Ventromedial hypothalamic stimulation glucose uptake in anesthetized rats
enhances peripheral
MAKOTO SUDO, YASUHIKO MINOKOSHI, AND TAKASHI SHIMAZU Department of Medical Biochemistry, School of Medicine, Ehime University, Shigenobu, Ehime 791-02, Japan
SUDO, MAKOTO, YASUHIKO MINOKOSHI, AND TAKASHI SHIMAN. Ventromedial hypothalamic stimulation enhancesperiphera1 glucose uptake in anesthetized rats. Am. J. Physiol. 261 (Endocrinol. Metab. 24): E298-E303, 1991.-Effects of &ctri-
tissues can be augmented by electrical stimulation of the ventromedial nucleus of the hypothalamus (VMH); VMH stimulation increases not only lipogenic and thermogenic activities (14) but also blood flow (10) of BAT through sympathetic activation of this tissue. Norepinephrine turnover in BAT and other sympathetically innervated organs is accelerated by VMH stimulation, indicating that the VMH is the major hypothalamic site intimately associated with sympathetic facilitation in peripheral tissues (18). To explore the role of the sympathetic nervous system in glucose uptake and utilization of peripheral tissues, we assessed in the present study the rate constant (Ki) of tissue glucose uptake during electrical and chemical (with glutamate) stimulation of the VMH, according to the 2-deoxy-D-glucose (2-DG) method (9,24) in anesthetized rats. The results indicated that VMH stimulation significantly increased Ki of net glucose uptake preferentially in BAT, heart, and skeletal muscles.
cal and chemical stimulation of the ventromedial (VMH) and lateral hypothalamic (LH) nuclei on glucoseuptake in periphera1 tissues were studied by the 2-deoxy-D-[3H]glucose (2[“H]DG) method in anesthetizedrats. Electrical stimulation of the VMH increasedthe rate constant of glucose uptake in brown adipose tissue (BAT; 8 times), heart (3 times), and skeletal muscles(1.5 times) but not in white adipose tissue, diaphragm, and brain, without detectable changesin plasma insulin levels. Chemical stimulation of the VMH by microinjection of L-glutamate also enhancedthe rate constant of glucoseuptake in BAT, heart, and skeletal musclespreferentially, which indicates that the enhancement of glucoseuptake in these tissuesis derived from activation of VMH neurons. The increasedrate of glucoseuptake in BAT in responseto VMH stimulation was effectively suppressedby surgical sympathetic denervation, suggestinga mediation of the sympathetic nerve in this effect. On the other hand, electrical stimulation of the LH had no appreciable effect on 2-[3H]DG uptake in any tissues.It is concludedthat glucoseuptake in certain peripheral METHODS tissuesis acceleratedselectively by activation of VMH neurons, the action of which is independentof plasmainsulin but which Animals and surgical procedures. Female Spragueis probably via the sympathetic nervous system. Dawley rats (Nihon Clea, Tokyo, Japan) weighing 200280 g were used. They were housed in plastic cages at 25 brown adiposetissue;skeletal muscle;sympathetic nerve; ven- t 1°C with a 12:12-h light-dark cycle (lights on from tromedial hypothalamic nucleus;L-glutamate 0700 h) and were given laboratory chow and water ad RECENTSTUDIEShave revealed that tissue glucose uptake
in vivo is mediated via both insulin-dependent and insulin-independent pathways (7, 12). Cold exposure and physical exercise are the two major stimuli in increasing glucose uptake via the insulin-independent pathways. Cold exposure, for instance, markedly enhances glucose uptake in peripheral tissues, particularly in skeletal muscles and brown adipose tissue (BAT) in conscious rats (8, 20, 25), in spite of the fact that it decreases plasma insulin levels. Physical exercise is also known to be a powerful stimulus to glucose utilization by skeletal muscles without an intervention of insulin secretion (17,26). Considering that cold exposure and physical exercise induce an activation of the sympathetic nervous system (3, 4), it might be possible that the insulin-independent glucose uptake in some tissues depends on the sympathetic nervous activity. We have demonstrated in previous studies that sympathetic nervous activity in BAT and other peripheral E298
0193-1849/91
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libitum. Under pentobarbital sodium anesthesia (50 mg/ kg ip), bipolar electrodes made of insulated 100 pm wire were implanted unilaterally into the VMH or lateral hypothalamic area (LH) of rats according to the atlas of Pellegrino et al. (16). The stereotaxic coordinates used were: AP5.8, L0.7, and H9.5 for the VMH; AP5.8, L2.0, and H9.0, for the LH. The electrodes were connected to a small plug, which was then anchored to the skull with acrylic dental cement. Double-walled cannulas, which had outer and inner diameters of 0.4 and 0.2 mm, respectively, were also stereotaxically implanted into the unilateral VMH and were anchored firmly to the skull. The animals were used after 2 wk of recovery. The position of the tip of the electrodes or cannulas was verified microscopically in brain sections made when the experiments were completed. On the day of experiments the rats were anesthetized with pentobarbital sodium (50 mg/kg ip) and were implanted with a cardiac catheter via the right external jugular vein as described previously (19). In addition, unilateral sympathetic denervation of the interscapular
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HYPOTHALAMIC
EFFECTS
ON
BAT was performed by severing four branches of the nerves to the right side of the BAT as described (15). The rats were then transferred onto a heat pad kept at 37OC. Electrical and chemical stimulation of the hypothalamic nuclei and injection of radiotracers. One hour after the
operations described above, electrical or chemical stimulation was delivered. Electrical stimuli consisted of 30s trains of monophasic square pulses (0.1-0.3 ms in duration, 6 V amplitude, and 50 Hz frequency) applied to the VMH or LH once every minute. For chemical stimulation, 100 nmol of monosodium L-glutamate dissolved in 1~1 saline solution were injected into the VMH through the implanted cannulas using the Hamilton microsyringe. To obtain a steady-state level of plasma glucose concentration during the experimental period (see Figs. 1 and 2), the hypothalamus had been stimulated electrically or chemically for 15 min before injection of radiotracers. After a 15min period of the hypothalamic stimulation, each rat was injected with 50 &i of 2deoxy-D-[3H]glucose (2-[3H]DG) and 5 &i of [‘“Clsucrose (ICN Radiochemicals, Irvine, CA) dissolved in 0.4 ml saline solution through the cardiac catheter. The catheter was immediately flushed with 0.3 ml of saline, and the hypothalamic stimulation was continued for another 15min period. Control rats for electrical stimulation had similarly implanted electrodes, but no electrical stimuli were applied, and controls for chemical stimulation were injected with 1 ~1 of saline into their VMH. Sampling. Blood samples (0.1 ml) were taken at min -15, -12, -9, -6, -3, 0, 5, 7, 9, 11, 13, and 15 before and after injection of the tracers and were replaced with an equivalent volume of saline. As soon as the last blood sample was obtained, pentobarbital sodium (100 mg/kg) was injected through the cardiac catheter, and the rats were quickly decapitated. The following tissues and organs were carefully dissected and freed of all extraneous materials as soon as possible: brain (cortex), heart (left ventricle), diaphragm, parametrial white adipose tissue (WAT), interscapular BAT, and skeletal muscles [quadriceps, gastrocnemius, soleus, and extensor digitorum longus (EDL)]. All tissues were rapidly frozen in liquid nitrogen, weighed, and solubilized in 0.5 ml of 0.5 N NaOH at 80°C for 1 h. The solution was added with 1 ml of 3% perchloric acid and centrifuged. The radioactivities of 3H and 14C in 1 ml of the supernatant were measured in a liquid scintillation fluid (ACS-II; Amersham) using Tri-Carb scintillation counter (Hewlett-Packard; model 300 C). Plasma samples were also analyzed for radioactivities of 2-[3H]DG and [ 14C]sucrose as well as for glucose levels (by a specific glucose oxidase method). Additionally, plasma samples before and after hypothalamic stimulation were analyzed for insulin levels by radioimmunoassay (insulin RIA kit; Midori-Jyuji, Tokyo, Japan). Measurement of Ki of net tissue uptake of 2-r3H]DG.
