BRAIN RESEARCH

ELSEVIER Brain Research 684 (1995) 47-55

Research report

Four-day hyperinsulinemia in euglycemic conditions alters local cerebral glucose utilization in specific brain nuclei of freely moving rats

Patrick Doyle *, Isabelle Cusin, Fran~oise Rohner-Jeanrenaud, Bernard Jeanrenaud FaculJ~ of Medicine, University of Geneva, 64, avenue de la Roseraie, 1211 Geneva 4, Switzerland

Accepted 14 March 1995

Abstract

Although insulin is a well known regulator of peripheral tissue glucose metabolism, there is little agreement over its effects on brain glucose metabolism. Several investigators report that peripheral insulin may enter the brain via several routes. The presence of insulin receptors specific to brain, coupled to diverse reports of the effect of acute insulin administration on brain glucose use, led us to carry out a 4-day hyperinsulinemic euglycemic clamp in freely moving rats with subsequent labelled 2-deoxyglucose metabolic mapping studies. It was found that after 4 days of peripheral insulin infusion, several brain regions (Anterior Hypothalamic area, Suprachiasmatic nucleus, Basolateral Amygdaloid nucleus, Supramammillary bodies, Medial Geniculate nucleus and Locus Coeruleus) had an altered local cerebral glucose utilization. Upon subsequent analysis of their anatomical and functional connections it is proposed that insulin may regulate an integrated circuit of pathways within the central nervous system.

Keywords: Cerebral glucose; Peripheral hyperinsulinemia; Central nervous system; Sympathetic regulation

1. Introduction

Insulin is a well established regulator of glucose utiliza- tion and metabolism in peripheral tissues such as muscle and adipose tissue [26]. In contrast, the brain is generally believed to be unresponsive to the stimulatory actions of insulin on glucose metabolism although glucose is its main source of energy. However, there is considerable evidence suggesting insulin as a peripheral afferent energy signal to the central nervous system (for review see [44]). In particu- lar, dose-dependent entry of circulating insulin into the CNS has been reported for several species [43,48] and different possible routes of entry of peripheral insulin to the brain have been proposed [38]. These involve uptake by the circumventricular organs (CVO) [55,58], transport to the cerebrospinal fluid (CSF) via the CVO's [56] or insulin receptor mediated intraceUular transport through the blood-brain barrier (BBB) [9,13,14]. It is unlikely that insulin is produced within the brain as earlier proposed [17,18,58]. Yet, there are distinct brain insulin receptors

* Corresponding author. Laborar~oires de Recherches M&aboliques, 64 avenue de la Roseraie, CH-1211 Geneva 4, Switzerland. Fax: (41) (22) 347-5979.

0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)00402-5

with a widespread distribution throughout the CNS. High- est densities have been reported to be in the olfactory bulb, hypothalamus, cerebral cortex, and hippocampus [2,18,21,52,571.

However, a link between peripheral insulin and brain glucose utilization has yet to be clearly defined, as differ- ent studies have led to divergent conclusions. In humans, Hertz et al. [20] showed that an i.v. infusion of insulin caused an increased glucose uptake whith a concomitant increased back flux of glucose from brain to blood, some- what masking any potential effect of insulin on brain glucose utilization.

H o m e t al. [22] viewed the brain as a negative control in an euglycemic-hyperinsulinemic clamp study in which there was no effect of insulin on rat brain glucose utiliza- tion over 60 min. The central nervous uptake of 3-0-meth- ylglucose during peripheral hyperinsulinemia in the rat has been shown to decrease (including a decrease in rate constants for the glucose transporter) but a concomitant increase in the distribution space for the hexose was also measured, resulting in a negligible effect of insulin on the brain glucose utilization index over 90 rain [35].

For investigating insulin effects on brain glucose metabolism studies should be carried out at euglycemia under steady-state conditions. Three recent studies fulfill-

48 P. Doyle et al. /Brain Research 684 (1995) 47-55

Table 1 Glycemia and insulinemia of saline-infused and euglycemic insulin-infused FA/? rats

Saline-infused rats (n = 6) Start of the experiment ( - 5 min.) End of experiment (45 rain.)

glucose insulin glucose insulin

Insulin-infused rats (n = 6)

(mM) (ng/ml) (rag/100 ml) (ng/ml) 5.67 + 0.1 1.6 + 0.1 5.7 + 0.1 1.5 + 0.2 5.9 + 0.5 4.4 + 0.2 5.3 ___ 0.5 4.6 -t- 0.2

There was no significant difference between saline-infused controls and insulin-infused rats.

ing this criteria have been carried out in rats, all yielding different results: Grundstein et al. [16] reported that an euglycemic-hyperinsulinemic clamp for 2 h, at the rela- tively low glycemia of 4 mM, resulted in significant decreases in 2-deoxyglucose utilization in cortex, hypo- thalamus and locus coeruleus. A similar study by Marfaing et al. [32] carried out at 4.7 mM glycemia also showed a tendency for hyperinsulinemia to lower local cerebral glu- cose utilization (LCGU) but these changes did not reach statistical significance. In contrast, during an euglycemic- hyperinsulinemic clamp at 7 mM over 60 min. Lucignani et al [31] found discrete, selective increases in LCGU in hypothalamic nuclei with the exception of the lateral area that had a decreased cerebral glucose use of 11%. To sum up, there is a general disparity of the results obtained on the effects of insulin on brain glucose utilization; these results are obtained almost entirely over an acute time course of up to 3 h and more importantly (except in one case) in anesthetized rats.

