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Effect of Chemical Stimulation of the Dorsomedial Hypothalamic Nucleus on Blood Plasma Glucose, Triglycerides and Free Fatty Acids in Rats
Brain Research Bulletin, Vol. 42, No. 3, pp. 195–198, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/97 $17.00 / .00
PII S0361-9230(96)00225-0
Effect of Chemical Stimulation of the Dorsomedial Hypothalamic Nucleus on Blood Plasma Glucose, Triglycerides and Free Fatty Acids in Rats CA´SSIA THAI¨S B. V. ZAIA,*1 LUIS C. J. GAZIRI,* DIMAS A. M. ZAIA,† EDSON DELATTRE,‡ MIRIAM S. DOLNIKOFF,\ and CESAR TIMO-IARIA§ *Departamento de Cieˆncias Fisiolo´gicas and †Departamento de Quı´mica, Universidade Estadual de Londrina; Campus Universita´rio, Londrina, Brasil; ‡Departamento de Fisiologia, Universidade Estadual de Campinas; \Departamento de Fisiologia, Universidade Federal de Sa˜o Paulo; and §Faculdade de Medicina, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil [Received 14 February 1996; Accepted 9 July 1996] ABSTRACT: The effects of chemical stimulation of the dorsomedial hypothalamic nucleus (DMH) on blood plasma concentration of glucose, triglycerides, insulin, and free fatty acids (FFA) were investigated in anesthetized adult Wistar rats. Microinjection of 12.5 nmol of norepinephrine into the DMH increased blood plasma concentration of glucose and FFA, decreased triglycerides, and did not change plasma insulin within 5 min; after 20 min, blood glucose and FFA reached control values. Microinjection of epinephrine (12.5 nmol) into the DMH also increased blood plasma glucose concentration and decreased triglycerides after 5 min. These effects are probably mediated by beta-adrenergic mechanisms, because they were prevented by beta-adrenergic antagonist propranolol, but not by alpha-adrenergic antagonist prazosin. Microinjection into the DMH of glutamate, dopamine, or acetylcholine failed to cause any change in those metabolic parameters, corroborating the hypothesis that the DMH is part of a beta-adrenergic pathway involved in short-term modulation of the availability of glucose and FFA. Copyright Q 1997 Elsevier Science Inc.
that stimulation of either the VMH or the LHA can affect glycemia and free fatty acids (FFA) mobilization, this study was undertaken to test the hypothesis that the DMH is also part of neural pathways related to those hypothalamic areas whose excitation causes short-term changes in blood glucose, triglycerides, and FFA concentrations. METHODS Adult male Wistar rats (n Å 117), weighing about 150 g, were used in the present study. The animals were kept under controlled temperature (23 { 17C) and illumination (12 h cycle) in the animal quarter, and had free access to water and standard laboratory diet (Purina) until the moment of the experiment. Fifteen minutes before the experiment, the animals were anesthetized with sodium pentobarbital (40 mg/kg) and then placed in a stereotaxic instrument. A unilateral (right side) guiding cannula (22 gauge) bearing an inner stylette was placed 0.5 mm above the DMH (AP: 4.4 mm; L: 0.3 mm; H: 6.6 mm) or dorsal portion of the caudate nucleus (CD, AP: 8.6 mm; L: 2.4 mm; H: 1.8 mm), according to the stereotaxic coordinates of Ko¨nig and Klippel [13]. Microinjection of drugs was performed through a needle (30 gauge) connected to a Hamilton syringe; the injection needle exceeded the tip of the guiding cannula by 0.5 mm. The drugs were dissolved in sterile saline immediately before use and microinjected starting 1 min after implantation of the cannula; a total volume of 1 ml was injected in 2 min. The animals were killed by decapitation at 5, 10, or 20 min after the end of the microinjection. Blood samples were collected into heparinized test tubes and centrifuged; the plasma was frozen at 0207C and stored for further use. Saline was used as a microinjection control drug. The plasmatic concentrations of glucose and triglycerides were measured by the oxidase peroxidase method [10,28]. Plasmatic insulin was measured by radioimmunoassay [8] using rat insulin as standard (Novo Research Institute, Denmark). The
KEY WORDS: Hypothalamus, Hyperglycemia, Adrenergic blockers, Norepinephrine, Epinephrine
INTRODUCTION Chemical stimulation of the ventromedial nucleus (VMH) of the hypothalamus [16,21], lateral hypothalamic (LHA) area [20], or paraventricular nucleus (PVN) of hypothalamus [1,12,15], electrolytic lesion of the VMH [30], as well as local cytoglucopenia in the medial forebrain bundle area [27] or the dorsomedial nucleus (DMH) of hypothalamus [29], have been reported to cause changes in blood glucose concentration. Electrical [18] or adrenergic [23] stimulation of the VMH or LHA also interferes with hormonal release and fat mobilization. Because there are direct connections between the DMH and each of those hypothalamic areas [25,26], and considering the claims
1 To whom correspondence should be addressed. C. T. B. V. Zaia, Departamento de Cieˆncias Fisiolo´gicas; Universidade Estadual de Londrina; Campus Universita´rio; Londrina, PR, 86051-970, Brasil.
