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Lipolytic activity in human adipose tissue
Life Sciences Vol . 3, pp. 851-855, 1964. Pergamon Press, Inc. Printed in the United States .
LIPOLYTIC ACTIVITY IId HUMAN ADIPOSE TISSUE J . David Schmatz Department of Medicine State University of New York at Buffalo and the Buffalo General Hospital Buffalo, N .Y . 14203
(Received 19 June 1964) A number of lipolytic activities have been demonstrated to be present in animal adipose Cissue (1-8), and physiological roles have been suggested (9-11) . Although human adipose tissue has not been studied as extensively as animal adipose tissue, demonstrated .
lipoprotein lipase (12) and other activities (2,13) have been This communication presents data which not only confirm the
existence of lipolytic activity in human subcutaneous adipose tissue, but also suggest that at least 2 enzymatic activities are present,
as evidenced
by separate in vitro pH, substrate and temperature requirements, and a difference in stability of the activities on standing . Methods Samples of human subcutaneous adipose tissue were obtained surgically and homogenized in 0 .15M KC1, 160 mg/ml .
The homogenate was centrifuged at 1000 xg
for 10 minutes, and the liquid layer tested for activity by a modification of a previously described method (14,15) .
Lipolysis was determined by a rise in free
fatty acids (FFA) which were extracted (16) and titrated automatically (17) . The results obtained during the linear phase of lipolytis have been expressed as~tEq . of FFA released per gram of tissue per hour . Results and Discussion At 37 ° C it was found that 2 different pH optima were obtained depending upon the substrate used .
Olive oil, cottonseed oil, and triolein were hydrolyzed over
851
852
LIPOLYTIC ACTIVITY IN HUMAN ADIPOSE TISSUE
Vol. 3, No . 8
a broad pH range with an optimum around pH 7 while tributyrin, ~eene 20, 60, and 80* were hydrolyzed best at pH 8 .5 . aentative of the difference .
Curves A and B of Figure 1 are repre-
In other experiments the activity represented by
Curve A has been shown to decrease between pH 6 and 7 .
At 47 ° C the optimal pH
for hydrolysis of tributyrin shifted fr°m 8 .5 to 8 .0 (Fig .
1, Curve C) .
FIG . 1 pH Curves A. B. C.
Olive oil substrate at 37°C Tributyrin substrate at 37°C Tributyrin substrate at 47°C
Six times as much fatty acid resulted from the hydrolysis of Tween 20 as from Tween 60 or 80, all of which exhibited an optimum pH of 8 .5 at 37°C .
While
the degree of hydrolysis appears to be related to the chain length of the esterified fatty acid, the optimum pH for hydrolysis does not appear to be so related . *1~eens 20, 60 and 80 are the respective monolaurate, monoatearate and monooleate eaters of polyoxyethylene sorbitan, manufactured by the Atlas Chemical Industries Inc ., Wilmington, Delaware .
Vol. 3, No. 8
LIPOLYTIC ACTNITY IN HUMAN ADIPOSE TLSSUE
853
The pH optimum obtained with emulsions of olive oil, cottonseed oil and triolein in contrast to that noted with tributyrin and various Tweena may represent a difference in "lipolytic" and "esterolytic" activities as suggested by Deanuellea (18) . In addition to pH and substrate differences further indication of 2 lipolytic activities was obtained by studying the assay systems at various temperatures .
Maximum hydrolysis occurred at 37°C with olive oil (Fig . 2, Curve A),
and at 47 °C with tributyrin (Fig . 2, Curve B) .
"c
FIG . 2 Temperature Curves A. B.
Olive oil substrate at pH 7 .0 Tributyrin substrate at pH 8 .5
On prolonged standing at 20°C a difference in stability of the two activities was apparent (Table 1), further suggesting the presence of 2 enzymatic activities . In conclusion 2 in vitro lipolytic activities have been demonstrated in human subcutaneous adipose tissue (Table 2) .
Whether or not they represe~it the
854
LIPOLYTIC ACTIVITY IN HUMAN ADIPOSE TISSUE
Vol. 3, No. 8
existence of 2 enzymes may be clarified by attempts at physical separation .
TAB LE 1 Percent of Original Activity
Days
Olive Oil
Tributyrin
0
100
100
1
82
97
4
61
100
7
17
85
The tissue preparation was kept at 20° C and assayed for both activities initially and at stated intervals up to 7 days . TABLE 2 Summary of Two Lipolytic Activities
Optimum pH
7 .0
8 .0
Optimum Temperature
37°C
47°C
Substrates
Olive Oil Cottonseed Oil Triolein
Tributyrin Tweens 20, 60, 80
Activity at 20°C
Unstable
Stable
Acknowledgements This work was supported in part by a Buswell Fellowship from the State University of New York at Buffalo and Grants AM-06872-01 and AM-05581-03 from the National Institute of Arthritis and Metabolic Diseases . The author wishes to thank Mr . John M . Buchheit for technical assistance, his surgical colleagues for continued support, Dr . Edmund Klein for advice and Dr . Thomas Vecchio of Upjohn Co . for supplying the olive oil as "Clearing factor emulsion" .