The mathematical model used to calculate the rate of 2[3H]DG uptake had been previously reported by Horn et al. (9). This model is now only available to provide an estimate of the net rate of 2-DG uptake over a finite period of time for a wide variety of peripheral tissues. It
TISSUE
GLUCOSE
UPTAKE
E299
has been demonstrated that the uptake rates of 2-[3H]DG were already maximal after 10 min in most tissues and that the loss of label from the tissues (transport out before phosphorylation) was negligible until 30 min after injection (9). Therefore, we chose 15 min as the optimum time for death in the present experiments. This also ensured that pharmacokinetic analysis of the glucose analogue was calculated from the linear portion of the elimination curve of 2- [3H]DG in plasma for all groups of animals. Ki of net tissue uptake of 2-[3H]DG was calculated using the following equation K i- -
C*K, C, (1 - e+ft)
where C is the intracellular concentration of 2- [3H]DG (disintegrations per min per mg tissue) at death, Kp is Ki of plasma disappearance of 2-[3H]DG, CpOis the extrapolated plasma 2- [3H]DG concentration at time 0, and t is the duration of the test, i.e., 15 min. Kp was evaluated from the slope provided by a linear regression analysis after logarithmic conversion of the data and was calculated using the equation Kp = slope X -2.303. C was calculated from the radioactivities of 3H and 14C in the plasma and tissues as reported previously (9, 25). Statistics. All values are expressed as means t SE. The effects of the different treatments on all data were evaluated with factorial analysis of variance. When a significant effect was found, these results were further compared with the Newman-Keuls multiple range test. A best-fit line for plasma levels of 2- [3H]DG was provided by linear regression analysis from which the corresponding slope was obtained. The difference was considered significant if P < 0.05. RESULTS
Effects of electrical and chemical stimulation of the hypothalamus on plasma glucose and insulin levels and plasma disappearance of 2-r3H]DG. When the VMH was
stimulated electrically or chemically with L-glutamate, the plasma levels of glucose increased significantly (Figs. 1A and 2A, and Table 1) as reported previously (5, 22). In rats with electrical stimulation of the VMH, plasma glucose concentration reached a steady-state level of -180 mg/dl after 12 min of intermittent stimulation, and, judging from that, the rate of change in glucose was 0.3 t 0.5 mg l all-l . min-’ during VMH stimulation after 12 min (Fig. lA), whereas the rate was 0.5 t 0.4 mg* dl-lo min-’ in unstimulated controls. Similarly, the level reached a steady state of -155 mg/dl (the rate of change in glucose was within 0.2 mg*dl-’ emin-‘) 12 min after microinjection of glutamate into the VMH and remained increased during the additional period, whereas it did not change detectably after saline injection into the same hypothalamic region (Fig. 2A). In contrast, the plasma glucose levels remained fairly constant during electrical stimulation of the LH (Fig. lA, Table 1). Plasma insulin concentration did not change significantly after electrical and chemical stimulation of either the VMH or the LH (Table 1). The time courses of decrease in radioactivity of plasma
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HYPOTHALAMIC
E300
A
l
VMH
A LH
stimulation
EFFECTS ON TISSUE GLUCOSE UPTAKE T
T
-r
stimulation
l 0
Chemical Control
stimulation
of
VMH
0 Control
stim.
r/”
t ; chemical
- 15 - 10 Time
n 2 g 75,000
- 5
after
0
injection
B
-2 n
5
10
of Z- [3H] DG (mid
A
E 50,000 g
: 45
Time
’
1
I
I
I
I
5
7
9
11
13
15
after
injection
Time
n 2 g 75,000
VMH stimulation A LH stimulation 0 Control
A
- 15 - 10 - 5
15
l
of 2- [3H] DG (min)
FIG. 1. Effects of electrical stimulation of ventromedial hypothalamic nucleus (VMH) and lateral hypothalamic nucleus (LH) on plasma glucose levels (A) and 2-deoxy-D-[3H]glucose (2-[“HIDG) disappearance (B). VMH or LH of anesthetized rats was stimulated electrically for 30 min. Electrical stimuli consisting of 30-s trains of monophasic square pulses (0.1-0.3 ms in duration, 6 V amplitude, and 50 Hz frequency) were applied once every minute. After 15-min period of hypothalamic stimulation (time 0) when plasma glucose concentration attained steady-state level, each rat was injected through cardiac catheter with 50 &i of 2-[:‘H]DG and 5 &i of [‘4C]sucrose, and animal was killed as soon as last blood sample was obtained (15 min). Control rats had similarly implanted electrodes, but no electrical stimuli were applied. Results are means & SE for 6-10 rats.
2-[“H]DG after a single injection of the glucose analogue with or without hypothalamic stimulation are shown in Figs. 1B and 2B. When plotted on a semilogarithmic scale, the plasma 2-[3H]DG disappearance curves were linear in all groups of animals. Although the total area under the 2-[3H]DG disappearance curves tended to decrease in rats with either electrical (Fig. 1B) or chemical stimulation (Fig. 2B) of the VMH, as compared with respective controls, there was no significant difference in the rate of plasma 2-[3H]DG disappearance (I&) between the VMH-stimulated and control groups. Electrical stimulation of the LH also did not alter the rate of 2- [ “H]DG disappearance from plasma (Fig. 1B). Effects of electrical stimulation of the VMH and LH on tissue glucose uptake. Ki of net tissue glucose uptake
obtained under pentobarbital sodium anesthesia for brain, diaphragm, WAT, BAT, heart muscle, and skeletal muscles are given in Figs. 3 and 4. A comparison of the
stimulation
; 25,000 3 5 a. 0
after
injection
B
45
0 I
I
5
7
9
after
5
10
15
of 2- [3 HI DG (min)
l Chemical 0 Control
’
Time
0
stimulation
I
injection
11
of
VMH
I
I
13
15
of 2- r3H] DG (min)
FIG. 2. Effects of chemical stimulation of VMH on plasma glucose levels (A) and 2-[“H]DG disappearance (B). VMH of anesthetized rats was stimulated chemically by microinjection of 1~1 of saline containing 100 nmol of L-glutamate. To obtain steady-state level of plasma glucose concentration during study of 2-[3H]DG elimination, radiotracers (50 &i of 2-[“H]DG and 5 &i of [‘4C]sucrose) were injected intravenously 15 min after microinjection of L-glutamate (time 0). Animals were killed as soon as last blood sample was obtained (15 min). Control rats were given 1 ~1 of saline into their VMH. Results are means & SE for 6 rats.
