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Lesions of the Ventromedial Hypothalamus Reduce Postingestional Thermogenesis
Physiology & Behavior, Vol. 61, No. 5, pp. 687–691, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0031-9384/97 $17.00 / .00
PII S0031-9384(96)00520-3
Lesions of the Ventromedial Hypothalamus Reduce Postingestional Thermogenesis M. MONDA, 1 A. SULLO, AND B. DE LUCA Dipartimento di Fisiologia Umana e Funzioni Biologiche Integrate ‘‘Filippo Bottazzi’’, Seconda Universita` di Napoli, Via Costantinopoli 16, 80138 Napoli, Italy Received 12 August 1996; Accepted 9 October 1996
MONDA, M., A. SULLO, AND B. DE LUCA. Lesions of ventromedial hypothalamus reduce postingestional thermogenesis. PHYSIOL BEHAV 61(5) 687–691, 1997.—The aim of this experiment was to evaluate the effects of ventromedial hypothalamus lesions on the thermogenic changes that follow food intake. Four groups of six Sprague-Dawley male rats were used. Under anesthesia with pentobarbital, the animals in the first and second groups received lesions at the ventromedial hypothalamus, and animals in the third and fourth groups received sham lesions. Body weight and food intake were monitored daily until the experimental procedure began. Twenty days after lesion, oxygen consumption, firing rate of sympathetic nerves to interscapular brown adipose tissue (IBAT), and IBAT temperature were monitored for 45 min both before and after 5 g food intake in 24 h fasted rats from the first and third groups. The same variables were measured in the animals of the second and fourth groups 50 days after receiving the lesions. Lesion placements were histologically verified. The results showed that lesions produced hyperphagia and obesity. Firing rate of nerves to IBAT, IBAT temperature, and oxygen consumption increased after food intake in sham-lesioned rats. This increase was significantly reduced by the lesion at both the 20- and 50-day time points. These findings indicate that the ventromedial hypothalamus controls postingestional activation of sympathetic discharge to IBAT. The reduction of postingestional thermogenesis could be involved in the development of obesity induced by lesion of the ventromedial hypothalamus. q 1997 Elsevier Science Inc. Oxygen consumption
Brown adipose tissue
Sympathetic activity
BODY WEIGHT is regulated by food intake and energy expenditure through a homeostatic mechanism (18). Several physiological conditions induce metabolic adaptations that increase or minimize futile energy losses. Hyperalimentation causes an increase in energy expenditure (33), and underfeeding reduces energy expenditure (5). In rodents, during lactation a suppression of brown adipose tissue thermogenesis occurs as an adaptive mechanism to save energy (36). Exercise enhances energy expenditure and causes a sex-dependent change of food intake (31,32). When homeostasis is altered, obesity or leanness generally results ( 18 ) . Rats with lesions of the lateral hypothalamus have reduced body weight ( 10 ) . These animals make adjustments both in food intake ( 27,30 ) and expenditure ( 6,7 ) appropriate to the maintenance of a lower body weight. Lesions of the ventromedial hypothalamus ( VMH ) increase food intake ( 16,22 ) and decrease both resting metabolism ( 37 ) and motor activity ( 35 ) . Nonshivering thermogenesis (15) is activated by food ingestion, and this activation is independent of the caloric density of ingested food in the first minutes after food intake. This phase is
Thermogenesis
Ventromedial hypothalamus
known as postingestional preadsorbitive thermogenesis. Brown adipose tissue (BAT) is the organ responsible for evoking 35– 65% of the total increase in metabolic heat production during various conditions (9,26), including postprandial thermogenesis (2,8,11,12). BAT occurs as numerous discrete deposits, particularly in the interscapular region of the body, and it is under the peripheral neurogenic control of the sympathetic nervous system (1,13,21). The aim of this experiment was to evaluate the effects of VMH lesions on the sympathetic and thermogenic changes that follow food intake. METHOD
Animals Male Sprague-Dawley rats, housed in a room with controlled temperature (22 { 17C) and humidity (70%), were used throughout the experiment. They were bred in the laboratory and had an initial body weight of 280–300 g. Each was caged singly in a room with 06:00–18:00 light–dark cycle. Food and water were provided ad libitum.
1 Requests for reprints should be addressed to Marcellino Monda, M.D., Dipartimento di Fisiologia Umana, Via Costantinopoli 16, 80138 Napoli, Italy.
