Journal of the Autonomic Nervous System, 4 (1981) 195-205 Elsevier/North-Holland Biomedical Press

Research Papers

195

IWPOTHALAiiIC REGULAT EFFECT OF’HYPOTHALAJW

AKIRA TAKAHASHI and TAKASH’i SHIMAZU 1

Division of Neurochemistry, Pliychiatric Research Institute of Tokyo, Setagaya-ku, Tokyo 156, and Department of Medicat Biochemistry, Ekime University School of Medicine, Shigenobu, Ehime 791-W (Japan)

(Received November 5th, 1980) (Accepted March 7th, 1981)

Key words: ventromedial hypothalamic nucleus - lateral hypothalamic nucleus - sympathetic innervation - plasma glycerol - plasma free fatty acids - plasma lactate

ABSTRACT

In unanesthetized ratr,, electrical stimulation of the ventromedial hy- pothalamic nucleus (VMH) induced a marked increase in plasma concentra- tion of glycerol, but did not increase the plasma free fatty acid (FFA) con- centration, probably owing to a great elevation of plasma lactate which might inhibit the release of FFA Erom adipose tissue. In anesthetized rats, on stimulation of the VMH there was no remarkable increase in the plasma lac- tate, and the plasma glycerol and FFA concentrations were botk elevated markedly. Electrical stimulation of the lateral hypothalamic nucleus (LH), on the other hand, had no significant effects on plmma glycerol and FFA levels. Bilateral adrenodemedullation did not prevent the lipolytic response to VMH stimulation, although it reduced slightly the increment of plasma glycerol and FFA. However, the lipolytic response was completely blocked by previous treatment of tke animals with hexametkonium or propranolol, but not with pkentolamine. These results suggest that sympatketic innerva- tion of the adipose tissue is tke dominant factor involved in VMH-induced lipolysis in the rat, while the role of the adrenal medulla is subdominant; the effect of VMH stimulation is :mainly transmitted through the sympathetic nervous system to /3-adrenergic receptor of the adipose tissue.

-- 1 To whom reprint raquests should be addressed.

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INTRODUCTION

There is now ample evidence that the sympathetic nervous system partici- pates in regulation of lipolysis in mamm&. Adipose tissue is richly supplied by sympathetic nerve fibers, which are important in control of its metabolic activity, and the norepinephrine released from sympathetic nerve endings is of primary importance for the lipolysis in situations requiring increased sym- pathetic activity, such as during cold exposure, forced muscular activity, or starvation [6,17,26].

Recent evidence has also indicated that the ventromedial hypothalamic nucleus (VMH) is involved in sympathetic nervous regulation of fat mobiliza- tion in rabbits and rats [ 18,281. We hve shown that electrical stimulation of the VMH causes lipolysis in rabbits, detected by a marked increase in plasma glycerol level, whereas electrical stimulation of the lateral hypothalamic nucleus (LH) has no appreciable effect on fat mobilization [20]. More recently, Bray and Nishizawa have demonstrated that bilateral lesions of the VMH attenuate fat mobilization in rats after stress [25], and that unilateral abdominal sympathectomy or an ipsilateral VMH lesion impairs lipid mobili- zation during fasting but that an LH lesion is without effect [ 51.

In our previous experiments [ 203, however, electrical stimulation of the H in unanesthetized rabbits was not associated with an increase in plasma

free fatty acids (FFA) in spite of a marked elevation of plasma glycerol. Secondly, approximately 80% of plasma glycerol elevation elicited by VMH stimulation was eliminated by bilateral adrenalectomy, implicating that the adrenal medulla was principally involved in VMH-induced lipolysis and the sympathetic innervation of the adipose tissue was of secondary importance in thiis species. In the rabbit 5 epinephrine is 10 times more effective than norepinephrine in increasing plasma glycerol in vivo, although norepineph- rine is alss ,effective in lipolysis in vitro [ 213. In the rat, on the other hand, norepinephrine has lipolytic action as potent as epinephrine in vivo [ 6,271.

