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Effect of hypothalamic preparations on human omental adipose tissue in vitro
Effect of Hypothalamic Preparations on Human Omental Adipose Tissue In Vitro By Tommie
W. Redding
The lipolytic responses of rat and human adipose tissue to several pituitary hormones, catecholamines, and purified preparations from human and porcine hypothalami have been compared. Fragments of human omental adipose tissue, obtained at surgery, or rat epididymal adipose tissue were incubated in vitro for 2 hr in 2 ml of KrebsRinger bicarbonate medium containing 3% bovine albumin. Acetic acid extracts of human and porcine stalk median eminence tissue were purified by gel filtration on Sephadex G-25. Lipidmobilizing factor (LMF) activity emerged in an area occupied by peptides with a molecular weight of 30005000. This lipolytic fraction was found to be free of thyroid-stimulating hormone (TSH) and catecholamines. The ACTH content of this fraction was found to be less than IO mU/mg. Addition of 20-60 rg/ml of porcine or human LMF to the medium significantly increased lipolysis, as measured by glycerol release into the medium, from human and rat adipose tissue. Human
and Andrew
V. Schally
LMF elicited a greated lipolytic response in human omental adipose tissue than did the porcine LMF, but porcine LMF was more potent in rat epididymal adipose tissue. This suggests that a species specificity may exist for the lipolytic activity of hypothalamic lipid mobilizers. Responsiveness of human omental adipose tissue to adrenalin was lower than that of rat epididymal adipose tissue. Addition of corticotropin A (IO-20 Pglml), human growth hormone (10-30 pg/ml), and alpha or beta melanocyte-stimulating hormone (MSH) (10-30 &g/ml) had no effect on lipolysis when incubated with human omental adipose tissue. However, the addition of 153.0 mu/ml of human TSH significantly increased the release of glycerol from human omental adipose tissue. These data suggest that human hypothalamic extracts, like those of porcine hypothalamic extracts, contain a lipid-mobilizing factor or factors that may differ chemically from corticotropin, TSH, growth hormone, and the catecholamines.
W
E HAVE PREVIOUSLY REPORTED that a substance or substances found in extracts from the hypothalami of pigs, cattle, sheep, and humans stimulated lipolysis on incubation of adipose tissue from the mouse, rat, hamster, guinea pig, and rabbit.r A partial purification of this substance from porcine hypothalamic extracts was achieved by molecular sieving through Sephadex G-25 columns. The active lipolytic material emerged in an area occupied by peptides with a molecular weight of 3000-5000. This fraction was shown to be free of catecholamines, and its lipolytic activity could not be accounted for by contamination with thyroid-stimulating hormone (TSH), oxytocin, vasopressin, a- and /3-melanocyte-stimulating hormone (MSH), or From the Endocrine and Polypeptide Laboratories, Veterans Adminisfration Hospital, and fhe Department of Medicine, Tulane University School of Medicine, New Orleans, La. Received for publication November IS, 1971. Supported in parf by USPHS Grant AM 07467. Tommie W. Redding, B.S.: Radioisotope Chemist, Veterans Administration Hospital, New OrIeans, La. Andrew V. Schally, Ph.D.: Chief, Endocrine and Polypeptide Laboratories, Veterans Administration Hospital, and Professor of Medicine, Tulane University School of Medicine, New Orleans, La. Metabolism, Vol. 21, No. 6 (June),1972
499
500
REDDING
AND SCHALLY
hypothalamic-releasing hormones. Studies on the in vitro lipolytic activity of this purified hypothalamic fraction, designated lipid-mobilizing factor (LMF), indicated that it was again active in adipose tissue from the mouse, rat, hamster, guinea pig, and rabbit. Part of the lipid-mobilizing activity of this purified porcine hypothalamic LMF fraction was thought to be due to contamination with ACTH and/or analogues with adrenocorticotropic activity. The lipolytic effects of hypothalamic extracts on human adipose tissue in vitro have not been previously reported. Accordingly, we have investigated the responses of human omental tissue to purified materials with LMF activity derived from porcine and human hypothalami and compared them with the responses to other substances known to augment lipolysis in fat cells of other mammals. MATERIALS
Tissue
Preparation
and Incubation
AND
METHODS
Procedure
Human Tissue: Human omental adipose tissue was taken from male patients undergoing abdominal surgery for hemigastrectomies, cholecystectomies, and exploratory procedures under general anesthesia. It has been previously shown by James et al.2 that sex, type of surgical procedure, or anesthetic agent used does not effect basal or stimulated Iipolysis in human omental adipose tissue in vitro. The age range was 25-68 yr. All sampling procedures were between 8 a.m. and 12 noon. The omental adipose tissue samples were brought immediateiy to the laboratory in incubation medium maintained a\ 37°C. The medium consisted of bicarbonate-Krebs-Ringer buffer, pH 7.4, with 3% bovine albumin (fraction V) without glucose. The tissue was sliced into small fragments, washed with buffer, blotted dry, and weighed to the nearest 0.1 mg. Fragments, weighing 50-100 mg, were transferred to plastic vials containing 2 ml of buffer. Test substances dissolved in buffer or saline were then added, and the vials were incubated in a Dubnoff incubator for 2 hr at 37°C under 95% O? and 5% CO,. Th ree of four vials were used to test each substance. Rat Tissue: Male rats weighing 200-300 g were decapitated, and the epididymal fat pads removed ,sliced into 5&7o-mg pieces, washed with buffer, blotted dry, and incubated as described above. At the end of the incubation period, the fat tissue was removed, and aliouots of the media were taken for the determination of glycerol by Korn’s modification3 of the method of Lambert and Niesh.4 Results are expressed in terms of pmoles of glycerol released/g of tissue per hour incubation time * the standard error of the mean. Statistical evaluation of the data was performed using the Student’s t test.5 Table 1. Effect of Lipid-mobilizing on the Release of Glycerol Dose (/lg/ml Treatment
Control
(4)
Porcine
LMF (4)
Human
LMF (4)
medium)
20 60 180 20 40 180
Factor (LMF) From Human and Porcine Hypothalami From Rat Epididymal Adipose Tissue In Vitro
Glycerol production (pmoles/gR hr Z?SE)
0.03 + 0.03 10.45 11.65 11.29 1.69 6.91 9.63
‘Difference between control and experimental. tprobability as determined by Student’s t test. *Number of incubation vials.
