- Email: [email protected]
Effects of corticotropin releasing factor on genetically obese (fatty) rats
Physiology & Behavior, Vol. 45, pp. 565-570. © Pergamon Press plc, 1989. Printed in the U.S.A.
0031-9384/89 $3.00 + .00
Effects of Corticotropin Releasing Factor on Genetically Obese (Fatty) Rats K. ARASE, N. S. S H A R G I L L
A N D G. A. B R A Y 2
Department o f Medicine, University o f Southern California, School o f Medicine Section o f Diabetes~Clinical Nutrition, L A C / U S C Medical Center, Los Angeles, CA R e c e i v e d 7 J u n e 1988 ARASE, K., N. S. SHARGILL AND G. A. BRAY. Effects of corticotropin releasingfactor on genetically obese (fatty) rats. PHYSIOL BEHAV 45(3) 565-570, 1989.-Corticotropin releasing factor (CRF) has been administered into the third ventricle of lean and genetically obese Zucker fatty rats in both acute and chronic experiments. Following a single injection of CRF (5 #g or approximately 1 nmole) there was an acute reduction of food intake in both the lean and obese animals, but the effect was greater in the obese. This effect persisted for the first three hours but was no longer detectable in either lean or genetically obese animals at 6 hours. Binding of GDP to mitochondria from interscapular brown adipose tissue in 21-hour deprived animals was lower in the fatty rats than in the lean controls. The injection of CRF significantly increased GDP binding in both the lean and fatty rats. During chronic infusion of CRF into the third ventricle of fatty rats, there was a significant decrease in food intake in the obese rats and fall of body weight in both groups. The basal levels of GDP binding were significantly lower in the saline-infused fatty rats than in the saline-infused lean controls. The chronic infusion of CRF increased GDP binding in the fatty rats but not in the lean animals. The CRF-treated values for GDP binding in fatty rats however, remained significantly below the baseline values in the control animals. Chronic CRF infusion also significantly lowered glucose levels in the fatty rat. These studies are consistent with the hypothesis that CRF may be involved in the decreased food intake and increased sympathetic activity observed in genetically obese fatty rats following adrenalectomy. Brown fat
Purine nucleotide binding
Genetic obesity
C O R T I C O T R O P I N releasing factor (CRF) is a forty-one amino acid hypothalamic neuropeptide, which is thought to be the primary physiological stimulus for the release of A C T H from the pituitary (32,33). This control is exerted through projections from the paraventricular nucleus (PVN) to the median eminence which contains high concentrations of C R F (23, 30, 31). Adrenalectomy has been shown to increase the turnover of C R F in the PVN, and is also known to reduce food intake and slow weight gain in genetically obese rodents (6, 7, 12, 19, 24, 34, 35). C R F is also widely distributed in other brain regions that are not associated with control of pituitary function such as the nucleus accumbens, the preoptic hypothalamus, the lateral hypothalamus, the parabrachial nucleus and the d o r s o m o t o r nucleus of the vagus (23, 28, 30). This distribution suggests that C R F may have a role in modulating the function of other systems. Britton et al. (8) and Morley and Levine (16,20) have both reported that C R F is a potent anorectic agent following central administration in the rat. These authors suggested that this peptide might be involved in stress-induced anorexia (20). C R F acts within the central nervous system to increase sympathetic activity (9, 10, 15). The fact that adrenalectomy reverses the hyperphagia and increases sympathetic activity in fatty rats as well as increasing C R F led to the following experiments in which C R F was administered into the ventricular sys-
Sympathetic nervous system
Food intake
CRF
tem of the genetically obese Zucker fatty rat to examine the hypothesis that it might suppress food intake and activate the sympathetic nervous system of these animals. METHOD
Animals The twenty-eight lean male rats and twenty-nine obese male Zucker rats used in these experiments were kindly provided by Dr. M. R. C. Greenwood (Vassar College, Poughkeepsie, NY). In the acute experiment the rats were 6 weeks old when they arrived in the laboratory (lean: n=17, b.wt. 130-170 g; obese: n=18, b.wt. 160-240 g). Animals used in the chronic experiment were 8 weeks old (lean: n = l l , b.wt. 160-200 g; obese: n = l 1, b.wt. 200-280 g) when they were received. Animals were housed individually in wire-bottom cages in a room illuminated from 0600 to 1800 hr with an ambient temperature maintained at 22_+2°C. Rats had ad lib access to ground Purina Laboratory Chow (Ralston-Purina Company, St. Louis, MO) and tap water except as described elsewhere.
Experimental Procedures Cannula implantation. Animals were anesthetized with sodium pentobarbital (35 m g / k g IP). They were mounted in the
~This work was supported in part by grants DK 32018 and DK 31988 from the National Institutes of Health. 2Requests for reprints should be addressed to G. A. Bray, M.D., Section of Diabetes/Clinical Nutrition, 2025 Zonal Avenue OCD-252, Los Angeles, CA 90033.
