Rostromedial Hypothalamic Monoamine Changes in Response to Intravenous Infusions of Insulin and...

app p095 25-01-96 14:59:55

Appetite, 1996, 26, 1–20

Rostromedial Hypothalamic Monoamine Changes in Response

to Intravenous Infusions of Insulin and Glucose in Freely

Feeding Obese Zucker rats: a Microdialysis Study

M. OROSCO, C. ROUCH and S. NICOLAIDISNeurobiologie des Regulations, CNRS URA 1860, College de France, Paris

We have shown previously that intravenous infusions of insulin, known to induceglucoprivic hunger, and of insulin combined with glucose, known to inducesatiety, produce in the VMH and PVN of Wistar rats monoaminergic changesthat differ from those related to spontaneously occurring hunger and satiety,while the genetically obese Zucker rat is totally resistant to the behavioural effectsof insulin and insulin+glucose infusions. In the present study, the impact of theseinfusions on VMH and PVN monoamines in obese Zucker rats was assessedusing microdialysis. Monoaminergic changes (increase in DOPAC and 5-HIAAand decrease in DA and 5-HT) were quite similar in obese rats to those we foundin normal rats when insulin was infused. In contrast, changes in 5-HT or DA inresponse to insulin and glucose were quite different in the Zucker rat. Mono-aminergic changes related to meals were more dramatic in the Zucker rat and sowere able to reverse the background changes produced by the insulin infusion.These data confirm the idea that the effect on monoamines of spontaneouslyoccurring hunger and satiety is different from the effect on monoamines by insulinand glucose-induced hunger and satiety. The results show disturbances of theobese Zucker rat related both to insulin and to hypothalamic monoamines thatmay be involved in the hyperphagia and obesity of this model.

1996 Academic Press Limited

I

The role of insulin in the control of food intake and body weight regulation hasattracted considerable interest. The first investigation showing the direct impact ofinsulin on central structures used bilateral slow and prolonged insulin infusions intothe medial hypothalamus of the rat: such infusions resulted in hypophagia and,more importantly, in a permanent reduction of body weight (Nicolaıdis, 1978).Similar effects of intracerebroventricular (i.c.v.) and hypothalamic infusions havesince been observed in both the rat and the baboon (McGowan et al., 1990; Woodset al., 1979). These inhibitory effects using central administration of low doses ofinsulin do not contradict the feeding-promoting effect of higher doses of peripherally-administered insulin. Indeed, while hypoglycemia-producing doses of intravenous

The authors would like to thank Daniel Gripois and Marie-France Blouquit for the gift of Zuckerrats and Marie-Jo Meile for histological preparation.

Correspondence to: M. Orosco, Neurobiologie des Regulations, CNRS URA 1860, College de France,11 place Marcelin Berthelot, 75231 Paris Cedex 05, France.

0195–6663/96/010001+20 $12.00/0 1996 Academic Press Limited

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(i.v.) or subcutaneous (s.c.) insulin bring about glucoprivic feeding in both rats andhumans (Grossman & Stein, 1948; McKay et al., 1940), the same doses of insulininhibit feeding when combined with a glucose administration that just preventshypoglycemia but has no satiating effect by itself (Even & Nicolaıdis, 1986; Nicolaıdis& Rowland, 1976). The glucoprivic effect of insulin is therefore required to enhancefeeding (for review, see Grossman, 1986), while minute doses of insulin applied inthe brain or, most importantly, low doses of peripheral insulin that do not substantiallyreduce glycemia, produce the opposite effect, i.e. satiety (Nicolaıdis & Rowland,1976; Vanderweele et al., 1980).

The question that arises is to what extent abnormalities of peripheral insulinand central changes in insulin affect monoaminergic activity in the feeding-relatedstructures of the hypothalamus in animals that show abnormal feeding. The ge-netically obese, homozygotic Zucker rat (fa/fa) shows several abnormalities related toinsulin both peripherally and centrally. These disturbances include hyperinsulinemia(Zucker & Antoniades, 1972), high insulin concentration in the cerebrospinal fluid(CSF) compared to the lean rats (Fa/fa and Fa/Fa) but a lower ratio of CSF toplasma insulin (Stein et al., 1983), and low hypothalamic insulin content (Baskin etal., 1985). Recently, in a microdialysis study in the obese rat, we reported a lowbackground hypothalamic insulin content, as compared to the lean rats, as well asan enhanced increase in hypothalamic insulin in the obese rats in response tointravenously infused insulin (Gerozissis et al., 1993). Other altered responses toinsulin are observed in the obese Zucker rat, such as peripheral resistance to insulin(Stern et al., 1975) and a reduced inhibitory effect on feeding of centrally administeredinsulin (Ikeda et al., 1986). We also showed recently that insulin infusion did notbring about glucoprivic feeding and that no inhibitory effect was produced by acombined insulin and glucose infusion that reliably suppresses feeding in the normalrat (Orosco et al., 1994a).