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Apparatus for Oxygen (O2 ) Consumption Measurement Resting O2 consumption was determined with an indirect calorimeter. The closed circuit apparatus used was an adaptation of Benedict and MacLeod’s calorimeter (3). Air was continuously circulated through a drying column (CaSO4 Drierite), a respiratory chamber with a 2.5-L and CO2 trap (soda lime), by a peristaltic pump at a rate of 2 L/min. The O2 reservoir was a 1L cylindrical metal bell, which fit in a concentric cylinder filled with water, forming an air-tight seal. The cylinder, graduated to 5 mL, was connected to the respiratory chamber. The temperature in the respiratory chamber was maintained constant by circulating water and was monitored by an internal thermometer. The volume of O2 consumed was corrected for temperature and pressure and was expressed as milliliters of O2 /min/kg b.wt.0.75 (19). Apparatus for Registration of Sympathetic Activity The firing rate of nerves to interscapular BAT (IBAT) was recorded by a pair of silver wire electrodes. The electrical pulses were amplified by a condenser-coupled amplifier and were filtered by bandpath filters (NeuroLog System, Digitimer). The raw pulses were displayed on a oscilloscope (Tektronix) and sent to a window discriminator. Square waves from the discriminator were sent to an analog–digital converter (DAS system, Keithley) and stored on a computer (Personal Computer AT, IBM) every 5 s. Furthermore, a rate meter with a reset time of 5 s was used to observe the time course of the nerve activity recorded by a pen recorder (Vitatron). Because the signal-to-noise ratio depended on the number of nerve filaments and the condition of contact between nerve and electrodes, the basal burst rates were different for each rat. The threshold level of the event detector was fixed during the experiment at 50% of the peaks of the largest pulses and above background noise. VMH lesion The lesion-making electrode consisted of a 250-mm-diameter stainless-steel wire insulated except for 0.5 mm at the tip. Under pentobarbital anesthesia (50 mg/kg b.wt.), the electrode was bilaterally inserted into the rats at the following coordinates: 0.2 mm posterior to Bregma, 1.0 mm lateral to the midline, and 9.8 mm below the surface of the skull, according to the atlas of Pellegrino et al. (28). A current of 1.5 mA was applied through the electrode for 15 s; a rectal cathode served as the remote electrode. The sham lesion was made with the same procedure as the lesion, except for the application of current.
On the 20th day, O2 consumption, firing rate of nerves to IBAT, and IBAT temperature were monitored for 45 min both before and after a presentation of 5 g standard food for 5 min. The amount of ingested food was measured. The animals had been kept fasting for 24 h before food presentation. The experimental procedure was carried out in all animals at the same times (18:00–20:00). Histology At the end of the experiment, the rats were injected with an overdose of pentobarbital and intracardially perfused with physiological saline, followed by a 10% solution of phosphate buffered formalin. The brains were removed and fixed in formalin. Afterward, the brains were cut into blocks that were placed in heptane at 0307C and later sliced in 75-mm sections (29). These microtome sections were subsequently stained with cresyl violet. RESULTS
Figure 1 shows the percentage of changes in firing rate of nerves to IBAT. Food ingestion increased the firing rate, which peaked at 5 min in the sham-lesioned rats. This increase was reduced in the lesioned animals both 20 days and 50 days after the lesion. Analysis of variance showed significant effects for lesion, F (1, 20) Å 24.923, p õ 0.01, time, F (6, 120) Å 31.265, p õ 0.01, and Lesion 1 Time, F (6, 120) Å 5.408, p õ 0.01. There were no group differences in baseline levels of sympathetic activity. Examples of changes in actual firing rate of the nerves to IBAT are reported in Fig. 2. Figure 3 illustrates TIBAT changes. Food intake caused a rise that peaked at 10 min in the sham-lesioned rats. This increase was reduced in the lesioned animals both 20 days and 50 days after the lesion. Analysis of variance showed significant effects for lesion, F (1, 20) Å 30.948, p õ 0.01, time, F (6, 120) Å 29.252, p õ 0.01, and Lesion 1 Time, F (6, 120) Å 2.868, p õ 0.05]. Furthermore, the changes in the firing rate of IBAT nerves preceded the changes in temperature. Figure 4 illustrates changes in O2 consumption. Food intake induced an increase, with a peak at 10 min in the sham-lesioned rats. The VMH lesion reduced the increase induced by food intake both 20 days and 50 days after lesion. Analysis of variance showed significant effects for lesion, F (1, 20) Å 21.075, p õ
Procedure Twelve animals received lesions in the VMH, and another 12 served as sham-operated controls. Food intake and body weight were measured daily. Half the animals in each group were tested at 20 days and half at 50 days following surgery. Four days before the experiment, the rats were anesthetized with pentobarbital (50 mg/kg b.wt.). IBAT was exposed, and intercostal nerves entering the right side of IBAT were identified. Nerve filaments were isolated from the central cut end of these nerve bundles under a dissecting microscope to position a pair of silver wire electrodes. The entire preparation was embedded into a small silicone mold and covered with silicone gel (WackerChemie, Munchen, Germany) that hardened sufficiently within 1 h to stabilize the preparation and to provide electrical insulation against the surrounding tissue. The silver wire electrodes were connected with a pin cemented to cranial theca by four screws.
