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Lipolysis in canine subcutaneous adipose tissue following release of endogenous histamine
EUROPEAN JOURNALOF PHARMACOLOGY13 (1971) 254-258. NORTH-HOLLANDPUBLISHINGCOMPANY
L I P O L Y S I S IN C A N I N E S U B C U T A N E O U S A D I P O S E T I S S U E F O L L O W I N G RELEASE OF ENDOGENOUS HISTAMINE Bertil B. FREDHOLM and Marianne FRISK-HOLMBERG Department of Pharmacology, Karolinska Institutet, 104 01 Stockholm, Sweden
Accepted 7 September 1970
Received 23 June 1970
B.B. FREDHOLM and M. FRISK-HOLMBERG,Lipolysis in canine subcutaneous adipose tissue following release of endogenous histamine, European J. Pharmacol. 13 (1971) 254-258. Compound 48/80 and tubocurarine, which are known to release mast cell histamine in vitro, increased the outflow of histamine from blood-perfused canine subcutaneous adipose tissue in situ. Histamine release was always accompanied by increased lipolysis. Histamine by itself evoked lipolysis both in vitro and in vivo. Second and subsequent injections of either compound 48/80 or tubocurarine were much less effective in releasing histamine and lipolytic products, indicating tachyphylaxis; cross tachyphylaxis between compound 48/80 and tubocurarine also occurred, the results suggest that adipose tissue mast cells might be of importance in the regulation of lipid mobilization by virtue of their histamine content. Compound 48/80 Tubocurarine
Lipolysis Histamine release
1. INTRODUCTION Intra-arterial injection of histamine causes release of free fatty acids (FFA) and glycerol from canine subcutaneous adipose tissue (Fredholm, Meng and Rosell, 1968) and mesenteric adipose tissue (Ballard and Rosell, 1969) in situ. The potent mast cell degranulating substance compound 48/80 (Mclntosh and Paton, 1949) is also effective. The lipolytic effect of the latter compound was supposed to be secondary to the release of histamine from stores in the adipose tissue, but a direct lipolytic effect of compound 48/80 was not ruled out. In the present experiments compound 48/80 and tubocurarine, which are believed to release mast cell histamine by an active degranulation mechanism (Frisk-Holmberg and Uvn~is, 1969)were administered to subcutaneous adipose tissue. The time course of the release of histamine and the products of lipolysis (glycerol and FFA) following injection of these substances was studied. In addition, the effects of hista-
Adipose tissue
mine and the histamine releasing agents were tested on adipose tissue incubated in vitro.
2. MATERIALS AND METHODS The experiments were conducted on female mongrel dogs, anaesthetized with sodium pentobarbital 25 mg/kg. The subcutaneous adipose tissue, situated in the inguinal region was isolated as described earlier by Rosell (1966). In 13 dogs the adipose tissue was perfused with the dog's own blood either at a constant flow by means of a perfusion apparatus (Renkin and Rosell, 1962) or by auto-perfusion. Systemic arterial or perfusion pressure was measured with a Statham pressure transducer and blood flow with a drop counter. All recordings were made on a Grass polygraph. Samples of venous and arterial blood were drawn into ice-cooled centrifuge tubes. Aliquots of plasma
B.B.Fredholm, M.Frisk-Holmberg, Lipolysis in canine subcutaneous adipose tissue were taken for enzymatic analysis of glycerol (Laurell and Tibbling, 1966) and the titrimetic analysis of FFA (Trout, Estes and Friedberg, 1960). Histamine was determined in 2 ml blood, which was immediately added to tubes containing an equal amount of ice-cold 0.6 N perchloric acid. Histamine was extracted into isoamyl alcohol and determined by a modification (Frisk-Holmberg, Lluch and Rosell, to be published) o f the method described by Anton and Sayre (1969). The net release or uptake of FFA, glycerol and histamine by the adipose tissue was calculated from the arterio-venous concentration difference multiplied by the blood or plasma flow. The results are expressed per 100 g tissue. Histamine dihydrochloride, tubocurarine and compound 48/80 (AB Leo, H~ilsingborg, Sweden) were dissolved in isotonic saline immediately before administration. The injections were made in a volume of 0.1 to 0.25 ml. Infusions were given at the rate of 0.08 ml/min. In 7 dogs, pieces of subcutaneous adipose tissue ( 1 - 3 g) were homogenized in 15 rnl 0.4 N perchloric acid. These extracts were analyzed for their histamine content (Shore, Burkhalter and Cohn Jr., 1959). Adipose tissue from 7 dogs was removed and washed in Krebs-Ringer bicarbonate buffer at body temperature. Approximately 1 g of adipose tissue, in pieces o f about 1 mm a, was incubated in plastic vessels containing 7 ml Krebs-Ringer bicarbonate buffer pH 7.4 with 10 mM glucose and 3% human serum albumin (Kabi, Stockholm, Sweden). The amounts of histamine and glycerol in the incubation medium were measured before and one hour after the addition of histamine, compound 48/80 and tubocurarine.
