THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

Vol. 260, No. 29, Issue of December 15, pp. 15880-15884,1985 Printed in U.S.A.

Insulin Receptors and Insulin Modulation of Norepinephrine Uptake in Neuronal Cultures from Rat Brain*

(Received for publication, March 25,1985)

Frederick T. Boyd, Jr.$, Derrel W. Clarke#Tl, Thomas F. Muther 11, and Mohan K. Raizada$** From the Departments of $Physiology, §Pediatrics, and llPharmacology and Therapeutics, The University of Florida, Gainesuille, Florida 32610

Neuronal cells from 1-day-old rat brain in primary culture have been utilized in the present study to char- acterize insulin-binding sites and a possible action of insulin on these cells. Binding of 1251-insulin to neu- ronal cultures was 90% specific and time-dependent and reached equilibrium in 120 min. Specific binding was reversible with greater than 90% of binding dis- sociable within 120 min with a tllz of dissociation of 15 min. Various insulin analogues competed for 1251- insulin binding to neuronal cultures according to their known biological potencies. Scatchard analysis of com- petition data yielded a typical curvilinear plot provid- ing a class of high affinity (Kd = 11 nM) and low affinity (Kd = 65 nM) binding sites. Light microscopic autora- diographic analysis of 1251-insulin bound to neuronal cultures revealed the presence of silver grains predom- inantly on the neurites with occasional occurrence on the cell soma. Insulin had no effect on neuronal 2- deoxyglucose uptake in contrast with our previous findings demonstrating a 2-fold stimulation of 2-dGlc uptake into astrocyte glial cells from rat brain (Clarke, D. W., Boyd, F. T., Jr., Kappy, M. S., and Raizada, M. K. (1984) J. Biol. Chem. 259, 11672-11675). Incu- bation of neuronal cultures with insulin caused a dose- dependent inhibition of [3H]norepinephrine uptake with significant inhibition occurring at 1.67 X 10-l’ M. These findings demonstrate that: 1) neuronal cells in primary culture possess specific insulin receptors which are predominantly located on neurites and 2) insulin modulates monoamine uptake in these cultures which suggests that insulin may modulate neural sig- naling via specific neuronal insulin receptors.

The central nervous system has been considered insulin- independent because of the limited penetration of pancreatic insulin into the brain. However, with the demonstration of insulin and insulin-binding sites in the brain (1-8), the pos- sible role of insulin in brain metabolism and function needs to be reconsidered. Studies of classical actions of insulin in the brain, such as stimulation of glucose uptake, have yielded conflicting results (9-16). However, several studies have sug- gested that brain insulin plays a role in neuromodulation. Barbaccia et al. (17) suggested a role for insulin as a neuro-

*This research was supported by National Institutes of Health Grant HD16722. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

7 Supported by National Institutes of Health Research Fellowship Grant l-F32-AM07247.

** Established Investigator of the American Heart Association. To whom correspondence should be addressed.

modulator interacting with the dopamine system active in olfactory bulbs. Sauter et al. (,18) reported that insulin at high doses stimulates release of dopamine, norepinephrine, and epinephrine from hypothalamic slices. Saller and Chiodo (19) have demonstrated changes in the firing rates of nigrostriatal dopamine neurons by insulin, and Palovcik et al. (20) have demonstrated changes in firing rates of hippocampal neurons by insulin.

All of these studies have been complicated by the use of mixed populations of neurons, glia, and endothelial cells from the central nervous system. Thus, any effect of insulin might thus be the result of changes in cellular function of any of these cell types. We have developed culture techniques which allow us to prepare relatively homogeneous populations of either neurons or astrocyte glial cells and look at the cell- specific actions of insulin. The cultured astrocyte glial cells possess specific insulin receptors, and glucose uptake into them is stimulated %fold via action of insulin at these recep- tors (21). We have also shown that neuronal cultures from 1- day-old rat brains contain catecholamines (22) and that these cultures possess specific uptake mechanisms for catechol- amines which are inhibited by specific norepinephrine (NE1) uptake blockers, such as maprotiline, but not by serotonin uptake inhibitors (23).