195 / 2a2d 2250 Mp 195 Monday Dec 16 01:19 PM EL–BRB (v. 42, no. 3) 2250
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concentration of FFA was measured by Falholt method [9]. Immediately after decapitation, the brains were removed and kept in 10% formalin. One week later, Nissl-stained (cresyl violet) sections (10 mm) were prepared for identification of the site of placement of the cannula. Only the animals in which the target area was reached were considered. The following drugs purchased from Sigma Chem. Co. (St. Louis, MO) were used: L-epinephrine bitartrate, L-norepinephrine-D-bitartrate, dopamine hydrochloride, acetylcholine chloride, L-glutamic acid monosodium salt, L-propranolol hydrochloride, and prazosin hydrochloride. Blood plasma glucose, triglycerides, and insulin levels obtained for each experimental group (Tables 1 and 2) were compared by using one-way analysis of variance (ANOVA) followed by multiple comparisons using Student–Newman–Keuls (SNK) procedure. Student’s t-test was used to compare blood plasma glucose, triglycerides, and free fatty acids levels (Fig. 1) obtained for saline and norepinephrine groups at each time after microinjection into the DMH. Means were considered significantly different for p õ 0.05. RESULTS Microinjection of 12.5 nmol of norepinephrine (NE) or epinephrine (E) into the DMH caused a significant increase in blood plasma glucose concentration, F(7, 39) Å 18.58, p õ 0.001, and decrease in the triglycerides, F (7, 39) Å 3.52, p õ 0.005, after 5 min (Table 1), compared with saline control group, whereas 2.5 nmol of epinephrine or 5 nmol of NE failed to cause any significant changes in blood plasma glucose concentration and triglycerides (Table 1). Microinjection into the DMH of dopamine (DA), monosodium glutamate (MSG), or acetylcholine (ACh) caused neither significant changes in blood plasma glucose concentration nor in triglycerides 5 min postinjection (Table 1). A previous (5 min) microinjection of the beta-adrenergic antagonist propranolol into the DMH prevented the increase in blood plasma glucose concentration, F (3, 23) Å 23.72, p õ 0.0001, and the decrease in triglycerides, F(3, 23) Å 6.13, p õ 0.003, caused by the microinjection of NE or E, but the preinjection of the alpha-adrenergic antagonist prazosin or of saline TABLE 1 BLOOD PLASMA GLUCOSE, TRIGLYCERIDES, AND INSULIN CONCENTRATIONS 5 MIN AFTER MICROINJECTION OF DRUGS INTO THE DORSOMEDIAL HYPOTHALAMIC NUCLEUS (DMH)
Groups
Saline (9) NE 5.0 nmol (4) NE 12.5 nmol (7) E 2.5 nmol (4) E 12.5 nmol (6) DA 12.5 nmol (5) MSG 12.5 nmol (6) ACh 12.5 nmol (6)
Glucose mg/dl
113.2 { 119.9 { 136.0 { 111.2 { 134.3 { 117.9 { 113.5 { 112.8 {
1.3 2.5 1.5* 2.8 3.7* 2.4 2.4 2.7
Triglycerides mg/dl
86.9 { 85.8 { 58.7 { 81.3 { 53.3 { 91.8 { 90.4 { 89.5 {
6.6 18.4 3.5† 2.4 6.6† 13.7 9.1 3.2
Insulin ng/ml
2.9 { 3.0 { 3.0 { 2.7 { 2.2 { 1.8 { 2.9 { 3.2 {
0.4 0.5 1.1 0.5 0.5 0.2 0.3 0.4
Values given are mean { SEM. Number of animals is in parenthesis. The drugs microinjected (1 ml/2 min) are: norepinephrine (NE), epinephrine (E), dopamine (DA), monosodium glutamate (MSG), and acetylcholine (ACh). * NE and E groups statistically different (p õ 0.001) from other glucose levels. † NE and E groups statistically different (p õ 0.005) from other triglycerides levels.
TABLE 2 BLOOD PLASMA GLUCOSE, TRIGLYCERIDES, AND INSULIN CONCENTRATIONS 5 MIN AFTER MICROINJECTION OF DRUGS INTO THE DORSOMEDIAL HYPOTHALAMIC NUCLEUS (DMH) PRETREATED WITH ADRENERGIC ANTAGONISTS
Groups
Saline / NE 12.5 nmol (7) PR / NE 12.5 nmol (7) PR / E 12.5 nmol (7) PZ / E nmol (6)
Glucose mg/dl
136.0 { 113.1 { 113.5 { 137.9 {
1.5 1.6* 3.0* 4.6
Triglycerides mg/dl
43.8 { 76.9 { 71.4 { 45.4 {
3.4 4.8† 11.9† 2.7
Insulin ng/ml
3.1 { 2.8 { 4.3 { 2.9 {
1.0 0.5 0.9 0.6
Values given are mean { SEM. Number of animals is in parenthesis. The drugs microinjected (1 ml/2 min) are norepinephrine (NE), epinephrine (E), propranolol (PR), or saline microinjected 5 min before microinjection of NE or E, and prazosin (PZ) microinjected 5 min before microinjection of E. * Different from Saline / NE and PZ / E groups ( p õ 0.0001) for blood plasma glucose levels. † Different from Saline / NE and PZ / E groups ( p õ 0.003) for blood plasma triglycerides levels.
