Physiology& Behavior,Vol.52, pp. 713-716, 1992

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Glucose Utilization and Insulin Binding in Discrete Brain Areas of Obese Rats P. M A R F A I N G - J A L L A T , ~ C. L E V A C H E R , Y. C A L A N D O , * L. P I C O N A N D L. P E N I C A U D

Laboratoire de Physiopathologie de la Nutrition, URA 307 C N R S and *Laboratoire de Physiologie et Physiopathologie Cdrdbrovasculaire, Inserm U182, Universitd Paris VII, Paris France Received 15 October 1991 MARFAING-JALLAT, P., C. LEVACHER, Y. CALANDO, L. PICON AND L. PENICAUD. Glucose utilization and insulin binding in discrete brain areas of obese rats. PHYS1OL BEHAV 52(4) 713-716, 1992.--The present study was carried out to determine whether geneticallyobese Zucker rats present changesin brain glucose utilizationand/or insulin binding when compared to their lean counterparts. Glucoseutilizationin the whole brain, determined by measurementof 2-deoxy(l-3H)glucose-6-phosphate, was significantlylower in obese than in lean Zucker rats. In order to precise the structure involved, we then used quantitative autoradiography methods after either (l-t4C) 2-deoxyglucose injection or '25I-insulinincubation. In obese rats, local cerebral glucose utilization(LCGU) was significantlydecreased in the externalplexiform layer (-37%, p < 0.05), in the lateral hypothalamus (-23%, p < 0.05), and in the basolateral amygdaloid nucleus (-30%, p < 0.05). In contrast, no difference in specific insulin binding was found between the two genotypes in any of the areas studied. These results are consistent with some data showinga decrease of LCGU in hyperinsulinemic rats. All together, these data show perturbations of glucose utilization, particularly in structures linked to the regulation of body weight and food intake in obese Zucker rats. Autoradiography

Insulin binding

Glucoseutilization

THE possibility that insulin may have a neuromodulatory role in the central nervous system is supported by various findings. First, significant insulin content and insulin binding sites have been described in the brain (1). Second, a number of central insulin actions has been described, such as modulation of firing of neurons, of uptake and release of neurotransmitters, and of ion transport as well as an important growth-promoting role in brain development [for review see (1)]. Third, insulin induces physiological and behavioral responses when infused into the brain. Indeed, infusion of insulin: 1. increases plasma insulin concentrations inducing a decrease of plasma glucose levels via a stimulation of the vagal nerves (24); 2. inhibits feeding resulting in body weight loss(3,19), probably via its action on turnover of hypothalamic catecholamines [for review see (1,14)]. There are evidences that plasma insulin has access to cerebrospinal fluid, and parallel changes in CSF and plasma insulin have been described (22,27). High concentrations of insulin binding sites have equally been identified in many nuclei of the brain (10). The regulation of insulin binding in the brain is still unclear, particularly in regard to its relation with plasma insulin concentration. Indeed, the obese Zucker rats, which display a number of metabolic and hormonal defects including hyperinsulinemia, present, in comparison with their lean littermates, a

Obeserats

reduction of insulin binding in the hypothalamus (7,16). But Wilcox et al. (26) found no reduction of insulin binding in any structure of the brain of obese Zucker rats. On the contrary, a slight increase in insulin binding was described in the arcuate and dorsomedial nuclei of the obese genotypes (26). Moreover, we have recently shown that insulin binding was significantly enhanced in the hypothalamus of rats submitted to a 2-hour euglycemic hyperinsulinemic clamp (15). Whereas the insulin effect on glucose utilization on peripheral tissues is well documented, the action of the hormone on this parameter in the brain is still unclear. Both no effect or a decreased glucose utilization has been reported in rats made hyperinsulinemic during a short period of time (9,15). However, to our knowledge, no data are available in long-term hyperinsulinemic rats. The present study was designed to determine both glucose utilization and insulin binding in specific brain areas in genetically lean (Fa/Fa) and obese (fa/fa) Zucker rats. We measured these two parameters by quantitative autoradiography methods in some nuclei involved in body weight regulation. METHOD Female homozygous lean (Fa/Fa, n = 11) and obese (fa/fa, n = 1 1) Zucker rats were obtained from our own breeding. They were housed in a room where a 12-hour light-dark cycle was

Requests for reprints should be addressed to Dr. P. Marfaing-JaUat,Laboratoire de Physiopathologiede la Nutrition, URA 307 CNRS, Universit6 Paris Vll, 2 Place Jussieu, 75251 Paris Cedex 05, France.