f IT f f f k
0.63 1.40 1.30 0.13 0.77 0.74
A’
Pt
-
-
10.42 11.53 11.26 1.66 6.88 9.60
0.001 0.005 0.001 0.001 0.001 0.061
HUMAN OMENTAL ADIPOSE TISSUE
Preparation
501
of Human Hypothalamic
Concentrates
The ventral portion of the hypothalamus between the mammillary bodies, including portions of the pituitary stalk, was removed from the brain during routine autopsy. These fragments weighed between 400-1000 mg and were obtained from unselected males and females between SO-80 yr of age. There was a time lapse of 20-48 hr between death and excision of tissues, which were kept frozen until lyophilized. Three-hundred thirty-nine fragments, weighing 47.5 g after lyophilization, were defatted and extracted according to the procedure developed for porcine hypothalami. 6.’ Partial purification of glacial acetic acid concentrates of porcine and human hypothalamic extract was achieved by molecular sieving on Sephadex G-25 using a column 7.5 X 150 cm and 1 M acetic acid as the solvent, as we have previously reported. 7.8 The ACTH activity of fractions from Sephadex was determined in hypophysectomized rats by measuring the increase in plasma corticosterone.9 The ACTH ccntent of human LMF was found to be less than 10 mU/mg. Corticotropin A (SO U/mg), a gift from Dr. W. F. White of Abbott Laboratories, was dissolved with 0.01 M HCI and diluted with buffered media for use in experiments. Human TSH and human growth hormone were obtained from the Endocrinology Study Section of the National Institute of Arthritis and Metabolic Disease. Alpha and beta MSH were prepared as described previously8 and represent pure samples with maximal activities (alpha were MSH, 1.5 X 107 U/mg; beta MSH, 0.6 X 107 U/mg). Ad renalin and noradrenalin purchased from commercial sources. The TSH content of this LMF fraction was determined by bioassay as previously described.1 RESULTS
Table 1 shows a dose response of rat epididymal fat tissue to porcine and human LMF. It can be seen that the response to porcine LMF is maximal at 60 pg/ml medium. Human LMF at the same doses was less lipolytic than the porcine material, suggesting either a species specificity of that human hypothalamic extract contained less of this lipolytic substance. Dose responses of human omental adipose tissue to human and porcine LMF and to adrenalin are shown in Table 2. A significant stimulation of Table 2. Effect of Lipid-mobilizing Factor (LMF) From Human and Porcine Hypothalami and Adrenaline on the Release of Glycerol From Human Omental Adipose Tissue In Vitro Dose (rP/ml Treatment Control (3)
Glycerol Production hr f SE)
@moles/g/Z
-
Human LMF (3)s
Porcine LMF (3)*
Adrenalin
medium)
(3)$
Control (5)* Human LMF (5)$
A’
Pt
1.10 + 0.08
-
-
20 80 180 20 60 180 0.1 1.0 10.0
3.60 -c 0.89 7.01 + 210 2.99 t: 0.62 2.81 f 0.20 2.08 + 0.33 3.04 +- 0.28 2.77 2 0.42 3.82 + 0.30 4.51 f 0.55
2.50 5.91 1.89 1.71 0.98 1.94 1.67 2.72 3.41
0.025 0.05 0.05 0.005 0.05 0.005 0.025 0.005 0.005
20 60 180
1.50 + 0.18 2.83 f 0.15 3.72 -c 0.11 3.06 -c 0.48
1.33 2.22 1.56
0.005 0.001 0.025
‘Difference between control and experimental. tprobabilty as determined by Student’s t test. *Number of incubation vials.
502
REDDING
Table 3. Effect of Various Substances on the Release of Glycerol Omental Adipose Tissue In Vitro Dose Treatment
(pg/ml)
Experiment A Control (3)* Human LMF (3)$ Noradrenalin (3)* Corticotropin A (3)* Experiment B Control (3)* Noradrenalin (3)$ Corticotropin A Experiment C Control (3)* Human LMF (3)$ Adrenalin (3)$ Noradrenalin (3)$
Glycerol Production (fimoles/g/2 hr f SE)
-
0.498 0.949 1.555 0.719
50 5 10
5 10
20 10 5
2 -c f r
0.032 0.15 0.12 0.11
AND SCHALLY
From Human
Pt
A’
-
-
0.451 1.057 0.221
0.025 0.001 N.S.
-
-
0.220 2 0.12 1.29 r 0.18 1.12 f 0.78
1.07 0.90
0.01 N.S.
0.94 3.34 2.22 1.75
2.40 1.28 0.81
0.001 0.025 0.05
+ If: k k
0.03 0.13 0.32 0.025
-
*Difference between control and experimental. tprobability determined by Student’s t test. *Number of incubation vials.
lipolysis was seen with all the doses tested. In contrast to results obtained with rat adipose tissue, the human LMF appeared to be at least as active as porcine LMF with human omental adipose tissue. Adrenalin caused a significant release of glycerol into the media at a dose of 0.1 pglml medium. Although further testing is required, these data suggest that species specificity may be involved in the lipolytic response of human omental adipose tissue to human and porcine LMF. Table 3 shows the effect of adrenalin, noradrenalin, corticotropin, and human LMF on the release of glycerol from human omental adipose tissue in three different experiments. Corticotropin A failed to elicit a lipolytic response in any of the experiments. However, adrenalin and noradrenalin, along with human LMF, significantly increased the release of glycerol into the media. Table 4. Lipolytic
Control
Activity of Some Hormones When Incubated Human Omental Adipose Tissue
Treatment
Dose (fig/ml)
Glycerol Production (wmoles/g/2 hr ?z SE)
A*
(4)t
-
1.10 rt 0.15
-
10 20 20 3.6 mU 50 10
1.04 1.46 1.48 2.44 2.23 2.52
alpha MSH (4)$ beta MSH (4)$ Corlicotropin A (4)$ Human TSH (4)$ Human LMF (4)$ Noradrenalin (4)$
*Difference between control and experimental. tprobability as determined by Student’s t test. *Number of incubation vials.