The obese Zucker rat also shows abnormalities related to monoamines; wereported previously a reduced activity of both dopamine and serotonin in the obeseas compared to its lean congener. This reduced DA and 5-HT activity was accountedfor by lower levels of the metabolites DOPAC and 5-HIAA in several brain regionsincluding hypothalamus, hippocampus and striatum (Orosco et al., 1986).

Interactions between peripheral insulin and brain monoamine metabolism havepreviously been examined using brain homogenates. Peripheral administration ofinsulin results in an increase in striatal dopamine (Kwok & Juorio, 1986) and inwhole brain serotonin (Juszkiewicz, 1985; Mackenzie & Trulson, 1978a). This actionmay occur via increased monoamine synthesis caused by the enhancement of uptakeof precursor amino-acids into the brain (Fernstrom & Wurtman, 1972; Mackenzie& Trulson, 1978b; Tagliamonte et al., 1975). Alternatively, it may occur via changesin monoamines produced by the glucoregulatory action of insulin (Holmes et al.,1989).

We showed recently that the monoaminergic changes brought about by ma-nipulations of insulin in normal rats seldom reflect its behavioral effects (hunger,satiation and satiety) and seem rather related to the metabolic effects of the peptide(Orosco & Nicolaıdis, 1994) because the changes observed were completely differentfrom those accompanying spontaneous occurrence of hunger, satiation and satiety(Orosco & Nicolaıdis, 1992). As both of these effects are disturbed in the obeseZucker rat, it was interesting to investigate whether these disturbances were relatedto an abnormal action of insulin. We intravenously infused either insulin or insulin

3INSULIN AND GLUCOSE ON MONOAMINES IN ZUCKER RATS

plus glucose while assaying VMH-PVN changes in DA, DOPAC, 5-HT and 5-HIAAusing microdialysis in freely moving rats.

In the present study, we compared monoamine changes in obese Zucker ratswith those in Wistar rats because this strain is prototypical of normality and themost often used in this type of investigation, and because both heterozygotic (Fa/fa) and “normal” homozygotic (Fa/Fa) non-obese Zucker rats show some of theabnormalities maximally expressed in the homozygotic fa/fa obese rats. For example,monoamine and metabolite levels from a number of dissected brain areas are lowerin the non-obese Zucker than in the Wistar rat (Orosco et al., 1981; 1985).

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Animals

Female 16-week-old Zucker obese (fa/fa) were used. They were bred and gen-erously provided by the Laboratoire d’Endocrinologie, Universite Paris-Sud, Orsay,France. Upon arrival in the laboratory, all of the rats were housed individually incylinder Plexiglas cages conceived to allow stressless chronic infusions in the un-restrained animal in a temperature-controlled room (24±1°C), with lights on from0600 to 1800 hrs. Food and water were available ad libitum.

Surgery

Rats were anesthetized with pentobarbital (Sanofi, 50 mg/kg) after pretreatmentwith muscle relaxant, xylasine (Rompun, Bayer).

The indwelling venous catheter was implanted according to a previously describedtechnique (Nicolaıdis et al., 1974). Briefly, a silicone rubber (Silastic) catheter, filledwith viscous polyvinylpyrrolidone solution (40% w/v), was inserted in the rightauricular cavity via the right external jugular vein approximately 5 mm before itpasses under the clavicle. The vein was tied around the catheter and held in placeby further sutures. The other end of the catheter was pulled subcutaneously througha slit in the skin at the top of the skull.