Table 1 The histamine content of canine subcutaneous adipose tissue.
Dog no.
1
2 3 4 5 6 7
Drug
Histamine content (ug/g)
16 18 72 54 19 8 9 40 38 8 10
48/80 48/80 48/80
2.2 1.9 0.47 0.57 1.17 3.7 0.72 1.01 0.39 2.70 0.98
48/80
1.72 + 0.42 (8) 0.70 + 0.20 (3)
carbonate solution with glucose and human serum albumin, adipose tissue released glycerol into the incubation medium at a rate of 0.74 + 0.06/amoles/ g/hour (n=12). This rate of glycerol release was increased by the addition o f histamine ( 0 . 0 7 1.33/ag/ml) to the incubation medium (fig. 1). Even when no histamine was added, the incubation medi-
DO0g~o~
IL i n c ~ ¢ ~ in
~p40.01 I #c0.01
7S.
3. RESULTS
3.1. In vitro experiments During a 60 rain incubation in Krebs-Ringer bi-
Adipose tissue weight (g)
Mean + S.E. Mean + S.E.
50, The content of endogenous histamine in adipose tissue is given in table 1. The content was 1.72 + 0.42 /ag/g (mean -+ S.E.) in eight adipose tissue specimens. In three dogs the injection of 50/~g 48/80 i.a. decreased the content to 0.70 + 0.20/ag/g.
255
g.,:,
~¢ 0.0¢
b.,
,'.o
(~Iml}
Fig. 1. The relationship between the percentage increase in rate of glycerol release and amount of histamine added to the incubation medium as measured after incubation (mean + S.E.).
256
B.B. Fredholrn,
M.Frisk-Holmherg,
Lipolysis
urn contained measurable amounts of histamine (0.07 f 0.06 pg/ml; n=12). Addition of compound 48/80 (0.07-50 pg/ml) or tubocurarine (7.1500 pg/ml) did not affect glycerol release, nor was there any significant change in the histamine concentration of the incubation fluid.
in canine subcutaneous
adipose
0-a
30.
*
effect of intra-arterial injection of histamine and 48/80 on blood flow and histamine and glycerol adipose tissue (37 g) perfused with blood with free flow. Blood pressure remained constant Hg throughout the experiment.