+ k r + f -c
0.04 0.08 0.13 0.26 0.11 0.45
0.06 0.38 0.38 1.34 1.13 1.42
With
Pt
N.S. N.S. N.S. 0.025 0.005 0.05
HUMAN
OMENTAL
ADIPOSE
TISSUE
503
Both alpha and beta MSH are found in significant amounts in the hypoalso contains MSH thalamus of several species. l”*ll Human hypothalamus activity.12 Therefore, it was of interest to determine the effect of these substances, along with human LMF and TSH, on the release of glycerol from human omental adipose tissue. Table 4 indicates that both alpha and beta MSH, at these doses, were without lipolytic activity on human omental adipose tissue. Human TSH and LMF and noradrenalin, but not corticotropin A, increased glycerol release from human omental adipose tissue. It has been previously reported that growth hormone is a potent lipolytic agent when incubated with rat adipose tissue.13 Addition of human growth hormone (10-20 pg/ml) or corticotropin A to the incubation medium failed to increase the release of glycerol from human omental adipose tissue as shown in Table 5. Adrenalin, noradrenalin, and human LMF again showed significant lipolytic activity. Human pituitary fractions containing TSH, but not growth hormone (GH) or ACTH, have been shown to increase lipolysis when incubated with human adipose tissue. 14,15 In Table 6, two additional experiments (A and B) indicate that glycerol release was enhanced when human omental adipose tissue was incubated in the presence of 1.5-3.0 mu/ml human TSH, human LMF, or adrenalin. Neither corticotropin A nor MSH significantly increased glycerol release. Since human TSH was found to be lipolytic in these experiments, it was necessary to determine the TSH content of our hypothalamic lipid-mobilizing substance. Table 7 shows the absence of TSH activity of our human LMF fraction when tested at 10 to SO times the active lipolytic dose. Analysis of this fraction for catecholamines is shown in Table 8. When tested at 1 and 5 mg on an alumina column followed by fluorometric determination, no catecholamines could be detected. Table 5. Effect of Human Growth Hormone, Human LMF, Corticotropin A and Adrenaline on the Release of Glycerol From Human Omental Adipose Tissue In Vitro Treatment
Experiment A Control (3) Human growth hormone (3)* Human LMF (3)$ Adrenalin (3)* Experiment B Control (4)* Corticotropin A (4)* Human LMF (4)* Human growth hormone (4)* Noradrenalin (4)$
Dose kg/ml)
Glycerol Production @moles/g/2 hr f SE)
-
0.82 f 0.16
-
-
10
1.14 f 0.11
0.32
N.S.
60 5
1.83 k 0.05 1.99 + 0.29
1 .Ol 1.17
0.025 0.05
-
1.30 -c 0.26
-
10 60 20
1.48 f 0.43 2.38 f 0.17 2.02 r 0.47
0.18 1.08 0.72
N.S. 0.025 N.S.
10
3.74 -c 0.38
2.44
0.005
*Difference between control and experimental. tprobability determined by Student’s t test. *Number of incubation vials.
A’
Pt
-
504
REDDING
AND SCHALLY
Table 6. Effect of Various Hormones on the Release of Glycerol From Human Omental Adipose Tissue In Vitro Dose Treatment
kg/ml)
Experiment A Control (3) Beta MSH (3)* Human
LMF (3)$
Human TSH (3)* Adrenalin (3)$ Experiment B Control (3)* alpha MSH (3)$ Human LMF (3)$ Corticotropin A (3)$ Nordadrenalin (3)$
Glycerol Production @moles/g/2 hr -c SE)
10 30 20 60 1.5 mu. 3.0 mu. 10
10 40 10 10
Pt
A’
0.66 1.06 1.06 1.41 2.66 1.90 1.60 3.08
f f f f f -c f -c
0.06 0.01 0.09 0.07 0.15 0.18 0.20 0.39
-
-
0.22 0.20 0.55 1.82 1.04 0.94 2.22
N.S. N.S. 0.01 0.001 0.01 0.025 0.01
0.76 1.51 2.10 0.70 2.84
-c + r f It
0.31 0.73 0.22 0.35 0.25
0.75 1.34 0.06 2.08
-
N.S. 0.025 N.S. 0.01
‘Difference between control and experimental. tprobability as determined by Student’s t test. *Number of incubation vials.
Table 7. Absence
Substance
Saline (5)t Human LMF (5)t Sephadex Human LMF (5)t Sephadex Bovine TSH (5)t
of TSH Activity in Human Hypothalamic Fraction From Sephadex
LMF
*V Release from Thyroid Gland of Mice. Changes Blood lz51 levels at 2 hr (cpm r SE)
Dose (&mouse)
P’
200
-42 -50
f 26 f 28
N.S. N.S.
1000
-13
+ 31
N.S.
0.2 mU 0.8 mU
492 -c 119 1464 f 265
0.01 0.005
“Student’s t test. tNumber of assay animals
Table 6. Catecholamine
Substance
Human LMF fraction from Sephadex
Content of Human Hypothalamic Fraction From Sephadex Amount Applied to Alumina Column (mg)
1.0 5.0
Lipid-Mobilizing
Catecholamine Present as Norepinephrine (ng)
<0.05 (Not detectable) <0.5 (Not detectable)
HUMAN
OMENTAL
ADIPOSE
505
TISSUE
DISCUSSION
Most of our knowledge regarding the lipolytic activity of pituitary hormones, glucagon, adrenalin, noradrenalin, and other substances is derived from experiments that utilize the adipose tissue of the rat. It is tempting to assume that all these findings are applicable to human adipose tissue; however, the assessment of natural and synthetic lipolytic substances has been complicated by species variability. It may be necessary to test each lipolytic agent on human adipose tissue before drawing any conclusions as to the anticipated lipolytic activity in humans. Hypothalamic extracts are known to be contaminated with significant lo-l2 Sephadex gel filtration does amounts of ACTH-like peptides and ACTH. not clearly separate ACTH from ACTH-like polypeptides. Since the adipose tissue from mice, rats, hamsters, guinea pigs, and rabbits responds to ACTH, it was concluded that lipolytic response seen in these species to our porcine LMF preparation may be due, in part, to ACTH or ACTH-like analogues. Although the ACTH content of our human LMF was found to be less than IO mU/mg, addition of an equivalent or greater amount of corticotropin to the incubation medium of human omental adipose tissue failed to elicit a lipolytic response. Addition of human growth hormone, a potent lipolytic agent on rat adipose tissue, I3 also failed to increase the release of glycerol from human omental adipose tissue. Bjorntorp et a1.16 have also failed to show a definite lipolytic response with ACTH and GH in human adipose tissue. Furthermore, Mosinger et al. 17s1* have been unable to show a response to ACTH in human mesenteric adipose tissue when incubated with phosphate buffer alone, but they did detect a response when 20% human serum was added to the buffered medium. Raben and HollenberglQ reported that neither ACTH nor growth hormone of any origin produced a definite lipolytic effect on human adipose tissue in vitro. On the other hand, several investigators reported that human pituitary fractions containing TSH but not GH or ACTH had lipolytic activity when incubated with human adipose tissue.14J5 Our data indicate a definite adipokinetic activity of human TSH when incubated with human omental adipose tissue (Tables 4 and 6). However, when tested by bioassay, our hypothalamic LMF fraction was shown to be devoid of TSH. From these data, it would seem that the lipid-mobilizing activity of our human hypothalamic material cannot be accounted for solely by contamination with ACTH, TSH, and growth hormone. Human omental adipose tissue was not responsive to either ACTH or human growth hormone, and TSH was not present as a contaminant. Human LMF elicited a greater response in human adipose tissue than did the porcine material, although porcine LMF was more potent in rat adipose tissue. This suggests that a species specificity may exist for the lipolytic activity of hypothalamic lipid mobilizers. Numerous investigators have reported the isolation of substances with lipid-mobilizing activity from the anterior pituitary2@2s of various species. Whether our hypothalamic LMF is the same or similar to these materials is not known. Our experiments with porcine LMF suggest that it is a peptide whose molecular weight is between 3000 and 5000. Work is in progress to characterize further this substance from human and porcine hypothalami.