FIG. 1. Means { SE of percentage of changes in firing rate of nerves innervating IBAT in VMH-lesioned or sham-lesioned rats 20 or 50 days after surgery. Food presentation was at time 0.
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FIG. 3. Means { SE of changes in IBAT temperature of VMH-lesioned or sham-lesioned rats 20 or 50 days after surgery. Food presentation was at time 0.
were 1.20 { 0.31 g for the sham-lesioned rats and 11.75 { 0.72 g for the lesioned rats. An increase of 2.03 { 0.34 g for shamlesioned rats and 12.50 { 0.56 g for lesioned rats was noted after 50 days. The analysis of variance showed a statistical difference across groups, F (3, 20) Å 180.001, p õ 0.01. The Sheffe` test, used as a post hoc test, showed a difference (p õ 0.05) between sham-lesioned and lesioned animals, both 20 and 50 days after lesion. The changes in body weight are shown in Fig. 6. The increases were 7.83 { 1.58 g for sham-lesioned animals and 65.50 { 3.06 g for lesioned rats after 20 days. In the sham-lesioned and lesioned animals, an increase of 10.03 { 7.04 g and 67.83 { 6.49 g, respectively, was registered after 50 days. The analysis of variance revealed the difference across groups, F (3, 20) Å 149.261, p õ 0.01. The post hoc test showed a difference ( p õ 0.05) between sham-lesioned and lesioned animals 20 and 50 days after lesion. The strain of rats utilized in this experiment was inbred, which caused a body weight gain lower than that of noninbred animals. Figure 7 illustrates an example of a VMH lesion. Darkened areas represent the extent of the lesion from stereotaxis planes
FIG. 2. Actual firing rate changes in a rat 20 days after sham lesion (A) or VMH lesion (B) and in a rat 50 days after sham lesion (C) or VMH lesion (D). The arrow indicates the time of food presentation.
0.01, time, F(6, 120) Å 59.761, p õ 0.01, and Lesion 1 Time, F(6, 120) Å 13.722, p õ 0.01. The amount of food ingested after 5 min presentation was 4.35 { 0.06 g in the first group, 4.21 { 0.08 in the second group, 4.32 { 0.07 in the third group, and 4.29 { 0.11 in the fourth group. The analysis of variance did not show any significant difference across the groups. The 5-min food intake was identical in all groups because the rats had been fasted for 24 h. The difference between the values of 24-h food intake, measured before and 20 or 50 days after the sham lesion or lesion, are indicated in Fig. 5. The increases in food intake after 20 days
FIG. 4. Means { SE of changes in oxygen consumption of VMH-lesioned or sham-lesioned rats 20 or 50 days after surgery. Food presentation was at time 0.
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FIG. 5. Means { SE of changes in 24-h food intake of VMH-lesioned or sham-lesioned rats 20 or 50 days after surgery.
FIG. 6. Means { SE of changes in body weight of VMH-lesioned or sham-lesioned rats 20 or 50 days after surgery.
29 – 33 ( 28 ) . The area with maximum damage is in plane 31. Lesions did not extend to the peri-VMH area, and their size was identical. For this reason, any relationship between lesion size and magnitude of metabolic response was not found.
could induce an increase of total amount of ingested food. This reduction may be another factor in the induction of obesity by the VMH lesion. In other words, the increased body weight of
DISCUSSION
Thermogenesis is under the control of the vegetative nervous system. Experimental evidence demonstrates that IBAT is a major organ of nonshivering thermogenesis, at least in rodents ( 15 ) . Many investigators have shown that activation of sympathetic nerves supplying IBAT causes a rise in temperature of this tissue ( 14,17,34 ) . We have clearly demonstrated that a modification of sympathetic activity causes change in IBAT activity ( 20,23 – 25 ) . The findings of the present experiment show that the VMH lesion modifies postingestional changes in the sympathetic discharge of nerves to IBAT, IBAT temperature, and O2 consumption, which demonstrates that the VMH is strongly involved in the control of sympathetic and thermogenic reactions due to food ingestion. In the present experiment, we obtained direct evidence of the activation of the sympathetic nervous system and of its involvement in the control of nonshivering thermogenesis. The findings of this experiment are the first to show that the VMH lesion modifies postingestional increase in the firing rate of the sympathetic nerves to IBAT. IBAT weight was not measured. The marked metabolic alterations could have resulted in loss of IBAT and degeneration of their sympathetic supply, which could have resulted in a smaller response. The thermogenic response was reduced 20 and 50 days after the lesion. This observation suggests that the VMH lesion induces a long-term modification in the sympathetic and thermogenic reaction to food intake. A long-term reduction of postingestional thermogenesis may contribute to obesity induced by a VMH lesion through a decrease in energy expenditure induced by reduced sympathetic activity. There is a close relationship between the sympathetic activity and food intake. Some evidence seems to suggest that an increase in sympathetic discharge reduces food intake ( 4 ) . Thus, a reduction in the sympathetic response after food intake
FIG. 7. Planes 29–33 from the stereotaxis atlas of Pellegrino et al. (28). Darkened areas represent extent of VMH lesion. The top section corresponds with plane 29. See (28) for abbreviations.