Our aim was therefore to measure the local cerebral glucose utilization of awake, freely moving rats after a longer-term (4-day) insulin infusion, at a maintained eug- lycemia, to ensure an entry of peripheral insulin to the CNS and subsequently locate insulin metabolically regulat- able brain sites.

2. Material and methods

Lean female heterozygote Zucker (FA/? ) rats, weigh- ing approximately 200 g were used throughout the study. The animals were housed in individual cages. Control and insulin-treated (2 units/day) rats maintained in eulgycemia by glucose infusion (see below) had free access to water and a standard laboratory chow (Lacta 299; Provimi-Lacta S.A., Cossonay, Switzerland). Daily measurement of food intake (controls: 21.6 + 1.1 g; insulin-treated: 13.01 _+ 0.3 g, P < 0.002) and caloric intake (controls: 86.3 _+ 4.4 kcal; insulin-treated: 91.3 + 3.2 kcal, NS) were performed.

Rats were anesthetized with Zoletil 20, (Reading Labo- ratories) and implanted with osmotic minipump(s) (Alzet 2001; Alza Corp., Palo Alto, CA, USA) delivering either saline or 2 units of insulin per day (U-400 human insulin; Hoe 21 PH; Hoechst AG, Frankfurt am Main, Germany) at a rate of 1 /~l/h for 4 days. Two chronic catheters were then placed, one in the femoral artery and one into the

femoral vein. These catheters were routed subcutaneously and externalized between the shoulders. To maintain nor- moglycemia in rats treated with insulin, rats were infused with a glucose solution (250 m g / m l ) containing 0.8% penicillin and 0.4% heparin via the chronic femoral artery catheter. Control rats were infused with isotonic saline containing 0.8% penicillin and 0.4% heparin. These infu- sions were performed through the artery for maintaining the permeability of the catheter during the 4-day treatment. This was done by way of a home-made device consisting of a swivel and an oscillating arm allowing the animals to move freely. Blood samples were taken from the tail vein at 09.00 a.m. and 17.00 p.m. to measure plasma insulin and glucose levels [6]. The rate of glucose infusion was monitored daily to maintain euglycemia (controls: 7.1 + 0.2 mM; insulin-infused: 6.8 + 0.2 mM, NS) and saline infu- sion was adjusted accordingly.

On the day of the experiment, glucose or saline infusion was switched to the femoral vein catheter, leaving the arterial catheter free for blood sampling. Rats were without food for 3 h. The following method has been described previously in detail [8]. At the start of the Sokoloff experi- ment [47] and following an i.v. bolus injection of 14C-2-de- oxy-D-glucose (125 /~Ci/kg b.wt.), blood samples were taken every minute for 3 min, then at 6, 10, 20, 30 and 45 min to ensure that a precise plasma labelled 2-deoxyglu- cose decay curve was obtained. Experiments in which plasma glucose concentration varied systematically more than 10% during the procedure were excluded because of violation of the theoretical requirement of a constant plasma glucose concentration [47]. Glucose levels were subse- quently measured in plasma samples by means of a glu- cose oxidase kit (Boehringer, Mannheim, Germany) and 14C 2-D-deoxyglucose concentration was determined in plasma using a liquid scintillation counter (Rackbeta Counter 1217 LKB Wallaw, Paris). Plasma insulin was measured by radioimmunoassay using rat insulin as stan- dard [19]. At 45 min the rats were decapitated, the brain was removed rapidly and placed on a cold plate which had been previously frozen to - 6 0 ° C, freezing the brains within one minute. All brains were then stored at - 80 ° C until sectioned. Brain sections, precisely 20 /xm in thick- ness, were prepared in a Reichert-Jung (Cambridge Instru- ments Inc., Buffalo NY, USA) cryostat maintained at a - 18 ° C object holder-temperature. The brain sections were picked up on microscope slides and placed immediately on

P. Doyle et al. /Brain Research 684 (1995) 47-55 49

a hot plate (Medax: Nagel Gmbh Kiel, Germany) at 60 ° C for at least 10 min and then placed in an X-ray cassette. A set of 14C methylmethacrylate standards which included a blank and a set of progressively increasing 14C concentra- tions were also placed in the cassette. The method of calibration has been described elsewhere [47].