565
566 Kopf stereotaxic apparatus with the incisor bar raised 1.5 mm above the interaural line. In this position a burr hole was drilled at coordinates A-P 6.0 mm anterior to the interaural line and an 18 rnm long 28 gauge stainless steel cannula was inserted for the acute experiment and a 13 mm long 22 gauge cannula was similarly inserted in the midline into the third ventricle for the chronic experiment. Cannulas were lowered 6.8-7.5 mm below the cortical surface at the midline of the sagittal sinus. The cannula was securely cemented with dental cement and three screws anchored it to the skull. A temporary stainless steel wire obturator of 0.1 mm diameter was inserted into the 28 gauge cannula and a temporary 28 gauge cannula was inserted into the 22 gauge cannula and cemented in place until the time of each injection for feeding or for measurement of GDP binding. The top of the cannula on the animals head was covered with a small cap to prevent scratching the cannula. Animals were allowed to recover from this cannulation surgery for at least 7 days before further testing or surgery. At the end of the experiment, rats were sacrificed and methylene blue dye was infused into the ventricular cannula to document its patency and placement. GDP binding. Interscapular brown adipose tissue (IBAT) was dissected from the surrounding muscle and white adipose tissue for preparation of mitochondria. The mitochondria were isolated, washed and incubated in the presence of 5 mM 8-3H GDP (specific activity 6.64 Ci/mmole, New England Nuclear, Boston, MA) as previously described (14). Specific binding was assessed as that displaceable by 200/zmoles of cold GDP.
Experimental Protocols Acute experiment. The acute effects of CRF on food intake and GDP binding were measured 7 days apart. The rats were deprived of food for 21 hours (1200-0900 hr) before each test. The temporary stainless steel wire was removed and polyethylene tubing (Clay Adams, Parsippany, N J) (PE-10) which had been filled with the test solution was connected directly to a 28 gauge guide cannula. Infusion was begun between 0855 and 0900 hr in unanesthetized and unrestrained rats using a Hamilton microsyringe attached to an infusion pump. Rats were infused with 5/zg of CRF (approximately 1 nmole) (human/rat CRF, Peninsula Laboratories Inc., Belmont, CA) or physiologic saline in a volume of 5 t~l. After the infusion, food was immediately introduced into the animal's cage and food intake was measured at 1, 3 and 6 hours. At the end of the experiment a temporary stainless steel wire was reinserted and cemented in place. Seven days later this identical program was repeated on the same animals. After infusion rats were placed in their individual cages from which all food had been removed. One hour after the infusion rats were killed by cervical decapitation. Interscapular brown adipose tissue was dissected from the surrounding muscle and white adipose tissue for preparation of IBAT mitochondria. Chronic experiment. An Alzet osmotic pump (Alza Corporation, Stanford, CA; model No. 2001) was implanted with rats under methoxyfluorane anesthesia. The minipump had been previously filled with a test solution and placed in 0.9% saline at 37°C for 4 hours. The minipump was connected to a 28 gauge infusion cannula by PE-60 polyethylene tubing filled with the test solution. The minipump was inserted underneath the skin of the animal's back, and the infusion cannula was placed in the ventricular guide cannula and cemented in place. Ampicillin (50 mg/kg, IM) was administered for 3 days after implantation. The Alzet minipumps were filled with either sterile saline, 0.15 M saline, or CRF dissolved in 0.15 M saline.
ARASE, SHARGILL AND BRAY 6
O~ 5
I
[-~ SALINEI m27J CRF
5_
ILl 4 3
8 o LL
.EAN
OBESE
0-1
Hr
CLC
LEAN
OBESE
1 - 3 Hrs
LEAN 3 -6
OBESE Hrs
FIG. 1. Effects of CRF on food intake of lean and fatty rats. Doses of 5 #g of CRF or a comparable volume of saline were injected into the third cerebral ventricle of 8 or 9 rats. Food intake was measured at 1, 3 and 6 hours following injection. Data are mean_+SEM.
CRF was delivered at a rate of 4.8/zg/day for 6 days. The test solutions were infused at 1 #l/hr. Daily food intake and body weights were measured between 0800 and 0830 hr. After 6 days the rats were killed by cervical decapitation. Blood was collected and serum separated and stored at - 2 0 ° C for further analysis. Selected organs were removed, trimmed and weighed.
Analytical Procedures Serum glucose was measured using a Beckman glucose analyzer and corticosterone by a competitive binding assay (22).