failed to prevent those effects of NE or E (Table 2). Microinjection of propranolol alone (n Å 5) into the DMH did not change blood plasma glucose (112.5 { 2.2 mg/dl) or triglycerides (89.8 { 4.5 mg/dl) levels 5 min after microinjection compared with the saline (Table 1) control group. Microinjection of NE (12.5 nmol) or saline (S, n Å 5) into the caudate nucleus (NE-CD, n Å 6) caused no change in blood plasma glucose concentration (NE-CD Å 115.3 { 1.2 mg/dl; S Å 117.0 { 3.3 mg/dl) or in triglycerides (NE-CD Å 92.3 { 7.4 mg/dl; S Å 87.8 { 3.8 mg/ dl) 5 min after microinjection. None of the drugs microinjected into the DMH caused significant changes in the level of plasma insulin 5 min postinjection (Tables 1 and 2). The blood plasma glucose concentration, t(11) Å 15.83, p õ 0.0001, and the triglycerides, t(11) Å 3.07, p õ 0.01, were still different from the saline control group 10 min after the microinjection of NE (12.5 nmol) into the DMH, and reached control values after twenty minutes (Fig. 1). Free fatty acids increased, t(11) Å 3.04, p õ 0.009, 5 min after injection of NE (12.5 nmol) into the DMH and reached control values 10 min after the microinjection (Fig. 1). DISCUSSION The main finding of this report is an increase in blood plasma glucose concentration and a decrease in the triglycerides 5 min after the microinjection of either NE or E into the DMH, and the inhibition of these effects by previous microinjection into the DMH of the beta-adrenergic antagonist propranolol, but not by the alpha-adrenergic antagonist prazosin. These observations suggest that the hyperglycemia and the lower plasma concentration of triglycerides observed after microinjection of NE or E into the DMH are specific and mediated by beta-adrenergic mechanisms, a conclusion that is further corroborated by the finding that microinjection of other putative neurotransmitters (DA, MSG, ACh) into the DMH or microinjection of NE (12.5 nmol) into the caudate nucleus were ineffective in causing changes in blood plasma glucose concentration and triglycerides. A previous investigation failed to observe changes of blood plasma glucose concentration after injection of NE into the DMH [7], but this disagreement with the present results can be ascribed to the use of a too low amount of NE (0.2 mg; about 1.2 nmol) in that previous study. In agreement with this interpretation, we
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STIMULATION OF THE DORSOMEDIAL NUCLEUS
197 have observed that the microinjection of 5.0 nmol of NE or 2.5 nmol of E caused no significant change in blood plasma glucose concentration. We observed that the concentration of FFA was increased 5 min after microinjection of NE (12.5 nmol) into the DMH. This result agrees with a previous report [4] of an increase in FFA after NE injection into the lateral brain ventricle and an inhibition of this response by previous administration of propranolol. The injection of NE into the DMH caused an increase in blood plasma glucose concentration within 5 min but no concomitant change in the blood plasma concentration of insulin. Analogous results were observed in studies of other hypothalamic nuclei. Infusion of NE into the lateral hypothalamic area (LHA) caused an increase in blood plasma concentration of insulin and FFA but no change in blood plasma glucose concentration, whereas infusion of NE into the VMH increased all three substances [23]. On the other hand, infusion of E into the LHA increased FFA, insulinemia, and glycemia, but its infusion into the VMH increased only FFA and insulinemia [23]. Electrical stimulation of the VMH caused a rapid activation of liver phosphorilase-a (within 30 s), and caused a decrease in insulinemia within 5 min [19]. Injection of NE into the paraventricular nucleus (PVN) caused an increase in blood plasma glucose concentration and a small but significant decrease in insulinemia within 5 min; both responses were abolished by ganglionic blockade, suggesting that they were mediated through the splanic nerves [12]; on the other hand, increase in neuropeptide Y into the PVN, by microinjection of dibutyryl adenosine 3 *,5 *-monophosphate into the third ventricle, did not change insulin or glycemia after 2 h [2]. Altogether these observations support the hypothesis that several hypothalamic nuclei (LHA, VMH, PVN, DMH), through neural pathways to the liver, endocrine pancreas, and adipose tissue, participate in a system capable of producing rapid adjustments in the offer of fuel to the organism. Because the DMH has efferent connections with sympathetic neurons of the lower medulla oblongata and spinal cord [6,22,25], these connections might explain