For the sake of verification, other animals (n=2) underwent a more laboriouspreparation. They were implanted with two venous catheters in addition to themicrodialysis device. One catheter was located in the right jugular, i.e. down-streamin blood flow, and was used for infusions of insulin and glucose; the second catheterwas located in the left jugular, i.e. up-stream, and was used to collect blood samplesfor glycemia assays. As a result, substances injected down-stream could not mixwith the collected blood.

Once the venous catheters were secured, the animal was placed in a stereotaxicframe (Kopf Instruments). A guide cannula (Carnegie) for the microdialysis probewas aimed at the space lining both the PVN at the upper part and the VMH at thelower part once the probe was inserted. The coordinates of the guide tip were(according to the atlas of Paxinos & Watson, 1986)−1·9 mm anterior, 0·5 mm lateraland 7 mm ventral to bregma. The dialysis probe protruded 2 mm beyond the guide-tube, and its tip reached a point 9 mm ventral to bregma.

The guide cannula, the venous catheter and the securing device (screw) werefixed to the skull with stainless steel screws and dental cement.

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Although by 5 days after the operation, the animals showed normal sleep, feedingand body weight gain, at least 1 week was allowed for postoperative recovery beforethe experiments began. During this period, the animals were accustomed to theexperimental conditions in their home cages: they were permanently connected tothe infusion and dialysis system of catheters to be used in the experiment, protectedby a metal sheath and kept out of the animals’ reach by means of a counterbalancedbeam. This arrangement allowed normal movements.

All experiments were performed in animals’ own home cages.

Microdialysis Procedure

The microdialysis membranes (Carnegie) were 2 mm long, with a diameter of0·5 mm and a 20 000 molecular weight cut-off. According to our in vitro calibrationtest, the relative recovery was around 10% for both monoamines and their metabolites.

The perfusion was performed with a Ringer type solution containing 147 m Na+,2·3 m Ca2+, 4 m K+ and 155·6 m Cl−. A system of catheters connected to a two-way swivel placed on the beam allowed perfusion of fluid into the probe and recoveryof the perfusates. The flow rate (2 ll/min) allowed the collection of 30 ll samplesevery 15 min into microvials contained in a refrigerated microcollector (Carnegie)controlled by a computer.

Analysis of Dialysates

The dialysates were analysed by means of reverse-phase liquid chromatographywith electrochemical detection (Albedo 100, Europhor) at a potential of 750 mV.The chromatographic system consisted of a 20 ll sample loop leading to a 10 cmcolumn (Europhor) with a 3·2 mm internal diameter and 3 l C-18 packing. Themobile phase consisted of an acetate buffer containing 100 l EDTA, 1 m octane-sulfonic acid and 6% v/v acetronitrile at pH 3·1. The compounds analysed weredopamine (DA), dihydroxyphenylacetic acid (DOPAC), 5-hydroxy-tryptamine (5-HT) and 5-hydroxyindolacetic acid (5-HIAA).

Experimental Protocol

At 0900 hrs the dialysis probe was inserted through the guide and the intravenousinfusion of the vehicle was set up by connecting a plastic tube to the head-piece ofthe indwelling catheter through one channel of the two-way swivel and by switchingon the pump. The following three procedures were applied in 12 rats at a randomorder on three successive days:

Insulin Infusion. After collecting four baseline dialysis samples during the vehicle(control) intravenous infusion (1400–1500 hrs), 1 IU of insulin (Actrapid, Novo) ina volume of 1 ml, was then infused over one hour (1500–1600 hrs). Dialysis sampleswere collected until 1800 hrs. This procedure was adapted from the work of Evenand Nicolaıdis (1986).

Meal patterns and the amount of powder chow consumed (accuracy±0·1 g and±1 min) were monitored by means of a specially designed food cup suspended ona strain gauge connected to a potentiometric recorder (Nicolaıdis et al., 1979) duringthe 4 h period of the experiment. The parameters measured were, total food intake,


number of meals, meal size, intermeal interval and latency to the onset of the firstmeal.

Combined Infusion of Insulin and Glucose. This intravenous infusion differed inthat two simultaneous infusions of two different solutions had to be performed. Thiswas made possible by connecting a metallic Y-shaped cannula to one channel of thetwo-way swivel.

