipolytic response was produced within 30 min. Lipolysis was linear for at least 1 hr and continued to increase for the 2-hr period of stimulation. Little or no additional lipolysis occurred from 2 to 4 hr in fat pads stimulated for a 4-hr period. In the studies reported below, a 1-hr period of incubation was employed. To relate the lipolytic response to the activity of the sympathetic nervous system, various drugs were employed which affect sympathetic function. The data in Table 2 show the effects of adrenergic blocking agents and indicate that fat pads taken from animals treated with dibenamine or ergotamine were less responsive to the lipolysis induced by electrical stimulation. Addition of DCB, an analogue of the beta adrenergic blocking agent dichloroisoproterenol (DCI), to the incubation medium produced some stimulation of lipolysis, but completely prevented any additional lipolytic response to subsequent electrical stimulation. Indeed, the slight lipolytic effect produced by DCB tended to decrease in the fat pad which was stimulated. Table 3 shows the effects of compounds which are known to influence sympathetic function without having typical adrenergic blocking activity. Pretreatment of the animals with syrosingopine or BW 392C60 effectively blocked (P
398
B.
Y '
WEISS and
R. P. MAICKEL
I
IO
20
30
40
50
60
70
80
90
100
110
120
DURATION OF STIMULATION(mm) FIG. 1. Electrical stimulation of rat epididymal fat pad: Time-Response. Rat epididymal fat pads were incubated in Krebs-Ringer phosphate buffer (PH 7.4) containing 5% bovine serum albumin. Nerves supplying the fat pads were stimulated with square wave impulses (1 msec; 5 V; 5O/sec). FFA and glycerol released into the medium were determined as described in Methods.
n - - - - - n glycerol; 0-0
TABLE 2.
No. of fat pads
FFA.
EFFECT OF ADRENERGICBLOCKING AGENTS ON RESPONSEOF EPIDIDYMALFAT PAD TO ELECTRICAL STIMULATION
Pretreatment
Added to medium
FFA
released @Eq/g/hr)
Non-stimulated
f S.E.)
Stimulated
Change due to electrical stimulation
6 6
Solvent Dibenamine
-
0.19&0.07 0*12*oGI
0~41*0~11 0.21 *o*os
0.22*0.12 0~09f0~02
6 6
Saline Ergotamine
-
0*07*0.04 0.41 kO.04
0.67f0.19 0*74&0*12
0.6OrtO.20 0*33&0*11
-
Saline DCB
0.07*0.04 0.73*0*11
0.49&0*09 055*0*17
0*42&-0.12 -0*18&0*21*
Rat epididymal fat pads were incubated for 1 hr in Krebs-Ringer phosphate buffer (pH 7.4) containing 5 % bovine serum albumin. Nerves supplying fat pads were stimulated with square wave impulses (1 msec; 5 V; SO/se@. FFA released in the medium were determined as described in Methods. Dibenamine HCl: 25 mg/kg i.p.; 44 hr and 20 hr prior to sacrifice. Ergotamine tartrate: 5 mg/kg Lp.; 1 hr prior to sacrifice. DCB: 1-(2’,4’-dichlorophenyl)-l-hydroxy-2-(t-butylamino) ethane HCl (lO-SM). *P < 0.05 compared with changes caused by electrical stimulation in control tissues.
399
Sympathetic nervous control of adipose tissue lipolysis
TABLE 3. EFFJXTOF SYMPATHOLYTICS ON RESPONSE OF EPIDIJXMALFAT PAD TO ELECTRICALSTBKJLATION Change
No. of
FFA released (rEq/g/hr)&S.E.
fat pads
Pretreatment
10
Non-stimulated
Stimulated
due to electrical stimulation
10
Solvent Syrosingopine
0.20f0.03 0.14f0.03
O-48*0.07 0*22&0.03
0.28f0.06 0.08 *0.04*
:
Saline BW 392C60
0.16f0.05 0.11+0.02
0.45 jzo.07 0.12*0.03
0.29f0.10 O-01&0.02*
4 4
Saline Bretylium
0.17f0.08 O.l7=tO*O6
0.48kO.13 0.39*0.07
0.31&0*13 0*22+oGI
Experimental
conditions and statistical notations were the same as those outlined in Table 2.
Syrosingopine: 3 mg/kg i.p. ; 22 hr and 5 hr prior to sacrifice (all rats were demedullated 3 weeks prior to use). BW 392C60: N-orthochlorobenzyl-N’,N”-dimethylguanidine 20 mg/kg i.p.; 4 hr prior to sacrifice. Bretylium tosylate: 40 mg/kg i.p.; 24 hr and 2.5 hr prior to sacrifice. *P < 0.05 compared with change caused by electrical stimulation in control tissues.
the fat pads to electrical stimulation. Bretylium was considerably less potent in this regard. Increased responsiveness of fat pads to electrical stimulation was produced by the addition of three structurally dissimilar compounds (Table 4). Pretreatment of rats with pargyline, a non-hydrazine monoamine oxidase inhibitor (TAYLOR et al., 1960), increased the lipolytic response to electrical stimulation by about 45%. Preincubation of the fat pads with either theophylline, a compound which inhibits phosphodiesterase, or the cholinergic blocking agent, atropine, also caused an enhanced release of FFA induced by nervous stimulation. TABLE 4.
