0013-7227/79/1046-1709$02.00/0 Endocrinology Copyright © 1979 by The Endocrine Society
Vol. 104, No. 6 Printed in U.S.A.
Somatostatin: Central Nervous System Actions on Glucoregulation MARVIN BROWN, JEAN RIVIER, AND WYLIE VALE Peptide Biology Laboratory, The Salk Institute, La Jolla, California 92037
hyperglycemia. Other analogs of SRIF and various unrelated peptides were found to be ineffective in reducing bombesininduced hyperglycemia. des-AA1' 2' "• 5> 12' 13-[D-Trp8]SRIF prevented the hyperglycemia induced by surgical stress or by ic administration of /?-endorphin or carbacol. des-AA1' 2p 4> 5l 12' 13[D-Trp8]SRIF given ic did not prevent hyperglycemia induced by systemic administration of epinephrine, arginine, or glucagon. These studies suggest that SRIF and its analogs may act within the brain to affect glucoregulation. (Endocrinology 104: 1709, 1979)
ABSTRACT. Somatostatin (SRIF) has been tested for its actions on the central nervous system to affect glucoregulation. In doses ineffective when given systemically, SRIF and SRIF analogs given intracisternally (ic) reduce hyperglycemia and hyperglucagonemia after ic bombesin administration. The SRIF analog, des-AA1-2> 4> 5> 12> 13-[D-Trp8]SRIF, decreases plasma insulin and elevates plasma glucose and glucagon when given systemically. However, when given ic, this peptide prevents the rise in glucose and glucagon after ic bombesin administration and is 10 times more potent than SRIF in reducing bombesin-induced
T
HE PRESENCE in mammalian brain of oligopeptides possessing a variety of biological effects has led to studies designed to assess what role these peptides might play in brain functions. One of these peptides, somatostatin (SRIF) may potentially modify an animal's ability to regulate carbohydrate metabolism, as determined by a variety of pharmacological studies. SRIF could alter hepatic glucose output by a direct effect on the liver (1-3) or by altering pancreatic insulin and glucagon secretion (4), which in turn would affect hepatic glucose output. SRIF could also alter the pituitary secretion of GH (5) which, secondarily, might affect carbohydrate metabolism. Recently, Unger et al. (6) have suggested that SRIF may play a role in controlling nutrient entry from the gut. Demonstration of the anatomic distribution of SRIF throughout the central nervous system (CNS) and gastrointestinal tract (7) and the observation that there exist multiple potential sites of action of SRIF to affect glucoregulation at extra-CNS sites provide a rationale to question whether there exist glucoregulatory SRIF sites of action within the CNS. Bombesin is a tetradecapeptide originally isolated from the skin of the frog Bombina bombina (8). A peptide with bombesin-like activity has also been found in mammalian intestine (9,10) and brain (11-13), as determined by RIA and immunofluorescence methods. Bombesin has been reported to increase blood pressure; to stimulate Received August 28, 1978. Address requests for reprints to: Dr. Marvin R. Brown, Peptide Biology Laboratory, The Salk Institute, P. O. Box 1809, La Jolla, California 92037.
the secretion of gastric acid, gastrin, and cholecystokinin; to stimulate exocrine pancreatic secretion; and to produce contraction of the gall bladder (14). In addition, we have recently demonstrated that bombesin and bombesin-like peptides cause the central nervous system to lower body temperature (15) and inhibit cold-induced TSH secretion in the rat (16). Bombesin administered intracranially has been demonstrated to promote rapid development of hyperglycemia in rats (17), probably mediated through effects on the CNS. Development of hyperglycemia is dependent on intact adrenal glands, is associated with acute elevation of plasma glucagon and absolute or relative lowering of plasma insulin, and is prevented by systemic administration of SRIF or the a-adrenergic antagonist phentolamine (Brown, M., unpublished observations) (17). Another peptide, /?-endorphin, given intracranially has also been reported to acutely elevate blood glucose (18, 19). We report here studies designed to assess possible actions of SRIF, analogs of SRIF, and various neuropeptides and neurotransmitters that may affect the CNS to influence glucoregulation independently or to modify bombesin-, /?-endorphin-, carbacol-, or stress-induced changes in glucoregulation. Materials and Methods Adult male Sprague-Dawley CD rats, obtained from Charles River, were fed Purina rat chow and water ad libitum. Animals were housed in temperature- and humidity-controlled quarters with 14 h of light (0600-2000 h) and 10 h of dark. Peptides were administered in 10-/U volumes into the cisterna magnum with
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BROWN, RIVIER, AND VALE
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a Hamilton microliter syringe after the lightly ether-anesthetized animals were mounted on the ear bars of a stereotaxic apparatus. Blood samples for determination of glucose, insulin, and glucagon were collected by decapitation. Rectal temperatures were recorded using a Thermoprobe (Yellow Springs Instrument Co., Yellow Springs, OH). Peptides were synthesized using solid phase methodology (20, 21) and were dissolved in artificial cerebrospinal fluid by methods previously described (15). Plasma glucose concentrations were determined by the glucose oxidase method using a Beckman glucose analyzer (Beckman Instrument Co., Palo Alto, CA). Plasma insulin and glucagon were determined by RIAs previously described (22). 30K Glucagon antiserum was a gift from Dr. Roger Unger (V.A. Hospital, Dallas, TX). Glucagon standard and rat insulin were kindly donated by the Eli Lilly Co. (Indianapolis, IN). 125ILabeled insulin was obtained from New England Nuclear Corp. (Boston, MA) and 125I-labeled glucagon was obtained from Nuclear Medical Laboratories, (Dallas, TX). All experiments were carried out in randomized block design with five animals per group. After analyses of variance, differences between treatment groups were determined by the multiple range tests of Dunnett and Duncan using the computer program EXBIOL. Potency estimates were determined from data obtained from four- and six-point bioassays using the computer program HUBA.
