LIPOLYTIC ACTIVITY OF ADIPOSE TISSUE IN MAN AND RAT*

Transcription

1 LIPOLYTIC ACTIVITY OF ADIPOSE TISSUE IN MAN AND RAT* BY ALBERT E. RENOLDt AND ALEXANDER MARBLE (From the George F. Baker Clinic, New England Deaconess Hospital, Boston) (Received for publication, November 10, 1949) In the course of a study of the factors involved in the production of lipodystrophies (fat atrophies and hypertrophies) by the subcutaneous injection of insulin (1, 2), it was considered pertinent to investigate the lipolytic activity of adipose tissue. Whereas data are numerous concerning lipases and esterases of other origin (3), references to the lipolytic enzymatic activity of adipose tissue are rather scanty. Quagliarello (4) in 1932 described an enzymatic splitting of both triolein and tributyrin by an extract obtained from fatty tissue of the dog. The activity of dried adipose tissue was found to be l/200 that of the same weight of dried pancreas but 20 times that of the same weight of dried liver. Using a histochemical method, Gomori (5, 6) found evidence of lipolytic activity in the adipose tissue of the rat and rabbit, but none in that of other animals, and only very occasionally in man. The lipolytic activity of adipose tissue in man was noted and briefly discussed by Marble and Smith (2) and its presence indicated by Oesterreicher (7). Methods Extraction-In the albino rat, tissue was taken from the fat pads of the groin and the interscapular region. In the human subjects, the subcutaneous and articular adipose tissue was obtained from amputated legs by careful dissection from visible veins and fascia. Immediately after the death of the animal, or after amputation, the excised tissue was placed in a glass container surrounded with ice; all subsequent procedures were performed at or near 0. The tissue was minced in a Waring blendor twice with 5 volumes of acetone and twice with 5 volumes of ether, then washed with ether on a filter, dried in partial vacuum in a cooled desiccator, and stored in a desiccator at 4. The preparation of the dried tissue was completed within 2 hours of removal from the body. In the process of extraction, the dry tissue was incubated for 1 hour at room temperature with 0.2 M borate buffer at ph 8.2 in the proportion of 500 mg. of tissue to 8 cc. of buffer for tissue of human origin, and * This work was aided by a grant from the American Cyanamid Company and the Diabetic Fund. t Holder of a fellowship of the Swiss-American Center of Medical Exchange. 367

2 368 LIPOLYTIC ACTIVITY OF ADIPOSE TISSUE 250 mg. to 8 cc. of buffer for that from rats. After centrifugation the slightly opalescent supernatant fluid was removed and used as an enzyme preparation. The protein content of the extract varied from 0.5 to 1.5 per cent and the non-protein nitrogen between 23 and 26 mg. per 100 cc. (fourteen extracts). Determination of Lipolytic Activity-In the method of Archibald (8), used throughout the study, a mixture of polyoxyalkylene derivatives of an ester of 1 mole of fatty acid (90 per cent lauric acid) per mole of sorbitan (Tween 20) is used as a substrate. This substrate is water-soluble. The free fatty acids formed are separated with ether and titrated. For routine determinations no activators or inhibitors of any kind were used. TABLE I Influence of ph of Extracting Buffer on Extraction of Enzyme Preparation* ph of extracting buffer At i&q. fatty acid formed * Extraction of the dry, defatted tissue (female rat, group weighing 120 to 160 sm.). tthe activity is expressed in milliequivalents of fatty acid formed per hour at ph 8.2 and 37. A, extract of 31 mg. of dry, defatted tissue; B, amount of extract containing 0.5 mg. of nitrogen. For each determination a blank value was obtained by incubating separately the extract and the buffered substrate. For each new tissue the destruction of all lipolytic activity by heat was confirmed by placing the extract in a boiling water bath for 30 seconds. The lipolytic activity is expressed in milliequivalents of fatty acids formed per hour at 37. Two values are given for every tissue: The first relates the activity to a standard amount (31 mg.) of dry, defatted tissue. The second value relates the activity to the extract containing a standard amount of extracted nitrogen (0.5 mg.);2 as the non-protein nitrogen was found to vary only between 23 and 26 mg. per 100 cc. in fourteen different extracts, this value can be considered to express the activity of a standard 1 Atlas Powder Company, Wilmington, Delaware. *The standard amounts used were chosen arbitrarily as corresponding to the 0.6 cc. of extract used: 31 mg. of dry, defatted tissue yielded 0.5 cc. of extract, and the average nitrogen content of this amount of extract was 0.5 mg. Bt

