0013-7227/79/1055-1262$02.00/0 Endocrinology Copyright © 1979 by The Endocrine Society
Vol. 105, No. 5 Printed in U.S.A.
Preparation and Properties of Hypothalamic Factors Capable of Altering Pancreatic Hormone Release in Vitro* J. H. MOLTZ,f R. E. DOBBS, S. M. McCANN, AND C. P. FAWCETT Department of Physiology, University of Texas Health Science Center at Dallas, Dallas, Texas 75235
ABSTRACT. Insulin release-inhibiting and glucagon-releasing activities previously detected in media in which rat ventromedial hypothalamic tissue had been incubated have now been detected in acid/acetone extracts of rat hypothalamic tissue. The activities from the two sources have identical responses to treatment with various proteolytic enzymes. They were treated with trypsin, chymotrypsin, carboxypeptidase, or pepsin and then tested in a pancreatic islet system for their ability to inhibit insulin and stimulate glucagon release. The insulin release-inhibiting activity was abolished by chymotrypsin, trypsin, carboxypeptidase, and pepsin treatment, whereas the glucagon-releasing activity was eliminated by carboxypeptidase and pepsin, was unaffected by trypsin, and was enhanced by chymotrypsin. These results
T
indicate not only the peptidic nature of the two activities but also show that both extract and incubate preparations appear to contain the same two peptide agents. Gel filtration on Sephadex G-25 demonstrated that the insulin release-inhibiting and glucagon-releasing activities were associated with two distinct entities. The insulin release-inhibiting activity was eluted soon after the void volume, whereas the glucagon-releasing activity was considerably retarded; each was distinguished from neurotensin and substance P. While no physiological significance can yet be assigned to these two peptides for the control of insulin and glucagon secretion, their existence provides further evidence for a possible neurohormonal pathway in the regulation of the endocrine pancreas. (Endocrinology 105: 1262, 1979)
HE PHYSIOLOGICAL role of the hypothalamus in the control of endocrine function, especially that of the anterior pituitary, has been examined for many years (1). Only recently, however, has the hypothalamus been thought of as a regulatory center for the endocrine pancreas. Several mechanisms have been proposed for the hypothalamic control of insulin and glucagon release. Frohman and coworkers (2) have postulated that the hyperinsulinemia associated with lesions of the ventromedial hypothalamus (VMH) is mediated via a direct neural pathway by way of the vagus nerve and that the inhibition of insulin release in response to the rising glucose levels associated with electrical stimulation of the VMH is due to increased sympathetic tone mediated via adrenal catecholamines (3). Another theory not involving a direct neural pathway has been put forth by Idahl and Martin (4). They explored the possibility that the hyperinsulinemia associated with a lesion in the VMH was due to a humoral factor rather than to the involvement of the autonomic nervous system. An insulin-releasing factor of ventrolateral hypothalamic origin was described by these workers, which led to the hypoth-
esis of a neurohormonal pathway from the hypothalamus to the pancreas (5). While seeking further evidence for the existence of a neurohormonal pathway, we were unable to confirm the existence of an insulin-releasing factor from the ventrolateral hypothalamus but detected insulin release-inhibiting and glucagon-releasing activities in incubates of VMH which were effective both in vivo (6) and in vitro (7). Our experiments were designed to obtain further support for a hypothalamic-pancreatic neurohormonal pathway by defining the chemical nature of these two activities through the use of proteolytic enzymes. Furthermore, it was necessary to show that insulin release-inhibiting and glucagon-releasing activities could be extracted from hypothalamic tissue. We also examined the possibility that these two activities are due to separate entities.
Received June 15, 1978. * This work was supported by NIH Grants AM-10073 and AM21163. f To whom requests for reprints should be addressed.
Preparation of VMH incubate
Materials and Methods The hypothalami used in the preparation of the tissue incubation (VMH) were obtained from large male Sprague-Dawley rats (400-450 g).
