M. Orosco

Spontaneous feeding-related monoaminergic changes in the rostromedial hypothalamus revealed by microdialysis

The activity of hypothalamic monoamines in response spontaneous feeding was investigated using the in vivo technique of brain microdialysis together with the instrumental recording of feeding pattern. The simultaneous variations of dopamine (DA), serotonin (5-HT), and their respective metabolites, DOPAC and 5-HIAA, were measured in the rostromedian hypothalamus, where the probe was located between the PVN and VMH. Throughout the experiment, the changes in DOPAC followed a mirror image of those in DA: DA regularly increased, reaching its zenith within the 15-min sample collected during the meal before returning to the same level as just before the meal. Following a premeal plateau, both 5-HT and 5-HIAA increased as soon as the beginning of feeding; 5-HT reached its zenith during the meal while 5-HIAA showed a more delayed and prolonged increase. When a new meal was initiated, 60 to 70 min later, a similar monoaminergic pattern was observed again. These data suggest that building up hunger is announced by an ascending slope of DA and setting up of satiation is concomitant with a descending slope of DA. Concerning serotonergic changes, the sharp 5-HT release during the meal would be a signal of satiation (transient preabsorptive fullness) while the longer-lasting increase in 5-HIAA, reflecting 5-HT synthesis, would be associated with satiety (more persistent postabsorptive state substituting satiation). These data partially confirm and extend previous pharmacological studies as well as the findings on deprivation-induced, imposed meals. They suggest a possible causal relation between monoaminergic changes and behavioral initiatives.

Specific inhibition of GLUT2 in arcuate nucleus by antisense oligonucleotides suppresses nervous control of insulin secretion

We previously demonstrated the presence of the glucose transporter GLUT2 in specific brain areas which are mainly involved in the control of fuel metabolism and feeding behavior, i.e., nuclei of the hypothalamus and of the anterior brainstem. We hypothesized that GLUT2 acts as a glucose sensor in these areas, as already described in pancreatic beta cells. In order to test this hypothesis, we injected antisense unmodified oligodeoxynucleotide (ODN) to GLUT2 into the arcuate nucleus. Antisense ODN efficiency on GLUT2 protein level was assessed on pancreatic islets in culture and they were shown to induce a 66% decrease in GLUT2 protein. Bilateral injections of GLUT2 antisense ODNs were performed twice daily over a two-day period in rats. Antisense ODNs induced a significant decline in body weight gain although total daily food intake was unchanged when compared both to control groups and to the period before treatment. Twenty hours after the last injection, anaesthetized rats received, via a catheter inserted into the carotid artery and directed towards the brain, a minute glucose load that by itself does not modify systemic blood glucose level but which induces increased insulinemia. This insulin response was completely abolished only in antisense-treated rats. These findings provide the first evidence for a physiological role of GLUT2 in the brain and support the hypothesis that this transporter is involved in a glucose sensing

Feeding-related immunoreactive insulin changes in the PVN-VMH revealed by microdialysis

The presence of insulin in the brain and its anorectic effect when centrally infused are well-established today. The question of physiological and dynamic changes in brain insulin in relation to meals is still unanswered and addressed here. Immunoreactive insulin (IRI) was measured using a sensitized RIA in 30-min microdialysates from VMH and PVN nuclei during and after a scheduled meal in male Wistar rats. We indeed observed elevations in hypothalamic IRI during the first 30 min of 1-h meals with a progressive return towards premeal levels in spite of a robust satiety. When the rats were accustomed to the scheduled meals, an anticipatory rise in IRI was found in the hypothalamus, but not in the plasma, during the 30 min preceding the due time of the meal whether the meal was presented or not. This anticipatory rise was proportional to the number of repeated scheduled meals. These results first suggest that hypothalamic IRI changes reflect in some instances those in the plasma although there are exceptions that cannot be accounted for by a simple plasma-brain tissue delivery. Besides, hypothalamic IRI can hardly be proposed as a satiety signal. The present data suggest a role in satiation rather than in satiety or, perhaps, in the inhibition of the behavioral response of feeding that can include the anticipatory rise.