The studies to be described were designed to investigate more fully the role of the sympathetic innervation and of the adrenal medulla in the rat, and to explore the cause of absence of plasma FFA response to VMH stimulation in unanesthetized animals.

Animals and surgical procedures Female Wistar rats, weighing 21.0-250 g, we:ce used. They were main-

tained in a temperature-controlled (25 + 1°C) room with a 12 h light cycle ts on 8.00-20.00 h), and on laboratory chow and ats were anesthetized with sodium pentob

neally) and placed in a stereotaxic apparatus. insulated 3.60 p wire with a bared tip, were im unilaterally in the

or LH of each rat, as described previously cted to a small plug which was then anchor

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to tnz 9cull by screws ana acrylic resin. The position of tht! tip of the elec- e was verified microscopically in brain sections made after the experi-

ments. Two weeks after implantation of the electrodes, each rat was provided,

under pentobarbital anesthesia, with a heart catheter (a silicone tubing having o .d. of 1 .O mm and i.d. of 0.5 mm) inserted from the right anterior jugular vein and positioned in the right atrium. The residual segment of the silicone tubing was passed under the skin and pulled out from back of the neck. This method allows frequent sampling of the blood without disturbing the animals’ behavior in unanesthetized rats.

In 15 rats with the implanted VMHelectrodes, bilateral adrenodemedulla- tion was carried out by the dorsal approach, directly after implantation of the heart catheter.

Electrical stimulation of the hypothalamus and blood sampling One week after implantation of the heart catheter, electrical stimuli, con-

sisting of monophasic square pulses of 0.3 ms duration at 100 Hz with a current of 0.2 mA (for unanesthetized rats) or 0.3 mA (for rats anesthetized with pentobarbital, 40 mg/kg), were applied to the hypothalamus of rats. The stimulating current was increased for the anesthetized- rats, in con- sideration of weak response under anesthesia. Control rats had similarly implanted electrodes but no electrical stimuli were applied. An interval timer was connected to the stimulator to allow repeated application of stimuli for 30 s periods, once every minute during the 20-30 min period.

Approximately 0.3 ml samples of blood were withdrawn from the heart catheter into heparinized syringes, at intervals before and after hypothalamic stimulation. After each sample was taken, 0.3 ml of isotonic saline was injected through the catheter to reduce the effect of blood removal. The blood samples were transferred into ‘chilled centrifuge tubes containing a trace amount of solid heparin, which did not affect the subsequent assays. Plasma was then separated by centrifugation and kept frozen until assayed. Sampling of the blood was performed between 13.00 and 15.00 h.

Administration of blocking agents Hexamethonium bromide (Nakarai Chemical, Tokyo), propranolol hydro-

chloride (Sigma Chemical) and phentolamine hydrochloride (Ciba Geigy, Japan) were dissolved in isotonic saline and injected subcutaneously 30 min before electrical stimulation of the VMH, at the doses of 40 mg/kg, 10 mg/kg and 5 mg/kg, respectively.

Assay of plasma Plasma concentrations of glycerol, hrec fatty acids (FFA), lactate, and

glucose were determinted by tha methods of Wieland [ 361, Falholt et al. [9 J, Barker and Summerson [ 31, and Bergmeyer and Bernt [ 41, respectively. Glycerokinase and glycerophosphate dehydrogenase for the glycerol assay, and glucose oxidase for the glucose assay, were purchased from Boehringer

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Mannheim. Statistical analyses of the data were made by Student’s E-test. Differences were considered significant when P < 0.05.