Endo • 1979 Vol 104 • No 6
from the brain and exert an effect on the endocrine pancreas to inhibit the secretion of glucagon, an analog of SRIF devoid of pancreatic a-cell (glucagon inhibitory) activity was used (23). This analog of SRIF, des-AA1'2> 4> 5'12- 13-[D-Trp8]SRIF, was markedly more potent than SRIF in reducing bombesin-induced hyperglycemia (Fig. 1). Neither SRIF nor des-AA1' 2l 4l 5> n' 13-[D-Trp8]SRIF alone significantly altered plasma glucose in this experiment. Similar doses of these peptides given systemically did not affect bombesin-induced hyperglycemia. Figure 2 demonstrates the specificity of des-AA1'2> 4> 5> 12> 13 -[D-Trp8]SRIF within the brain to prevent bombesininduced hyperglycemia. In panel A, bombesin was administered ic while 100 jug [D-Trp8]SRIF (a SRIF analog more potent than SRIF in inhibiting the secretion of GH, insulin, and glucagon) and 100 fig des-AA1' 2' 4> 5> 12> 13 -[D-
Trp8]SRIF were given sc. As we have previously reported, des-AA1' 2> 4> 5- 12- 13-[D-Trp8]SRIF and other insulin-selective analogs, when given systemically, produce hyperglycemia resulting from the lowering of plasma insulin and the elevation of plasma glucagon (23). Bombesin-induced hyperglycemia is prevented by systemic administration of [D-Trp8]SRIF, probably because
Results and Discussion Figure 1 demonstrates hyperglycemia 60 min after intracisternal (ic) administration of 100 ng bombesin, and attenuation of this hyperglycemia by coadministration of SRIF. Higher doses of SRIF given systemically transiently reduce hyperglycemia mediated by bombesin, probably by inhibition of pancreatic glucagon secretion (data not shown). To eliminate the possibility that SRIF given ic might prevent bombesin-induced hyperglycemia by leakage
300 (f) O O
z>
_J
400
C9
250 -
if)
4> 5| 12> 13 -[DTrp8]SRIF given systemically accentuated the hyperglycemic effects of bombesin. Figure 2B demonstrates a comparison of the effects of [D-Trp8]SRIF and des-AA1' 2,4.5> i2, i3.j- D . T r p 8j S R I F £yen i c m i n fl u e n c i n g bombesininduced hyperglycemia. One microgram of [D-Trp8]SRIF administered ic reduced bombesin-induced hyperglycemia; this was similar to its effect when given sc. In contrast to its effect when given sc, 1 /xg des-AA1'2> 4> 5> 12> 13 -[D-Trp8]SRIF administered ic prevented bombesin-induced hyperglycemia. The low dose requirement of SRIF and its analogs and the specific effect of des-AA1'2l 4l 5> 12> 13 -[D-Trp8]SRIF of reversing bombesin-induced hyperglycemia when given ic strongly support the hypothesis that SRIF exerts actions within the brain to prevent hyperglycemia secondary to ic bombesin administration. Analogs of SRIF found inactive in other SRIF bioassay systems were also demonstrated to be ineffective in preventing bombesin-induced hyperglycemia (Table 1). [DPhe6]SRIF, des-Trp8-SRIF, and [D-Pheu]SRIF (all given ic) show low potencies in inhibiting bombesin-induced hyperglycemia as well as in inhibiting pituitary secretion of GH in vitro and pancreatic secretion of glucagon and insulin in vivo. Enhanced potency of des-AA1'2> 4> 5i 12> 13SRIF, des-AA1'2-4- 5- 12' 13-[D-Trp8]SRIF, and des-AA1'2' 4, 5. i2, i3.|- D . Trp 8 j D _c ys 14 ]SRIF to affect bombesin-induced hyperglycemia compared to their actions on GH, insulin, and glucagon could be explained by the increased penetration and. distribution of these peptides into the
brain due to their enhanced lipophilic properties or reduced size. Other neuropeptides, angiotensin II, bradykinin, CLIP, LHRH, a-MSH, substance P, and TRF, did not significantly alter bombesin-induced hyperglycemia. Bombesin given ic has previously been reported to produce hyperglycemia associated with suppression of plasma insulin and elevation of plasma glucagon (17). Intracisternal administration of des-AA1' 2> 4> 5l 12> 13 -[DTrp8]SRIF prevented bombesin-induced hyperglycemia and hyperglucagonemia (Fig. 3). In this experiment, bombesin did not produce an absolute reduction of plasma insulin; however, plasma insulin levels did not rise in a fashion appropriate for the blood glucose concentration, thereby suggesting relative inhibition of plasma insulin secretion. Figure 3 also demonstrates the ability of bombesin to inhibit the rise of plasma insulin produced by
TABLE 1. Potency relation to SRIF of several peptides
Peptide
SRIF [D-Phe6]SRIF [D-Trp8]SRIF (DT8» SRIF) des-Trp8-SRIF [D-Phe u ]SRIF des-AA'-2-4'512'13-SR,IF des-AA 1 ' 2 ' 451213 -[D-Trp 8 ]SRIF (0DT8-SRIF) des-AA 1 ' 2 4 5 I 2 1 3 -[D-
Prevent bombesininduced hyperglycemia 100 l2 ' l3 [D-Trp8]SRIF (1 jug). **, P < 0.01 compared to control.
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the ic administration of des-AA1-2> 4> 5-12> 13-[D-Trp8]SRIF, and although the mechanism by which these various changes in plasma insulin, glucagon, and glucose occur remains to be seen, bombesin-induced changes in insulin, glucagon, and glucose depend on an intact adrenal gland (17). It has been suggested that bombesin's actions on the CNS to promote development of hyperglycemia is secondary to increased sympathetic adrenal outflow, resulting in increased catecholamine release, which elevates plasma glucagon and reduces plasma insulin (17). SRIF could theoretically interrupt this pathway within the CNS or stimulate efferent sympathetic or parasympathetic pathways, altering the adrenal, pancreas, or liver directly. Demonstration of the action of SRIF to prevent bombesin-induced hyperglycemia led to the question of whether SRIF or its analogs might prevent hyperglycemia due to other types of stimuli. /?-Endorphin and /Mipotropin6i-9i (10 jug) given ic elevate plasma glucose (Fig. 4). Similar to bombesin, this hyperglycemia requires an intact adrenal (Brown, M., unpublished observations). This rise in plasma glucose is prevented by ic administration of 1 jug des-AA1'2> 4l 5l 12> 13[D-Trp8]SRIF (Fig. 4). Bombesin and /?-endorphin, although not structurally similar, share several common CNS actions, such as production of hypothermia, hyper-
250 -
o VEHICLE
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glycemia, and analgesia. In addition, bombesin-induced hypothermia is partially reversible by the opiate receptor antagonist naloxone. Since neither opiate peptides (24) nor bombesin (Lazarus, L., personal communication) interfere in specific synaptosomal receptor assays for each other, it is unlikely that these peptides directly exert their actions via a single receptor. While SRIF reverses bombesin-induced hyperglycemia and hypothermia, SRIF reverses only the hyperglycemia and not the hypothermia induced by /?-endorphin, thus suggesting that the actions of these peptides may be pharmacologically dissociable (Brown, M., unpublished observations). In addition, /8-endorphin-induced hyperglycemia is reversed by the opiate receptor antagonist naloxone, while bombesin-induced hyperglycemia is not (Brown, M., unpublished observations). Hyperglycemia after ether anesthesia and placement of a jugular venous catheter and reduction of this rise in plasma glucose by des-AA1- % 4-5- 12- 13-[D-Trp8]SRIF are demonstrated in Fig. 5. Such data provide the rationale to question the possibility that SRIF might modulate CNS pathways involved in physiological glucoregulation. In the course of studies designed to evaluate the role of various neurotransmitters in the action of peptide modification of glucoregulation, carbacol given ic was noted to be potent in elevating plasma glucose. Similar to bombesin and /8-endorphin, carbacol given ic does not produce hyperglycemia in the absence of intact adrenal glands (Brown, M., unpublished observations). Figure 6 demonstrates the rise in plasma glucose 60 min after ic administration of various doses of carbacol and the prevention of this hyperglycemia by ic administration of atropine and des-AA1-2-4) 5*12> 13-[D-Trp8]SRIF. The time course of development of carbacol-induced hyperglycemia is shown in Fig. 7. Carbacol appears to produce an
0DT8-SS /9-ENDOR.
