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KsJ mice: modulation by the opiate antagonist, Nalmefene
Brain Research, 445 (1988) 262-267 Elsevier
262 BRE 13418
Regional brain glucose uptake in genetically diabetic C57BL/KsJ mice: modulation by the opiate antagonist, Nalmefene David R. Garris 1 and Mary Ellen Michel 2'* tDepartment of Anatomy, ClevelandResearch Laboratory, Kansas City, MO 64131 (U.S.A.) and 2Key Pharmaceuticals, Inc., Miami, FL 33269-0670 (U.S.A.)
(Accepted 22 September 1987) Key words: Diabetes; Brain glucose uptake; C57BL/KsJ mouse; Nalmefene; Opiate antagonist
A novel opiate antagonist, Nalmefene (0.5 or 5.0 mg/kg/day) was tested for its ability to modulate regional brain glucose uptake rates in genetically diabetic C57BL/KsJ mice, which normally exhibit a depressed CNS carbohydrate metabolism relative to agematched controls. Daily Nalmefene treatment had no effect on circulating blood glucose levels in either normal or diabetic mice over a 7-week test period. However, all brain regions, except the olfactory bulbs, exhibited normalized glucose uptake rates in diabetic mice relative to controls. These data suggest a role for opiate antagonists in the modulation of CNS glucose metabolism during hyperglycemic states. INTRODUCTION Changes in neuronal structure, function and metabolism are recognized to occur in association with type I (insulin-dependent) and type II (non-insulindependent) diabetic conditions 1.Sa°a4-19`37. In the genetically obese-diabetic C57BL/KsJ (db/db) mouse, premature neuronal degeneration TM, depressed glucose uptake rates 19, impaired hypothalamic-pituitary regulation 24 and a decline in neuronal responsiveness to steroid hormones uses4 are some of the recognized changes associated with the expression of the hyperglycemic-hyperinsulinemic state in this mufine model 6'7. In turn, changes in peripheral nerve 22' 33-36 pancreatic6.7, hepatic6.7, renal4,21.29 and reproductive tract structure and function ~3.2° are also recognized to occur following the onset of the diabetic condition. Recent studies have indicated that some of the neuronal and peripheral tissue changes associated with the expression of the db/db mutation may be therapeutically corrected s,9a2. In addition, opiate antagonists have recently been demonstrated to
modulate circulating glucose levels under experimentally induced hyperglycemic conditions 23. These studies suggest that neuroactive substances may be used therepeutically for the normalization of peripheral tissue and neuronal glucose utilization rates in diabetics. Nalmefene (Nalmetrene: 6-methylene naltrexone) is a newly developed opiate antagonist with a recognized CNS site of action 25,31.32. Nalmefene has been demonstrated to effectively reduce insulin resistance and hyperinsulinemia in obese human subjects without significant side-effects and to modulate centrally regulated food and water intake rates in obese rats 31. In addition, Nalmefene blocks the estrogeninduced increase in prolactin release from rat pituitaries and enhances luteinizing hormone release 25, indicating that it effectively modulates CNS neuroendocrine activity 2.3. To date, however, the ability of Nalmefene to modify such metabolic parameters as regional brain glucose uptake or utilization rates has not been determined. Considering that depressed CNS glucose uptake is recognized as an early altera-
* Present address: Division of Stroke & Trauma, NINCDS, Bethesda, MD 20892, U.S.A. Correspondence: D.R. Garris, Department of Anatomy, Cleveland Research Laboratory, 6401 Rockhili Road, Kansas City, MO 64131, U.S.A.
263 tion in genetically diabetic C57BL/KsJ mice 19, the capability of reversing or delaying this CNS impairment with Nalmefene therapy was of interest. The present studies were undertaken to determine the effects of Nalmefene on modulating the diabetes-associated depression of regional brain glucose uptake rates relative to age-matched controls and peripheral changes in systemic blood glucose levels. MATERIALS AND METHODS
Animals Adult, female C57BL/KsJ mice (Jackson Laboratory, Bar Harbor, ME) were used in these studies. Each normal (+/?) mouse was age-matched with a genetically diabetic (db/db) litter mate. All mice were housed 5 per cage under controlled environmental (23 °C) conditions, with an established photoperiod of 12 h light/day (lights on 07.00 h). Blood glucose levels (Autotechnicon method) and body weights were determined weekly for all mice. Animals exhibiting both obesity (>30 g) and hyperglycemia (>250 mg/dl) relative to controls (18-25 g and 80-110 mg/dl) were regarded as overt diabetics.
