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Reduced sympathetic nervous system activity in rats with ventromedial hypothalamic lesions
Life Sciences, Vol. 30, pp. 913-920 Printed in the U.S.A.
Pergamon Press
REDUCED SYMPATHETIC NERVOUS SYSTEM ACTIVITY IN RATS WITH VENTROMEDIAL HYPOTHALAMIC LESIONS Jerry G. Vander Tuig, Allen W. Knehans and Dale R. Romsos Department of Food Science and Human Nutrition Michigan State University East Lansing, MI 48824 (Received in final form January 13, 1982) Summary To determine if alterations in sympathetic nervous system (SNS) activity occur in rats with ventromedial hypothalamic (VMH) lesions, norepinephrine ( N E ) turnover rates were examined in various tissues of lesioned and control, weanling rats. VMHlesioned rats fed a high-carbohydrate diet ad libitum for 4 weeks following surgery were not hyperphagic, but they gained 50% more body energy than control rats. VMH lesions extended the half-life of 3H-NE in interscapular brown adipose tissue (BAT) by 42%, in abdominal white adipose tissue (WAT) by 201%, in heart by 61% and in pancreas by 85%, and reduced total NE turnover (ng/organ/br) in BAT (38%), WAT (57%), heart (30%) and pancreas (53%). Reduced SNS activity in BAT is consistent with the decreased energy expenditure (heat production) and increased energy efficiency observed in VMH-lesioned rats. In WAT, decreased SNS activity coupled with hyperinsulinemia would facilitate energy storage as fat by reducing lipid mobilization. In the pancreas, reduced SNS activity would contribute to byperinsulinemia. These results support the hypothesis that VMH lesions decrease SNS activity in several organs. This change in autonomic tone is very likely a major factor in the development of obesity in VMH-lesioned animals. Obesity that develops in animals with ventromedial hypothalamic (VMH) lesions is not simply the result of overeating because of damage to a neural satiety center. Young adult, VMH-lesioned rats that are prevented from overeating (1-3) and weanling, VMH-lesioned rats that are normophagic (4-6) still gain more body energy than control rats. Therefore, these VMH-lesioned rats must expend less energy than control rats. The metabolic basis for tbls reduced energy expenditure in normophagic, VMH-lesioned rats has not been established. Recently, a hypothesis involving the autonomic nervous system has been proposed to explain the development of hypothalamic obesity. Powley (7) has suggested that many of the behavioral and metabolic changes observed in VMH-lesioned animals are the result of enhanced "cephalic reflexes" associated
Supported by NIH AM 15847. Article Number 10051.
Michigan
Agriculture
Experiment
0024-3205/82/110913-08503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
Station
Journal
914
VMH Lesions and Sympathetic Activity
Vol. 30, No. ii, 1982
with food stimuli. He proposes that VMH lesions remove the "damping" of these reflexes, increasing positive feedback responses such as salivary, gastric and pancreatic secretions. Bray and York (8,9) have modified these ideas to suggest that VMH lesions result in an imbalanced autonomic nervous system with decreased sympathetic outflow. Reduced sympathetic nervous system (SNS) activity could contribute to several metabolic changes associated with hypothalamic obesity, including hyperinsulinemia (3,10,11), less ability to mobilize lipid stores (12), and decreased thermogenesis (6). Conflicting reports have appeared concerning SNS activity in VMH-lesioned animals. Reduced salivary gland weight (I0) and impaired ability to mobilize fatty acids when stressed (12) provide indirect evidence for suppressed SNS activity in VMH-lesioned rats. But data obtained with a more direct indicator of SNS activity, norepinephrine (NE) turnover (13), suggest that SNS activity is not suppressed in mice with gold thioglucose VMH lesions (14). NE turnover in hearts, which was the only tissue examined in these VMH-damaged mice, was similar to that in hearts of control mice. However, the gold thioglucose-treated mice, unlike control mice, failed to suppress SNS activity in their hearts during fasting (14). Thus, the status of the SNS in VMH-lesioned animals is presently unresolved. The purpose of this study was to assess SNS activity in several tissues (brown adipose tissue, white adipose tissue, heart and pancreas) that are likely to be involved in the development of obesity in VMH-lesioned rats. NE turnover rate was used as an indicator of SNS activity (13). Weanling rats were lesioned because they become obese without overeating (4-6), thus avoiding the confounding effects of hyperphagia on SNS activity (15,16). Methods Female Sprague-Dawley rats at 21 days of age were obtained from Harlan Industries, Inc., Cumberland, Indiana. Rats were housed in individual cages with raised, wire mesh floors at a room temperature of 23+1 ° . Room lights were on 12 hours per day. A high-carbohydrate diet (17~ and water were available ad libitum throughout the experiment. After an initial adjustment period of 1 week, rats were divided by weight into 3 groups. One group received bilateral, electrolytic lesions in the VMH and another group served as sham-operated, control rats. The third group was killed at the time of surgery to provide initial body energy values. Rats were anesthetized with sodium pentobarbital (35 mg/kg body weight, i.p.) and were also given approximately 1 mg atropine methyl nitrate (i.p.) to reduce respiratory problems during surgery and recovery from the anesthetic. Lesions, stereotaxically positioned (David Kopf Instruments, Tujunga, california) using coordinates of Bernardis and Skelton (18), were produced by passing 1.5 mA of anodal current for 10 seconds through a stainless steel electrode which was insulated except for 0.5 mm at the tip. Sham-operated rats were treated similarly except no current was passed through the electrode. Following surgery, all rats received (i.m.) 20,000 units of penicillin G. VMH-lesioned and sham-operated rats were maintained for 4 weeks after surgery. During that time food intake and body weights were measured. Total body energy was measured in the initial group of rats killed at the time of surgery, and in 8 VMH-lesioned and 8 sham-operated rats killed 4 weeks after surgery. To measure body energy, gastrointestinal contents were removed, carcasses were homogenized, and aliquots were combusted in a bomb calorimeter (19). Body energy of lesioned and control rats did not include energy of
Vol. 30, No. ii, 1982
tissues removed adipose tissue, pancreas).