the hyperglycemia, increase in FFA and decrease in triglycerides observed after stimulation of the DMH. There is considerable evidence that the ventromedial nucleus and the lateral area of the hypothalamus have important regulatory functions related to food intake [30] and the modulatory control of glycemia [11,16,19] and insulinemia [11,30]; the paraventricular nucleus also participates in the modulation of food intake [3,14], glycemia, and insulinemia [15,24]. The possible participation of the DMH on these homeostatic functions is less well characterized, although such participation should be expected from the existence of direct connections between the DMH and each one of those three hypothalamic nuclei [26]. Moreover, studies have shown the presence of glucose sensitive neurons in the DMH [5,17], suggesting that it might exert a control over the other hypothalamic nuclei. In the present study, we have presented evidence that the DMH is also part of that hypothalamic system whose activation can cause rapid increases in glycemia and fat mobilization. ACKNOWLEDGEMENTS
FIG. 1. Blood plasma concentrations of glucose (mg/dl), free fatty acids (FFA, mmol/dl) and triglycerides (mg/dl) 5, 10 or 20 min after microinjection of norepinephrine (slanted rule column NE 12.5 nmol) or saline (open column) into the DMH. Values are mean { SEM. Number of animals is in parenthesis. *Different from saline group (p õ 0.01). **Different from saline group (p õ 0.004). ***Different from saline group (p õ 0.0001).
The authors are thankful to Mr. Ideval Azarias de Souza for technical assistance, and to Dr. Cla´udia B. R. Martinez for statistic helping. This work was supported by grants from CNPq 401456/87-0/BF/FV, CONCITEC-PR 022088/1, and CPG-UEL 259206/94, Brazil. C.T.B.V.Z. thanks for research fellowship from CNPq 523767/94-3.
REFERENCES 1. Akabayashi, A.; Zaia, C. T. B. V.; Silva, I.; Chae, H. J.; Leibowitz, S. F. Neuropeptide Y in the arcuate nucleus is modulated by alterations in glucose utilization. Brain Res. 621:343–348; 1993.
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2. Akabayashi, A.; Zaia, C. T. B. V.; Gabriel, S. M.; Silva, I.; Cheung, W. K.; Leibowitz, S. F. Intracerebroventricular injection of dibutyryl cyclic adenosine 3 *,5 *-monophosphate increases hypothalamic levels of neuropeptide Y. Brain Res. 660:323–328; 1994. 3. Akabayashi, A.; Zaia, C. T. B. V.; Koenig, J. I.; Gabriel, S. M.; Silva, I.; Leibowitz, S. F. Diurnal rhythm of galanin-like immunoreactivity in the paraventricular and suprachiasmatic nuclei and other hypothalamic areas. Peptides 15:1437–1444; 1994. 4. Barbosa, M. C.; Migliorini, R. H. Free fatty acid mobilization in rats following intracerebroventricular norepinephrine. Am. J. Physiol. 242:E248–E252; 1982. 5. Bernardis, L. L.; Bellinger, L. L. The dorsomedial hypothalamic nucleus revisited: 1986 update. Brain Res. Rev. 12:321–381; 1987. 6. Coyle, J. T.; Snyder, S. H. Catecholamines. In: Siegel, G. J.; Albers, R. W.; Agranoff, B. W.; Katzman, R., eds. Basic neurochemistry. Boston: Little, Brown and Company; 1981:205–217. 7. De Jong, A.; Strubbe, J. H.; Steffens, A. B. Hypothalamic influence on insulin and glucagon release in the rat. Am. J. Physiol. 233:E380– E388; 1977. 8. Desbuquois, B.; Aurbach, G. D. Use of polyethylene glycol to separate free and antibody-bound peptide hormones in radioimmunoassays. J. Clin. Endocrinol. 33:732–738; 1971. 9. Falholt, K.; Lund, B.; Falholt, W. An easy colorimetric micromethod for routine determination of free fatty acids in plasma. Clin. Chim. Acta 46:105–111; 1973. 10. Fossati, P.; Principe, L. Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide. Clin. Chem. 28:2077–2080; 1982. 11. Frohman, L. A.; Bernardis, L. L. Effect of hypothalamic stimulation on plasma glucose, insulin, and glucagon levels. Am. J. Physiol. 221:1596–1603; 1971. 12. Ionescu, E.; Coimbra, C. C.; Walker, C. D.; Jeanrenaud, B. Paraventricular nucleus modulation of glycemia and insulinemia in freely moving lean rats. Am. J. Physiol. 257:R1370–R1376; 1989. 13. Ko¨nig, J. F. R.; Klippel, R. A. The rat brain. A stereotaxic atlas of the forebrain and lower parts of the brain stem. Baltimore: Williams and Wilkins; 1963. 14. Leibowitz, S. F. Hypothalamic paraventricular nucleus: Interaction between a2-noradrenergic system and circulating hormones and nutrients in relation to energy balance. Neurosci. Biobehav. Rev. 12:101–109; 1988. 15. Leibowitz, S. F.; Sladek, C.; Spencer, L.; Tempel, D. Neuropeptide Y, epinephrine and norepinephrine in the paraventricular nucleus: stimulation of feeding and the release of corticosterone, vasopressin and glucose. Brain Res. Bull. 21:905–912; 1988.