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VMH-lesioned animals may be caused by a reduction of satiety signals and a decrease in postingestional energy expenditure.
ACKNOWLEDGMENTS
This study was supported by a grant from the Italian National Research Council.
REFERENCES 1. Amaro, S.; Monda, M.; De Luca, B. EEG arousal, sympathetic activity and brown adipose tissue thermogenesis after conditioned taste aversion. Physiol. Behav. 60:71–75; 1996. 2. Amaro, S.; Monda, M.; Pellicano, M. P.; Cioffi, L. A.; De Luca, B. Postprandial thermogenesis and conditioned taste aversion or preference. Physiol. Behav. 56:463–470; 1994. 3. Benedict, F. G.; Mc Leod, M. The heat production of the albino rat. J. Nutr. 1:346–366; 1928. 4. Bray, G. A. Reciprocal relation between the sympathetic nervous system and food intake. Brain Res. Bull. 27:517–520; 1991. 5. Corbett, S. W.; Stern, J. S.; Keesey, R. E. Energy expenditure in rats with diet-induced obesity. Am. J. Clin. Nutr. 44:173–180; 1986. 6. Corbett, S. W.; Wilterdink, E. J.; Keesey, R. E. Resting oxygen consumption in over- and under-fed rats with lateral hypothalamic lesions. Physiol. Behav. 35:971–977; 1985. 7. De Luca, B.; Monda, M.; Amaro, S.; Pellicano, M. P. Heat production and motor deficit in rats lesioned in globus pallidus, entopeduncular nucleus and lateral hypothalamus. Physiol. Behav. 45:119– 126; 1989. 8. De Luca, B.; Monda, M.; Amaro, S.; Pellicano, M. P.; Cioffi, L. A. Lack of diet-induced thermogenesis following lesions of paraventricular nucleus in rats. Physiol. Behav. 46:685–691; 1989. 9. De Luca, B.; Monda, M.; Amaro, S.; Pellicano, M. P.; Cioffi, L. A. Thermogenic changes following frontal neocortex stimulation. Brain Res. Bull. 22:1003–1007; 1989. 10. De Luca, B.; Monda, M.; Pellicano, M. P.; Zenga, A. Cortical control of thermogenesis induced by lateral hypothalamic lesion and overeating. Am. J. Physiol. 253:R626–R633; 1987. 11. De Luca, B.; Monda, M.; Sullo, A. Changes in eating behavior and thermogenic activity following inhibition of nitric oxide formation. Am. J. Physiol. 268:R1533–R1538; 1995. 12. Foster, D. O. Quantitative contribution of brown adipose tissue to overall metabolism. Can. J. Biochem. Cell. Biol. 62:618–622; 1984. 13. Foster, D. O.; Depocas, F.; Zaror-Behrens, G. Unilaterality of the sympathetic innervation of each pad of rat interscapular brown adipose tissue. Can. J. Physiol. Pharmacol. 60:107–113; 1982. 14. Geloen, A.; Trayhurn, P. Regulation of the level of uncoupling protein in brown adipose tissue by insulin requires the mediation of the sympathetic nervous system. FEBS Lett. 267:265–267; 1990. 15. Himmis-Hagen, J. Non-shivering thermogenesis. Brain Res. Bull. 12:151–160; 1984. 16. Hoebel, B. G.; Teitelbaum, P. Weight regulation in normal and hypothalamic hyperphagic rats. J. Comp. Physiol. Psychol. 61:189– 193; 1966. 17. Kawate, R.; Talan, M. I.; Engel, B. T. Sympathetic nervous activity to brown adipose tissue increases in cold-tolerant mice. Physiol. Behav. 55:921–925; 1994. 18. Keesey, R. E.; Powley, T. L. The regulation of body weight. Annu. Rev. Psychol. 37:109–133; 1986. 19. Kleiber, M. The fire of life. An introduction to animal energetics. Huntington, NY: R. E. Kreiger Publishing Company; 1975.