Treatment Nalmefene was dissolved in saline to obtain injection doses of 0.5 and 5.0 mg/kg/0.2 ml. Physiological saline served as the vehicle control for all sham-injections. Animals received daily s.c. injections of Nalmefene or saline between 08.00 and 09.00 h for 7 weeks. The last blood glucose determination and injection occurred 24 h prior to the determination of brain glucose uptake rates.
Regional brain glucose uptake Using radiolabeled 2-deoxy-[1,2-3H(N)]glucose (2-DG) as a substrate marker, regional brain glucose uptake rates were determined as previously described tg. In brief, age-matched control and diabetic mice, treated with either saline or Nalmefene (0.5 or 5.0 mg/kg doses) received a 10 ~Ci dose of 2.DG i.p. between 08.00 and 10.00 h. At 30 rain postinjection, each mouse was perfused with 10 ml of saline, the brains rapidly removed and frozen. At the time of perfusion, a 50/~! sample of blood was collected from the orbital sinus for the estimation of circulating 2DG levels, which was then used for the calculation of
glucose specific activity. Subsequently, each brain was microdissected into specific regions as described previously n, weighed, solubilized and the radioactivity estimated. Using the individual specific activities of 2-DG, regional brain glucose uptake rates were calculated for each animal and expressed as /~mol/g tissue/30 min ~9. The established control values from each group of specific brain regions were used to represent 100% of expected glucose uptake rates, and the saline- or Naimefene-treated diabetic values were compared on a percentage basis to control values. All values exhibiting a P ~<0.05 from control values (Student's t-test) were accepted as denoting significant intergroup differences.
Experimental protocol Age-matched control (+17) and diabetic (dbldb) mice between 8 and 16 weeks of age received daily s.c. injections of saline or Nalmefene for 7 weeks. Body weights and blood glucose valves were monitored on a weekly basis. Regional brain glucose uptake rates were determined after 7 weeks of treatment as previously described 19. All values were expressed as/~mol glucose/g tissue/30 min. Changes in db/db glucose uptake rates were expressed relative to established control values. RESULTS Neither saline nor Nalmefene injections had any significant effect on the basal, circulating blood glucose levels in either control (+/?) or diabetic (dbldb) mice (Fig. 1). Regardless of treatment, control mice exhibited blood glucose levels of approximately 100 mg/dl throughout the study and over a typical 24-h period (Fig. 1). In contrast, all diabetic mice had blood glucose values that ranged between 300 and 400 mg/dl (Fig. 1). All diabetic mice exhibited significantly elevated blood glucose levels relative to agematched controls (Fig. 1). Regional brain glucose uptake rates in control mice are represented in Table 1. Such areas as the olfactory bulbs and frontal cortex exhibked the highest glucose uptake rates in these studies, while most other areas with higher white/gray matter ratios (e.g. pons) were lower. When expressed as the percentage of control values, many of the diabetic brain areas exhibited a marked depression in 2-DG utilization rates
264 Control
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Time (hours
Brain Region
a/(~/?)-S ..... .............. f ] ' ( - + l ? ) - N
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Fig. 2. Regional brain glucose uptake rates in diabetic (dbldb) mice are represented as the percentage of age-matched control (+/?) values. All values are represented as group means (+ S.E.M.), with significant (P ~< 0.05) intergroup differences denoted by an asterisk.
post-injection)
Fig. 1, Representative 24-h blood glucose (mg/dl) levels in control (+/?) and diabetic (db/db) C57BL/KsJ mice following either physiological saline (S) or Nalmefene (N; combined 0.5 or 5.0 mg/kg/day) treatment at 0 h are indicated. All values are represented as group mean (4- S.E.M.) for 4-6 animals per group. No significant differences resulted from drug therapy over the 24-h period in either group. All db/db blood glucose levels were significantly (P ~ 0.05) elevated over corresponding control levels.
areas, including the hippocampus, midbrain, pons, cerebellum and pituitary were found to have similar glucose uptake rates as those of controls. The dia betes-associated depression of regional 2-DG uptake 400.
2so;. (Fig. 2). Such areas as the olfactory bulbs, frontal cortex, septal area, amygdala, hypothalamus and medulla all exhibited a significant reduction in uptake rates relative to control rates. However, many
TABLE I
Regional brain glucose uptake in C57BL/KsJ (+ ?) female m&e All values are represented as group mean.s (_+ S.E.M.) for 16week-old mice (n = 3). Nalmefene treatment had no effect on regional brain glucose uptake rates in controls.