--a *
IO 0
T(ELEbSE
GLICEROL FFb
:
h’. 0
Fig. 2. The compound release in (pcv 39.5) at 150 mm
* 0
+.llOle~lm~n
4o 100
20
3.2. In vivo experiments 3.2.1. Injections of compound 48180 and tubocurarine Intra-arterial injection of 2.5 pg compound 48/80 caused a marked release of glycerol and histamine whereas lower doses were ineffective (fig. 2). Repeated injections of the same amount of compound 48/80 had a decreasing effect on glycerol or histamine release. This was not due to tachyphylaxis to histamine since histamine was effective before and after compound 48/80 (fig. 2). Injections of 0.5 and 2 mg tubocurarine after two 50 pg doses of compound 48/80 did not release histamine (fig. 3). Intra-arterial injection of 0.5 mg tubocurarine produced changes in lipolytic rate and histamine release similar to those seen with compound 48/80 (fig. 4). Upon repeated administration of tubocurarine the response was diminished and subsequent administration of 100 pg compound 48/80 had very little effect
tissue
L.
*
TIME
,rn,“l
0
*
*
:-**a*
30
90
60
I*
120
** io
** * * .
*
3%
. l
IOOQ HE.TIMINE
.
RELEASE
05 . 0
IcA
.,:,0
%#-a
*aA
*
*._
.& 5oPa
d-
Y
LCL_._
wd”r.ma ,a&,,, 0.3m-g
2.0rnQ
Fig. 3. The effect of compound 48/80 and tubocurarine in adipose tissue (26 g) perfused with defibrinated blood (pcv 36.0) at a constant rate (4.7 ml/min/lOO g).
on glycerol or histamine release. These results indicate that cross-tachyphylaxis occurred between compound 48180 and tubocurarine. 3.2.2. Infusion of compound 48/80 and tubocurarine Fig. 5 illustrates the effect of infusing compound 48/80 at different rates in 4 dogs. After a resting 48/80 was infused for about period, compound 30 min at rates of 1, 7.5, 24 and 50 pg/min respectively. The resting blood flow showed individual
Fig. 4. The effect of tubocurarine and compound 48/80 in adipose tissue (25 g) perfused with blood (pcv 40) with free flow. Blood pressure dropped from 150 mm Hg initially to 125 mm Hg at the end of the experiment.
B.B.Fredholm, M.Frisk-Holmberg, Lipolysis in canine subcutaneous adipose tissue INFUSION IOOg BLOOD
I
COMPOUND 4 8 / 8 0
...............
41 ........... I 1
TIME(mini
---
. ,
0
,
I0
20
?.e
"
30
~.YCER~ 3.01
~g
2.0.
HIRELEASE STA~IN~ ! LO.
Fig. 5. The effect of infusion of 1 *; 7.5 o; 24 • and 50 • •g 48/80/min in adipose tissue (20, 40, 38, 35 g respectively) perfused with blood (pcv 35, 40, 40, 32 respectively) with free flow. The blood pressure remained essentially constant during the infusion in each dog. ml/min
BLOOD FLOW
/Jmoles/minloog 4 t GLYCEROL RELEASE 2
TIME (rnin)
o
ju~l/min IO0 g
iI
o
5
io
HISTAMINE RELEASE
' ~ 8 0 0 j u g d- tubocurorine/min ---.
Fig. 6. The effect of tubocurarine in adipose tissue (40 g) perfused with blood (per 38) with free flow. The blood pressure dropped from 160 to 130 mm Hg during the course of experiment.
257
variations within the normal range ( 3 . 2 - 1 9 m l / min/100 g) reported for canine subcutaneous adipose tissue (Fredholm, 1970). It is seen that with the lowest infusion-rate compound 48/80 had no effect on blood flow, tipolysis or histamine release; with the higher infusion rates blood flow, histamine release and lipolytic rate increased. There was no clearcut dose response relationship since approximately the same amounts of histamine were released during the infusion period by 7.5, 24 and 50/ag compound 48/80/rain. However, the higher the infusion rate the earlier maximal release occurred. Histamine release decreased with time in spite of continued infusion of compound 48/80. Fig. 6 illustrates an experiment in which 800 jug tubocurarine/min was infused. The result is similar to those reported above for compound 48/80. The maximal rate of histamine release was obtained very rapidly following the start of the infusion, whereas the glycerol release increased more slowly, reaching maximal values when the histamine release rates had returned to normal.