Hypothalamic tissue corresponding to the ventromedial region (15 mg wet wt) was placed in Gey and Gey medium (1-3 1262
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HYPOTHALAMIC FACTORS AND THE PANCREAS ml) (8) containing 5.5 mM glucose and 0.1% bovine serum albumin and incubated for 30 min at 37 C in an atmosphere of 95% O2-5% C0 2 , as described previously (7). Preparation of extracts Hypothalami were obtained from normal female rats after sacrifice by decapitation. The brain was rapidly frozen on dry ice, and the hypothalamus was removed and freeze-dried. Fifty hypothalami were homogenized in 15 ml acetone-0.01 N HCl (80:20, vol/vol) to a uniform consistency and centrifuged at 2000 X g (J-21, Beckman Instruments, Palo Alto, CA) for 15 min. The supernatant was decanted, and the extraction was repeated on the residue. The two supernatants were pooled and extracted three times with petroleum ether (boiling point 40-60 C). The organic phase was discarded, and the aqueous phase was concentrated by rotary evaporation and was then lyophilized. The residue was dissolved in the appropriate volume of Gey and Gey medium (8) for assay in the pancreatic islet system, as previously described (7), or in 0.2 N acetic acid for gel filtration on columns of Sephadex G-25. In either case, a small amount of insoluble material was removed by centrifugation.
1263
Pancreatic islet system Islets were prepared by collagenase digestion of the male rat pancreas, as previously described (7). Quadruplicate vials containing 10 islets in Gey and Gey medium (1 ml) plus glucose (5.5 mM) and albumin (0.1%) were preincubated for 1 h. This medium was replaced with medium containing the test samples and Trasylol (1000 klU/ml). The incubation was continued at 37 C under O2 (95%)-CO2 (5%) for a further hour, during which aliquots were removed every 20 min for hormone assay. These assay conditions were arrived at on the basis of the results of preliminary experiments in which preincubation time, glucose concentration, and sampling times were varied in order to select a protocol which would permit measurement of effects on insulin and glucagon release in a single assay. Pancreatic hormone assays In all of the above experiments, insulin was assayed using the method of Yalow and Berson (9), as modified by Herbert et al. (10). Glucagon was measured using the RIA procedure and 30K antibody of Faloona et al. (11). The RIAs of neurotensin and substance P content of the column fractions were kindly conducted by Dr. R. Birnbaum in the laboratory of Dr. S. Leeman.
Enzyme experiments Experiments designed to test the effect of various proteolytic enzymes on the agents responsible for insulin release-inhibiting and the glucagon-releasing activities were performed on the hypothalamic incubate and the hypothalamic extract. In separate experiments, carboxypeptidase [35 U/mg; 5 fig; Nutritional Biochemicals Corp. (NBC)], trypsin (10,000 benzoylarginine ethyl ester U/mg; 1 jug; NBC), or chymotrypsin (45 benzoyltyrosine ethyl ester U/mg; 1 /ig; NBC) in Gey and Gey medium (100 /xl) were added to 1 ml incubate or extract. After 2 h at room temperature, Trasylol (1000 klU) and albumin (0.1%) in Gey and Gey medium were added, and the sample was added to the pancreatic islets which had already undergone the 60min preincubation period. Aliquots were removed for RIA of insulin and glucagon after 20, 40, and 60 min. Pepsin (2500 U/ mg; NBC) treatment of the hypothalamic incubate was performed by adding the enzyme solution (1 /ig) to a sample whose pH had been lowered to 2.5 by the addition of 0.01 N HCl. After 2 h at 37 C, the pH was raised to 7.4 with 0.01 N NaOH before the addition of Trasylol and albumin. Throughout these experiments, the appropriate control samples (enzyme alone, incubate or extract alone, and medium extract blank) were treated identically. Gel filtration Lyophilized extract equivalent to 50 hypothalami, prepared as described above, was dissolved in 0.2 N acetic acid and applied to a column (50 X 2 cm; exclusion volume, 68 ml) of Sephadex G-25 which had been previously equilibrated with 0.2 N acetic acid. Aliquots (0.4 ml) from consecutive fractions (4 ml) were pooled in groups throughout the eluate, lyophilized, and dissolved in Gey and Gey medium for testing in the islet system.
