- Email: [email protected]
Insulin co-injection suppresses the thermogenic response to glutamate microinjection into the VMH in rats
326
Brain Research, 527 (1990) 326-329 Elsevier
BRES 24275
Short Communications
Insulin co-inject n suppmuel the ther microinjecUon into the
ic in rats
to pJtmlmte
Shimon Amir, Alessandra Schiavetto and Robin Pollock Centerfor Studies in Behavioral Neurobiology, Departmentof Psychology, Concordia University, Montreal, Que. (Canada)
(Accepted 29 May 1990) Key words: Insulin; Glutamate; Ventromediai hypothalamic nucleus; Brown adipose tissue; Thermogenesis; Rat
Selective stimulation of ventromedial hypothalamic nucleus (VMH) neurons by microinjection of the excitatory amino acid glutamate sharply increased interscapular brown adipose tissue (IBAT) and core temperatures in urethane-anaesthetized rats. This effect was blocked by co-injection of insulin (0.1-1/~g) though not an inactive insulin analog, T N B 3 insulin. Injection of insulin (1/~g) into the contralateral VMH or systemic administration of insulin (1/~g) had no effect on the thermogenic response to intra-VMH glutamate. These results complement those showing that intra-VMH insulin suppresses the basal firing rate of sympathetic nerves to IBAT and diminishes cold-induced nonshivering thermogenesis in BAT and add support to the view that insulin functions as an inhibitory signal on VMH neurons controlling thermogenesis in BAT.
Heat generation in brown adipose tissue (BAT) is influenced by insulin, but the mechanisms mediating the effect of insulin on BAT are incompletely understood 27. Insulin can influence heat production directly by influencing BAT metabolism 34, as well as indirectly, via effects in the central nervous system. An important central nervous system region concerned with neural control of BAT thermogenesis is the ventromedial hypothalamic nucleus (VMH) 25. In previous experiments, injection of insulin into the VMH in rats was found to suppress the basal firing rate of sympathetic nerves supplying BAT, suggesting that insulin has a direct inhibitory action on the neural outflow from the VMH to BAT 3°,32. However, the effect of intra-VMH insulin on heat production in BAT induced either by physiological or neural stimuli was not reported. Recently, we have shown that injection of insulin into the VMH suppresses cold-induced stimulation of BAT thermogenesis in rats 2. In the present study we examined the effect of intraV M H insulin on the thermogenic response resulting from direct stimulation of V M H neurons with the excitatory amino acid glutamate. In a previous study, we demonstrated that injection of glutamate into the VMH activates BAT thermogenesis via the sympathetic outflow, leading to a sharp increase in interscapular BAT (IBAT) and core temperatures 4. We report here that insulin co-injection blocks the thermogenic effect of intra-VMH
injection of glutamate in a direct and specific manner. Normally fed, male Wistar rats (275-325 g) were anaesthetized intraperitoneally with urethane (1.4 g/kg) and mounted in a stereotaxic instrument equipped with a thermostatically-controlled heating blanket calibrated to keep body temperature above 36 °C. A small incision was made 5 cm posterior to the scapulae and a thermistor probe (YSI 409B) was inserted and placed under the right lobe of IBAT. A second thermistor probe (YSI 401) was inserted 5 cm into the rectum. A 28 gauge needle connected to a Hamilton microsyringe containing glutamate (1 M), insulin (bovine insulin; Sigma) or glutamate plus insulin was then introduced into the right V M H using the following coordinates from the Paxinos and Watson 22 atlas: AP -2.5, L 0.6, V 9'.5. The position of the needles was later verified histologically. Interscapular BAT and core temperatures were monitored continuously using YSI telethermometers (model 41) connected to a flat bed recorder. Intra-VMH injection of saline (0.25 /~1, n = 4) or insulin (0.1, 0.5 or 1/~g, in 0.25/~1 saline; n = 4 for each dose of insulin) had no effect on basal IBAT or core temperatures of urethane-anaesthetized rats (data not shown). In contrast, intra-VMH injection of insulin suppressed the thermogenic response to intra-VMH co-injection of glutamate. Fig. 1 shows examples of the effect of injecting glutamate alone (0.25 ~tl of 1 M
Correspondence: S. Amir, Center for Studies in Behavioral Neurobiology, Department of Psychology, Concordia University, 1455 de Maisonneuve Blvd. West, Montreal, Que., Canada H3G IMS.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
327 I BAT •
1.50.
T GLU
I C;LU
r GLU
T I GLU INS, I Jig ( CONTRALATERALVMH)
1.00.