Autoradiographs were prepared from these sections di- rectly in an X-ray cassette with Kodak blue sensitive Medical X-Ray Film, Type SB-5 (Kodak CAT 175 3045 semicoated film). The exposure time was 1 week. The autoradiographs provided a pictorial representation of the relative 14C concentrations in various cerebral structures. When analyzing the autoradiographs and to ensure optimal localization of specific nuclei, the same sections were stained with Cresyl violet and superimposed on the image of the section of interest. For this purpose, a flicker-align- ment process coupled to channel-linking contained within the software of the MCID M1 Image analysis program (Imaging Research Inc., Ont., Canada) was used. Thus, each section provided its own histological landmark when cross-referred to the stereotaxic atlas of Paxinos and Wat- son which was also refe:rred to for nomenclature [39]. Analysis of autoradiograms to give a converted optical density measurement of /a.mol/100 g min -1 glucose uti- lization was carried out using the rate constants of Sokoloff [47] incorporated in the MCID MI Image analyser system. This was based on a previous report [31] that LCGU obtained when calculated using the corrected rate constants of Namba et al [35] for hyperinsulinemia were no different from the results obtained using the rate constants of Sokoloff et al. [47]. Total rat brain uptake values were calculated as a weighted mean i.e. sum of all the pixel density values divided by the number of pixels, to give an impression of overall utilization of glucose.

The effect of 4-day hyperinsulinemia compared to saline infused controls was tested statistically initially by ANOVA and then post-hoe by Student's t-test for unpaired data taking into account the Bonferroni adjustment for multiple analyses. Areas were considered different with a P < 0.05. all values are provided so the reader may be directed to areas of interest by the individual post-hoc t-values.

3. Results

During the experimental period of 45 min. the plasma glucose concentration remained stable within + 10% in the insulin-treated rats and did not differ significantly from saline-infused controls. Plasma insulin levels were signifi- cantly higher (3 fold) in the insulin treated group com- pared to respective control rats ( P < 0.01) (Table 1).

3.1. Local cerebral glucose utilization (LCGU)

The effects of a 4 day hyperinsulinemic euglycemic clamp on LCGU was examined in 59 brain structures (Table 2). Insulin treatment did not change the total LCGU after 4 days when expressed as a weighted mean (saline controls 68.2 + 4.4 vs. insulin-treated rats 72.0 + 4.0 /zmol/100 g /min , NS, n = 6 rats per group) (see Meth- ods). Insulin treatment was shown not to change the overall utilization of glucose compared to saline controls by ANOVA.

Of the 59 cerebral structures examined, rats that under- went 4-day hyperinsulinemia in the presence of steady-state euglycemic conditions showed by post-hoc analysis, statis- tically significant changes in six brain regions. They were, namely, Anterior Hypothalamic area ( + 34%), Suprachias- matic nucleus ( - 2 7 % ) , Basolateral Amygdaloid nucleus ( - 30%), Supramammillary bodies ( + 19%), Medial Geniculate nucleus ( + 32%), and Locus Coeruleus ( - 15%) These changes were observed reproducibly in 5, 5, 6, 5, 4 and 6 rats out of 6 respectively. (See Figs. 1 and 2, Table 2).

4. Discussion

The results of this study show that, after 4 days, hyper- insulinemia under normoglycemic steady-state conditions has no effect on total local cerebral glucose utilization (LCGU), but on the LCGU of specific brain nuclei. Namely, Anterior hypothalamus, Supramammillary bodies, Basolateral Amygdala, Suprachiasmatic nucleus, Medial

Fig. 1. (see following pages). A: representative autoradiograms (below) of a saline infused control, left (this example Medial Geniculate LCGU, 93.0 #mols/100 g. min-i) and 4-day hyperinsulinemic-euglycemic clamp, right (this example Medial Geniculate LCGU, 118.0 ~mol/100 g. min-1) rat with Cresyl-violet histologically stained slices (above) of the same slice. The arrow refers to the Medial Geniculate nucleus which had an increased LCGU after euglycemic hyperinsulinemia of 4 "days duration (Table 2). B: format as above, arrow refers to Supramammillary bodies (SUM) which also had an increased LCGU after 4 days euglycemic-hyperinsulinemia right, (this example, SuM LCGU 98.0/zmol/100 g. min-1) when compared to saline infused controls, left (this example, SuM LCGU 82.0 p, mol/100 g. min-1). As can be seen in this example the thalamic nuclei tended to have an increased LCGU but were beyond the limits of statistical significance, for mean values see Table 2. It should be noted that these representations of the original autoradingraphs have been 'normalized' to allow direct visual comparison within the same calibration scale. This process (part of the MCID MI software: see Methods) changes the image data rendering pseudoimages. In this case the autoradiographs are not over-exposed but have been manipulated to allow comparison within the same scale.