In the present study, neuronal cultures have been used to characterize neuronal insulin receptors and to investigate insulin’s action on neuronal cells. The effect of insulin on 2- deoxy-D-glucose uptake into neurons was studied to determine whether our previous findings in astrocyte glial cultures were limited to this cell type. Our findings show that insulin does not affect neuronal glucose uptake and that this action may be specific for astrocyte glial cells within the central nervous system to regulate local glucose availability. In addition, we investigated the effect of insulin on the well-characterized NE uptake system in neuronal cultures. Insulin is capable of inhibiting NE uptake at very low concentrations which indi- cates an action on high affinity insulin receptors. These findings suggest that insulin may exert neuromodulatory ef- fects directly on neurons by decreasing the inactivation of synaptic transmitter by reuptake.

EXPERIMENTAL PROCEDURES

Materials One-day-old Sprague-Dawley rats were obtained from our breeding

colony. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, and horse serum were purchased from GIBCO, Grand Island, NY. Deoxyribonuclease I and 1 X crystallized trypsin (190 units/mg) were from Worthington. Bovine serum albumin (Fraction V), poly-

’ The abbreviations used are: NE, norepinephrine; DMEM, Dul- becco’s modified Eagle’s medium; PBS, phosphate-buffered saline; HEPES, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid.

15880

Insulin Receptors and Modulation of Norepinephrine Uptake 15881

L-lysine (M, = 150,000), cytosine arabinoside, pargyline HCl, L- ascorbic acid, and (-)-norepinephrine were from Sigma. LiquiscintR was from National Diagnostics; (-)-[7-3H]norepinephrine (specific activity 16.9-24.0 Ci/mmol) and 2-deoxy-~-[G-~H]glucose (specific activity 8.0 Ci/mmol) were from New England Nuclear. Crystalline porcine insulin was purchased from Elanco Products Co., Indianap- olis, IN. Bz6-labeled '251-porcine insulin was kindly provided by Dr. Bruce Frank of Eli Lilly Laboratories, Indianapolis, IN.

Methods Preparation of Neuronal Cultures-Neuronal cells in primary cul-

tures were prepared as described previously from brains of l-day-old rats (2). Briefly, brains were removed from the cranium at the level of the medulla oblongata and placed in isotonic salt solution. Brains were carefully cleaned of pia mater and blood vessels, minced, and dissociated with mild trypsin treatment. Dissociated cells were sus- pended in DMEM containing 10% fetal bovine serum and sedimented at 500 x g for 10 min. After washing the pellet once, the cells were suspended in DMEM containing 10% fetal bovine serum. Cells were then plated at a density of 8 X lo6 cells/dish onto Falcon 60-mm plastic tissue culture dishes which had been coated with poly-L-lysine. After 3 days in culture, the medium was replaced with DMEM containing 10% horse serum with 10 y M cytosine arabinoside to inhibit the multiplication of dividing cells, predominantly glial cells. After 48 h, medium was replaced with DMEM containing 10% horse serum. Cells were maintained in culture for 14 days before being used for experiments. These cultures have previously been shown to consist of greater than 80% neurons (2).

Measurement of 1251-Insulin Biding-Specific binding of lZ5I-la- beled insulin to membrane receptors was measured in neuronal cul- tures by the procedure described previously (24). Briefly, cultures were rinsed twice, 4 ml each, with phosphate-buffered saline (PBS), pH 7.4. Triplicate cultures were incubated at 24 "C with 1.0 ml of HEPES buffer, pH 7.4 (110 mM HEPES, 30 mM NaC1, 10 mM glucose), containing 150,000 cpm of 1251-insulin labeled at the B,, position and 1.6% bovine serum albumin to determine total binding. In addition, triplicate cultures were also incubated with 16.6 y~ unlabeled porcine insulin in the reaction mixture to determine non- specific binding. In other experiments, varying concentrations of insulin or other peptides were added. Following incubation, the cul- tures were rapidly washed four times with a total volume of 20 ml of ice-cold PBS. The attached cells were dissolved in 1.0 ml of 0.2 N NaOH and transferred to tubes. The dishes were rinsed with 1 ml of water, which was combined with original samples. Radioactivity was determined in a Beckman 5500 y-counter with a counting efficiency of 73% for lZ5I. Specific binding was calculated by subtracting non- specific binding obtained in the presence of an excess of unlabeled insulin from total binding. The protein content of each sample was determined in duplicate by the method of Lowry et al. (25).

