Alterations in the Serotonin and Dopamine Pathways by Cystathionine Beta Synthase Overexpression in Murine Brain.

Cystathionine beta synthase (CBS) is one of the 225 genes on chromosome 21 (HSA 21) that are triplicated in persons with trisomy 21 (Down syndrome). Although most triplicate HSA21 genes have their orthologous genes on murine chromosome 16, the murine ortholog of hCBS is on murine chromosome 17 and thus is not present in the well-studied Ts65Dn mouse model of trisomy 21. Persons with trisomy 21 (T21) present deficits in neurotransmission and exhibit early brain aging that can partially be explained by monoamine neurotransmitter alterations. We used transgenic mice for the hCBS gene, which overexpress the CBS protein in various brain regions, to study if CBS overexpression induces modifications in the monoamine neurotransmitters in the hypothalamus, thalamus, hippocampus, and striatum from transgenic and control female and male mice aged 3-4 months and 11-12 months. Sex, age, and brain area each influenced neurotransmitter levels. Briefly, the serotonin pathway was modified by CBS overexpression in various brain areas in female mice but not in male mice. The dopamine pathway was modified in brain regions according to sex and age. These results may allow us to better understand the role of the transsulfuration pathway and especially CBS overexpression in the metabolism of biogenic amines and the catecholamine catabolism in persons with trisomy 21.

Dietary triglycerides act on mesolimbic structures to regulate the rewarding and motivational aspects of feeding.

Circulating triglycerides (TGs) normally increase after a meal but are altered in pathophysiological conditions, such as obesity. Although TG metabolism in the brain remains poorly understood, several brain structures express enzymes that process TG-enriched particles, including mesolimbic structures. For this reason, and because consumption of high-fat diet alters dopamine signaling, we tested the hypothesis that TG might directly target mesolimbic reward circuits to control reward-seeking behaviors. We found that the delivery of small amounts of TG to the brain through the carotid artery rapidly reduced both spontaneous and amphetamine-induced locomotion, abolished preference for palatable food and reduced the motivation to engage in food-seeking behavior. Conversely, targeted disruption of the TG-hydrolyzing enzyme lipoprotein lipase specifically in the nucleus accumbens increased palatable food preference and food-seeking behavior. Finally, prolonged TG perfusion resulted in a return to normal palatable food preference despite continued locomotor suppression, suggesting that adaptive mechanisms occur. These findings reveal new mechanisms by which dietary fat may alter mesolimbic circuit function and reward seeking.

Comparative effects of Citrullus colocynthis, sunflower and olive oil-enriched diet in streptozotocin-induced diabetes in rats.

Citrullus colocynthis (colocynth) seeds are traditionally used as antidiabetic medication in Mediterranean countries. The present study evaluated the differential effects of diets enriched with C. colocynthis, sunflower or olive oils on the pancreatic beta-cell mass in streptozotocin (STZ)-induced diabetes in rats. STZ injection induced rapid hyperglycaemia in all animals. However, 2 months later, hyperglycaemia was significantly less pronounced in the rats fed a C. colocynthis oil-enriched diet compared with other rat groups (7.9mM versus 12mM and 16mM with colocynth versus olive and sunflower oils, respectively). Assessment of insulin sensitivity using the homoeostasis model assessment (HOMA) method also indicated less insulin resistance in the rats fed a C. colocynthis oil-enriched diet versus the other rats. Finally, 2 months after STZ injection, the pancreatic beta-cell mass was similar in both the STZ-treated rats fed the colocynth oil-enriched diet and their controls fed the same diet. In contrast, the pancreatic beta-cell mass remained lower in the STZ-induced diabetic rats fed with olive oil- and sunflower oil-enriched diets compared with the C. colocynthis group. We conclude that C. colocynthis oil supplementation may have a beneficial effect by partly preserving or restoring pancreatic beta-cell mass in the STZ-induced diabetes rat model.

NADP-malate dehydrogenase gene evolution in Andropogoneae (Poaceae): gene duplication followed by sub-functionalization.