Brain region
Glucose uptake rate (l~M/g/30rain)
Olfactory bulbs Cortex (frontal) Septum Amygdala Hypothalamus Hippocampus Midbrain Pons Medulla Cerebellum Pituitary
5.7 + 0.2 5.8 + 0.5 4.9 + 0.4 4.1 + 0.3 4.6 + 0.2 4.1 + 0.3 4.6 + 0.8 3.9 + 0.4 4.2 4- 0.3 4.1 4- 0.6 4.6 4- 0.3
I - - I N a l m e f e n e 0.5 m g / k g / d a y E ' l N a l m e f e n e 5.0 m g / k g l d a y
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Brain Region Fig. 3. Effects of Nalmefem,e treatments (0.5 and 5.0 mg/kgi day) on regional brain glucose uptake rates in diabetic mice are represented as percentage of the corresponding control (+/?) values. All values are represented as group means (+ S.E.M.) for 3-4 animals per group, with significant (P ~ 0.05) differences from control levels denoted by an asterisk.
265 was effectively reversed by. both Nalmefene treatment doses (Fig. 3). When compared to control values, the glucose uptake rates of each diabetic brain region, except the olfactory bulbs, demonstrated normalized carbohydrate metabolism. No significant differences were found between the 0.5 and 5.0 mg/kg doses of Nalmefene with respect to their ability to normalize the db/db brain glucose uptake rates to control levels. Nalmefene had no apparent effect on modulating regional brain glucose uptake rates in control mice. DISCUSSION The results of the present studies indicate that the novel opiate antagonist, Nalmefene, is capable of modulating the rate of glucose uptake in several brain areas of genetically diabetic C57BL/KsJ mice. The diabetes-associated depression in glucose uptake rates was normalized to control levels in all brain regions, except the olfactory bulbs. In contrast, this drug had no effect on circulating blood glucose concentrations. These data suggest that the opiate antagonist may either have a direct, stimulatory effect on neuronal glucose uptake rates or that it may serve to antagonize an opioid-associated depression of CNS metabolism in diabetics. The ability of Nalmefene to accentuate carbohydrate utilization in such steroid target sites as the amygdala, hypothalamus and septum also suggests that these various CNS loci, which are recognized to participate in neuroendocrine regulatory functions 14'lsag, may be classified as primary CNS sites of action for the drug 3l'32. It is recognized that naloxone, another opiate antagonist, is also steroid-dependent with respect to its modulation of gonadotrophin release 23. In addition, estrogenic steroids are capable of binding to CNS opiate receptors 27. These findings, and those of the present study, suggest that opiate antagonists may serve as CNS stimulants with respect to such functions as neuronal metabolism or hormonal responsivity. In turn, these data suggest that opioids may elicit their wellrecognized analgesic effects by depressing normal neuronal activity and metabolism at selected target sites in the CNS. To date, the regional levels of CNS opioids have not been reported in this mutant model which would serve to indicate if elevated opiate levels are associated with the depressed CNS glucose
uptake rates. Recent studies have indicated that intracerebroventricular administration of naloxone can effectively reduce circulating blood glucose levels following CNS glucodeprivation e3. In the present studies, the systemic administration of Nalmefene was not found to influence circulating blood glucose levels. In addition, preliminary studies (Garris, unpublished observations) have suggested that Nalmefene has no significant effect on peripheral tissue (i.e. lung, heart, pancreas, spleen, liver, kidney, ovary and fat) glucose uptake rates in diabetic or normal mice. The different responses elicited by these opiate antagonist drugs are probably related to the specific experimental models used as well as the method of treatment. In addition, the lack of effect of the opiate antagonists on peripheral tissue metabolism may be related to the insulin-dependent nature of glucose transport in peripheral tissues as compared to the non-insulin-dependent mode of glucose uptake by the CNS 26'2s. Since the peripheral hyperglycemic condition was not corrected by Nalmefene therapy, the CNS normalization of glucose uptake rates in diabetic mice further suggests that this drug is specific for CNS sites of action. The exact mechanism(s) through which these modulations in brain glucose utilization rates are achieved remain to be elucidated. Previous reports have detailed various changes in neuronal structure and function in the C57BL/KsJ (db/db) mouse lla2"14"lS"w. Both age- and diabetes-related changes have been reported to occur with the expression of the genetic mutation and the subsequent exacerbation of the diabetic condition 6"7as'19. The earliest changes that have been recognized with respect to CNS alterations following the overt expression of diabetes is an impaired glucose uptake rate 19. Similar findings regarding amino acid metabolism have also been reported for other diabetic models 3°. Subsequently, in several brain regions, a depressed neuronal responsivity to hormonal modulation has been recognized to occur ila4 and, with an extended duration of the hyperglycemic condition, premature neuronal degeneration occurs TM. Recent evidence has supported the results of the present studies by indicating that some steroid hormones can also effectively restore or maintain normal CNS glucose uptake rates in diabetic mice t2, similar to the actions of Nalmefene. It remains to be determined if
266 these diverse therapies utilize similar or i n d e p e n d e n t modes of action to elicit these corrective effects and if long term t r e a t m e n t can either correct, delay or prevent the deleterious effects of the db/db m u t a t i o n on CNS structure and function. In summary, the present studies are the first indication that the novel opiate antagonist, N a l m e f e n e ,
may have m o d u l a t i n g effects on regional brain glucose u p t a k e rates in diabetic mice. M a n y of the areas responding to t h e r a p y are also recognized to contain high CNS levels of natural opioid pe~tides. These data suggest a potential role for opiate agonists and antagonists in m o d u l a t i n g the expression of the obes i t y - d i a b e t e s s y n d r o m e in this m u r i n e model.