VMH Lesions and Sympathetic Activity
915
for norepinephrine turnover analysis (interscapular brown approximately 1 g of white adipose tissue, the heart and
Brains of lesioned rats were fixed in buffered 10% formalin, embedded in paraffin and 6u sections were stained with cresyl violet. Only rats that had bilateral symmetrical lesions within the VMH area and elevated amounts of abdominal adipose tissue were used. Norepinepbrine (NE) turnover rates were measured in lesioned and control rats at the end of the 4-week study. Young and Landsberg have established that NE turnover rates provide valid indicators of SNS activity in rats (15,16) and in gold thioglucose VMH-lesioned mice (14). Tritiated NE (levo-[7,8-3H(N)], New England Nuclear), diluted in 0.9% NaCI to provide an average dose of 261 uCi 3~H-NE/kg body weight, was injected (i.p.) in a total volume of 0.5 ml. Separate groups of rats were killed by cervical dislocation at 2, 6, 12 and 24 hours following the injection. Interscapular brown adipose tissue (BAT), approximately 1 g of abdominal white adipose tissue (WAT), the heart and pancreas were rapidly removed and frozen between pieces of dry ice. Tissues were stored frozen on dry ice until assayed for NE. NE was assayed using a modification of the HPLC method of Refshauge et al. (20). Tissues were homogenized in 0.4 N perchloric acid with sodium metablsulfite and EDTA added as antioxidants, and dihydroxybenzylamine added as an internal standard. Following centrifugation, 0.5 M tris-HCl, pH 8.6 was added to the supernatant and NE was adsorbed on alumina. After washing the alumina with water, NE was eluted with 0.1 N perchloric acid and injected into an HPLC system with a Whatman Partisil-10 ODS-3 reversed phase column. NE and internal standard were quantitated with electrochemical detection (Bioanalytical Systems, West Lafayette, IN). The NE was collected and counted to determine specific activity of 3H-NE. Half-lives and fractional turnover rates (k) of NE were calculated from regression equations of 3H-NE specific activity plotted against time after injection of tracer. Slopes of regression lines were compared using the variance estimated for the difference between slopes (21). Total NE turnover per organ was calculated as the product of fractional turnover (k) and endogenous NE per organ. Individual means were compared using Student's t test. Results TABLE I Weight Gain, Energy Intake and Energy Gain of Sham-Operated and VMH-Lesioned Rats Sham-operated Body weight gain, g/4 weeks Metabolizable energy intake, kcal/4 weeks Body energy gain, kcal/4 weeks
134 +
3
1638 + 32 346 ~ 26
VMH-lesioned 125 +
5*
1688 + 51 518 ~ 47*
Means + S~M for 24 sham-operated and 18 VMH-lesioned, weanling rats. Initial body weights averaged 76 + 2 g. Body energy gain was calculated for 8 rats from each group. An asterisk (*) indicates a significant difference (P<0.05) between sham-operated and VMH-lesioned rats.
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Body weight gain, metabolizable energy intake and body energy gain of sham-operated and VMH-lesioned, weanling rats are presented in Table I. VMH-lesioned rats gained slightly less body weight than sham-operated rats despite similar ad libltum metabolizable energy intake by both groups. VMH-lesioned rats, however, gained 50% more body energy during the 4-week study because they accumulated more fat (Table II) and presumably less lean tissue than control rats. Final body energy values for sham-operated and VMH-lesioned rats were 459 + 30 and 642 + 47 kcal (mean + SEM), respectively. Gross energy efficiency (b~dy energy gain~metabolizable e n e r g y intake x 100) was 22 + 1% in sham-operated rats and 32 + 2% in VMH-lesioned rats (P<0.001). Since food intake was not increased, greater energy retention by VMH-lesioned rats must have resulted from a concomitant reduction in energy expenditure. NE turnover data from interscapular BAT, abdominal WAT, heart and pancreas are summarized in Table II and Figure i. NE turnover was measured in BAT because BAT is an important organ for tbermoregulatory heat production in rats (22), and because we bad previously observed that NE turnover was suppressed in BAT of obese (ob/ob) mice (23). Interscapular BAT of VMH-lesioned rats was heavier than that of sham-operated rats, but NE levels were similar in BAT of both groups (Table II). Increased BAT weight in VMH-lesioned rats was likely because of greater lipid content. The half-life of 3H-NE in BAT of VMH-lesioned rats averaged 7.9 hours compared with 5.6 hours in control rats (Figure I). These values correspond to fractional turnover rates (k) of 8.8 1.3 and 12.5 + 1.1% per hour (P<0.05) for VMH-lesioned and control rats, respectively. Total NE turnover in the interscapular BAT depot was decreased 38% by VMH lesions (Table II).