4. DISCUSSION Canine subcutaneous adipose tissue contained about 1.7 tzg/g histamine, which is similar to the amounts found by Stock and Westermann (1963) in white adipose tissue from other species and locations. It has been concluded that histamine in white adipose tissue is predominantly located in mast ceils (Bieck, Stock and Westermann, 1967), which are abundant in white adipose tissue (Sheldon, 1965), especially around blood vessels. Histamine is lipolytic in canine subcutaneous adipose tissue if injected in amounts greater than 0.5/ag i.a. (Fredholm et al., 1968). The whole subcutaneous fat pad contained 30.7-+ 2.5 g histamine. If released locally therefore, these amounts would be able to stimulate lipolysis. In the present experiments, concentrations of histamine below 0.1/ag/ml significantly increased lipolysis in canine subcutaneous adipose tissue in vitro. Adipose tissue histamine was released into blood when exposed in vivo to histamine releasing agents like tubocurarine and compound 48/80. Histamine release was always followed by an increased release of
258
B.B.Fredholm, M.Frisk-Holmberg, Lipolysis in canine subcutaneous adipose tissue
lipolytic products (glycerol and FFA); when histamine release was absent there was no lipolysis. Further, in vitro histamine, in concentrations similar to those released in vivo after perfusion of histamine releasing agents, was lipolytic. Repeated administration of either of the releasing agents caused a much smaller histamine and glycerol release. Similarly histamine release decreased despite continued infusion of releasing agents. This tachyphylaxis, which also occurred between the two agents, was probably due to the depletion of releasable histamine. In agreement with this was the finding that a single injection of compound 48/80 released about 6 0 p e r c e n t of adipose tissue histamine. In vitro compound 48/80 and tubocurarine did not increase the histamine or glycerol concentration in the incubation medium, even if added in very high concentrations. The reason for this is unknown. In conclusion the present results support the hypothesis that the histamine releasing agents are lipolytic b y virtue of their capacity to release endogenous histamine, and not by a direct action on the fat cells. Moreover, the results provide circumstantial evidence that mast-cells might be of importanc6 in the regulation of lipid metabolism.
ACKNOWLEDGMENTS This study was supported by grants from the Swedish Medical Research Council (B 70-40X-2553-02) and Karolinska Instituter. The skilful technical assistance of Miss Mona Engqvist and Miss Annkatrin Tunt]ord is gratefully acknowledged.
REFERENCES Anton, H.A. and D. Sayre, 1969, A modified fluorimetric procedure for tissue histamine and its distribution in
various animals, J. Pharmacol. Exptl. Therap. 166, 285292. Ballard, K. and S. RoseU, 1969, The unresponsiveness of lipid metabolism in canine mesentric adipose tissue to biogenic amines and to sympathetic nerve stimulation, Acta Physiol. Scand. 77,442-448. Bieck, P., K. Stock and E. Westermann, 1967, U-ber die Bedeutung des Serotonins im Fettgewebe, Arch. Exptl. Pathol. Pharmakol. 256, 218-236. Fredholm, B.B., 1970, Studies on the sympathetic regulation of circulation and metabolism in isolated canine subcutaneous adipose tissue, Acta Physiol. Scand. Suppl. 354. Fredholm, B.B., H.C. Meng and S. Rosell, 1968, Release of free fatty acids from canine subcutaneous tissue by histamine and compound 48/80, Life Sci. 7, 1209-1215. Frisk-Holmberg, M., S. Lhich and S. Rosell, Metabolic and vascular effects of chlorpromazine in canine subcutaneous adipose tissue, to be published. Frisk-Holmberg, M. and B. Uvn~is, 1969, The mechanism of histamine release from isolated rat peritoneal mast cells induced by tubocurarine, Acta Physiol. Scand. 