50 P. Doyle et al. / Brain Research 684 (1995) 47-55

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52 P. Doyle et al. / Brain Research 684 (1995) 47-55

Table 2 Local cerebral glucose utilization of discrete brain areas of F A / ? rats treated with a 4-day hyperinsulinemic-euglycemic clamp and respective saline treated controls. Values are mean of at least four bilateral measurements taken from at least 6 rats of each group 5: standard error of the mean

Brain region Saline Four-day Post-hoc Student's P of control insulin t-value significance LCGU LCGU

/,~mol/lO0 g / ra in

Motor cortex 108.9 5:8.0 Somatosensory cortex 112.4 5:7.0 Accumbens nucleus 74.8 + 5.5 Cingulate cortex 107.0 + 7.4 Corpus callosum (genu) 26.7 5:2.3 Lateral septal nucleus

dorsal part 41.6 5:4.6 intermediate part 46.8 + 3.5 ventral part 46.8 5:2.3

Caudate putamen 86.8 5:4.6 Medial septal nucleus 63.6 + 3.4 Nuclear vertical limb of diagonal band

dorsal part 61.4 + 4.9 ventral part 60.1 5:3.9

Optic nerve (2n) 29.4 + 3.1 Olfactory bulb 47.8 + 2.7 Corpus callosum 28.7 5:2.4 Ventral pallidum 58.0 + 4.6 Nucleus horizontal limb diagonal band 62.5 + 2.8 Optic chiasm 27.2 + 4.1 Bed nucleus of stria terminalis 51.9 5:2.7 Globus pallidus 47.6 + 4.1 Lateral hypothalamic area 56.9 + 1.7 Paraventricular thalamic nucleus, anterior part 60.6 5:6.9 Anterior hypothalamic area 33.5 + 3.6 Suprachiasmatic nucleus 47.5 ± 1.2 Paraventricular hypothalamic nucleus

magnocellular part 40.3 + 2.5 parvocellular part 39.1 + 1.5

Field CA3 of Ammon ' s Horn 60.8 + 1.9 Dentate gyrns 41.9 + 4.0 Arcuate nucleus 48.2 5:1.8 Medial amygdaloid nucleus 64.2 + 7.2 Basolateral amygdaloid nucleus 96.2 5:4.7 Ventroposterior thalamic nucleus lateral part 73.3 + 5.2 Ventrolateral thalamic nucleus 82.7 5:5.0 Gelatinosus nucleus of the thalamus 97.5 + 4.9 Ventromedial hypothalamic nucleus 43.7 5:2.4 Median eminence 45.2 + 5.1 Mediodorsal thalamic nucleus 88.6 5:4.4 Dorsomedial hypothalamic nucleus 46.1 + 3.6 Dorsal hypothalamic area 41.1 5:4.6 Laterodorsal thalamic nucleus 87.5 + 6.8 Lateral habenular nucleus 90.4 + 6.3 Dorsomedial hypothalamic nucleus, compact part 50.7 + 6.2 Ventromedial hypothalamic nucleus

dorsomedial part 47.5 + 4.6 central part 49.6 5:4.5 ventrolateral part 49.5 5:4.3

Field CA1 of Ammon 's born 63.7 5:4.2 Field CA2 of Ammon 's horn 74.4 5:4.6 Field CA4 of Ammon ' s horn 65.3 5:3.1 Hippocampal fissure 91.5 5:1.5 Lateral amygdaloid nucleus 80.8 5:5.1 Fornix 55.9 5:3.9 Substantia nigra, reticular part 72.9 5:5.3

117.1 + 8.4 0.71 0.50 119.2 + 10.6 0.54 0.60 84.9 + 5.1 1.62 0.14 121.6 + 18.5 1.02 0.33 27.4 + 4.5 0.15 0.88

43.6 + 3.9 0.32 0.76 52.1 + 5.2 0.84 0.42 51.8 + 5.5 0.82 0.43 101.1 + 9.8 1.32 0.22 62.5 + 4.3 0.19 0.86

71.5 + 5.1 1.52 0.16 70.7 + 5.9 1.49 0.17 22.3 + 2.9 1.46 0.18 42.4 + 4.3 0.22 0.92 24.7 + 6.3 1.57 0.15 63.7 + 9.3 0.46 0.66 81.2 + 7.4 1.77 0.12 30.2 + 7.1 0.34 0.74 63.8 + 12.2 0.78 0.46 65.8 + 17.2 0.84 0.43 52.2 + 5.2 0.72 0.50 51.0 + 5.1 1.10 0.30 45.0 + 2.4 1.88 0.03 * I" 37.4 + 3.2 2.93 0.02 * $

47.3 + 5.4 1.18 0.27 46.4 + 8.3 0.87 0.41 76.6 + 8.1 1.73 0.12 58.3 + 6.2 1.48 0.18 51.4 + 2.6 1.03 0.31 72.4 + 5.2 0.95 0.37 67.0 + 3.8 2.42 0.03 * ~, 81.3 + 6.6 0.95 0.37 88.9 + 5.8 0.82 0.43 104.6 + 7.1 0.73 0.48 45.7 + 2.5 0.59 0.57 38.3 + 1.5 1.30 0.22 99.4 -I- 7.3 1.10 0.30 49.6 ± 5.0 0.16 0.88 46.9 + 3.9 0.98 0.22 96.9 + 7.8 0.85 0.41 106.4 + 11.0 1.27 0.23 49.2 + 4.5 0.21 0.84

50.5 5:6.7 0.33 0.75 46.8 5:5.6 0.35 0.73 48.1 ± 3.3 0.26 0.80 70.9 + 6.3 0.96 0.36 80.1 + 6.9 0.68 0.51 66.4 ± 5.4 0.19 0.86 93.8 5:7.4 0.25 0.81 90.8 ± 6.0 1.19 0.27 57.4 5:6.7 0.17 0.87 68.6 5:5.9 0.51 0.63