Spontaneous feeding-related monoamine changes in rostromedial hypothalamus of the obese Zucker rat: A microdialysis study

Using microdialysis in freely moving rats, we have been able to observe changes in monoamines from the ventromedial and paraventricular hypothalamic nuclei before, during, and after spontaneous feeding. Because the genetically obese Zucker rat shows abnormalities related both to feeding and monoamines, it was of interest to investigate possible particularities in the monoaminergic variations around spontaneous feeding. The profile of changes in Zucker rats was grossly similar to that of Wistar rats: increases in 5-hydroxy-tryptamine (5-HT), 5-hydroxyindolacetic acid (5-HIAA), and dopamine (DA), and decrease in dihydroxyphenylacetic acid (DOPAC). However, the release in monoamines (5-HT and DA) was more dramatic and longer-lasting than in Wistar rats, while the changes in the metabolites were proportionnally less pronounced. This suggests that high concentrations of these feeding-related amines are released and remain in the synaptic cleft of the obese rat, possibly because they are required in larger amounts to bring about satiety. The hyperphagia of the obese Zucker rat may therefore be the result of a resistance to these prandially released satiety-promoting neurosubstances.

Basal and hyperinsulinemia-induced immunoreactive hypothalamic insulin changes in lean and genetically obese Zucker rats revealed by microdialysis

Lean and genetically obese Zucker rats were implanted with permanent intravenous catheters and a guide cannula was aimed at the region of the ventromedial (VMH) and paraventricular (PVN) nuclei to measure immunoreactive insulin collected by means of microdialysis. Preliminary experiments assessed the validity of a novel assay of insulin in microdialysates by a sensitized radioimmunoassay technique. This method was then used to measure basal levels of insulin and those induced by i.v. infusion of 0.5 U of insulin over 30 min in both lean and obese rats. Basal hypothalamic immunoreactive insulin levels were lower in the obese rats than in the lean Zucker rats. When insulin was infused i.v. for 30 min, hypothalamic immunoreactive insulin showed an increase in the 30-60 min sample, which was twice as great in the obese rats. Two facts suggest that the insulin found in the microdialysates was of cerebral, not vascular origin: the short latency in the response and the finding that the response was greater in obese rats.

Brain insulin response to feeding in the rat is both macronutrient and area specific

Using microdialysis, we showed recently that hypothalamic immuno-reactive insulin (IRI) levels increased after a meal of chow and decreased in response to a fat meal. In the present study, we have compared extracellular hypothalamic and extrahypothalamic basal IRI levels and investigated the effect of meals composed exclusively of either carbohydrates (85% starch, 15% sucrose) or casein on both plasma and medial hypothalamic (PVN-VMH) insulin. The response of IRI to a carbohydrate meal was also investigated in the cerebellum. Basal hypothalamic IRI was twofold higher in the hypothalamus as compared to the cerebellum (33 +/- 4 and 15 +/- 2 pg/mL, respectively). Hypothalamic IRI increased twofold in response to the carbohydrate meal (72 +/- 15 pg/mL) but remained unchanged during the casein meal. No IRI change was found in the cerebellum after a meal of carbohydrates (16 +/- 2 pg/mL). Insulinemia was increased by both the carbohydrate and the casein meal. However, the protein-induced increase was less pronounced (maximum + 359% compared to 1650% for carbohydrates). The present data show a dual specificity of brain insulin response to feeding; in addition to the macronutrient specific variations, a regional specificity was also observed. Taken together with previous observations, the present data are in favor of an involvement of PVN-VMH insulin in the control of feeding and macronutrient-specific appetites.

Insulin Responses to a Fat Meal in Hypothalamic Microdialysates and Plasma

In a recent microdialysis study in freely-behaving rats, we observed changes in immunoreactive insulin (IRI) in hypothalamic dialysates after a meal of standard laboratory chow. These changes did not always parallel plasma insulin variations, suggesting a partial independence from peripheral insulin. In the present study, we have attempted to assess the profile of medial hypothalamus (VMPH-PVN) extracellular insulin and peripheral insulin before and after a fat meal (lard). In contrast to the increase we previously observed with chow meals, hypothalamic extracellular IRI decreased during the fat meal and fell to 60% 30 min after the meal. Plasma insulin levels did not change. The intake of the lard meal, provided in unlimited amounts, was much larger in calories than the intake of a chow meal under the same conditions. However, when rats were offered a meal of chow after they had eaten a meal averaging 6.7 g of fat (61 calories), they immediately began eating the chow. Thus, the meal of fat produced no general satiation. On the contrary, the rats consumed a second chow meal only after a delay of approximately 40 min after the first one. The present data, in conjunction with our previous observations with chow fed rats, suggest that the level of extracellular hypothalamic IRI may decrease independently of plasma insulin levels and may, at least partially, account for the observed lack of satiation.