16. Matsushita, H.; Shimazu, T. Chemical coding of the hypothalamic neurones in metabolic control. II. Norepinephrine-sensitive neurones and glycogen breakdown in liver. Brain Res. 183:79–87; 1980. 17. Oomura, Y.; Yoshimatsu, H. Neural network of glucose monitoring system. J. Auton. Nerv. Syst. 10:359–372; 1984. 18. Shimazu, T. Central nervous system regulation of liver and adipose tissue metabolism. Diabetologia 20:343–356; 1981. 19. Shimazu, T.; Fukuda, A.; Ban, T. Reciprocal influences of the ventromedial and lateral hypothalamic nuclei on blood glucose level and liver glycogen content. Nature 210:1178–1179; 1966. 20. Shimazu, T.; Matsushita, H.; Ishikawa, K. Cholinergic stimulation of the rat hypothalamus: Effects on liver glycogen synthesis. Science 194:535–536; 1976. 21. Shimazu, T.; Matsushita, H.; Ishikawa, K. Hypothalamic control of liver glycogen metabolism in adult and aged rats. Brain Res. 144:343–352; 1978. 22. Shirokawa, T.; Nakamura, S. Antidromic activation of rat dorsomedial hypothalamic neurons from locus coeruleus and median eminence. Brain Res. Bull. 18:291–295; 1987. 23. Steffens, A. B.; Damsma, G. L; Van Der Gugten, J.; Luiten, P. G. M. Circulating free fatty acids, insulin, and glucose during chemical stimulation of hypothalamus in rats. Am. J. Physiol. 247:E765– E771; 1984. 24. Tempel, D. L.; Leibowitz, S. F. Galanin inhibits insulin and corticosterone release after injection into the PVN. Brain Res. 536:353– 357; 1990. 25. Ter Horst, G. J.; Luiten, P. G. M. The projections of the dorsomedial hypothalamic nucleus in the rat. Brain Res. Bull. 16:231–248; 1986. 26. Ter Horst, G. J.; Luiten, P. G. M. Phaseolus vulgaris leuco-agglutinin tracing of intrahypothalamic connections of the lateral, ventromedial, dorsomedial and paraventricular hypothalamic nuclei in the rat. Brain Res. Bull. 18:191–203; 1987. 27. Timo–Iaria, C. Glucoreceptor system: from control of glycemia to feeding behavior. News Physiol. Sci. 5:46–49; 1990. 28. Trinder, P. Determination of blood-glucose using an oxidase peroxidase system with a non-carcinogenic chromogen. Ann. Clin. Biochem. 6:24–29; 1969. 29. Yoshimatsu, H.; Egawa, M.; Bray, G. A. Adrenal sympathetic nerve activity in response to hypothalamic injections of 2-deoxy-D-glucose. Am. J. Physiol. 261:R875–R881; 1991. 30. Zaia, C. T. B. V.; Oller do Nascimento, C. M. P.; Timo-Iaria, C.; Dolnikoff, M. S. Time course of insulin, corticosterone, and metabolic changes caused by lesion of the ventromedial hypothalamus in the rat. Physiol. Behav. 39:707–714; 1987.
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Effect of Chemical Stimulation of the Dorsomedial Hypothalamic Nucleus on Blood Plasma Glucose, Triglycerides and Free Fatty Acids in Rats CA´SSIA THAI¨S B. V. ZAIA,*1 LUIS C. J. GAZIRI,* DIMAS A. M. ZAIA,† EDSON DELATTRE,‡ MIRIAM S. DOLNIKOFF,\ and CESAR TIMO-IARIA§ *Departamento de Cieˆncias Fisiolo´gicas and †Departamento de Quı´mica, Universidade Estadual de Londrina; Campus Universita´rio, Londrina, Brasil; ‡Departamento de Fisiologia, Universidade Estadual de Campinas; \Departamento de Fisiologia, Universidade Federal de Sa˜o Paulo; and §Faculdade de Medicina, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil [Received 14 February 1996; Accepted 9 July 1996] ABSTRACT: The effects of chemical stimulation of the dorsomedial hypothalamic nucleus (DMH) on blood plasma concentration of glucose, triglycerides, insulin, and free fatty acids (FFA) were investigated in anesthetized adult Wistar rats. Microinjection of 12.5 nmol of norepinephrine into the DMH increased blood plasma concentration of glucose and FFA, decreased triglycerides, and did not change plasma insulin within 5 min; after 20 min, blood glucose and FFA reached control values. Microinjection of epinephrine (12.5 nmol) into the DMH also increased blood plasma glucose concentration and decreased triglycerides after 5 min. These effects are probably mediated by beta-adrenergic mechanisms, because they were prevented by beta-adrenergic antagonist propranolol, but not by alpha-adrenergic antagonist prazosin. Microinjection into the DMH of glutamate, dopamine, or acetylcholine failed to cause any change in those metabolic parameters, corroborating the hypothesis that the DMH is part of a beta-adrenergic pathway involved in short-term modulation of the availability of glucose and FFA. Copyright Q 1997 Elsevier Science Inc.