Statistical Evaluation A two-way ANOVA was carried out on food intake and GDP binding values in the acute experiment. Three-way ANOVA with one repeat measure was carried out on food intake and body weight values in the chronic experiment. Other data in the chronic experiment were examined by two-way ANOVA. Individual comparisons were done by the NeumanKeuls test. All data are expressed as mean_+SEM. RESULTS
Acute Experiment Data on food intake following the infusion of CRF into the third ventricle of lean and obese fatty rats are presented in Fig. 1. The infusion of 5/~g (1 nmole) at CRF at 0900 decreased food intake in both lean and fatty rats during the first one hour in food-deprived rats, F(I,24)=201.8, p<0.01. The suppression of food intake during the first hour was greater in the obese animals (88.5% below control values) than in the lean animals (73.4% below control values). This suppressive effect of CRF on food intake continued during the next two hours, F(1,24)= 4.53, p<0.05. However, there was no significant difference during the period 3-6 hours after injecting CRF. Data on GDP binding are shown in Table 1. The mean value for the obese animals was lower than the mean for the lean animals but this difference was not statistically significant. One hour after the infusion, the animals treated with CRF had significantly higher values for ODP binding compared to saline-
CRF AND GENETIC OBESITY
569
ature and GDP-binding by brown adipose tissue. Since the injection of C R F into the third ventricle increases G D P binding in both the lean and fatty rats, this suggests that the efferent control systems for sympathetic activity are intact in fatty rats. A similar suggestion has been made by Holt et aL (13,14) who have reported that electrical stimulation of ventromedial, supra optic and anterior hypothalamus will produce comparable increments in temperature of brown adipose tissue in lean and genetically obese fatty rats. The present studies have demonstrated that either chronic or acute treatment with C R F reduces food intake and increases sympathetic activity. This reciprocal relationship between food intake and sympathetic activity is consistent with several previous observations from our laboratory (1-5, 25-27). In spontaneously feeding rats, the firing rate of sympathetic nerves to brown adipose tissue was inversely related to the food intake over the preceding four hours (25). Similarly, experimental maneuvers which decrease food intake such as injections o f fenfluramine or L H lesions are associated with increased sympathetic activity measured by norepinephrine turnover in brown adipose tissue, by G D P binding to mitochondria from brown adipose tissue or by electrical firing rate of nerves supplying brown adipose tissue (1,4). Conversely, experimental maneuvers, which increase food intake such as V M H lesions or injections of 2-deoxyglucose, are associated with a decrease in sympathetic activity measured by any of the indices noted above (3, 5, 26, 27). These sets of observations suggest that sympathetic activity and food intake may be part of a feedback control system.
A rise in C R F concentrations may play a role in the reversal o f genetic obesity by adrenalectomy. It is now well established that complete adrenalectomy reduces food intake and increases sympathetic activity in obese mice (6, 24, 34), diabetes mice (6) and fatty rats (6, 12, 19, 35). Data o f McLaughlin et al. (18) also suggest that C R F may be involved in regulation of fat stores. When fatty rats were immunized against C R F weight gains were decreased during the first 1-4 weeks and food intake was decreased between weeks 5 and 8. After removing corticosterone which is the endogenous feedback inhibitor for corticotropin release, the concentration o f C R F in the paraventricular nucleus rises (28). The neurons in the paraventricular nucleus which contain C R F may cosecrete either cholecystokinin or vasopressin (29). All of these peptides have been demonstrated to reduce food intake, but increased secretion of C R F would be a more likely candidate since it is the most potent. Increased C R F concentrations in the paraventricular nucleus may reduce food intake or the C R F may be released into the cerebroventricular system and transported to other hypothalamic sites where it can reduce food intake and increase sympathetic activity. LeFeuvre et aL (15) found that C R F increased G D P binding when 1.2 nmol was injected into the PVN, arguing that this may be the site of action. Measurements of electrical firing rate of sympathetic nerves to brown adipose tissue in our laboratory have shown that C R F increases firing when injected into the preoptic area but not the PVN (Egawa, Yoshimatsu and Bray, unpublished observations). Thus more information is needed to provide a complete picture.
REFERENCES
1. Arase, K.; Sakaguchi, T.; Bray, G. A. Effect of fenfluramine on sympathetic firing rate. Pharmacol. Biochem. Behav. 29(3):675680; 1988. 2. Arase, K.; York, D. A.; Shimizu, H.; Shargill, N.S.; Bray, G. A. Effects of corticotropin-releasing factor on food intake and brown adipose tissue thermogenesis in rats. Am. J. Physiol. 225:E255E259; 1988. 3. Arase, K.; York, D. A.; Bray, G. A. Corticosterone inhibition of the intracerebroventricular effect of 2-deoxy-D-glucose on brown adipose tissue thermogenesis. Physiol. Behav. 40(4):489-495; 1987. 4. Arase, K.; Sakaguchi, T.; Bray, G. A. Lateral hypothalamic lesions and activity of the sympathetic nervous system. Life Sci. 41(5):657662; 1987. 5. Arase, K.; Shargill, N. S.; Bray, G. A. Effects of intraventricular infusion of corticotropin releasing factor on VMH-lesioned obese rats. Am. J. Physiol., in press; 1989. 6. Bray, G. A. Regulation of energy balance: Studies on genetic, hypothalamic and dietary obesity. Proc. Nutr. Soc. 41:95-108; 1982. 7. Bray, G. A. Obesity-A disease of nutrient or energy balance? Nutr. Rev. 45(2):33-43; 1987. 8. Britton, D.; Koob, G.; Rivier, J.; Vale, W. lntraventricular corticotropin-releasing factor enhances behavioral effects of novelty. Life Sci. 31:363-367; 1982. 9. Brown, M. R.; Fisher, L. A.; Spiess, J.; Rivier, J.; Rivier, C.; Vale, W. Corticotropin releasing factor: actions on the sympathetic nervous system and metabolism. Endocrinology 111:928-931; 1982. 10. Brown, M. D.; Fisher, L. A. Corticotropin releasing factor: effects on the autonomic nervous system and visceral systems. Fed. Proc. 44:243-248; 1985. 11. Himms-Hagen, J. Brown adipose tissue metabolism and thermogenesis. Annu. Rev. Nutr. 5:69-94; 1985. 12. Holt, S.; York, D. A. The effect of adrenalectomy on GDP binding to brown adipose tissue mitochondria of obese rats. Biochem. J. 208:819-822; 1982.