506
REDDING
AND SCHALLY
ACKNOWLEDGMENT We are greatly indebted to E. B. Ferguson, Jr., M.D., for his helpful editorial advice on this manuscript. We also wish to thank Mrs. T. Taylor for her technical assistance in performing these experiments. 1. Redding, T. W., and Schally, A. V.: A lipid-mobilizing factor from the hypothalamus. Metabolism 19 :641,1970. 2. James, R. C., Burns, T. W., and Chase, G. R.: Lipolysis of human adipose tissue cells: Influence of donor factors. J. Lab. Clin. Med. 77:254, 1971. 3. Korn, E. D.: Clearing factor, a heparinactivated lipoprotein lipase. J. Biol. Chem. 215:1, 1955. 4. Lambert, M., and Neish, A. C.: Rapid method for the estimation of glycerol in fermentation solution. Canad. J. Res. 28 (Section B) :83, 1950. S. Snedecor, G. W.: Statistical Methods. Ames, Iowa, Iiowa State College Press, 1957. 6. Schally, A. V., Bowers, C. Y., and Redding, T. W.: Presence of thyrotropic hormone-releasing factor (TRF) in porcine hypothalamus. Proc. Sot. Exp. Biol. Med. 121:718, 1966. 7. -, Redding, T. W., Bowers, C. Y., and Barrett, J. F.: Isolation and properties of thyrotropin-releasing hormone. J. porcine Biol. Chem. 244:4077, 1969. 8. Porath, J., and Schally, A. V. : Gel filtration of posterior pituitary hormones. Endocrinology 70:738,1962. 9. Guillemin, R., Clayton, G. W., Smith, J. D., and Libcomb, H. S.: Measurement of the corticosteroids in rat plasma: Physiological validation of a method. Endocrinology 63:399, 1958. 10. Schally, A. V., Lipscomb, H. S., Long, J. M., Dear, W. E’., and Guillemin, R.: Chromatography and hormonal activity of dog hypothalamus. Endocrinology 70 ~78, 1962. 11. Guillemin, R., Schally, A. V., Lipscomb, H. S., Anderson, R. N., and Long, J. M.: On the presence of hog hypothalamus of /3-corticotropin-releasing fractions, a- and /3-MSH, adrenocorticotropin, lysine vasopressin, and oxytocin. Endocrinology 7o:471, 1962. 12. Schally, A. V., Arimura, A., Bowers, C. Y., Wakabayashi, I., Kastin, A. J., Redding, T. W., Mittler, J. C., Nair, R. M. G., Pizzolato, P., and Segal, A. J.: Purification of hypothalamic releasing hormones of hu-
man origin. J. Clin. Endocr. 31:291, 1970. 13. Fain, J. N., Kovacev, V. P., and Scow, R.: Effect of growth hormone and dexamethasone on lipolysis and metabolism in isolated fat cells of the rat. J. Biol. Chem. 240:3522. 1964. 14. Burns, T. W., Hales, C. N., and Stockwell-Hartree, A.: Observations of the lipolytic activity of human serum and pituitary fractions in vitro. J. Endocr. 39:213, 1967. 15. Holland, M.: The effect of bovine anterior pituitary extract on lipid mobilization in the rat and man. J. Endocr. 44307, 1969. 16. Bjorntorp, I’., Karlsson, M., and Hovden, A.: Quantitative aspects of lipolysis and reesterification in human adipose tissue in vitro. Acta Med. Stand. l&35:89,1969. 17. Mosinger, B., Kuhn, E., and Kujalova, V.: Action of adipokinetic hormone on human adipose tissue in vitro. J. Lab. Clin. Med. 66~380, 1965. 18. Kujalova, V., Kuhn, E., and Mosinger, B.: Lipolytic activity of human serum on human adipose tissue in vitro. In Proceedings of the International Union of Physiologic Sciences, XXIV International Congress, Washington, D. C., 1968. Abstract 746. 19. Raben, M. S., and Hollenberg, C. H.: Zn Wolsteinholme, G. E. W., and Connor, C. M. G. (Eds.): Ciba Foundation Colloquium, Endocrinology, Vol. 13. Boston, Little, Brown, 1960, p. 89. 20. Friesen, H., Barrett, S., and Ashwood, E. B.: Metabolic effect of two peptides from the anterior pituitary gland. Endocrinology 70579, 1962. 21. Raben, M. S., Landolt, R., Smith, F. A., Hoffmann, K., and Yama, H.: Adipokinetic activity of synthetic peptides related to corticotropin. Nature (London) 189:681, 1961. 22. Rudman, D., Seidman, F., Brown, S. S., and Hersch, R. L.: Adipokinetic activity of porcine fraction H in the rabbit, guinea pig, rat, and mouse. Endocrinology 70:233, 1962. 23. Lohmay, P., and Li, C. H.: Biological properties of ovine P-lipotropic hormone. Endocrinology 82 ~898,1968.