TABLE 1. Effects of hypothalamic stimulation on plasma glucose and insulin levels in anesthetized rats Electrical Stimulation
Plasma glucose, Prestimulation Poststimulation Plasma insulin, Prestimulation Poststimulation
VMH Chemical Stimulants
Control
VMH
LH
Saline
103k6 llOt5
108t5 189tll*
lOlt6 111&7
119t13 119&8
3223 3Ok3
33t2 31k2
34k6 36k4
321k3 32k2
Glutamate
mg/dl 115t6 156&15*
pU/ml 3722 35t2
Values are means * SE for 6-10 rats. VMH, ventromedial hypothalamic nucleus; LH, lateral hypothalamic nucleus. Experimental conditions are as in Figs. 1 and 2. * Plasma concentration after hypothalamic stimulation (15 min) was significantly different from that before stimulation (-15 min) at P C 0.01.
Ki values among these tissues revealed that of brain, heart muscle, and diaphragm (from x 10D3/min) were one order of magnitude the Ki of skeletal muscles, BAT, and WAT
the basal Ki 14.6 to 28.3 higher than (from 1.6 to
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HYPOTHALAMIC
EFFECTS
ON
TISSUE
7.7 X 10B3/min). Electrical stimulation of the VMH increased glucose uptake in BAT, heart,, and skeletal muscles preferentially. In BAT and heart muscle, the Ki values were increased by electrical stimulation of the VMH about eight times (from 2.9 to 24.4 X 10B3/min) and three times (from 18.7 to 56.4 X 10D3/min), respectively (Fig. 3). The increased value of Ki in BAT after VMH stimulation was completely suppressed by local sympathetic denervation, and the value in the denervated side of BAT was not significantly different from the control value. The increased Ki values after VMH stimulation were also found in four different types of skeletal muscles (Fig. 4). The EDL and soleus muscles have been known to be mainly composed of fast-twitch glycolytic and slowtwitch oxidative fibers, respectively, and the quadriceps and gastrocnemius muscles are composed of mixed-type fibers. Electrical stimulation of the VMH enhanced the Ki values of these skeletal muscles by -1.5 times (P < 0.01 in all of the skeletal muscles investigated). In contrast, electrical stimulation of the VMH had no appreciable effects on the Ki values in WAT, diaphragm, and brain (Fig. 4). Electrical stimulation of the LH, on the other hand, did not appreciably affect 2- [3H]DG uptake in any tissues investigated (Figs. 3 and 4).
I]
GLUCOSE Control
E301
UPTAKE VA
Quadri
LH
VMH
stimulation
Gastro
Soleus
stimulation
EDL
Effect of chemical stimulation of the VMH on glucose uptake in peripheral tissues. To confirm that neurons of
the VMH are specifically concerned in hypothalamic regulation of glucose uptake in peripheral tissues, we next examined the effect of chemical stimulation of the VMH by microinjection of glutamate, a well-known excitatory neurotransmitter in the central nervous system (6). Microinjection of L-glutamate (100 nmol in 1 ~1 saline) into the VMH significantly increased the Ki values in skeletal muscles to the similar extent to those seen after electrical stimulation (from 7.1 to 7.6 X 10B3/min in controls to from 10.6 to 11.2 X 10e3/min in rats with glutamate injection) (Fig. 5). Chemical stimulation of the VMH a Is0 increased the Ki values in BAT and heart BAT
Heart
30
x.*.
.*. x
60
T 20
Diaphragm
Brain
c 0.01.
muscle about 3 and 2.5 times, respectively. The effect on the Ki value in BAT was likewise abolished by local sympathetic denervation. However, chemical stimulation of the VMH with glutamate did not significantly alter the Ki values in brain, diaphragm, and WAT, as compared with the saline-injected controls (data not shown). DISCUSSION
10
Innervated
Denervated 0
Control
a
LH
stimulation
VMH
stimulation
3. Effect of electrical stimulation of hypothalamus on rate constant of 2-[“H]DG uptake in vivo in brown adipose tissue (BAT) and heart in anesthetized rats. Rate constant of 2-[‘H]DG uptake was calculated as described in METHODS. One hour before start of electrical stimulation, right side of interscapular BAT of anesthetized rats was surgically denervated, and electrical stimuli were given intermittently to VMH or LH. Other experimental conditions are as in Fig. 1. Results are means t SE for 6 rats. * Significantly different from controls without stimulation at P < 0.01. FIG.
WAT
4. Effect of electrical stimulation of hypothalamus on rate constant of 2-[:‘H]DG uptake in vivo in skeletal muscles, white adipose tissue (WAT), diaphragm, and brain in anesthetized rats. Four different hindlimb muscles, quadriceps (quadri), gastrocnemius (gastro), soleus, and extensor digitorum long-us (EDL) were sampled. Parametrial WAT and cerebral cortex were sampled for WAT and brain, respectively. Experimental conditions are as for Fig. 1. Results are means t SE for 6 rats. * Significantly different from controls without stimulation at P FIG.
The present study demonstrates for the first time that glucose uptake and utilization in BAT, heart, and skeletal muscles are markedly increased by selective stimulation of the VMH. Because it has already been established that hepatic glucose output can be affected by electrical and chemical stimulation of the hypothalamus (2l), the present findings indicate that not only glucose production but also its utilization in certain tissues are accelerated by stimulation of the VMH. By contrast, stimulation of the LH has no significant effect on glucose uptake in any tissues examined, although it has been known to enhance glycogen synthesis in the liver (21). The question of whether the effect of VMH stimulation on tissue glucose uptake originates from excitation of cell bodies of VMH neurons or from activation of axons
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E302
HYPOTHALAMIC I[
Quadri
Saline
Gastro
m
BAT
T
ON TISSUE
L- glutamate
EDL
Soleus
3%
EFFECTS
Heart %
80 60 40 20
0
Denervated Innervated
0
FIG. 5. Effect of chemical stimulation of VMH on rate constant of 2[“H]DG uptake in vivo in skeletal muscles, BAT, and heart muscle. Four different hindlimb muscles, quadriceps, gastrocnemius, soleus, and EDL were sampled. One hour before microinjection of L-glutamate (100 nmol) into VMH, right side of interscapular BAT of anesthetized rats was surgically denervated. Control rats were injected with same volume (1 ~1) of saline into VMH. Experimental conditions are as for Fig. 2. Results are means ~frSE for 6 rats. * Significantly different from saline controls at P < 0.01.
