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Aims/hypothesis Drug and surgical-based therapies in type 2 diabetes are associated with altered gut microbiota architecture. Here we investigated the role of the gut microbiome in improved glucose homeostasis following bariatric surgery. Methods We carried out gut microbiome analyses in gastrectomised (by vertical sleeve gastrectomy [VSG]) rats of the Goto–Kakizaki (GK) non-obese model of spontaneously occurring type 2 diabetes, followed by physiological studies in the GK rat. Results VSG in the GK rat led to permanent improvement of glucose tolerance associated with minor changes in the gut microbiome, mostly characterised by significant enrichment of caecal Prevotella copri. Gut microbiota enrichment with P. copri in GK rats through permissive antibiotic treatment, inoculation of gut microbiota isolated from gastrectomised GK rats, and direct inoculation of P. copri, resulted in significant improvement of glucose tolerance, independent of changes in body weight. Plasma bile acids were increased in GK rats following inoculation with P. copri and P. copri-enriched microbiota from VSG-treated rats; the inoculated GK rats then showed increased liver glycogen and upregulated expression of Fxr (also known as Nr1h4), Srebf1c, Chrebp (also known as Mlxipl) and Il10 and downregulated expression of Cyp7a1. Conclusions Our data underline the impact of intestinal P. copri on improved glucose homeostasis through enhanced bile acid metabolism and farnesoid X receptor (FXR) signalling, which may represent a promising opportunity for novel type 2 diabetes therapeutics.

Dietary n-3 polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), are known to influence glucose homeostasis. We recently showed that Elovl2 expression in beta cells, which regulates synthesis of endogenous DHA, was associated with glucose tolerance and played a key role in insulin secretion. The present study aimed to examine the role of the very long chain fatty acid elongase 2 (ELOVL2)/DHA axis on the adverse effects of palmitate with high glucose, a condition defined as glucolipotoxicity, on beta cells. We detected ELOVL2 in INS-1 beta cells and mouse and human islets using quantitative PCR and western blotting. Downregulation and adenoviral overexpression of Elovl2 was carried out in beta cells. Ceramide and diacylglycerol levels were determined by radio-enzymatic assay and lipidomics. Apoptosis was quantified using caspase-3 assays and poly (ADP-ribose) polymerase cleavage. Palmitate oxidation and esterification were determined by [U-14C]palmitate labelling. We found that glucolipotoxicity decreased ELOVL2 content in rodent and human beta cells. Downregulation of ELOVL2 drastically potentiated beta cell apoptosis induced by glucolipotoxicity, whereas adenoviral Elovl2 overexpression and supplementation with DHA partially inhibited glucolipotoxicity-induced cell death in rodent and human beta cells. Inhibition of beta cell apoptosis by the ELOVL2/DHA axis was associated with a decrease in ceramide accumulation. However, the ELOVL2/DHA axis was unable to directly alter ceramide synthesis or metabolism. By contrast, DHA increased palmitate oxidation but did not affect its esterification. Pharmacological inhibition of AMP-activated protein kinase and etomoxir, an inhibitor of carnitine palmitoyltransferase 1 (CPT1), the rate-limiting enzyme in fatty acid β-oxidation, attenuated the protective effect of the ELOVL2/DHA axis during glucolipotoxicity. Downregulation of CPT1 also counteracted the anti-apoptotic action of the ELOVL2/DHA axis. By contrast, a mutated active form of Cpt1 inhibited glucolipotoxicity-induced beta cell apoptosis when ELOVL2 was downregulated. Our results identify ELOVL2 as a critical pro-survival enzyme for preventing beta cell death and dysfunction induced by glucolipotoxicity, notably by favouring palmitate oxidation in mitochondria through a CPT1-dependent mechanism.

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.

International audience; Trisomy 21 (T21) or Down syndrome (DS) is the most common genetic disorder associated with intellectual disability and affects around 5 million persons worldwide. Neuroanatomical phenotypes associated with T21 include slight reduction of brain size and weight, abnormalities in several brain areas including spines dysgenesis, dendritic morphogenesis, and early neuroanatomical characteristics of Alzheimer's disease. Monoamine neurotransmitters are involved in dendrites development, functioning of synapses, memory consolidation, and their levels measured in the cerebrospinal fluid, blood, or brain areas that are modified in individuals with T21. DYRK1A is one of the recognized key genes that could explain some of the deficits present in individuals with T21. We investigated by high-performance liquid chromatography with electrochemical detection the contents and processing of monoamines neurotransmitters in four brain areas of female and male transgenic mice for the Dyrk1a gene (mBactgDyrk1a). DYRK1A overexpression induced dramatic deficits in the serotonin contents of the four brain areas tested and major deficits in dopamine and adrenaline contents especially in the hypothalamus. These results suggest that DYRK1A overexpression might be associated with the modification of monoamines content found in individuals with T21 and reinforce the interest to target the level of DYRK1A expression as a therapeutic approach for persons with T21.