IBAT/BEFORE CORF_JBEFORE IBAT/AFTER CORE/AFTER
0.50. T GLU
T GLU+INS,0.1 Jag 0.00,
A
T otu
CONTRALATERAL
T T GLU
SYSTEMIC
TNB3
INSULIN
i
T GLU
~" GLU+INS,O.S~g
T GLU
T GLU+INS, 1Ug
INS, lug (SYSTEMIC)
T GLU
1" GLU+TNB3INS, 1 ~(J
i ' c L_ 10 min
Fig. 1. A typical example of the effect of intra-VMH injection of glutamate (0.25/~1 of 1 M solution), glutamate plus insulin (INS; 0.1, 0.5 or 1/~g) injected into the same VMH site, glutamate plus insulin (1/~g) injected into the contralateral VMH, glutamate plus systemic insulin (1 lzg) or glutamate plus TNB 3 insulin injected into the same VMH site (1 /~g) on IBAT and core temperatures in urethaneanaesthetized rats. In all experiments animals were first treated with glutamate alone; 60 min later they were given a second injection of glutamate together with one of the above insulin treatments.
solution) or glutamate plus insulin (0.1, 0.5 or 1/~g) into the V M H on IBAT and core temperatures. Injecting glutamate into the V M H led to a sharp and rapid increase in I B A T temperature, which was maximal at 8-12 min and returned to preinjection value after 25-35 min. Intra-VMH glutamate also increased core temperature;
150.
IBAT/BEFORE
10 o .
CORE,BEFORE IBAT/AFTER CORE/AFTER
? <]
0.50
•
0.00 • NCNE
0.1 u.g
0.5 u.g
Fig. 3. The effect of contralateral or systemic injection of insulin (1 /~g) or co-injection of TNB3 insulin (1/~g) on the changes in IBAT and core temperature (mean + SEM) induced by injection of glutamate (0.25 /A of 1 M solution) into the VMH in urethaneanaesthetized rats. Each group consisted of 8 rats.
1 I.tg
INSULIN
Fig. 2. The effect of intra-VMH insulin on the changes in IBAT and core temperature (mean __+ S.E.M.) induced by co-injection of glutamate (0.25/~1 of 1 M solution) in urethane-anaesthetized rats. Animals (n -- 10/group) were first given an intra-VMH injection of glutamate ('BEFORE'). Sixty min later they were reinjected with glutamate plus one of three doses of insulin ('AFTER'). The asterisks indicate significant difference from the corresponding 'BEFORE' value (t-test, P < 0.001).
the change in core temperature was less pronounced and never exceeded the increase in I B A T temperature. A second injection of glutamate, given 60 min after the first glutamate injection had a similar effect on I B A T and core temperature. In a separate series, co-injection of insulin (0.1 /~g) slightly, though significantly, attenuated the increase in IBAT and core temperatures induced by a second injection of glutamate into the VMH. Higher doses of insulin (0.5 and 1/~g), tested in separate groups of animals further suppressed the increase in IBAT and core temperatures induced by coinjected glutamate (Fig. 2). Intra-VMH injection of insulin (1/~g) had no effect on the increase in IBAT and core temperatures induced by glutamate injection into the contralateral V M H (Fig. 1). Similarly, systemic injection of insulin (1/~g) 5 min before injecting glutamate into the V M H had no effect on the increase in IBAT and core temperature (Fig. 1). Finally, intra-VMH injection of a biologically inactive insulin analog, TNB 3 insulin (1/~g), had no effect on the increase in IBAT and core temperatures induced by co-injection of glutamate (Fig. 1). The data on the effect of insulin injection into the contralateral VMH, of systemic injection of insulin, or of co-injection of TNB 3 insulin on the thermogenic response to intra-VMH injection of glutamate are shown in Fig. 3. The main function of BAT is to generate heat in response to thermoregulatory or metabolic challenges imposed by cold or overfeeding 5,9,26. The production of heat depends on activation of the sympathetic nervous system 16 and there is now ample evidence that the V M H is important for transducing thermal and metabolic signals to sympathetic activity and heat production in B A T 10'13-15'20'23'25"29'35. The V M H is also considered to be an important site of autonomic action of insulin. The VMH contains high concentration of insulin receptors 6,8 and insulin-sensitive neurons 21, and injection of insulin
328 into the VMH has been shown to influence the regulation of pancreatic insulin secretion and glucose production by the liver 12'36'37'38. More recent studies have shown that insulin might act in the VMH to modulate sympathetic efferent mechanisms controlling heat production in BAT. In these experiments, injection of insulin into the VMH in rats suppressed the basal firing rate of sympathetic nerves to BAT 3°. Insulin appeared to exert its inhibitory action via direct effect on VMH neurons, as prior selective destruction of VMH cells with the neurotoxin kainic acid prevented the effect of insulin on sympathetic activity. In a subsequent study, we found that injection of insulin into the VMH could attenuate cold-induced stimulation of BAT thermogenesis 2. Intra-VMH insulin had no effect on the thermogenic response to systemic norepinephrine administration, indicating that the effect was due to inhibitory action upon VMH neural elements concerned with communicating thermal signals to BAT and not due to activation of an inhibitory mechanism. More recently, we demonstrated that selective stimulation of VMH neurons with glutamate activates BAT thermogenesis in rats 4. This effect was mediated by the