Comparison of metabolic activities of orbital fat with those of other adipose tissues

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1 Comparison of metabolic activities of orbital fat with those of other adipose tissues Eta Aronovsky, Ruth Levarl, Walter Kornblueth, and Ernst Wertheimer The lipid metabolism of various adipose tissues from cats (orbital, paw, subcutaneous, and omental) was studied in vitro. The total content of free fatty acid (FFA) after incubation u>as greater in subcutaneous fat from fasted, cats as compared to that in fed cats, but fasting did not significantly affect the content of FFA in orbital or paw fat. Incubating in the presence of adrenaline and noradrenalin stimulated the release of FFA in all the adipose tissues studied, but to different degrees. The effect of noradrenalin on the FFA content of orbital fat was very small, while that on paw fat was slightly greater. Subcutaneous fat showed the greatest stimulation by noradrenalin. Adrenalin caused only a small increase in the release of FFA in orbital fat, but a great increase in paw fat. The uptake of labeled palmitic acid, by omentum was rapid, and, instant esterification occurred. However, these processes were exceedingly slow in orbital fat and intermediate in rate in paw fat. A consistant finding with respect to all the parameters studied was that orbital fat is considerably less metabolically active than other adipose tissues. M..any studies have been carried out on the metabolism of epididymal fat because of its thinness and symmetry. These characteristics make it possible to use the tissue with minimum handling; moreover, the same animal may serve as both the experimental and the control subject. However, it is doubtful whether the metabolic behavior of such specialized adipose tissue is typical of other stores of body fat. It is more reasonable to suppose that each type From the Department of Biochemistry, the Hebrew University-Hadassah Medical School, and the Department of Ophthalmology, Mayer de Rothschild-Hadassah University Hospital, Jerusalem, Israel. This study was supported by Fight for Sight Grant-in-Aid No. G-229 (C-4) of the National Council to Combat Blindness, Inc., New York, N. Y. of adipose tissue possesses specific metabolic patterns in accordance with its specialized function. With the exception of epididymal fat there is only scant knowledge concerning the metabolic characteristics of other adipose tissues. Subcutaneous fat, for instance, is less active than either omentum or epididymal fat, 1 while the intrascapular brown fat appears to be very active." Paw fat does not accumulate glycogen upon refeeding. a In addition, it has been observed that in a state of extreme emaciation, when the fat of some tissues has completely disappeared, fat could still be detected in certain adipose tissues such as Bichat adipose tissue. The present communication describes studies on the accumulation and mobilization of orbital fat. Since this fat mainly has a mechanical function, its metabolism 259

2 260 Aronovsky et al. estigntrvt! Ophthalmology June 1963 is compared, on the one hand, with that of another mechanical fat (paw fat) and, on the other hand, with that of adipose tissues whose metabolic activity is known to be high (omentum and subcutaneous fat). Materials and methods Stray cats of both sexes weighing 2 to 3 kilograms were used. They were kept in cages and fed on milk and bread ad libitum for at least one week prior to the experiment. The term "fasted" cats refers to animals from which food, but not water, was withheld for 3 consecutive days. "Refed" cats were fasted for 4 consecutive days and fed for 1 day. "Fed" and "refed" cats received sugared milk (50 gram per 100 ml.) approximately 4 hours prior to their sacrifice. The cats were anesthesized by intraperitoneal injection of 5 per cent sodium pentobarbiturate (1 ml. per kilogram) and were killed by bleeding. The orbital fat was removed after enucleation of the globe. This fat consisted of two kinds: a