Introduction et but de l’etude Des etudes montrent que l’accumulation de lipides dans l’hypothalamus serait responsable de l’installation d’une lipotoxicite centrale. Ce phenomene pourrait jouer un role dans l’apparition d’une insulinoresistance peripherique et du diabete de type 2 en deregulant le controle nerveux de l’homeostasie glucidique. L’accumulation des ceramides est impliquee dans le developpement d’une lipotoxicite des tissus peripheriques. L’objectif de cette etude est de determiner le role du metabolisme des ceramides au niveau hypothalamique dans l’installation d’une insulinoresistance centrale et d’etudier les mecanismes impliques. Nous avons determine le role du metabolisme des ceramides hypothalamiques dans la deregulation de l’homeostasie glucidique induite par l’obesite. Materiel et methodes L’installation d’une insulinoresistance centrale est etudiee a l’aide d’approches in vitro, en utilisant des cellules hypothalamiques de souris GT1-7 traitees avec 1 mM de palmitate (acide gras sature) pendant 24 h. L’insulinoresistance est mesuree par quantification d’Akt phosphorylee (western blot). Les ceramides sont quantifies par lipidomique, l’expression d’ARNm codant pour les genes de la voie de synthese de novo des ceramides par qRT-PCR. Des rats Zucker obeses sont perfuses avec la myriocine (inhibiteur de la synthese de novo des ceramides) en ICV pendant 21 jours. Des tests de sensibilite a l’insuline et de tolerance au glucose sont realises. A la fin du traitement, ils recoivent une injection ICV d’insuline, l’insulinoresistance hypothalamique est mesuree et les ceramides sont quantifies. Les ilots de Langerhans sont isoles pour des tests de secretion d’insuline in vitro. Resultats et analyse statistique Nous avons mis en evidence une insulinoresistance dans la lignee hypothalamique GT1-7 traitees avec le palmitate qui s’accompagne d’une accumulation de ceramides. En presence de myriocine, les ceramides ne sont plus accumules et le l’insulinoresistance induite par le palmitate est contre-carree. L’action insulinoresistante des ceramides a la peripherie est connue pour etre mediee par la PKC ζ. En utilisant un inhibiteur et un virus contre PKC ζ, nous avons egalement montre que le palmitate n’est plus capable d’induire une insulinoresistance et ce malgre la presence d’une accumulation de ceramides. Chez le rat Zucker obese, nous mise en evidence une accumulation de ceramides hypothalamiques qui est contre-carree par la myriocine. Ceci est associe avec une amelioration de la sensibilite a l’insuline dans l’hypothalamus. De facon, interessante, ces animaux ameliorent leur tolerance au glucose associe a une augmentation de la secretion d’insuline. Les ilots de Langerhans isoles a partir de ces rats presentent une capacite secretoire augmentee lors du traitement avec la myriocine. Conclusion Au final, notre etude revele que la lipotoxicite hypothalamique est associee a une accumulation de ceramides dans cette structure, responsable de l’installation d’une insulinoresistance. Ces resultats mettent egalement en evidence le role cle du metabolisme des ceramides au niveau de l’hypothalamus dans la deregulation du controle nerveux de l’homeostasie glucidique induit par l’obesite.

Brain lipid sensing is necessary to regulate energy balance. Lipoprotein lipase (LPL) may play a role in this process. We tested if hippocampal LPL regulated energy homeostasis in rodents by specifically attenuating LPL activity in the hippocampus of rats and mice, either by infusing a pharmacological inhibitor (tyloxapol), or using a genetic approach (adeno-associated virus expressing Cre-GFP injected into Lpl (lox/lox) mice). Decreased LPL activity by either method led to increased body weight gain due to decreased locomotor activity and energy expenditure, concomitant with increased parasympathetic tone (unchanged food intake). Decreased LPL activity in both models was associated with increased de novo ceramide synthesis and neurogenesis in the hippocampus, while intrahippocampal infusion of de novo ceramide synthesis inhibitor myriocin completely prevented body weight gain. We conclude that hippocampal lipid sensing might represent a core mechanism for energy homeostasis regulation through de novo ceramide synthesis.