Statistical methods Statistical differences between the means of the treatment groups were determined by use of Student's nonpaired t test. In those instances where results were expressed as a percent of control, the difference between means was analyzed using the nonparametric Mann-Whitney U test. Results
Effect of proteolytic enzymes on the VMH activity Figure 1 shows the effect of trypsin (1 jug/ml) and chymotrypsin (1 jug/ml) on the insulin release-inhibiting and glucagon-releasing activities present in the VMH incubate. Those islets incubated in the VMH incubate showed significantly reduced insulin release at 40 (P < 0.001) and 60 min (P < 0.005) compared to those islets incubated in the control medium. Treatment of the VMH incubate with trypsin or chymotrypsin abolished the suppression of insulin release, and there was no significant difference in insulin released from the islets incubated in the control plus enzyme groups and those incubated in the VMH plus enzyme group. In the same experiment, glucagon release from the islets incubated in the VMH medium showed a significant increase (P < 0.0005) at all collection times compared to the control islets. Treatment of the VMH incubate with trypsin had no significant effect on its glucagon-releasing activity. The VMH plus chymotrypsin treatment group, however, showed a significant increase (P < 0.0005) in glucagon release compared to that released from islets treated with the VMH incubate alone. Neither enzyme had any sig-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 10:46 For personal use only. No other uses without permission. . All rights reserved.
Endo • 1979 Vol 105 • No 5
MOLTZ ET AL.
1264
50 |
..
40
VMH » Trypsin VMH • Chymotrysin Control • Trypsin Control • Chymotrysir Control
a. .£ 30 D _C
20 10 40 Incubation Time(min)
0
60
VMH (p
Vol. 105, No. 5 Printed in U.S.A.
Preparation and Properties of Hypothalamic Factors Capable of Altering Pancreatic Hormone Release in Vitro* J. H. MOLTZ,f R. E. DOBBS, S. M. McCANN, AND C. P. FAWCETT Department of Physiology, University of Texas Health Science Center at Dallas, Dallas, Texas 75235
ABSTRACT. Insulin release-inhibiting and glucagon-releasing activities previously detected in media in which rat ventromedial hypothalamic tissue had been incubated have now been detected in acid/acetone extracts of rat hypothalamic tissue. The activities from the two sources have identical responses to treatment with various proteolytic enzymes. They were treated with trypsin, chymotrypsin, carboxypeptidase, or pepsin and then tested in a pancreatic islet system for their ability to inhibit insulin and stimulate glucagon release. The insulin release-inhibiting activity was abolished by chymotrypsin, trypsin, carboxypeptidase, and pepsin treatment, whereas the glucagon-releasing activity was eliminated by carboxypeptidase and pepsin, was unaffected by trypsin, and was enhanced by chymotrypsin. These results
T
indicate not only the peptidic nature of the two activities but also show that both extract and incubate preparations appear to contain the same two peptide agents. Gel filtration on Sephadex G-25 demonstrated that the insulin release-inhibiting and glucagon-releasing activities were associated with two distinct entities. The insulin release-inhibiting activity was eluted soon after the void volume, whereas the glucagon-releasing activity was considerably retarded; each was distinguished from neurotensin and substance P. While no physiological significance can yet be assigned to these two peptides for the control of insulin and glucagon secretion, their existence provides further evidence for a possible neurohormonal pathway in the regulation of the endocrine pancreas. (Endocrinology 105: 1262, 1979)
HE PHYSIOLOGICAL role of the hypothalamus in the control of endocrine function, especially that of the anterior pituitary, has been examined for many years (1). Only recently, however, has the hypothalamus been thought of as a regulatory center for the endocrine pancreas. Several mechanisms have been proposed for the hypothalamic control of insulin and glucagon release. Frohman and coworkers (2) have postulated that the hyperinsulinemia associated with lesions of the ventromedial hypothalamus (VMH) is mediated via a direct neural pathway by way of the vagus nerve and that the inhibition of insulin release in response to the rising glucose levels associated with electrical stimulation of the VMH is due to increased sympathetic tone mediated via adrenal catecholamines (3). Another theory not involving a direct neural pathway has been put forth by Idahl and Martin (4). They explored the possibility that the hyperinsulinemia associated with a lesion in the VMH was due to a humoral factor rather than to the involvement of the autonomic nervous system. An insulin-releasing factor of ventrolateral hypothalamic origin was described by these workers, which led to the hypoth-