13. Holt, S. J.; Wheal, H. V.; York, D. A. Hypothalamic control of brown adipose tissue in Zucker Lean and obese rats. Effect of electrical stimulation of the ventromedial nucleus and other hypothalamic centres. Brain Res. 405:227-233; 1987. 14. Holt, S. J.; Wheal, H. V.; York, D. A. Response of brown adipose tissue to electrical stimulation of hypothalamic centres in intact and adrenalectomized Zucker rats. Neurosci. Lett. 84:6367; 1987. 15. LeFeuvre, R. A.; Rothwell, N. J.; Stock, M. J. Activation of brown adipose tissue in response to central injection of corticotropin releasing hormone in the rat. Neuropharmacology 16:12171221; 1987. 16. Levine, A. S.; Rogers, B.; Kneip, J.; Grace, M.; Morley, J. E. Effect of centrally administered corticotropin-releasing factor (CRF) on multiple feeding paradigms. Neuropharmacology 22:337339; 1983. 17. Lupien, J. R.; Bray, G. A. Nicotine increases thermogenesis in brown adipose tissue in rats. Pharmacol. Biochem. Behav. 29(1): 33-37; 1988. 18. McLaughlin, C. L.; Stern, J. S.; Baile, C. A. Weight gain and food intake in corticotropin releasing factor immunized Zucker rats. Physiol. Behav. 41:171-178; 1987. 19. Marchington, D.; Rothwell, N. J.; Stock, M. J.; York, D. A. Energy balance, diet-induced thermogenesis and brown adipose tissue in lean and obese (fa/fa) Zucker rats after adrenalectomy. J. Nutr. 113:1395-1402; 1983. 20. Morley, J. E.; Levine, A. S. Corticotropin-releasing factor, grooming and ingestive behaviors. Life Sci. 31:1459-1464; 1982. 21. Morley, J. E. Neuropeptide regulation of appetite and weight. Endocr. Rev. 8(3):256-287; 1987. 22. Murphy, B. E. P. Some studies of the protein-binding of steroids and their application to the routine micro and ultramicro measurements of various steroids in body fluids by competitive proteinbinding radioassay. J. Clin. Endocrinol. Metab. 27:973-990; 1967.
570 23. Petrusz, P.; Merchenthaler, I.; Maderdrut, J. L.; Heitz, P. U. Central and peripheral distribution of corticotropin-releasing factor. Fed. Proc. 44:229-235; 1985. 24. Saito, M.; Bray, G. A. Adrenalectomy and food restriction in the genetically obese (ob/ob) mouse. Am. J. Physiol. 246:R20-R25; 1984. 25. Sakaguchi, T.; Takahashi, M.; Bray, G. A. Diurnal changes in sympathetic activity: relation to food intake and to insulin injected into the ventromedial or suprachiasmatic nucleus. J. Clin. Invest. 32(1):282-286; 1988. 26. Sakaguchi, T.; Bray, G. A. Intrahypothalamic injection of insulin decreases firing rate of sympathetic nerves. Proc. Natl, Acad. Sci. USA 84:2012-2014; 1987. 27. Sakaguchi, T.; Bray, G. A. The effect of intrahypothalamic injections of glucose on sympathetic efferent firing rate. Brain Res. Bull. 18:591-595; 1987. 28. Sawchenko, P. E. Evidence for a local site of action for glucosteroid in inhibiting CRF and vasopressin expression in the paraventricular nucleus. Brain Res. 403:213-224; 1987. 29. Sawchenko, P. E.; Swanson, L. W. Localization, colocalization, and plasticity of corticotropin-releasing factor immunoreactivity in rat brain. Fed. Proc. 44:221-227; 1985.
ARASE, SHARGILL AND BRAY 30. Swanson, L. W.; Sawchenko, P. E.; Rivier, J.; Vale, W. W. Organization of ovine corticotropin-releasing factor immunoreactive cells and fibers in the rat brain: an immunohistochemical study. Neuroendocrinology 36(3):165-186; 1983. 31. Swanson, L. W.; Sawchenko, P. E. Hypothalamic integration: organization of the paraventricular and supraoptic nuclei. Annu. Rev. Neurosci. 6:269-324; 1983. 32. Vale, W.; Rivier, C.; Brown, M. R.; Spiess, J.; Koob, G.; Swanson, L.; Bilezikjian, L.; Bloom, F.; Rivier, J. Chemical and biological characterization of corticotropin releasing factor. Recent Prog. Horm. Res. 39:245-270; 1983. 33. Vale, W.; Spiess, J.; Rivier, C.; Rivier, J. Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and beta-endorphin. Science 213:1394-1397; 1981. 34. vander Tuig, J. G.; Ohshima, K.; Yoshida, T.; Romsos, D. R.; Bray, G. A. Adrenalectomy increases norepinephrine turnover in brown adipose tissue of obese (ob/ob) mice. Life Sci. 30:14231432; 1985. 35. Yukimura, Y.; Bray, G. A. Effects of adrenalectomy on body weight and the size and number of fat cells in the Zucker (fatty) rat. Endocr. Res. 5(3):189-198; 1978. 36. York, D. A. Neural activity in hypothalamic and genetic obesity. Proc. Nutr. Soc. 46:105-117; 1987.