The same amount of insulin as in experiment 1 (1 IU in 1 ml) was infused togetherwith 5·1 g of glucose (1500–1600 hrs). Six millilitres of the glucose solution were soadministered over 2 h (1500–1700 hrs). This amount of glucose solution and thistime of infusion, longer than the insulin infusion, were necessary to prevent aninsulin-induced hypoglycemia during and after the perfusion. The insulin/glucoseratio used in the present experiment has been previously shown to block feeding(Even and Nicolaıdis, 1986).

As in the above protocol, feeding patterns were recorded.Control Experiments. The aqueous vehicle was infused for the whole period of

the experiment (4 h) and feeding behaviour was monitored and assays performed asin the other experimental paradigms.

Glycemia

Blood glucose levels were assessed with a glucose analyser using the glucoseoxidase method, before, during and after insulin and insulin+glucose infusions inthe home cage of the two animals bearing two venous catheters. Samples of blood(0·2 ml) were obtained from the up-stream chronic catheter located in the jugularvein and infusions were derived through the down-stream atrial catheter. The durationof glycemic patterns was chosen in order to cover the period of changes observedin microdialysates and to remove minimum amount of blood.

This low number of animals, due to the difficulty in keeping the two venouscatheters preparation potent, was sufficient to obtain reproducible results and toshow the otherwise expected differences with the Wistar rats’ data that we observedpreviously.

Histology

At the end of each experiment, the animals were given a lethal dose of pentobarbitaland were perfused through the heart with saline followed by 10% formol. The brainwas removed, further hardened in 10% formol, and was cut into serial coronal sectionsat 60 lm. Following glycerine staining, they were observed at low magnification byan observer unaware of the data obtained to check for the position of the dialysisprobe (Fig. 1). The animals whose brain did not show a correct placement of theguide and the probe (n=4) were discarded by the independent observer. The datafrom eight animals out of the 12 were thus included in this study.

We previously observed (Orosco and Nicolaıdis, 1994), and we confirmed in thepresent study, that the results were similar whether the position of the microdialysisprobe was more caudal than average, i.e. in the VMH or more rostral than average,i.e. in the PVN or as intended, between these two nuclei. As a result, all the datawere pooled together.

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F 1. Coronal section at the level of anterior hypothalamus depicting the locationof the microdialysis probe aimed at the space between the paraventricular nucleus (PVN,antero-superior part) and the ventromedian hypothalamus (VMH, postero-inferior part).

Statistics

Mean basal levels in 20 ll of analysed dialysates were the following: DA, 25 pg;DOPAC, 20 pg; 5-HT, 2 pg and 5-HIAA, 1200 pg. As is usual in experiments suchas this one, the percent change from the mean of the four baseline samples wascalculated for each animal for DA, DOPAC, 5-HT and 5-HIAA. The results werethen expressed as mean percentage change±SEM at each time and the statisticalsignificance was calculated by a paired Student’s t-test, each animal (n=8 withcorrect location of the probe) being its own control.

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Effect of Insulin Infusion on Food Intake, Glycemia and Medial HypothalamicMonoamines

As shown recently (Orosco et al., 1994a), insulin, in contrast to what it usuallydoes in normal rats, did not stimulate feeding in the obese Zucker rat. One or twomeals of about 1 g each took place during the whole experimental period but duringthe same period, Zucker rats that did not receive insulin also ingested 2 g in twomeals (Table 1).

Glycemia decreased during insulin infusion and returned to normal levels as soonas the infusion period ended (Table 2).

Insulin infusion induced an early, 10% (1st 15 min aliquot) to 40% (2nd and 3rdaliquot), increase in DOPAC levels, still reaching 16 to 33% during the 45 min post-infusion and beginning to return to baseline levels during the postinfusion hour (Fig.2). When a meal took place during the infusion, DOPAC started decreasing duringthe 15 min sample corresponding to the meal, further decreased in the next 15 minfollowing the meal and increased again thereafter (Fig. 3 for characteristic individual


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F 2. Changes in DA, DOPAC, 5-HT and 5-HIAA in 15 min microdialysate samples,during and after intravenous insulin (1 IU during 1 hr). Results are expressed as meanpercentage change from baseline±SEM (n=8). ∗P<0·05, ∗∗P<0·01 compared to baseline.