region has been resolved of passage in this hypothalamic with L- *glutamate, which bY using chemica 1 stimulation is known as the excitatory neurotransmitter in the central nervous system (6). This kind of study has revealed that chemical stimulation of the VMH reproduces the effects similar to those observed after electrical stimulation, providing evidence for the participation of specific neurons of the VMH in the hypothalamic regulation of tissue glucose uptake. The slightly less effects on glucose uptake and the blood glucose levels observed with chemical stimulation than with electrical stimulation of the VMH can be due to a submaximal stimulation of the VMH neurons. The effect of VMH stimulation on tissue glucose uptake is most probably mediated by the sympathetic nerve activity because the VMH is regarded as a hypothalamic component of the sympathetic nervous system (18, 21). Indeed, the increase in Ki of glucose uptake in BAT in response to electrical and chemical stimulation of the VMH was effectively prevented by surgical sympathetic denervation of BAT. Similarly, our previous studies have shown that lipogenic and thermogenic responses of BAT to VMH stimulation are almost completely abolished by local sympathetic denervation (14). In keeping with these
GLUCOSE
UPTAKE
observations, Marette and Bukowiecki (13) recently reported that physiological concentrations of norepinephrine enhanced glucose transport in isolated BAT, indicating the presence of non-insulin-mediated pathways for increasing glucose uptake in BAT. In the present study, we did not examine the possible effect of denervation on the increased glucose uptake in muscles after VMH stimulation. However, this might be mediated likewise by the sympathetic nervous system, since skeletal muscles have been recognized to be supplied with noradrenergic sympathetic axons that are distributed to the muscle spindles and extrafusal muscle fibers (2). It has also been shown that sodium and potassium transport in rat skeletal muscle during hypokalemia is regulated by the sympathetic nervous system and that this regulation (suppression of the Na+-K+ pump) is achieved by the apparent release of norepinephrine on muscle after nerve activity (1). Although glucose is known to stimulate its own transport across the plasma membrane, the accelerated rate of glucose uptake in certain tissues in response to VMH stimulation seems unlikely to be simply the result of mass action of the elevated blood glucose on the basis of the following reasons. First, Ki of 2-DG uptake per se more than the glucose metabolic rate was increased by VMH stimulation. Second, the increase in Ki was observed preferentially in BAT, heart, and skeletal muscles but not in white adipose tissue, diaphragm, and brain; if the effect were induced by the mass action of the elevated blood glucose, the Ki value would have been increased in all tissues. Third, the accelerated rate of glucose uptake in BAT was almost totally suppressed in the denervated side of the tissue, despite the presence of hyperglycemia during electrical and chemical stimulation of the VMH. The most likely cause of the accelerated glucose uptake by particular tissues is an enhanced rate of membrane transport of this substrate. However, further experiments are needed to ensure that glucose transporters are actually activated by stimulation of the VMH and efferent sympathetic nerves, particularly in muscles. The effect of VMH stimulation is not mediated by insulin ., because plasma insulin conce ntration ha .S been shown to remain unchanged or rather suppressed d uring VMH stimulation (5,23). In fact, electrical and chemical stimulation of the VMH did not increase the plasma levels of insulin (Table 1). Instead, the results of the present study indicate that the effect of VMH stimulation on tissue glucose uptake is probably mediated by the sympathetic nerve and thus depends on an insulinindependent mechanism of glucose transport that may operate simultaneously in the insulin-sensitive tissues like muscle and BAT but not in WAT and diaphragm. The observed effects on tissue glucose uptake could conceivably be related, at least in part, to a change in blood flow. Indeed, because VMH stimulation has been reported to increase regional blood flows of BAT and skeletal muscle (lo), a possible opening of the capillary beds may contribute to the accelerated glucose uptake observed in these tissues after VMH stimulation. Similarly, the contribution of blood flow to an increased glucose uptake in leg skeletal muscle has recently been observed both in lean and obese subjects with regard to
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HYPOTHALAMIC
EFFECTS
ON TISSUE
insulin action (11). Finally, although, in the present study, the VMH was stimulated under anesthesia of the animals to prevent muscular activity, the possibility remains that VMH stimulation increases intracellular glucase disposal by accelerating glycolysis and glucose oxidation in skeletal muscles and other sympathetically innervated tissues, which could also contribute to the enhanced glucose uptake in these tissues on VMH stimulation. We thank Drs. T. Shinomiya and H. Shibata for helpful discussion in initiating this study and M. Miyoshi for typing the manuscript. This work was supported by a Grant-in-Aid for scientific research from the Ministry of Education, Science, and Culture of Japan and by a Grant from the Health Science Promotion Foundation. Address for reprint requests: T. Shimazu, Dept. of Medical Biochemistry, Ehime Univ. School of Medicine, Shigenobu, Ehime 701-02, Japan. Received 29 May 1990; accepted in final form 29 April 1991. REFERENCES N. Sodium pump in skeletal muscle: central nervous system-induced suppression by a-adrenoreceptors. Science Wash. DC 213: 1252-1254,198l. BARKER, D., AND M. SAITO. Autonomic innervation of receptors and muscle fibres in cat skeletal muscle. Proc. R. Sot. Lond. B Biol. Sci. B212: 317-332,198l. CHRISTENSEN, N. J., AND H. GALBO. Sympathetic nervous activity during exercise. Annu. Reu. Physiol. 45: 139-153, 1983. DULLOO, A. G., J. B. YOUNG, AND L. LANDSBERG. Sympathetic nervous system responses to cold exposure and diet in rat skeletal muscle. Am. J. Physiol. 255 (Endocrinol. 1Metab. 18): E180-E188, 1988. FROHMAN, L. A., AND L. L. BERNARDIS. Effect of hypothalamic stimulation on plasma glucose, insulin, and glucagon levels. Am. J. Physiol. 221: 1596-1603, 1971. GOODCHILD, A. K., R. A. L. DAMPNEY, AND R. BANDLER. A method for evoking physiological responses by stimulation of cell bodies, but not axons of passage, within localized regions of the central nervous system. J. Neurosci. Methods 6: 351-363, 1982. GOTTESMAN, I., L. MANDARINO, AND J. GERICH. Estimation and kinetic analysis of insulin-independent glucose uptake in human subjects. Am. J. Physiol. 244 (Endocrinol. Metab. 7): E632-E635, 1983. GRECO-PEROTTO, R., D. ZANINETTI, F. ASSIMACOPOULOS-JEANNET, E. BOBBIONI, AND B. JEANRENAUD. Stimulatory effect of cold adaptation on glucose utilization by brown adipose tissue. Relationship with changes in the glucose transporter system. J. Biol. AKAIKE,
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F. G., C. J. GOODNER, AND M. A. BERRIE. A [3H]2-deoxyglucose method for comparing rates of glucose metabolism and insulin responses among rat tissues in vivo. Validation of the model and the absence of an insulin effect on brain. Diabetes 33: 141-152, 1984.
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10. IWAI, M., N. S.