Light Microscopic Autorudiography-Binding of 1251-insulin to neu- ronal cultures was localized by light microscopic autoradiography. Insulin was bound to neuronal cultures as described above except cultures were incubated with 500,000 cpm of '251-insulin/dish. Follow- ing the incubation, cultures were washed twice with ice-cold PBS and were then fixed with 3.7% glutaraldehyde in PBS, pH 7.2, for 30 min at 4 "C. After fixation, cultures were washed twice with PBS and then dehydrated through graded alcohols and air-dried. The dishes were coated with liquid nuclear emulsion (Ilford K5), diluted 1:l with distilled water containing 2% glycerol, according to the method of Rogers (26). Plates were dried overnight over silica gel a t room temperature and then stored at 5 "C in light tight boxes. After 3 weeks of exposure, the emulsions were developed for 2 min in D-19 diluted 1:l with water, rinsed with water, and fixed for 2 min with 20% sodium thiosulfate. The dishes were finally washed for 10 min with distilled water, and the cells were examined by phase-contrast microscopy.

Determination of 2-Deoxy-D-glucose Uptake-The uptake of 2-dGlc was studied using neuronal cells attached to 60-mm culture dishes (21). Growth medium was removed, and cells were washed twice with DMEM without serum and incubated in the same medium for 24 h at 37 "C in humidified 5% coz/95% air. Cultures were washed twice with PBS, pH 7.4, and incubated with indicated concentrations of insulin for 5 min at 37 "C. This was followed by addition of 0.5 mM 2-dGlc and 1 pCi of 2-deoxy-~-[G-~H]glucose. Incubation was contin- ued for an additional 5 min at 37 "C. Cells were washed four times with ice-cold PBS and digested with 0.5 ml of 0.2 N NaOH. Aliquots

of the cell digest were taken for protein determination and for liquid scintillation counting.

fH]Norepinephrine Uptake Experiments-Uptake of [3H]NE into cultured neurons was determined in cells attached to 60-mm culture dishes. Growth medium was removed and cells were washed once with PBS, 37 "C, pH 7.4, containing CaClz (0.75 mM), MgClZ (0.75 mM), and glucose (30 mM). Uptake was determined by incubating cultures with 1.0 ml of PBS containing pargyline (100 pM), ascorbate (200 y ~ ) , and 0.1 y~ (-)-[3H]NE for 2 min. Previous studies (23) have shown that inclusion of 100 ~ L M pargyline in the uptake reaction almost completely prevents deamination of NE to dihydroxyphenyl- glycol after NE is taken up into the cells. Following the incubation, the reaction mixture was removed and dishes were washed twice with ice-cold PBS, pH 7.4. Cells were dissolved in 1.0 ml of 0.1 N NaOH and were transferred to plastic tubes. Each dish was rinsed with 1.0 ml of distilled water which was combined with the original sample. A 1.0-ml aliquot of each sample was combined with 10 ml of LiquiscintR, and radioactivity was determined in an LKB Liquid Scintillation counter (model 1215) with a counting efficiency of 57% for tritium. [3H]NE uptake was expressed as pmol/mg of protein.

To determine the effect of insulin on catecholamine uptake, cul- tures were pretreated with various concentrations of unlabeled por- cine insulin for 10 min. The same concentration of insulin was included in the reaction mixture during the uptake period. The time course of insulin's effect on NE uptake in neuronal cultures was studied by preincubating cultures with a maximally effective concen- tration (167 nM) for varying times from 0 to 60 min.

RESULTS

Kinetic and Autoradiographic Characterization of Neuronal Insulin Binding-Binding of 1251-insulin to neuronal cultures was time-dependent. The time course of 1251-insulin binding to neuronal cultures showed that equilibrium binding was reached after 2 h at 24 "C (Fig. la). The tllz of association was 35 min. Total binding of 1251-insulin to neuronal cultures