CONTROL /9-ENDOR. + 0DT8-SS
100
0DT8-SS
10
20 30 40 50 TIME (minutes) ETHER + CATHETER PLACEMENT
°t
60
FIG. 4. Changes in plasma glucose levels in rats after ether anesthesia and placement of an indwelling jugular vein catheter. This rise in plasma glucose is prevented by des-AA'12> 4> 5'12> 13-[D-Trp8]SRIF (1 jug) given ic immediately after etherization. *, P < 0.05; **, P < 0.01 (compared to zero time sample).
120k 10 15 TIME (min.)
20
FIG. 5. Intracisternally administered /8-endorphin (10 fig) produces hyperglycemia that is reversed by des-AA1-2'4-6-l2> l3-[D-Trp8]SRIF (1
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SRIF: CNS ACTIONS ON GLUCOREGULATION 300
r
0 CARBACOL
200 P 4l 5> 12' 13[D-Trp8]SRIF given ic (Fig. 8). The rise in plasma glucagon after sc administration of epinephrine, des-AA1' 2l 4,5, i2, ^.[o.Trp^SRIF, and arginine is also not altered by ic administration of des-AA1' 2' 4' 5' 12- 13-[D-Trp8]SRIF. These results suggest that SRIF CNS actions to influence glucoregulation do not significantly affect glucogenic actions of substances that act directly to affect pancreatic insulin and glucagon secretion or hepatic glucose production. Thus, these results are consistent with the idea of des-AA1'2'4'5> 12> 13-[D-Trp8]SRIF acting within the brain to interrupt efferent signals which results in the change of pancreatic insulin and glucagon secretion and hepatic glucose production. Certainly, a likely mechanism is that SRIF may decrease adrenal catecholamine production via a CNS action. Acute elevation of blood glucose levels after various forms of stress may be regulated in part by the CNS. CNS-mediated elevation of blood glucose or enhancement of glucose production may be of adaptive importance to insure that the critical dependence of brain metabolism on the presence of adequate glucose (availability) is fulfilled. The piqure experiments of Bernard first demonstrated brain influences on glucoregulation (25), although the mechanisms by which this puncture of the floor of the fourth ventricle results in hyperglycemia TABLE 2. Effects of atropine on bombesin- and carbacol-induced hyperglycemia Treatment Control Bombesin (100 ng) Carbacol (10 jug) Atropine 10 /ig 100 ng Bombesin + atropine l/*g 10 M g 100 jug
Plasma glucose (mg/dl) 160 ± 8 252 ± 27 346 ± 33 170 ± 12 164 ± 6 159 ± 11 250 ± 15 264 ± 23 255 ± 15
Carbacol + atropine l/*g 10 fig 100 jug
194 ± 8 172 ± 8 177 ± 10
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BROWN, RIVIER, AND VALE
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300UJ 12> 13-[D-Trp8]SRIF given via the same route prevents the rise in plasma epinephrine produced by bombesin (30). We conclude that bombesin acts within the brain to increase sympathetic outflow and increase adrenomedullary epinephrine secretion, which in turn lowers plasma insulin and elevates plasma glucagon, resulting in elevation of plasma glucose. SRIF or des-AA1 • 2,4,5,12,13_[D.Trp8]SRIF prevents this hyperglycemia via a CNS action to prevent adrenomedullary epinephrine secretion. We speculate that the hyperglycemia after surgical stress or after the administration of carbacol or /8-endorphin, as described in the paper, is prevented by des-AA1'2l 4-5l 12> 13-[D-Trp8]SRIF acting within the CNS to decrease adrenomedullary epinephrine secretion.
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Acknowledgments We wish to thank Roberta Alien, Alice Heinig, Ginny Page, Karen von Dessonneck, Ron Kaiser, and Robert Galyean for their excellent technical assistance and Laurie Taylor and Sue Hebert for the editing and typing of this paper.
150-
D
CONTROL
| _ 12
0DT8-SS
5 12> 13
FIG. 8. Effect of ic administration of des-AA ' ' "• ' -[D-Trp8]SRIF (1 fig) development of hyperglycemia after administration of 100 ng bombesin given ic; epinephrine (20 fig), des-AA1'% "' 5| 12> 13-[D-Trp8]SRIF (100 fig), and arginine (200 mg) given sc; and glucagon (10 fig) given iv. Blood samples were collected 30 min after treatments.