76, 335339. Laurell, S. and G. Tibbling, 1966, An enzymatic fluorimetric micromethod for the determination of glycerol, Clin. Chim. Acta 13,317-322. Mclntosh, F.C. and W.D.M. Paton, 1949, The liberation of histamine by certain organic bases, J. Physiol. (London) 109, 190-219. Renkin, E. and S. RoseU, 1962, The influence of sympathetic adrenergic vasoconstrictor nerves on transport of diffusible solutes from blood to tissues in skeletal muscle, Acta Physiol. Scand. 67,343-351. Rosell, S., 1966, Release of free fatty acid from subcutaneous adipose tissue in dogs following sympathetic nerve stimulation, Acta Physiol. Scand. 67, 343-351. Sheldon, H., 1965, Morphology of adipose tissue; a microscopic anatomy of fat, in: Handbook of Physiology, Vol. 5, ed. A.E. Renold and G.F. Cahill Jr. (American Physiological Society, Washington D.C.) pp. 125 - 139. Shore, P., A. Burkhalter and W. Cohn Jr., 1959, A method for the fluorimetric assay of histamine in tissue, J. Pharmacol. Exptl. Therap. 127, 182-186. Stock, K. and E. Westermann, 1963, Concentration of norepinephrine, serotonin and histamine, and of amine metabolizing enzymes in mammalian adipose tissue, J. Lipid Res. 4, 297-310. Trout, D.L., H. Estes and S.J. Friedberg, 1960, Titration of free fatty acids of plasma: A study of current methods and a new modification, J. Lipid Res. 1,199-202.
L I P O L Y S I S IN C A N I N E S U B C U T A N E O U S A D I P O S E T I S S U E F O L L O W I N G RELEASE OF ENDOGENOUS HISTAMINE Bertil B. FREDHOLM and Marianne FRISK-HOLMBERG Department of Pharmacology, Karolinska Institutet, 104 01 Stockholm, Sweden
Accepted 7 September 1970
Received 23 June 1970
B.B. FREDHOLM and M. FRISK-HOLMBERG,Lipolysis in canine subcutaneous adipose tissue following release of endogenous histamine, European J. Pharmacol. 13 (1971) 254-258. Compound 48/80 and tubocurarine, which are known to release mast cell histamine in vitro, increased the outflow of histamine from blood-perfused canine subcutaneous adipose tissue in situ. Histamine release was always accompanied by increased lipolysis. Histamine by itself evoked lipolysis both in vitro and in vivo. Second and subsequent injections of either compound 48/80 or tubocurarine were much less effective in releasing histamine and lipolytic products, indicating tachyphylaxis; cross tachyphylaxis between compound 48/80 and tubocurarine also occurred, the results suggest that adipose tissue mast cells might be of importance in the regulation of lipid mobilization by virtue of their histamine content. Compound 48/80 Tubocurarine
Lipolysis Histamine release
1. INTRODUCTION Intra-arterial injection of histamine causes release of free fatty acids (FFA) and glycerol from canine subcutaneous adipose tissue (Fredholm, Meng and Rosell, 1968) and mesenteric adipose tissue (Ballard and Rosell, 1969) in situ. The potent mast cell degranulating substance compound 48/80 (Mclntosh and Paton, 1949) is also effective. The lipolytic effect of the latter compound was supposed to be secondary to the release of histamine from stores in the adipose tissue, but a direct lipolytic effect of compound 48/80 was not ruled out. In the present experiments compound 48/80 and tubocurarine, which are believed to release mast cell histamine by an active degranulation mechanism (Frisk-Holmberg and Uvn~is, 1969)were administered to subcutaneous adipose tissue. The time course of the release of histamine and the products of lipolysis (glycerol and FFA) following injection of these substances was studied. In addition, the effects of hista-
Adipose tissue
mine and the histamine releasing agents were tested on adipose tissue incubated in vitro.