T~TE
il
Norepinephrine (NE) Turnover in Brown Adipose Tissue (BAT), White Adipose Tissue (WAT), Heart and Pancreas of Sham-Operated and VMH-Lesioned Rats
Tissue BAT Sham-operated vMH-lesioned WAT Sham-operated VMH-lesioned
Organ weight (m9)
348 ~ 14 520 + 26*
5,361 + 269 11,449 + 683*
Endogenous NE (ng/organ)
NE turnover rate
(n~/or~an/hr)
544 ~ 20 484 + 27
68 ~ 4 42 + 2*
155 + ii 189 + 36
14 + 1 6 + i*
Heart Sham-operated VMH-lesioned
796 + 15 651 + 15"
986 + 44 1,117 + 77*
71 + 3 50 + 2*
Pancreas Sham-operated VMH-lesioned
602 + 23 453 + 23*
322 + 14 268 + 17"
32 + 1 15 + i*
Means ~ S~M for 24 sham-operated and 18 VMH-lesioned rats. An asterisk (*) indicates a significant difference (P< 0.05) between sham-operated and VMH-lesioned rats. WAT weight includes all dissectible abdominal fat.
Vol. 30, No. ii, 1982
VMH Lesions and Sympathetic Activity
Brown Adipose Tissue
917
White Adipose Tissue
jVM.
•
lO~..............
10=
1/2 - 2 3 3hr
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~
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HOURS FIG. 1 Effect of VMH lesions on norepinephrine (NE) turnover in BAT, WAT, heart and pancreas. Data points represent means ~ SEM for 4 to 6 rats. Respective r values for BAT, WAT, heart and pancreas were 0.87, 0.41, 0.84, and 0.71 for VMR-lesioned rats and 0.93, 0.74, 0.93 and 0.90 for sham-operated rats. For each tissue, the slope calculated for VMH-lesioned rats was significantly different (P<0.05) from that calculated for sham-operated rats.
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NE turnover rates were also measured in W A T because of the importance of the SNS in mobilizing lipids from fat stores (12), and to compare NE turnover in WAT and in BAT. Fat samples were taken from gonadal fat pads in VMH-lesioned and sham-operated rats. Total WAT organ weight was defined as total dissectable abdominal fat, excluding mesenterlc fat pads. VMH-lesioned rats had more than twice as much abdominal fat as control rats, but NE content in WAT was comparable in lesioned and control rats (Table II). NE half-llfe was 7.7 hours in WAT from control rats, but was tripled to 23.3 hours by VMH lesions (Figure i). As a result, fractional turnover (k) of NE in WAT averaged 9.0 + 2.0% per hour in control rats, but only 3.0 + 1.8% per hour (P <0.05) in VMH---lesioned rats. Total WAT NE turnover in VMH~lesioned rats was less than half that observed in sham-operated rats (Table II). Since the metabolic rate and SNS activity in hearts of normal rats respond in parallel to cold exposure, overfeeding and fasting (15,16), NE turnover was also investigated in hearts of sham-operated and VMH-lesioned rats. Hearts of lesioned rats weighed less, but contained 13% more NE, than hearts of control rats (Table If). NE half-life averaged 9.7 and 15.5 hours in hearts of sham-operated and VMH-lesioned rats, respectively (Figure i). Corresponding fractional turnover rates were 7.2 + 0.6 and 4.5 + 0.7% per hour (P<0.01). Total heart NE turnover was 30% less in VMH-lesioned rats. Turnover of NE in the pancreas was examined because the SNS affects insulin secretion ( 8 , 9 ) and because hyperinsulinemia is associated with hypothalamic obesity (3,10,11). Both pancreas weight and NE levels were decreased similarly in VMH-lesioned rats (Table If). Consequently, NE concentration per mg of pancreas was unaffected by VMH lesions. NE half-life averaged 6.9 hours in control pancreas, but was increased to 12.8 hours in VMH-lesioned rats (Figure i). Fractional NE turnover (k) in pancreas decreased from 10.0 + 1.1% per hour in control rats to 5.4 + 1.3% per hour (P < 0.02) in VMH-lesioned rats. Total NE turnover in the pancreas of vMHlesioned rats was less than half that observed in sham-operated rats (Table If). Discussion VMH lesions in weanling rats reduced NE turnover in every tissue examined (BAT, WAT, heart and pancreas). Because NE turnover has been demonstrated to be a valid indicator of SNS activity (14-16), these results suggest that SNS activity is reduced in these organs of VMH-lesioned animals. Concomitant acceleration of SNS activity and energy expenditure during cold exposure and overfeeding, and decreased SNS activity and energy expenditure during fasting (15,16), suggest a relationship between the SNS and energy balance. Our observations of decreased SNS activity and energy expenditure (6) in VMH-lesioned, weanling rats are in agreement with these studies and suggest an important role for the SNS in the development of increased energy efficiency and subsequent obesity in VMH-lesioned rats. Because the temperature at which the rats in this study were housed (23 ° ) was below their thermoneutral zone (24), altered thermoregulatory heat production in the VMB-lesioned rats may have been a factor in their increased efficiency of energy retention as it is in obese (ob/ob) mice housed below their thermoneutral zone (19). NE turnover in BAT of VMH-lesioned rats was reduced 38% in the present study. This compares with a 70% reduction in NE turnover in BAT of obese (ob/ob) mice housed at 25 ° (23). BAT is the major organ responsible for thermoregulatory beat production in rats (22). Since the SNS regulates heat production in BAT (25), it seems reasonable to conclude that reduced NE turnover in BAT of VMH-lesioned rats and obese (ob/ob) mice housed at 23-25 ° contributes to their increased efficiency of energy
Vol. 30, No. ii, 1982
VMH Lesions and Sympathetic Activity
919