RESULTS

Effects of electrical stimulation of the VMH and LH on plasma levels of glycerol, FFA and lactate in unanesthetized and anesthetized rats

Electrical stimulation of the VMH in unanesthetized rats caused a marked increase in plasma glycerol concentration: its level increased progressively with the time of YXH stimulation, and reached about 2.5 times the initial level at 30 min (Fig. 1). Plasma FFA concentration was expected to be increased by stimulation of the VMH, because FFA is another hydrolytic product of tr&lycerides. However, it was not elevated by VMH stimulation, possibly owing to enhanced utilization of FFA by peripheral tissues or to inhibition of FFA release from adipose tissue into the circulation. As reported previously [ 20,29,31], plasma glucose concentration also increased on VMH stimulation, its peak being attained earlier (in IO min) than that of glycerol level (data not shown). Electrical stimulation of the LH, on the other hand, had no appreciable effects on plasma glycerol, FFA and glucose concentrations.

0 IO 20 30

Time of stimulation (min 1

Fig. 1. Effects of electrical stimulation of the VMH and LH on plasma levels of glycerol and FFA in unanesthetized rats. Blood samples were obtained from rats at the times indicated during intermittent electrical stimulation of the V control rats without electrical stimulation (0). Numbers of VMH, n = 6; LH, n = 6;control, n = 5. Each point is mean k from the value before stimulation.

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Plasma lactate level, which is reported to affect the release of FFA from adipose tissue [l&23], was expected to increase after VMH stimulation, since electrical stimulation of the VMH in unanesthetized rats elicited excitement and muscu exercise. In fact, the concentration of plasma lac- tate increased rapidly d markedly with stimulation of the VMH in un- anesthetized rats (Fig. 2): the maximum level of about E&fold incre,ase was

S-10 min after the onset of stimulation, and the level declined &&wards. By contrast, in pentobarbital-anesthetized rats, there

was no remarkable incre in the plasma lactate on VMH stimulation. The effects of VMH stimulation on plasma glycerol and FFA concentra-

tions were then investigated in anesthetized rats, and the results are shown in Fig. 3. In these rats, the b ma glycerol and FFA were s’hghtly lower than the levels in unanesthetized rat+ probably reflecting the condition free from emotional and postural stress. On electrical stimulation of the VMH, both 1 1s increased remarkably: the increase in plasma gly- cerol level was in ro proportion to the time of stimulation, like that seen in unanesthetized rats, and the increase in plasma FFA level was also propor- tional to the time during the first 10 min; both glycerol and FFA levels declined gradually after cessation of the stimulation. These results indicate that stimulation of the VMH induces lipolysis in adipose tissue. The absence of increase of plasma FFA in unanesthetized rats is probably due to the marked elevation of plasma lactate which may inhibit the release of FFA into the circulation.

Role of the adrenal medulla in lipolysis induced by VMH stimulation

Fig. 3 also shows the effect of adrenodemedullation on VMH-induced lipolysis in anesthetized rats. The basal levels of plasma glycerol and FFA did not change appreciably one week after bilateral adrenodemedullation. The increases in plasma glycerol and FFA in response to VMH stimulation were not prevented by adrenodemedullation, although the magnitude of the increases was insignificantly reduced. At the end of 20 min stimulation, the increments of plasma glycerol and FFA in rats with adrenodemedullation were 71% and 77%, respectively, of those in intact rats. For confirmation, the effect of adrenodemedullation on the lzesponse of plasma glycerol to VMH stimulation was also studied in unanesthetized rats (Fig. 4). The plasma glycerol level in demedullated rats likewise respond+ed to the stimula- tion, but the plasma FFA did not. Thus, the e.drenal medullary secretion of catecholamines seems not to be a principal factor involved in lipolysis after VMH stimulation, but is rather a minor factor in the rat.

Effects of adrenergic blocking agents on lipolytic response to VMH stimula- tion

To examine whether VMH-induced lipolysis is mediated by the sympa- thetic nervous system, adrenergic, blocking agents were administered periph-

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i. 0 IO 20 30

Time IminI

Fig. 2. Changes in plasma lactate level on electrical stimulation of the VMH in unanesthe- tized and anesthetized rats. In anesthetized rats, sodium pentobarbital (40 mg/kg) was injected intraperitoneally 20 min before start of the experiment. Solid blocks on the abscissae indicate duration of intermittent stimulation of the VMH. Points plre means + S.E. of values in 5 rats. * Significantly different from the value before stimulation.