HELL, AND T. SHIMAZU. Effect of ventromedial hypothalamic stimulation on blood flow of brown adipose tissue in rats. Pfluegers Arch. 410: 44-47, 1987. 11 LAAKSO, M., S. V. EDELMAN, G. BRECHTEL, AND A. D. BARON. ’ Decreased effect of insulin to stimulate skeletal muscle blood flow in obese man. A novel mechanism for insulin resistance. J. CZin. Inuest. 85: 1844-1852, 1990. LAVELLE-JONES, M., M. H. SCOTT, 0. KOLTERMAN, A. R. MOOSA, 12. AND J. M. OLEFSKY. Non-insulin-mediated glucose uptake predominates in postabsorptive dogs. Am. J. Physiol. 252 (Endocrinol. Metab. 15): E660-E666, 1987. 13. MARETTE, A., AND L. J. BUKOWIECKI. Stimulation of glucose transport by insulin and norepinephrine in isolated rat brown adipocytes. Am. J. Physiol. 257 (Cell Physiol. 26): C714-C721,1989. 14. MINOKOSHI, Y., M. SAITO, AND T. SHIMAZU. Sympathetic denervation impairs responses of brown adipose tissue to VMH stimulation. Am. J. Physiol. 251 (Regulatory Integrative Comp. Physiol. 20): R1005-R1008,1986. 15. MINOKOSHI, Y., M. SAITO, AND T. SHIMAZU. Metabolic and morphological alterations of brown adipose tissue after sympathetic denervation in rats. J. Auton. New. Syst. 15: 197-204, 1986. 16. PELLEGRINO, L. J., A. S. PELLEGRINO, AND A. J. CUSHMAN. A Stereotaxic Atlas of the Rat Brain. New York: Plenum, 1979. 17. RICHTER, E. A., T. PLOUG, AND H. GALBO. Increased muscle glucose uptake after exercise. No need for insulin during exercise. Diabetes 34: 1041-1048, 1985. AND T. SHIMAZU. Accelerated norepi18. SAITO, M., Y. MINOKOSHI, nephrine turnover in peripheral tissues after ventromedial hypothalamic stimulation in rats. Bruin Res. 481: 298-303, 1989. 19. SAITO, M., Y. MINOKOSHI, AND T. SHIMAZU. Metabolic and sympathetic nerve activities of brown adipose tissue in tube-fed rats. Am. J. Physiol. 257 (Endocrinol. Metab. 20): E374-E378, 1989. 20. SHIBATA, H., F. P~~RUSSE, A. VALLERAND, AND L. J. BUKOWIECKI. Cold exposure reverses inhibitory effects of fasting on peripheral glucose uptake in rats. Am. J. Physiol. 257 (Regulatory Integrative Comp. Physiol. 26): R96-RlOl, 1989. 21. SHIMAZU, T. Neuronal regulation of hepatic glucose metabolism in mammals. Diabetes Metab. Rev. 3: 185-206, 1987. 22 SHIMAZU, T., A. FUKUDA, AND T. BAN. Reciprocal influence of the . ventromedial and lateral hypothalamic nuclei on blood glucose level and liver glycogen content. Nature Lond. 210: 1178-1179, 23 1966. SHIMAZU, T., AND K. ISHIKAWA. Modulation by the hypothalamus of glucagon and insulin secretion in rabbits: studies with electrical and chemical stimulation. Endocrinology 108: 605-611, 1981. 24* SOKOLOFF, L., M. REIVICH, C. KENNEDY, M. H. DES ROSIERS, C. S. PATLAK, K. D. PETTIGREW, 0. SAKURADA, AND M. SHINOHARA. The [14C]deoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure and normal values in the conscious and anesthetized albino rat. J. Neurochem. 28: 897-916, 1977. 25. VALLERAND, A. L., F. PI~RUSSE, AND L. J. BUKOWIECKI. Cold exposure potentiates the effect of insulin on in vivo glucose uptake. Am. J. Physiol. 253 (Endocrinol Metab. 16): E179-E186, 1987. 26. WALLBERG-HENRIKSSON, H., AND J. 0. HOLLOSZY. Contractile activity increases glucose uptake by muscle in severely diabetic rats. J. Appl. Physiol. 57: 1045-1049, 1984. l
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enhances peripheral
MAKOTO SUDO, YASUHIKO MINOKOSHI, AND TAKASHI SHIMAZU Department of Medical Biochemistry, School of Medicine, Ehime University, Shigenobu, Ehime 791-02, Japan
SUDO, MAKOTO, YASUHIKO MINOKOSHI, AND TAKASHI SHIMAN. Ventromedial hypothalamic stimulation enhancesperiphera1 glucose uptake in anesthetized rats. Am. J. Physiol. 261 (Endocrinol. Metab. 24): E298-E303, 1991.-Effects of &ctri-
tissues can be augmented by electrical stimulation of the ventromedial nucleus of the hypothalamus (VMH); VMH stimulation increases not only lipogenic and thermogenic activities (14) but also blood flow (10) of BAT through sympathetic activation of this tissue. Norepinephrine turnover in BAT and other sympathetically innervated organs is accelerated by VMH stimulation, indicating that the VMH is the major hypothalamic site intimately associated with sympathetic facilitation in peripheral tissues (18). To explore the role of the sympathetic nervous system in glucose uptake and utilization of peripheral tissues, we assessed in the present study the rate constant (Ki) of tissue glucose uptake during electrical and chemical (with glutamate) stimulation of the VMH, according to the 2-deoxy-D-glucose (2-DG) method (9,24) in anesthetized rats. The results indicated that VMH stimulation significantly increased Ki of net glucose uptake preferentially in BAT, heart, and skeletal muscles.
cal and chemical stimulation of the ventromedial (VMH) and lateral hypothalamic (LH) nuclei on glucoseuptake in periphera1 tissues were studied by the 2-deoxy-D-[3H]glucose (2[“H]DG) method in anesthetizedrats. Electrical stimulation of the VMH increasedthe rate constant of glucose uptake in brown adipose tissue (BAT; 8 times), heart (3 times), and skeletal muscles(1.5 times) but not in white adipose tissue, diaphragm, and brain, without detectable changesin plasma insulin levels. Chemical stimulation of the VMH by microinjection of L-glutamate also enhancedthe rate constant of glucoseuptake in BAT, heart, and skeletal musclespreferentially, which indicates that the enhancement of glucoseuptake in these tissuesis derived from activation of VMH neurons. The increasedrate of glucoseuptake in BAT in responseto VMH stimulation was effectively suppressedby surgical sympathetic denervation, suggestinga mediation of the sympathetic nerve in this effect. On the other hand, electrical stimulation of the LH had no appreciable effect on 2-[3H]DG uptake in any tissues.It is concludedthat glucoseuptake in certain peripheral METHODS tissuesis acceleratedselectively by activation of VMH neurons, the action of which is independentof plasmainsulin but which Animals and surgical procedures. Female Spragueis probably via the sympathetic nervous system. Dawley rats (Nihon Clea, Tokyo, Japan) weighing 200280 g were used. They were housed in plastic cages at 25 brown adiposetissue;skeletal muscle;sympathetic nerve; ven- t 1°C with a 12:12-h light-dark cycle (lights on from tromedial hypothalamic nucleus;L-glutamate 0700 h) and were given laboratory chow and water ad RECENTSTUDIEShave revealed that tissue glucose uptake
in vivo is mediated via both insulin-dependent and insulin-independent pathways (7, 12). Cold exposure and physical exercise are the two major stimuli in increasing glucose uptake via the insulin-independent pathways. Cold exposure, for instance, markedly enhances glucose uptake in peripheral tissues, particularly in skeletal muscles and brown adipose tissue (BAT) in conscious rats (8, 20, 25), in spite of the fact that it decreases plasma insulin levels. Physical exercise is also known to be a powerful stimulus to glucose utilization by skeletal muscles without an intervention of insulin secretion (17,26). Considering that cold exposure and physical exercise induce an activation of the sympathetic nervous system (3, 4), it might be possible that the insulin-independent glucose uptake in some tissues depends on the sympathetic nervous activity. We have demonstrated in previous studies that sympathetic nervous activity in BAT and other peripheral E298
0193-1849/91
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libitum. Under pentobarbital sodium anesthesia (50 mg/ kg ip), bipolar electrodes made of insulated 100 pm wire were implanted unilaterally into the VMH or lateral hypothalamic area (LH) of rats according to the atlas of Pellegrino et al. (16). The stereotaxic coordinates used were: AP5.8, L0.7, and H9.5 for the VMH; AP5.8, L2.0, and H9.0, for the LH. The electrodes were connected to a small plug, which was then anchored to the skull with acrylic dental cement. Double-walled cannulas, which had outer and inner diameters of 0.4 and 0.2 mm, respectively, were also stereotaxically implanted into the unilateral VMH and were anchored firmly to the skull. The animals were used after 2 wk of recovery. The position of the tip of the electrodes or cannulas was verified microscopically in brain sections made when the experiments were completed. On the day of experiments the rats were anesthetized with pentobarbital sodium (50 mg/kg ip) and were implanted with a cardiac catheter via the right external jugular vein as described previously (19). In addition, unilateral sympathetic denervation of the interscapular