EFPECTOPVAR~OUSDRUGSONRESPONSEOFEPIDIDYMALFATPADTOELECTRICALSTIMULA~ON
No. of fat pads
Pretreatment
6 6
Saline MO 911
Stimulated
Change due to electrical stimulation
0.08 f 0.03 0.25 kO.08
064&0.17 1*22+0*21
0*56+0.16 0.97f0.21
FFA released bEq/g/hr) Added to medium -
Non-stimulated
f S.E.
8 8
-
Saline Atropine
0.12+0.03 0*11*0*03
0*47*0*09 O-72&0.06
0*35&0*08 0.61 rtO.06,
8 8
-
Saline Theophylline
O-28&0*07 0*42*0.06
0*53&0.03 0.89f0.08
0*25&0*04 0~47*0*04t
Experimental
conditions were the same as those outlined in Table 2.
MO 911: Pargyline - 50 mg/kg i.p.; 1.5 hr prior to sacrifice. Atropine sulfate: lo-’ M. Theophylline: 4 x lo-” M. *P < @05 compared
with changes caused by electrical stimulation in control tissues.
tP < 0.01 compared
with changes caused by electrical stimulation in control tissues.
B. WEISSand R. P. MAICKEL
400 Table
5 shows
that fat pads from
medium in response to electrical controls
(P
whereas
adrenalectomized
stimulation
fat
pads
as compared
from
rats release
less FFA
into
the
with fat pads from sham operated
cortisone-treated
rats
release
significantly
(P
8
Stimulated
Change due to electrical stimulation
FFA released &Eq/g/hr)&S.E. Animal condition
Treatment
8
Sham operated Adrenalectomized
8 8
Non operated Non operated
Non-stimulated
-
0~41*0*04 056&0.07
1*05*0*12 0.84f0.11
064&0*10 0.28~0*11*
Saline Cortisone
0.22f0.05 0*29&-0.11
0.54f0.11 1*22*0*17
0*32&0.11 0.93 f0.06t
Rats were adrenalectomized 5 days prior to use. For the first 4 days, they were maintained on 5 % glucose1% saline in their drinking water; on the last day they were given tap water. All rats were given free access to food. Experimental conditions were the sameras those outlined in Table 2. Cortisone acetate: 50 mg/kg s.c.; 19 hr and 3 hr prior to sacrifice. *P
DISCUSSION The early
observations
but also terminating recent
studies.
conclusive
However,
anatomical
FAWCETT, 1954). in adipose
adipose
disposed
not only along
blood
confirmed
that
they are sympathetic
recently
reviewed
and physiological
is as yet lacking
lipolysis upon the administration
is based
largely
of catecholamine
or in vitro or the increase in plasma FFA observed when animals are subjected
Thus,
it was demonstrated
the adrenergic
blocking
that epididymal
influences upon
the
either in vivo to “stressful”
situations. The purpose of this study was to provide more direct pharmacological that white adipose tissue responds functionally like one which has a sympathetic tion.
origin,
(SIDMAN and
evidence for autonomic
by BRODIE et al. (1965),
vessels by more
since these nerves have not been traced to their central
proof
of increased
being
tissue cells have been adequately
The pharmacological
tissue,
observations
of nerve fibers
among
evidence innerva-
fat pads from rats which had received
agents,
dibenamine or ergotamine, were less responsive to the Complete inhibition of the response was seen stimulation.