sympathetic system, as it could be prevented by pharmacological interventions that either block neurotransmission in sympathetic ganglia (i.e. chlorisondamine chloride) or inhibit norepinephrine stimulation of fladrenergic receptors (i.e., propranolol). In the present study, we found that intra-VMH co-injection of insulin at doses shown to reduce basal sympathetic activity in BAT or to diminish cold-induced stimulation of BAT thermogenesis (i.e. 0.5 and 1 ~tg)2'3° strongly attenuated the increase in IBAT temperature induced by intra-VMH injection of glutamate. Moreover, we obtained evidence suggesting that insulin exerts its inhibitory action via specific receptors. The possibility that insulin blocked the thermogenic response to intra-VMH glutamate by acting outside the insulin receptor complex was excluded by the demonstration that coinjection of TNB 3 insulin, an inactive insulin analog modified at regions of the molecule necessary for receptor interaction, had no effect on the thermogenic response to intra-VMH injection of glutamate. Finally, we determined that the inhibitory action involves direct effect on the neural elements stimulated by glutamate. The possibility that the suppressive action was mediated by an inhibitory efferent pathway from the VMH or that it was secondary to central or peripheral action on glucoregulation37 were excluded by the fact that neither the injection of insulin into the contralateral VMH nor systemic administration of the hormone had any effect. Taken together, the results show that insulin can block in a direct and specific manner VMH neural elements whose stimulation by glutamate causes sympathetic activation of thermoge-
nesis in BAT. These results, viewed in conjunction with earlier reports that intra-VMH injection of insulin suppresses basal sympathetic activity and cold-induced thermogenesis in BAT, are consistent with the notion that insulin acts in the VMH to modulate neural mechanisms regulating the outflow of thermogenic signals to BAT. The idea that insulin interacts with central mechanisms involved in the regulation of BAT thermogenesis is further supported by studies on the effect of injecting insulin into hypothalamic regions outside the VMH. A number of extra VMH regions have now been implicated in the central control of BAT thermogenesis, including the suprachiasmatic nucleus, paraventricular nucleus and lateral hypothalamic area 1"3'1s, and it has been shown that injecting insulin into these sites can modify sympathetic activity in BAT. For example, insulin injection into the suprachiasmatic nucleus was found to suppress basal sympathetic activity in BAT during the day, and to increase sympathetic activity during the night 3z. Injecting insulin into the paraventricular nucleus suppressed sympathetic activity in BAT 31. Insulin injection into the lateral hypothalamic area had a small stimulatory effect on sympathetic activity in BAT 33. Furthermore, injecting insulin into the cerebroventricular system suppressed basal firing rate of sympathetic nerves to BAT in a manner similar to that reported after intra-VMH injection of insulin. When insulin was coinjected with glucose, which in itself increases sympathetic activity, it appeared to exert an excitatory action on sympathetic activityH. Finally, it has been shown that insulin injection into the systemic circulation influences BAT thermogenesis in experimental animals; the effect of systemic insulin appeared to be mediated, in part, by the sympathetic system7'17"19"27"28'34. These diverse findings on the effect of centrally or systemically administered insulin, taken together with evidence that insulin from the systemic circulation can gain access to the central nervous system and affect the function of hypothalamic neurons 24, further support the view that insulin may function as a neuroendocrine signal to hypothalamic mechanisms regulating sympathetic activity and heat production in BAT. In summary, the present experiments demonstrate that injection of insulin into the VMH significantly attenuates the increase in IBAT and core temperatures that results from microinjection of glutamate into the same VMH site. These results further support the notion that insulin may function as an inhibitory signal on VMH neurons involved in the control of BAT. This study was supported by a grant from the Natural Sciences and Engineering Council of Canada.
329 1 Amir, S., Stimulation of the paraventricular nucleus with glutamate activates interscapular brown adipose tissue thermogenesis in rats, Brain Research, 508 (1990) 152-155. 2 Amir, S., Lagiorgia, M. and Pollock, R., Intra-ventromedial hypothalamic injection of insulin suppresses brown fat thermogenesis in the anaesthetized rat, Brain Research, 480 (1989) 340-343. 3 Amir, S., Shizgal, P. and Rompr6, P.-P., Glutamate injection into the suprachiasmatic nucleus stimulates brown fat thermogenesis in the rat, Brain Research, 498 (1989) 140-144. 4 Amir, S., Intra-ventromedial hypothalamic injection of glutamate stimulates brown adipose tissue thermogenesis in the rat, Brain Research, 511 (1990) 341-344. 5 Barnard, T., Brown adipose tissue as an effector of nonshivering thermogenesis, Experientia, 33 (1977) 1124-1126. 