3 A. E. RENOLD AND A. hlarble 369 amount of extracted protein. This was thought to eliminate differences of extraction present even with standard conditions and evidenced by the variations in protein content. Total nitrogen and non-protein nitrogen values were obtained by micro-kjeldahl procedures. For all ph determinations a Beckman ph meter was used, and the ph indicated in the tables is always the ph at the beginning of the incubation, determined separately in each incubated sample. To evaluate the conditions of extraction, the activity of a number of extracts obtained at different temperatures and after times of extraction varying between 10 minutes and 22 hours was determined. It was evident that time and temperature had but little influence on the extraction. However, room temperature was slightly more favorable and there was indication that extraction time over 6 hours should be avoided. The ph during extraction was of great importance, with a definite optimum in the neighborhood of ph 8.0 (see Table I). The activity of the extract of a given amount of dried, defatted tissue was found to equal or surpass the activity of the equivalent amount of dried tissue itself, added directly to the incubation mixture. (For this comparison the samples were continuously shaken during incubation.) The standard deviation for a single determination of lipolytic activity, obtained from twelve different extracts of the same tissue, was f m.eq. Results Lipolytic Activity of Adipose Tissue of Man and Rat-As illustrated by Fig. 1, A and B, the relation between the amount of enzyme present and the milliequivalents of acid formed can be considered linear up to a titration value of 0.04 m.eq. of fatty acid formed. At this point the ph of the sample is approximately 0.3 lower than at the beginning of the incubation, and is therefore getting outside the optimum ph range. If higher titration values are obtained, adequate dilution of the enzyme preparation with extracting buffer will bring the value into the desired range of linear relationship. The actual activity of the extract can then be calculated by taking into consideration the dilution used. The ph optimum (Fig. 1, D) is in agreement with the findings of Quagliarello (4). The lipolytic activity of the dry tissue, stored in a desiccator at 4, has in no case shown a decrease exceeding 10 per cent over a period of 4 months. The lipolytic activity of the extract stored.at 4 is stable up to a period averaging 3 weeks. Relative degree of lipolytic activity of rat organs, as measured by Archibald s method, was obtained by comparing it with the lipolytic activity of similar

4 370 LIPOLYTJC ACTIVITY OF ADIPOSE I ISSUE extracts of other organs. This was done separately for male and female rats. There was, however, no appreciable sex difference in the lipolytic activity of the tissues examined. The values obtained were within a range sufficiently narrow to justify their report as averages; these are represented graphically in Fig. 2. M,Eq. cl012 ;~fj@/ j/ll as LL 0.1 Q cc EXTRACT c c EXTRACT MeEq. M.Eq :@/ I Ez(q Q aoio If GO Go 10.0 TIME IN MINUTES I IO.0 PH FIQ. 1. Lipolytic activity of adipose tissue. In A and B is shown the relation of the amount of extract present to the amount of ether-soluble acid liberated from the substrate per hour at ph 8.2 and 37. The amount of acid liberated is expressed in milliequivalents. A, extract of human adipose tissue (female patient J. P.). B, extract of rat sdipose tissue (female, group weighing 220 to 250 pm.). The lipolytic activity of rat adipose tissue is significantly greater than that of human adipose tissue even if the nitrogen content of the extract is taken into consideration. C, the relation of the time of incubation with an extract of human adipose tissue to the amount of ether-soluble acid liberated from the substrate at ph 8.2 and 37. D, the relation of the lipolytic activity of an extract of human adipose tissue to the ph of the reaction mixture at 37. Lipolytic Activity of Human Subcutaneous and Articular Adipose Tissue in Four Unselected Non-Diabetic and Ten Diabetic Patients-In the four non-diabetic patients subcutaneous fat could be obtained only in the course of various surgical procedures. In the diabetic patients subcutaneous and intra-articular subpatellar fat was taken from amputated legs. The results for the four non-diabetic and the ten diabetic patients are

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6 372 LIPOLYTIC ACTIVITY OF ADIPOSE TISSUE TABLE III Lipolytic Activity of Adipose Tissue (Subcutaneous and Articulur) of Five Mats and Five Female Diabetic Patients* - MaI.2 patients F.3lUll~ patients M.eqiofGd Atj Bt _- At Bt B. S. Subcutaneous N. M. Subcutaneous J. P. I Articular Articular S. G. Subcutaneous L. A. Subcutaneous I Articular Articular F. G. Subcutaneous M. B. Subcutaneous Articular I R. S. Subcutaneous Articular Articular E. B. Subcutaneous L. D. Subcutaneous I Articular Articular _ Average, subcutaneous f0.003$ f0.005$ articular f *0.001# - * Extraction of the dry, defatted tissue with 0.2 M borate buffer at ph 8.2 and room temperature for 1 hour. t Activity, see foot-note to Table I. t Standard error of the mean (o/d:). TABLE IV Lipolylic Activity of Brown (Interscapular) and White (Subcutaneous) Fat of Rats al Different A 2s and after a Period of Fasting* Female I Male Female Rats fasted 72 hrs.. Weight P Fatty tissue extracted At acid _-- Bt White Brown White Brown White Brown White Brown White and brown * Extraction of the dry, defatted tissue with 0.2 M borate buffer at ph 8.2 and room temperature for 1 hour. t Activity, see foot-note to Table I.