2. MATERIALS AND METHODS The experiments were conducted on female mongrel dogs, anaesthetized with sodium pentobarbital 25 mg/kg. The subcutaneous adipose tissue, situated in the inguinal region was isolated as described earlier by Rosell (1966). In 13 dogs the adipose tissue was perfused with the dog's own blood either at a constant flow by means of a perfusion apparatus (Renkin and Rosell, 1962) or by auto-perfusion. Systemic arterial or perfusion pressure was measured with a Statham pressure transducer and blood flow with a drop counter. All recordings were made on a Grass polygraph. Samples of venous and arterial blood were drawn into ice-cooled centrifuge tubes. Aliquots of plasma
B.B.Fredholm, M.Frisk-Holmberg, Lipolysis in canine subcutaneous adipose tissue were taken for enzymatic analysis of glycerol (Laurell and Tibbling, 1966) and the titrimetic analysis of FFA (Trout, Estes and Friedberg, 1960). Histamine was determined in 2 ml blood, which was immediately added to tubes containing an equal amount of ice-cold 0.6 N perchloric acid. Histamine was extracted into isoamyl alcohol and determined by a modification (Frisk-Holmberg, Lluch and Rosell, to be published) o f the method described by Anton and Sayre (1969). The net release or uptake of FFA, glycerol and histamine by the adipose tissue was calculated from the arterio-venous concentration difference multiplied by the blood or plasma flow. The results are expressed per 100 g tissue. Histamine dihydrochloride, tubocurarine and compound 48/80 (AB Leo, H~ilsingborg, Sweden) were dissolved in isotonic saline immediately before administration. The injections were made in a volume of 0.1 to 0.25 ml. Infusions were given at the rate of 0.08 ml/min. In 7 dogs, pieces of subcutaneous adipose tissue ( 1 - 3 g) were homogenized in 15 rnl 0.4 N perchloric acid. These extracts were analyzed for their histamine content (Shore, Burkhalter and Cohn Jr., 1959). Adipose tissue from 7 dogs was removed and washed in Krebs-Ringer bicarbonate buffer at body temperature. Approximately 1 g of adipose tissue, in pieces o f about 1 mm a, was incubated in plastic vessels containing 7 ml Krebs-Ringer bicarbonate buffer pH 7.4 with 10 mM glucose and 3% human serum albumin (Kabi, Stockholm, Sweden). The amounts of histamine and glycerol in the incubation medium were measured before and one hour after the addition of histamine, compound 48/80 and tubocurarine.
Table 1 The histamine content of canine subcutaneous adipose tissue.
Dog no.
1
2 3 4 5 6 7
Drug
Histamine content (ug/g)
16 18 72 54 19 8 9 40 38 8 10
48/80 48/80 48/80
2.2 1.9 0.47 0.57 1.17 3.7 0.72 1.01 0.39 2.70 0.98
48/80
1.72 + 0.42 (8) 0.70 + 0.20 (3)
carbonate solution with glucose and human serum albumin, adipose tissue released glycerol into the incubation medium at a rate of 0.74 + 0.06/amoles/ g/hour (n=12). This rate of glycerol release was increased by the addition o f histamine ( 0 . 0 7 1.33/ag/ml) to the incubation medium (fig. 1). Even when no histamine was added, the incubation medi-
DO0g~o~
IL i n c ~ ¢ ~ in
~p40.01 I #c0.01
7S.
3. RESULTS
3.1. In vitro experiments During a 60 rain incubation in Krebs-Ringer bi-
Adipose tissue weight (g)
Mean + S.E. Mean + S.E.
50, The content of endogenous histamine in adipose tissue is given in table 1. The content was 1.72 + 0.42 /ag/g (mean -+ S.E.) in eight adipose tissue specimens. In three dogs the injection of 50/~g 48/80 i.a. decreased the content to 0.70 + 0.20/ag/g.