Copyright 0 1991 the American Physiological Society
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HYPOTHALAMIC
EFFECTS
ON
BAT was performed by severing four branches of the nerves to the right side of the BAT as described (15). The rats were then transferred onto a heat pad kept at 37OC. Electrical and chemical stimulation of the hypothalamic nuclei and injection of radiotracers. One hour after the
operations described above, electrical or chemical stimulation was delivered. Electrical stimuli consisted of 30s trains of monophasic square pulses (0.1-0.3 ms in duration, 6 V amplitude, and 50 Hz frequency) applied to the VMH or LH once every minute. For chemical stimulation, 100 nmol of monosodium L-glutamate dissolved in 1~1 saline solution were injected into the VMH through the implanted cannulas using the Hamilton microsyringe. To obtain a steady-state level of plasma glucose concentration during the experimental period (see Figs. 1 and 2), the hypothalamus had been stimulated electrically or chemically for 15 min before injection of radiotracers. After a 15min period of the hypothalamic stimulation, each rat was injected with 50 &i of 2deoxy-D-[3H]glucose (2-[3H]DG) and 5 &i of [‘“Clsucrose (ICN Radiochemicals, Irvine, CA) dissolved in 0.4 ml saline solution through the cardiac catheter. The catheter was immediately flushed with 0.3 ml of saline, and the hypothalamic stimulation was continued for another 15min period. Control rats for electrical stimulation had similarly implanted electrodes, but no electrical stimuli were applied, and controls for chemical stimulation were injected with 1 ~1 of saline into their VMH. Sampling. Blood samples (0.1 ml) were taken at min -15, -12, -9, -6, -3, 0, 5, 7, 9, 11, 13, and 15 before and after injection of the tracers and were replaced with an equivalent volume of saline. As soon as the last blood sample was obtained, pentobarbital sodium (100 mg/kg) was injected through the cardiac catheter, and the rats were quickly decapitated. The following tissues and organs were carefully dissected and freed of all extraneous materials as soon as possible: brain (cortex), heart (left ventricle), diaphragm, parametrial white adipose tissue (WAT), interscapular BAT, and skeletal muscles [quadriceps, gastrocnemius, soleus, and extensor digitorum longus (EDL)]. All tissues were rapidly frozen in liquid nitrogen, weighed, and solubilized in 0.5 ml of 0.5 N NaOH at 80°C for 1 h. The solution was added with 1 ml of 3% perchloric acid and centrifuged. The radioactivities of 3H and 14C in 1 ml of the supernatant were measured in a liquid scintillation fluid (ACS-II; Amersham) using Tri-Carb scintillation counter (Hewlett-Packard; model 300 C). Plasma samples were also analyzed for radioactivities of 2-[3H]DG and [ 14C]sucrose as well as for glucose levels (by a specific glucose oxidase method). Additionally, plasma samples before and after hypothalamic stimulation were analyzed for insulin levels by radioimmunoassay (insulin RIA kit; Midori-Jyuji, Tokyo, Japan). Measurement of Ki of net tissue uptake of 2-r3H]DG.
The mathematical model used to calculate the rate of 2[3H]DG uptake had been previously reported by Horn et al. (9). This model is now only available to provide an estimate of the net rate of 2-DG uptake over a finite period of time for a wide variety of peripheral tissues. It
TISSUE
GLUCOSE
UPTAKE
E299
has been demonstrated that the uptake rates of 2-[3H]DG were already maximal after 10 min in most tissues and that the loss of label from the tissues (transport out before phosphorylation) was negligible until 30 min after injection (9). Therefore, we chose 15 min as the optimum time for death in the present experiments. This also ensured that pharmacokinetic analysis of the glucose analogue was calculated from the linear portion of the elimination curve of 2- [3H]DG in plasma for all groups of animals. Ki of net tissue uptake of 2-[3H]DG was calculated using the following equation K i- -
C*K, C, (1 - e+ft)
where C is the intracellular concentration of 2- [3H]DG (disintegrations per min per mg tissue) at death, Kp is Ki of plasma disappearance of 2-[3H]DG, CpOis the extrapolated plasma 2- [3H]DG concentration at time 0, and t is the duration of the test, i.e., 15 min. Kp was evaluated from the slope provided by a linear regression analysis after logarithmic conversion of the data and was calculated using the equation Kp = slope X -2.303. C was calculated from the radioactivities of 3H and 14C in the plasma and tissues as reported previously (9, 25). Statistics. All values are expressed as means t SE. The effects of the different treatments on all data were evaluated with factorial analysis of variance. When a significant effect was found, these results were further compared with the Newman-Keuls multiple range test. A best-fit line for plasma levels of 2- [3H]DG was provided by linear regression analysis from which the corresponding slope was obtained. The difference was considered significant if P < 0.05. RESULTS
Effects of electrical and chemical stimulation of the hypothalamus on plasma glucose and insulin levels and plasma disappearance of 2-r3H]DG. When the VMH was
stimulated electrically or chemically with L-glutamate, the plasma levels of glucose increased significantly (Figs. 1A and 2A, and Table 1) as reported previously (5, 22). In rats with electrical stimulation of the VMH, plasma glucose concentration reached a steady-state level of -180 mg/dl after 12 min of intermittent stimulation, and, judging from that, the rate of change in glucose was 0.3 t 0.5 mg l all-l . min-’ during VMH stimulation after 12 min (Fig. lA), whereas the rate was 0.5 t 0.4 mg* dl-lo min-’ in unstimulated controls. Similarly, the level reached a steady state of -155 mg/dl (the rate of change in glucose was within 0.2 mg*dl-’ emin-‘) 12 min after microinjection of glutamate into the VMH and remained increased during the additional period, whereas it did not change detectably after saline injection into the same hypothalamic region (Fig. 2A). In contrast, the plasma glucose levels remained fairly constant during electrical stimulation of the LH (Fig. lA, Table 1). Plasma insulin concentration did not change significantly after electrical and chemical stimulation of either the VMH or the LH (Table 1). The time courses of decrease in radioactivity of plasma
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HYPOTHALAMIC
E300
A
l
VMH
A LH
stimulation
EFFECTS ON TISSUE GLUCOSE UPTAKE T
T
-r
stimulation
l 0
Chemical Control
stimulation
of
VMH
0 Control
stim.
r/”
t ; chemical
- 15 - 10 Time
n 2 g 75,000
- 5
after
0
injection
B
-2 n
5
10
of Z- [3H] DG (mid
A
E 50,000 g
: 45
Time
’
1
I
I
I
I
5
7
9
11
13
15
after
injection
Time
n 2 g 75,000
VMH stimulation A LH stimulation 0 Control
A
- 15 - 10 - 5
15
l
of 2- [3H] DG (min)
FIG. 1. Effects of electrical stimulation of ventromedial hypothalamic nucleus (VMH) and lateral hypothalamic nucleus (LH) on plasma glucose levels (A) and 2-deoxy-D-[3H]glucose (2-[“HIDG) disappearance (B). VMH or LH of anesthetized rats was stimulated electrically for 30 min. Electrical stimuli consisting of 30-s trains of monophasic square pulses (0.1-0.3 ms in duration, 6 V amplitude, and 50 Hz frequency) were applied once every minute. After 15-min period of hypothalamic stimulation (time 0) when plasma glucose concentration attained steady-state level, each rat was injected through cardiac catheter with 50 &i of 2-[:‘H]DG and 5 &i of [‘4C]sucrose, and animal was killed as soon as last blood sample was obtained (15 min). Control rats had similarly implanted electrodes, but no electrical stimuli were applied. Results are means & SE for 6-10 rats.