20. Monda, M.; Amaro, S.; De Luca, B. Non-shivering thermogenesis during prostaglandin E1 fever in rats: role of cerebral cortex. Brain Res. 651:148–154; 1994. 21. Monda, M.; Amaro, S.; De Luca, B. Septal lesions block sympathetic activation following frontal cortex stimulation in the rat. Acta Physiol. Scand. 155:275–282, 1995. 22. Monda, M.; Amaro, S.; De Luca, B. The influence of exercise on energy balance changes induced by ventromedial hypothalamic lesion in the rat. Physiol. Behav. 54:1057–1061; 1993. 23. Monda, M.; Amaro, S.; Sullo, A.; De Luca, B. Lateral hypothalamic lesion induces sympathetic stimulation and hyperthermia by activating synthesis of cerebral prostaglandins. Prostaglandins 51:169– 178; 1996. 24. Monda, M.; Amaro, S.; Sullo, S.; De Luca, B. Nitric oxide reduces the thermogenic changes induced by lateral hypothalamic lesion. J. Physiol. (Paris) 88:347–352; 1994. 25. Monda, M.; Amaro, S.; Sullo, A.; De Luca, B. Nitric oxide reduces the thermogenic changes induced by prostaglandin E1. Brain Res. Bull. 38:489–493; 1995. 26. Monda, M.; Sullo, A.; De Luca, E. N G-methyl-L-arginine increases the hyperthermic effects of prostaglandin E1. J. Physiol. (Paris) 90:79–83; 1996. 27. Monda, M.; Sullo, A.; De Luca, E.; Pellicano, M. P. Lysine acetylsalycilate modifies aphagia and thermogenic changes induced by lateral hypothalamic lesion. Am. J. Physiol. 271:R1638–R1642; 1996. 28. Pellegrino, L. J.; Pellegrino, A. S.; Cushman, A. J. A stereotaxic atlas of the rat brain. New York: Plenum Press; 1979. 29. Pieri, L.; Hoffman, D. Improved rapid method to check electrode localizations in the brain. Physiol. Behav. 15:113–115; 1975. 30. Powley, T. L.; Keesey, R. E. Relationship of body weight to the lateral hypothalamic feeding syndrome. J. Comp. Physiol. Psychol. 70:25–36; 1970. 31. Richard, D.; Arnold, J. Influence of exercise training in the regulation of energy balance. J. Obesity Weight Regul. 6:212–214; 1987. 32. Richard, D.; Rochon, L.; Deshaies, Y. Effects of exercise training on energy balance of ovariectomized rats. Am. J. Physiol. 253:R740–R745, 1987. 33. Rothwell, N. J.; Stock, M. J. Energy expenditure of cafeteria-fed rats determined from measurements of energy balance and indirect calorimetry. J. Physiol. (Lond.) 328:371–377; 1982. 34. Sakaguchi, T.; Takahashi, M.; Bray, G. A. Lateral hypothalamus and sympathetic firing rate. Am. J. Physiol. 255:R507–R512; 1988. 35. Tokunaga, K.; Matsuzawa, Y.; Fujioka, S.; Kobatake, T.; Keno, Y.; Odaka, H.; Matsuo, T.; Tarui, S. PVN-lesioned obese rats maintain ambulatory activity and its circadian rhythm. Brain Res. Bull. 26:393–396; 1991. 36. Trayhurn, P.; Richard, D. Brown adipose tissue thermogenesis and the energetics of pregnancy and lactation in rodents. Biochem. Soc. Trans. 13:826–827; 1985. 37. Vilberg, T. R.; Keesey, R. E. Reduced energy expenditure after ventromedial hypothalamic lesions in female rats. Am. J. Physiol. 247:R183–R188; 1984.