Vol. 9, No. 8
LIPOLYTIC ACTIVITY IN HUMAN ADIPOSE TISSUE
855
References 1.
G . Quagliariello,and G . Scoz, Arch . Di Sc . Biol . , 1~7 , 513 (1932) .
2.
A .E . Renold,and A, Marble, J . Biol . Chem . ,
3.
E .D . Korn, and T .W . Quigley, Biochim . Biophys . Acts ,
4.
W .S . Lynn, Jr ., and N .C . Perryman, J . Biol . Chem . , 235,
5.
M .A . Rizack, J . Biol . Chem . , 236, 657
6.
C .H . Hollenberg, M .S . Raben, and E,B . Astwood, Endocrinol . , 68, 589 (1961) .
7.
L . Mashburn, Studies of Lipolvtic Enzvmea, Ph .D . dissertation, Duke University,
185 , 367 (1950) . 18,
143 (1955) . 1912 (1960) .
(1961) .
1961 .
8.
M . Vaughan, J .E . Berger, and D . Steinberg, J . Biol . Chem . , 239, 401 (1964) .
9.
M . Rodbell, J . Biol . Chem . , 235, 1613, 1960 .
10 .
A . Bezman, J .M . Felts, and R .J . Havel, J . Lipid Research , 3, 427 (1962) .
11 .
E . Wertheimer, and E . Shafrir, Recent Prou . Hormone Res . , 16, 467 (1960) .
12 .
P .J . Nestel, and R .J . Havel, Proc . Soc . Exptl . Biol . Med . ,
13 .
E . Gorin, and E . Shafrir, Biochim & Biophvs Acta , 84, 24 (1964) .
14 .
J .D, Schnatz, and R .H . Williams, Metab . , _, 349 (1962) .
15 .
M . Bier, in S .P . Colowick,
109, 985 (1962) .
and N .O . Kaplan (Eds .), Methods in EnzymoloAy ,
Vol . I, Academic Press, New York, 1955, p . 629 . 16 .
V .P, Dole, J . Clin . Invest . , _, 150 (1956) .
17 .
J .D . Schnatz, J . Lipid Research , in press .
18 .
P . Desnuelle, in F .F . Nord (Ed .), Advances in EnzymoloRV , Vol . 23, Interscience Publishers, Inc ., New York, 1961, p . 129 .
LIPOLYTIC ACTIVITY IId HUMAN ADIPOSE TISSUE J . David Schmatz Department of Medicine State University of New York at Buffalo and the Buffalo General Hospital Buffalo, N .Y . 14203
(Received 19 June 1964) A number of lipolytic activities have been demonstrated to be present in animal adipose Cissue (1-8), and physiological roles have been suggested (9-11) . Although human adipose tissue has not been studied as extensively as animal adipose tissue, demonstrated .
lipoprotein lipase (12) and other activities (2,13) have been This communication presents data which not only confirm the
existence of lipolytic activity in human subcutaneous adipose tissue, but also suggest that at least 2 enzymatic activities are present,
as evidenced
by separate in vitro pH, substrate and temperature requirements, and a difference in stability of the activities on standing . Methods Samples of human subcutaneous adipose tissue were obtained surgically and homogenized in 0 .15M KC1, 160 mg/ml .
The homogenate was centrifuged at 1000 xg
for 10 minutes, and the liquid layer tested for activity by a modification of a previously described method (14,15) .
Lipolysis was determined by a rise in free
fatty acids (FFA) which were extracted (16) and titrated automatically (17) . The results obtained during the linear phase of lipolytis have been expressed as~tEq . of FFA released per gram of tissue per hour . Results and Discussion At 37 ° C it was found that 2 different pH optima were obtained depending upon the substrate used .
Olive oil, cottonseed oil, and triolein were hydrolyzed over
851
852
LIPOLYTIC ACTIVITY IN HUMAN ADIPOSE TISSUE
Vol. 3, No . 8
a broad pH range with an optimum around pH 7 while tributyrin, ~eene 20, 60, and 80* were hydrolyzed best at pH 8 .5 . aentative of the difference .
Curves A and B of Figure 1 are repre-
In other experiments the activity represented by
Curve A has been shown to decrease between pH 6 and 7 .
At 47 ° C the optimal pH
for hydrolysis of tributyrin shifted fr°m 8 .5 to 8 .0 (Fig .
1, Curve C) .
FIG . 1 pH Curves A. B. C.
Olive oil substrate at 37°C Tributyrin substrate at 37°C Tributyrin substrate at 47°C
Six times as much fatty acid resulted from the hydrolysis of Tween 20 as from Tween 60 or 80, all of which exhibited an optimum pH of 8 .5 at 37°C .