2-[“H]DG after a single injection of the glucose analogue with or without hypothalamic stimulation are shown in Figs. 1B and 2B. When plotted on a semilogarithmic scale, the plasma 2-[3H]DG disappearance curves were linear in all groups of animals. Although the total area under the 2-[3H]DG disappearance curves tended to decrease in rats with either electrical (Fig. 1B) or chemical stimulation (Fig. 2B) of the VMH, as compared with respective controls, there was no significant difference in the rate of plasma 2-[3H]DG disappearance (I&) between the VMH-stimulated and control groups. Electrical stimulation of the LH also did not alter the rate of 2- [ “H]DG disappearance from plasma (Fig. 1B). Effects of electrical stimulation of the VMH and LH on tissue glucose uptake. Ki of net tissue glucose uptake
obtained under pentobarbital sodium anesthesia for brain, diaphragm, WAT, BAT, heart muscle, and skeletal muscles are given in Figs. 3 and 4. A comparison of the
stimulation
; 25,000 3 5 a. 0
after
injection
B
45
0 I
I
5
7
9
after
5
10
15
of 2- [3 HI DG (min)
l Chemical 0 Control
’
Time
0
stimulation
I
injection
11
of
VMH
I
I
13
15
of 2- r3H] DG (min)
FIG. 2. Effects of chemical stimulation of VMH on plasma glucose levels (A) and 2-[“H]DG disappearance (B). VMH of anesthetized rats was stimulated chemically by microinjection of 1~1 of saline containing 100 nmol of L-glutamate. To obtain steady-state level of plasma glucose concentration during study of 2-[3H]DG elimination, radiotracers (50 &i of 2-[“H]DG and 5 &i of [‘4C]sucrose) were injected intravenously 15 min after microinjection of L-glutamate (time 0). Animals were killed as soon as last blood sample was obtained (15 min). Control rats were given 1 ~1 of saline into their VMH. Results are means & SE for 6 rats.
TABLE 1. Effects of hypothalamic stimulation on plasma glucose and insulin levels in anesthetized rats Electrical Stimulation
Plasma glucose, Prestimulation Poststimulation Plasma insulin, Prestimulation Poststimulation
VMH Chemical Stimulants
Control
VMH
LH
Saline
103k6 llOt5
108t5 189tll*
lOlt6 111&7
119t13 119&8
3223 3Ok3
33t2 31k2
34k6 36k4
321k3 32k2
Glutamate
mg/dl 115t6 156&15*
pU/ml 3722 35t2
Values are means * SE for 6-10 rats. VMH, ventromedial hypothalamic nucleus; LH, lateral hypothalamic nucleus. Experimental conditions are as in Figs. 1 and 2. * Plasma concentration after hypothalamic stimulation (15 min) was significantly different from that before stimulation (-15 min) at P C 0.01.
Ki values among these tissues revealed that of brain, heart muscle, and diaphragm (from x 10D3/min) were one order of magnitude the Ki of skeletal muscles, BAT, and WAT
the basal Ki 14.6 to 28.3 higher than (from 1.6 to
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HYPOTHALAMIC
EFFECTS
ON
TISSUE
7.7 X 10B3/min). Electrical stimulation of the VMH increased glucose uptake in BAT, heart,, and skeletal muscles preferentially. In BAT and heart muscle, the Ki values were increased by electrical stimulation of the VMH about eight times (from 2.9 to 24.4 X 10B3/min) and three times (from 18.7 to 56.4 X 10D3/min), respectively (Fig. 3). The increased value of Ki in BAT after VMH stimulation was completely suppressed by local sympathetic denervation, and the value in the denervated side of BAT was not significantly different from the control value. The increased Ki values after VMH stimulation were also found in four different types of skeletal muscles (Fig. 4). The EDL and soleus muscles have been known to be mainly composed of fast-twitch glycolytic and slowtwitch oxidative fibers, respectively, and the quadriceps and gastrocnemius muscles are composed of mixed-type fibers. Electrical stimulation of the VMH enhanced the Ki values of these skeletal muscles by -1.5 times (P < 0.01 in all of the skeletal muscles investigated). In contrast, electrical stimulation of the VMH had no appreciable effects on the Ki values in WAT, diaphragm, and brain (Fig. 4). Electrical stimulation of the LH, on the other hand, did not appreciably affect 2- [3H]DG uptake in any tissues investigated (Figs. 3 and 4).
I]
GLUCOSE Control
E301
UPTAKE VA
Quadri
LH
VMH
stimulation
Gastro
Soleus
stimulation
EDL
Effect of chemical stimulation of the VMH on glucose uptake in peripheral tissues. To confirm that neurons of
the VMH are specifically concerned in hypothalamic regulation of glucose uptake in peripheral tissues, we next examined the effect of chemical stimulation of the VMH by microinjection of glutamate, a well-known excitatory neurotransmitter in the central nervous system (6). Microinjection of L-glutamate (100 nmol in 1 ~1 saline) into the VMH significantly increased the Ki values in skeletal muscles to the similar extent to those seen after electrical stimulation (from 7.1 to 7.6 X 10B3/min in controls to from 10.6 to 11.2 X 10e3/min in rats with glutamate injection) (Fig. 5). Chemical stimulation of the VMH a Is0 increased the Ki values in BAT and heart BAT
Heart
30
x.*.
.*. x
60
T 20
Diaphragm
Brain
c 0.01.
muscle about 3 and 2.5 times, respectively. The effect on the Ki value in BAT was likewise abolished by local sympathetic denervation. However, chemical stimulation of the VMH with glutamate did not significantly alter the Ki values in brain, diaphragm, and WAT, as compared with the saline-injected controls (data not shown). DISCUSSION
10
Innervated
Denervated 0
Control
a
LH
stimulation
VMH
stimulation
3. Effect of electrical stimulation of hypothalamus on rate constant of 2-[“H]DG uptake in vivo in brown adipose tissue (BAT) and heart in anesthetized rats. Rate constant of 2-[‘H]DG uptake was calculated as described in METHODS. One hour before start of electrical stimulation, right side of interscapular BAT of anesthetized rats was surgically denervated, and electrical stimuli were given intermittently to VMH or LH. Other experimental conditions are as in Fig. 1. Results are means t SE for 6 rats. * Significantly different from controls without stimulation at P < 0.01. FIG.
WAT
4. Effect of electrical stimulation of hypothalamus on rate constant of 2-[:‘H]DG uptake in vivo in skeletal muscles, white adipose tissue (WAT), diaphragm, and brain in anesthetized rats. Four different hindlimb muscles, quadriceps (quadri), gastrocnemius (gastro), soleus, and extensor digitorum long-us (EDL) were sampled. Parametrial WAT and cerebral cortex were sampled for WAT and brain, respectively. Experimental conditions are as for Fig. 1. Results are means t SE for 6 rats. * Significantly different from controls without stimulation at P FIG.