that stimulation of either the VMH or the LHA can affect glycemia and free fatty acids (FFA) mobilization, this study was undertaken to test the hypothesis that the DMH is also part of neural pathways related to those hypothalamic areas whose excitation causes short-term changes in blood glucose, triglycerides, and FFA concentrations. METHODS Adult male Wistar rats (n Å 117), weighing about 150 g, were used in the present study. The animals were kept under controlled temperature (23 { 17C) and illumination (12 h cycle) in the animal quarter, and had free access to water and standard laboratory diet (Purina) until the moment of the experiment. Fifteen minutes before the experiment, the animals were anesthetized with sodium pentobarbital (40 mg/kg) and then placed in a stereotaxic instrument. A unilateral (right side) guiding cannula (22 gauge) bearing an inner stylette was placed 0.5 mm above the DMH (AP: 4.4 mm; L: 0.3 mm; H: 6.6 mm) or dorsal portion of the caudate nucleus (CD, AP: 8.6 mm; L: 2.4 mm; H: 1.8 mm), according to the stereotaxic coordinates of Ko¨nig and Klippel [13]. Microinjection of drugs was performed through a needle (30 gauge) connected to a Hamilton syringe; the injection needle exceeded the tip of the guiding cannula by 0.5 mm. The drugs were dissolved in sterile saline immediately before use and microinjected starting 1 min after implantation of the cannula; a total volume of 1 ml was injected in 2 min. The animals were killed by decapitation at 5, 10, or 20 min after the end of the microinjection. Blood samples were collected into heparinized test tubes and centrifuged; the plasma was frozen at 0207C and stored for further use. Saline was used as a microinjection control drug. The plasmatic concentrations of glucose and triglycerides were measured by the oxidase peroxidase method [10,28]. Plasmatic insulin was measured by radioimmunoassay [8] using rat insulin as standard (Novo Research Institute, Denmark). The
KEY WORDS: Hypothalamus, Hyperglycemia, Adrenergic blockers, Norepinephrine, Epinephrine
INTRODUCTION Chemical stimulation of the ventromedial nucleus (VMH) of the hypothalamus [16,21], lateral hypothalamic (LHA) area [20], or paraventricular nucleus (PVN) of hypothalamus [1,12,15], electrolytic lesion of the VMH [30], as well as local cytoglucopenia in the medial forebrain bundle area [27] or the dorsomedial nucleus (DMH) of hypothalamus [29], have been reported to cause changes in blood glucose concentration. Electrical [18] or adrenergic [23] stimulation of the VMH or LHA also interferes with hormonal release and fat mobilization. Because there are direct connections between the DMH and each of those hypothalamic areas [25,26], and considering the claims
1 To whom correspondence should be addressed. C. T. B. V. Zaia, Departamento de Cieˆncias Fisiolo´gicas; Universidade Estadual de Londrina; Campus Universita´rio; Londrina, PR, 86051-970, Brasil.