W. Redding
The lipolytic responses of rat and human adipose tissue to several pituitary hormones, catecholamines, and purified preparations from human and porcine hypothalami have been compared. Fragments of human omental adipose tissue, obtained at surgery, or rat epididymal adipose tissue were incubated in vitro for 2 hr in 2 ml of KrebsRinger bicarbonate medium containing 3% bovine albumin. Acetic acid extracts of human and porcine stalk median eminence tissue were purified by gel filtration on Sephadex G-25. Lipidmobilizing factor (LMF) activity emerged in an area occupied by peptides with a molecular weight of 30005000. This lipolytic fraction was found to be free of thyroid-stimulating hormone (TSH) and catecholamines. The ACTH content of this fraction was found to be less than IO mU/mg. Addition of 20-60 rg/ml of porcine or human LMF to the medium significantly increased lipolysis, as measured by glycerol release into the medium, from human and rat adipose tissue. Human
and Andrew
V. Schally
LMF elicited a greated lipolytic response in human omental adipose tissue than did the porcine LMF, but porcine LMF was more potent in rat epididymal adipose tissue. This suggests that a species specificity may exist for the lipolytic activity of hypothalamic lipid mobilizers. Responsiveness of human omental adipose tissue to adrenalin was lower than that of rat epididymal adipose tissue. Addition of corticotropin A (IO-20 Pglml), human growth hormone (10-30 pg/ml), and alpha or beta melanocyte-stimulating hormone (MSH) (10-30 &g/ml) had no effect on lipolysis when incubated with human omental adipose tissue. However, the addition of 153.0 mu/ml of human TSH significantly increased the release of glycerol from human omental adipose tissue. These data suggest that human hypothalamic extracts, like those of porcine hypothalamic extracts, contain a lipid-mobilizing factor or factors that may differ chemically from corticotropin, TSH, growth hormone, and the catecholamines.
W
E HAVE PREVIOUSLY REPORTED that a substance or substances found in extracts from the hypothalami of pigs, cattle, sheep, and humans stimulated lipolysis on incubation of adipose tissue from the mouse, rat, hamster, guinea pig, and rabbit.r A partial purification of this substance from porcine hypothalamic extracts was achieved by molecular sieving through Sephadex G-25 columns. The active lipolytic material emerged in an area occupied by peptides with a molecular weight of 3000-5000. This fraction was shown to be free of catecholamines, and its lipolytic activity could not be accounted for by contamination with thyroid-stimulating hormone (TSH), oxytocin, vasopressin, a- and /3-melanocyte-stimulating hormone (MSH), or From the Endocrine and Polypeptide Laboratories, Veterans Adminisfration Hospital, and fhe Department of Medicine, Tulane University School of Medicine, New Orleans, La. Received for publication November IS, 1971. Supported in parf by USPHS Grant AM 07467. Tommie W. Redding, B.S.: Radioisotope Chemist, Veterans Administration Hospital, New OrIeans, La. Andrew V. Schally, Ph.D.: Chief, Endocrine and Polypeptide Laboratories, Veterans Administration Hospital, and Professor of Medicine, Tulane University School of Medicine, New Orleans, La. Metabolism, Vol. 21, No. 6 (June),1972
499
500
REDDING
AND SCHALLY
hypothalamic-releasing hormones. Studies on the in vitro lipolytic activity of this purified hypothalamic fraction, designated lipid-mobilizing factor (LMF), indicated that it was again active in adipose tissue from the mouse, rat, hamster, guinea pig, and rabbit. Part of the lipid-mobilizing activity of this purified porcine hypothalamic LMF fraction was thought to be due to contamination with ACTH and/or analogues with adrenocorticotropic activity. The lipolytic effects of hypothalamic extracts on human adipose tissue in vitro have not been previously reported. Accordingly, we have investigated the responses of human omental tissue to purified materials with LMF activity derived from porcine and human hypothalami and compared them with the responses to other substances known to augment lipolysis in fat cells of other mammals. MATERIALS
Tissue
Preparation
and Incubation
AND
METHODS
Procedure
Human Tissue: Human omental adipose tissue was taken from male patients undergoing abdominal surgery for hemigastrectomies, cholecystectomies, and exploratory procedures under general anesthesia. It has been previously shown by James et al.2 that sex, type of surgical procedure, or anesthetic agent used does not effect basal or stimulated Iipolysis in human omental adipose tissue in vitro. The age range was 25-68 yr. All sampling procedures were between 8 a.m. and 12 noon. The omental adipose tissue samples were brought immediateiy to the laboratory in incubation medium maintained a\ 37°C. The medium consisted of bicarbonate-Krebs-Ringer buffer, pH 7.4, with 3% bovine albumin (fraction V) without glucose. The tissue was sliced into small fragments, washed with buffer, blotted dry, and weighed to the nearest 0.1 mg. Fragments, weighing 50-100 mg, were transferred to plastic vials containing 2 ml of buffer. Test substances dissolved in buffer or saline were then added, and the vials were incubated in a Dubnoff incubator for 2 hr at 37°C under 95% O? and 5% CO,. Th ree of four vials were used to test each substance. Rat Tissue: Male rats weighing 200-300 g were decapitated, and the epididymal fat pads removed ,sliced into 5&7o-mg pieces, washed with buffer, blotted dry, and incubated as described above. At the end of the incubation period, the fat tissue was removed, and aliouots of the media were taken for the determination of glycerol by Korn’s modification3 of the method of Lambert and Niesh.4 Results are expressed in terms of pmoles of glycerol released/g of tissue per hour incubation time * the standard error of the mean. Statistical evaluation of the data was performed using the Student’s t test.5 Table 1. Effect of Lipid-mobilizing on the Release of Glycerol Dose (/lg/ml Treatment
Control
(4)
Porcine
LMF (4)
Human
LMF (4)
medium)
20 60 180 20 40 180
Factor (LMF) From Human and Porcine Hypothalami From Rat Epididymal Adipose Tissue In Vitro
Glycerol production (pmoles/gR hr Z?SE)
0.03 + 0.03 10.45 11.65 11.29 1.69 6.91 9.63
‘Difference between control and experimental. tprobability as determined by Student’s t test. *Number of incubation vials.