120

g 100

H 5 5 80

2 60 X

I 8 40

a

20

40 80 120 160 0 40 80

TIME (MIN)

r

B

I-r

120

FIG. 1. Association and dissociation of '261-insulin binding to neuronal cells cultured from rat brain. a, time course of lZ5I- insulin binding to neuronal cells cultured from rat brain. Cultures of neuronal cells were incubated at 24 "C with 150,000 cpm/ml BZ6- labeled 1251-insulin in triplicate with or without 16.7 yM unlabeled insulin for the indicated time periods. Cultures were washed with ice- cold PBS and dissolved with 0.2 N NaOH. Results are expressed as the percentage of radioactivity bound at equilibrium. Data are given as mean & S.E. of triplicate determinations and are representative of three experiments. Specific binding at 120 min was 5.42% of total radioactivity per mg of protein. b, time course of 1251-insulin dissocia- tion from binding sites on neuronal cultures. Triplicate neuronal cultures were incubated at 24 "C with 150,000 cpm/ml Bze-labeled '=I-insulin for 150 min. They were washed once and incubated with 16.7 FM unlabeled insulin for the indicated time periods. Cultures were washed with ice-cold PBS and dissolved, and radioactivity was counted as described under "Experimental Procedures."

15882 Insulin Receptors and Modulation of Norepinephrine Uptake

at equilibrium was 6.3 f 0.1% (mean S.E./mg of protein), and nonspecific binding was 0.9 f 0.1%. Greater than 90% of bound 1251-insulin could be dissociated within 2 h after addi- tion of a large excess (16.6 pM) of unlabeled porcine insulin (Fig. lb). The tlp of dissociation of 1251-insulin was 15 min.

Binding of '251-insulin to neuronal cultures was saturable and specific for insulin. Cells were incubated with 1251-insulin in the presence of increasing concentrations of unlabeled porcine insulin for 2 h (Fig. 2). The IC50 for inhibition of 1251- insulin binding by porcine insulin was 16.7 nM. These data were analyzed by the method of Scatchard (28) and results in a curvilinear plot (Fig. 3). The apparent affinity constants were calculated using a model for two classes of binding sites. The apparent high affinity site has a dissociation constant (&) of 11 nM, and the low affinity site has a Kd of 65 nM. The binding capacity (Bo) was 1.8 pmol/mg of protein.

Competition for 1251-insulin binding by other peptides struc- turally or physiologically related to insulin showed competi-

1 1 1 0 9 8 7 6

-LOG INSULIN CONCENTRATION (MI

FIG. 2. Competition-inhibition curve of insulin binding to neuronal cultures from rat brain. Neuronal cultures were incu- bated at 24 "C for 120 min with 150,000 cpm/ml Bzs-labeled lZ5I-

were then washed with ice-cold PBS, dissolved in 0.2 N NaOH, and insulin and increasing concentrations of unlabeled insulin. Cultures

counted as described under "Experimental Procedures." Results are expressed as the percentage of total 1251-insulin specifically bound per mg of protein and are the mean of triplicate determinations.

tion for binding proportional to their biological activity at peripheral insulin receptors (Fig. 4). The order of potency in displacing 1251-insulin was: chicken insulin > human insulin = porcine insulin > proinsulin > guinea pig insulin. Growth hormone and glucagon did not compete for insulin binding.

Autoradiographic microscopy was performed to determine whether 1251-insulin was binding to neurons or to the small percentage of glial cells which persist in these cultures. Ex- posure of IIford K5 emulsion for 3 weeks to '251-insulin-labeled neuronal cultures revealed discrete grains specifically associ- ated with neurons in these cultures (Fig. 5). Neurites had a higher density of grains than the neuronal soma (Fig. 5, a and b). Not all neurons exhibited autoradiographic grains (Fig. 5c). No specific localizaton of label occurred when 1251-insulin was bound to cultures in the presence of 16.6 p~ unlabeled porcine insulin.

Effects of Insulin on 2-Deoxy-D-glucose Uptake and Norep- inephrine Uptake into Neuronal Cultures-Incubation of neu- ronal cultures with increasing concentrations of insulin for 5 min resulted in no change in 2-dGlc uptake (Fig. 6). The basal level of 2-dGlc uptake into these cultures was 5 nmol/mg of protein/min.

Incubation of neuronal cultures with 169 nM insulin re- sulted in a time-dependent decrease in the uptake of i3H]NE. The effect was evident by 5 min of preincubation, reached maximum inhibition by 10 min, and remained unchanged over a 1-h time course. Preincubation of neuronal cultures with various concentrations of insulin for 10 min resulted in a dose-dependent reduction in NE uptake (Fig. 7). A 30% inhibition of NE uptake was observed at 1.67 X lo7 M insulin.