Fig. 3. Effects of electrical stimulation of the VMH on plasma glycerol and FFA levels in ) and adrenal demedullated ( ) rats under anesthesia. The levels of control ani-

mals (0) without electrical stimulation are also given. Bilateral adrenodemedullation was performed 1 week before stimulation. Solid blocks on the abscissae show duration of intermittent stimulation of the VMH. Points are means f S.E. of values in 5 animals. * Significantly different from the value before stimulation.

Time of stimulation hhl

Fig. 4. Effect of bilateral adrenodemedullation on the response of plasma glycerol to VMH stimulation in unanesthetized rats. Intermittent stimulation of the out during 30 min. Numbers of animals in each group were: intact with V

6; adrenodemedullated with VMH stimulation ( ), n = 5; intact control (O), n = 5. Each point is mean f SE. * Significantly different from the value before stimulation.

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erally. When the rats were treated with hexamethonium (40 mg/kg) (a ganglionic blocker), 30 min before hypoth in plasma glycerol and FFA in response

mic stimulation, the increases VMH stimulation were com-

pletely: blocked (Fig. 5). i&ration of hexamethonium alone had no signif&mt effects on the gests that the effect of VM

levels of glycerol and FFA. This result sug-

via sympathetic ganglia. ulation is mediated by the peripheral nerves

Moreover, pretreatment of the animals with propranok0 (10 mg/kg), a P-adrenergic blocking agent, almost completely suppre induced by VMH stimulation

the lipokysis (Fi .6). On the other hand, p~\sious injection

of phentolamine (5 mg/kg), an cu-adrenergic blocking agent, did not inhibit

5

Time (mid Time (mln) Fig. 5. Effect of hexamethonium on lipolytic response to stimulation of the V anesthetized rats. Hexamethonium (Hexa, 40 mg/kg) was injected subcutaneously 30 min before onset of VMH stimulation, Numbers in each group were: VMH stirnula- tion after treatment w h hexamethonium , VMH stimulation without hexa - methonium treatment ( ), n = 6; hexameth tment alone (A), n = 5. Each point is mean f SE. Solid blocks on the abscissae show duration of intermittent stir lulation of the VMH. * Significantly different from the value before stimulation.

Fig. 6. Effects of propranolol and phentolamine on lipoiytic response to stinnxlation of the VMH in anesthetized rats. Propranolol (Prop, 10 mg/kg) or phentolan ine (Phen,

ly 30 min before electrical stimulation of the VMH. were: VMH stimulation with propranolol treatment one (a), n = 4; VMH stimuiation with phentolamine

entolamine treatment alone (A), n = 4. Each point is mean * S.E. Solid blocks on the abscissae indicate duration of intermittent stimulation of the VMH. * Significantly different from the value before stimulation.

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the increase of plasma glycerol after VMH stimulation, although it reduced slightly the increment of plasma FF A in 20 min of stimulation. The reduced response of plasma FFA in phentolamine-treated rats may be related to a possible rise in plasma insulin which can stimulate esterificsltion of FFA and a consequent reduction in FFA release. The plasma insulin level is reported to be suppressed during VMH stimulation despite the rise in glucose level, but its suppressicn can be relieved by c-adrenergic blockage [ 141. Also, administration ~;-f ?hentolamine alone slightly increased the basal levels of plasma FFA and glycerol, which might be related to the decrease (about 20%) of plasma glucose concentration (data not shown.).

DISCUSSION

This study demonstrates that electrical stimulation of the VMH, but not the LH, causes lipolysis in rats, mainly through activation of the sympathetic innervation of adipose tissue and acting on P-adrenergic receptor. These findings complement and extend previous observations on the role of the central nervous system in fat mobilization [15,16,34], and support the recent ‘view that the ventromedial region of the hypothalamus acts as a regulatolry center for fat mobilization by modulating activation of the sym- pathetic nervous system [ 18,281, although a variant model of a potential role of the VMH in regulating sympathetic activity has been proposed [ 371.