Dietary supplement may potentially help to fight obesity and other metabolic disorders such as insulin-resistance and low-grade inflammation. The present study aimed to test whether supplementation with Agaricus blazei murill (ABM) extract could have an effect on diet-induced obesity in rats. Wistar rats were fed with control diet (CD) or high-fat diet (HF) and either with or without supplemented ABM for 20 weeks. HF diet-induced body weight gain and increased fat mass compared to CD. In addition HF-fed rats developed hyperleptinemia and insulinemia as well as insulin resistance and glucose intolerance. In HF-fed rats, visceral adipose tissue also expressed biomarkers of inflammation. ABM supplementation in HF rats had a protective effect against body weight gain and all study related disorders. This was not due to decreased food intake which remained significantly higher in HF rats whether supplemented with ABM or not compared to control. There was also no change in gut microbiota composition in HF supplemented with ABM. Interestingly, ABM supplementation induced an increase in both energy expenditure and locomotor activity which could partially explain its protective effect against diet-induced obesity. In addition a decrease in pancreatic lipase activity is also observed in jejunum of ABM-treated rats suggesting a decrease in lipid absorption. Taken together these data highlight a role for ABM to prevent body weight gain and related disorders in peripheral targets independently of effect in food intake in central nervous system.

Obesity-related diseases such as diabetes and dyslipidemia result from metabolic alterations including the defective conversion, storage and utilization of nutrients, but the central mechanisms that regulate this process of nutrient partitioning remain elusive. As positive regulators of feeding behaviour, agouti-related protein (AgRP) producing neurons are indispensible for the hypothalamic integration of energy balance. Here, we demonstrate a role for AgRP-neurons in the control of nutrient partitioning. We report that ablation of AgRP-neurons leads to a change in autonomic output onto liver, muscle and pancreas affecting the relative balance between lipids and carbohydrates metabolism. As a consequence, mice lacking AgRP-neurons become obese and hyperinsulinemic on regular chow but display reduced body weight gain and paradoxical improvement in glucose tolerance on high-fat diet. These results provide a direct demonstration of a role for AgRP-neurons in the coordination of efferent organ activity and nutrient partitioning, providing a mechanistic link between obesity and obesity-related disorders.

International audience; Feeding behavior is exquisitely regulated by homeostatic and hedonic neural substrates that integrate energy demand as well as the reinforcing and rewarding aspects of food. Understanding the net contribution of homeostatic and reward-driven feeding has become critical because of the ubiquitous source of energy-dense foods and the consequent obesity epidemic. Hypothalamic agouti-related peptide-secreting neurons (AgRP neurons) provide the primary orexigenic drive of homeostatic feeding. Using models of neuronal inhibition or ablation, we demonstrate that the feeding response to a fast ghrelin or serotonin receptor agonist relies on AgRP neurons. However, when palatable food is provided, AgRP neurons are dispensable for an appropriate feeding response. In addition, AgRP-ablated mice present exacerbated stress-induced anorexia and palatable food intake--a hallmark of comfort feeding. These results suggest that, when AgRP neuron activity is impaired, neural circuits sensitive to emotion and stress are engaged and modulated by food palatability and dopamine signaling.

International audience; Recent studies reported the impact of leptin on peripheral insulin sensitivity and glucose utilization. However, little is known concerning the effect of central leptin on hypothalamic and hepatic insulin efficiency. This study aimed to determine the consequence of chronic intra-cerebroventricular (ICV) leptin or murine leptin antagonist (MLA) infusion on hypothalamic and hepatic insulin signaling pathways, in rats. A 2-week central leptin infusion enhanced insulin-dependent Akt phosphorylation in the liver without changing PTP-1B protein expression, associated to insulin receptor (IR) upregulation and reduced IRS-1 phosphorylation on Ser302 residue. In the hypothalamus, a chronic ICV leptin infusion induced PTP-1B associated with a specific decrease in insulin-dependent Akt phosphorylation. In contrast, a chronic MLA infusion did not alter IR and PTP-1B expressions in hypothalamus and liver. Our results underline a brain leptin-dependent increase in hepatic insulin efficiency as mirrored by IR up-regulation, increased insulin-dependent Akt phosphorylation and reduced IRS-1 phosphorylation on Ser302 residue.