255
g.,:,
~¢ 0.0¢
b.,
,'.o
(~Iml}
Fig. 1. The relationship between the percentage increase in rate of glycerol release and amount of histamine added to the incubation medium as measured after incubation (mean + S.E.).
256
B.B. Fredholrn,
M.Frisk-Holmherg,
Lipolysis
urn contained measurable amounts of histamine (0.07 f 0.06 pg/ml; n=12). Addition of compound 48/80 (0.07-50 pg/ml) or tubocurarine (7.1500 pg/ml) did not affect glycerol release, nor was there any significant change in the histamine concentration of the incubation fluid.
in canine subcutaneous
adipose
0-a
30.
*
effect of intra-arterial injection of histamine and 48/80 on blood flow and histamine and glycerol adipose tissue (37 g) perfused with blood with free flow. Blood pressure remained constant Hg throughout the experiment.
--a *
IO 0
T(ELEbSE
GLICEROL FFb
:
h’. 0
Fig. 2. The compound release in (pcv 39.5) at 150 mm
* 0
+.llOle~lm~n
4o 100
20
3.2. In vivo experiments 3.2.1. Injections of compound 48180 and tubocurarine Intra-arterial injection of 2.5 pg compound 48/80 caused a marked release of glycerol and histamine whereas lower doses were ineffective (fig. 2). Repeated injections of the same amount of compound 48/80 had a decreasing effect on glycerol or histamine release. This was not due to tachyphylaxis to histamine since histamine was effective before and after compound 48/80 (fig. 2). Injections of 0.5 and 2 mg tubocurarine after two 50 pg doses of compound 48/80 did not release histamine (fig. 3). Intra-arterial injection of 0.5 mg tubocurarine produced changes in lipolytic rate and histamine release similar to those seen with compound 48/80 (fig. 4). Upon repeated administration of tubocurarine the response was diminished and subsequent administration of 100 pg compound 48/80 had very little effect
tissue
L.
*
TIME
,rn,“l
0
*
*
:-**a*
30
90
60
I*
120
** io
** * * .
*
3%
. l
IOOQ HE.TIMINE
.
RELEASE
05 . 0
IcA
.,:,0
%#-a
*aA
*
*._
.& 5oPa
d-
Y
LCL_._
wd”r.ma ,a&,,, 0.3m-g
2.0rnQ
Fig. 3. The effect of compound 48/80 and tubocurarine in adipose tissue (26 g) perfused with defibrinated blood (pcv 36.0) at a constant rate (4.7 ml/min/lOO g).
on glycerol or histamine release. These results indicate that cross-tachyphylaxis occurred between compound 48180 and tubocurarine. 3.2.2. Infusion of compound 48/80 and tubocurarine Fig. 5 illustrates the effect of infusing compound 48/80 at different rates in 4 dogs. After a resting 48/80 was infused for about period, compound 30 min at rates of 1, 7.5, 24 and 50 pg/min respectively. The resting blood flow showed individual
Fig. 4. The effect of tubocurarine and compound 48/80 in adipose tissue (25 g) perfused with blood (pcv 40) with free flow. Blood pressure dropped from 150 mm Hg initially to 125 mm Hg at the end of the experiment.
B.B.Fredholm, M.Frisk-Holmberg, Lipolysis in canine subcutaneous adipose tissue INFUSION IOOg BLOOD
I
COMPOUND 4 8 / 8 0
...............
41 ........... I 1
TIME(mini
---
. ,
0
,
I0
20
?.e
"
30
~.YCER~ 3.01
~g
2.0.
HIRELEASE STA~IN~ ! LO.
Fig. 5. The effect of infusion of 1 *; 7.5 o; 24 • and 50 • •g 48/80/min in adipose tissue (20, 40, 38, 35 g respectively) perfused with blood (pcv 35, 40, 40, 32 respectively) with free flow. The blood pressure remained essentially constant during the infusion in each dog. ml/min
BLOOD FLOW
/Jmoles/minloog 4 t GLYCEROL RELEASE 2
TIME (rnin)
o
ju~l/min IO0 g
iI
o
5
io
HISTAMINE RELEASE
' ~ 8 0 0 j u g d- tubocurorine/min ---.