f IT f f f k
0.63 1.40 1.30 0.13 0.77 0.74
A’
Pt
-
-
10.42 11.53 11.26 1.66 6.88 9.60
0.001 0.005 0.001 0.001 0.001 0.061
HUMAN OMENTAL ADIPOSE TISSUE
Preparation
501
of Human Hypothalamic
Concentrates
The ventral portion of the hypothalamus between the mammillary bodies, including portions of the pituitary stalk, was removed from the brain during routine autopsy. These fragments weighed between 400-1000 mg and were obtained from unselected males and females between SO-80 yr of age. There was a time lapse of 20-48 hr between death and excision of tissues, which were kept frozen until lyophilized. Three-hundred thirty-nine fragments, weighing 47.5 g after lyophilization, were defatted and extracted according to the procedure developed for porcine hypothalami. 6.’ Partial purification of glacial acetic acid concentrates of porcine and human hypothalamic extract was achieved by molecular sieving on Sephadex G-25 using a column 7.5 X 150 cm and 1 M acetic acid as the solvent, as we have previously reported. 7.8 The ACTH activity of fractions from Sephadex was determined in hypophysectomized rats by measuring the increase in plasma corticosterone.9 The ACTH ccntent of human LMF was found to be less than 10 mU/mg. Corticotropin A (SO U/mg), a gift from Dr. W. F. White of Abbott Laboratories, was dissolved with 0.01 M HCI and diluted with buffered media for use in experiments. Human TSH and human growth hormone were obtained from the Endocrinology Study Section of the National Institute of Arthritis and Metabolic Disease. Alpha and beta MSH were prepared as described previously8 and represent pure samples with maximal activities (alpha were MSH, 1.5 X 107 U/mg; beta MSH, 0.6 X 107 U/mg). Ad renalin and noradrenalin purchased from commercial sources. The TSH content of this LMF fraction was determined by bioassay as previously described.1 RESULTS
Table 1 shows a dose response of rat epididymal fat tissue to porcine and human LMF. It can be seen that the response to porcine LMF is maximal at 60 pg/ml medium. Human LMF at the same doses was less lipolytic than the porcine material, suggesting either a species specificity of that human hypothalamic extract contained less of this lipolytic substance. Dose responses of human omental adipose tissue to human and porcine LMF and to adrenalin are shown in Table 2. A significant stimulation of Table 2. Effect of Lipid-mobilizing Factor (LMF) From Human and Porcine Hypothalami and Adrenaline on the Release of Glycerol From Human Omental Adipose Tissue In Vitro Dose (rP/ml Treatment Control (3)
Glycerol Production hr f SE)
@moles/g/Z
-
Human LMF (3)s
Porcine LMF (3)*
Adrenalin
medium)
(3)$
Control (5)* Human LMF (5)$
A’
Pt
1.10 + 0.08
-
-
20 80 180 20 60 180 0.1 1.0 10.0
3.60 -c 0.89 7.01 + 210 2.99 t: 0.62 2.81 f 0.20 2.08 + 0.33 3.04 +- 0.28 2.77 2 0.42 3.82 + 0.30 4.51 f 0.55
2.50 5.91 1.89 1.71 0.98 1.94 1.67 2.72 3.41
0.025 0.05 0.05 0.005 0.05 0.005 0.025 0.005 0.005
20 60 180
1.50 + 0.18 2.83 f 0.15 3.72 -c 0.11 3.06 -c 0.48
1.33 2.22 1.56
0.005 0.001 0.025
‘Difference between control and experimental. tprobabilty as determined by Student’s t test. *Number of incubation vials.
502
REDDING
Table 3. Effect of Various Substances on the Release of Glycerol Omental Adipose Tissue In Vitro Dose Treatment
(pg/ml)
Experiment A Control (3)* Human LMF (3)$ Noradrenalin (3)* Corticotropin A (3)* Experiment B Control (3)* Noradrenalin (3)$ Corticotropin A Experiment C Control (3)* Human LMF (3)$ Adrenalin (3)$ Noradrenalin (3)$
Glycerol Production (fimoles/g/2 hr f SE)
-
0.498 0.949 1.555 0.719
50 5 10
5 10
20 10 5
2 -c f r
0.032 0.15 0.12 0.11
AND SCHALLY
From Human
Pt
A’
-
-
0.451 1.057 0.221
0.025 0.001 N.S.
-
-
0.220 2 0.12 1.29 r 0.18 1.12 f 0.78
1.07 0.90
0.01 N.S.
0.94 3.34 2.22 1.75
2.40 1.28 0.81
0.001 0.025 0.05
+ If: k k
0.03 0.13 0.32 0.025
-
*Difference between control and experimental. tprobability determined by Student’s t test. *Number of incubation vials.
lipolysis was seen with all the doses tested. In contrast to results obtained with rat adipose tissue, the human LMF appeared to be at least as active as porcine LMF with human omental adipose tissue. Adrenalin caused a significant release of glycerol into the media at a dose of 0.1 pglml medium. Although further testing is required, these data suggest that species specificity may be involved in the lipolytic response of human omental adipose tissue to human and porcine LMF. Table 3 shows the effect of adrenalin, noradrenalin, corticotropin, and human LMF on the release of glycerol from human omental adipose tissue in three different experiments. Corticotropin A failed to elicit a lipolytic response in any of the experiments. However, adrenalin and noradrenalin, along with human LMF, significantly increased the release of glycerol into the media. Table 4. Lipolytic
Control
Activity of Some Hormones When Incubated Human Omental Adipose Tissue
Treatment
Dose (fig/ml)
Glycerol Production (wmoles/g/2 hr ?z SE)
A*
(4)t
-
1.10 rt 0.15
-
10 20 20 3.6 mU 50 10
1.04 1.46 1.48 2.44 2.23 2.52
alpha MSH (4)$ beta MSH (4)$ Corlicotropin A (4)$ Human TSH (4)$ Human LMF (4)$ Noradrenalin (4)$
*Difference between control and experimental. tprobability as determined by Student’s t test. *Number of incubation vials.