retention. In agreement with these observations, Seydoux et al. (26) demonstrated that the sensitivity and response of BAT from VMH-lesioned rats to norepinephrine is also decreased when compared with that of control rats. In addition to reduced thermoregulatory heat production by BAT, other factors are likely to be involved in the enhanced efficiency of energy retention in VMH-lesioned rats and genetically obese (ob/ob) mice. VMH-lesioned rats acutely exposed to 30° still consume less oxygen than control rats (unpublished observations) and obese mice housed at 33° (within the thermoneutral zone) still exhibit a higher efficiency of energy retention than lean mice (19). One possibility is that the lower numbers of Na+,K+-ATPase enzyme units found in skeletal muscle of VMH-lesioned rats (6) and obese mice (27,28) contribute to increased efficiency of energy retention in these animals. The fractional turnover rate of NE in WAT was reduced from 9% to 3% per hour by VMH lesions. These results support earlier suggestions, based on less direct measures, that SNS activity is suppressed in WAT of VMH-lesioned rats (12). This reduction, coupled with hyperinsulinemia characteristic of these rats (3,10,11), would suppress lipolysis and facilitate lipid a~cumulation in WAT. NE turnover rates in heart were reduced 30% by VMH lesions. These results appear at variance with reports of no suppression of NE turnover in hearts of obese (oh/oh) mice (23) or of gold thioglucose-treated mice (14); however, the differences may simply reflect the stage of development at which the animals were examined. VMH-lesioned rats in the present study were not overweight and d a lower metabolic rate than control rats, whereas the 8-week-old obese mice were 60% overweight (23) and had a metabolic rate per animal as high as that of control mice. Presumably the gold thioglucose-lesioned mice, which were at least 4 standard deviations above the mean weight of the control mice (14), also had a metabolic rate at least as high as that of the control mice. If NE turnover in the heart is linked to metabolic rate, then the differences between weanling, VMH-lesioned rats and the young adult obese mice in the other studies (14,23) would be expected. Measurement of NE turnover in hearts of adult hyperphagic and overweight VMH-lesioned rats and in young normophagic obese (oh/oh) mice would help resolve this apparent discrepancy. The autonomic hypothesis proposed to explain the development of hypothalamic obesity emphasizes the role of hyperinsulinemia resulting from reduced sympathetic outflow to the pancreas (8,9). In the present study, pancreatic NE turnover rates were markedly reduced in VMH-lesioned rats. These data support the hypothesis which proposes that by reducing SNS activity in pancreas, VMH lesions enhance the parasympathetic stimulation of insulin secretion mediated by the vagus nerve (8,9,11). From the data reported here, it can be concluded that VMH lesions in weanling rats decrease NE turnover rates in BAT, WAT, heart and pancreas. This decreased SNS activity probably contributes to reduced energy expenditure by BAT, accumulation of excess lipid by WAT, and hyperinsulinemia. Thus, decreased sympathetic outflow to these sympathetically innervated organs following VMH lesions is likely a major factor in the development of bypothalamic obesity.
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Acknowledgments The authors wish to thank H. Wells for technical assistance, preparation of the graph and D. Klein for typing the manuscript.
C. Oberg
for
References I. 2. 3. 4. 5. 6. 7. 8. 9. 10. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
P.W. BAN, Am. J. Physiol. 215 1343-1350 (1968). L.M. VAN PUTTEN, D.W. VAN BEKKUM and A. QUERIDO, Metabol. 4 68-74 (1955). J.E. COX and T.L. POWLEY, Am. J. Physiol. 240 E566-E572 (1981). P.W. HAN, C.H. LIN, K.C. CHU, J.Y. MU and A.C. LIU, Am. J. Physiol. 209 627-631 (1965). L.L. BERNARDIS and L.A. FROHMAN, J. Comp. Neurol. 141 107-115 (1971). J.G. VANDER TUIG, A.M. FLYNN and D.R. ROMSOS, Proc. Soc. Exp. Biol. Med. 167 475-479 (1981). T.L. POWLEY, Psychol Rev. 84 89-126 (1977). G.A. BRAY and D.A. YORK, Physiol Rev. 59 719-809 (1979). G.A. BRAY, S. INOUE and Y. NISHIZAWA, Diabetologia 20 366-377 (1981). S. INOUE, L.A. CAMPFIELD and G.A. BRAY, Am. J. Physiol. 233 RI62-RI68 (1977). H.R. BERTHOUD and B. JEANRENAUD, Endocrinol. 10_.5 146-151 (1979). Y. NISHIZAWA and G.A. BRAy, J. Clln. Invest. 61 714-721 (1978). N.H. NEFF, T.N. TOZER, W. HAMMER, E. COSTA and B.B. BRODIE, J. Pharmacol. Exp. Ther. 160 48-52 (1968). J.B. YOUNG and L. LANDSBERG, J. Clin. Invest. 65 1086-1094 (1980). L. LANDSBERG and J.B. YOUNG, N. Engl. J. Med. 298 1295-1301 (1978). J.B. YOUNG and L. LANDSBERG, Am. J. Physiol. 236 E524-E533 (1979). P.Y. LIN, D.R. ROMSOS, J.G. VANDER TUIG and G.A. LEVEILLE, J. Nutr. 109 1143-1153 (1979). L.L. BERNARDIS and F.R. SKELTON, Am. J. Anat. 116 69-74 (1965). J.G. VANDER TUIG, D.R. ROMSOS and G.A. LEVEILLE, J. Nutr. ii0 35-41 (1980). C. REFSHAUGE, P.T. KISSINGER, R. DREILING, L. BLANK, R. FREEMAN and R.N. ADAMS, Life Sci. 14 311-322 (1974). J.L. GILL, Design and Analysis of Experiments in the Animal and Medical Sciences, vol. i, p. 106, Iowa State University Press, Ames, IA. (1978). D.O. FOSTER and M.L. FRYDMAN, Can J. Physiol. Pharmacol. 56 110-122 (1978). A.W. KNEHANS and D.R. ROMSOS, Fed. Proc. 40 888 (1981). M. KLEIBER, The Fire of Life: An Introduction to Animal Ener@etics, p. 249, R.E. Krieger Publishing Company, Huntington, New York (1975). L. BUKOWIECKI, N. FOLLEA, A. PARADIS and A. COLLET, Am. J. Physiol. 238 E552-E563 (1980). J. SEYDOUX, F.R. JEANRENAUD, F.A. JEANNET, B. JEANRENAUD and L. GIRARDIER, Pflugers Arch. 390 1-4 (1981). M.H. LIN, J.G. VANDER TUIG, D.R. ROMSOS, T. AKERA and G.A. LEVEILLE, Am. J. Physiol. 237 E 265-E272 (1979). M.H. LIN, J.G. VANDER TUIG, D.R. ROMSOS, T. AKERA and G.A. LEVEILLE, Am. J. Physiol. 238 EI93-EI99 (1980).