0031-9384/89 $3.00 + .00
Effects of Corticotropin Releasing Factor on Genetically Obese (Fatty) Rats K. ARASE, N. S. S H A R G I L L
A N D G. A. B R A Y 2
Department o f Medicine, University o f Southern California, School o f Medicine Section o f Diabetes~Clinical Nutrition, L A C / U S C Medical Center, Los Angeles, CA R e c e i v e d 7 J u n e 1988 ARASE, K., N. S. SHARGILL AND G. A. BRAY. Effects of corticotropin releasingfactor on genetically obese (fatty) rats. PHYSIOL BEHAV 45(3) 565-570, 1989.-Corticotropin releasing factor (CRF) has been administered into the third ventricle of lean and genetically obese Zucker fatty rats in both acute and chronic experiments. Following a single injection of CRF (5 #g or approximately 1 nmole) there was an acute reduction of food intake in both the lean and obese animals, but the effect was greater in the obese. This effect persisted for the first three hours but was no longer detectable in either lean or genetically obese animals at 6 hours. Binding of GDP to mitochondria from interscapular brown adipose tissue in 21-hour deprived animals was lower in the fatty rats than in the lean controls. The injection of CRF significantly increased GDP binding in both the lean and fatty rats. During chronic infusion of CRF into the third ventricle of fatty rats, there was a significant decrease in food intake in the obese rats and fall of body weight in both groups. The basal levels of GDP binding were significantly lower in the saline-infused fatty rats than in the saline-infused lean controls. The chronic infusion of CRF increased GDP binding in the fatty rats but not in the lean animals. The CRF-treated values for GDP binding in fatty rats however, remained significantly below the baseline values in the control animals. Chronic CRF infusion also significantly lowered glucose levels in the fatty rat. These studies are consistent with the hypothesis that CRF may be involved in the decreased food intake and increased sympathetic activity observed in genetically obese fatty rats following adrenalectomy. Brown fat
Purine nucleotide binding
Genetic obesity
C O R T I C O T R O P I N releasing factor (CRF) is a forty-one amino acid hypothalamic neuropeptide, which is thought to be the primary physiological stimulus for the release of A C T H from the pituitary (32,33). This control is exerted through projections from the paraventricular nucleus (PVN) to the median eminence which contains high concentrations of C R F (23, 30, 31). Adrenalectomy has been shown to increase the turnover of C R F in the PVN, and is also known to reduce food intake and slow weight gain in genetically obese rodents (6, 7, 12, 19, 24, 34, 35). C R F is also widely distributed in other brain regions that are not associated with control of pituitary function such as the nucleus accumbens, the preoptic hypothalamus, the lateral hypothalamus, the parabrachial nucleus and the d o r s o m o t o r nucleus of the vagus (23, 28, 30). This distribution suggests that C R F may have a role in modulating the function of other systems. Britton et al. (8) and Morley and Levine (16,20) have both reported that C R F is a potent anorectic agent following central administration in the rat. These authors suggested that this peptide might be involved in stress-induced anorexia (20). C R F acts within the central nervous system to increase sympathetic activity (9, 10, 15). The fact that adrenalectomy reverses the hyperphagia and increases sympathetic activity in fatty rats as well as increasing C R F led to the following experiments in which C R F was administered into the ventricular sys-
Sympathetic nervous system
Food intake
CRF
tem of the genetically obese Zucker fatty rat to examine the hypothesis that it might suppress food intake and activate the sympathetic nervous system of these animals. METHOD
Animals The twenty-eight lean male rats and twenty-nine obese male Zucker rats used in these experiments were kindly provided by Dr. M. R. C. Greenwood (Vassar College, Poughkeepsie, NY). In the acute experiment the rats were 6 weeks old when they arrived in the laboratory (lean: n=17, b.wt. 130-170 g; obese: n=18, b.wt. 160-240 g). Animals used in the chronic experiment were 8 weeks old (lean: n = l l , b.wt. 160-200 g; obese: n = l 1, b.wt. 200-280 g) when they were received. Animals were housed individually in wire-bottom cages in a room illuminated from 0600 to 1800 hr with an ambient temperature maintained at 22_+2°C. Rats had ad lib access to ground Purina Laboratory Chow (Ralston-Purina Company, St. Louis, MO) and tap water except as described elsewhere.
Experimental Procedures Cannula implantation. Animals were anesthetized with sodium pentobarbital (35 m g / k g IP). They were mounted in the
~This work was supported in part by grants DK 32018 and DK 31988 from the National Institutes of Health. 2Requests for reprints should be addressed to G. A. Bray, M.D., Section of Diabetes/Clinical Nutrition, 2025 Zonal Avenue OCD-252, Los Angeles, CA 90033.