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.

Differents elements exterieurs au systeme nerveux central (SNC) peuvent influencer son fonctionnement et sa reponse a des agressions. D’une part, l’alimentation, selon sa composition, peut augmenter ou diminuer les risques de neurodegenerescence. D’autre part, des etudes epidemiologiques et des experiences sur des modeles animaux montrent que le surpoids et l’obesite augmentent le risque de developper des maladies neurodegeneratives (Alzheimer, Parkinson…) ainsi que la susceptibilite a des neurodegenerescences aigues (traumatisme, ischemie, chimique…). La neurogenese constitutive ou induite dans le SNC adulte represente un espoir therapeutique permettant de compenser les pertes liees aux phenomenes neurodegeneratifs. Cependant, meme s’il a ete montre que l’alimentation peut affecter la production de nouveaux neurones, l’influence de l’exces de tissu adipeux sur la neurogenese n’est pas encore determinee.

It is now clearly demonstrated that fatty acids (FAs) may modulate neural control of energy homeostasis and specifically affect both insulin secretion and action. Indeed, pancreatic β-cells receive rich neural innervation and FAs induce important changes in autonomic nervous activity. We previously reported that chronic infusion of lipids decreased sympathetic nervous system activity and led to exaggerated glucose-induced insulin secretion (GIIS), as would be expected from the known inhibitory effect of sympathetic splanchnic nerve activity on insulin secretion. Intracarotid infusion of lipids that do not change plasma FA concentrations also lead to increased GIIS. This effect of FAs on GIIS was prevented by inhibition of β-oxidation. It is noteworthy that a single intracarotid injection of oleic acid also induced a transient increase in plasma insulin without any change in plasma glucose, suggesting that FAs per se can regulate neural control of insulin secretion. Finally, using whole cell current clamp recordings in hypothalamic slices and calcium imaging in dissociated hypothalamic neurons, we identified a hypothalamic subpopulation of neurons either excited (13%) or inhibited (6%) by FAs. Thus, FAs per se or their metabolites modulate neuronal activity, as a means of directly monitoring ongoing fuel availability by central nervous system nutrient-sensing neurons involved in the regulation of insulin secretion.

International audience; 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.

In this study, we have investigated the frequencies of TAP1 and TAP2 alleles in a group of 226 persons, living in La Reunion Island, consisting of 70 patients with insulin-dependent diabetes mellitus (IDDM) and most of their first degree relatives ( i.e. , 156 parents and full sibling subjects) and previously HLA DQB1, DQA1, and DRB1 genotyped. The population of this island is constituted by a particular structure of highly crossbreeding people. Interestingly, the new TAP2*0104 allele, previously discovered by our team in Reunion Island, was found to be increased in the IDDM population and the calculated HRR was relatively high (HRR = 3.3). This result seems to be due to a positive linkage disequilibrium between TAP2*0104 allele and the highly diabetogenous DQB1* 0201–DQA1* 0501–DRB1 0301 haplotype (HRR = 9), which suggests that TAP2*0104 cannot be considered as an additional predispositional factor, but more as a genetic susceptibility marker of IDDM. In addition, we show that minor alleles (TAP2D, *0102, *0103, *0104) are associated with a restricted number of HLA DQ-DR haplotypes and each of them exhibits a preferential linkage with one particular haplotype. In contrast with other alleles, and despite a HRR value close to 1, we show that TAP2*0102 allele contributes significantly to a drastic reduction of the diabetogenic effect of DQB1*0201–DQA1*0301.1–DRB*0701 haplotype. Indeed, this haplotype, which is usually preferentially transmitted to affected children, is dominantly transmitted to healthy children when it is associated with TAP2*0102. Therefore, this allele seems to contribute to genetic protection to IDDM.

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.

In previous experiments, we described a decrease in extracellular hypothalamic serotonin, 5-hydroxy-tryptamine (5-HT), in response to ingestion of a lard meal. This effect was related to the low satiating potency of lard as compared with other nutrients. Since the composition in fatty acids might be important for these effects, the purpose of this study was to analyze the neurochemical and behavioral responses to ingestion of different sources of fat. Unique meals of three margarines used in human feeding were compared with a meal of lard with regard to their effects on hypothalamic 5-HT, satiety, anxiety-like behavior and sucrose consumption. A vegetable margarine with a high content in saturated fatty acids also decreased hypothalamic 5-HT while ingestion of a sunflower oil and an olive oil enriched margarines, both high in polyunsaturated fatty acids, did not affect significantly 5-HT levels. However, these two last ones were not the most satiating. The olive oil margarine induced a tendency to an anxiety-like behavior while lard increased sucrose consumption. Thus, ingestion of fats may alter specifically behavioral responses. The involvement of 5-HT is likely in the case of lard ingestion but probably not for the other sources of fat.