Electrical stimulation of the VMH is thought to promote the release of catec’holamines from sympathetic nerve endings and1 the adrenal medulla [ 10,221, because the VMH is presumed to be part of the sympathetic neural component of :‘:ne hypothalamus [ 1,2,28]. We have shown previously that in the adult rabbit apprp?ximatek,r 80% of plasma glycerol elevation elicited by YMH stimulation is mediated by epinephrine released from the adrenal medulla through sympathetic innervation of this organ [ 20,221. Similarly, hyperuricemia induced by VMH stimulation in rats has been reported to depend in large part on the release of epinephrine from the adrenal medulla [ 331. However, the present findings indicate that in the rat the principal fac- tor involved in lipolysis after VMH stimulation is probably the sympathetic innervation of adipose tissue, and the adrenal medullary hormone is a minor factor, because adrenodemedullation impaired the VMH-induced lipolysis only about 2O---30% (Figs. 3 and 4). This difference in the mechanism of VMH-induced lipolysis may be due to species difference between the rabbit and rat. In the rabbit, rgrepinephrine has little lipolytic activity in vivo [ 213, whereas in the rat it has a potent lipolytic activity [ 273.

There is a good deal of evidence that direct stimulation of the sympathetic nerves causes fat mobilization from the adipose tissue. Electrical stimulation of the sympathetic nerve supply of the epididymal fat pads incubated in vitro results in rapid release of FFA and glycerol into the i [ 8,355. Fredholm and Rose11 [ 123 have shown that st~n~u~~t~~ pathetic nerves to subcutaneous adipose tissue increases th glycerol and FFA from the tissue perfused in situ, and

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stimulation is inhibited by P-adrenergic blocking agents and potentiated by cw adrenergic blockade. Their results are consistent with our present obserja- tions that the increased lipolysis after electrical stimulation of the V blocked by P-receptor blocking agent as w2ll as ganglionic blocking agent, but not by ~vreceptor blocking agent (Figs. 5 and 6). These results suggest t’btat the effect of VMH stimulation on lipolysis is mainly transmitted through the sympathetic nervous system to p-adrenergic receptor of the adipose tissue.

Electrical stimulation of the VMH also increases plasma level of glucagon [13,30] that is known to have lipolytic action on adipose tissue in vitro. However, a fairly large dose of gluc;xon is required for this effect, Con- versely, intravenous injection of glucegon into dogs decreases the plasma FFA level, which is probably attributed to an increased utilization of glucose consequent to the concomitant hyperglycemia induced by this hormone 1241.

Studies by Miller and co-workers [ 19,231 have indicated that lactate has a direct inhibitory effect on the release of FFA from adipose tissue in vivo: the effect does not require the presence of insulin and is independent of the concentration of plasma glucose. It has also been reported that increase of plasma lactate in concentrations such as those occurring during muscular exercise and shock, suppresses the rate of FF’A release from the subcutane- ous adipose tissue in situ without significantly affecting the glycerol release upon nerve stimulation [ll]. In our experiments, plasma lactate level was greatly increased by VMH stimulation in unanesthetized rats. However, in anesthetized rats, the plasma lactate was not so markedly increased due to immobility (Fig. 2). On VMH stimulation the plasma FF 2 level was found to increase in anesthetized rats, but not in unanesthetized rats (Figs. 1,3). This finding is compatible with our previous observation that VMH-induced lipolysis in unanesthetized rabbits is not accompanied by plasma FFA eleva- tion [ 201. Hence, it seems likely that the plasma lactate elevation after VMH stimulation inhibits the release of FFA from adipose tissue in unanesthetized animals, although the mechanism by which lactate suppresses the release of FFA remains to be established. In addition, anesthesia may change the hemodynamic response to stimulation of the VMH. It has been suggested that changes in blood flow through adipose tissue alter the rate of removal of FFA [7].

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