565
566 Kopf stereotaxic apparatus with the incisor bar raised 1.5 mm above the interaural line. In this position a burr hole was drilled at coordinates A-P 6.0 mm anterior to the interaural line and an 18 rnm long 28 gauge stainless steel cannula was inserted for the acute experiment and a 13 mm long 22 gauge cannula was similarly inserted in the midline into the third ventricle for the chronic experiment. Cannulas were lowered 6.8-7.5 mm below the cortical surface at the midline of the sagittal sinus. The cannula was securely cemented with dental cement and three screws anchored it to the skull. A temporary stainless steel wire obturator of 0.1 mm diameter was inserted into the 28 gauge cannula and a temporary 28 gauge cannula was inserted into the 22 gauge cannula and cemented in place until the time of each injection for feeding or for measurement of GDP binding. The top of the cannula on the animals head was covered with a small cap to prevent scratching the cannula. Animals were allowed to recover from this cannulation surgery for at least 7 days before further testing or surgery. At the end of the experiment, rats were sacrificed and methylene blue dye was infused into the ventricular cannula to document its patency and placement. GDP binding. Interscapular brown adipose tissue (IBAT) was dissected from the surrounding muscle and white adipose tissue for preparation of mitochondria. The mitochondria were isolated, washed and incubated in the presence of 5 mM 8-3H GDP (specific activity 6.64 Ci/mmole, New England Nuclear, Boston, MA) as previously described (14). Specific binding was assessed as that displaceable by 200/zmoles of cold GDP.
Experimental Protocols Acute experiment. The acute effects of CRF on food intake and GDP binding were measured 7 days apart. The rats were deprived of food for 21 hours (1200-0900 hr) before each test. The temporary stainless steel wire was removed and polyethylene tubing (Clay Adams, Parsippany, N J) (PE-10) which had been filled with the test solution was connected directly to a 28 gauge guide cannula. Infusion was begun between 0855 and 0900 hr in unanesthetized and unrestrained rats using a Hamilton microsyringe attached to an infusion pump. Rats were infused with 5/zg of CRF (approximately 1 nmole) (human/rat CRF, Peninsula Laboratories Inc., Belmont, CA) or physiologic saline in a volume of 5 t~l. After the infusion, food was immediately introduced into the animal's cage and food intake was measured at 1, 3 and 6 hours. At the end of the experiment a temporary stainless steel wire was reinserted and cemented in place. Seven days later this identical program was repeated on the same animals. After infusion rats were placed in their individual cages from which all food had been removed. One hour after the infusion rats were killed by cervical decapitation. Interscapular brown adipose tissue was dissected from the surrounding muscle and white adipose tissue for preparation of IBAT mitochondria. Chronic experiment. An Alzet osmotic pump (Alza Corporation, Stanford, CA; model No. 2001) was implanted with rats under methoxyfluorane anesthesia. The minipump had been previously filled with a test solution and placed in 0.9% saline at 37°C for 4 hours. The minipump was connected to a 28 gauge infusion cannula by PE-60 polyethylene tubing filled with the test solution. The minipump was inserted underneath the skin of the animal's back, and the infusion cannula was placed in the ventricular guide cannula and cemented in place. Ampicillin (50 mg/kg, IM) was administered for 3 days after implantation. The Alzet minipumps were filled with either sterile saline, 0.15 M saline, or CRF dissolved in 0.15 M saline.
ARASE, SHARGILL AND BRAY 6
O~ 5
I
[-~ SALINEI m27J CRF
5_
ILl 4 3
8 o LL
.EAN
OBESE
0-1
Hr
CLC
LEAN
OBESE
1 - 3 Hrs
LEAN 3 -6
OBESE Hrs
FIG. 1. Effects of CRF on food intake of lean and fatty rats. Doses of 5 #g of CRF or a comparable volume of saline were injected into the third cerebral ventricle of 8 or 9 rats. Food intake was measured at 1, 3 and 6 hours following injection. Data are mean_+SEM.
CRF was delivered at a rate of 4.8/zg/day for 6 days. The test solutions were infused at 1 #l/hr. Daily food intake and body weights were measured between 0800 and 0830 hr. After 6 days the rats were killed by cervical decapitation. Blood was collected and serum separated and stored at - 2 0 ° C for further analysis. Selected organs were removed, trimmed and weighed.
Analytical Procedures Serum glucose was measured using a Beckman glucose analyzer and corticosterone by a competitive binding assay (22).