esis of a neurohormonal pathway from the hypothalamus to the pancreas (5). While seeking further evidence for the existence of a neurohormonal pathway, we were unable to confirm the existence of an insulin-releasing factor from the ventrolateral hypothalamus but detected insulin release-inhibiting and glucagon-releasing activities in incubates of VMH which were effective both in vivo (6) and in vitro (7). Our experiments were designed to obtain further support for a hypothalamic-pancreatic neurohormonal pathway by defining the chemical nature of these two activities through the use of proteolytic enzymes. Furthermore, it was necessary to show that insulin release-inhibiting and glucagon-releasing activities could be extracted from hypothalamic tissue. We also examined the possibility that these two activities are due to separate entities.
Received June 15, 1978. * This work was supported by NIH Grants AM-10073 and AM21163. f To whom requests for reprints should be addressed.
Preparation of VMH incubate
Materials and Methods The hypothalami used in the preparation of the tissue incubation (VMH) were obtained from large male Sprague-Dawley rats (400-450 g).
Hypothalamic tissue corresponding to the ventromedial region (15 mg wet wt) was placed in Gey and Gey medium (1-3 1262
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 10:46 For personal use only. No other uses without permission. . All rights reserved.
HYPOTHALAMIC FACTORS AND THE PANCREAS ml) (8) containing 5.5 mM glucose and 0.1% bovine serum albumin and incubated for 30 min at 37 C in an atmosphere of 95% O2-5% C0 2 , as described previously (7). Preparation of extracts Hypothalami were obtained from normal female rats after sacrifice by decapitation. The brain was rapidly frozen on dry ice, and the hypothalamus was removed and freeze-dried. Fifty hypothalami were homogenized in 15 ml acetone-0.01 N HCl (80:20, vol/vol) to a uniform consistency and centrifuged at 2000 X g (J-21, Beckman Instruments, Palo Alto, CA) for 15 min. The supernatant was decanted, and the extraction was repeated on the residue. The two supernatants were pooled and extracted three times with petroleum ether (boiling point 40-60 C). The organic phase was discarded, and the aqueous phase was concentrated by rotary evaporation and was then lyophilized. The residue was dissolved in the appropriate volume of Gey and Gey medium (8) for assay in the pancreatic islet system, as previously described (7), or in 0.2 N acetic acid for gel filtration on columns of Sephadex G-25. In either case, a small amount of insoluble material was removed by centrifugation.
1263
Pancreatic islet system Islets were prepared by collagenase digestion of the male rat pancreas, as previously described (7). Quadruplicate vials containing 10 islets in Gey and Gey medium (1 ml) plus glucose (5.5 mM) and albumin (0.1%) were preincubated for 1 h. This medium was replaced with medium containing the test samples and Trasylol (1000 klU/ml). The incubation was continued at 37 C under O2 (95%)-CO2 (5%) for a further hour, during which aliquots were removed every 20 min for hormone assay. These assay conditions were arrived at on the basis of the results of preliminary experiments in which preincubation time, glucose concentration, and sampling times were varied in order to select a protocol which would permit measurement of effects on insulin and glucagon release in a single assay. Pancreatic hormone assays In all of the above experiments, insulin was assayed using the method of Yalow and Berson (9), as modified by Herbert et al. (10). Glucagon was measured using the RIA procedure and 30K antibody of Faloona et al. (11). The RIAs of neurotensin and substance P content of the column fractions were kindly conducted by Dr. R. Birnbaum in the laboratory of Dr. S. Leeman.