Pergamon Press
REDUCED SYMPATHETIC NERVOUS SYSTEM ACTIVITY IN RATS WITH VENTROMEDIAL HYPOTHALAMIC LESIONS Jerry G. Vander Tuig, Allen W. Knehans and Dale R. Romsos Department of Food Science and Human Nutrition Michigan State University East Lansing, MI 48824 (Received in final form January 13, 1982) Summary To determine if alterations in sympathetic nervous system (SNS) activity occur in rats with ventromedial hypothalamic (VMH) lesions, norepinephrine ( N E ) turnover rates were examined in various tissues of lesioned and control, weanling rats. VMHlesioned rats fed a high-carbohydrate diet ad libitum for 4 weeks following surgery were not hyperphagic, but they gained 50% more body energy than control rats. VMH lesions extended the half-life of 3H-NE in interscapular brown adipose tissue (BAT) by 42%, in abdominal white adipose tissue (WAT) by 201%, in heart by 61% and in pancreas by 85%, and reduced total NE turnover (ng/organ/br) in BAT (38%), WAT (57%), heart (30%) and pancreas (53%). Reduced SNS activity in BAT is consistent with the decreased energy expenditure (heat production) and increased energy efficiency observed in VMH-lesioned rats. In WAT, decreased SNS activity coupled with hyperinsulinemia would facilitate energy storage as fat by reducing lipid mobilization. In the pancreas, reduced SNS activity would contribute to byperinsulinemia. These results support the hypothesis that VMH lesions decrease SNS activity in several organs. This change in autonomic tone is very likely a major factor in the development of obesity in VMH-lesioned animals. Obesity that develops in animals with ventromedial hypothalamic (VMH) lesions is not simply the result of overeating because of damage to a neural satiety center. Young adult, VMH-lesioned rats that are prevented from overeating (1-3) and weanling, VMH-lesioned rats that are normophagic (4-6) still gain more body energy than control rats. Therefore, these VMH-lesioned rats must expend less energy than control rats. The metabolic basis for tbls reduced energy expenditure in normophagic, VMH-lesioned rats has not been established. Recently, a hypothesis involving the autonomic nervous system has been proposed to explain the development of hypothalamic obesity. Powley (7) has suggested that many of the behavioral and metabolic changes observed in VMH-lesioned animals are the result of enhanced "cephalic reflexes" associated
Supported by NIH AM 15847. Article Number 10051.
