Kinetic quantification of protein polymer nanoparticles using non-invasive imaging.

Protein polymers are repetitive amino acid sequences that can assemble monodisperse nanoparticles with potential applications as cancer nanomedicines. Of the currently available molecular imaging methods, positron emission tomography (PET) is the most sensitive and quantitative; therefore, this work explores microPET imaging to track protein polymer nanoparticles over several days. To achieve reliable imaging, the polypeptides were modified by site-specific conjugation using a heterobifunctional sarcophagine chelator, AmBaSar, which was subsequently complexed with (64)Cu. AmBaSar/(64)Cu was selected because it can label particles in vivo over periods of days, which is consistent with the timescales required to follow long-circulating nanotherapeutics. Using an orthotopic model of breast cancer, we observed four elastin-like polypeptides (ELPs)-based protein polymers of varying molecular weight, amino acid sequence, and nanostructure. To analyze this data, we developed a six-compartment image-driven pharmacokinetic model capable of describing their distribution within individual subjects. Surprisingly, the assembly of an ELP block copolymer (78 kD) into nanoparticles (R(h) = 37.5 nm) minimally influences pharmacokinetics or tumor accumulation compared to a free ELP of similar length (74 kD). Instead, ELP molecular weight is the most important factor controlling the fate of these polymers, whereby long ELPs (74 kD) have a heart activity half-life of 8.7 hours and short ELPs (37 kD) have a half-life of 2.1 hours. These results suggest that ELP-based protein polymers may be a viable platform for the development of multifunctional therapeutic nanoparticles that can be imaged using clinical PET scanners.

Early changes in insulin secretion and action induced by high-fat diet are related to a decreased sympathetic tone.

To evaluate the relationship between the development of obesity, nervous system activity, and insulin secretion and action, we tested the effect of a 2-mo high-fat diet in rats (HF rats) on glucose tolerance, glucose-induced insulin secretion (GIIS), and glucose turnover rate compared with chow-fed rats (C rats). Moreover, we measured pancreatic and hepatic norepinephrine (NE) turnover, as assessment of sympathetic tone, and performed hypothalamic microdialysis to quantify extracellular NE turnover. Baseline plasma triglyceride, free fatty acid, insulin, and glucose concentrations were similar in both groups. After 2 days of diet, GIIS was elevated more in HF than in C rats, whereas plasma glucose time course was similar. There was a significant increase in basal pancreatic NE level of HF rats, and a twofold decrease in the fractional turnover constant was observed, indicating a change in sympathetic tone. In ventromedian hypothalamus of HF rats, the decrease in NE extracellular concentration after a glucose challenge was lower compared with C rats, suggesting changes in overall activity. After 7 days, insulin hypersecretion persisted, and glucose intolerance appeared. Later (2 mo), there was no longer insulin hypersecretion, whereas glucose intolerance worsened. At all times, HF rats also displayed hepatic insulin resistance. On day 2 of HF diet, GIIS returned to normal after treatment with oxymetazoline, an alpha(2A)-adrenoreceptor agonist, thus suggesting the involvement of a low sympathetic tone in insulin hypersecretion in response to glucose in HF rats. In conclusion, the HF diet rapidly results in an increased GIIS, at least in part related to a decreased sympathetic tone, which can be the first step of a cascade of events leading to impaired glucose homeostasis.

Extracellular hypothalamic serotonin and plasma amino acids in response to sequential carbohydrate and protein meals.

In previous studies, we showed that carbohydrate and protein ingestion, respectively, increased and decreased hypothalamic extracellular serotonin and the plasma ratio tryptophan over its competitor amino acids (Trp/LNAAs), reflecting serotonin synthesis. Serotonin levels returned towards baseline 2 h after either meal while the ratio remained altered. The question addressed is the ability of serotonin to respond expectedly to a second meal of the alternate nutrient. Rats were fed with sequential meals of either carbohydrates first and then casein 2 h later or in reverse order. Hypothalamic serotonin was measured using microdialysis. Permanent blood sampling allowed to track in parallel plasma amino acids. A carbohydrate meal increased hypothalamic serotonin, so did a subsequent casein meal. Conversely, following a casein meal that reduced serotonin, a carbohydrate meal also decreased it. The plasma ratio Trp/LNAAs was enhanced by a carbohydrate meal and remained high for 2h. A subsequent casein meal reversed this change but the ratio remained higher than basal values. A first casein meal reduced the ratio that was not increased again by a subsequent carbohydrate meal. It is obvious that ingestion of specific nutrients induce long-lasting metabolic and neurochemical variations that prevent subsequent changes to occur. The lack of expected changes to a second meal addresses again the hypothesis of alternate appetites for carbohydrates and proteins driven by serotonin changes.