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F 3. Characteristic individual profiles of changes in DOPAC in 15 min micro-dialysate samples, during and after intravenous insulin (1 IU over 1 h) infusion. Spontaneouslyoccurring feeding episodes are indicated. Results are expressed as percentage change frombaseline.

profiles). In contrast, DA levels decreased during and after the end of the infusion(down to −70%) (Fig. 2). A trend towards recovery of baseline DA levels wasobserved during and even sometimes just before the occurrence of a meal, withoutreaching positive values (Fig. 4 for characteristic individual profiles). Levels of 5-HIAA, the metabolite of 5-HT, rose in a pattern similar to that of DOPAC (Fig.2). This rise was further increased during feeding (Fig. 5 for characteristic individualprofiles). The level of 5-HT itself fell during the infusion period (Fig. 2). However,the occurrence of a meal during the infusion period resulted in a superimposed risein 5-HT that lasted after the termination of the meal (Fig. 6 for characteristicindividual profiles). Whenever meals occurred during insulin infusion, the changespreviously observed in response to spontaneous meals (Orosco et al., 1994b) weresuperimposed over the background insulin-induced monoamine and metabolitechanges.


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F 4. Characteristic individual profiles of changes in DA in 15 min microdialysatesamples, during and after intravenous insulin (1 IU over 1 h) infusion. Spontaneously occurringfeeding episodes are indicated. Results are expressed as percentage change from baseline.

Effects of Combined Infusion of Insulin and Glucose on Food Intake and MedialHypothalamic Monoamines

Contrary to their action in normal rats, insulin and glucose infused together didnot suppress feeding in Zucker rats (Table 1). This observation is in agreement withour recently published findings (Orosco et al., 1994a).

Glycemia dramatically increased during the infusion of insulin+glucose followedby glucose alone. Hyperglycemia did not return to normal levels during the wholeexperimental period (Table 2).

Infusion of both insulin and glucose induced effects similar to those observedwith insulin alone regarding the monoamine metabolites DOPAC and 5-HIAA:

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F 5. Characteristic individual profiles of changes in 5-HIAA in 15 min micro-dialysate samples, during and after intravenous insulin (1 IU over 1 h) infusion. Spontaneouslyoccurring feeding episodes are indicated. Results are expressed as percent change from baseline.

DOPAC increased even more than in response to insulin alone (up to 160%) untilthe end of glucose infusion (that lasted 1 h longer than insulin infusion) and thenslowly returned toward basal levels (Fig. 7). Here again, the occurrence of a mealbrought about a transient decrease in the regular rise of DOPAC (Fig. 8 forcharacteristic individual profiles). The levels of 5-HIAA also increased, but no morethan in response to insulin infusion alone (Fig. 7) and with the same tendency towarda further increase whenever a meal occurred (Fig. 9 for characteristic individualprofiles). As for the profile of changes in the monoamines (DA and 5-HT) themselves,


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F 6. Characteristic individual profiles of changes 5-HT in 15 min microdialysatesamples, during and after intravenous insulin (1 IU over 1 h) infusion. Spontaneously occurringfeeding episodes are indicated. Results are expressed as percentage change from baseline.

T 1Number of meals, total and per meal intake (g) during and after infusions of vehicle,insulin (1 IU during 1 h) and insulin+glucose (insulin: 1 IU over 1 h and glucose: 5·1 gover 2 h) in obese Zucker rats. All the recordings were performed between 1400 and

1800 hrs

Vehicle Insulin Insulin+Glucose

Number of meals 2·2±0·2 1·7±0·2 1·7±0·2Total food intake (g) 2·1±0·2 1·5±0·4 1·9±0·6Intake per meal (g) 0·9±0·3 0·8±0·2 1·1±0·3

it was different from that observed when insulin was infused alone: DA increasedonly transiently and 5-HT did not change significantly (Fig. 7). The occurrence ofa meal further boosted the rise in DA (Fig. 10 for characteristic individual profiles)and induced a large and long-lasting increase in 5-HT (Fig. 11 for characteristicindividual profiles).

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F 7. Changes in DA, DOPAC, 5-HT and 5-HIAA in 15 min microdialysate samples,during and after concomitant intravenous insulin and glucose infusion (insulin: 1 IU over 1 hand glucose: 5·1 g over 2 h). Results are expressed as percentage change from baseline±SEM(n=8). ∗P<0·05; ∗∗P<0·01; ∗∗∗P<0·001 compared to baseline.