Michigan
Agriculture
Experiment
0024-3205/82/110913-08503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
Station
Journal
914
VMH Lesions and Sympathetic Activity
Vol. 30, No. ii, 1982
with food stimuli. He proposes that VMH lesions remove the "damping" of these reflexes, increasing positive feedback responses such as salivary, gastric and pancreatic secretions. Bray and York (8,9) have modified these ideas to suggest that VMH lesions result in an imbalanced autonomic nervous system with decreased sympathetic outflow. Reduced sympathetic nervous system (SNS) activity could contribute to several metabolic changes associated with hypothalamic obesity, including hyperinsulinemia (3,10,11), less ability to mobilize lipid stores (12), and decreased thermogenesis (6). Conflicting reports have appeared concerning SNS activity in VMH-lesioned animals. Reduced salivary gland weight (I0) and impaired ability to mobilize fatty acids when stressed (12) provide indirect evidence for suppressed SNS activity in VMH-lesioned rats. But data obtained with a more direct indicator of SNS activity, norepinephrine (NE) turnover (13), suggest that SNS activity is not suppressed in mice with gold thioglucose VMH lesions (14). NE turnover in hearts, which was the only tissue examined in these VMH-damaged mice, was similar to that in hearts of control mice. However, the gold thioglucose-treated mice, unlike control mice, failed to suppress SNS activity in their hearts during fasting (14). Thus, the status of the SNS in VMH-lesioned animals is presently unresolved. The purpose of this study was to assess SNS activity in several tissues (brown adipose tissue, white adipose tissue, heart and pancreas) that are likely to be involved in the development of obesity in VMH-lesioned rats. NE turnover rate was used as an indicator of SNS activity (13). Weanling rats were lesioned because they become obese without overeating (4-6), thus avoiding the confounding effects of hyperphagia on SNS activity (15,16). Methods Female Sprague-Dawley rats at 21 days of age were obtained from Harlan Industries, Inc., Cumberland, Indiana. Rats were housed in individual cages with raised, wire mesh floors at a room temperature of 23+1 ° . Room lights were on 12 hours per day. A high-carbohydrate diet (17~ and water were available ad libitum throughout the experiment. After an initial adjustment period of 1 week, rats were divided by weight into 3 groups. One group received bilateral, electrolytic lesions in the VMH and another group served as sham-operated, control rats. The third group was killed at the time of surgery to provide initial body energy values. Rats were anesthetized with sodium pentobarbital (35 mg/kg body weight, i.p.) and were also given approximately 1 mg atropine methyl nitrate (i.p.) to reduce respiratory problems during surgery and recovery from the anesthetic. Lesions, stereotaxically positioned (David Kopf Instruments, Tujunga, california) using coordinates of Bernardis and Skelton (18), were produced by passing 1.5 mA of anodal current for 10 seconds through a stainless steel electrode which was insulated except for 0.5 mm at the tip. Sham-operated rats were treated similarly except no current was passed through the electrode. Following surgery, all rats received (i.m.) 20,000 units of penicillin G. VMH-lesioned and sham-operated rats were maintained for 4 weeks after surgery. During that time food intake and body weights were measured. Total body energy was measured in the initial group of rats killed at the time of surgery, and in 8 VMH-lesioned and 8 sham-operated rats killed 4 weeks after surgery. To measure body energy, gastrointestinal contents were removed, carcasses were homogenized, and aliquots were combusted in a bomb calorimeter (19). Body energy of lesioned and control rats did not include energy of
Vol. 30, No. ii, 1982
tissues removed adipose tissue, pancreas).
VMH Lesions and Sympathetic Activity
915
for norepinephrine turnover analysis (interscapular brown approximately 1 g of white adipose tissue, the heart and
Brains of lesioned rats were fixed in buffered 10% formalin, embedded in paraffin and 6u sections were stained with cresyl violet. Only rats that had bilateral symmetrical lesions within the VMH area and elevated amounts of abdominal adipose tissue were used. Norepinepbrine (NE) turnover rates were measured in lesioned and control rats at the end of the 4-week study. Young and Landsberg have established that NE turnover rates provide valid indicators of SNS activity in rats (15,16) and in gold thioglucose VMH-lesioned mice (14). Tritiated NE (levo-[7,8-3H(N)], New England Nuclear), diluted in 0.9% NaCI to provide an average dose of 261 uCi 3~H-NE/kg body weight, was injected (i.p.) in a total volume of 0.5 ml. Separate groups of rats were killed by cervical dislocation at 2, 6, 12 and 24 hours following the injection. Interscapular brown adipose tissue (BAT), approximately 1 g of abdominal white adipose tissue (WAT), the heart and pancreas were rapidly removed and frozen between pieces of dry ice. Tissues were stored frozen on dry ice until assayed for NE. NE was assayed using a modification of the HPLC method of Refshauge et al. (20). Tissues were homogenized in 0.4 N perchloric acid with sodium metablsulfite and EDTA added as antioxidants, and dihydroxybenzylamine added as an internal standard. Following centrifugation, 0.5 M tris-HCl, pH 8.6 was added to the supernatant and NE was adsorbed on alumina. After washing the alumina with water, NE was eluted with 0.1 N perchloric acid and injected into an HPLC system with a Whatman Partisil-10 ODS-3 reversed phase column. NE and internal standard were quantitated with electrochemical detection (Bioanalytical Systems, West Lafayette, IN). The NE was collected and counted to determine specific activity of 3H-NE. Half-lives and fractional turnover rates (k) of NE were calculated from regression equations of 3H-NE specific activity plotted against time after injection of tracer. Slopes of regression lines were compared using the variance estimated for the difference between slopes (21). Total NE turnover per organ was calculated as the product of fractional turnover (k) and endogenous NE per organ. Individual means were compared using Student's t test. Results TABLE I Weight Gain, Energy Intake and Energy Gain of Sham-Operated and VMH-Lesioned Rats Sham-operated Body weight gain, g/4 weeks Metabolizable energy intake, kcal/4 weeks Body energy gain, kcal/4 weeks
134 +
3
1638 + 32 346 ~ 26
VMH-lesioned 125 +
5*
1688 + 51 518 ~ 47*
Means + S~M for 24 sham-operated and 18 VMH-lesioned, weanling rats. Initial body weights averaged 76 + 2 g. Body energy gain was calculated for 8 rats from each group. An asterisk (*) indicates a significant difference (P<0.05) between sham-operated and VMH-lesioned rats.