6 Baskin, D.G., Figlewicz, D.P., Woods, S.C., Porte, D. Jr. and Dorsa, D.M., Insulin in the brain, Annu. Rev. Physiol., 49 (1987) 335-347. 7 Christin, L., Nacht, C.-A., Vernet, O., Ravussin, E., Jequier, E. and Acheson, K.J., Insulin: its role in the thermic effect of glucose, J. Clin. Invest., 77 (1986) 1747-1755. 8 Havrankova, J., Brownstein, M. and Roth, J., Insulin and insulin receptors in rodent brain, Diabetologia, 20 (1981) 268-273. 9 Himms-Hagen, J. and Phil, D., Thermogenesis in brown adipose tissue as an energy buffer, New Engl. J. Med., 311 (1984) 1549-1558. 10 Holt, S.J., Wheal, H.V. and York, D.A., Hypothalamic control of brown adipose tissue in Zucker lean and obese rats. Effect of electrical stimulation of the ventromedial nucleus and other hypothalamic centres, Brain Research, 405 (1987) 227-233. 11 Holt, S.J. and York, D.A., Integration of intracerebroventricular insulin and glucose in the regulation of the activity of sympathetic efferent nerves to brown adipose tissue in lean and obese Zucker rats, Brain Research, 500 (1989) 384-388. 12 Iguchi, A., Durleson, P.D. and Szabo, A.J., Decrease in plasma glucose concentration after microinjection of insulin into VMH, Am. J. Physiol., 240 (1981) E95-E100. 13 Imai-Matsumura, K., Matsumura, K. and Nakayama, T., Involvement of ventromedial hypothalamus in brown adipose tissue thermogenesis induced by preoptic cooling in rats, Jpn. J. Physiol., 34 (1984) 939-943. 14 Imai-Matsumura, K., Matsumura, K., Tsai, C.-L. and Nakayama, T., Thermal responses of ventromedial hypothalamic neurons in vivo and in vitro, Brain Research, 445 (1988) 193-197. 15 Iwai, M., Shinomiya Hell, N. and Shimazu, T., Effect of ventromediai hypothalamic stimulation on blood flow of brown adipose tissue in rats, Pflager's Arch., 410 (1987) 44-47. 16 Landsberg, L., Saville, M.E. and Young, J.B., Sympathoadrenal system and regulation of thermogenesis, Am. J. Physiol., 247 (1984) E181-E189. 17 Larue-Acbaglotis, C., Goubern, M. and Laury, M.CI., Concomitant food intake and adipose tissue responses under chronic insulin infusion in rats, Physiol. Behav., 44 (1988) 95-100. 18 Lupien, J., Tokunaga, K., Kemnitz, J.W., Groos, E. and Bray, G.A., Lateral hypothalamic lesions and fenfluramine increase thermogenesis in brown adipose tissue, Physiol. Behav., 38
(1986) 15-20. 19 Menendez, J.A. and Atrens, D,M., Insulin increases energy expenditure and respiratory quotient in the rat, Pharmacol. Biochem. Behav., 34 (1989) 765-768. 20 Niijima, A., Rohner-Jeanrenaud, E and Jeanrenaud, B., Role of ventromedial hypothalamus on sympathetic efferents of brown adipose tissue, Am. J. Physiol., 247 (1984) R650-R654. 21 Oomura, Y. and Kita, H., Insulin acting as a neuromodulator through the hypothalamus, Diabetologia, Suppl. 20 (1981) 290-298. 22 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney, 1982. 23 Perkins, M.N., Rothwell, N.J., Stock, M.J. and Stone, T.W., Activation of brown adipose tissue thermogenesis by the ventromedial hypothalamus, Nature, 289 (1981) 401-402. 24 Posner, B.I., Insulin interaction with the central nervous system: nature and possible significance, Proc. Nutr. Soc., 46 (1987) 97-103. 25 Rothwell, N.J., Central control of brown adipose tissue, Proc. Nutr. Soc., 48 (1989) 197-206. 26 Rothwell, N.J. and Stock, M.J., A role for brown adipose tissue in diet-induced thermogenesis, Nature, 281 (1979) 31-35. 27 Rothwell, N.J. and Stock, M.J., Insulin and thermogenesis, Int. J. Obesity, 12 (1988) 93-102. 28 Rothwell, N.J., Stock, M.J. and Tedstone, A.E., Effects of ciglitazone on energy balance, thermogenesis and brown fat activity in the rat, Mol. Cell. Endocrinol., 57 (1987) 253-257. 29 Saito, M., Minokoshi, Y. and Shimazu, T., Brown adipose tissue after ventromedial hypothalamic lesions in rats, Am. J. Physiol., 248 (1985) E20-E25. 30 Sakaguchi, T. and Bray, G.A., Intrahypothalamic injection of insulin decreases firing rate of sympathetic nerves, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 2012-2014. 31 Sakaguchi, T. and Bray, G.A., Sympathetic activity following paraventricular injections of glucose and insulin, Brain Res. Bull., 21 (1988) 25-29. 32 Sakaguchi, T., Takahashi, M. and Bray, G.A., Diurnal changes in sympathetic activity: relation to food intake and to insulin injected into the ventromedial or suprachiasmatic nucleus, J. Clin. Invest., 82 (1988) 282-286. 33 Sakaguchi, T., Takahashi, M. and Bray, G.A., Lateral hypothalamus and sympathetic firing rate, Am. J. Physiol., 255 (1988) R507-R512. 34 Shibata, H., P6russe, F. and Bukowiecki, L.J., The role of insulin in nonshivering thermogenesis, Can. J. Physiol. Pharmacol., 65 (1986) 152-158. 35 Shimazu, T. and Takahashi, A., Stimulation of hypothalamic nuclei has differential effects on lipid synthesis in brown and white adipose tissue, Nature, 284 (1980) 62-63. 36 Szabo, A.J. and Szabo, O., Influence of the insulin sensitive central nervous system glucoregulator receptor on hepatic glucose metabolism, J. Physiol., 253 (1975) 121-133. 37 Szabo, A.J. and Szabo, O., Insulin injected into CNS structures or into the carotid artery: effect on carbohydrate homeostasis of the intact animal, Adv. Metabol. Disorders, 10 (1983) 385-399. 38 Szabo, O. and Szabo, A.J., Evidence for an insulin-sensitive receptor in the central nervous system, Am. J. Physiol., 223 (1972) 1349-1353.