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Lesions of the Ventromedial Hypothalamus Reduce Postingestional Thermogenesis M. MONDA, 1 A. SULLO, AND B. DE LUCA Dipartimento di Fisiologia Umana e Funzioni Biologiche Integrate ‘‘Filippo Bottazzi’’, Seconda Universita` di Napoli, Via Costantinopoli 16, 80138 Napoli, Italy Received 12 August 1996; Accepted 9 October 1996
MONDA, M., A. SULLO, AND B. DE LUCA. Lesions of ventromedial hypothalamus reduce postingestional thermogenesis. PHYSIOL BEHAV 61(5) 687–691, 1997.—The aim of this experiment was to evaluate the effects of ventromedial hypothalamus lesions on the thermogenic changes that follow food intake. Four groups of six Sprague-Dawley male rats were used. Under anesthesia with pentobarbital, the animals in the first and second groups received lesions at the ventromedial hypothalamus, and animals in the third and fourth groups received sham lesions. Body weight and food intake were monitored daily until the experimental procedure began. Twenty days after lesion, oxygen consumption, firing rate of sympathetic nerves to interscapular brown adipose tissue (IBAT), and IBAT temperature were monitored for 45 min both before and after 5 g food intake in 24 h fasted rats from the first and third groups. The same variables were measured in the animals of the second and fourth groups 50 days after receiving the lesions. Lesion placements were histologically verified. The results showed that lesions produced hyperphagia and obesity. Firing rate of nerves to IBAT, IBAT temperature, and oxygen consumption increased after food intake in sham-lesioned rats. This increase was significantly reduced by the lesion at both the 20- and 50-day time points. These findings indicate that the ventromedial hypothalamus controls postingestional activation of sympathetic discharge to IBAT. The reduction of postingestional thermogenesis could be involved in the development of obesity induced by lesion of the ventromedial hypothalamus. q 1997 Elsevier Science Inc. Oxygen consumption
Brown adipose tissue
Sympathetic activity
BODY WEIGHT is regulated by food intake and energy expenditure through a homeostatic mechanism (18). Several physiological conditions induce metabolic adaptations that increase or minimize futile energy losses. Hyperalimentation causes an increase in energy expenditure (33), and underfeeding reduces energy expenditure (5). In rodents, during lactation a suppression of brown adipose tissue thermogenesis occurs as an adaptive mechanism to save energy (36). Exercise enhances energy expenditure and causes a sex-dependent change of food intake (31,32). When homeostasis is altered, obesity or leanness generally results ( 18 ) . Rats with lesions of the lateral hypothalamus have reduced body weight ( 10 ) . These animals make adjustments both in food intake ( 27,30 ) and expenditure ( 6,7 ) appropriate to the maintenance of a lower body weight. Lesions of the ventromedial hypothalamus ( VMH ) increase food intake ( 16,22 ) and decrease both resting metabolism ( 37 ) and motor activity ( 35 ) . Nonshivering thermogenesis (15) is activated by food ingestion, and this activation is independent of the caloric density of ingested food in the first minutes after food intake. This phase is
Thermogenesis
Ventromedial hypothalamus
known as postingestional preadsorbitive thermogenesis. Brown adipose tissue (BAT) is the organ responsible for evoking 35– 65% of the total increase in metabolic heat production during various conditions (9,26), including postprandial thermogenesis (2,8,11,12). BAT occurs as numerous discrete deposits, particularly in the interscapular region of the body, and it is under the peripheral neurogenic control of the sympathetic nervous system (1,13,21). The aim of this experiment was to evaluate the effects of VMH lesions on the sympathetic and thermogenic changes that follow food intake. METHOD
Animals Male Sprague-Dawley rats, housed in a room with controlled temperature (22 { 17C) and humidity (70%), were used throughout the experiment. They were bred in the laboratory and had an initial body weight of 280–300 g. Each was caged singly in a room with 06:00–18:00 light–dark cycle. Food and water were provided ad libitum.
1 Requests for reprints should be addressed to Marcellino Monda, M.D., Dipartimento di Fisiologia Umana, Via Costantinopoli 16, 80138 Napoli, Italy.
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Apparatus for Oxygen (O2 ) Consumption Measurement Resting O2 consumption was determined with an indirect calorimeter. The closed circuit apparatus used was an adaptation of Benedict and MacLeod’s calorimeter (3). Air was continuously circulated through a drying column (CaSO4 Drierite), a respiratory chamber with a 2.5-L and CO2 trap (soda lime), by a peristaltic pump at a rate of 2 L/min. The O2 reservoir was a 1L cylindrical metal bell, which fit in a concentric cylinder filled with water, forming an air-tight seal. The cylinder, graduated to 5 mL, was connected to the respiratory chamber. The temperature in the respiratory chamber was maintained constant by circulating water and was monitored by an internal thermometer. The volume of O2 consumed was corrected for temperature and pressure and was expressed as milliliters of O2 /min/kg b.wt.0.75 (19). Apparatus for Registration of Sympathetic Activity The firing rate of nerves to interscapular BAT (IBAT) was recorded by a pair of silver wire electrodes. The electrical pulses were amplified by a condenser-coupled amplifier and were filtered by bandpath filters (NeuroLog System, Digitimer). The raw pulses were displayed on a oscilloscope (Tektronix) and sent to a window discriminator. Square waves from the discriminator were sent to an analog–digital converter (DAS system, Keithley) and stored on a computer (Personal Computer AT, IBM) every 5 s. Furthermore, a rate meter with a reset time of 5 s was used to observe the time course of the nerve activity recorded by a pen recorder (Vitatron). Because the signal-to-noise ratio depended on the number of nerve filaments and the condition of contact between nerve and electrodes, the basal burst rates were different for each rat. The threshold level of the event detector was fixed during the experiment at 50% of the peaks of the largest pulses and above background noise. VMH lesion The lesion-making electrode consisted of a 250-mm-diameter stainless-steel wire insulated except for 0.5 mm at the tip. Under pentobarbital anesthesia (50 mg/kg b.wt.), the electrode was bilaterally inserted into the rats at the following coordinates: 0.2 mm posterior to Bregma, 1.0 mm lateral to the midline, and 9.8 mm below the surface of the skull, according to the atlas of Pellegrino et al. (28). A current of 1.5 mA was applied through the electrode for 15 s; a rectal cathode served as the remote electrode. The sham lesion was made with the same procedure as the lesion, except for the application of current.