Variations in extracellular monoamines in the prefrontal cortex and medial hypothalamus after modafinil administration: a microdialysis study in rats.

The role of brain amines in mediating the effects of the wake-promoting agent modafinil, used in the treatment of sleepiness associated with narcolepsy is still uncertain. Therefore we studied the effects of modafinil on extracellular serotonin (5-HT), dopamine (DA) and noradrenaline (NA), in rat prefrontal cortex and in the medial hypothalamus area. Modafinil (128 mg/kg i.p.) significantly increased waking in the first 4 h of EEG sleep recording. This cortical and behavioral activation was associated with an initial increase in extracellular 5-HT, DA and NA during the first 60 min following modafinil administration. In the prefrontal cortex, 5-HT release remained high for 3 h after modafinil administration. In contrast, in the hypothalamus, only NA release was enhanced while DA and 5-HT levels remained low. In a first step, modafinil may generate waking partly via cortical monoamine release, particularly DA and 5-HT, and also hypothalamic NA. In a second step, maintenance of waking might depend on hypothalamic NA.

Macronutrient-induced cascade of events leading to parallel changes in hypothalamic serotonin and insulin.

Extracellular serotonin (5-HT) and insulin from hypothalamic PVN-VMH region follow parallel changes in response to specific macronutrient ingestion. Possible independent or causal mechanisms have been investigated. A common primary event might be pancreatic insulin secretion for both insulin entry into the brain and 5-HT synthesis through variations in the ratio of tryptophan over competitor amino acids. The steps of this cascade were found to account only partly for the changes in hypothalamic 5-HT and insulin. The central consequences of these metabolic effects may be modulated directly at the hypothalamic level. For instance, we observed a positive relation between the changes in insulin and 5-HT and the satiating potency of each nutrient. In addition, a direct action of dexfenfluramine on insulin has been found at the hypothalamic level showing that an activation of the serotonergic system immediately enhances insulin levels. This central event may be an important step in a cascade of events triggered by macronutrient ingestion leading to common hypothalamic insulin and 5-HT changes involved in feeding regulation.

[Variations of hypothalamic and cortical prostaglandins and monoamines reveal transitions in arousal states: microdialysis study in the rat]. / Variations des prostaglandines et des monoamines hypothalamiques et corticales révélatrices de la transition entre états de vigilance: étude en microdialyse chez le rat.

Brain microdialysis coupled with EEG recording allowed us to track dynamic neurochemical changes every 3 or 6 minutes in relation to sleep/wake cycles. We chose to investigate prostaglandins (PG) and monoamines (catecholamines, serotonin and metabolites) because of their respective role in the states of vigilance, mainly suggested by pharmacological approaches, and because of the known interactions between PGs and monoamines. We focused on the paraventricular and ventromedial nuclei of the hypothalamus for their involvement in the relationships between feeding, metabolic rate and sleep, and the prefrontal cortex for its role in vigilance. These studies revealed a few changes in prostaglandin or monoamine levels as a function of a given state of vigilance. In particular, serotonin levels were higher during wakefulness than during sleep in both hypothalamus and cortex. Both hypothalamic and cortical PGE2 levels were higher during wakefulness than during slow wave sleep and still higher during paradoxical sleep in the cortex. Cortical PGD2 showed an exactly reverse profile of PGE2. These changes are in agreement with the described awaking action of PGE2 and with the hypnogenic action of PGD2. Our most informative findings were the sequential changes around transitions from one state to another that allow to predict the moment of onset of both sleep and wakefulness. Both in hypothalamus and in cortex, ondulatory patterns of PGE2 were encountered around the transitions between states. PGE2 was high in the middle of wakefulness, then regularly dropped announcing the occurrence of sleep, where the drop persisted before giving place to a rise in prediction to the next period of wakefulness. A similar profile was also observed for cortical serotonin, but its low levels reached a plateau during sleep. Cortical dopamine levels showed sudden and dramatic drops during short periods of wakefulness closely surrounding slow wave sleep. In some instances, as in the case of PGE2, similar profiles of variations could be found both in the hypothalamus and cortex. But in most cases, different and even opposite profiles were encountered in those two structures. Interestingly, in some instances, the pattern of changes in both prostaglandins and monoamines were similar, as for example between hypothalamic PGE2 and dopamine as well as between cortical PGE2 and serotonin. These similarities support the idea of the suggested interaction between prostaglandins and monoamines, in particular concerning their involvement in the regulation of sleep/wake cycles.