P. Doyle et al. /Brain Research 684 (1995) 47-55 53

Geniculate nucleus and Locus Coeruleus. As we have described elsewhere [7] to establish an understanding of why such selective effects of insulin only occur in 6 areas of the 59 studied, it is useful to establish how these areas may be connected and determine the possible neurophysio- logical role of a resulting 'circuit' and determine the outcome of such selectiw, • changes reflecting functional alterations. For instance, it was found that after 4 days of insulin-treatment rats had a decreased LCGU at the level of the Locus Coeruleus. The Locus Coeruleus (LC) con- tains half of the total number of noradrenaline synthesizing neurons and therefore is by far the most important nora- drenergic center in the brain [36]. The decreased LCGU seen in LC may therefore be of functional relevance. LC efferent connections make up two main ascending fibre systems, a large dorsal noradrenerglc bundle and a rela- tively small rostral limb of the periventricular pathway [24]. The former pathway traverses the midbrain, eventu- ally reaching the hypothalamus. The mesencephalic areas of these efferent terminations include reciprocal formations with the Septal nuclei, Hippocampal and Amygdaloid re- gions [24,51].

The hypothalamus receives noradrenergic input from the Locus Coeruleus [23,30,42] mostly arising from the latter of the two noradrenergic systems (Nb: Anterior hypothalamic area, increased LCGU after 4-day hyperinsu- linemia). Also, it was fourtd that common regions associ- ated with the terminations of LC efferents displayed vari- ous changes in LCGU after insulin treatment. The main telencephalic areas associated with noradrenergic bundle terminations are: (a) the central and basal nuclei of the Amygdala [5,10,25] (Basal Amygdaloid nucleus LCGU was found to be decreased after insulin administration); (b) the anterior regions of the Olfactory nucleus [11,28,50,53], (i.e. closely associated areas such as the Suprachiasmatic nucleus had a decreased LCGU after insulin treatment Table 2); (c) the nucleus of the diagonal band [15,29,34] (Table 2); (d) the hippocampal formation (Dentate Gyms, areas of Ammon's Horn) [15], (Table 2); (e) other neocor- tical areas beyond the scope of this paper [11,15,28,29,34,50,53].

When one considers noradrenerglc fibres innervating the above areas in the context of the results obtained, it

seems plausible to propose a role of exogenous insulin in regulating a functional circuit, originating a t /or centering upon the Locus Coeruleus. Even more so, insulin has been previously implicated in sympathetic nervous regulation in man [1,27,40] and rats, mostly in the periphery [33,37,41,46]. It has also been shown that chronic insulin administration given intracerebro-ventricularly reduces the mRNA of the transporter of noradrenaline at the level of the Locus Coeruleus [12] although we stress that compar- isons betwee peripheral and central sympathetic regulation should be made with caution.

The present study was carried out in conscious freely moving rats receiving exogenous insulin for 4 days, in light of evidence that insulin is found in rat CSF in proportion to plasma levels [45,48,49]. Delivery of circu- lating insulin to the brain has been hypothesized to occur relatively slowly through the blood-CSF barrier (Choroid plexus), with subsequent uptake from CSF into brain tissue (i.e. more than 90 min) [4,43]. Another relatively slow entry point of insulin to the brain is via the blood-brain barrier (BBB), [3,9,38]. These saturable transport systems for insulin do not exclude that non-saturable processes participate in the delivery of peripheral insulin to certain brain areas [54]. Therefore, it should be stressed that after 4 days of peripheral hyperinsulinemia significant amounts of insulin should have reached the brain of awake rats. This may not have been the case in short-term hyperinsu- linemic-euglycemic clamp studies on previously anes- thetized rats [16,22,31,32]. It is interesting to note that the brain areas reported here with altered glucose uptake do not coincide with areas of high insulin receptor density [2,18,21,52,57]. This suggests a neuroregulatory role of insulin besides that of its normal ligand-receptor interac- tion with respect to glucose metabolism. Although, the insulin-responsive glucose transporter Glut 4 is not the abundant transporter isoform for glucose in brain [26]. Throughout the experimental period both glucose and in- sulin plasma levels were 'clamped' in steady-state condi- tions, hence the possibility of afferent glucose/insulin sensor signals to the CNS via the vagus would be minimal. This is perhaps further substantiated by the lack of effect of 4 days insulin-treatment on VMH and PVN glucose uptake. Whereas, although the anterior hypothalamic area

Table 2 (continued)

Brain region Saline Four-day Post-hoc Student's P of control insulin t-value significance LCGU LCGU

/~mol/lO0 g /min

Mammillary bodies 102.9 + 5.4 102.3 + 3.6 Supramammillary bodies 84.1 + 2.5 100.1 + 5.9 Entorhinal cortex 62.2 + 3.1 56.4 + 5.4 Medial geniculate nucleus 91.4 + 5.5 120.8 + 8.5 Subicuhm 67.8 + 4.4 71.8 + 6.3 Anterior pituitary 54.1 + 3.3 48.4 + 4.6 Locus coeruleus 102.7 5:4.7 87.7 5:4.9