+ k r + f -c
0.04 0.08 0.13 0.26 0.11 0.45
0.06 0.38 0.38 1.34 1.13 1.42
With
Pt
N.S. N.S. N.S. 0.025 0.005 0.05
HUMAN
OMENTAL
ADIPOSE
TISSUE
503
Both alpha and beta MSH are found in significant amounts in the hypoalso contains MSH thalamus of several species. l”*ll Human hypothalamus activity.12 Therefore, it was of interest to determine the effect of these substances, along with human LMF and TSH, on the release of glycerol from human omental adipose tissue. Table 4 indicates that both alpha and beta MSH, at these doses, were without lipolytic activity on human omental adipose tissue. Human TSH and LMF and noradrenalin, but not corticotropin A, increased glycerol release from human omental adipose tissue. It has been previously reported that growth hormone is a potent lipolytic agent when incubated with rat adipose tissue.13 Addition of human growth hormone (10-20 pg/ml) or corticotropin A to the incubation medium failed to increase the release of glycerol from human omental adipose tissue as shown in Table 5. Adrenalin, noradrenalin, and human LMF again showed significant lipolytic activity. Human pituitary fractions containing TSH, but not growth hormone (GH) or ACTH, have been shown to increase lipolysis when incubated with human adipose tissue. 14,15 In Table 6, two additional experiments (A and B) indicate that glycerol release was enhanced when human omental adipose tissue was incubated in the presence of 1.5-3.0 mu/ml human TSH, human LMF, or adrenalin. Neither corticotropin A nor MSH significantly increased glycerol release. Since human TSH was found to be lipolytic in these experiments, it was necessary to determine the TSH content of our hypothalamic lipid-mobilizing substance. Table 7 shows the absence of TSH activity of our human LMF fraction when tested at 10 to SO times the active lipolytic dose. Analysis of this fraction for catecholamines is shown in Table 8. When tested at 1 and 5 mg on an alumina column followed by fluorometric determination, no catecholamines could be detected. Table 5. Effect of Human Growth Hormone, Human LMF, Corticotropin A and Adrenaline on the Release of Glycerol From Human Omental Adipose Tissue In Vitro Treatment
Experiment A Control (3) Human growth hormone (3)* Human LMF (3)$ Adrenalin (3)* Experiment B Control (4)* Corticotropin A (4)* Human LMF (4)* Human growth hormone (4)* Noradrenalin (4)$
Dose kg/ml)
Glycerol Production @moles/g/2 hr f SE)
-
0.82 f 0.16
-
-
10
1.14 f 0.11
0.32
N.S.
60 5
1.83 k 0.05 1.99 + 0.29
1 .Ol 1.17
0.025 0.05
-
1.30 -c 0.26
-
10 60 20
1.48 f 0.43 2.38 f 0.17 2.02 r 0.47
0.18 1.08 0.72
N.S. 0.025 N.S.
10
3.74 -c 0.38
2.44
0.005
*Difference between control and experimental. tprobability determined by Student’s t test. *Number of incubation vials.
A’
Pt
-
504
REDDING
AND SCHALLY
Table 6. Effect of Various Hormones on the Release of Glycerol From Human Omental Adipose Tissue In Vitro Dose Treatment
kg/ml)
Experiment A Control (3) Beta MSH (3)* Human
LMF (3)$
Human TSH (3)* Adrenalin (3)$ Experiment B Control (3)* alpha MSH (3)$ Human LMF (3)$ Corticotropin A (3)$ Nordadrenalin (3)$
Glycerol Production @moles/g/2 hr -c SE)
10 30 20 60 1.5 mu. 3.0 mu. 10
10 40 10 10
Pt
A’
0.66 1.06 1.06 1.41 2.66 1.90 1.60 3.08
f f f f f -c f -c
0.06 0.01 0.09 0.07 0.15 0.18 0.20 0.39
-
-
0.22 0.20 0.55 1.82 1.04 0.94 2.22
N.S. N.S. 0.01 0.001 0.01 0.025 0.01
0.76 1.51 2.10 0.70 2.84
-c + r f It
0.31 0.73 0.22 0.35 0.25
0.75 1.34 0.06 2.08
-
N.S. 0.025 N.S. 0.01
‘Difference between control and experimental. tprobability as determined by Student’s t test. *Number of incubation vials.
Table 7. Absence
Substance
Saline (5)t Human LMF (5)t Sephadex Human LMF (5)t Sephadex Bovine TSH (5)t
of TSH Activity in Human Hypothalamic Fraction From Sephadex
LMF
*V Release from Thyroid Gland of Mice. Changes Blood lz51 levels at 2 hr (cpm r SE)
Dose (&mouse)
P’
200
-42 -50
f 26 f 28
N.S. N.S.
1000
-13
+ 31
N.S.
0.2 mU 0.8 mU
492 -c 119 1464 f 265
0.01 0.005
“Student’s t test. tNumber of assay animals
Table 6. Catecholamine
Substance
Human LMF fraction from Sephadex
Content of Human Hypothalamic Fraction From Sephadex Amount Applied to Alumina Column (mg)
1.0 5.0
Lipid-Mobilizing
Catecholamine Present as Norepinephrine (ng)
<0.05 (Not detectable) <0.5 (Not detectable)
HUMAN
OMENTAL
ADIPOSE
505
TISSUE
DISCUSSION
Most of our knowledge regarding the lipolytic activity of pituitary hormones, glucagon, adrenalin, noradrenalin, and other substances is derived from experiments that utilize the adipose tissue of the rat. It is tempting to assume that all these findings are applicable to human adipose tissue; however, the assessment of natural and synthetic lipolytic substances has been complicated by species variability. It may be necessary to test each lipolytic agent on human adipose tissue before drawing any conclusions as to the anticipated lipolytic activity in humans. Hypothalamic extracts are known to be contaminated with significant lo-l2 Sephadex gel filtration does amounts of ACTH-like peptides and ACTH. not clearly separate ACTH from ACTH-like polypeptides. Since the adipose tissue from mice, rats, hamsters, guinea pigs, and rabbits responds to ACTH, it was concluded that lipolytic response seen in these species to our porcine LMF preparation may be due, in part, to ACTH or ACTH-like analogues. Although the ACTH content of our human LMF was found to be less than IO mU/mg, addition of an equivalent or greater amount of corticotropin to the incubation medium of human omental adipose tissue failed to elicit a lipolytic response. Addition of human growth hormone, a potent lipolytic agent on rat adipose tissue, I3 also failed to increase the release of glycerol from human omental adipose tissue. Bjorntorp et a1.16 have also failed to show a definite lipolytic response with ACTH and GH in human adipose tissue. Furthermore, Mosinger et al. 17s1* have been unable to show a response to ACTH in human mesenteric adipose tissue when incubated with phosphate buffer alone, but they did detect a response when 20% human serum was added to the buffered medium. Raben and HollenberglQ reported that neither ACTH nor growth hormone of any origin produced a definite lipolytic effect on human adipose tissue in vitro. On the other hand, several investigators reported that human pituitary fractions containing TSH but not GH or ACTH had lipolytic activity when incubated with human adipose tissue.14J5 Our data indicate a definite adipokinetic activity of human TSH when incubated with human omental adipose tissue (Tables 4 and 6). However, when tested by bioassay, our hypothalamic LMF fraction was shown to be devoid of TSH. From these data, it would seem that the lipid-mobilizing activity of our human hypothalamic material cannot be accounted for solely by contamination with ACTH, TSH, and growth hormone. Human omental adipose tissue was not responsive to either ACTH or human growth hormone, and TSH was not present as a contaminant. Human LMF elicited a greater response in human adipose tissue than did the porcine material, although porcine LMF was more potent in rat adipose tissue. This suggests that a species specificity may exist for the lipolytic activity of hypothalamic lipid mobilizers. Numerous investigators have reported the isolation of substances with lipid-mobilizing activity from the anterior pituitary2@2s of various species. Whether our hypothalamic LMF is the same or similar to these materials is not known. Our experiments with porcine LMF suggest that it is a peptide whose molecular weight is between 3000 and 5000. Work is in progress to characterize further this substance from human and porcine hypothalami.