A potential role of central insulin in learning and memory related to feeding.

1. Hypothalamic insulin (HI) is well known for its role in feeding regulation. In addition, its concentration is modified in response to meals. Recent studies suggest that brain insulin participates in memory processes, possibly through stimulation by glucose. 2. The present microdialysis study focused on local in vivo regulation of HI by glucose and on the effects of aging on HI, since aging is characterized by deterioration of memory, body weight regulation, and central glucose utilization. Glucose (8 mM) infused for 5 min increased extracellular HI levels rapidly, by 4.6-fold, and cerebellar insulin levels by 0.4-fold only, suggesting a specific area-dependent regulation of HI by glucose. Neither insulinemia nor glycemia were affected, suggesting a central mechanism. The same dose of glucose induced a modest (0.4-fold), delayed (45 min) increase in hypothalamic serotonin, suggesting that the effect of glucose on HI is independent of a previously defined local serotonin-induced insulin release. HI levels in old normal weight rats were half the levels of young rats. In genetically old obese (fa/fa) Zucker rats, HI concentration was 30% of that in young normal rats, suggesting a deterioration of HI availability when aging and obesity are combined. 3. The above results, in line with recent considerations on a potential role of central insulin in learning and memory, suggest particular effects of HI on feeding and memory and probably on a specific "memory for food."

Effects of pure macronutrient ingestion on plasma tryptophan and large neutral amino acids.

The role of tryptophan and its competitor large neutral amino acids, proposed earlier for serotonin synthesis following carbohydrate or protein ingestion, was reassessed in relation to a recent study investigating serotonin release, including the so far unknown effects of fats. In the present study, meals of either carbohydrates, casein, or lard, were supplied to rats for 30 min and blood samples collected every 15 min to follow the changes in plasma large neutral amino acids. In response to carbohydrates, amino acid levels fell and the ratio tryptophan over sum of other amino acids increased. Following casein ingestion, all amino acids were enhanced, tryptophan somewhat less, leading to a decreased ratio. The lard meal induced a slight decrease in some amino acids while the ratio remained constant. Only in response to casein, and partly to carbohydrates, did a consistent relation appear between the previously observed serotonin changes and the ratio. These data suggest that a relationship between the ratio and the previously observed serotonin changes is not always encountered because the release is not obligatorily coupled to synthesis and is subject to behavioral influences. It remains that serotonin release is affected by the composition of the meal through peripheral metabolic mechanisms.

Activation of hypothalamic insulin by serotonin is the primary event of the insulin-serotonin interaction involved in the control of feeding.

In previous experiments, we reported a close parallelism in the responses of both serotonin (5-HT) and insulin in the hypothalamic PVN-VMH region of freely-moving rats during feeding. Thus, hypothalamic 5-HT and insulin may participate, independently or in interaction, in the control of carbohydrate and fat ingestion. The precedence of the activation of one or the other substance remained to be investigated. In adult male Wistar rats, (a) dexfenfluramine was administered to the PVN-VMH region by reverse microdialysis (80 microM for 10 min) while local insulin was assessed; (b) insulin was locally infused (400 mU for 10 min) through the tip of the dialysis probe while 5-HT was measured. Dexfenfluramine immediately increased 5-HT release, and also extracellular insulin levels (+102%). This activation of insulin by serotonin is actually a central effect since neither insulinemia nor glycemia were affected. Conversely, insulin enhanced 5-HT release (+81%), but only 45 min after the beginning of its infusion. Noradrenaline, dopamine and metabolites were slightly or not at all modified by insulin. These data demonstrate that an interaction does exist between insulin and 5-HT in the VMH-PVN area. Because of the delay of 5-HT response to insulin, an activation of the serotonergic system would be the causal event acting immediately on insulin, and not the contrary. Whatever the exact mechanism of this interaction, it seems to be a link in a larger cascade of events involving numerous neurotransmitters and peptides leading to the regulation of feeding.

Neuromodulation of the prefrontal cortex during sleep: a microdialysis study in rats.