On the 20th day, O2 consumption, firing rate of nerves to IBAT, and IBAT temperature were monitored for 45 min both before and after a presentation of 5 g standard food for 5 min. The amount of ingested food was measured. The animals had been kept fasting for 24 h before food presentation. The experimental procedure was carried out in all animals at the same times (18:00–20:00). Histology At the end of the experiment, the rats were injected with an overdose of pentobarbital and intracardially perfused with physiological saline, followed by a 10% solution of phosphate buffered formalin. The brains were removed and fixed in formalin. Afterward, the brains were cut into blocks that were placed in heptane at 0307C and later sliced in 75-mm sections (29). These microtome sections were subsequently stained with cresyl violet. RESULTS
Figure 1 shows the percentage of changes in firing rate of nerves to IBAT. Food ingestion increased the firing rate, which peaked at 5 min in the sham-lesioned rats. This increase was reduced in the lesioned animals both 20 days and 50 days after the lesion. Analysis of variance showed significant effects for lesion, F (1, 20) Å 24.923, p õ 0.01, time, F (6, 120) Å 31.265, p õ 0.01, and Lesion 1 Time, F (6, 120) Å 5.408, p õ 0.01. There were no group differences in baseline levels of sympathetic activity. Examples of changes in actual firing rate of the nerves to IBAT are reported in Fig. 2. Figure 3 illustrates TIBAT changes. Food intake caused a rise that peaked at 10 min in the sham-lesioned rats. This increase was reduced in the lesioned animals both 20 days and 50 days after the lesion. Analysis of variance showed significant effects for lesion, F (1, 20) Å 30.948, p õ 0.01, time, F (6, 120) Å 29.252, p õ 0.01, and Lesion 1 Time, F (6, 120) Å 2.868, p õ 0.05]. Furthermore, the changes in the firing rate of IBAT nerves preceded the changes in temperature. Figure 4 illustrates changes in O2 consumption. Food intake induced an increase, with a peak at 10 min in the sham-lesioned rats. The VMH lesion reduced the increase induced by food intake both 20 days and 50 days after lesion. Analysis of variance showed significant effects for lesion, F (1, 20) Å 21.075, p õ
Procedure Twelve animals received lesions in the VMH, and another 12 served as sham-operated controls. Food intake and body weight were measured daily. Half the animals in each group were tested at 20 days and half at 50 days following surgery. Four days before the experiment, the rats were anesthetized with pentobarbital (50 mg/kg b.wt.). IBAT was exposed, and intercostal nerves entering the right side of IBAT were identified. Nerve filaments were isolated from the central cut end of these nerve bundles under a dissecting microscope to position a pair of silver wire electrodes. The entire preparation was embedded into a small silicone mold and covered with silicone gel (WackerChemie, Munchen, Germany) that hardened sufficiently within 1 h to stabilize the preparation and to provide electrical insulation against the surrounding tissue. The silver wire electrodes were connected with a pin cemented to cranial theca by four screws.
FIG. 1. Means { SE of percentage of changes in firing rate of nerves innervating IBAT in VMH-lesioned or sham-lesioned rats 20 or 50 days after surgery. Food presentation was at time 0.