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.

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."

Reproductive function is highly dependent on nutritional input. We recently provided evidence that the unsaturated ω6 fatty acid (FA), linoleic acid (linoleic), interferes with transcription and secretion of the gonadotropin LH, highlighting the existence of a lipid sensing in pituitary gonadotropes. Here, we show, using a combination of in vivo and in vitro models, that linoleic differentially regulates Lhb and Fshb expression. Central exposure of rats to linoleic over 7 days was associated with increase of Lhb but not Fshb transcript levels. Consistently, exposure of rat pituitary cells or LβT2 cells to linoleic increased Lhb, whereas it dramatically decreased Fshb transcript levels without affecting its stability. This effect was also induced by ω9 and ω3-polyunsaturated FA but not by saturated palmitic acid. Analysis of the underlying mechanisms in LβT2 cells using small interfering RNA revealed that early growth response protein 1 mediates linoleic stimulation of Lhb expression. Furthermore, we demonstrated that linoleic counteracts activin and bone morphogenetic protein-2 stimulation of Fshb expression. Using Western blotting and Smad-responsive reporter gene assays, linoleic was shown to decrease basal Smad2/3 phosphorylation levels as well as activin- and bone morphogenetic protein-2-dependent activation of Smad, uncovering a new FA-sensitive signaling cascade. Finally, the protein phosphatase magnesium-dependent 1A was shown to mediate linoleic inhibition of basal Smad phosphorylation and Fshb expression, identifying protein phosphatase magnesium-dependent 1A as a new target of FA in gonadotropes. Altogether, this study provides a novel mechanism by which FAs target gene expression and underlines the relevant role of pituitary gonadotropes in mediating the effects of nutritional FA on reproductive function.

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.

Several methods for protein determination have been described. In almost all cases, a detection reagent is involved (Coomassie Blue reagent, bicinchoninic acid, Folin reagent). Proteins quantification determination by measurements in the U.V. region1 have been abandonned because of the weak sensitivity of the apparatus (concentrations of the order of 100 μg/ml) and because of the lack of precision at low wavelengths (far U.V.). Owing to the progress in the performance of equipments, (i) we have been able to show that the reliability of current equipments allowed measurements in the more sensitivity range of proteins (190 to 220 nm) and beyond (220 to 1100 nm) and (ii) we have hence calculated the correlation factors between absorbance values and wavelengths for 17 proteins. It was found that 190 nm and 277 nm were the best wavelengths in far U.V. region for the protein quantification (correlation factor respectively of 0.69 and 0.62). The concentrations of 16 proteins were predicted at 190 nm and...

The inhibition of palmito (Acanthophoenix rubra) polyphenol oxidase (PPO) is reported. Recently, two forms of palmito PPO were partially purified by hydrophobic chromatography. Inhibitory effects of various carboxylic acids on these two forms have been studied. Both forms showed identical behaviour towards the inhibitors studied. Cinnamic acid was found to have the greatest inhibitory effect (Ki = 0·06 mM). When the inhibitory effects of acids from the benzoic acid family and from the cinnamic acid family were compared, it was found that acids which possess a double bond between the benzene ring and the carboxylic function showed the highest inhibitory effect. This inhibitory effect was decreased by substitutions on the benzene ring. The influence of pH on the inhibitory effect of carboxylic acids on palmito PPO has also been investigated. Ki decreased with a decrease in pH. This effect is due to the fact that it is only the undissociated form (AH) of carboxylic acids that is responsible for inhibition of PPO. Inhibition constants (Ki for the AH form have been recalculated and were found to remain constant in the pH range studied (for benzoic acid Ki = 0·14 mM, for cinnamic acid Ki = 0·019 mM, for sorbic acid Ki = 0·15 mM).