7 A. E. RENOLD AND A. MARBLE 373 of fasting. No correlation between lipolytic activity and sex, age, or food intake is apparent. In the young rat the brown interscapular fat (12) shows less lipolytic activity than the white fat. DISCUSSION From the data presented it appears justifiable to conclude that lipolytic activity of an enzymatic nature with a ph optimum at 8.2 is present in the adipose tissue of man and rat. The level of this activity is characteristic and surprisingly constant in any given tissue. This is in agreement with the uniformity of results obtained with the histochemical lipase determinations by Gomori (5), using an analogous water-soluble substrate, but is at variance with the greater scattering of values obtained with other methods in which the substrate must be emulsified and in which no separation of the fatty acids precedes their titration (9). In rats the lipolytic activity is approximately 6 times as great as in humans. In the rat the lipolytic activity of adipose tissue per unit of weight of dry, defatted tissue is equivalent to approximately one-third of that of the pancreas and one-half that of the liver. Of the organs examined, pancreas, liver, kidney, lung, and testis showed lipolytic activity; only muscle and spleen showed practically none. Of particular importance is the lack of lipolytic activity found in muscle and spleen, as this makes unlikely the possibility that the activity found in adipose tissue is derived from its content of blood, blood vessels, or some other generally distributed tissue. These results apply, of course, only to the substrate used (Tween 20). However, since they agree with earlier results obtained with other substrates (9) and since they show a marked activity of both pancreas and liver, the substrate used seems to be well adapted to detect any kind of lipolytic activity. In man the lipolytic activity of subcutaneous adipose tissue is approximately the same in normal subjects and in diabetic patients receiving amounts of insulin adequate for clinical control of diabetes. Our studies included, in the diabetics, the subpatellar, intra-articular fat; this adipose tissue belongs to a physiologically distinct group in which the storage function of fat seems to be subordinated to its mechanical function (plantar fat, the sucking pad of the new-born, retroorbital fat, etc.). In starvation this type of fat is characterized by its resistance to mobilization (10). It has further been demonstrated by early tagging methods with fat stains that the rate of turnover in this type of fat must be lower than in subcutaneous or abdominal fat (11). The lipolytic activity of the subpatellar, intra-articular fat was found in both males and females to be only one-third that of the subcutaneous fat. The lipolytic activity in the male diabetic is consistently and surpris-

8 374 LIPOLYTIC ACTIVITY OF ADIPOSE TISSUE ingly about one-fourth that in the female diabetic, both in subcutaneous and in articular fat. In the four unselected, nondiabetic patients examined the difference was less striking, but still distinct. Other than sex, no factor which might account for the difference between the two groups was found. The difference noted may possibly be correlated with the clinical finding that the atrophic type of lipodystrophy due to insulin is found frequently in children and women but much less frequently in adult males (2). Quagliarello (4) described an increased autolytic fat splitting in the fat of fasting dogs compared to fed dogs. In the rat we found no increase of lipolytic activity in the tissue of animals fasted for 72 hours compared with activity of the tissues of fed animals. In fact, lipolytic activity was somewhat lower in the fasted animals. In conclusion, it should be emphasized that, while the lipolytic activity of a given group of adipose tissues is surprisingly constant, different groups of fatty tissue show quite clearly a number of distinct, reproducible differences which suggest that this lipolytic activity may have a distinct physiological function. The nature of this function is not apparent from data now available. SUMMARY 1. Lipolytic activity of an enzymatic nature was demonstrated in the subcutaneous adipose tissue of both man and rat. In the rat such activity was approximately one-third that of the pancreas and one-half that of the liver. 2. In man the lipolytic activity of intra-articular fat amounted to only about one-third that of the subcutaneous fat. 3. In man the lipolytic activity of both subcutaneous and articular fat in males was consistently less than in females. In diabetic males it amounted to only about one-fourth of the corresponding activity in tissues of diabetic females. 4. In the rat there was no correlation between lipolytic activity and sex, age, or food intake. In the young rat the activity of the brown fat was less than that of the white fat. We are indebted to Dr. Gerhard Schmidt and Dr. Siegfried Thannhauser for their interest and stimulating suggestions. BIBLIOGRAPHY 1. Depisch, F., Klin. Wochschr., 6, 1965 (1926). 2. Marble, A., and Smith, R. M., Proc. Am. Diabetes Awn., 2, 173 (1942). 3. Bamann, E., and My&&k, K., Methoden der Fermentfomchung, Leipzig (1941).

9 A. E. RENOLD AND A. MARBLE Quagliarello, G., and Scoz, G., Arch. SC. biol., 17, 513 (1932). 5. Gomori, G., Arch. Path., 41, 121 (1946). 6. Gomori, G., Proc. Sot. Exp. Biol. and Med., 68, 362 (1945). 7. Oesterreicher, D. L., Thesis, University of Western Ontario (1947). 8. Archibald, R. M., J. Biol. Chem., 166, 443 (1946). 9. Virtanen, A. I., 2. physiol. Chem., 219, 1 (1933). 10. Miillendorf, W. V., Handbuch der mikroskopische Anatomie des Menschen, Berlin, 123 (1920). 11. Gage, S. H., and Fish, P. A., Am. J. Anut., 34.1 (1924). 12. Wertheimer, B., Physiol. Rev., 28, 451 (1943).

10 LIPOLYTIC ACTIVITY OF ADIPOSE TISSUE IN MAN AND RAT Albert E. Renold and Alexander Marble J. Biol. Chem. 1950, 185: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at tml#ref-list-1