Ontogeny of brain monoamines in lean and obese female Zucker rats

Differences in monoamine and metabolite levels were previously observed between lean and obese 16 week-old female Zucker rats. In order to test whether these variations exist initially in young animals, catecholamines (CA), serotonin (5-HT) and some of their metabolites were assayed in brain areas of Zucker rats between 5 and 16 weeks of age. The levels of most compounds in all areas studied increased with age in both groups. At 5 weeks of age, there was no difference between lean and obese rats. At 8 weeks, a reduction of dopamine (DA) metabolites appeared in the striatum and cortex of obese rats and persisted up to 16 weeks. A reduction of the 5-HT metabolite, 5-hydroxyindolacetic acid (5-HIAA), occurred at 8 weeks only. At 16 weeks, increases of DA and 5-HT in the hypothalamus and decreases of norepinephrine (NE), 5-HT and 5-HIAA in the hippocampus appeared. These data suggest that the development of obesity occurs before any monoamine alterations.

Serotoninergic modulation of sodium appetite in the rat

There are two mechanisms leading to an enhancement of salt intake: one is induced by a sodium deficit and the other is need-free. The serotonin involvement in need-induced and/or need-free sodium appetite is interesting to consider because related drugs are already used against another cardiovascular risk factor, obesity. The effect of dexfenfluramine (1.5 or 3 mg/kg), an anorectic drug enhancing 5-HT transmission, and of metergoline (2 or 4 mg/kg), a 5-HT antagonist, was assessed in need-induced (depletion-induced), subsequent need-free, and spontaneous sodium appetite. Dexfenfluramine (3 mg/kg) decreased by 75% to 90% the depletion-induced intake of an aversive 3% NaCl solution, as well as the spontaneous intake of a less aversive 1.8% NaCl solution. Water intake was not diminished under these conditions. Metergoline significantly increased salt intake in need-free conditions in rats with either a history of three previous depletions or not. These results confirm the involvement of serotonin in sodium appetite and extend this involvement to both need-induced (natriorexis) and need-free (natriophilia) conditions. The metergoline experiments also suggest that 5-HT exerts a tonic inhibition on salt intake.

Changes in hypothalamic prostaglandin E2 may predict the occurrence of sleep or wakefulness as assessed by parallel EEG and microdialysis in the rat

Prostaglandin (PG) E2 is produced by mammalian hypothalamus and when administered exogenously prolongs wakefulness. In order to study the relation of endogenous hypothalamic PGE2 to sleep and wakefulness, we have used microdialysis in freely moving rats associated with EEG recording. Male Wistar rats were implanted with three cortical electrodes and with a guide cannula for microdialysis in the space between the paraventricular nucleus (PVN) and the ventromedial hypothalamus (VMH). PGE2 was measured by RIA in 3- or 6-min dialysates 15 days after surgery, when sleep patterns were normal again and PGE2 production stabilised. PGE2 levels were significantly higher during wakefulness (601 +/- 35 pg/ml, 5 experiments, 35 samples) than during slow-wave sleep (487 +/- 24 pg/ml, 5 experiments, 49 samples). Samples corresponding to paradoxical sleep showed a tendency towards higher PGE2 values compared to slow-wave sleep but lower compared to wakefulness. In epochs of wakefulness or sleep lasting at least 12 min, high PGE2 levels in the middle of wakefulness regularly dropped, thus announcing the occurrence of sleep. During sleep, PGE2 first went on dropping and then reincreased towards the values that characterize early periods of wakefulness. In its turn, this reincrease in PGE2 announced the end of sleep and the imminent occurrence of wakefulness. It is the first study to our knowledge showing that the evolvement in endogenous PG profile may predict the occurrence of sleep or wakefulness.