remains undetermined. Stimulation of various hypothalamic areas results in the modification of blood levels of glucose, insulin, and glucagon. Stimulation of the ventrolateral hypothalamus facilitates insulin secretion and lowers blood glucose (26), while stimulation of the ventromedial hypothalamus inhibits insulin secretion and elevates plasma levels of glucagon and glucose (27). These changes may result from alteration in hypothalamic sympathetic and parasympathetic outflow, which has been suggested to directly alter the secretion of insulin and glucagon by the endocrine pancreas (28). Alternatively, direct sympathetic innervation of the liver may potentially influence hepatic glucose production (29). Although acute changes in blood glucose, insulin, and glucagon after various types of stress, exercise, and starvation could be mediated by glucoregulatory pathways emerging from the brain, the role that the CNS might play in physiological regulation of glucose metabolism remains undefined. Whether bombesin, /?-endorphin, or SRIF are physiologically involved in or activate pathways which might mediate some of these mechanically, electrically, or phys-
References 1. Oliver, J. R., and S. R. Wagle, Studies on the inhibition of insulin release, glycogenolysis and gluconiagenesis by somatostatin in the rat islet of Langerhans in isolated hepatocytes, Biochem Biophys Res Commun 62: 772, 1975. 2. Sacks, H., K. Waligora, J. Matthews, and B. Pimstone, Inhibition by somatostatin of glucagon induced glucose release from the isolated perfused rat liver, Diabetes 26: 22,1977 (Abstract). 3. Sacca, L., and R. Sherwin, Somatostatin (SRIF) alters sensitivity to glucagon and epinephrine independent of insulin and glucagon availability, Diabetes 26: 23, 1977 (Abstract). 4. Brown, M., and W. Vale, Somatostatin: five years of progress, Biomedicine 28: 93, 1978. 5. Pfeiffer, E. F., C. Missner, W. Beischer, W. Kerner, and S. Raptis, The anti-diabetic action of somatostatin assessed by the artificial 0-cell, Metabolism (Suppl 1) 27: 1415, 1978. 6. Unger, R. H., E. Ipp, V. Schusdziarra, L. Orci, Hypothesis: physiologic role of pancreatic somatostatin and the contribution of D-cell disorders to diabetes mellitus, Life Sci 20: 2081,1977. 7. Vale, W., C. Rivier, and M. Brown, Regulatory peptides of the hypothalamus, Annu Rev Physiol 29: 473, 1977. 8. Anastasi, A., V. Erspamer, and M. Bucci, Isolation and structure of bombesin and alytesin, two analogous active peptides from the skin of the European amphibians Bombina and Alytes, Experientia 27: 166, 1971. 9. Polak, J. M., R. Hobbs, S. R. Bloom, E. Solcia, and A. G. E. Pearse, Distribution of a bombesin-like peptide in human gastrointestinal tract, Lancet 2: 1109, 1976. 10. Walsh, J. H., and A. L. Holmquist, Radioimmunoassay of bombesin peptides: identification of bombesin-like immunoreactivity in vertebrate gut extracts, Gastroenterology 70: 948, 1976 (Abstract 90). 11. Brown, M., J. Rivier, R. Kobayashi, and W. Vale, Neurotensin-like and bombesin-like peptides: CNS distribution and actions, In Bloom, S. R. (ed.), Gut Hormones, Churchill-Livingstone, Edinburgh, 1978, pp. 550-558. 12. Villarreal, J., J. Rivier, and M. Brown, Evidence for a bombesin-
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SRIF: CNS ACTIONS ON GLUCOREGULATION
13. 14.
15. 16. 17. 18. 19. 20. 21.
like substance in hypothalamus: chemical and immune-logical characterization, Endocrinology 102: 390, 1978 (Abstract). Polak, J. M., A. E. Bishop, M. A. Ghatei, S. R. Bloom, and M. Brown, Bombesin-Like Immunoreactivity in the Rat Brain, The Endocrine Society, United Kingdom, in press. Erspamer, V., and P. Melchiorri, Actions of bombesin on secretions and motility of the gastrointestinal tract, In Thompson, J. C. (ed.), Gastrointestinal Hormones, University of Texas Press, Austin, 1975, pp. 575-589. Brown, M., J. Rivier, and W. Vale, Bombesin: potent effects on thermoregulation in the rat, Science 196: 998, 1977. Brown, M., J. Rivier, A. Wolfe, and W. Vale, TRF and bombesin: actions on thermoregulation and TSH secretion in rats, Endocrinology 100: 279, 1977 (Abstract). Brown, M., J. Rivier, and W. Vale, Bombesin affects the central nervous system to produce hyperglycemia in rats, Life Sci 21: 1729, 1977. Feldberg, W., Pharmacology of the central actions of endorphins, In Bloom, S. R. (ed.), Gut Hormones, Churchill-Livingstone, Edinburgh, 1978, p. 495. Brown, M., and W. Vale, Somatostatin (SS): central nervous system (CNS) actions on glucose and temperature regulation, Endocrinology 102: 779, 1978 (Abstract). Rivier, J., Somatostatin. Total solid phase synthesis, J Am Chem Soc 96: 2986, 1974. Rivier, J., and M. Brown, Bombesin, bombesin analogs and related peptides: effects on thermoregulation, Biochemistry 17: 1766,1978.
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22. Brown, M., J. Rivier, and W. Vale, Biological activity of somatostatin and somatostatin analogs on inhibition of arginine-induced insulin and glucagon release in the rat, Endocrinology 98: 336, 1976. 23. Brown, M., W. Vale, and J. Rivier, Insulin selective somatostatin (SS) analogs, Diabetes (Suppl 1) 26: 29, 1977. 24. Moody, T., C. Pert, J. Rivier, and M. Brown, Bombesin: specific binding to rat brain membranes, Proc Natl Acad Sci USA 75: 5372, 1978. 25. Bernard, C, Chiens rendus diabetques, C R Soc Biol (Paris) 1: 60, 1849. ' 26. Steffens, A. B., G. J. Mogenson, and J. A. F. Stevenson, Blood glucose, insulin, and free fatty acids after stimulation and lesions of the hypothalamus, Am J Physiol 222: 1446, 1972. 27. Frohmann, L., and L. Bernardis, Effects of hypothalamic stimulation on plasma glucose, insulin and glucagon levels, Am J Physiol 221: 1596, 1971. 28. Woods, S. C, and D. Porte, Neurocontrol of the endocrine pancreas, Physiol Rev 54: 596, 1974. 29. Shimazu, R., and S. Ogasawara, Effects of hypothalamic stimulation on gluconeogenesis and glycolysis in rat liver, Am J Physiol 228: 1787, 1975. 30. Brown, M., and D. Fisher, Central nervous system influence on glucoregulation, Clin Res 27: 84, 1979 (Abstract). 31. Vale, W., J. Rivier, N. Ling, and M. Brown, Biological and immunological activities and applications of somatostatin analogs, Metabolism 27: 1391, 1978 (Abstract 9).