Brain Research, 527 (1990) 326-329 Elsevier
BRES 24275
Short Communications
Insulin co-inject n suppmuel the ther microinjecUon into the
ic in rats
to pJtmlmte
Shimon Amir, Alessandra Schiavetto and Robin Pollock Centerfor Studies in Behavioral Neurobiology, Departmentof Psychology, Concordia University, Montreal, Que. (Canada)
(Accepted 29 May 1990) Key words: Insulin; Glutamate; Ventromediai hypothalamic nucleus; Brown adipose tissue; Thermogenesis; Rat
Selective stimulation of ventromedial hypothalamic nucleus (VMH) neurons by microinjection of the excitatory amino acid glutamate sharply increased interscapular brown adipose tissue (IBAT) and core temperatures in urethane-anaesthetized rats. This effect was blocked by co-injection of insulin (0.1-1/~g) though not an inactive insulin analog, T N B 3 insulin. Injection of insulin (1/~g) into the contralateral VMH or systemic administration of insulin (1/~g) had no effect on the thermogenic response to intra-VMH glutamate. These results complement those showing that intra-VMH insulin suppresses the basal firing rate of sympathetic nerves to IBAT and diminishes cold-induced nonshivering thermogenesis in BAT and add support to the view that insulin functions as an inhibitory signal on VMH neurons controlling thermogenesis in BAT.
Heat generation in brown adipose tissue (BAT) is influenced by insulin, but the mechanisms mediating the effect of insulin on BAT are incompletely understood 27. Insulin can influence heat production directly by influencing BAT metabolism 34, as well as indirectly, via effects in the central nervous system. An important central nervous system region concerned with neural control of BAT thermogenesis is the ventromedial hypothalamic nucleus (VMH) 25. In previous experiments, injection of insulin into the VMH in rats was found to suppress the basal firing rate of sympathetic nerves supplying BAT, suggesting that insulin has a direct inhibitory action on the neural outflow from the VMH to BAT 3°,32. However, the effect of intra-VMH insulin on heat production in BAT induced either by physiological or neural stimuli was not reported. Recently, we have shown that injection of insulin into the VMH suppresses cold-induced stimulation of BAT thermogenesis in rats 2. In the present study we examined the effect of intraV M H insulin on the thermogenic response resulting from direct stimulation of V M H neurons with the excitatory amino acid glutamate. In a previous study, we demonstrated that injection of glutamate into the VMH activates BAT thermogenesis via the sympathetic outflow, leading to a sharp increase in interscapular BAT (IBAT) and core temperatures 4. We report here that insulin co-injection blocks the thermogenic effect of intra-VMH
injection of glutamate in a direct and specific manner. Normally fed, male Wistar rats (275-325 g) were anaesthetized intraperitoneally with urethane (1.4 g/kg) and mounted in a stereotaxic instrument equipped with a thermostatically-controlled heating blanket calibrated to keep body temperature above 36 °C. A small incision was made 5 cm posterior to the scapulae and a thermistor probe (YSI 409B) was inserted and placed under the right lobe of IBAT. A second thermistor probe (YSI 401) was inserted 5 cm into the rectum. A 28 gauge needle connected to a Hamilton microsyringe containing glutamate (1 M), insulin (bovine insulin; Sigma) or glutamate plus insulin was then introduced into the right V M H using the following coordinates from the Paxinos and Watson 22 atlas: AP -2.5, L 0.6, V 9'.5. The position of the needles was later verified histologically. Interscapular BAT and core temperatures were monitored continuously using YSI telethermometers (model 41) connected to a flat bed recorder. Intra-VMH injection of saline (0.25 /~1, n = 4) or insulin (0.1, 0.5 or 1/~g, in 0.25/~1 saline; n = 4 for each dose of insulin) had no effect on basal IBAT or core temperatures of urethane-anaesthetized rats (data not shown). In contrast, intra-VMH injection of insulin suppressed the thermogenic response to intra-VMH co-injection of glutamate. Fig. 1 shows examples of the effect of injecting glutamate alone (0.25 ~tl of 1 M
Correspondence: S. Amir, Center for Studies in Behavioral Neurobiology, Department of Psychology, Concordia University, 1455 de Maisonneuve Blvd. West, Montreal, Que., Canada H3G IMS.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
327 I BAT •
1.50.
T GLU
I C;LU
r GLU
T I GLU INS, I Jig ( CONTRALATERALVMH)
1.00.
IBAT/BEFORE CORF_JBEFORE IBAT/AFTER CORE/AFTER
0.50. T GLU
T GLU+INS,0.1 Jag 0.00,
A
T otu
CONTRALATERAL
T T GLU
SYSTEMIC
TNB3
INSULIN
i
T GLU
~" GLU+INS,O.S~g
T GLU
T GLU+INS, 1Ug
INS, lug (SYSTEMIC)
T GLU
1" GLU+TNB3INS, 1 ~(J
i ' c L_ 10 min
Fig. 1. A typical example of the effect of intra-VMH injection of glutamate (0.25/~1 of 1 M solution), glutamate plus insulin (INS; 0.1, 0.5 or 1/~g) injected into the same VMH site, glutamate plus insulin (1/~g) injected into the contralateral VMH, glutamate plus systemic insulin (1 lzg) or glutamate plus TNB 3 insulin injected into the same VMH site (1 /~g) on IBAT and core temperatures in urethaneanaesthetized rats. In all experiments animals were first treated with glutamate alone; 60 min later they were given a second injection of glutamate together with one of the above insulin treatments.
solution) or glutamate plus insulin (0.1, 0.5 or 1/~g) into the V M H on IBAT and core temperatures. Injecting glutamate into the V M H led to a sharp and rapid increase in I B A T temperature, which was maximal at 8-12 min and returned to preinjection value after 25-35 min. Intra-VMH glutamate also increased core temperature;
150.