DISCUSSION

These experiments show that neuronal cultures from rat brain possess insulin-binding sites which fulfill the major requirements of receptors. The binding of insulin to the neuronal insulin receptor is saturable, rapid, and reversible. Insulin binding is displaced by other peptides in proportion to the degree with which they are physiologically active at peripheral insulin receptors. Insulin receptors have been au- toradiographically localized on both the neuronal soma and

2.5 1

0.5 t

1 400 800 1200

BOUND ffMoc/MG PROTEIN)

FIG. 3. Scatchard plot of 1Z51-insulin binding to neuronal cultures from rat brain. Data obtained from the competition- inhibition curve plotted in Fig. 2 were replotted by the method of Scatchard (27). This curve was analyzed according to a two-site model of receptor binding and resulted in a high affinity Kd of 11 nM and low affinity Kd of 65 nM with a Bo of 1.8 pmol/mg of protein.

0.1 10 1000

HORMONE (nM) FIG. 4. Specificity of '251-insulin binding to neuronal cul-

tures from rat brain. Neuronal cultures were incubated in triplicate with 150,000 cpm/ml Bz6-labeled 1251-insulin and various concentra- tions of porcine insulin (O), human insulin (O), porcine proinsulin (A), guinea pig insulin (A), growth hormone (O), and glucagon (0). Cultures were washed with ice-cold PBS, dissolved in 0.2 N NaOH, and counted as described in the text. Results are expressed as the percentage of maximal specifically bound radioactivity and are the mean of triplicate determinations.

Insulin Receptors and Modulation of Norepinephrine Uptake 15883

FIG. 5. Light microscopic autoradiography of Iz6I-insulin binding to neuronal cultures from rat brain. Developed autora- diograms were observed under light microscopy and photographed. a, autoradiographic grains over neurites; b, neuronal soma as well as neurites labeled with autoradiographic grains; c, neurons with no specific autoradiographic labeling. Bars represent 10 p ~ .

neurites which are kinetically similar to specific insulin re- ceptors characterized in other tissues.

In contrast with our previous findings (20) which demon- strated a 2-fold stimulation of 2-dGlc uptake into cultured

I I 20 40 00 80 100 107

INSULINW"

FIG. 6. Dose response of the effect of insulin on 2-deoxy-D- glucose uptake by neuronal cultures from rat brain. Serum- deprived neuronal cultures were incubated for 5 min at 37 "C with the indicated concentrations of porcine insulin. After this incubation, 0.5 mM 2-deoxy-~-[G-~H]glucose was added and the incubation was continued for 5 min. Cultures were dissolved in 0.2 N NaOH and counted as described in the text. Data are given as mean * S.E. of triplicate determinations. No statistically significant difference was observed between untreated cultures and cultures treated with various concentrations of insulin as well as among the treated groups as determined by ANOVA followed by Neuman-Keuls test.

1 I 1 1 I

I I I I 10 0 8

h u h (-LIXJ M 1

FIG. 7. Dose response of the effect of insulin on norepineph- rine uptake into neuronal cultures from rat brain. Neuronal cultures were preincubated for 10 min at 37 "C with the indicated concentrations of insulin. Cultures were then washed once with warm PBS and incubated for 2 min at 37 "C with 0.1 p~ [3H]norepinephrine in the presence of 100 p~ pargyline, 200 p~ ascorbate, and the indicated concentrations of insulin. After this uptake period, cultures were washed twice with ice-cold PBS, dissolved in 0.1 N NaOH, and counted for radioactivity. Inhibition of total t3H]norepinephrine up- take was statistically significant by 1.67 X 10"' M (ANOVA p < 0.05, significantly different from untreated cells at a level of 1% by Neu- man-Keuls test).

astrocyte glial cells by physiological concentrations of insulin, we found no effect of insulin on 2-dGlc uptake into neurons. However, we have shown the basal level of 2-dGlc uptake into neurons to be 25 times higher than basal uptake into astrocyte glia in culture (5.0 uersus 0.2 nmol/min/mg of protein (21)). This difference may have masked changes in astrocyte 2-dGlc uptake regulated by insulin in previous in uiuo experiments. It has been demonstrated that peripheral insulin stimulates glucose flux from the blood to the brain (28). This may be due to action on vascular epithelial cells or on astrocyte glia. Astrocyte glial cells are thought to make up the blood brain

15884 Insulin Receptors and Modulation of Norepinephrine Uptake

barrier and are anatomically suited for a role in controlling local availability of metabolic substrates in the brain. Our findings regarding sensitivity of astrocyte glial cells but not neurons to stimulation of 2-dGlc uptake by insulin support this role for astrocytes. Local availability of glucose in the brain may be regulated by actions of peripheral insulin on certain components of the blood brain barrier and brain insulin on others.