Fig. 6. The effect of tubocurarine in adipose tissue (40 g) perfused with blood (per 38) with free flow. The blood pressure dropped from 160 to 130 mm Hg during the course of experiment.
257
variations within the normal range ( 3 . 2 - 1 9 m l / min/100 g) reported for canine subcutaneous adipose tissue (Fredholm, 1970). It is seen that with the lowest infusion-rate compound 48/80 had no effect on blood flow, tipolysis or histamine release; with the higher infusion rates blood flow, histamine release and lipolytic rate increased. There was no clearcut dose response relationship since approximately the same amounts of histamine were released during the infusion period by 7.5, 24 and 50/ag compound 48/80/rain. However, the higher the infusion rate the earlier maximal release occurred. Histamine release decreased with time in spite of continued infusion of compound 48/80. Fig. 6 illustrates an experiment in which 800 jug tubocurarine/min was infused. The result is similar to those reported above for compound 48/80. The maximal rate of histamine release was obtained very rapidly following the start of the infusion, whereas the glycerol release increased more slowly, reaching maximal values when the histamine release rates had returned to normal.
4. DISCUSSION Canine subcutaneous adipose tissue contained about 1.7 tzg/g histamine, which is similar to the amounts found by Stock and Westermann (1963) in white adipose tissue from other species and locations. It has been concluded that histamine in white adipose tissue is predominantly located in mast ceils (Bieck, Stock and Westermann, 1967), which are abundant in white adipose tissue (Sheldon, 1965), especially around blood vessels. Histamine is lipolytic in canine subcutaneous adipose tissue if injected in amounts greater than 0.5/ag i.a. (Fredholm et al., 1968). The whole subcutaneous fat pad contained 30.7-+ 2.5 g histamine. If released locally therefore, these amounts would be able to stimulate lipolysis. In the present experiments, concentrations of histamine below 0.1/ag/ml significantly increased lipolysis in canine subcutaneous adipose tissue in vitro. Adipose tissue histamine was released into blood when exposed in vivo to histamine releasing agents like tubocurarine and compound 48/80. Histamine release was always followed by an increased release of
258
B.B.Fredholm, M.Frisk-Holmberg, Lipolysis in canine subcutaneous adipose tissue
lipolytic products (glycerol and FFA); when histamine release was absent there was no lipolysis. Further, in vitro histamine, in concentrations similar to those released in vivo after perfusion of histamine releasing agents, was lipolytic. Repeated administration of either of the releasing agents caused a much smaller histamine and glycerol release. Similarly histamine release decreased despite continued infusion of releasing agents. This tachyphylaxis, which also occurred between the two agents, was probably due to the depletion of releasable histamine. In agreement with this was the finding that a single injection of compound 48/80 released about 6 0 p e r c e n t of adipose tissue histamine. In vitro compound 48/80 and tubocurarine did not increase the histamine or glycerol concentration in the incubation medium, even if added in very high concentrations. The reason for this is unknown. In conclusion the present results support the hypothesis that the histamine releasing agents are lipolytic b y virtue of their capacity to release endogenous histamine, and not by a direct action on the fat cells. Moreover, the results provide circumstantial evidence that mast-cells might be of importanc6 in the regulation of lipid metabolism.
ACKNOWLEDGMENTS This study was supported by grants from the Swedish Medical Research Council (B 70-40X-2553-02) and Karolinska Instituter. The skilful technical assistance of Miss Mona Engqvist and Miss Annkatrin Tunt]ord is gratefully acknowledged.
REFERENCES Anton, H.A. and D. Sayre, 1969, A modified fluorimetric procedure for tissue histamine and its distribution in
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