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Vol. 104, No. 6 Printed in U.S.A.
Somatostatin: Central Nervous System Actions on Glucoregulation MARVIN BROWN, JEAN RIVIER, AND WYLIE VALE Peptide Biology Laboratory, The Salk Institute, La Jolla, California 92037
hyperglycemia. Other analogs of SRIF and various unrelated peptides were found to be ineffective in reducing bombesininduced hyperglycemia. des-AA1' 2' "• 5> 12' 13-[D-Trp8]SRIF prevented the hyperglycemia induced by surgical stress or by ic administration of /?-endorphin or carbacol. des-AA1' 2p 4> 5l 12' 13[D-Trp8]SRIF given ic did not prevent hyperglycemia induced by systemic administration of epinephrine, arginine, or glucagon. These studies suggest that SRIF and its analogs may act within the brain to affect glucoregulation. (Endocrinology 104: 1709, 1979)
ABSTRACT. Somatostatin (SRIF) has been tested for its actions on the central nervous system to affect glucoregulation. In doses ineffective when given systemically, SRIF and SRIF analogs given intracisternally (ic) reduce hyperglycemia and hyperglucagonemia after ic bombesin administration. The SRIF analog, des-AA1-2> 4> 5> 12> 13-[D-Trp8]SRIF, decreases plasma insulin and elevates plasma glucose and glucagon when given systemically. However, when given ic, this peptide prevents the rise in glucose and glucagon after ic bombesin administration and is 10 times more potent than SRIF in reducing bombesin-induced
T
HE PRESENCE in mammalian brain of oligopeptides possessing a variety of biological effects has led to studies designed to assess what role these peptides might play in brain functions. One of these peptides, somatostatin (SRIF) may potentially modify an animal's ability to regulate carbohydrate metabolism, as determined by a variety of pharmacological studies. SRIF could alter hepatic glucose output by a direct effect on the liver (1-3) or by altering pancreatic insulin and glucagon secretion (4), which in turn would affect hepatic glucose output. SRIF could also alter the pituitary secretion of GH (5) which, secondarily, might affect carbohydrate metabolism. Recently, Unger et al. (6) have suggested that SRIF may play a role in controlling nutrient entry from the gut. Demonstration of the anatomic distribution of SRIF throughout the central nervous system (CNS) and gastrointestinal tract (7) and the observation that there exist multiple potential sites of action of SRIF to affect glucoregulation at extra-CNS sites provide a rationale to question whether there exist glucoregulatory SRIF sites of action within the CNS. Bombesin is a tetradecapeptide originally isolated from the skin of the frog Bombina bombina (8). A peptide with bombesin-like activity has also been found in mammalian intestine (9,10) and brain (11-13), as determined by RIA and immunofluorescence methods. Bombesin has been reported to increase blood pressure; to stimulate Received August 28, 1978. Address requests for reprints to: Dr. Marvin R. Brown, Peptide Biology Laboratory, The Salk Institute, P. O. Box 1809, La Jolla, California 92037.
the secretion of gastric acid, gastrin, and cholecystokinin; to stimulate exocrine pancreatic secretion; and to produce contraction of the gall bladder (14). In addition, we have recently demonstrated that bombesin and bombesin-like peptides cause the central nervous system to lower body temperature (15) and inhibit cold-induced TSH secretion in the rat (16). Bombesin administered intracranially has been demonstrated to promote rapid development of hyperglycemia in rats (17), probably mediated through effects on the CNS. Development of hyperglycemia is dependent on intact adrenal glands, is associated with acute elevation of plasma glucagon and absolute or relative lowering of plasma insulin, and is prevented by systemic administration of SRIF or the a-adrenergic antagonist phentolamine (Brown, M., unpublished observations) (17). Another peptide, /?-endorphin, given intracranially has also been reported to acutely elevate blood glucose (18, 19). We report here studies designed to assess possible actions of SRIF, analogs of SRIF, and various neuropeptides and neurotransmitters that may affect the CNS to influence glucoregulation independently or to modify bombesin-, /?-endorphin-, carbacol-, or stress-induced changes in glucoregulation. Materials and Methods Adult male Sprague-Dawley CD rats, obtained from Charles River, were fed Purina rat chow and water ad libitum. Animals were housed in temperature- and humidity-controlled quarters with 14 h of light (0600-2000 h) and 10 h of dark. Peptides were administered in 10-/U volumes into the cisterna magnum with
1709
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BROWN, RIVIER, AND VALE
1710
a Hamilton microliter syringe after the lightly ether-anesthetized animals were mounted on the ear bars of a stereotaxic apparatus. Blood samples for determination of glucose, insulin, and glucagon were collected by decapitation. Rectal temperatures were recorded using a Thermoprobe (Yellow Springs Instrument Co., Yellow Springs, OH). Peptides were synthesized using solid phase methodology (20, 21) and were dissolved in artificial cerebrospinal fluid by methods previously described (15). Plasma glucose concentrations were determined by the glucose oxidase method using a Beckman glucose analyzer (Beckman Instrument Co., Palo Alto, CA). Plasma insulin and glucagon were determined by RIAs previously described (22). 30K Glucagon antiserum was a gift from Dr. Roger Unger (V.A. Hospital, Dallas, TX). Glucagon standard and rat insulin were kindly donated by the Eli Lilly Co. (Indianapolis, IN). 125ILabeled insulin was obtained from New England Nuclear Corp. (Boston, MA) and 125I-labeled glucagon was obtained from Nuclear Medical Laboratories, (Dallas, TX). All experiments were carried out in randomized block design with five animals per group. After analyses of variance, differences between treatment groups were determined by the multiple range tests of Dunnett and Duncan using the computer program EXBIOL. Potency estimates were determined from data obtained from four- and six-point bioassays using the computer program HUBA.