0.09 0.93 2.60 0.03 * 1' 0.24 0.78 2.91 0.02 * 1' 0.48 0.66 0.98 0.35 2.06 0.03 * ~,

54 P. Doyle et al. / Brain Research 684 (1995) 47-55

has not been reported to be glucose- and/or insulin-sensi- tive, the increase in LCGU, which has been reported elsewhere [31] may be a result of secondary changes from other hypothalamic regions with reciprocal connections, such as the VMH [31]. Based on the data presented we therefore propose that insulin acts as a humoral signal modulating central nervous system (CNS) activity within an integrated group of regions.

Acknowledgements

This work was supported by Grant 32.26405.89 of the Swiss National Science Foundation (Berne, Switzerland) and by a grant-in-aid of Eli Lilly and Company, Indianapo- lis, Indiana, USA. The excellent technical assistance of Ms P. Arboit is gratefully acknowledged. We are also indebted to Ms F. Touabi for secretarial preparation of the manuscript and Mr. P. Germann for photographic and figure preparation. The authors are members of the Geneva-Diabetes-Group.

References

[1] Anderson, E.A., Hoffman, R.P., Balon, T.W., Sinkey, C.A. and Mark, A.L., Hyperinsulinemia produces both sympathetic neural activation and vasodilation in normal humans, J. Clin. Invest., 87 (1991) 2246-2252.

[2] Baskin, D.G., Brewitt, B., Davidson, D.A., Carp, E.S., Paquette, T., Figlewicz, D.P., Levellen, T.K., Graham, M.K., Woods, S.C. and Dorsa, D.M., Quantitative autoradiographic evidence for insulin receptors in the choroid plexus of the rat brain, Diabetes, 35 (1986) 246-249.

[3] Baskin, D.G., Wilcox, B.J., Figlewicz, D.P. and Dorsa, D.M., Insulin and insulin-like growth factors in the CNS, Trends Neurosci, 11 (1988) 107-111.

[4] Baskin, D.G., Woods, S.C., West, D.B., Van Houten, M., Posner, B.I., Dorsa, D.M. and Porte Jr., D., Immunocytochemical detection of insulin in rat hypothalamus and its possible uptake from cere- brospinal fluid, Endocrinology, 113 (1983) 1818-1825.

[5] Blackstad, T.W., Fuxe, K. and H6kfelt, T., Noradrenaline nerve terminals in the hippocampal region of the rat and the guinea pig, Z. Zellforsch., 78 (1967) 463-473.

[6] Cusin, I., Terrettaz, J., Rohner-Jeartrenaud, F. and Jeanrenaud, B., Metabolic consequences of hyperinsulinaemia imposed on normal rats on glucose handling by white adipose tissue, muscles and liver, Biochem. J., 267 (1990) 99-103.

[7] Doyle, P., Guillaume-Gentil, C., Rohner-Jeanrenand, F. and Jeanre- naud, B., Effects of corticosterone administration on local cerebral glucose utilization of rats, Brain Res., in press.

[8] Doyle, P., Rohner-Jeanrenaud, F. and Jeanrenand, B., Local cerebral glucose utilization in brains of lean and genetically obese (fa/fa) rats, Am. J. PhysioL, 264 (1993) E29-E36.

[9] Duffy, K.R. and Pardridge, W.M., Blood-brain barrier transcytosis of insulin in developing rabbits, Brain Res., 420 (1987) 32-38.

[10] Fallon, J.H., Koziell, D.A. and Moore, R.Y., Catecholamine innerva- tion of the basal forebrain. II. Amygdala, suprarhinal cortex and entorhinal cortex, J. Comp. Neurol., 180 (1978) 509-532.

[11] Fallon, J.H. and Moore, R.Y., Catecholamine innervation of the basal forebrain. III. Olfactory bulb, anterior olfactory nuclei, olfac- tory tubercle and periform cortex, J. Comp. Neurol., 180 (1978) 533-544.

[12] Figlewicz, D.P., Szot, P., Israel, P.A. and Payne, C., Insulin reduces norepinephfine transporter mRNA in vivo in rat locus coeruleus, Brain Res., 602 (1993) 161-164.

[13] Frank, H.J. and Pardridge, W.M., Insulin binding to brain microves- sels, Adv. Metab. Disord., 10 (1983) 291-302.

[14] Frank, H.J. and Pardfidge, W.M., Binding and internalization of insulin and insulin-like growth factors by isolated brain microves- sels, Diabetes, 35 (1986) 654-657.

[15] Gatter, K.C. and Powell, T.P.S., The projection of the locus coeruleus upon the neocortex in the macaque monkey, Neuroscience, 2 (1977) 441-445.

[16] Grunstein, H.S., James, D.E., Storlien, L.H., Smythe, G.A. and Kraegen, E.W., Hyperinsulinemia suppresses glucose utilization in specific brain regions: in vivo studies using the euglycaemic clamp in the rat, Endocrinology, 116 (1985) 604-610.