506
REDDING
AND SCHALLY
ACKNOWLEDGMENT We are greatly indebted to E. B. Ferguson, Jr., M.D., for his helpful editorial advice on this manuscript. We also wish to thank Mrs. T. Taylor for her technical assistance in performing these experiments. 1. Redding, T. W., and Schally, A. V.: A lipid-mobilizing factor from the hypothalamus. Metabolism 19 :641,1970. 2. James, R. C., Burns, T. W., and Chase, G. R.: Lipolysis of human adipose tissue cells: Influence of donor factors. J. Lab. Clin. Med. 77:254, 1971. 3. Korn, E. D.: Clearing factor, a heparinactivated lipoprotein lipase. J. Biol. Chem. 215:1, 1955. 4. Lambert, M., and Neish, A. C.: Rapid method for the estimation of glycerol in fermentation solution. Canad. J. Res. 28 (Section B) :83, 1950. S. Snedecor, G. W.: Statistical Methods. Ames, Iowa, Iiowa State College Press, 1957. 6. Schally, A. V., Bowers, C. Y., and Redding, T. W.: Presence of thyrotropic hormone-releasing factor (TRF) in porcine hypothalamus. Proc. Sot. Exp. Biol. Med. 121:718, 1966. 7. -, Redding, T. W., Bowers, C. Y., and Barrett, J. F.: Isolation and properties of thyrotropin-releasing hormone. J. porcine Biol. Chem. 244:4077, 1969. 8. Porath, J., and Schally, A. V. : Gel filtration of posterior pituitary hormones. Endocrinology 70:738,1962. 9. Guillemin, R., Clayton, G. W., Smith, J. D., and Libcomb, H. S.: Measurement of the corticosteroids in rat plasma: Physiological validation of a method. Endocrinology 63:399, 1958. 10. Schally, A. V., Lipscomb, H. S., Long, J. M., Dear, W. E’., and Guillemin, R.: Chromatography and hormonal activity of dog hypothalamus. Endocrinology 70 ~78, 1962. 11. Guillemin, R., Schally, A. V., Lipscomb, H. S., Anderson, R. N., and Long, J. M.: On the presence of hog hypothalamus of /3-corticotropin-releasing fractions, a- and /3-MSH, adrenocorticotropin, lysine vasopressin, and oxytocin. Endocrinology 7o:471, 1962. 12. Schally, A. V., Arimura, A., Bowers, C. Y., Wakabayashi, I., Kastin, A. J., Redding, T. W., Mittler, J. C., Nair, R. M. G., Pizzolato, P., and Segal, A. J.: Purification of hypothalamic releasing hormones of hu-
man origin. J. Clin. Endocr. 31:291, 1970. 13. Fain, J. N., Kovacev, V. P., and Scow, R.: Effect of growth hormone and dexamethasone on lipolysis and metabolism in isolated fat cells of the rat. J. Biol. Chem. 240:3522. 1964. 14. Burns, T. W., Hales, C. N., and Stockwell-Hartree, A.: Observations of the lipolytic activity of human serum and pituitary fractions in vitro. J. Endocr. 39:213, 1967. 15. Holland, M.: The effect of bovine anterior pituitary extract on lipid mobilization in the rat and man. J. Endocr. 44307, 1969. 16. Bjorntorp, I’., Karlsson, M., and Hovden, A.: Quantitative aspects of lipolysis and reesterification in human adipose tissue in vitro. Acta Med. Stand. l&35:89,1969. 17. Mosinger, B., Kuhn, E., and Kujalova, V.: Action of adipokinetic hormone on human adipose tissue in vitro. J. Lab. Clin. Med. 66~380, 1965. 18. Kujalova, V., Kuhn, E., and Mosinger, B.: Lipolytic activity of human serum on human adipose tissue in vitro. In Proceedings of the International Union of Physiologic Sciences, XXIV International Congress, Washington, D. C., 1968. Abstract 746. 19. Raben, M. S., and Hollenberg, C. H.: Zn Wolsteinholme, G. E. W., and Connor, C. M. G. (Eds.): Ciba Foundation Colloquium, Endocrinology, Vol. 13. Boston, Little, Brown, 1960, p. 89. 20. Friesen, H., Barrett, S., and Ashwood, E. B.: Metabolic effect of two peptides from the anterior pituitary gland. Endocrinology 70579, 1962. 21. Raben, M. S., Landolt, R., Smith, F. A., Hoffmann, K., and Yama, H.: Adipokinetic activity of synthetic peptides related to corticotropin. Nature (London) 189:681, 1961. 22. Rudman, D., Seidman, F., Brown, S. S., and Hersch, R. L.: Adipokinetic activity of porcine fraction H in the rabbit, guinea pig, rat, and mouse. Endocrinology 70:233, 1962. 23. Lohmay, P., and Li, C. H.: Biological properties of ovine P-lipotropic hormone. Endocrinology 82 ~898,1968.