Endo • 1979 Vol 104 • No 6
from the brain and exert an effect on the endocrine pancreas to inhibit the secretion of glucagon, an analog of SRIF devoid of pancreatic a-cell (glucagon inhibitory) activity was used (23). This analog of SRIF, des-AA1'2> 4> 5'12- 13-[D-Trp8]SRIF, was markedly more potent than SRIF in reducing bombesin-induced hyperglycemia (Fig. 1). Neither SRIF nor des-AA1' 2l 4l 5> n' 13-[D-Trp8]SRIF alone significantly altered plasma glucose in this experiment. Similar doses of these peptides given systemically did not affect bombesin-induced hyperglycemia. Figure 2 demonstrates the specificity of des-AA1'2> 4> 5> 12> 13 -[D-Trp8]SRIF within the brain to prevent bombesininduced hyperglycemia. In panel A, bombesin was administered ic while 100 jug [D-Trp8]SRIF (a SRIF analog more potent than SRIF in inhibiting the secretion of GH, insulin, and glucagon) and 100 fig des-AA1' 2' 4> 5> 12> 13 -[D-
Trp8]SRIF were given sc. As we have previously reported, des-AA1' 2> 4> 5- 12- 13-[D-Trp8]SRIF and other insulin-selective analogs, when given systemically, produce hyperglycemia resulting from the lowering of plasma insulin and the elevation of plasma glucagon (23). Bombesin-induced hyperglycemia is prevented by systemic administration of [D-Trp8]SRIF, probably because
Results and Discussion Figure 1 demonstrates hyperglycemia 60 min after intracisternal (ic) administration of 100 ng bombesin, and attenuation of this hyperglycemia by coadministration of SRIF. Higher doses of SRIF given systemically transiently reduce hyperglycemia mediated by bombesin, probably by inhibition of pancreatic glucagon secretion (data not shown). To eliminate the possibility that SRIF given ic might prevent bombesin-induced hyperglycemia by leakage
300 (f) O O
z>
_J
400
C9
250 -
if)
4> 5| 12> 13 -[DTrp8]SRIF given systemically accentuated the hyperglycemic effects of bombesin. Figure 2B demonstrates a comparison of the effects of [D-Trp8]SRIF and des-AA1' 2,4.5> i2, i3.j- D . T r p 8j S R I F £yen i c m i n fl u e n c i n g bombesininduced hyperglycemia. One microgram of [D-Trp8]SRIF administered ic reduced bombesin-induced hyperglycemia; this was similar to its effect when given sc. In contrast to its effect when given sc, 1 /xg des-AA1'2> 4> 5> 12> 13 -[D-Trp8]SRIF administered ic prevented bombesin-induced hyperglycemia. The low dose requirement of SRIF and its analogs and the specific effect of des-AA1'2l 4l 5> 12> 13 -[D-Trp8]SRIF of reversing bombesin-induced hyperglycemia when given ic strongly support the hypothesis that SRIF exerts actions within the brain to prevent hyperglycemia secondary to ic bombesin administration. Analogs of SRIF found inactive in other SRIF bioassay systems were also demonstrated to be ineffective in preventing bombesin-induced hyperglycemia (Table 1). [DPhe6]SRIF, des-Trp8-SRIF, and [D-Pheu]SRIF (all given ic) show low potencies in inhibiting bombesin-induced hyperglycemia as well as in inhibiting pituitary secretion of GH in vitro and pancreatic secretion of glucagon and insulin in vivo. Enhanced potency of des-AA1'2> 4> 5i 12> 13SRIF, des-AA1'2-4- 5- 12' 13-[D-Trp8]SRIF, and des-AA1'2' 4, 5. i2, i3.|- D . Trp 8 j D _c ys 14 ]SRIF to affect bombesin-induced hyperglycemia compared to their actions on GH, insulin, and glucagon could be explained by the increased penetration and. distribution of these peptides into the
brain due to their enhanced lipophilic properties or reduced size. Other neuropeptides, angiotensin II, bradykinin, CLIP, LHRH, a-MSH, substance P, and TRF, did not significantly alter bombesin-induced hyperglycemia. Bombesin given ic has previously been reported to produce hyperglycemia associated with suppression of plasma insulin and elevation of plasma glucagon (17). Intracisternal administration of des-AA1' 2> 4> 5l 12> 13 -[DTrp8]SRIF prevented bombesin-induced hyperglycemia and hyperglucagonemia (Fig. 3). In this experiment, bombesin did not produce an absolute reduction of plasma insulin; however, plasma insulin levels did not rise in a fashion appropriate for the blood glucose concentration, thereby suggesting relative inhibition of plasma insulin secretion. Figure 3 also demonstrates the ability of bombesin to inhibit the rise of plasma insulin produced by
TABLE 1. Potency relation to SRIF of several peptides
Peptide
SRIF [D-Phe6]SRIF [D-Trp8]SRIF (DT8» SRIF) des-Trp8-SRIF [D-Phe u ]SRIF des-AA'-2-4'512'13-SR,IF des-AA 1 ' 2 ' 451213 -[D-Trp 8 ]SRIF (0DT8-SRIF) des-AA 1 ' 2 4 5 I 2 1 3 -[D-
Prevent bombesininduced hyperglycemia 100 l2 ' l3 [D-Trp8]SRIF (1 jug). **, P < 0.01 compared to control.
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BROWN, RIVIER, AND VALE
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the ic administration of des-AA1-2> 4> 5-12> 13-[D-Trp8]SRIF, and although the mechanism by which these various changes in plasma insulin, glucagon, and glucose occur remains to be seen, bombesin-induced changes in insulin, glucagon, and glucose depend on an intact adrenal gland (17). It has been suggested that bombesin's actions on the CNS to promote development of hyperglycemia is secondary to increased sympathetic adrenal outflow, resulting in increased catecholamine release, which elevates plasma glucagon and reduces plasma insulin (17). SRIF could theoretically interrupt this pathway within the CNS or stimulate efferent sympathetic or parasympathetic pathways, altering the adrenal, pancreas, or liver directly. Demonstration of the action of SRIF to prevent bombesin-induced hyperglycemia led to the question of whether SRIF or its analogs might prevent hyperglycemia due to other types of stimuli. /?-Endorphin and /Mipotropin6i-9i (10 jug) given ic elevate plasma glucose (Fig. 4). Similar to bombesin, this hyperglycemia requires an intact adrenal (Brown, M., unpublished observations). This rise in plasma glucose is prevented by ic administration of 1 jug des-AA1'2> 4l 5l 12> 13[D-Trp8]SRIF (Fig. 4). Bombesin and /?-endorphin, although not structurally similar, share several common CNS actions, such as production of hypothermia, hyper-
250 -
o VEHICLE
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glycemia, and analgesia. In addition, bombesin-induced hypothermia is partially reversible by the opiate receptor antagonist naloxone. Since neither opiate peptides (24) nor bombesin (Lazarus, L., personal communication) interfere in specific synaptosomal receptor assays for each other, it is unlikely that these peptides directly exert their actions via a single receptor. While SRIF reverses bombesin-induced hyperglycemia and hypothermia, SRIF reverses only the hyperglycemia and not the hypothermia induced by /?-endorphin, thus suggesting that the actions of these peptides may be pharmacologically dissociable (Brown, M., unpublished observations). In addition, /8-endorphin-induced hyperglycemia is reversed by the opiate receptor antagonist naloxone, while bombesin-induced hyperglycemia is not (Brown, M., unpublished observations). Hyperglycemia after ether anesthesia and placement of a jugular venous catheter and reduction of this rise in plasma glucose by des-AA1- % 4-5- 12- 13-[D-Trp8]SRIF are demonstrated in Fig. 5. Such data provide the rationale to question the possibility that SRIF might modulate CNS pathways involved in physiological glucoregulation. In the course of studies designed to evaluate the role of various neurotransmitters in the action of peptide modification of glucoregulation, carbacol given ic was noted to be potent in elevating plasma glucose. Similar to bombesin and /8-endorphin, carbacol given ic does not produce hyperglycemia in the absence of intact adrenal glands (Brown, M., unpublished observations). Figure 6 demonstrates the rise in plasma glucose 60 min after ic administration of various doses of carbacol and the prevention of this hyperglycemia by ic administration of atropine and des-AA1-2-4) 5*12> 13-[D-Trp8]SRIF. The time course of development of carbacol-induced hyperglycemia is shown in Fig. 7. Carbacol appears to produce an
0DT8-SS /9-ENDOR.