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Body weight gain, metabolizable energy intake and body energy gain of sham-operated and VMH-lesioned, weanling rats are presented in Table I. VMH-lesioned rats gained slightly less body weight than sham-operated rats despite similar ad libltum metabolizable energy intake by both groups. VMH-lesioned rats, however, gained 50% more body energy during the 4-week study because they accumulated more fat (Table II) and presumably less lean tissue than control rats. Final body energy values for sham-operated and VMH-lesioned rats were 459 + 30 and 642 + 47 kcal (mean + SEM), respectively. Gross energy efficiency (b~dy energy gain~metabolizable e n e r g y intake x 100) was 22 + 1% in sham-operated rats and 32 + 2% in VMH-lesioned rats (P<0.001). Since food intake was not increased, greater energy retention by VMH-lesioned rats must have resulted from a concomitant reduction in energy expenditure. NE turnover data from interscapular BAT, abdominal WAT, heart and pancreas are summarized in Table II and Figure i. NE turnover was measured in BAT because BAT is an important organ for tbermoregulatory heat production in rats (22), and because we bad previously observed that NE turnover was suppressed in BAT of obese (ob/ob) mice (23). Interscapular BAT of VMH-lesioned rats was heavier than that of sham-operated rats, but NE levels were similar in BAT of both groups (Table II). Increased BAT weight in VMH-lesioned rats was likely because of greater lipid content. The half-life of 3H-NE in BAT of VMH-lesioned rats averaged 7.9 hours compared with 5.6 hours in control rats (Figure I). These values correspond to fractional turnover rates (k) of 8.8 1.3 and 12.5 + 1.1% per hour (P<0.05) for VMH-lesioned and control rats, respectively. Total NE turnover in the interscapular BAT depot was decreased 38% by VMH lesions (Table II).
T~TE
il
Norepinephrine (NE) Turnover in Brown Adipose Tissue (BAT), White Adipose Tissue (WAT), Heart and Pancreas of Sham-Operated and VMH-Lesioned Rats
Tissue BAT Sham-operated vMH-lesioned WAT Sham-operated VMH-lesioned
Organ weight (m9)
348 ~ 14 520 + 26*
5,361 + 269 11,449 + 683*
Endogenous NE (ng/organ)
NE turnover rate
(n~/or~an/hr)
544 ~ 20 484 + 27
68 ~ 4 42 + 2*
155 + ii 189 + 36
14 + 1 6 + i*
Heart Sham-operated VMH-lesioned
796 + 15 651 + 15"
986 + 44 1,117 + 77*
71 + 3 50 + 2*
Pancreas Sham-operated VMH-lesioned
602 + 23 453 + 23*
322 + 14 268 + 17"
32 + 1 15 + i*
Means ~ S~M for 24 sham-operated and 18 VMH-lesioned rats. An asterisk (*) indicates a significant difference (P< 0.05) between sham-operated and VMH-lesioned rats. WAT weight includes all dissectible abdominal fat.
Vol. 30, No. ii, 1982
VMH Lesions and Sympathetic Activity
Brown Adipose Tissue
917
White Adipose Tissue
jVM.
•
lO~..............
10=
1/2 - 2 3 3hr
t1/2=7"Thr
A O~ e-
1
10=-
lO =
•
fVMH
r
E 1D *>
, w
ld
ld Sham J
~
.......
tl/2 =5.6hr
O
o u
0 a.
2
6
12
I I
24
~
Heart c ":" ,,C
10 3
I
6
,~
I
0
24
Pancreas
v.. ][-,:q -~_~
l Shr
O.
I
12
•, o F . . = . . . ~ - ~ . "
C
Z
2
~d
10 =
s ~ tl/2 = 6.9hr
t1/2=9.7hr
ld
I 2
I 6
I 12
k 24
I 2
I 6
1 12
I 24
HOURS FIG. 1 Effect of VMH lesions on norepinephrine (NE) turnover in BAT, WAT, heart and pancreas. Data points represent means ~ SEM for 4 to 6 rats. Respective r values for BAT, WAT, heart and pancreas were 0.87, 0.41, 0.84, and 0.71 for VMR-lesioned rats and 0.93, 0.74, 0.93 and 0.90 for sham-operated rats. For each tissue, the slope calculated for VMH-lesioned rats was significantly different (P<0.05) from that calculated for sham-operated rats.