[17] Havrankova, J., Browstein, M.J. and Roth, J., Concentrations of insulin and of insulin receptors in the brain are independent of peripheral insulin levels. Studies of obese and streptozotocin treated rodents, J. Clin. Invest., 64 (1979) 636-642.

[18] Havrankova, J., Schmechel, D., Roth, J. and Brownstein, M.J., Identification of insulin in the rat brain, Proc. Natl. Acad. Sci. USA, 75 (1978) 5737-5741.

[19] Herbert, V., Lau, ICS., Gottlieb, C.W. and Bleicher, S.J., Coated charcoal immunoassay of insulin, J. Clin. Endocrinol., 25 (1965) 1375-1384.

[20] Hertz, M.H., Paulson, O.B., Barry, D.I., Sandahl, J. and Svendsen, P.A., Insulin increases glucose transfer across the blood brain barrier in man, J. Clin. Invest., 67 (1981) 597-604.

[21] Hill, J.M., Lesniak, M.A., Pert, C.B. and Roth, J., Autoradiographic localization of insulin receptors in rat brain: prominence in olfactory and limbic areas, Neuroscience, 17 (1986) 1127-1138.

[22] Horn, F.G., Goodner, C.J. and Berrie, A.M., A (3H)2-Deoxyglucose method for comparing rates of glucose metabolism and insulin responses among rat tissues in vivo. Validation of the model and the absence of an insulin effect on brain, Diabetes, 33 (1984) 141-152.

[23] Jacobwitz, D.M., Fluorescence microscopic mapping of CNS nor- epinephrine systems in the rat forebrain. In W.E. Stumpf and L.D. Grant (Eds.), AnatomicalNeuroendocrinology, Karger, Basel, 1975, pp. 368-380.

[24] Jones, B.E. and Friedman, L., Atlas of catecholamine perikarya, varicosities and pathways in the brainstem of the cat, J. Comp. Neurol., 215 (1983) 382-396.

[25] Jones, B.E. and Moore, R.Y., Ascending projections of the locus coeruleus in the rat. II. Autoradiographic study, Brain Res., 127 (1977) 23-53.

[26] Kahn, B.B., Facilitative glucose transporters: regulatory mechanisms and dysregulation in diabetes, J. Clin. Invest., 89 (1992) 1367-1374.

[27] Lembo, G., Napoli, R., Capaldo, B., Rendina, V., Iaccarino, G., Volpe, M., Trimarco, B. and Sacca, L., Abnormal sympathetic overactivity evoked by insulin in the skeletal muscle of patients with essential hypertension, J. Clin. Invest., 90 (1992) 24-29.

[28] Lindvall, O., Bj6rklund, A. and Divac, I., Organization of catechol- amines neurons projecting to the frontal cortex in the rat, Brain Res., 142 (1978) 1-24.

[29] Lindvail, O. and Stenevi, U., Dopamine and noradrenaline neurons projecting to the septal area in the rat, Cell Tissue Res., 190 (1978) 383-407.

[30] Loy, R., Koziell, D.A., Lindsey, J.D. and Moore, R.Y., Noradrener- gic innervation of the adult rat hippocampal formation, J. Comp. Neurol., 189 (1980) 699-710.

[31] Luciguani, G., Namba, H., Nehlig, A., Porrino, L.J., Kennedy, C. and Sokoloff, L., Effects of insulin on local cerebral glucose utiliza- tion in the rat, J. Cereb. Blood Flow Metab., 7 (1987) 309-314.

[32] Marfaing, P., P6nicaud, L., Broer, Y., Mraovitch, S., Calando, Y. and Picon, L., Effect of hyperinsulinemia on local cerebral insulin binding and glucose utilization in normogiycemic awake rats, Neu- rosci. Lett., 115 (1990) 279-285.

P. Doyle et al. /Brain Research 684 (1995) 47-55 55

[33] Menendez, J.A. and Atrens, D.M., Insulin and the paraventricular hypothalamus: modulation of energy balance, Brain Res., 555 (1991) 193-201.

[34] Moore, R.Y., Catecholamine innervation of the basal forebrain. I. The septal area, J. Comp. Neurol., 177 (1978) 665-684.

[35] Namba, H., Lucignani, G., Nehlig, A., Patlak, C., Pettigrew, K., Kennedy, C. and Sokoloff, L.,, Effects of insulin on hexose transport across blood brain barrier in normoglycemia, Am. J. Physiol., 252 (1987) E299-E303.

[36] Nieuwenhuys, R., Voogd, J. and Van Huijzen, A., The Human Central Nervous System, Springer-Verlag, Heidelberg, 1988,

[37] Oomura, Y., Glucose as a regulator of neuronal activity, Adv. Metab. Disord., 10 (1983) 31-65.

[38] Pardridge, W.M., Receptor-mediated peptide transport through the blood-brain barrier, Endocr. Rev., 7 (1986) 314-330.