REFERENCES
Depopulation of the ventromedial hypothalamic nucleus in the diabetic Chinese hamster, Acta Neuropathol., 56 (1982) 63-66. 17 Garris, D.R., Smith, C., Davis, D., Diani, A. and Gerritsen, G., Morphometric evaluation of the hypothalamicovarian axis of the ketonuric, diabetic Chinese hamster: relationship to the reproductive cycle, Diabetologia, 23 (1982) 275-279. 18 Garris, D.R., West, R.L. and Coleman, D.L., Morphometric analysis of medial basal hypothalamic neuronal degeneration in diabetes (db/db) mutant C57BL/KsJ mice: relation to age and hyperglycemia, Dev. Brain Res., 20 (1985) 161-168. 19 Garris, D.R., Williams, S.K., Coleman, D.L. and Morgan, C.R., Glucose utilization by the mouse brain: influence of age and diabetes, Dev. Brain Res., 15 (1984) 141-146. 20 Garris, D.R., Williams, S.K. and West, L., Morphometric evaluation of diabetes-associated, ovarian atrophy in the C57BL/KsJ mouse: relationship to age and ovarian function, Anat. Rec., 211 (1985)434-443. 21 Gartner, K., Glomerular hyperfiltration during the onset of diabetes in mice, Diabetologia, 15 (1979) 59-63. 22 Hawker, J.S., Ambrose, W.W., Yates, P.E., Koch, G.G. and Carson, K.A., Peripheral neuropathy in mouse diabetes mellitus, Acta Neuropathol., 51 (1980) 145-154. 23 Ipp, E., Garberoglio, C., Richter, H., Moossa, A.R. and Rubenstein, A.H., Naloxone decreases centrally induced hyperglycemia in dogs, evidence for an opioid role in glucose homeostasis, Diabetes, 33 (1984) 619-621. 24 Johnson, L.M. and Sidman, R.L., A reproductive endocrine profile in the diabetes (db/db) mutant mouse, Biol. Reprod., 20 (1979) 552-559. 25 Johnson, M.D. and Crowley, W.R., Effects of opiate antagonists on serotonin turnover and on luteinizing hormone and prolactin secretion in estrogen- or morphine-treated rats, Neuroendocrinology, 38 (1984) 322-327. 26 Klip, A., Regulation of glucose transport by insulin and non-hormonal fact.ors, Life Sci., 31 (1969) 2537-2548. 27 LaBella, F.S., Kim, R.S. and Templeton, J., Opiate receptor binding activity of 17-alpha estrogenic steroids, Life Sci., 23 (1978) 1797-1804. 28 Lautala, P. and Martin, J.M., Glucose metabolism in rat hypothalamus, Acta Endocrinol., 98 (1981) 481-487. 29 Like, A.A., Lavine, R., Pattenbarger, L. and Chick, W., Studies in the diabetic mutant mouse. VI. Evolution of glomerular lesions and associated proteinuria, Am. J. Pathol., 66 (1972) 193-207. 30 McCall, A.L., Millington, W.R. and Wurtman, R.J., Metabolic fuel and amino acid transport into the brain in experimental diabetes meilitus, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 5406-5410. 31 McLaughlin, C.L. and Baile, C.A., Naimefene decreases
1 Bestetti, G. and Rossi, G.L., Hypothalamic lesions in rats with long-term streptozotocin-induced diabetes mellitus. A semiquantitative light and electron microscopic study, Acta Neuropathol., 52 (1980) 119-127. 2 Bhanott, R. and Wilkinson, M., Opiatergic control of LH secretion is eliminated by gonadectomy, Endocrinology, 112 (1983) 399-401. 3 Bhanott, R. and Wilkinson, M., The inhibitory effect of opiates on gonadotrophin secretion is dependent upon gonadal steroids, J. Endocrinol., 102 (1984) 133-141. 4 Bower, C., Brown, B.M., Steffes, M.W., Vernier, R.L. and Mauer, S.N., Studies of the glomerular mesangium and the juxtaglomerular apparatus in the genetically diabetic mouse, Lab. lnvest., 43 (1980) 333-346. 