The present study demonstrates for the first time that glucose uptake and utilization in BAT, heart, and skeletal muscles are markedly increased by selective stimulation of the VMH. Because it has already been established that hepatic glucose output can be affected by electrical and chemical stimulation of the hypothalamus (2l), the present findings indicate that not only glucose production but also its utilization in certain tissues are accelerated by stimulation of the VMH. By contrast, stimulation of the LH has no significant effect on glucose uptake in any tissues examined, although it has been known to enhance glycogen synthesis in the liver (21). The question of whether the effect of VMH stimulation on tissue glucose uptake originates from excitation of cell bodies of VMH neurons or from activation of axons
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E302
HYPOTHALAMIC I[
Quadri
Saline
Gastro
m
BAT
T
ON TISSUE
L- glutamate
EDL
Soleus
3%
EFFECTS
Heart %
80 60 40 20
0
Denervated Innervated
0
FIG. 5. Effect of chemical stimulation of VMH on rate constant of 2[“H]DG uptake in vivo in skeletal muscles, BAT, and heart muscle. Four different hindlimb muscles, quadriceps, gastrocnemius, soleus, and EDL were sampled. One hour before microinjection of L-glutamate (100 nmol) into VMH, right side of interscapular BAT of anesthetized rats was surgically denervated. Control rats were injected with same volume (1 ~1) of saline into VMH. Experimental conditions are as for Fig. 2. Results are means ~frSE for 6 rats. * Significantly different from saline controls at P < 0.01.
region has been resolved of passage in this hypothalamic with L- *glutamate, which bY using chemica 1 stimulation is known as the excitatory neurotransmitter in the central nervous system (6). This kind of study has revealed that chemical stimulation of the VMH reproduces the effects similar to those observed after electrical stimulation, providing evidence for the participation of specific neurons of the VMH in the hypothalamic regulation of tissue glucose uptake. The slightly less effects on glucose uptake and the blood glucose levels observed with chemical stimulation than with electrical stimulation of the VMH can be due to a submaximal stimulation of the VMH neurons. The effect of VMH stimulation on tissue glucose uptake is most probably mediated by the sympathetic nerve activity because the VMH is regarded as a hypothalamic component of the sympathetic nervous system (18, 21). Indeed, the increase in Ki of glucose uptake in BAT in response to electrical and chemical stimulation of the VMH was effectively prevented by surgical sympathetic denervation of BAT. Similarly, our previous studies have shown that lipogenic and thermogenic responses of BAT to VMH stimulation are almost completely abolished by local sympathetic denervation (14). In keeping with these
GLUCOSE
UPTAKE
observations, Marette and Bukowiecki (13) recently reported that physiological concentrations of norepinephrine enhanced glucose transport in isolated BAT, indicating the presence of non-insulin-mediated pathways for increasing glucose uptake in BAT. In the present study, we did not examine the possible effect of denervation on the increased glucose uptake in muscles after VMH stimulation. However, this might be mediated likewise by the sympathetic nervous system, since skeletal muscles have been recognized to be supplied with noradrenergic sympathetic axons that are distributed to the muscle spindles and extrafusal muscle fibers (2). It has also been shown that sodium and potassium transport in rat skeletal muscle during hypokalemia is regulated by the sympathetic nervous system and that this regulation (suppression of the Na+-K+ pump) is achieved by the apparent release of norepinephrine on muscle after nerve activity (1). Although glucose is known to stimulate its own transport across the plasma membrane, the accelerated rate of glucose uptake in certain tissues in response to VMH stimulation seems unlikely to be simply the result of mass action of the elevated blood glucose on the basis of the following reasons. First, Ki of 2-DG uptake per se more than the glucose metabolic rate was increased by VMH stimulation. Second, the increase in Ki was observed preferentially in BAT, heart, and skeletal muscles but not in white adipose tissue, diaphragm, and brain; if the effect were induced by the mass action of the elevated blood glucose, the Ki value would have been increased in all tissues. Third, the accelerated rate of glucose uptake in BAT was almost totally suppressed in the denervated side of the tissue, despite the presence of hyperglycemia during electrical and chemical stimulation of the VMH. The most likely cause of the accelerated glucose uptake by particular tissues is an enhanced rate of membrane transport of this substrate. However, further experiments are needed to ensure that glucose transporters are actually activated by stimulation of the VMH and efferent sympathetic nerves, particularly in muscles. The effect of VMH stimulation is not mediated by insulin ., because plasma insulin conce ntration ha .S been shown to remain unchanged or rather suppressed d uring VMH stimulation (5,23). In fact, electrical and chemical stimulation of the VMH did not increase the plasma levels of insulin (Table 1). Instead, the results of the present study indicate that the effect of VMH stimulation on tissue glucose uptake is probably mediated by the sympathetic nerve and thus depends on an insulinindependent mechanism of glucose transport that may operate simultaneously in the insulin-sensitive tissues like muscle and BAT but not in WAT and diaphragm. The observed effects on tissue glucose uptake could conceivably be related, at least in part, to a change in blood flow. Indeed, because VMH stimulation has been reported to increase regional blood flows of BAT and skeletal muscle (lo), a possible opening of the capillary beds may contribute to the accelerated glucose uptake observed in these tissues after VMH stimulation. Similarly, the contribution of blood flow to an increased glucose uptake in leg skeletal muscle has recently been observed both in lean and obese subjects with regard to
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HYPOTHALAMIC
EFFECTS
ON TISSUE
insulin action (11). Finally, although, in the present study, the VMH was stimulated under anesthesia of the animals to prevent muscular activity, the possibility remains that VMH stimulation increases intracellular glucase disposal by accelerating glycolysis and glucose oxidation in skeletal muscles and other sympathetically innervated tissues, which could also contribute to the enhanced glucose uptake in these tissues on VMH stimulation. We thank Drs. T. Shinomiya and H. Shibata for helpful discussion in initiating this study and M. Miyoshi for typing the manuscript. This work was supported by a Grant-in-Aid for scientific research from the Ministry of Education, Science, and Culture of Japan and by a Grant from the Health Science Promotion Foundation. Address for reprint requests: T. Shimazu, Dept. of Medical Biochemistry, Ehime Univ. School of Medicine, Shigenobu, Ehime 701-02, Japan. Received 29 May 1990; accepted in final form 29 April 1991. REFERENCES N. Sodium pump in skeletal muscle: central nervous system-induced suppression by a-adrenoreceptors. Science Wash. DC 213: 1252-1254,198l. BARKER, D., AND M. SAITO. Autonomic innervation of receptors and muscle fibres in cat skeletal muscle. Proc. R. Sot. Lond. B Biol. Sci. B212: 317-332,198l. CHRISTENSEN, N. J., AND H. GALBO. Sympathetic nervous activity during exercise. Annu. Reu. Physiol. 45: 139-153, 1983. DULLOO, A. G., J. B. YOUNG, AND L. LANDSBERG. Sympathetic nervous system responses to cold exposure and diet in rat skeletal muscle. Am. J. Physiol. 255 (Endocrinol. 1Metab. 18): E180-E188, 1988. FROHMAN, L. A., AND L. L. BERNARDIS. Effect of hypothalamic stimulation on plasma glucose, insulin, and glucagon levels. Am. J. Physiol. 221: 1596-1603, 1971. GOODCHILD, A. K., R. A. L. DAMPNEY, AND R. BANDLER. A method for evoking physiological responses by stimulation of cell bodies, but not axons of passage, within localized regions of the central nervous system. J. Neurosci. Methods 6: 351-363, 1982. GOTTESMAN, I., L. MANDARINO, AND J. GERICH. Estimation and kinetic analysis of insulin-independent glucose uptake in human subjects. Am. J. Physiol. 244 (Endocrinol. Metab. 7): E632-E635, 1983. GRECO-PEROTTO, R., D. ZANINETTI, F. ASSIMACOPOULOS-JEANNET, E. BOBBIONI, AND B. JEANRENAUD. Stimulatory effect of cold adaptation on glucose utilization by brown adipose tissue. Relationship with changes in the glucose transporter system. J. Biol. AKAIKE,
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