The optimal temperature of palmito (Acanthophoenix rubra) polyphenol oxidase (PPO) is 30 degrees C. The Arrhenius activation energy was calculated to be 5.41 kJ mol(-1). Standard enthalpy of the reaction is -60.99 kJ mol(-1). At 25 degrees C, standard free energy and standard entropy were, respectively, 16.75 kJ mol(-1) and -260.87 J mol(-1) K(-1). The enzyme heated at temperatures above 30 degrees C loses its activity. Fifty percent inhibition is reached in 18 min at 70 degrees C, in 8 min at 75 degrees C, and in 2.5 min at 80 degrees C. The kinetics of the thermal irreversible denaturation of this enzyme is characterized by two steps: N leads to X(Td) leads to D, where N represents the native form, X represents an intermediate form, the structure of which depends on the deactivation temperature Td, and D is the completely denatured form of the enzyme. Our experimental results rule out a two-isoenzyme (with varying heat sensitivity) model. The thermodynamic parameters of the irreversible denaturation of the intermediate form were 102.70 and 97.10 kJ mol(-1) for activation enthalpy and activation energy, respectively, and 16.85 J mol(-1) K(-1) for activation entropy at 60 degrees C. Furthermore, this paper describes the effect of pH on the activity of the PPO. Studies were carried out with 4-methylcatechol and pyrogallol as substrates. The pH profile was not a function of the nature of the substrate assayed. The pH optimum was 5.2. The plot of logVmax app vs pH indicates that the oxidation of the substrates depended of the ionization of two groups in the enzyme-substrate complex with apparent pK values of 3.06 and 7.29 and 3.44 and 7.12, respectively, for 4-methylcatechol and pyrogallol. The very slight differences between the values suggest the existence of only one site on the molecule for both substrates.

Dietary supplement may potentially help to fight obesity and other metabolic disorders such as insulin-resistance and low-grade inflammation. The present study aimed to test whether supplementation with Agaricus blazei murill (ABM) extract could have an effect on diet-induced obesity in rats.Wistar rats were fed with control diet (CD) or high-fat diet (HF) and either with or without supplemented ABM for 20 weeks.HF diet-induced body weight gain and increased fat mass compared to CD. In addition HF-fed rats developed hyperleptinemia and insulinemia as well as insulin resistance and glucose intolerance. In HF-fed rats, visceral adipose tissue also expressed biomarkers of inflammation. ABM supplementation in HF rats had a protective effect against body weight gain and all study related disorders. This was not due to decreased food intake which remained significantly higher in HF rats whether supplemented with ABM or not compared to control. There was also no change in gut microbiota composition in HF supplemented with ABM. Interestingly, ABM supplementation induced an increase in both energy expenditure and locomotor activity which could partially explain its protective effect against diet-induced obesity. In addition a decrease in pancreatic lipase activity is also observed in jejunum of ABM-treated rats suggesting a decrease in lipid absorption.Taken together these data highlight a role for ABM to prevent body weight gain and related disorders in peripheral targets independently of effect in food intake in central nervous system.

International audience; Serotonin and insulin are key regulators of homeostatic mechanisms in the hypothalamus. However, in type 2 diabetes, the hypothalamic responsiveness to serotonin is not clearly established. We used a diabetic model, the Goto Kakizaki (GK) rats, to explore insulin receptor expression, insulin and serotonin efficiency in the hypothalamus and liver by means of Akt phosphorylation. Insulin or dexfenfluramine (stimulator of serotonin) treatment induced Akt phosphorylation in Wistar rats but not in GK rats that exhibit down-regulated insulin receptor. Studies in a neuroblastoma cell line showed that serotonin-induced Akt phosphorylation is PI3-kinase dependent. Finally, in response to food intake, hypothalamic serotonin release was reduced in GK rats, indicating impaired responsiveness of this neurotransmitter. In conclusion, hypothalamic serotonin as insulin efficiency is impaired in diabetic GK rats. The insulin-serotonin cross-talk and impairment observed is one potential key modification in the brain during the onset of diabetes.

We have synthesized a series of non-glycosidically linked di- and trisaccharide monoethers of the type (3-5)-D-xylan, by a one-pot reaction between the alcohol ROH and 3,5-anhydro-l,2-O-isopropylidene-α-D-xylofuranose, in the presence of KOH and toluene-DMSO at 80 °C.