While
the degree of hydrolysis appears to be related to the chain length of the esterified fatty acid, the optimum pH for hydrolysis does not appear to be so related . *1~eens 20, 60 and 80 are the respective monolaurate, monoatearate and monooleate eaters of polyoxyethylene sorbitan, manufactured by the Atlas Chemical Industries Inc ., Wilmington, Delaware .
Vol. 3, No. 8
LIPOLYTIC ACTNITY IN HUMAN ADIPOSE TLSSUE
853
The pH optimum obtained with emulsions of olive oil, cottonseed oil and triolein in contrast to that noted with tributyrin and various Tweena may represent a difference in "lipolytic" and "esterolytic" activities as suggested by Deanuellea (18) . In addition to pH and substrate differences further indication of 2 lipolytic activities was obtained by studying the assay systems at various temperatures .
Maximum hydrolysis occurred at 37°C with olive oil (Fig . 2, Curve A),
and at 47 °C with tributyrin (Fig . 2, Curve B) .
"c
FIG . 2 Temperature Curves A. B.
Olive oil substrate at pH 7 .0 Tributyrin substrate at pH 8 .5
On prolonged standing at 20°C a difference in stability of the two activities was apparent (Table 1), further suggesting the presence of 2 enzymatic activities . In conclusion 2 in vitro lipolytic activities have been demonstrated in human subcutaneous adipose tissue (Table 2) .
Whether or not they represe~it the
854
LIPOLYTIC ACTIVITY IN HUMAN ADIPOSE TISSUE
Vol. 3, No. 8
existence of 2 enzymes may be clarified by attempts at physical separation .
TAB LE 1 Percent of Original Activity
Days
Olive Oil
Tributyrin
0
100
100
1
82
97
4
61
100
7
17
85
The tissue preparation was kept at 20° C and assayed for both activities initially and at stated intervals up to 7 days . TABLE 2 Summary of Two Lipolytic Activities
Optimum pH
7 .0
8 .0
Optimum Temperature
37°C
47°C
Substrates
Olive Oil Cottonseed Oil Triolein
Tributyrin Tweens 20, 60, 80
Activity at 20°C
Unstable
Stable
Acknowledgements This work was supported in part by a Buswell Fellowship from the State University of New York at Buffalo and Grants AM-06872-01 and AM-05581-03 from the National Institute of Arthritis and Metabolic Diseases . The author wishes to thank Mr . John M . Buchheit for technical assistance, his surgical colleagues for continued support, Dr . Edmund Klein for advice and Dr . Thomas Vecchio of Upjohn Co . for supplying the olive oil as "Clearing factor emulsion" .
Vol. 9, No. 8
LIPOLYTIC ACTIVITY IN HUMAN ADIPOSE TISSUE
855
References 1.
G . Quagliariello,and G . Scoz, Arch . Di Sc . Biol . , 1~7 , 513 (1932) .
2.
A .E . Renold,and A, Marble, J . Biol . Chem . ,
3.
E .D . Korn, and T .W . Quigley, Biochim . Biophys . Acts ,
4.
W .S . Lynn, Jr ., and N .C . Perryman, J . Biol . Chem . , 235,
5.
M .A . Rizack, J . Biol . Chem . , 236, 657
6.
C .H . Hollenberg, M .S . Raben, and E,B . Astwood, Endocrinol . , 68, 589 (1961) .
7.
L . Mashburn, Studies of Lipolvtic Enzvmea, Ph .D . dissertation, Duke University,
185 , 367 (1950) . 18,
143 (1955) . 1912 (1960) .
(1961) .
1961 .
8.
M . Vaughan, J .E . Berger, and D . Steinberg, J . Biol . Chem . , 239, 401 (1964) .
9.
M . Rodbell, J . Biol . Chem . , 235, 1613, 1960 .
10 .
A . Bezman, J .M . Felts, and R .J . Havel, J . Lipid Research , 3, 427 (1962) .
11 .
E . Wertheimer, and E . Shafrir, Recent Prou . Hormone Res . , 16, 467 (1960) .
12 .
P .J . Nestel, and R .J . Havel, Proc . Soc . Exptl . Biol . Med . ,
13 .
E . Gorin, and E . Shafrir, Biochim & Biophvs Acta , 84, 24 (1964) .
14 .
J .D, Schnatz, and R .H . Williams, Metab . , _, 349 (1962) .
15 .
M . Bier, in S .P . Colowick,
109, 985 (1962) .
and N .O . Kaplan (Eds .), Methods in EnzymoloAy ,
Vol . I, Academic Press, New York, 1955, p . 629 . 16 .
V .P, Dole, J . Clin . Invest . , _, 150 (1956) .
17 .
J .D . Schnatz, J . Lipid Research , in press .
18 .
P . Desnuelle, in F .F . Nord (Ed .), Advances in EnzymoloRV , Vol . 23, Interscience Publishers, Inc ., New York, 1961, p . 129 .