T 2Changes in plasma glucose levels (mg/dl), before, duringand after intravenous infusions of insulin (1 IU during 1 hinfusion) and insulin+glucose (insulin: 1 IU over 1 h and

glucose: 5·1 g over 2 h) in obese Zucker rats

Time Insulin Insulin+Glucose

Basal 100 ± 2 100 ± 220 min 59·5± 3·5 345 ±5040 min 65·5± 2·5 395·5± 0·560 min 83 ±20 414·5±18·580 min 100·5± 2·5 427·5± 5·5

D

The purpose of this study was to investigate, in the obese Zucker rat, theeffect on both rostromedial hypothalamic monoamines and feeding behaviour ofmanipulations of glucose metabolism. In addition, our purpose was to see whetherpossible disturbances in central monoamines might account for the excessive intakeor for the resistance to insulin that characterize these genetically obese rats. Becauseit was less well established, one of the insulin resistance-related properties of theobese Zucker rat, i.e. the lack of both insulin-induced glucoprivic feeding andinsulin+glucose-induced satiety (Orosco et al., 1994a) was first verified in the presentexperiment. In effect, in the obese Zucker rat, the meals we observe in the course ofthe infusions are similar in size and frequency to those we observe in vehicle-infusedsubjects. Because feeding remains uninfluenced by all of the above infusions, it ispossible to consider all meals as spontaneously occurring events. Therefore, theobserved changes in hypothalamic monoamines are related to two parallel events,i.e. the background effect induced by the i.v. insulin and glucose infusions and theoccasional, superimposed spontaneous feeding episodes.

It is well-established that in the obese Zucker rat, insulin does not lower glycemiaas much as it does in Wistar rats (Orosco & Nicolaıdis, 1994) and to some extentin lean Zucker rats (Zucker & Antoniades, 1972). This phenomenon was verified inthe present experiment. Similarly, insulin resistance in the obese rat, accounts forthe dramatic hyperglycemia in response to the coinfusion of insulin and glucose asopposed to normoglycemia that is observed in normal rats receiving the sameinfusions (Even and Nicolaıdis, 1986). These findings confirm that the obese Zuckerrat is not responsive to insulin as far as the clearance and utilization of glucose isconcerned.

To assess the possible peculiarities of the obese rat’s monoamine changes inresponse to glucose and insulin infusion, it is necessary to compare the presentfindings with those recently collected in Wistar rats under the same conditions(Orosco & Nicolaıdis, 1994). Most of the monoamines respond similarly in both theobese Zucker and Wistar rats. Insulin infusion results in a decrease in DA and 5-HT and in an increase in DOPAC and 5-HIAA. The increases in both metabolitesDOPAC and 5-HIAA are in agreement with similar increases previously observedin brain tissue homogenates (Orosco et al., 1991) and in the CSF (Danguir et al.,

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F 8. Characteristic individual profiles of changes in DOPAC in 15 min micro-dialysate samples, during and after concomitant intravenous insulin and glucose infusion(insulin: 1 IU over 1 h and glucose: 5·1 g over 2 h). Spontaneously occurring feeding episodesare indicated. Results are expressed as percentage change from baseline.

1984). These metabolite increases seem to reflect an enhanced synthesis of the aminesdue to an increased transport of the precursor amino-acids, tryptophan and tyrosine,into the brain (Fernstrom & Wurtman, 1972). Indeed, these increases in metaboliteswere reported to reflect the same metabolic steps whether they are measured inmicrodialysates or in ex vivo tissues, because DOPAC and 5-HIAA represent thefirst step of monoamine catabolism and as such their variations have identicalmeaning whether they are measured directly after their formation (homogenates) orafter being released in the extracellular space (dialysates) (Carboni et al., 1992;Westerink et al., 1987). Contrary to their respective metabolites, the active amines


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F 9. Characteristic individual profiles of changes in 5-HIAA in 15 min micro-dialysate samples, during and after concomitant intravenous insulin and glucose infusion(insulin: 1 IU over 1 h and glucose: 5·1 g over 2 h). Spontaneously occurring feeding episodesare indicated. Results are expressed as percentage change from baseline.