5 Bray, G.A. and York, D.A., Hypothalamic and genetic obesity in experimental animals: an autonomic and endocrine hypothesis, Physiol. Rev., 59 (1979) 719-809. 6 Coleman, D.L., Diabetes-obesity syndromes in mice, Diabetes, 31 Suppl. 1 (1982) 1-6. 7 Coleman, D.L., Obese and diabetes: two mutant genes causing diabetes-obesity syndromes in mice, Diabetologia, 14 (1978) 141-148. 8 Coleman, D.L., Leiter, E.H. and Schwizer, R.W., Therapeutic effects of dehydroepiandrosterone (DHEA) in diabetic mice, Diabetes, 31 (1982) 830-833. 9 Coleman, D.L., Schwizer, R.W. and Leiter, E . H , Effect of genetic background on the therapeutic effects of dehydroepiandrosterone (DHEA) in diabetes-obesity mutants and in aged normal mice, Diabetes, 33 (1984) 26-32. 10 DeJong, R.N., CNS manifestation of diabetes mellitus, Postgrad. Med., 61 (1977) 101-107. 11 Garris, D.R., Depressed progesterone accumulation by the brain and peripheral tissues of diabetic C57BL/KsJ mice: normalization by estrogen therapy, Horm. Res., 25 (1987) 37-48. 12 Garris, D.R., Glucose utilization in the diabetic mouse brain: therapeutic effects of ovarian steroids, insulin and an opiate antagonist, Fed. Proc., 44 (1985) 1656. 13 Garris, D.R., Diabetes-associated alterations in uterine structure in the C57BL/KsJ mouse: relationship to changes in estradiol accumulation, circulating ovarian steroid levels and age, Anat. Rec., 211 (1985) 414-419. 14 Garris, D.R. and Coleman, D.L., Diabetes-associated changes in estradiol accumulation in the aging C57BL/KsJ mouse brain, Neurosci. Lett., 49 (1984) 285-290. 15 Garris, D.R., Coleman, D.L. and Morgan, C.R., Age and diabetes-related changes in glucose uptake and estradiol accumulation in the C57BL/KsJ mouse, Diabetes, 34 (1985) 47-52. 16 Garris, D.R., Diani, A.R., Smith, C. and Gerritsen, G.,
267 meal size, food and water intake and weight gain in Zucker rats. Pharmacol. Biochem. Behav., 19 (1983) 235-240. 32 Michel, M.E., Bolger, G. and Weissman, B.A., Binding of a new opiate antagonist, Nalmefene, to rat brain membranes, Pharmacologist. 26 (1984) 20 I. 33 Moore, S.A., Peterson, R.G., Fellow, D.L., Cartwright, T.R. and O'Connor, B.L., Reduced sensory and motor conductance velocity in 24-week old diabetic (C57BL/KsJ db/db) mice, Exp. Neurol.. 70 (1980) 548-555. 34 Olsson, Y., Soderbergh, J.S., Sourander, P. and Angervail, L., A patho-anatomical study of the central and peripheral nervous system in diabetes of early onset and long
duration, Pathol. Eur., 3 (1968) 62-79. 35 Reske-Nielson, E., Lundback, K. and Rafaelson, O.J.+ Pathological changes in the central and peripheral nervous system of young long-term diabetes, Diabetologia. 1 (1965) 233-241. 36 Sima, A.F. and Robertson, D.M., Peripheral neuropathy in the mutant diabetes mouse BL/KsJ db/db+ Acta Neuropathol., 41 (1979) 85-89. 37 Smith-West, C. and Garris, D.R., Diabetes-associated hypothalamic neuronal depopulation in the aging Chinese hamster, Dev. Brain Res., 9 (1983) 385-389.
262 BRE 13418
Regional brain glucose uptake in genetically diabetic C57BL/KsJ mice: modulation by the opiate antagonist, Nalmefene David R. Garris 1 and Mary Ellen Michel 2'* tDepartment of Anatomy, ClevelandResearch Laboratory, Kansas City, MO 64131 (U.S.A.) and 2Key Pharmaceuticals, Inc., Miami, FL 33269-0670 (U.S.A.)