soft type around the optic nerve, and a harder type within the orbital cavity which appeared to be present in a globular arrangement. Histologically, there was little difference between these two fats; that around the optic nerve showed smaller lobules containing less connective tissue. The fat around the optic nerve was preferred for use in the experiments, but whenever the amount was not sufficient, both kinds of fat were used and divided equally in each vessel. The paw fat was collected by making a deep incision into the paw pad; the fat tissue was then cut from within. Care was taken to include as little connective tissue as possible. The possibility of slight contamination with connective tissue could not be excluded, but since the results of the experiments with paw fat were as consistent as those with the other adipose tissues, this contingency was not considered to be a disturbing factor. Subcutaneous fat was taken from between the hind legs. Tissues were removed immediately after bleeding and were weighed, rinsed in saline (at room temperature), and placed in the incubation medium. Release of FFA. The medium consisted of 1.3 ml. of 5 per cent bovine albumin (fraction V)* dissolved in 0.9 per cent NaCl and adjusted to ph 7.4 with NaOH; those experiments where Adrenalin or noradrenalin was added to the medium are indicated in the text. Incubation was carried out for 2 hours at 37 C. with gentle agitation. At the end of the intubation period an aliquot of the medium was removed, placed in 5 ml. of Dole's mixture,* extracted, and separated. In order to remove any shorter chain organic acid which might have been present, the heptane phase was washed with a "blank lower phase." 5 FFA content was then measured by titrating it with 0.02N NaOH, with an ethanolic solution of Nile blue as the indicator. The tissue was then removed from the incubation medium, washed in saline, dried, and homogenized in Tenbrock glass homogenizers containing 6 ml. of Dole's extraction mixture. A 5 ml. aliquot was then taken and treated exactly as above. The results of the experiments with the use of Adrenalin or noradrenalin are expressed as percentage of the control, and the significance is calculated according to Student's method for correlating samples, since the experimental and control tissues were taken from the same animal. 0 Experiments showing the effect of fasting are calculated similarly, but with the use of Student's method for small samples, 0 since these tissues were obviously from different animals. Uptake and esterification of palmitate. The following incubation mixture was used for these experiments: 1.2 ml. of calcium-free Krebs-Ringer phosphate buffer 7 ph 7.4 containing 5 per cent bovine albumin, 0.12 to 0.21 ^c per milliliter of 1-C 14 palmitic acid," 1.4 to 1.6 /imole per milliliter of unlabeled carrier, and 5.2 to 8.3 wnole per milliliter of glucose. A series of 6 to 8 vessels were incubated for different periods of time; FFA in the medium and in the tissue as well as tissue glycerides were extracted, isolated, titrated, and counted for radioactivity as described by Kerpel and associates. 5 Nitrogen in the tissue was determined by a modification of Lang's method 9 after it was defatted, as described by Itzhaki and Wertheimer. 10 Results Effect of fasting on FFA content. Subcutaneous adipose tissue from fasted cats contained about twice as much FFA as that from fed cats (Table I). However, there was very little difference in the FFA content of the medium under these conditions. The FFA content of paw fat of fasted cats was only slightly higher than that of fed cats. In orbital fat the FFA in the medium and in the tissue was (insignificantly) lower in fasted cats than in fed ones. - *: Pentex Corporation, Kanknke "The Radiochemical Centre, Amersham, Buckinghamshii England.