IBAT/BEFORE
10 o .
CORE,BEFORE IBAT/AFTER CORE/AFTER
? <]
0.50
•
0.00 • NCNE
0.1 u.g
0.5 u.g
Fig. 3. The effect of contralateral or systemic injection of insulin (1 /~g) or co-injection of TNB3 insulin (1/~g) on the changes in IBAT and core temperature (mean + SEM) induced by injection of glutamate (0.25 /A of 1 M solution) into the VMH in urethaneanaesthetized rats. Each group consisted of 8 rats.
1 I.tg
INSULIN
Fig. 2. The effect of intra-VMH insulin on the changes in IBAT and core temperature (mean __+ S.E.M.) induced by co-injection of glutamate (0.25/~1 of 1 M solution) in urethane-anaesthetized rats. Animals (n -- 10/group) were first given an intra-VMH injection of glutamate ('BEFORE'). Sixty min later they were reinjected with glutamate plus one of three doses of insulin ('AFTER'). The asterisks indicate significant difference from the corresponding 'BEFORE' value (t-test, P < 0.001).
the change in core temperature was less pronounced and never exceeded the increase in I B A T temperature. A second injection of glutamate, given 60 min after the first glutamate injection had a similar effect on I B A T and core temperature. In a separate series, co-injection of insulin (0.1 /~g) slightly, though significantly, attenuated the increase in IBAT and core temperatures induced by a second injection of glutamate into the VMH. Higher doses of insulin (0.5 and 1/~g), tested in separate groups of animals further suppressed the increase in IBAT and core temperatures induced by coinjected glutamate (Fig. 2). Intra-VMH injection of insulin (1/~g) had no effect on the increase in IBAT and core temperatures induced by glutamate injection into the contralateral V M H (Fig. 1). Similarly, systemic injection of insulin (1/~g) 5 min before injecting glutamate into the V M H had no effect on the increase in IBAT and core temperature (Fig. 1). Finally, intra-VMH injection of a biologically inactive insulin analog, TNB 3 insulin (1/~g), had no effect on the increase in IBAT and core temperatures induced by co-injection of glutamate (Fig. 1). The data on the effect of insulin injection into the contralateral VMH, of systemic injection of insulin, or of co-injection of TNB 3 insulin on the thermogenic response to intra-VMH injection of glutamate are shown in Fig. 3. The main function of BAT is to generate heat in response to thermoregulatory or metabolic challenges imposed by cold or overfeeding 5,9,26. The production of heat depends on activation of the sympathetic nervous system 16 and there is now ample evidence that the V M H is important for transducing thermal and metabolic signals to sympathetic activity and heat production in B A T 10'13-15'20'23'25"29'35. The V M H is also considered to be an important site of autonomic action of insulin. The VMH contains high concentration of insulin receptors 6,8 and insulin-sensitive neurons 21, and injection of insulin
328 into the VMH has been shown to influence the regulation of pancreatic insulin secretion and glucose production by the liver 12'36'37'38. More recent studies have shown that insulin might act in the VMH to modulate sympathetic efferent mechanisms controlling heat production in BAT. In these experiments, injection of insulin into the VMH in rats suppressed the basal firing rate of sympathetic nerves to BAT 3°. Insulin appeared to exert its inhibitory action via direct effect on VMH neurons, as prior selective destruction of VMH cells with the neurotoxin kainic acid prevented the effect of insulin on sympathetic activity. In a subsequent study, we found that injection of insulin into the VMH could attenuate cold-induced stimulation of BAT thermogenesis 2. Intra-VMH insulin had no effect on the thermogenic response to systemic norepinephrine administration, indicating that the effect was due to inhibitory action upon VMH neural elements concerned with communicating thermal signals to BAT and not due to activation of an inhibitory mechanism. More recently, we demonstrated that selective stimulation of VMH neurons with glutamate activates BAT thermogenesis in rats 4. This effect was mediated by the sympathetic system, as it could be prevented by pharmacological interventions that either block neurotransmission in sympathetic ganglia (i.e. chlorisondamine chloride) or inhibit norepinephrine stimulation of fladrenergic receptors (i.e., propranolol). In the present study, we found that intra-VMH co-injection of insulin at doses shown to reduce basal sympathetic activity in BAT or to diminish cold-induced stimulation of BAT thermogenesis (i.e. 0.5 and 1 ~tg)2'3° strongly attenuated the increase in IBAT temperature induced by intra-VMH injection of glutamate. Moreover, we obtained evidence suggesting that insulin exerts its inhibitory action via specific receptors. The possibility that insulin blocked the thermogenic response to intra-VMH glutamate by acting outside the insulin