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Activity
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ii, 1982
NE turnover rates were also measured in W A T because of the importance of the SNS in mobilizing lipids from fat stores (12), and to compare NE turnover in WAT and in BAT. Fat samples were taken from gonadal fat pads in VMH-lesioned and sham-operated rats. Total WAT organ weight was defined as total dissectable abdominal fat, excluding mesenterlc fat pads. VMH-lesioned rats had more than twice as much abdominal fat as control rats, but NE content in WAT was comparable in lesioned and control rats (Table II). NE half-llfe was 7.7 hours in WAT from control rats, but was tripled to 23.3 hours by VMH lesions (Figure i). As a result, fractional turnover (k) of NE in WAT averaged 9.0 + 2.0% per hour in control rats, but only 3.0 + 1.8% per hour (P <0.05) in VMH---lesioned rats. Total WAT NE turnover in VMH~lesioned rats was less than half that observed in sham-operated rats (Table II). Since the metabolic rate and SNS activity in hearts of normal rats respond in parallel to cold exposure, overfeeding and fasting (15,16), NE turnover was also investigated in hearts of sham-operated and VMH-lesioned rats. Hearts of lesioned rats weighed less, but contained 13% more NE, than hearts of control rats (Table If). NE half-life averaged 9.7 and 15.5 hours in hearts of sham-operated and VMH-lesioned rats, respectively (Figure i). Corresponding fractional turnover rates were 7.2 + 0.6 and 4.5 + 0.7% per hour (P<0.01). Total heart NE turnover was 30% less in VMH-lesioned rats. Turnover of NE in the pancreas was examined because the SNS affects insulin secretion ( 8 , 9 ) and because hyperinsulinemia is associated with hypothalamic obesity (3,10,11). Both pancreas weight and NE levels were decreased similarly in VMH-lesioned rats (Table If). Consequently, NE concentration per mg of pancreas was unaffected by VMH lesions. NE half-life averaged 6.9 hours in control pancreas, but was increased to 12.8 hours in VMH-lesioned rats (Figure i). Fractional NE turnover (k) in pancreas decreased from 10.0 + 1.1% per hour in control rats to 5.4 + 1.3% per hour (P < 0.02) in VMH-lesioned rats. Total NE turnover in the pancreas of vMHlesioned rats was less than half that observed in sham-operated rats (Table If). Discussion VMH lesions in weanling rats reduced NE turnover in every tissue examined (BAT, WAT, heart and pancreas). Because NE turnover has been demonstrated to be a valid indicator of SNS activity (14-16), these results suggest that SNS activity is reduced in these organs of VMH-lesioned animals. Concomitant acceleration of SNS activity and energy expenditure during cold exposure and overfeeding, and decreased SNS activity and energy expenditure during fasting (15,16), suggest a relationship between the SNS and energy balance. Our observations of decreased SNS activity and energy expenditure (6) in VMH-lesioned, weanling rats are in agreement with these studies and suggest an important role for the SNS in the development of increased energy efficiency and subsequent obesity in VMH-lesioned rats. Because the temperature at which the rats in this study were housed (23 ° ) was below their thermoneutral zone (24), altered thermoregulatory heat production in the VMB-lesioned rats may have been a factor in their increased efficiency of energy retention as it is in obese (ob/ob) mice housed below their thermoneutral zone (19). NE turnover in BAT of VMH-lesioned rats was reduced 38% in the present study. This compares with a 70% reduction in NE turnover in BAT of obese (ob/ob) mice housed at 25 ° (23). BAT is the major organ responsible for thermoregulatory beat production in rats (22). Since the SNS regulates heat production in BAT (25), it seems reasonable to conclude that reduced NE turnover in BAT of VMH-lesioned rats and obese (ob/ob) mice housed at 23-25 ° contributes to their increased efficiency of energy
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VMH Lesions and Sympathetic Activity
919
retention. In agreement with these observations, Seydoux et al. (26) demonstrated that the sensitivity and response of BAT from VMH-lesioned rats to norepinephrine is also decreased when compared with that of control rats. In addition to reduced thermoregulatory heat production by BAT, other factors are likely to be involved in the enhanced efficiency of energy retention in VMH-lesioned rats and genetically obese (ob/ob) mice. VMH-lesioned rats acutely exposed to 30° still consume less oxygen than control rats (unpublished observations) and obese mice housed at 33° (within the thermoneutral zone) still exhibit a higher efficiency of energy retention than lean mice (19). One possibility is that the lower numbers of Na+,K+-ATPase enzyme units found in skeletal muscle of VMH-lesioned rats (6) and obese mice (27,28) contribute to increased efficiency of energy retention in these animals. The fractional turnover rate of NE in WAT was reduced from 9% to 3% per hour by VMH lesions. These results support earlier suggestions, based on less direct measures, that SNS activity is suppressed in WAT of VMH-lesioned rats (12). This reduction, coupled with hyperinsulinemia characteristic of these rats (3,10,11), would suppress lipolysis and facilitate lipid a~cumulation in WAT. NE turnover rates in heart were reduced 30% by VMH lesions. These results appear at variance with reports of no suppression of NE turnover in hearts of obese (oh/oh) mice (23) or of gold thioglucose-treated mice (14); however, the differences may simply reflect the stage of development at which the animals were examined. VMH-lesioned rats in the present study were not overweight and d a lower metabolic rate than control rats, whereas the 8-week-old obese mice were 60% overweight (23) and had a metabolic rate per animal as high as that of control mice. Presumably the gold thioglucose-lesioned mice, which were at least 4 standard deviations above the mean weight of the control mice (14), also had a metabolic rate at least as high as that of the control mice. If NE turnover in the heart is linked to metabolic rate, then the differences between weanling, VMH-lesioned rats and the young adult obese mice in the other studies (14,23) would be expected. Measurement of NE turnover in hearts of adult hyperphagic and overweight VMH-lesioned rats and in young normophagic obese (oh/oh) mice would help resolve this apparent discrepancy. The autonomic hypothesis proposed to explain the development of hypothalamic obesity emphasizes the role of hyperinsulinemia resulting from reduced sympathetic outflow to the pancreas (8,9). In the present study, pancreatic NE turnover rates were markedly reduced in VMH-lesioned rats. These data support the hypothesis which proposes that by reducing SNS activity in pancreas, VMH lesions enhance the parasympathetic stimulation of insulin secretion mediated by the vagus nerve (8,9,11). From the data reported here, it can be concluded that VMH lesions in weanling rats decrease NE turnover rates in BAT, WAT, heart and pancreas. This decreased SNS activity probably contributes to reduced energy expenditure by BAT, accumulation of excess lipid by WAT, and hyperinsulinemia. Thus, decreased sympathetic outflow to these sympathetically innervated organs following VMH lesions is likely a major factor in the development of bypothalamic obesity.
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VMH Lesions and Sympathetic
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Acknowledgments The authors wish to thank H. Wells for technical assistance, preparation of the graph and D. Klein for typing the manuscript.
C. Oberg
for
References I. 2. 3. 4. 5. 6. 7. 8. 9. 10. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
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