713

714 maintained. Fhey were studied at 10 weeks of age. The rats were catheterized under ketamine chloride anesthesia (12.5 mg/100 g body weight, IP; lmalg~ne M~rieux, Lyon, France). A catheter was inserted into the right jugular vein, led under the skin, and connected at the top of the head to a metal tube fixed on the skull by dental cement. Experiments were performed only when animals had regained their initial body weight, usually 2 to 3 days after surgery, Measurement of whole brain glucose utilization was made according to Ferr6 et al. (6). Briefly, a tracer dose of 2-deoxy ( l3H)glucose (30 #Ci; 20 Ci/mmol, CEA, Saclay, France) was injected as a bolus, and blood was collected at various time intervals during 60 minutes for determination of blood glucose and 2DG radioactivity. At the end of this period, rats were killed and the brain rapidly removed, and the 2DG (l-3H)glucose-6 phosphate content was then determined as described previously (6). Measurement of local cerebral glucose utilization was initiated by the intravenous injection of the nonmetabolisable glucose analog 2-deoxy ( 1-14C)glucose (20 #Ci, specific activity 50 mCi/ mmol CEA, France) as a bolus. Blood was collected at various time intervals up to 60 minutes after 2DG injection for determination of blood glucose concentration and 2DG radioactivity. At the end of this period, rats were killed by cervical dislocation and the brain removed and rapidly frozen in isopentane cooled to -40°C. Serial coronal sections (20 #m thickness) were obtained with a cryostat at - 15oC, and were subsequently analyzed for the measure of 2-deoxy l-'4C glucose content. Autoradiograms were prepared by exposing the sections with X-ray film (Kodak SB 5) in light-tight X-ray cassettes for approximately 7 days. Local cerebral tissue concentration of L4C was determined by quantitative densitometry (Pericolor Numelec) by reference to eight calibrated ~4C standards ranging from 40 to 1069 nCi/g exposed together with the brain sections. Six to eight measurements of optical density were made for each brain area examined in each animal. Glucose utilization was calculated by means of SokolotFs operational equation (21). Another group of rats (eight Fa/Fa, and eight fa/fa) was analyzed for insulin binding sites by means of autoradiography as described elsewhere (4). Briefly, after a preincubation of the coronal sections of 20 um in KRP-BSA 1% during 1 hour, slices were incubated during 16 hours with '25I-insulin (specific activity: 1200 tzCi/ug). Both preincubation and incubation were performed at 4°C. The specific binding was calculated by the difference between the binding with or without 3.10 .5 M native insulin. Autoradiograms were obtained by apposition of the sections on LKB 3H-ultrafilm during about 7 days. The quantitative analysis was performed by means of a Biocom densitometer. The optical densities were referred to five calibrated ~25Istandards (3,7 to 31.2 fmol/mg protein) of brain homogenates developed and fixed in the same conditions of exposure as the experimental films. Our criteria was to obtain six to ten readings for each structure for each animal.

Analytical Methods Blood samples (50 tzl) for determination of glucose level and 2DG specific radioactivity were deproteinized with 250 ~1 of ZnSO4 (2.71%) and 250 ~zlof Ba(OH)2 (2.62%) and immediately centrifuged. An aliquot of the supernatant was used for the determination of glucose concentration using a glucose oxydase kit (Boerhinger, Meylan, France). Another aliquot was counted in a liquid scintillation spectrometer for the determination of blood 2-deoxy (l-3H)glucose or 2-deoxy (1-'4C) glucose. Plasma insulin was determined by a radioimmunoassay using rat insulin as standards.