(Accepted 22 September 1987) Key words: Diabetes; Brain glucose uptake; C57BL/KsJ mouse; Nalmefene; Opiate antagonist
A novel opiate antagonist, Nalmefene (0.5 or 5.0 mg/kg/day) was tested for its ability to modulate regional brain glucose uptake rates in genetically diabetic C57BL/KsJ mice, which normally exhibit a depressed CNS carbohydrate metabolism relative to agematched controls. Daily Nalmefene treatment had no effect on circulating blood glucose levels in either normal or diabetic mice over a 7-week test period. However, all brain regions, except the olfactory bulbs, exhibited normalized glucose uptake rates in diabetic mice relative to controls. These data suggest a role for opiate antagonists in the modulation of CNS glucose metabolism during hyperglycemic states. INTRODUCTION Changes in neuronal structure, function and metabolism are recognized to occur in association with type I (insulin-dependent) and type II (non-insulindependent) diabetic conditions 1.Sa°a4-19`37. In the genetically obese-diabetic C57BL/KsJ (db/db) mouse, premature neuronal degeneration TM, depressed glucose uptake rates 19, impaired hypothalamic-pituitary regulation 24 and a decline in neuronal responsiveness to steroid hormones uses4 are some of the recognized changes associated with the expression of the hyperglycemic-hyperinsulinemic state in this mufine model 6'7. In turn, changes in peripheral nerve 22' 33-36 pancreatic6.7, hepatic6.7, renal4,21.29 and reproductive tract structure and function ~3.2° are also recognized to occur following the onset of the diabetic condition. Recent studies have indicated that some of the neuronal and peripheral tissue changes associated with the expression of the db/db mutation may be therapeutically corrected s,9a2. In addition, opiate antagonists have recently been demonstrated to
modulate circulating glucose levels under experimentally induced hyperglycemic conditions 23. These studies suggest that neuroactive substances may be used therepeutically for the normalization of peripheral tissue and neuronal glucose utilization rates in diabetics. Nalmefene (Nalmetrene: 6-methylene naltrexone) is a newly developed opiate antagonist with a recognized CNS site of action 25,31.32. Nalmefene has been demonstrated to effectively reduce insulin resistance and hyperinsulinemia in obese human subjects without significant side-effects and to modulate centrally regulated food and water intake rates in obese rats 31. In addition, Nalmefene blocks the estrogeninduced increase in prolactin release from rat pituitaries and enhances luteinizing hormone release 25, indicating that it effectively modulates CNS neuroendocrine activity 2.3. To date, however, the ability of Nalmefene to modify such metabolic parameters as regional brain glucose uptake or utilization rates has not been determined. Considering that depressed CNS glucose uptake is recognized as an early altera-
* Present address: Division of Stroke & Trauma, NINCDS, Bethesda, MD 20892, U.S.A. Correspondence: D.R. Garris, Department of Anatomy, Cleveland Research Laboratory, 6401 Rockhili Road, Kansas City, MO 64131, U.S.A.
263 tion in genetically diabetic C57BL/KsJ mice 19, the capability of reversing or delaying this CNS impairment with Nalmefene therapy was of interest. The present studies were undertaken to determine the effects of Nalmefene on modulating the diabetes-associated depression of regional brain glucose uptake rates relative to age-matched controls and peripheral changes in systemic blood glucose levels. MATERIALS AND METHODS
Animals Adult, female C57BL/KsJ mice (Jackson Laboratory, Bar Harbor, ME) were used in these studies. Each normal (+/?) mouse was age-matched with a genetically diabetic (db/db) litter mate. All mice were housed 5 per cage under controlled environmental (23 °C) conditions, with an established photoperiod of 12 h light/day (lights on 07.00 h). Blood glucose levels (Autotechnicon method) and body weights were determined weekly for all mice. Animals exhibiting both obesity (>30 g) and hyperglycemia (>250 mg/dl) relative to controls (18-25 g and 80-110 mg/dl) were regarded as overt diabetics.
Treatment Nalmefene was dissolved in saline to obtain injection doses of 0.5 and 5.0 mg/kg/0.2 ml. Physiological saline served as the vehicle control for all sham-injections. Animals received daily s.c. injections of Nalmefene or saline between 08.00 and 09.00 h for 7 weeks. The last blood glucose determination and injection occurred 24 h prior to the determination of brain glucose uptake rates.
Regional brain glucose uptake Using radiolabeled 2-deoxy-[1,2-3H(N)]glucose (2-DG) as a substrate marker, regional brain glucose uptake rates were determined as previously described tg. In brief, age-matched control and diabetic mice, treated with either saline or Nalmefene (0.5 or 5.0 mg/kg doses) received a 10 ~Ci dose of 2.DG i.p. between 08.00 and 10.00 h. At 30 rain postinjection, each mouse was perfused with 10 ml of saline, the brains rapidly removed and frozen. At the time of perfusion, a 50/~! sample of blood was collected from the orbital sinus for the estimation of circulating 2DG levels, which was then used for the calculation of
glucose specific activity. Subsequently, each brain was microdissected into specific regions as described previously n, weighed, solubilized and the radioactivity estimated. Using the individual specific activities of 2-DG, regional brain glucose uptake rates were calculated for each animal and expressed as /~mol/g tissue/30 min ~9. The established control values from each group of specific brain regions were used to represent 100% of expected glucose uptake rates, and the saline- or Naimefene-treated diabetic values were compared on a percentage basis to control values. All values exhibiting a P ~<0.05 from control values (Student's t-test) were accepted as denoting significant intergroup differences.