To test the hypothesis that biogenic amines of the prefrontal cortex are involved in state-dependent cortical and behavioural activation, changes in extracellular levels of serotonin (5-HT), dopamine (DA), and noradrenaline (NA) were determined during the sleep-wake cycle in freely moving rats using microdialysis probes with parallel EEG recording. Serotonin gradually increased up to 450% during wakefulness (W) as compared to slow wave sleep (SWS), before decreasing toward stable levels during the next episode of SWS. Dopamine and its metabolite homovanillic acid (HVA) were reduced during W as compared to SWS. Although contradictory with the generally admitted enhancement of DA activity related to vigilance, this may be due to the particular role of DA neurons in the prefrontal cortex. However, DA and HVA showed dramatic changes announcing the transition between SWS and W. During paradoxical sleep (PS), DA and 5-HT showed complex changes, the direction of which depended on whether PS was followed by SWS or W. Biogenic amines of the prefrontal cortex are probably involved in cortical and behavioural activation.

Synergistic effect of arcuate and raphe nuclei graft to alleviate insulinemia and obesity in Zucker rats.

The arcuate nucleus (AN) of the hypothalamus is a key area in which endocrine messages are relayed to the brain, while midbrain raphe nucleus (RN) is the source of brain serotonin. Both nuclei contribute to the central mechanism of energy homeostasis. This experiment aimed to determine the impact of AN and RN grafts on insulinemia and obesity in diabetic rats. AN and RN were dissected from 15-day (Fa/Fa) lean embryos and grafted separately or together into the third brain ventricle of obese (fa/fa) male Zucker rats. Histological analysis showed the functional maturity of grafts, which were vascularized, contained neurons reinnervating the periventricular hypothalamus and hypophysis, and expressed neuropeptide Y and enzymes for dopamine and serotonin synthesis. Three months after transplantation, the rats grafted with AN or RN had a lower body weight gain compared to sham-operated rats (19% and 17%, respectively). Rats grafted with AN together with RN gained significantly less body weight than rats grafted with AN or RN separately (31% vs. sham-operated rats), and showed a decreased plasma insulin concentration (132 +/- 33 microU/ml) vs. sham-operated rats (459 +/- 108 microU/ml, p < 0.05). A synergistic effect on alleviating obesity and insulinemia by double AN and RN grafts suggests that both these nuclei are functionally interrelated in maintaining energy homeostasis, and normal functioning of both nuclei is altered during obesity.

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.

Determination, using microdialysis, of hypothalamic serotonin variations in response to different macronutrients.

In response to a chow meal in rats, we observed previously in PVN-VMH dialysates, an increase in serotonin (5-HT) that could be related to satiety or to metabolic consequences of the composition of the meal. Indeed, carbohydrates are admitted to increase 5-HT synthesis while proteins decrease it, but the time course and mechanisms of these effects were not known. For that purpose, pure carbohydrates, proteins, or fats were offered for 30 min and the changes in 5-HT from PVN-VMH dialysates were followed. Carbohydrates (85% starch + 15% sucrose) enhanced 5-HT levels as soon as the first 15 min of feeding, with a maximum 60 min later. Conversely, protein ingestion induced in the second 15 min of the meal, a decrease in 5-HT that lasted 2 h. During a fat meal (lard), 5-HT levels also decreased at the beginning of the meal and remained low during 45 min. The present data reassess the previous theories on the serotonergic effects of specific macronutrient ingestion. The effect of a fat meal on 5-HT levels had never been described so far. The increase in 5-HT in response to a carbohydrate meal is further specified. The 5-HT decrease induced by proteins, in agreement with the previous theories, is better explained now by using pure protein diets and extracellular 5-HT assay. However, all the changes observed start too early to be only metabolic in origin. Other mechanisms may occur, including the release of 5-HT in response to a meal to induce satiety.

Dopamine turnover in the mediobasal hypothalamus in rat fetuses.

In this study, the dopamine turnover in the mediobasal hypothalamus, the key compartment of the neuroendocrine regulation of reproduction, was evaluated in fetal male and female rats. High-performance liquid chromatography with electrochemical detection was used to measure 3,4-dihydroxyphenylalanine, dopamine and 3,4-dihydroxyphenylacetic acid in the mediobasal hypothalamus of fetuses on the 21st day of intrauterine development and in primary cell culture (cell extracts and culture medium) of the same brain region, explanted at the 17th fetal day and maintained for seven days. The same technique was applied to determine dopamine release from fetal neurons of the mediobasal hypothalamus in response to an excess of K+ in the perifusion system or in culture. L-3,4-Dihydroxyphenylalanine, dopamine and 3,4-dihydroxyphenylacetic acid were detected both ex vivo and in culture. The ratios of the concentrations of L-3,4-dihydroxyphenylalanine/dopamine and 3,4-dihydroxyphenylacetic acid/dopamine were significantly higher in vitro than ex vivo, showing a lower rate of dopamine production and a higher rate of its degradation in the experiments in vitro. Moreover, it has been demonstrated that an excess of K+, i.e. a membrane depolarization, resulted in a highly increased release of dopamine in the perifusion system and in culture. The dopaminergic activity in the developing mediobasal hypothalamus showed sexual dimorphism that was manifested in a greater concentration of 3,4-dihydroxyphenylalanine and dopamine, at least in cell extracts of cultures, as well as in a higher rate of dopamine release, both in the perifusion system and in culture in males compared to females. Thus, dopamine is synthesized and released in response to a membrane depolarization in the mediobasal hypothalamus of rats as early as the end of intrauterine development, suggesting its contribution to the inhibitory control of pituitary prolactin secretion.