MARFAING-JAI~I_AI [il .\i• Results are presented as means values _+SEM. Mann-Whitney (:-test was used for statistical analysis. RESU[/I S

Blood glucose concentrations were not different in lean Fa/ Fa (96 mg/dl) and obese fa/fa rats (101 mg/dl). Plasma insulin was about tenfold higher in obese (350 _+ 62 #U/ml) than in lean rats (31 ± 8 #U/ml, p < 0.001). Whole brain glucose utilization was significantly lower (p < 0.01) in obese (35 ± I/~mol/ 100 g/mira n - 5) than in lean (47 _+ 2 #mol/100 g/rain; n 4) rats. The various sites studied by quantitative autoradiography are illustrated by the diagram of Fig. 1. Local cerebral glucose utilization of the lean rats ranged from 26.8 +_ 1.4 #mol/lO0 g/ min in the arcuate nucleus to 55 _+ 3 /~mol/lO0 g/rain in the external plexiform layer (EPL) of the olfactory bulbs (Fig. 2). These values are in agreement with the values obtained in Wistar rats in a previous experiment (15). In the obese Zucker rats, a significant lower glucose utilization was observed in the EPL, in the lateral hypothalamus, and in the basolateral amygdaloid nucleus (p < 0.05). Densitometric measurements of the insulin binding sites showed that specific binding sites minus nonspecific binding in the presence of 3.10 5 M unlabelled insulin was 88% of the total binding. Values of the lean rats ranged from 9.5 +_ 0.5 fmol/mg protein in the EPL to 6.2 _+ 0.3 fmol/mg protein in the lateral hypothalamus. In obese rats, the values of the same structures were 10.8 _+ 0.7 and 6.7 + 1.9 fmol/mg protein. No significant differences were found between lean and obese genotypes in any of the areas studied (Fig. 2). DISCUSSION

These data demonstrate that glucose utilization is significantly decreased in the brain of obese Zucker rats, this decrease being more pronounced in some brain areas such as the external plexiform layer of the olfactory bulbs, the lateral hypothalamus, and the basolateral amygdaloid nucleus. The significance of this defect in term of cause-and-effect relationship to obesity cannot be safely established, since in the present work obesity was fully developed. But such a decrease has to be related to the observation made by Levin and Sullivan who reported, prior to the induction of obesity, a reduction of 2-deoxyglucose uptake in the hypothalamus of Sprague-Dawley rats prone to diet-induced obesity (DIO) when compared to diet-resistant rats (DR) (14). Thus, in this later model, the rats presents some preexisting difference which could cause the further changes in weight gain. Thus, in two different models of obesity, there is a trend towards a reduction of glucose utilization in some brain areas. Although the effect of insulin on brain glucose utilization are still controversial, there are some data showing a decreased glucose utilization or oxydation under insulin exposure in brain. This has been reported either in vivo during euglycemic hyperinsulinemic clamp (9,17) or in vitro (13). It is still not clear whether this is due to a direct effect of insulin as in peripheral tissues or an indirect effect by a neuromodulatory action of insulin on these cerebral structures. However, it should be recalled that insulin has been shown to penetrate from the circulation into the cerebral spinal fluid (CSF) (20,22,25,27). On the other hand, it has been shown that obese Zucker rats have higher CSF insulin levels than their lean littermates (23). Although, obese Zucker rats present a resistance to the effect of insulin, both peripherally and centrally, this is not observed in every tissue and for all metabolic pathways (8,11,12,18). Thus, in term of

BRAIN GLUCOSE UTILIZATION IN OBESE RATS

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FIG. 1. Diagram of the brain sites in which glucose utilization and insulin binding were determined. EPL, external plexiform layer; IGR, inter granular layer;CA 1, field of Ammon's horn; BLA, basolateralamygdaloid; VMH, ventromedial hypothalamus; LH, lateral hypothalamus; PE, periventricular hypothalamus; SOL, tractus solitaris nucleus; 10, dorso motor nucleus of the vagus.

glucose utilization, whereas in 3-month-old obese Zucker rats muscles are insulin resistant, white adipose tissue is still normosensitive (12,18). It is then tempting to speculate that the decreased glucose utilization observed in the brain of obese Zucker rats could be related to their high insulin concentrations in plasma and/or CSF. In peripheral tissues (muscles, white adipose tissue, liver), high concentrations of insulin lead to a decreased insulin receptors number, a phenomenon known as down regulation. Such a lower insulin binding has been reported in the olfactory bulbs and hypothalamus of the chronically hyperinsulinemic obese