Statistical Evaluation A two-way ANOVA was carried out on food intake and GDP binding values in the acute experiment. Three-way ANOVA with one repeat measure was carried out on food intake and body weight values in the chronic experiment. Other data in the chronic experiment were examined by two-way ANOVA. Individual comparisons were done by the NeumanKeuls test. All data are expressed as mean_+SEM. RESULTS
Acute Experiment Data on food intake following the infusion of CRF into the third ventricle of lean and obese fatty rats are presented in Fig. 1. The infusion of 5/~g (1 nmole) at CRF at 0900 decreased food intake in both lean and fatty rats during the first one hour in food-deprived rats, F(I,24)=201.8, p<0.01. The suppression of food intake during the first hour was greater in the obese animals (88.5% below control values) than in the lean animals (73.4% below control values). This suppressive effect of CRF on food intake continued during the next two hours, F(1,24)= 4.53, p<0.05. However, there was no significant difference during the period 3-6 hours after injecting CRF. Data on GDP binding are shown in Table 1. The mean value for the obese animals was lower than the mean for the lean animals but this difference was not statistically significant. One hour after the infusion, the animals treated with CRF had significantly higher values for ODP binding compared to saline-
CRF AND GENETIC OBESITY
569
ature and GDP-binding by brown adipose tissue. Since the injection of C R F into the third ventricle increases G D P binding in both the lean and fatty rats, this suggests that the efferent control systems for sympathetic activity are intact in fatty rats. A similar suggestion has been made by Holt et aL (13,14) who have reported that electrical stimulation of ventromedial, supra optic and anterior hypothalamus will produce comparable increments in temperature of brown adipose tissue in lean and genetically obese fatty rats. The present studies have demonstrated that either chronic or acute treatment with C R F reduces food intake and increases sympathetic activity. This reciprocal relationship between food intake and sympathetic activity is consistent with several previous observations from our laboratory (1-5, 25-27). In spontaneously feeding rats, the firing rate of sympathetic nerves to brown adipose tissue was inversely related to the food intake over the preceding four hours (25). Similarly, experimental maneuvers which decrease food intake such as injections o f fenfluramine or L H lesions are associated with increased sympathetic activity measured by norepinephrine turnover in brown adipose tissue, by G D P binding to mitochondria from brown adipose tissue or by electrical firing rate of nerves supplying brown adipose tissue (1,4). Conversely, experimental maneuvers, which increase food intake such as V M H lesions or injections of 2-deoxyglucose, are associated with a decrease in sympathetic activity measured by any of the indices noted above (3, 5, 26, 27). These sets of observations suggest that sympathetic activity and food intake may be part of a feedback control system.
A rise in C R F concentrations may play a role in the reversal o f genetic obesity by adrenalectomy. It is now well established that complete adrenalectomy reduces food intake and increases sympathetic activity in obese mice (6, 24, 34), diabetes mice (6) and fatty rats (6, 12, 19, 35). Data o f McLaughlin et al. (18) also suggest that C R F may be involved in regulation of fat stores. When fatty rats were immunized against C R F weight gains were decreased during the first 1-4 weeks and food intake was decreased between weeks 5 and 8. After removing corticosterone which is the endogenous feedback inhibitor for corticotropin release, the concentration o f C R F in the paraventricular nucleus rises (28). The neurons in the paraventricular nucleus which contain C R F may cosecrete either cholecystokinin or vasopressin (29). All of these peptides have been demonstrated to reduce food intake, but increased secretion of C R F would be a more likely candidate since it is the most potent. Increased C R F concentrations in the paraventricular nucleus may reduce food intake or the C R F may be released into the cerebroventricular system and transported to other hypothalamic sites where it can reduce food intake and increase sympathetic activity. LeFeuvre et aL (15) found that C R F increased G D P binding when 1.2 nmol was injected into the PVN, arguing that this may be the site of action. Measurements of electrical firing rate of sympathetic nerves to brown adipose tissue in our laboratory have shown that C R F increases firing when injected into the preoptic area but not the PVN (Egawa, Yoshimatsu and Bray, unpublished observations). Thus more information is needed to provide a complete picture.
REFERENCES
1. Arase, K.; Sakaguchi, T.; Bray, G. A. Effect of fenfluramine on sympathetic firing rate. Pharmacol. Biochem. Behav. 29(3):675680; 1988. 2. Arase, K.; York, D. A.; Shimizu, H.; Shargill, N.S.; Bray, G. A. Effects of corticotropin-releasing factor on food intake and brown adipose tissue thermogenesis in rats. Am. J. Physiol. 225:E255E259; 1988. 3. Arase, K.; York, D. A.; Bray, G. A. Corticosterone inhibition of the intracerebroventricular effect of 2-deoxy-D-glucose on brown adipose tissue thermogenesis. Physiol. Behav. 40(4):489-495; 1987. 4. Arase, K.; Sakaguchi, T.; Bray, G. A. Lateral hypothalamic lesions and activity of the sympathetic nervous system. Life Sci. 41(5):657662; 1987. 5. Arase, K.; Shargill, N. S.; Bray, G. A. Effects of intraventricular infusion of corticotropin releasing factor on VMH-lesioned obese rats. Am. J. Physiol., in press; 1989. 6. Bray, G. A. Regulation of energy balance: Studies on genetic, hypothalamic and dietary obesity. Proc. Nutr. Soc. 41:95-108; 1982. 7. Bray, G. A. Obesity-A disease of nutrient or energy balance? Nutr. Rev. 45(2):33-43; 1987. 8. Britton, D.; Koob, G.; Rivier, J.; Vale, W. lntraventricular corticotropin-releasing factor enhances behavioral effects of novelty. Life Sci. 31:363-367; 1982. 9. Brown, M. R.; Fisher, L. A.; Spiess, J.; Rivier, J.; Rivier, C.; Vale, W. Corticotropin releasing factor: actions on the sympathetic nervous system and metabolism. Endocrinology 111:928-931; 1982. 10. Brown, M. D.; Fisher, L. A. Corticotropin releasing factor: effects on the autonomic nervous system and visceral systems. Fed. Proc. 44:243-248; 1985. 11. Himms-Hagen, J. Brown adipose tissue metabolism and thermogenesis. Annu. Rev. Nutr. 5:69-94; 1985. 12. Holt, S.; York, D. A. The effect of adrenalectomy on GDP binding to brown adipose tissue mitochondria of obese rats. Biochem. J. 208:819-822; 1982.