Experimental protocol Age-matched control (+17) and diabetic (dbldb) mice between 8 and 16 weeks of age received daily s.c. injections of saline or Nalmefene for 7 weeks. Body weights and blood glucose valves were monitored on a weekly basis. Regional brain glucose uptake rates were determined after 7 weeks of treatment as previously described 19. All values were expressed as/~mol glucose/g tissue/30 min. Changes in db/db glucose uptake rates were expressed relative to established control values. RESULTS Neither saline nor Nalmefene injections had any significant effect on the basal, circulating blood glucose levels in either control (+/?) or diabetic (dbldb) mice (Fig. 1). Regardless of treatment, control mice exhibited blood glucose levels of approximately 100 mg/dl throughout the study and over a typical 24-h period (Fig. 1). In contrast, all diabetic mice had blood glucose values that ranged between 300 and 400 mg/dl (Fig. 1). All diabetic mice exhibited significantly elevated blood glucose levels relative to agematched controls (Fig. 1). Regional brain glucose uptake rates in control mice are represented in Table 1. Such areas as the olfactory bulbs and frontal cortex exhibked the highest glucose uptake rates in these studies, while most other areas with higher white/gray matter ratios (e.g. pons) were lower. When expressed as the percentage of control values, many of the diabetic brain areas exhibited a marked depression in 2-DG utilization rates
264 Control
s°°1
O
100" :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: "::: ..........
E
== v c O ¢1 N
•
60,
'
i
' I/
40-
300~.~.
,,,,~
~
G) ¢) 0 O 20-
01111
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~ooJ ~.
. ~
Ol , 0 2
i 6
Time (hours
Brain Region
a/(~/?)-S ..... .............. f ] ' ( - + l ? ) - N
I 12
t 24
Fig. 2. Regional brain glucose uptake rates in diabetic (dbldb) mice are represented as the percentage of age-matched control (+/?) values. All values are represented as group means (+ S.E.M.), with significant (P ~< 0.05) intergroup differences denoted by an asterisk.
post-injection)
Fig. 1, Representative 24-h blood glucose (mg/dl) levels in control (+/?) and diabetic (db/db) C57BL/KsJ mice following either physiological saline (S) or Nalmefene (N; combined 0.5 or 5.0 mg/kg/day) treatment at 0 h are indicated. All values are represented as group mean (4- S.E.M.) for 4-6 animals per group. No significant differences resulted from drug therapy over the 24-h period in either group. All db/db blood glucose levels were significantly (P ~ 0.05) elevated over corresponding control levels.
areas, including the hippocampus, midbrain, pons, cerebellum and pituitary were found to have similar glucose uptake rates as those of controls. The dia betes-associated depression of regional 2-DG uptake 400.
2so;. (Fig. 2). Such areas as the olfactory bulbs, frontal cortex, septal area, amygdala, hypothalamus and medulla all exhibited a significant reduction in uptake rates relative to control rates. However, many
TABLE I
Regional brain glucose uptake in C57BL/KsJ (+ ?) female m&e All values are represented as group mean.s (_+ S.E.M.) for 16week-old mice (n = 3). Nalmefene treatment had no effect on regional brain glucose uptake rates in controls.
Brain region
Glucose uptake rate (l~M/g/30rain)
Olfactory bulbs Cortex (frontal) Septum Amygdala Hypothalamus Hippocampus Midbrain Pons Medulla Cerebellum Pituitary
5.7 + 0.2 5.8 + 0.5 4.9 + 0.4 4.1 + 0.3 4.6 + 0.2 4.1 + 0.3 4.6 + 0.8 3.9 + 0.4 4.2 4- 0.3 4.1 4- 0.6 4.6 4- 0.3
I - - I N a l m e f e n e 0.5 m g / k g / d a y E ' l N a l m e f e n e 5.0 m g / k g l d a y
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Brain Region Fig. 3. Effects of Nalmefem,e treatments (0.5 and 5.0 mg/kgi day) on regional brain glucose uptake rates in diabetic mice are represented as percentage of the corresponding control (+/?) values. All values are represented as group means (+ S.E.M.) for 3-4 animals per group, with significant (P ~ 0.05) differences from control levels denoted by an asterisk.