CONTROL /9-ENDOR. + 0DT8-SS
100
0DT8-SS
10
20 30 40 50 TIME (minutes) ETHER + CATHETER PLACEMENT
°t
60
FIG. 4. Changes in plasma glucose levels in rats after ether anesthesia and placement of an indwelling jugular vein catheter. This rise in plasma glucose is prevented by des-AA'12> 4> 5'12> 13-[D-Trp8]SRIF (1 jug) given ic immediately after etherization. *, P < 0.05; **, P < 0.01 (compared to zero time sample).
120k 10 15 TIME (min.)
20
FIG. 5. Intracisternally administered /8-endorphin (10 fig) produces hyperglycemia that is reversed by des-AA1-2'4-6-l2> l3-[D-Trp8]SRIF (1
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SRIF: CNS ACTIONS ON GLUCOREGULATION 300
r
0 CARBACOL
200 P 4l 5> 12' 13[D-Trp8]SRIF given ic (Fig. 8). The rise in plasma glucagon after sc administration of epinephrine, des-AA1' 2l 4,5, i2, ^.[o.Trp^SRIF, and arginine is also not altered by ic administration of des-AA1' 2' 4' 5' 12- 13-[D-Trp8]SRIF. These results suggest that SRIF CNS actions to influence glucoregulation do not significantly affect glucogenic actions of substances that act directly to affect pancreatic insulin and glucagon secretion or hepatic glucose production. Thus, these results are consistent with the idea of des-AA1'2'4'5> 12> 13-[D-Trp8]SRIF acting within the brain to interrupt efferent signals which results in the change of pancreatic insulin and glucagon secretion and hepatic glucose production. Certainly, a likely mechanism is that SRIF may decrease adrenal catecholamine production via a CNS action. Acute elevation of blood glucose levels after various forms of stress may be regulated in part by the CNS. CNS-mediated elevation of blood glucose or enhancement of glucose production may be of adaptive importance to insure that the critical dependence of brain metabolism on the presence of adequate glucose (availability) is fulfilled. The piqure experiments of Bernard first demonstrated brain influences on glucoregulation (25), although the mechanisms by which this puncture of the floor of the fourth ventricle results in hyperglycemia TABLE 2. Effects of atropine on bombesin- and carbacol-induced hyperglycemia Treatment Control Bombesin (100 ng) Carbacol (10 jug) Atropine 10 /ig 100 ng Bombesin + atropine l/*g 10 M g 100 jug
Plasma glucose (mg/dl) 160 ± 8 252 ± 27 346 ± 33 170 ± 12 164 ± 6 159 ± 11 250 ± 15 264 ± 23 255 ± 15
Carbacol + atropine l/*g 10 fig 100 jug
194 ± 8 172 ± 8 177 ± 10
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BROWN, RIVIER, AND VALE
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300UJ 12> 13-[D-Trp8]SRIF given via the same route prevents the rise in plasma epinephrine produced by bombesin (30). We conclude that bombesin acts within the brain to increase sympathetic outflow and increase adrenomedullary epinephrine secretion, which in turn lowers plasma insulin and elevates plasma glucagon, resulting in elevation of plasma glucose. SRIF or des-AA1 • 2,4,5,12,13_[D.Trp8]SRIF prevents this hyperglycemia via a CNS action to prevent adrenomedullary epinephrine secretion. We speculate that the hyperglycemia after surgical stress or after the administration of carbacol or /8-endorphin, as described in the paper, is prevented by des-AA1'2l 4-5l 12> 13-[D-Trp8]SRIF acting within the CNS to decrease adrenomedullary epinephrine secretion.
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Acknowledgments We wish to thank Roberta Alien, Alice Heinig, Ginny Page, Karen von Dessonneck, Ron Kaiser, and Robert Galyean for their excellent technical assistance and Laurie Taylor and Sue Hebert for the editing and typing of this paper.
150-
D
CONTROL
| _ 12
0DT8-SS
5 12> 13
FIG. 8. Effect of ic administration of des-AA ' ' "• ' -[D-Trp8]SRIF (1 fig) development of hyperglycemia after administration of 100 ng bombesin given ic; epinephrine (20 fig), des-AA1'% "' 5| 12> 13-[D-Trp8]SRIF (100 fig), and arginine (200 mg) given sc; and glucagon (10 fig) given iv. Blood samples were collected 30 min after treatments.