receptor complex was excluded by the demonstration that coinjection of TNB 3 insulin, an inactive insulin analog modified at regions of the molecule necessary for receptor interaction, had no effect on the thermogenic response to intra-VMH injection of glutamate. Finally, we determined that the inhibitory action involves direct effect on the neural elements stimulated by glutamate. The possibility that the suppressive action was mediated by an inhibitory efferent pathway from the VMH or that it was secondary to central or peripheral action on glucoregulation37 were excluded by the fact that neither the injection of insulin into the contralateral VMH nor systemic administration of the hormone had any effect. Taken together, the results show that insulin can block in a direct and specific manner VMH neural elements whose stimulation by glutamate causes sympathetic activation of thermoge-
nesis in BAT. These results, viewed in conjunction with earlier reports that intra-VMH injection of insulin suppresses basal sympathetic activity and cold-induced thermogenesis in BAT, are consistent with the notion that insulin acts in the VMH to modulate neural mechanisms regulating the outflow of thermogenic signals to BAT. The idea that insulin interacts with central mechanisms involved in the regulation of BAT thermogenesis is further supported by studies on the effect of injecting insulin into hypothalamic regions outside the VMH. A number of extra VMH regions have now been implicated in the central control of BAT thermogenesis, including the suprachiasmatic nucleus, paraventricular nucleus and lateral hypothalamic area 1"3'1s, and it has been shown that injecting insulin into these sites can modify sympathetic activity in BAT. For example, insulin injection into the suprachiasmatic nucleus was found to suppress basal sympathetic activity in BAT during the day, and to increase sympathetic activity during the night 3z. Injecting insulin into the paraventricular nucleus suppressed sympathetic activity in BAT 31. Insulin injection into the lateral hypothalamic area had a small stimulatory effect on sympathetic activity in BAT 33. Furthermore, injecting insulin into the cerebroventricular system suppressed basal firing rate of sympathetic nerves to BAT in a manner similar to that reported after intra-VMH injection of insulin. When insulin was coinjected with glucose, which in itself increases sympathetic activity, it appeared to exert an excitatory action on sympathetic activityH. Finally, it has been shown that insulin injection into the systemic circulation influences BAT thermogenesis in experimental animals; the effect of systemic insulin appeared to be mediated, in part, by the sympathetic system7'17"19"27"28'34. These diverse findings on the effect of centrally or systemically administered insulin, taken together with evidence that insulin from the systemic circulation can gain access to the central nervous system and affect the function of hypothalamic neurons 24, further support the view that insulin may function as a neuroendocrine signal to hypothalamic mechanisms regulating sympathetic activity and heat production in BAT. In summary, the present experiments demonstrate that injection of insulin into the VMH significantly attenuates the increase in IBAT and core temperatures that results from microinjection of glutamate into the same VMH site. These results further support the notion that insulin may function as an inhibitory signal on VMH neurons involved in the control of BAT. This study was supported by a grant from the Natural Sciences and Engineering Council of Canada.
329 1 Amir, S., Stimulation of the paraventricular nucleus with glutamate activates interscapular brown adipose tissue thermogenesis in rats, Brain Research, 508 (1990) 152-155. 2 Amir, S., Lagiorgia, M. and Pollock, R., Intra-ventromedial hypothalamic injection of insulin suppresses brown fat thermogenesis in the anaesthetized rat, Brain Research, 480 (1989) 340-343. 3 Amir, S., Shizgal, P. and Rompr6, P.-P., Glutamate injection into the suprachiasmatic nucleus stimulates brown fat thermogenesis in the rat, Brain Research, 498 (1989) 140-144. 4 Amir, S., Intra-ventromedial hypothalamic injection of glutamate stimulates brown adipose tissue thermogenesis in the rat, Brain Research, 511 (1990) 341-344. 5 Barnard, T., Brown adipose tissue as an effector of nonshivering thermogenesis, Experientia, 33 (1977) 1124-1126. 6 Baskin, D.G., Figlewicz, D.P., Woods, S.C., Porte, D. Jr. and Dorsa, D.M., Insulin in the brain, Annu. Rev. Physiol., 49 (1987) 335-347. 7 Christin, L., Nacht, C.-A., Vernet, O., Ravussin, E., Jequier, E. and Acheson, K.J., Insulin: its role in the thermic effect of glucose, J. Clin. Invest., 77 (1986) 1747-1755. 8 Havrankova, J., Brownstein, M. and Roth, J., Insulin and insulin receptors in rodent brain, Diabetologia, 20 (1981) 268-273. 