Zucker rats (7,16). Obviously, we did not confirm this result in the present study. This discrepancy may be due to differences in the experimental procedures, particularly because of the fact that we used autoradiographic measurements, whereas in the other studies, insulin binding was measured on membrane homogenates. However, it should be added that the absence of differences in insulin binding in the brain of obese Zucker rats or (ob/ob) mice was also reported in two other publications (10,26). This underlines the fact that the relationships between brain insulin receptors regulation and plasma insulin is still under debate. This is emphasized by the fact that acute exposure to

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FIG. 2. Glucose utilization and insulin binding in different brain areas of lean (n = 7, open bars) and obese (n = 6, hatched bars) Zucker rats. EPL, external plexiform layer; IGR, inter granular layer; CAI, fieldof Ammon's horn; BLA, basolateralamygdaloid; VMH, ventromedial hypothalamus; LH, lateral hypothalamus; PE, periventricular hypothalamus; SOL, nucleus tractus solitaris; 10, dorso motor nucleus of the vagus. Values are means + SEM. *p < 0.05.

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M A R F A I N G - J A I , L A T ET AL,

insulin during a 2-hour euglycemic hyperinsulinemic clamp, induces an increase of insulin binding in the hypothalamus (15). However, evidence from studies on cultured brain cells indicate that insulin receptors from glial cells down regulate in response to increased insulin concentrations, whereas those from neuronal cells do not (2,5).

In summary, this study shows a decreased glucose utilization in the brain of obese Zucker rats. Although one cannot exclude that this decrease could be due to an impairment in some specific structures, it could also be the result of a general perturbation in the brain of obese rats such as a decreased glucose transport at the level of the blood-brain barrier.

REFERENCES 1. Baskin, D. G.; Figlewicz, D. P.; Woods, S. C.; Porte, D.; Dorsa, D. M. Insulin in the brain. Annu. Rev. Physiol. 49:335-347; 1987. 2. Boyd, F. T.: Raisada, M. K. Effects of insulin and tunicamycin on neuronal insulin receptors in culture. Am. J. Physiol. 245:C283C287; 1983. 3. Briet: D. P.; Davis, J. D. Reduction of food intake and body weight by chronic intraventricular insulin infusion. Brain Res. Bull. 12: 571-575; 1984. 4. Broer, Y.; Lhiaubet, A, M.; Rosselin, G.; Rostene, W. Etude autoradiographique et quantitative des sites de liaison de l'insuline dans le cerveau de Rat. C. R. Acad. Sci. [III] 304:31-36; 1987. 5. Clarke, D. W.; Boyd, F. T.; Kappy, M. S.; Raizada, M. K. Insulin binds to specific receptors and stimulates 2 deoxy-glucose uptake in cultured glial cells from rat brain. J. Biol. Chem. 259:11672-11675; 1985. 6. Ferrr, P.; Leturque, A.; Burnol, A. F.; P4nicaud, L.; Girard, J. A method to quantify glucose utilization in vivo in skeletal muscle and white adipose tissue of the anesthetized rat. Biochem. J. 228: 103-110; 1985. 7. Figlewicz, D. P.; Dorsa, D. M.; Stein, L. J.; Baskin, D. G.; Paquette, T.; Greenwood, M. R. C.; Woods, S. C.; Porte, D. Jr. Brain and fiver insulin binding is decreased in Zucker rats carrying the "fa" gene. Endocrinology 117:1537-1543; 1985. 8. Figlewicz, D.; Szot, P.; Greenwood, M. R. C. Insulin stimulates inositol incorporation in hippocampus of lean but not obese Zucker rats. Physiol. Behav. 47:325-330; 1990. 9. Grundstein, H. S.; James, D. E.; Storlien, L. H.; Smythe, G. A.; Kraegen, E. W. Hyperinsulinemia suppresses glucose utilization in specific brain regions: In vivo studies using the euglycemic clamp in the rat. Endocrinology 116:604-610; 1985. 10. Havrankova, J.; Roth, J.; Browstein, M. J. Concentrations of insulin and insulin receptors in the brain are independent of peripheral insulin levels. J. Clin. Invest. 64:636-642; 1979. 11. Ikeda, H.; West, D. B.; Pustek, J. J.; Figlewicz, D. P.; Greenwood, M. R. C.; Porte, D., Jr,; Woods, S. C. Intraventricular insulin reduces food intake and body weight of lean but not obese Zucker rats. Appetite 7:381-386; 1986. 12. Jeanrenaud, B.; Halimi, S.; Van de Werve, G. Neuro-endocrine disorders seen as a triggers of the triad: Obesity-insulin resistance-abnormal glucose tolerance. Diabetes Metab. Rev. 1:261-291; 1985. 13. Lautala, P.; Martin, J. M. Glucose metabolism in rat hypothalamus. Acta Endocrinol. 98:481-487; 1981. 14. Levin, B. E,; Sullivan, A. C. Differences in saccharin-induced cerebral glucose utilization between obesity-prone and resistant rats. Brain Res. 488:221-232; 1989. 15. Marfaing, P.; Prnicaud, L.; Broer, Y.; Mraovitch, S.; Calando, Y.; Picon, L. Effects of hyperinsulinemia on local cerebral insulin binding