13. Holt, S. J.; Wheal, H. V.; York, D. A. Hypothalamic control of brown adipose tissue in Zucker Lean and obese rats. Effect of electrical stimulation of the ventromedial nucleus and other hypothalamic centres. Brain Res. 405:227-233; 1987. 14. Holt, S. J.; Wheal, H. V.; York, D. A. Response of brown adipose tissue to electrical stimulation of hypothalamic centres in intact and adrenalectomized Zucker rats. Neurosci. Lett. 84:6367; 1987. 15. LeFeuvre, R. A.; Rothwell, N. J.; Stock, M. J. Activation of brown adipose tissue in response to central injection of corticotropin releasing hormone in the rat. Neuropharmacology 16:12171221; 1987. 16. Levine, A. S.; Rogers, B.; Kneip, J.; Grace, M.; Morley, J. E. Effect of centrally administered corticotropin-releasing factor (CRF) on multiple feeding paradigms. Neuropharmacology 22:337339; 1983. 17. Lupien, J. R.; Bray, G. A. Nicotine increases thermogenesis in brown adipose tissue in rats. Pharmacol. Biochem. Behav. 29(1): 33-37; 1988. 18. McLaughlin, C. L.; Stern, J. S.; Baile, C. A. Weight gain and food intake in corticotropin releasing factor immunized Zucker rats. Physiol. Behav. 41:171-178; 1987. 19. Marchington, D.; Rothwell, N. J.; Stock, M. J.; York, D. A. Energy balance, diet-induced thermogenesis and brown adipose tissue in lean and obese (fa/fa) Zucker rats after adrenalectomy. J. Nutr. 113:1395-1402; 1983. 20. Morley, J. E.; Levine, A. S. Corticotropin-releasing factor, grooming and ingestive behaviors. Life Sci. 31:1459-1464; 1982. 21. Morley, J. E. Neuropeptide regulation of appetite and weight. Endocr. Rev. 8(3):256-287; 1987. 22. Murphy, B. E. P. Some studies of the protein-binding of steroids and their application to the routine micro and ultramicro measurements of various steroids in body fluids by competitive proteinbinding radioassay. J. Clin. Endocrinol. Metab. 27:973-990; 1967.
570 23. Petrusz, P.; Merchenthaler, I.; Maderdrut, J. L.; Heitz, P. U. Central and peripheral distribution of corticotropin-releasing factor. Fed. Proc. 44:229-235; 1985. 24. Saito, M.; Bray, G. A. Adrenalectomy and food restriction in the genetically obese (ob/ob) mouse. Am. J. Physiol. 246:R20-R25; 1984. 25. Sakaguchi, T.; Takahashi, M.; Bray, G. A. Diurnal changes in sympathetic activity: relation to food intake and to insulin injected into the ventromedial or suprachiasmatic nucleus. J. Clin. Invest. 32(1):282-286; 1988. 26. Sakaguchi, T.; Bray, G. A. Intrahypothalamic injection of insulin decreases firing rate of sympathetic nerves. Proc. Natl, Acad. Sci. USA 84:2012-2014; 1987. 27. Sakaguchi, T.; Bray, G. A. The effect of intrahypothalamic injections of glucose on sympathetic efferent firing rate. Brain Res. Bull. 18:591-595; 1987. 28. Sawchenko, P. E. Evidence for a local site of action for glucosteroid in inhibiting CRF and vasopressin expression in the paraventricular nucleus. Brain Res. 403:213-224; 1987. 29. Sawchenko, P. E.; Swanson, L. W. Localization, colocalization, and plasticity of corticotropin-releasing factor immunoreactivity in rat brain. Fed. Proc. 44:221-227; 1985.
ARASE, SHARGILL AND BRAY 30. Swanson, L. W.; Sawchenko, P. E.; Rivier, J.; Vale, W. W. Organization of ovine corticotropin-releasing factor immunoreactive cells and fibers in the rat brain: an immunohistochemical study. Neuroendocrinology 36(3):165-186; 1983. 31. Swanson, L. W.; Sawchenko, P. E. Hypothalamic integration: organization of the paraventricular and supraoptic nuclei. Annu. Rev. Neurosci. 6:269-324; 1983. 32. Vale, W.; Rivier, C.; Brown, M. R.; Spiess, J.; Koob, G.; Swanson, L.; Bilezikjian, L.; Bloom, F.; Rivier, J. Chemical and biological characterization of corticotropin releasing factor. Recent Prog. Horm. Res. 39:245-270; 1983. 33. Vale, W.; Spiess, J.; Rivier, C.; Rivier, J. Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and beta-endorphin. Science 213:1394-1397; 1981. 34. vander Tuig, J. G.; Ohshima, K.; Yoshida, T.; Romsos, D. R.; Bray, G. A. Adrenalectomy increases norepinephrine turnover in brown adipose tissue of obese (ob/ob) mice. Life Sci. 30:14231432; 1985. 35. Yukimura, Y.; Bray, G. A. Effects of adrenalectomy on body weight and the size and number of fat cells in the Zucker (fatty) rat. Endocr. Res. 5(3):189-198; 1978. 36. York, D. A. Neural activity in hypothalamic and genetic obesity. Proc. Nutr. Soc. 46:105-117; 1987.