3 Volume 2 Number 3 Comparison of metabolic activities of adipose tissues 261 Table I. Effect of fasting on fat mobilization (microequivalents FFA per gram wet weight) Diet No. of experiments Medium Tissue Subcutaneous adipose tissue Fed f Fasted Total. content of FFA Paw fat Fed Fasted Orbital fat Fed Fasted n, Calculated according to Student's method for small samples. (, Not significant Table II. The effect of Adrenalin and noradrenalin in vitro on FFA content of fasted cats J k i Adrenalin" pf No. of exp Control Experiment Total coi lent of FFA Effect Adipose tissue No. of exp. iieh Fl-A/Ginit eight wet Subcutaneous tissue Paw fat ±23 Orbital fat ± ± 6.5 "At final co of 37 per nilliliter. I Calculated according t j Student's method for coi related samples Control Noradrenalin' Experiment of FFA lieq FFA/Gm. wet weight Effect. 86 ± ±23 V ± Effect of adrenalin and noradrenalin in vitro on FFA content of adipose tissues from fasted cats. In the presence of Adrenalin and noradrenalin the total FFA content of the subcutaneous fat was increased by about 56 per cent and 86 per cent, respectively. The FFA content of paw fat rose by approximately 300 per cent in the presence of adrenalin, while in the presence of noradrenalin it rose by only 57 per cent. The increase of FFA in orbital fat in the presence of either of these hormones was slight, but significant (Table II). It seems, in general, that adipose tissues of cats are less responsive to these hormones than those from other animal species. Rate of uptake and esterification of 1-C' palmitic acid. Since adipose tissue of refed animals shows a greater metabolic activity than that of fasted animals, 11 refed cats were used for these experiments. The various adipose tissues differed markedly in their ability to take up and incorporate palmitic acid, in spite of their comparatively small differences in neutral fat content (Table III). During 6 hours of incuba-

4 262 Aronovsky et al. tstigntioe Ophthalmology lime 1963 Table III. Neutral fat and nitrogen content in omental, paw, and orbital adipose tissue of refed cats Neutral fat (% of wet weight) Nitrogen (ing./cm. wet weight) Adipose tissue Omental 54.8 ± ± 0.54 Paw 33.6 ± ± 2.71 Orbital 50.5 ± ± 0.64 tion, the uptake (per 100 mg. wet tissue) of labeled palmitate from the medium was 1.2 /j.mole in the omental fat, 0.69 /j.mole in the paw fat, and 0.18 pinole in the orbital fat (Fig. 1). Because the paw fat contained much more nitrogen than the other tissues (Table III), the palmitate uptake of the paw fat, when calculated on a nitrogen basis, was very similar to that of orbital fat. When the results were calculated on the basis of total lipids, the same relationship in the uptake of palmitic acid by the various adipose tissues was demonstrated. Only in the case of paw fat were the values somewhat higher, yet still lower than those of the omental fat. Thirty minutes after incubation, 4 per cent of the labeled palmitic acid inside the omental tissue was found in the form of FFA. At the same time, 91 per cent could be accounted for as neutral fat. It is apparent that this tissue esterified palmitate almost immediately on entrance. In orbital fat, after 30 minutes, 77 per cent of the labeled palmitate was found as FFA, and, even after 6 hours, 26 per cent was still present as FFA. The amount of radioactivity found as neutral fat after 30 minutes and 6 hours was IS per cent and 66 per cent, respectively. Thus, orbital fat incorporates FFA into neutral fat at a considerably lower rate than omentum. The rate of esterification in paw fat increases much more rapidly than that of orbital fat. Thirty minutes after incubation, 55 per cent of labeled palmitate is found as FFA, and 4 hours after incubation, only 10 per cent of free acids remain, the other 90 per cent having been transformed into neutral fats (Fig. 2). Fig. 1. Uptake of 1-C 1 ' 1 palmitic acid. 6)(4) 'I 16) 12) [ 2) (6) "I 1 V' (6I x. 161 V 31 (61 I (4) 1 (8) ( 1 Hours ) >') i (3 7~i' - Fig. 2.. Decrease in percentage of 1-C 11 palmitic acid in various adipose tissues during incubation. The percentage of radioactivity as FFA was calculated as the ratio of CPM in tissue FFA to total tissue radioactivity (xloo).