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

Using microdialysis, we showed recently that hypothalamic immunoreactive 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.

Combined effect of obesity and aging on feeding-induced monoamine release in the rostromedial hypothalamus of the Zucker rat.

OBJECTIVE: The obesity of the Zucker rat is associated with numerous metabolic and neurochemical disturbances involving the central transmitters regulating feeding behaviour. Among them, the release of satiety-related monoamines from the median hypothalamus in response to a meal is enhanced in obese, as compared to normal, rats as though larger amounts of these amines were necessary to bring about satiety in obese rats. Besides, the obese Zucker rat has often been described as shorter-living than its lean congener. One of the reasons for the shorter longevity of the obese rat was investigated in this study: it could be an aggravation of its obesity-related central disturbances with age. METHODS: We assessed the response to a meal of the hypothalamic monoamines, dopamine and serotonin, in young (four month old) and old (twelve month old) lean (Fa-Fa) and obese (fa-fa) Zucker rats. The in vivo technique of microdialysis was used to combine behavioural recordings and continuous neurochemical assays. RESULTS: The exacerbation of monoamine release observed in young obese rats in response to a meal was no longer found in old obese rats. Serotonin increase during a meal weakened with aging, especially in obese rats. Dopamine (DA) response to a meal was completely reversed in old obese rats, with a decrease instead of the increase observed in the three other groups. CONCLUSION: The decrease of monoaminergic response to a meal with age is apparently the opposite to the enhanced release related to obesity. However, this does not correspond to an amelioration of the hyperphagia of the obese rats with age, as we could observe in parallel behavioural experiments, but rather to a decrease in neurotransmitter metabolism and thus in neuronal functioning.

Prolactin secretion and its dopamine inhibitory control in rat fetuses.

This study has determined in rats the ontogenetic schedule of the onset of pituitary prolactin (PRL) synthesis and release as well as of the establishment of the dopamine (DA) inhibitory control of PRL secretion. RIA recognized PRL traces in the pituitary at the 18th embryonic day (E18), although a clearly detectable amount of this hormone was first measured at E20, suggesting the onset of PRL synthesis. The PRL level in the pituitary increased significantly by E22, in females to a higher extent than in males. Decapitation of fetuses did not cause any change in the PRL plasma level in males showing no PRL release from the pituitary until term. Conversely, there was a slight but significant fall of plasma PRL in decapitated females, suggesting PRL release from the pituitary. An inhibition of DA receptors on lactotropes of fetuses resulted in an increased level of plasma PRL at E20, but not at E18, while the pituitary content of PRL remained unchanged. The same treatment at E22 caused a significant increase of the PRL concentration in plasma and a concomitant fall in the pituitary that could be prevented by preliminary encephalectomy. These data show that the tuberoinfundibular DA system begins to inhibit PRL release from lactotropes between E20 and E22, completely arresting PRL release from the pituitary in males but not in females.

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'

Microdialysis and EEG in rats reveal cortical PGE2 changes during sleep and wakefulness.

Prostaglandin (PG) E2 production was assessed in freely moving rats using the technique of microdialysis in the prefrontal cortex associated with parallel cortical EEG recordings. PGE2 concentrations were 40% higher during wakefulness than during slow wave sleep. PGE2 values varied during wakefulness with a maximal increase in the middle of the stage and a drop towards lower values before the occurrence of slow wave sleep. These variations were similar to those observed previously in the rostromedial hypothalamus, where PGE2 concentration was 2.6 times lower than that in the cortex. These data document a positive correlation between cortical EEG activation and PGE2 levels. Taken together with pharmacological data on the awakening effect of centrally administered PGE2, these observations are in favor of an involvement of PGE2 in the generation of wakefulness.

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.