Enzyme experiments Experiments designed to test the effect of various proteolytic enzymes on the agents responsible for insulin release-inhibiting and the glucagon-releasing activities were performed on the hypothalamic incubate and the hypothalamic extract. In separate experiments, carboxypeptidase [35 U/mg; 5 fig; Nutritional Biochemicals Corp. (NBC)], trypsin (10,000 benzoylarginine ethyl ester U/mg; 1 jug; NBC), or chymotrypsin (45 benzoyltyrosine ethyl ester U/mg; 1 /ig; NBC) in Gey and Gey medium (100 /xl) were added to 1 ml incubate or extract. After 2 h at room temperature, Trasylol (1000 klU) and albumin (0.1%) in Gey and Gey medium were added, and the sample was added to the pancreatic islets which had already undergone the 60min preincubation period. Aliquots were removed for RIA of insulin and glucagon after 20, 40, and 60 min. Pepsin (2500 U/ mg; NBC) treatment of the hypothalamic incubate was performed by adding the enzyme solution (1 /ig) to a sample whose pH had been lowered to 2.5 by the addition of 0.01 N HCl. After 2 h at 37 C, the pH was raised to 7.4 with 0.01 N NaOH before the addition of Trasylol and albumin. Throughout these experiments, the appropriate control samples (enzyme alone, incubate or extract alone, and medium extract blank) were treated identically. Gel filtration Lyophilized extract equivalent to 50 hypothalami, prepared as described above, was dissolved in 0.2 N acetic acid and applied to a column (50 X 2 cm; exclusion volume, 68 ml) of Sephadex G-25 which had been previously equilibrated with 0.2 N acetic acid. Aliquots (0.4 ml) from consecutive fractions (4 ml) were pooled in groups throughout the eluate, lyophilized, and dissolved in Gey and Gey medium for testing in the islet system.
Statistical methods Statistical differences between the means of the treatment groups were determined by use of Student's nonpaired t test. In those instances where results were expressed as a percent of control, the difference between means was analyzed using the nonparametric Mann-Whitney U test. Results
Effect of proteolytic enzymes on the VMH activity Figure 1 shows the effect of trypsin (1 jug/ml) and chymotrypsin (1 jug/ml) on the insulin release-inhibiting and glucagon-releasing activities present in the VMH incubate. Those islets incubated in the VMH incubate showed significantly reduced insulin release at 40 (P < 0.001) and 60 min (P < 0.005) compared to those islets incubated in the control medium. Treatment of the VMH incubate with trypsin or chymotrypsin abolished the suppression of insulin release, and there was no significant difference in insulin released from the islets incubated in the control plus enzyme groups and those incubated in the VMH plus enzyme group. In the same experiment, glucagon release from the islets incubated in the VMH medium showed a significant increase (P < 0.0005) at all collection times compared to the control islets. Treatment of the VMH incubate with trypsin had no significant effect on its glucagon-releasing activity. The VMH plus chymotrypsin treatment group, however, showed a significant increase (P < 0.0005) in glucagon release compared to that released from islets treated with the VMH incubate alone. Neither enzyme had any sig-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 10:46 For personal use only. No other uses without permission. . All rights reserved.
Endo • 1979 Vol 105 • No 5
MOLTZ ET AL.
1264
50 |
..
40
VMH » Trypsin VMH • Chymotrysin Control • Trypsin Control • Chymotrysir Control
a. .£ 30 D _C
20 10 40 Incubation Time(min)
0
60
VMH (p