BACKGROUND AND AIMS: Plastid NADP-dependent malate dehydrogenase (MDH) catalyses the conversion of oxaloacetate to malate. In C4 plants, it is involved in photosynthetic carbon assimilation. In Poaceae, one NADP-MDH gene has been identified in rice (C3; Erhartoideae) and maize (C4; Panicoideae), whereas two tandemly repeated genes have been identified in Sorghum (C4; Panicoideae). In the present study, the molecular evolution of the NADP-MDH multigene family was investigated in order to analyse how the C4 isoform has evolved over a broader range of panicoid grasses. METHODS: Polymerase chain reaction (PCR)-based cloning was used to isolate cDNAs encoding NADP-MDHs from 15 species of Panicoideae. A gene phylogeny was reconstructed based on cDNA sequences using distance and maximum parsimony methods. Episodic selection along some branches of the phylogenetic tree was tested by analysing non-synonymous and synonymous rate ratios. Transcription of NADP-MDH genes was compared in green leaves of five accessions of Saccharum, Sorghum and Vetiveria using a semi-quantitative PCR approach. KEY RESULTS: Phylogenetic analyses of these data support the existence of two NADP-MDH gene lineages (NMDH-I and NMDH-II) in several Andropogoneae (i.e. Saccharum, Sorghum and Vetiveria). Episodic positive selection was shown along the basal branch of the NMDH-II clade. Three amino acid modifications allow the two gene lineages to be distinguished, suggesting a positive selection at these sites. In green leaves, we showed that the transcript accumulation was higher for NMDH-I than for NMDH-II. CONCLUSIONS: It is hypothesized that the maintenance of both NADP-MDH genes in some Andropogoneae is due to a partition of the original functions across both copies. NMDH-I probably corresponds to the C4 isoform as previously suggested. Nevertheless, some C4 species (e.g. maize) only have one gene which should be selected for its high expression level in leaves. This study confirms that gene duplicates have been recruited for C4 photosynthesis but are not required in every case.

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.

Assessment of the C(4) phosphoenolpyruvate carboxylase gene diversity in grasses (Poaceae).

C(4) phosphoenolpyruvate carboxylase (PEPC) is a key enzyme in the C(4) photosynthetic pathway. To analyze the diversity of the corresponding gene in grasses, we designed PCR primers to specifically amplify C(4) PEPC cDNA fragments. Using RT-PCR, we generated partial PEPC cDNA sequences in several grasses displaying a C(4) photosynthetic pathway. All these sequences displayed a high homology (78-99%) with known grass C(4) PEPCs. PCR amplification did not occur in two grasses that display the C(3) photosynthetic pathway, and therefore we assumed that all generated sequences corresponded to C(4) PEPC transcripts. Based on one large cDNA segment, phylogenetic reconstruction enabled us to assess the relationships between 22 grass species belonging to the subfamilies Panicoideae, Arundinoideae and Chloridoideae. The phylogenetic relationships between species deduced from C(4) PEPC sequences were similar to those deduced from other molecular data. The sequence evolution of the C(4) PEPC isoform was faster than in the other PEPC isoforms. Finally, the utility of the C(4) PEPC gene phylogeny to study the evolution of C(4) photosynthesis in grasses is discussed.

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.

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.

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.

Purification and characterization of a multienzymatic complex in sugarcane, a C4 plant.

We have discovered a multienzymatic complex in fresh young sugarcane leaves. This complex is constituted of three enzymes: PEPcase, NADP-MDH and malic enzyme. After successive molecular sieving chromatography, we have obtained a highly purified sample of the complex which has a molecular weight of 711 kDa. Its functional interest has been evaluated by comparing the kinetic properties of the enzymes in their free forms to those in their complexed form. We show that the association of the three enzymes leads to important changes in their respective kinetic properties.

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.

Inhibition of palmito polyphenoloxidase by halide salts.

The inhibitory properties of halide salts on palmito polyphenoloxidase (PPO) are described. Halide salts have the same inhibitory effect on the two forms of palmito PPO separated by hydrophobic chromatography. Fluoride and chloride ions showed a non-competitive, mixed type inhibition while bromide and iodide ions were found to be non-competitive inhibitors. A study of the Ki for the different halide salts showed that the smaller F- ion is a stronger inhibitor than I- and Br- and that Cl- has the highest Ki value. This suggests that the active site of the palmito PPO is not easily accessible. The inhibition by chloride and fluoride ion was found to be pH-dependent. The inhibitory effects of these ions increased with a decrease in pH. It is suggested that halide ions (X) could bind to either the protonated enzyme (EH) or the protonated substrate-enzyme complex (EHS) to yield inactive forms EHX and EHSX, respectively.

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.

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.