Disturbances of insulin or brain monoamine metabolism may play a role in the impaired regulation of food intake and body weight in the obese Zucker rat. We investigated a possible insulin-monoamine interaction by measuring monoamine levels in the hypothalamus and striatum of obese (fa-fa) and lean (Fa-Fa and Fa-fa) Zucker rats after peripheral insulin administration. The classically reported effects of insulin, i.e., increases in tryptophan, 5-hydroxy-indolacetic acid (5-HIAA) and dihydroxyphenylacetic acid (DOPAC) levels, were observed in the hypothalamus of Fa-Fa and Fa-fa rats, but not in obese fa-fa rats. Given the mechanism of action of insulin, this lack of effect in the obese rats may be related to the peripheral insulin resistance they exhibit. Furthermore, given the role of these monoaminergic systems, this reduced effect may be related to the impaired regulation of food intake and body weight. At 8 wk of age, however, insulin restored the decreased basal 5-HIAA levels observed in the obese rats. Increases in 5-HIAA levels following insulin administration appeared in the striatum of Fa-Fa rats only, suggesting that, as for brain insulin content, other central insulin-related disturbances may be related to the presence of the “fa” gene. In addition, certain effects of insulin on striatal dopamine release were observed in only the Fa-Fa and fa-fa rats, suggesting a particular disturbance related to the heterozygous character. This latter point calls for further investigations on the central dopaminergic effects of insulin.

In addition to its anorectic properties, dexfenfluramine may inhibit some manifestations of feeding-related reward. We attempted to verify this effect by measuring paw-lick latency on the hot plate test in rats conditioned to expect a palatable food. The involvement of variations in β-endorphinergic, dopaminergic and serotonergic systems was assessed. Despite an inherent effect of increasing paw-lick latency, dexfenfluramine (1.5 mg/kg IP) partly reversed the expectancy-induced increase in this latency. Saline-treated “expectant” rats displayed elevated plasma β-endorphin levels and reduced hypothalamic 5-HIAA/5-HT and DOPAC/DA ratios. Only the decrease in the DOPAC/DA ratio was reversed by dexfenfluramine, suggesting an involvement of the dopaminergic system in this dexfenfluramine-sensitive reward system.

International audience; Objective: Recently, an activation of the endocannabinoid system during obesity has been reported. More particularly, it has been demonstrated that hypothalamic levels of both endocannabinoids, 2‐arachidonoylglycerol and anandamide (N‐arachidonoylethanolamine), are up‐regulated in genetically obese rodents. Circulating levels of both endocannabinoids were also shown to be higher in obese compared with lean women. Yet, the direct production of endocannabinoids by human adipocytes has never been demonstrated. Our aim was to evaluate the ability of human adipocytes to produce endocannabinoids.Research Methods and Procedures: The production of endocannabinoids by human adipocytes was investigated in a model of human white subcutaneous adipocytes in primary culture. The effects of leptin, adiponectin, and peroxisome proliferator‐activated receptor (PPAR)‐γ activation on endocannabinoid production by adipocytes were explored. Endocannabinoid levels were determined by high‐performance liquid chromatography (HPLC)‐atmospheric pressure chemical ionization (APCI)‐mass spectrometry (MS) analysis, leptin and adiponectin secretion measured by enzyme‐linked immunosorbent assay (ELISA), and PPAR‐γ protein expression examined by Western blotting.Results: We show that 2‐arachidonoylglycerol, anandamide, and both anandamide analogs, N‐palmitoylethanolamine and N‐oleylethanolamine, are produced by human white subcutaneous adipocytes in concentrations ranging from 0.042 ± 0.004 to 0.531 ± 0.048 pM/mg lipid extract. N‐palmitoylethanolamine is the most abundant cannabimimetic compound produced by human adipocytes, and its levels are significantly down‐regulated by leptin but not affected by adiponectin and PPAR‐γ agonist ciglitazone. N‐palmitoylethanolamine itself does not affect either leptin or adiponectin secretion or PPAR‐γ protein expression in adipocytes.Discussion: This study has led to the identification of human adipocytes as a new source of endocannabinoids and related compounds. The biological significance of these adipocyte cannabimimetic compounds and their potential implication in obesity should deserve further investigations.