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FIG. 3. Means { SE of changes in IBAT temperature of VMH-lesioned or sham-lesioned rats 20 or 50 days after surgery. Food presentation was at time 0.
were 1.20 { 0.31 g for the sham-lesioned rats and 11.75 { 0.72 g for the lesioned rats. An increase of 2.03 { 0.34 g for shamlesioned rats and 12.50 { 0.56 g for lesioned rats was noted after 50 days. The analysis of variance showed a statistical difference across groups, F (3, 20) Å 180.001, p õ 0.01. The Sheffe` test, used as a post hoc test, showed a difference (p õ 0.05) between sham-lesioned and lesioned animals, both 20 and 50 days after lesion. The changes in body weight are shown in Fig. 6. The increases were 7.83 { 1.58 g for sham-lesioned animals and 65.50 { 3.06 g for lesioned rats after 20 days. In the sham-lesioned and lesioned animals, an increase of 10.03 { 7.04 g and 67.83 { 6.49 g, respectively, was registered after 50 days. The analysis of variance revealed the difference across groups, F (3, 20) Å 149.261, p õ 0.01. The post hoc test showed a difference ( p õ 0.05) between sham-lesioned and lesioned animals 20 and 50 days after lesion. The strain of rats utilized in this experiment was inbred, which caused a body weight gain lower than that of noninbred animals. Figure 7 illustrates an example of a VMH lesion. Darkened areas represent the extent of the lesion from stereotaxis planes
FIG. 2. Actual firing rate changes in a rat 20 days after sham lesion (A) or VMH lesion (B) and in a rat 50 days after sham lesion (C) or VMH lesion (D). The arrow indicates the time of food presentation.
0.01, time, F(6, 120) Å 59.761, p õ 0.01, and Lesion 1 Time, F(6, 120) Å 13.722, p õ 0.01. The amount of food ingested after 5 min presentation was 4.35 { 0.06 g in the first group, 4.21 { 0.08 in the second group, 4.32 { 0.07 in the third group, and 4.29 { 0.11 in the fourth group. The analysis of variance did not show any significant difference across the groups. The 5-min food intake was identical in all groups because the rats had been fasted for 24 h. The difference between the values of 24-h food intake, measured before and 20 or 50 days after the sham lesion or lesion, are indicated in Fig. 5. The increases in food intake after 20 days
FIG. 4. Means { SE of changes in oxygen consumption of VMH-lesioned or sham-lesioned rats 20 or 50 days after surgery. Food presentation was at time 0.
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FIG. 5. Means { SE of changes in 24-h food intake of VMH-lesioned or sham-lesioned rats 20 or 50 days after surgery.
FIG. 6. Means { SE of changes in body weight of VMH-lesioned or sham-lesioned rats 20 or 50 days after surgery.
29 – 33 ( 28 ) . The area with maximum damage is in plane 31. Lesions did not extend to the peri-VMH area, and their size was identical. For this reason, any relationship between lesion size and magnitude of metabolic response was not found.
could induce an increase of total amount of ingested food. This reduction may be another factor in the induction of obesity by the VMH lesion. In other words, the increased body weight of
DISCUSSION
Thermogenesis is under the control of the vegetative nervous system. Experimental evidence demonstrates that IBAT is a major organ of nonshivering thermogenesis, at least in rodents ( 15 ) . Many investigators have shown that activation of sympathetic nerves supplying IBAT causes a rise in temperature of this tissue ( 14,17,34 ) . We have clearly demonstrated that a modification of sympathetic activity causes change in IBAT activity ( 20,23 – 25 ) . The findings of the present experiment show that the VMH lesion modifies postingestional changes in the sympathetic discharge of nerves to IBAT, IBAT temperature, and O2 consumption, which demonstrates that the VMH is strongly involved in the control of sympathetic and thermogenic reactions due to food ingestion. In the present experiment, we obtained direct evidence of the activation of the sympathetic nervous system and of its involvement in the control of nonshivering thermogenesis. The findings of this experiment are the first to show that the VMH lesion modifies postingestional increase in the firing rate of the sympathetic nerves to IBAT. IBAT weight was not measured. The marked metabolic alterations could have resulted in loss of IBAT and degeneration of their sympathetic supply, which could have resulted in a smaller response. The thermogenic response was reduced 20 and 50 days after the lesion. This observation suggests that the VMH lesion induces a long-term modification in the sympathetic and thermogenic reaction to food intake. A long-term reduction of postingestional thermogenesis may contribute to obesity induced by a VMH lesion through a decrease in energy expenditure induced by reduced sympathetic activity. There is a close relationship between the sympathetic activity and food intake. Some evidence seems to suggest that an increase in sympathetic discharge reduces food intake ( 4 ) . Thus, a reduction in the sympathetic response after food intake
FIG. 7. Planes 29–33 from the stereotaxis atlas of Pellegrino et al. (28). Darkened areas represent extent of VMH lesion. The top section corresponds with plane 29. See (28) for abbreviations.
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VMH-lesioned animals may be caused by a reduction of satiety signals and a decrease in postingestional energy expenditure.
ACKNOWLEDGMENTS
This study was supported by a grant from the Italian National Research Council.
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