16. 17.

18, 19. 20.

21.

22.

23.

24.

25.

26.

27.

and glucose utilization in normoglycemic awake rats. Neurosci. Lett. 115:279-285; 1990. Melnyk, R. Decreased binding to hypothalamic insulin receptors in young genetically obese rats. Physiol. Behav. 40:237-241 ; 1987. Namba, H.; Lucignani, G.; Nehlig, A.; Patlak, C.; Pettigrew, K.; Kennedy, C.; Sokoloff, L. Effects of insulin on hexose transport across blood-brain barrier in normoglycemia. Am. J. Physiol. 25:E299E303; 1987. Prnicaud, L.; Ferrr, P. Development of insulin resistance during the course of obesity: Lessons from animal models, J. Obes. Weight Reg. 7:91-109; 1988. Porte, D., Jr,; Woods, S. C. Regulation of lbod intake and body weight by insulin. Diabetology 20:274-280; 1981. Radosevich, P. M.; Brooks, D. L.; Brown, L. L.; Williams, P. E.; Abumrad, N. N. Effects of insulin-induced hypoglycemia on plasma and cerebrospinal fluid levels of B endorphins, ACTH, cortisol, norepinephrine, insulin and glucose in the conscious dog. Brain Res. 458:325-338; 1988. Sokoloff, L.; Reivich, M.; Kennedy, C.; Des Rosiers, M. H.; Patlak, C. S.; Pettigrew, K. D.: Sakurada, O.; Shinohara, M. ~4C Deoxyglucose method for the measurement of local cerebral glucose utilization: Theory, procedure and normal values in the conscious and anesthetized albino rat. J. Neurochem. 28:897-916; 1977. Steffens, A. B.; Scheurink, A. J. W.; Porte, D.; Woods, S, C. Penetration of peripheral glucose and insulin into cerebrospinal fluid in rats. Am. J. Physiol. 255:R200-R204; 1988. Stein, L. J.; Dorsa, D. M.; Baskin, D. G.; Figlewicz, D. P.; lkeda, H.; Franckmann, S. P.; Greenwood, M. R. C.; Porte, D. Jr.; Woods, S. C. Immunoreactive insulin levels are elevated in the cerebrospinal fluid of genetically obese Zucker rats. Endocrinology 113:2299-2301: 1983. Taborsky, G. J., Jr.; Bergman, R. N. Effect of insulin, glucose, and 2-deoxyglucose infusion into the third cerebral ventricle of conscious dogs on plasma insulin, glucose and free fatty acids. Diabetes 29: 278-283; 1980. Wallum, B. J.; Porte, D., Jr.; Figlewicz, D. P.; Jacobsen, L.; Dorsa, D. Cerebrospinal fluid insulin levels increase during intravenous insulin infusion in man. J. Clin. Endocrinol. Metab. 64:190-194: 1987. Wilcox, B. J,: Corp, E. S.; Dorsa, D. M.; Figlewicz, D. P.; Greenwood, M. R. C.; Woods, S. C.: Baskin, D. G. Insulin binding in the hypothalamus of lean and genetically obese Zucker rats. Peptides 10: 1159-1164; 1989. Woods, S. C.; Porte, D. Relationship between plasma and cerebrospinal fluid insulin levels of dogs. Am. J. Physiol. 223:E331-E334; 1977.