lipolytic effects of electrical when DCB, a structural analogue of the beta adrenergic blocking agent, DCI, was added to the incubation medium. DCB was used because it elicits relatively slight positive lipolytic
action as compared with DC1 (LOVE et al., 1963a; 1963b). Similarly, the response to nerve stimulation was inhibited by pretreatment of the animals with either syrosingopine, which decreases the catecholamine stores in white adipose tissue to undetectable levels within 4 hr (STOCK and WESTERMANN, 1963), or with BW 392C60, which prevents the release of NE induced by electrical stimulation (BOURA and GREEN, 1963). The relative lack of effectiveness of bretylium may be due to the frequency with which the nerves were stimulated. GESSA et al. (1963), in studies involving the electrical stimulation of the splanchic nerve, have shown that bretylium blocked the response elicited by low frequency stimulation (4 c/s) but not
Sympathetic nervous control of adipose tissue lipolysis
401
that produced by high frequency stimulation (32 c/s). On the other hand, BW 392C60 blocked the response in both high and low frequency stimulatio. It has been shown (STOCK and WESTERMANN,1963) that inhibition of monoamine oxidase increases NE levels in adipose tissue. Administration of pargyline, a potent monoamine oxidase inhibitor, resulted in an increased lipolytic response to electrical stimulation, again indicating an adrenergic component to the lipolytic effects of nervous stimulation. Further evidence that the lipolytic response of rat epididymal fat pads is typically sympathetic is given from results with adrenalectomized rats. Adrenalectomy, which decreases the response to sympathetic stimulation in vivo (BRODIEet al., 1966), also decreases FFA release in response to electrical stimulation. Similarly, pretreatment of rats with corisone, which has been shown to increase the lipolytic response to catecholamines (SHAFRIRand KERPEL, 1964; DAVIESand WEISS,unpublished observations), increases the the lipolytic response to electrical stimulation of nerves supplying epididymal fat pads. In an attempt to gain further insight into the more discrete mechanisms involved in the sequence of reactions between the release of NE from nerve terminals and the lipolytic response, fat pads were incubated with theophylline prior to stimulation of the nerve fibers. This compound inhibits cyclic nucleotide phosphodiesterase prepared from beef heart (BUTCHERand SUTHERLAND,1962) as well as that prepared from rat adipose tissue (WEISSet al., 1966; BRODIEet al., 1966; HYNIEet al., 1966). Cyclic 3’,5’-AMP, the substrate for this enzyme, stimulates lipolysis in isolated fat cells and has been implicated as a mediator of the hormone-induced lipolytic response (MAICICEL et al., 1965; BUTCHWet al., 1965; WEISSet al., 1966). So far, the major evidence for a role of the cyclic nucleotide in lipolysis has been provided with studies of exogenously administered agents. However, the demonstration that theophylline increases the lipolysis caused by electrical stimulation indicates that catecholamines endogenously released from nerve endings may induce an increased formation of the cyclic nucleotide, thereby causing the lipolytic response. The stimulatory effect of atropine on the response of fat pads to electrical stimulation lends support to the possibility of a cholinergic influence on adipose tissue lipolysis. If there are cholinergic as well as adrenergic fibers innervating adipose tissue, then one would expect that the two systems would be antagonistic. Electrical stimulation of the entire neurovascular pedicle would then necessarily excite both types of fibers. By selectively inhibiting the cholinergic response with atropine, the restraining influence would be removed, and a greater lipolytic action would become manifest. The apparent reversal of the effect seen with DCB may also be explained by the existence of both types of fibers. DCB, by blocking only the stimulatory fibers, would thus unmask the effect of the inhibitory ones. The observation of SIDMANand FAWCETT(1954) that nerves entering fat bodies are mixed nerves carrying more than one type of fiber is in accord with this hypothesis. An alternate explanation for the decreased lipolytic response to electrical stimulation in the presence of DCB should be considered in light of the recent results of TURTLEand KIPNIS (1967), which indicate that alpha receptor stimulation decreases the concentration of cyclic 3’,5’-AMP in adipose tissue, whereas stimulation of beta receptors increases cyclic 3’,5’-AMP levels. Thus, the beta blocking drug, DCB, may have unmasked the alpha stimulating effect of sympathetic nervous stimulation, resulting in an inhibition of lipolysis.
402
B. WEISSand R. P. MAICKEL
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SHAFRIR, E. and KERPEL, S. (1964). Fatty acid esterification and release as related to the carbohydrate metabolism of adipose tissue: Effect of epinephrine, cortisol and adrenalectomy. Archs biochem. Biophys. 105:231-246. SIDMAN,R. L. and FAWCE~~, D. W. (1954). The effect of peripheral nerve section on some metabolic responses of brown adipose tissue in mice. Anat. Rec. 118:487-507. SDMAN,R. L., PERKMS, M. and WEINER, N. (1962). Noradrenaline and adrenaline content of adipose tissue. Nature, Lond. 193: 36. STOCK, K. and WESTERMANN,E. 0. (1963). Concentration of norepinephrine, serotonin, and histamine, and of amine-metabolizing enzymes in mammalian adipose tissue. J. Lipid Res. 4: 297-304. TAYLOR,J. D., WYKES, A. A., GLADISH,Y. C. and MARTIN, W. B. (1960). New inhibitor of monoamine oxidase. Nature, Lond. 187: 941-942. TURTLE,J. R. and Kmms, D. M. (1967). An adrenergic receptor mechanism for the control of cyclic 3’,5’adenosine monophosphate synthesis in tissues. Biochem. biophys. Res. Commun. 28: 797-802. WEISS,B., DAMES, J. I. and BRODIE,B. B. (1966). Evidence for a role of adenosine 3’,5’-monophosphate in adipose tissue lipolysis. Biochem. Pharmac. 15:1553-1561. WEISS, B. and MAICKEL, R. P. (1964). Pharmacological demonstration of sympathetic innervation of adipose tissue. Pharmacologist 6: 172. WENKE, M., MUHLBACHOVA,E., SCHUSTEROVA, D., ELISOVA,K. and HYNIE, S. (1964). Effect of directly acting sympathomimetic drugs on lipid metabolism in vitro. Znt. J. Neuropharmac. 3: 283-292. WIRSEN,C. (1964). Adrenergic innervation of adipose tissue examined by fluorescence microscopy. Nature, Land. 202: 913.