5 Comparison of metabolic activities of adipose tissues 263 Discussion When these results have been interpreted, it should be pointed out that the metabolic activity of adipose tissue of the cat is, on the whole, lower than that of the rat, the animal most frequently used for experimental work. Conventional laboratory animals could not be used for these experiments since they did not have a sufficient amount of orbital fat. Cats were found most suitable because they possessed a sufficient quantity of paw and orbital fat. It is common practice in studying the metabolism of adipose tissues to assume that results found in one adipose tissue apply to all adipose tissues in general. However, the data reported above indicate that the different adipose tissues used in this study possess different metabolic activities. Omental, epididymal, and mesenterial fat, which have high metabolic activity, have no 7nechanical function. Subcutaneous fat, which presumably possesses a supporting and insulating function, is metabolically less active. Least active is the fat from the orbit and paw, both of which serve mainly a mechanical function. 1 -' " These results might suggest that an inverse relationship exists between metabolic activity and mechanical function of the tissue. Mobilization of fat. The most striking outcome of these experiments is the fact that, during fasting, mobilization of orbital fat is not increased but, on the contrary, even slightly decreased. Under the same conditions, mobilization of paw fat is slightly, but not significantly, increased, while that of subcutaneous fat shows substantial increase. Another important finding is the influence of noradrenalin on FFA release. The effect on orbital fat is the least, that on paw fat is greater, and that on subcutaneous fat is the largest. Adrenalin also affects the release of subcutaneous fat more than that of orbital fat, but it has a particularly large effect on paw fat. With the assumption that the sympaticoadrenal system is one of the controlling mechanisms for fat mobilization, the different reactions to fasting of the three adipose tissues discussed above could be explained by their different responses to noradrenalin in vitro. A relationship between noradrenalin within the adipose tissue and fatty acid release has been established recently."" 17 The exceptionally large effect of Adrenalin on mobilization of paw fat is still unexplained. Accumulation of labeled palmitic acid and its incorporation into ghjcerides. The uptake of labeled palmitic acid and its esterification follow the same order of reactivity as other metabolic processes (discussed above) in these adipose tissues. The acid is taken up and incorporated immediately by the omental fat, but very slowly by orbital fat. The activity of paw fat is intermediate. The above experiments show that the influence of some factors on the mobilization of orbital and, to a lesser extent, paw fat is smaller than that on subcutaneous fat. The "mechanical" adipose tissues are also much slower in fat accumulation. It might be of interest to find out whether the "mechanical" adipose tissues also differ in other aspects of fat metabolism from the adipose tissues known to possess high metabolic activity. REFERENCES 1. Benjamin, W., Cellhorn, A., Wagner, M., and Kundel, H.: Effect of aging on lipid composition and metabolism in the adipose tissues of the rat, Am. J. Physiol. 201: 540, Wertheimer, E., and Shapiro, B.: The physiology of adipose tissue, Physiol. Rev. 28: 451, Hoffman, A., and Wertheimer, E.: Personal communication. 4. Dole, V. P.: Relation between nonesterified fatty acids in plasma and metabolism of glucose, J. Clin. Invest. 35: 150,, Dole, V. P., and Meinertz, H.: Microdetermination of long-chain fatty acids in plasma and tissues, J. Biol. Chem. 235; 2595, Paterson, D. D.: Statistical technique in agricultural research, New York, 1939, McGraw- Hill Book Co., Inc.

6 264 Aronovsky el al..stigutwe Ophthulmologv ]mie Umbreit, VV. W., Burris, R. H., and Staulfer, f. F.: Manonietric techniques and tissue metabolism, ed. 2, Minneapolis, 1949, Burgess Publishing Company. 8. Kerpel, S., Shafrir, E., and Shapiro, B.: Mechanism of fatty acid assimilation in adipose tissue, Biochem. ct biophys. acta 40: 495, Lang, C. A.: Simple microdetermination of Kjeldahl nitrogen in biological materials, Anal. Chem. 30: 1692, Itzhaki, S., and Wertheimer, E.: Metabolism of adipose tissue in vitro; nutritional factor and elfect of insulin, Endocrinology 61: 72, Shapiro, B., Chowers, I., and Rose, C: Fatty acid uptake and esterification in adipose tissue, Biochem. et biophys. acta 23: 115, Wertlieimer, E., and Shafrir, E.: Influence of hormones on adipose tissue as a center of fat metabolism, Recent Progr. Hormone Res. 16: 467, Stary, Z., and Tekman, S.: Metabolism of fat tissues of different anatomical location, Bull. Fac. Med. Istanbul 15: 257, Smith, R. L., Paoletti, R., and Brodie, B. B.: The identification and assay of noradrenalin in adipose tissue, Biochem. J. 82: 19p, Smith, R. L., Paoletti, R., and Brodie, B. B.: the essential role of noradrenaline in corticotropin-incluced fatty acid mobilization, Biochem. J. 84: 51p, Dury, A.: Effect of reserpine on fatty acid (FFA) mobilization, Fed. Proc. 21: 284, 1962 (absr.). 17. Paoletti, R., Smith, R. L., Maickel, R. P., and Brodie, B. B.: Identification and physiological role of norepinephrine in adipose tissue, Biochem. et biophys. res, commun. 5: 424, 1961.