remains undetermined. Stimulation of various hypothalamic areas results in the modification of blood levels of glucose, insulin, and glucagon. Stimulation of the ventrolateral hypothalamus facilitates insulin secretion and lowers blood glucose (26), while stimulation of the ventromedial hypothalamus inhibits insulin secretion and elevates plasma levels of glucagon and glucose (27). These changes may result from alteration in hypothalamic sympathetic and parasympathetic outflow, which has been suggested to directly alter the secretion of insulin and glucagon by the endocrine pancreas (28). Alternatively, direct sympathetic innervation of the liver may potentially influence hepatic glucose production (29). Although acute changes in blood glucose, insulin, and glucagon after various types of stress, exercise, and starvation could be mediated by glucoregulatory pathways emerging from the brain, the role that the CNS might play in physiological regulation of glucose metabolism remains undefined. Whether bombesin, /?-endorphin, or SRIF are physiologically involved in or activate pathways which might mediate some of these mechanically, electrically, or phys-
References 1. Oliver, J. R., and S. R. Wagle, Studies on the inhibition of insulin release, glycogenolysis and gluconiagenesis by somatostatin in the rat islet of Langerhans in isolated hepatocytes, Biochem Biophys Res Commun 62: 772, 1975. 2. Sacks, H., K. Waligora, J. Matthews, and B. Pimstone, Inhibition by somatostatin of glucagon induced glucose release from the isolated perfused rat liver, Diabetes 26: 22,1977 (Abstract). 3. Sacca, L., and R. Sherwin, Somatostatin (SRIF) alters sensitivity to glucagon and epinephrine independent of insulin and glucagon availability, Diabetes 26: 23, 1977 (Abstract). 4. Brown, M., and W. Vale, Somatostatin: five years of progress, Biomedicine 28: 93, 1978. 5. Pfeiffer, E. F., C. Missner, W. Beischer, W. Kerner, and S. Raptis, The anti-diabetic action of somatostatin assessed by the artificial 0-cell, Metabolism (Suppl 1) 27: 1415, 1978. 6. Unger, R. H., E. Ipp, V. Schusdziarra, L. Orci, Hypothesis: physiologic role of pancreatic somatostatin and the contribution of D-cell disorders to diabetes mellitus, Life Sci 20: 2081,1977. 7. Vale, W., C. Rivier, and M. Brown, Regulatory peptides of the hypothalamus, Annu Rev Physiol 29: 473, 1977. 8. Anastasi, A., V. Erspamer, and M. Bucci, Isolation and structure of bombesin and alytesin, two analogous active peptides from the skin of the European amphibians Bombina and Alytes, Experientia 27: 166, 1971. 9. Polak, J. M., R. Hobbs, S. R. Bloom, E. Solcia, and A. G. E. Pearse, Distribution of a bombesin-like peptide in human gastrointestinal tract, Lancet 2: 1109, 1976. 10. Walsh, J. H., and A. L. Holmquist, Radioimmunoassay of bombesin peptides: identification of bombesin-like immunoreactivity in vertebrate gut extracts, Gastroenterology 70: 948, 1976 (Abstract 90). 11. Brown, M., J. Rivier, R. Kobayashi, and W. Vale, Neurotensin-like and bombesin-like peptides: CNS distribution and actions, In Bloom, S. R. (ed.), Gut Hormones, Churchill-Livingstone, Edinburgh, 1978, pp. 550-558. 12. Villarreal, J., J. Rivier, and M. Brown, Evidence for a bombesin-
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SRIF: CNS ACTIONS ON GLUCOREGULATION
13. 14.
15. 16. 17. 18. 19. 20. 21.
like substance in hypothalamus: chemical and immune-logical characterization, Endocrinology 102: 390, 1978 (Abstract). Polak, J. M., A. E. Bishop, M. A. Ghatei, S. R. Bloom, and M. Brown, Bombesin-Like Immunoreactivity in the Rat Brain, The Endocrine Society, United Kingdom, in press. Erspamer, V., and P. Melchiorri, Actions of bombesin on secretions and motility of the gastrointestinal tract, In Thompson, J. C. (ed.), Gastrointestinal Hormones, University of Texas Press, Austin, 1975, pp. 575-589. Brown, M., J. Rivier, and W. Vale, Bombesin: potent effects on thermoregulation in the rat, Science 196: 998, 1977. Brown, M., J. Rivier, A. Wolfe, and W. Vale, TRF and bombesin: actions on thermoregulation and TSH secretion in rats, Endocrinology 100: 279, 1977 (Abstract). Brown, M., J. Rivier, and W. Vale, Bombesin affects the central nervous system to produce hyperglycemia in rats, Life Sci 21: 1729, 1977. Feldberg, W., Pharmacology of the central actions of endorphins, In Bloom, S. R. (ed.), Gut Hormones, Churchill-Livingstone, Edinburgh, 1978, p. 495. Brown, M., and W. Vale, Somatostatin (SS): central nervous system (CNS) actions on glucose and temperature regulation, Endocrinology 102: 779, 1978 (Abstract). Rivier, J., Somatostatin. Total solid phase synthesis, J Am Chem Soc 96: 2986, 1974. Rivier, J., and M. Brown, Bombesin, bombesin analogs and related peptides: effects on thermoregulation, Biochemistry 17: 1766,1978.
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22. Brown, M., J. Rivier, and W. Vale, Biological activity of somatostatin and somatostatin analogs on inhibition of arginine-induced insulin and glucagon release in the rat, Endocrinology 98: 336, 1976. 23. Brown, M., W. Vale, and J. Rivier, Insulin selective somatostatin (SS) analogs, Diabetes (Suppl 1) 26: 29, 1977. 24. Moody, T., C. Pert, J. Rivier, and M. Brown, Bombesin: specific binding to rat brain membranes, Proc Natl Acad Sci USA 75: 5372, 1978. 25. Bernard, C, Chiens rendus diabetques, C R Soc Biol (Paris) 1: 60, 1849. ' 26. Steffens, A. B., G. J. Mogenson, and J. A. F. Stevenson, Blood glucose, insulin, and free fatty acids after stimulation and lesions of the hypothalamus, Am J Physiol 222: 1446, 1972. 27. Frohmann, L., and L. Bernardis, Effects of hypothalamic stimulation on plasma glucose, insulin and glucagon levels, Am J Physiol 221: 1596, 1971. 28. Woods, S. C, and D. Porte, Neurocontrol of the endocrine pancreas, Physiol Rev 54: 596, 1974. 29. Shimazu, R., and S. Ogasawara, Effects of hypothalamic stimulation on gluconeogenesis and glycolysis in rat liver, Am J Physiol 228: 1787, 1975. 30. Brown, M., and D. Fisher, Central nervous system influence on glucoregulation, Clin Res 27: 84, 1979 (Abstract). 31. Vale, W., J. Rivier, N. Ling, and M. Brown, Biological and immunological activities and applications of somatostatin analogs, Metabolism 27: 1391, 1978 (Abstract 9).
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