[39] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordi- nates, Academic Press, New York, 1987,

[40] Rowe, J.W., Young, J.B., Minaker, K.L., Stevens, A.L., PaUotta, J. and Landsberg, L., Effect of insulin and glucose infusions on sympathetic nervous system activity in normal man, Diabetes, 30 (1981) 219-225.

[41] Sakaguchi, T. and Bray, G.A.. Intrahypothalamic injection of insulin decreases firing rate of sympathetic nerves, Proc. Natl. Acad. Sci. USA, 84 (1987) 2012-2014.

[42] Sawchenko, P.E. and Swanson, L.W., The organization of noradren- ergic pathways from the brainstem to the paraventricular and supraoptic nuclei in the rat, I~Irain Res. Rev., 4 (1982) 275-325.

[43] Schwartz, M.W., Bergman, R.N., Kahn, S.E., Taborsky, G.J., Fisher, L.D., Sipols, A.J., Woods, S.C., Steil, G.M. and Porte Jr., D., Evidence for entry of plasma insulin into cerebrospinal fluid through an intermediate compartment in dogs. Quantitative aspects and implications for transport, J. Clin. Invest., 88 (1991) 1272-1281.

[44] Schwartz, M.W., Figlewicz, D.P., Baskin, D.G., Woods, S.C. and Porte Jr., D., Insulin in the brain: a hormonal regulator of energy balance, Endocr. Rev., 13 (1992) 387-414.

[45] Schwartz, M.W., Sipols, A.J., Kahn, S.E., Lattemann, D.P., Taborsky Jr., G., Bergman, R.N., Woods, S.C. and Porte Jr., D., Kinetics and specificity of insulin uptake from plasma into cerebrospinal fluid, Am. J. Physiol., 259 (1990) E378-E383.

[46] Schwartz, M.W., Sipols, A.£r., Marks, J.L., Sanacora, G., White, J.D., Scheurinck, A., Kahn, S.E., Baskin, D.G., Woods, S.C., Figlewicz, D.P. and Porte Jr., D., Inhibition of hypothalamic neu- ropeptide Y gene expression by insulin, Endocrinology, 130 (1992) 3608-3616.

[47] Sokoloff, L., Reivich, M., Kennedy, C., Des Rosiers, M.H., Patlak, C.S., Pettigrew, K.D., Sakurada, O. and Shinohara, M., The [14C]de- oxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure and normal values in the conscious and anaesthetized albino rat, J. Neurochem., 28 (1977) 897-916.

[48] Steffens, A.B., Scheurink, A.J., Porte Jr., D. and Woods, S.C., Penetration of peripheral glucose and insulin into cerebrospinal fluid in rats, Am. J. PhysioL, 255 (24) (1988) R200-R204.

[49] Strubbe, J.H., Porte Jr., D. and Woods, S.C., Insulin responses and glucose levels in plasma and cerebrospinal fluid during fasting and refeeding in the rat, Physiol. Behav., 44 (1988) 205-208.

[50] Swanson, L.W. and Hartman, B.K., The central adrenergic system, an immunofluorescence study of the location of cell bodies and their efferent connections in the rat utilizing dopamine-beta-hydroxylase as a marker, J. Comp. Neurol., 163 (1975) 467-506.

[51] Tohyama, M., Maeda, T. and Shimizu, N., Detailed noradrenaline pathways of locus coeruleus neurons to the cerebral cortex with use of 6-hydroxydopa, Brain Res., 79 (1974) 139-144.

[52] Unger, J.W., McNeill, T.H., Moxley, R.T., White, M., Moss, A.M. and Livingston, J.N., Distribution of insulin receptor-like immuno- reactivity in the rat forebrain, Neuroscience, 31 (1989) 143-148.

[53] Ungerstedt, U., Stereotoxic mapping of the monoamine pathways in the rat brain, Acta Physiol. Scand., 367 Suppl. (1971) 1-49.

[54] Van Houten, M., Posner, B.I., Kopriwa, B.M. and Brawer, J.R., Insulin binding site localized to nerve terminals in rat median eminence and arcuate nucleus, Science, 207 (1980) 1081-1083.

[55] Van Houten, M., Posner, B.O., Kopriwa, B.M. and Brawer, J.R., Insulin binding sites in the rat brain: in vivo localization to the circumventricular organs by quantitative autoradiography, En- docrinology, 105 (1979) 666-672.

[56] Wallum, B.J., Taborsky, G.J., Porte Jr., D., Figlewicz, D.P., Jacob- sen, L., Beard, J.C., Ward, W.K. and Dorsa, D.M., Cerebrospinal fluid levels increase during intravenous insulin infusion in man, J. Clin. Endocrinol. Metab., 64 (1987) 190.

[57] Wertber, G.A., Hogg, A., Oidfield, B.J., McKinley, M.J., Figdor, R., Allen, A.M. and Mendelsohn, F.A., Localization and characteriza- tion of insulin receptors in rat brain and pituitary gland using in vitro autoradiography and computerized densitometry, Endocrinology, 121 (1987) 1562-1570.

[58] Yalow, R.S. and Eng, J., Insulin in the central nervous system, Adv. Metab. Disord., 10 (1983) 341-348.