Norepinephrine uptake has been well characterized in this culture system (23). NE uptake is sodium-, temperature-, time-, and concentration-dependent. Uptake is inhibited by specific inhibitors of NE uptake such as maprotiline and desmethylimipramine. Autoradiographic characterization of this uptake system has shown that NE uptake is specific to a subpopulation of neurons in culture. This implies that cul- tured neurons maintain specific norepinephrine uptake mech- anisms. The neuronal culture NE uptake system is identical to the in vivo uptake mechanism in these respects and is a good model system for investigating modulation of norepi- nephrine uptake into neurons.

We have used this system to study the effect of insulin on norepinephrine uptake. Insulin has effects in neuronal cul- tures at concentrations which suggest that the neuronal in- sulin receptor mediates physiological actions of brain insulin. Insulin inhibits specific norepinephirne uptake into neurons at a concentration as low as 1.67 X 10-l' M. This concentration of insulin binds less than 5% of total neuronal insulin recep- tors, which implies that neurons are very sensitive to insulin inhibition of NE uptake.

There are several indications that the neuronal insulin receptor is physiologically different from both peripheral in- sulin receptors and the astrocyte glial receptor. The neuronal insulin receptor is regulated differently than the peripheral and glial insulin receptor. Down-regulation of the peripheral insulin receptor by insulin is a universally recognized phe- nomenon. Insulin is capable of down-regulating the astrocyte glial insulin receptor.' However, the neuronal insulin receptor is not down-regulated by exposure to insulin (24). The neu- ronal insulin receptor is functionally different from the astro- cyte glial insulin receptor in that it does not mediate the classical metabolic effect of stimulation of 2-dGlc uptake. However, the neuronal insulin receptor does mediate changes in monoamine uptake which could be the basis for neuromod- ulatory effects of insulin. These differences between neuronal and glial insulin receptors suggest specialization for specific roles in the brain.

There is now sufficient evidence to support the conclusion that there is a brain insulin system which acts via specific neuronal and glial insulin receptors to modulate brain metab- olism and neuronal function. Insulin has been found in the brain in concentrations of 2-100 ng/g of tissue (1,6,7). These findings suggested that insulin is either made in the brain or concentrated from peripheral sources. However, we have dem- onstrated that insulin is found in neuronal cells in culture and is reduced by inhibition of protein synthesis (2,4), which indicated that the brain can synthesize insulin. Most conclu- sively, recent studies in our laboratories have found an mRNA species in neuronal cultures which hybridizes to cDNA probes for insulin, demonstrating that insulin is synthesized in neu- ronal cells? We have also observed that neuronal insulin is released under depolarizing conditions!

F. T. Boyd, Jr., D. W. Clarke, and M. K. Raizada, unpublished

D. L. Cooper, D. W. Clarke, M. K. Raizada, J. L. Stein, and G. S.

D. W. Clarke, F. T. Boyd, Jr., and M. K. Raizada, unpublished

observation.

Stein, manuscript submitted.

observations.

Insulin-binding sites have been demonstrated to be ubiq- uitous within the central nervous system (3, 5). Specific insulin receptors have been demonstrated on both astrocyte glial cells and neurons in culture. Astrocyte glial insulin receptors mediate classical physiological effects of insulin such as stimulation of glucose uptake and stimulation of macromolecule synthesis (21, 29). Neuronal insulin receptors mediate modulation of monoamine uptake mechanisms. In- sulin may also affect other neuromodulatory mechanisms which may account for previous reports of insulin modulating firing rates of neurons (17-20). These findings from cultured cells from rat brain suggest that specializations exist in the rat brain by which insulin can mediate changes in brain metabolism and neural function.

Acknowledgments-We thank Muriel Turlington and Lynn Holmes for their expert technical assistance.

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