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concentration of FFA was measured by Falholt method [9]. Immediately after decapitation, the brains were removed and kept in 10% formalin. One week later, Nissl-stained (cresyl violet) sections (10 mm) were prepared for identification of the site of placement of the cannula. Only the animals in which the target area was reached were considered. The following drugs purchased from Sigma Chem. Co. (St. Louis, MO) were used: L-epinephrine bitartrate, L-norepinephrine-D-bitartrate, dopamine hydrochloride, acetylcholine chloride, L-glutamic acid monosodium salt, L-propranolol hydrochloride, and prazosin hydrochloride. Blood plasma glucose, triglycerides, and insulin levels obtained for each experimental group (Tables 1 and 2) were compared by using one-way analysis of variance (ANOVA) followed by multiple comparisons using Student–Newman–Keuls (SNK) procedure. Student’s t-test was used to compare blood plasma glucose, triglycerides, and free fatty acids levels (Fig. 1) obtained for saline and norepinephrine groups at each time after microinjection into the DMH. Means were considered significantly different for p õ 0.05. RESULTS Microinjection of 12.5 nmol of norepinephrine (NE) or epinephrine (E) into the DMH caused a significant increase in blood plasma glucose concentration, F(7, 39) Å 18.58, p õ 0.001, and decrease in the triglycerides, F (7, 39) Å 3.52, p õ 0.005, after 5 min (Table 1), compared with saline control group, whereas 2.5 nmol of epinephrine or 5 nmol of NE failed to cause any significant changes in blood plasma glucose concentration and triglycerides (Table 1). Microinjection into the DMH of dopamine (DA), monosodium glutamate (MSG), or acetylcholine (ACh) caused neither significant changes in blood plasma glucose concentration nor in triglycerides 5 min postinjection (Table 1). A previous (5 min) microinjection of the beta-adrenergic antagonist propranolol into the DMH prevented the increase in blood plasma glucose concentration, F (3, 23) Å 23.72, p õ 0.0001, and the decrease in triglycerides, F(3, 23) Å 6.13, p õ 0.003, caused by the microinjection of NE or E, but the preinjection of the alpha-adrenergic antagonist prazosin or of saline TABLE 1 BLOOD PLASMA GLUCOSE, TRIGLYCERIDES, AND INSULIN CONCENTRATIONS 5 MIN AFTER MICROINJECTION OF DRUGS INTO THE DORSOMEDIAL HYPOTHALAMIC NUCLEUS (DMH)
Groups
Saline (9) NE 5.0 nmol (4) NE 12.5 nmol (7) E 2.5 nmol (4) E 12.5 nmol (6) DA 12.5 nmol (5) MSG 12.5 nmol (6) ACh 12.5 nmol (6)
Glucose mg/dl
113.2 { 119.9 { 136.0 { 111.2 { 134.3 { 117.9 { 113.5 { 112.8 {
1.3 2.5 1.5* 2.8 3.7* 2.4 2.4 2.7
Triglycerides mg/dl
86.9 { 85.8 { 58.7 { 81.3 { 53.3 { 91.8 { 90.4 { 89.5 {
6.6 18.4 3.5† 2.4 6.6† 13.7 9.1 3.2
Insulin ng/ml
2.9 { 3.0 { 3.0 { 2.7 { 2.2 { 1.8 { 2.9 { 3.2 {
0.4 0.5 1.1 0.5 0.5 0.2 0.3 0.4
Values given are mean { SEM. Number of animals is in parenthesis. The drugs microinjected (1 ml/2 min) are: norepinephrine (NE), epinephrine (E), dopamine (DA), monosodium glutamate (MSG), and acetylcholine (ACh). * NE and E groups statistically different (p õ 0.001) from other glucose levels. † NE and E groups statistically different (p õ 0.005) from other triglycerides levels.
TABLE 2 BLOOD PLASMA GLUCOSE, TRIGLYCERIDES, AND INSULIN CONCENTRATIONS 5 MIN AFTER MICROINJECTION OF DRUGS INTO THE DORSOMEDIAL HYPOTHALAMIC NUCLEUS (DMH) PRETREATED WITH ADRENERGIC ANTAGONISTS
Groups
Saline / NE 12.5 nmol (7) PR / NE 12.5 nmol (7) PR / E 12.5 nmol (7) PZ / E nmol (6)
Glucose mg/dl
136.0 { 113.1 { 113.5 { 137.9 {
1.5 1.6* 3.0* 4.6
Triglycerides mg/dl
43.8 { 76.9 { 71.4 { 45.4 {
3.4 4.8† 11.9† 2.7
Insulin ng/ml
3.1 { 2.8 { 4.3 { 2.9 {
1.0 0.5 0.9 0.6
Values given are mean { SEM. Number of animals is in parenthesis. The drugs microinjected (1 ml/2 min) are norepinephrine (NE), epinephrine (E), propranolol (PR), or saline microinjected 5 min before microinjection of NE or E, and prazosin (PZ) microinjected 5 min before microinjection of E. * Different from Saline / NE and PZ / E groups ( p õ 0.0001) for blood plasma glucose levels. † Different from Saline / NE and PZ / E groups ( p õ 0.003) for blood plasma triglycerides levels.