265 was effectively reversed by. both Nalmefene treatment doses (Fig. 3). When compared to control values, the glucose uptake rates of each diabetic brain region, except the olfactory bulbs, demonstrated normalized carbohydrate metabolism. No significant differences were found between the 0.5 and 5.0 mg/kg doses of Nalmefene with respect to their ability to normalize the db/db brain glucose uptake rates to control levels. Nalmefene had no apparent effect on modulating regional brain glucose uptake rates in control mice. DISCUSSION The results of the present studies indicate that the novel opiate antagonist, Nalmefene, is capable of modulating the rate of glucose uptake in several brain areas of genetically diabetic C57BL/KsJ mice. The diabetes-associated depression in glucose uptake rates was normalized to control levels in all brain regions, except the olfactory bulbs. In contrast, this drug had no effect on circulating blood glucose concentrations. These data suggest that the opiate antagonist may either have a direct, stimulatory effect on neuronal glucose uptake rates or that it may serve to antagonize an opioid-associated depression of CNS metabolism in diabetics. The ability of Nalmefene to accentuate carbohydrate utilization in such steroid target sites as the amygdala, hypothalamus and septum also suggests that these various CNS loci, which are recognized to participate in neuroendocrine regulatory functions 14'lsag, may be classified as primary CNS sites of action for the drug 3l'32. It is recognized that naloxone, another opiate antagonist, is also steroid-dependent with respect to its modulation of gonadotrophin release 23. In addition, estrogenic steroids are capable of binding to CNS opiate receptors 27. These findings, and those of the present study, suggest that opiate antagonists may serve as CNS stimulants with respect to such functions as neuronal metabolism or hormonal responsivity. In turn, these data suggest that opioids may elicit their wellrecognized analgesic effects by depressing normal neuronal activity and metabolism at selected target sites in the CNS. To date, the regional levels of CNS opioids have not been reported in this mutant model which would serve to indicate if elevated opiate levels are associated with the depressed CNS glucose
uptake rates. Recent studies have indicated that intracerebroventricular administration of naloxone can effectively reduce circulating blood glucose levels following CNS glucodeprivation e3. In the present studies, the systemic administration of Nalmefene was not found to influence circulating blood glucose levels. In addition, preliminary studies (Garris, unpublished observations) have suggested that Nalmefene has no significant effect on peripheral tissue (i.e. lung, heart, pancreas, spleen, liver, kidney, ovary and fat) glucose uptake rates in diabetic or normal mice. The different responses elicited by these opiate antagonist drugs are probably related to the specific experimental models used as well as the method of treatment. In addition, the lack of effect of the opiate antagonists on peripheral tissue metabolism may be related to the insulin-dependent nature of glucose transport in peripheral tissues as compared to the non-insulin-dependent mode of glucose uptake by the CNS 26'2s. Since the peripheral hyperglycemic condition was not corrected by Nalmefene therapy, the CNS normalization of glucose uptake rates in diabetic mice further suggests that this drug is specific for CNS sites of action. The exact mechanism(s) through which these modulations in brain glucose utilization rates are achieved remain to be elucidated. Previous reports have detailed various changes in neuronal structure and function in the C57BL/KsJ (db/db) mouse lla2"14"lS"w. Both age- and diabetes-related changes have been reported to occur with the expression of the genetic mutation and the subsequent exacerbation of the diabetic condition 6"7as'19. The earliest changes that have been recognized with respect to CNS alterations following the overt expression of diabetes is an impaired glucose uptake rate 19. Similar findings regarding amino acid metabolism have also been reported for other diabetic models 3°. Subsequently, in several brain regions, a depressed neuronal responsivity to hormonal modulation has been recognized to occur ila4 and, with an extended duration of the hyperglycemic condition, premature neuronal degeneration occurs TM. Recent evidence has supported the results of the present studies by indicating that some steroid hormones can also effectively restore or maintain normal CNS glucose uptake rates in diabetic mice t2, similar to the actions of Nalmefene. It remains to be determined if
266 these diverse therapies utilize similar or i n d e p e n d e n t modes of action to elicit these corrective effects and if long term t r e a t m e n t can either correct, delay or prevent the deleterious effects of the db/db m u t a t i o n on CNS structure and function. In summary, the present studies are the first indication that the novel opiate antagonist, N a l m e f e n e ,
may have m o d u l a t i n g effects on regional brain glucose u p t a k e rates in diabetic mice. M a n y of the areas responding to t h e r a p y are also recognized to contain high CNS levels of natural opioid pe~tides. These data suggest a potential role for opiate agonists and antagonists in m o d u l a t i n g the expression of the obes i t y - d i a b e t e s s y n d r o m e in this m u r i n e model.
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