9 Himms-Hagen, J. and Phil, D., Thermogenesis in brown adipose tissue as an energy buffer, New Engl. J. Med., 311 (1984) 1549-1558. 10 Holt, S.J., Wheal, H.V. and York, D.A., Hypothalamic control of brown adipose tissue in Zucker lean and obese rats. Effect of electrical stimulation of the ventromedial nucleus and other hypothalamic centres, Brain Research, 405 (1987) 227-233. 11 Holt, S.J. and York, D.A., Integration of intracerebroventricular insulin and glucose in the regulation of the activity of sympathetic efferent nerves to brown adipose tissue in lean and obese Zucker rats, Brain Research, 500 (1989) 384-388. 12 Iguchi, A., Durleson, P.D. and Szabo, A.J., Decrease in plasma glucose concentration after microinjection of insulin into VMH, Am. J. Physiol., 240 (1981) E95-E100. 13 Imai-Matsumura, K., Matsumura, K. and Nakayama, T., Involvement of ventromedial hypothalamus in brown adipose tissue thermogenesis induced by preoptic cooling in rats, Jpn. J. Physiol., 34 (1984) 939-943. 14 Imai-Matsumura, K., Matsumura, K., Tsai, C.-L. and Nakayama, T., Thermal responses of ventromedial hypothalamic neurons in vivo and in vitro, Brain Research, 445 (1988) 193-197. 15 Iwai, M., Shinomiya Hell, N. and Shimazu, T., Effect of ventromediai hypothalamic stimulation on blood flow of brown adipose tissue in rats, Pflager's Arch., 410 (1987) 44-47. 16 Landsberg, L., Saville, M.E. and Young, J.B., Sympathoadrenal system and regulation of thermogenesis, Am. J. Physiol., 247 (1984) E181-E189. 17 Larue-Acbaglotis, C., Goubern, M. and Laury, M.CI., Concomitant food intake and adipose tissue responses under chronic insulin infusion in rats, Physiol. Behav., 44 (1988) 95-100. 18 Lupien, J., Tokunaga, K., Kemnitz, J.W., Groos, E. and Bray, G.A., Lateral hypothalamic lesions and fenfluramine increase thermogenesis in brown adipose tissue, Physiol. Behav., 38
(1986) 15-20. 19 Menendez, J.A. and Atrens, D,M., Insulin increases energy expenditure and respiratory quotient in the rat, Pharmacol. Biochem. Behav., 34 (1989) 765-768. 20 Niijima, A., Rohner-Jeanrenaud, E and Jeanrenaud, B., Role of ventromedial hypothalamus on sympathetic efferents of brown adipose tissue, Am. J. Physiol., 247 (1984) R650-R654. 21 Oomura, Y. and Kita, H., Insulin acting as a neuromodulator through the hypothalamus, Diabetologia, Suppl. 20 (1981) 290-298. 22 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney, 1982. 23 Perkins, M.N., Rothwell, N.J., Stock, M.J. and Stone, T.W., Activation of brown adipose tissue thermogenesis by the ventromedial hypothalamus, Nature, 289 (1981) 401-402. 24 Posner, B.I., Insulin interaction with the central nervous system: nature and possible significance, Proc. Nutr. Soc., 46 (1987) 97-103. 25 Rothwell, N.J., Central control of brown adipose tissue, Proc. Nutr. Soc., 48 (1989) 197-206. 26 Rothwell, N.J. and Stock, M.J., A role for brown adipose tissue in diet-induced thermogenesis, Nature, 281 (1979) 31-35. 27 Rothwell, N.J. and Stock, M.J., Insulin and thermogenesis, Int. J. Obesity, 12 (1988) 93-102. 28 Rothwell, N.J., Stock, M.J. and Tedstone, A.E., Effects of ciglitazone on energy balance, thermogenesis and brown fat activity in the rat, Mol. Cell. Endocrinol., 57 (1987) 253-257. 29 Saito, M., Minokoshi, Y. and Shimazu, T., Brown adipose tissue after ventromedial hypothalamic lesions in rats, Am. J. Physiol., 248 (1985) E20-E25. 30 Sakaguchi, T. and Bray, G.A., Intrahypothalamic injection of insulin decreases firing rate of sympathetic nerves, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 2012-2014. 31 Sakaguchi, T. and Bray, G.A., Sympathetic activity following paraventricular injections of glucose and insulin, Brain Res. Bull., 21 (1988) 25-29. 32 Sakaguchi, T., Takahashi, M. and Bray, G.A., Diurnal changes in sympathetic activity: relation to food intake and to insulin injected into the ventromedial or suprachiasmatic nucleus, J. Clin. Invest., 82 (1988) 282-286. 33 Sakaguchi, T., Takahashi, M. and Bray, G.A., Lateral hypothalamus and sympathetic firing rate, Am. J. Physiol., 255 (1988) R507-R512. 34 Shibata, H., P6russe, F. and Bukowiecki, L.J., The role of insulin in nonshivering thermogenesis, Can. J. Physiol. Pharmacol., 65 (1986) 152-158. 35 Shimazu, T. and Takahashi, A., Stimulation of hypothalamic nuclei has differential effects on lipid synthesis in brown and white adipose tissue, Nature, 284 (1980) 62-63. 36 Szabo, A.J. and Szabo, O., Influence of the insulin sensitive central nervous system glucoregulator receptor on hepatic glucose metabolism, J. Physiol., 253 (1975) 121-133. 37 Szabo, A.J. and Szabo, O., Insulin injected into CNS structures or into the carotid artery: effect on carbohydrate homeostasis of the intact animal, Adv. Metabol. Disorders, 10 (1983) 385-399. 38 Szabo, O. and Szabo, A.J., Evidence for an insulin-sensitive receptor in the central nervous system, Am. J. Physiol., 223 (1972) 1349-1353.