failed to prevent those effects of NE or E (Table 2). Microinjection of propranolol alone (n Å 5) into the DMH did not change blood plasma glucose (112.5 { 2.2 mg/dl) or triglycerides (89.8 { 4.5 mg/dl) levels 5 min after microinjection compared with the saline (Table 1) control group. Microinjection of NE (12.5 nmol) or saline (S, n Å 5) into the caudate nucleus (NE-CD, n Å 6) caused no change in blood plasma glucose concentration (NE-CD Å 115.3 { 1.2 mg/dl; S Å 117.0 { 3.3 mg/dl) or in triglycerides (NE-CD Å 92.3 { 7.4 mg/dl; S Å 87.8 { 3.8 mg/ dl) 5 min after microinjection. None of the drugs microinjected into the DMH caused significant changes in the level of plasma insulin 5 min postinjection (Tables 1 and 2). The blood plasma glucose concentration, t(11) Å 15.83, p õ 0.0001, and the triglycerides, t(11) Å 3.07, p õ 0.01, were still different from the saline control group 10 min after the microinjection of NE (12.5 nmol) into the DMH, and reached control values after twenty minutes (Fig. 1). Free fatty acids increased, t(11) Å 3.04, p õ 0.009, 5 min after injection of NE (12.5 nmol) into the DMH and reached control values 10 min after the microinjection (Fig. 1). DISCUSSION The main finding of this report is an increase in blood plasma glucose concentration and a decrease in the triglycerides 5 min after the microinjection of either NE or E into the DMH, and the inhibition of these effects by previous microinjection into the DMH of the beta-adrenergic antagonist propranolol, but not by the alpha-adrenergic antagonist prazosin. These observations suggest that the hyperglycemia and the lower plasma concentration of triglycerides observed after microinjection of NE or E into the DMH are specific and mediated by beta-adrenergic mechanisms, a conclusion that is further corroborated by the finding that microinjection of other putative neurotransmitters (DA, MSG, ACh) into the DMH or microinjection of NE (12.5 nmol) into the caudate nucleus were ineffective in causing changes in blood plasma glucose concentration and triglycerides. A previous investigation failed to observe changes of blood plasma glucose concentration after injection of NE into the DMH [7], but this disagreement with the present results can be ascribed to the use of a too low amount of NE (0.2 mg; about 1.2 nmol) in that previous study. In agreement with this interpretation, we
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197 have observed that the microinjection of 5.0 nmol of NE or 2.5 nmol of E caused no significant change in blood plasma glucose concentration. We observed that the concentration of FFA was increased 5 min after microinjection of NE (12.5 nmol) into the DMH. This result agrees with a previous report [4] of an increase in FFA after NE injection into the lateral brain ventricle and an inhibition of this response by previous administration of propranolol. The injection of NE into the DMH caused an increase in blood plasma glucose concentration within 5 min but no concomitant change in the blood plasma concentration of insulin. Analogous results were observed in studies of other hypothalamic nuclei. Infusion of NE into the lateral hypothalamic area (LHA) caused an increase in blood plasma concentration of insulin and FFA but no change in blood plasma glucose concentration, whereas infusion of NE into the VMH increased all three substances [23]. On the other hand, infusion of E into the LHA increased FFA, insulinemia, and glycemia, but its infusion into the VMH increased only FFA and insulinemia [23]. Electrical stimulation of the VMH caused a rapid activation of liver phosphorilase-a (within 30 s), and caused a decrease in insulinemia within 5 min [19]. Injection of NE into the paraventricular nucleus (PVN) caused an increase in blood plasma glucose concentration and a small but significant decrease in insulinemia within 5 min; both responses were abolished by ganglionic blockade, suggesting that they were mediated through the splanic nerves [12]; on the other hand, increase in neuropeptide Y into the PVN, by microinjection of dibutyryl adenosine 3 *,5 *-monophosphate into the third ventricle, did not change insulin or glycemia after 2 h [2]. Altogether these observations support the hypothesis that several hypothalamic nuclei (LHA, VMH, PVN, DMH), through neural pathways to the liver, endocrine pancreas, and adipose tissue, participate in a system capable of producing rapid adjustments in the offer of fuel to the organism. Because the DMH has efferent connections with sympathetic neurons of the lower medulla oblongata and spinal cord [6,22,25], these connections might explain the hyperglycemia, increase in FFA and decrease in triglycerides observed after stimulation of the DMH. There is considerable evidence that the ventromedial nucleus and the lateral area of the hypothalamus have important regulatory functions related to food intake [30] and the modulatory control of glycemia [11,16,19] and insulinemia [11,30]; the paraventricular nucleus also participates in the modulation of food intake [3,14], glycemia, and insulinemia [15,24]. The possible participation of the DMH on these homeostatic functions is less well characterized, although such participation should be expected from the existence of direct connections between the DMH and each one of those three hypothalamic nuclei [26]. Moreover, studies have shown the presence of glucose sensitive neurons in the DMH [5,17], suggesting that it might exert a control over the other hypothalamic nuclei. In the present study, we have presented evidence that the DMH is also part of that hypothalamic system whose activation can cause rapid increases in glycemia and fat mobilization. ACKNOWLEDGEMENTS
FIG. 1. Blood plasma concentrations of glucose (mg/dl), free fatty acids (FFA, mmol/dl) and triglycerides (mg/dl) 5, 10 or 20 min after microinjection of norepinephrine (slanted rule column NE 12.5 nmol) or saline (open column) into the DMH. Values are mean { SEM. Number of animals is in parenthesis. *Different from saline group (p õ 0.01). **Different from saline group (p õ 0.004). ***Different from saline group (p õ 0.0001).
The authors are thankful to Mr. Ideval Azarias de Souza for technical assistance, and to Dr. Cla´udia B. R. Martinez for statistic helping. This work was supported by grants from CNPq 401456/87-0/BF/FV, CONCITEC-PR 022088/1, and CPG-UEL 259206/94, Brazil. C.T.B.V.Z. thanks for research fellowship from CNPq 523767/94-3.
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