DA and 5-HT were concomitantly decreased in our study. Although this result wasnot expected, it is not inconsistent with an enhanced synthesis of these aminesbecause, according to previously established microdialysis principles, the releasedrather than intracellular amines are measured in microdialysates (Imperato and DiChiara, 1984). Certainly, older studies that had used homogenates had concludedthat synthesis, metabolism and release were coupled processes. However, since thedevelopment of microdialysis, an increasing number of examples of uncoupled effects,especially concerning DA is being reported (Wood and Altar, 1988). In a recentstudy for example, it is shown that, after precursor loading, synthesis can be

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F 10. Characteristic individual profiles of changes in DA in 15 min microdialysatesamples, during and after concomitant intravenous insulin and glucose infusion (insulin: 1 IUover 1 h and glucose: 5·1 g over 2 h). Spontaneously occurring feeding episodes are indicated.Results are expressed as percentage change from baseline.

dissociated from release when measured by microdialysis but not by brain homogenateassays (Westerink and De Vries, 1991).

One of the findings in the present study is that the magnitude of the monoaminevariations is larger and their time-course shorter in the obese Zucker rat. Thiscomparison still holds when one takes into account the superimposition of the meal-induced monoamine changes which, as mentioned above, is clearly observable in theobese but not in normal rats. This peculiarity bringing about feeding-bound changes


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F 11. Characteristic individual profiles of changes in 5-HT in 15 min microdialysatesamples, during and after concomitant intravenous insulin and glucose infusion (insulin: 1 IUover 1 h and glucose: 5·1 g over 2 h). Spontaneously occurring feeding episodes are indicated.Results are expressed as percentage change from baseline.

that occur at random makes it necessary to assess individual rather than pooledpatterns.

In obese rats, the feeding-induced monoamine changes are similar to those thatwe observed in spontaneously feeding Wistar rats (Orosco and Nicolaıdis, 1992). Inparticular, they also show an increase in 5-HT, 5-HIAA and DA and decrease inDOPAC (Orosco et al., 1995). When these feeding-induced monoamine changesoccur in the obese Zucker rat during infusion of insulin or insulin and glucose,they are algebrically superimposable on the infusion-induced background levels,particularly when the infusion and the feeding effects are in opposite directions. Thisis not the case in Wistar rats where the feeding-induced monoamine changes duringinsulin infusion are totally blunted by the background insulin-induced changes(Orosco & Nicolaıdis, 1994).

The presence of hypothalamic monoamine changes in response to i.v. infusionof insulin demonstrates that at least the rostromedial hypothalamic monoamines inthe obese Zucker rat are not totally resistant to the effect of peripheral insulin. Thisfinding is in contrast to the total resistance of the obese Zucker rat towards the

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feeding effect of insulin (Orosco et al., 1994a) or to the lack of changes in striataldopamine after insulin injection (Orosco et al., 1991; 1992).

The obese Zucker rat shows some pecularities also in the effects of insulin andglucose infusion on levels of 5-HT and DA. The feeding-induced increase in 5-HT,which is exaggerated in the obese Zucker rat (Orosco et al., 1995), hides the decreasebrought about by insulin infusion. DA also increases slightly in response to theinfusion and even more so when feeding occurs.

In conclusion, the present investigation reveals a number of monoamine changesthat differ between the obese Zucker rat and the Wistar rat. However, some of thesedifferences are difficult to interpret with our present knowledge. It certainly appearsthat the obese rat is not completely resistant to the action of insulin on monoamines,and yet its feeding behaviour remains unaffected by these infusions. The obese ratis nevertheless less responsive than the Wistar rat since, in the obese, the insulin-induced monoamine changes are easily reversed by the occurrence of meals. Thus,we further show that the monoaminergic and the feeding effects of insulin areindependent from each other. As we pointed out previously (Orosco & Nicolaıdis,1994), changes in monoamines in the VMH and PVN area—that controls bothfeeding and metabolism—seem to express metabolic events rather than the behavioralstates of hunger and satiety. These metabolic effects are not simply related to glucoselevel or to glucose utilization but rather to a more global metabolic state of whichthe glucose utilization is only a part. Nevertheless, although in independent ways,the disturbances of both insulin and monoamines in the obese Zucker rat mayparticipate in the impairment of both food intake and body weight observed in thesemutants.

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Received 19 September 1994, revision 10 January 1995

Rostromedial Hypothalamic Monoamine Changes in Response to Intravenous Infusions of Insulin and...

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