1. High-fat diets, modify the neuroendocrine response and, when prolonged, result in positive energy balance and obesity. Little is known about the effects of fat on the mechanisms operating in the initial steps of the neural and endocrine disturbances. 2. The studies reported here were designed to access the impact of the consumption of a single exclusively animal fat meal (lard), 24 h following its ingestion a) on the response of the hypothalamic serotonergic system to a standard laboratory chow meal and b) on the circulating levels of glucose, insulin, and leptin. The release of serotonin in the extracellular medial hypothalamic space (including the paraventricular-PVN and ventromedian-VMH nuclei) was determined using electrochemical detection following HPLC in samples obtained in vivo by microdialysis, in nonanesthetized adult male Wistar rats. 3. A lard meal resulted in decreased hypothalamic serotonin release postprandially and attenuated (24 h later) the hypothalamic serotonin response that normally follows a balanced meal. 4. In permanently catheterized rats, postprandial glucose and insulin levels measured in samples obtained in vivo, were either not, or only slightly, modified after a lard meal, whereas plasma leptin levels were increased. Interestingly, 24 h after a meal, insulin and leptin levels were increased in those animals eating a fat meal compared with those eating chow. Next-day glucose levels remained identical after the absorption either of a chow, or a lard meal. 5. The changes induced by the fat meal on peripheral and central regulators of energy and glucose homeostasis represent either adaptive mechanisms or early alterations that could render the organism vulnerable to further insults.

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 α2A-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.

Among food proteins, alpha-lactalbumin (LAC) has the highest ratio of tryptophan (Trp) over its competitor amino acids. Consequently, contrary to casein (CAS), LAC ingestion increases Trp access to the brain leading to enhanced serotonin (5-HT) synthesis. As an index of serotonergic activity, we assessed extracellular 5-HT in response to LAC ingestion, using microdialysis, and performed behavioural tests in rats in order to characterise the suggested improvements of mood observed in humans after ingestion of this protein. Rats were fed with diets enriched either in LAC or CAS as control, acutely (30 min meals) or chronically (3 and 6 days). A 30 min LAC meal significantly increased 5-HT release in the medial hypothalamus. This effect disappeared after 3 and 6 days of diet. The basal premeal 5-HT levels were increasingly enhanced by the LAC diet. Compared to a CAS meal, LAC increased the percentage of time spent on the open arms of the elevated plus maze and the number of visits to the centre of the open field, suggesting an anxiolytic-like effect. A single LAC meal decreased sucrose consumption, while 3 or 6 days diets enhanced it, reflecting an appetitive and/or rewarding action. In conclusion, LAC ingestion induces anxiolytic-like and rewarding effects possibly related to serotonergic activation. Shifting transiently, the commonly consumed CAS-enriched to LAC-enriched diets may induce beneficial effects on mood.

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.

Introduction Des etudes recentes ont montre que l'accumulation de lipides dans l'hypothalamus serait responsable de l'installation d'une lipotoxicite centrale. Ce phenomene pourrait jouer un role dans l'apparition d'une insulinoresistance et du diabete en deregulant le controle nerveux de l'homeostasie glucidique. Il a ete montre que l'accumulation des ceramides joue un role preponderant dans le developpement d'une lipotoxicite au niveau des tissus peripheriques. Nos resultats preliminaires ont montre que la perfusion de saindoux, compose principalement d'acides gras satures, directement dans le cerveau, conduit a une insulino-resistance peripherique associee a une accumulation de ceramides dans l'hypothalamus. Materiels et methodes Afin etudier la chronologie qui existe entre l'installation d'une insulino-resistance centrale et peripherique, nous avons decide d'etudier les effets d'un acide gras sature, le palmitate sur l'insulino-resistance centrale a l'aide d'approches in vitro (cellules hypothalamiques) et in vivo (rat). Resultats Nous avons pu mettre en evidence un phenomene d'insulino-resistance centrale in vitro en reponse au palmitate qui s'accompagne d'une accumulation de ceramides. Certains ARNm codant pour les enzymes de la voie de synthese de novo des ceramides etant augmentes a la suite de ces traitements, nous avons decide d'inhiber cette voie avec la myriocine. En presence de myriocine, ce phenomene d'insulino-resistance centrale est partiellement contrecarre. Chez le rat, nous avons pu observer le meme phenomene d'insulino-resistance centrale en reponse a une perfusion dans la carotide de palmitate. Cette insulino-resistance s'accompagne a son tour d'une accumulation de ceramides dans l'hypothalamus. Conclusion Au final, notre etude revele que les acides gras satures induisent une accumulation de ceramides dans l'hypothalamus. De facon interessante, cette accumulation est responsable de l'installation d'une insulino-resistance hypothalamique. Ces resultats mettent en evidence le role cle du metabolisme des ceramides dans la deregulation du controle nerveux de l'homeostasie glucidique observe au cours de l'obesite.

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.

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.

We found out the existence in fresh young leaves of sugarcane of a PEPcase-MDH(NADP)-Malic enzyme multienzymatic complex. The enzyme was purified as follows

We found out the existence in fresh young leaves of sugarcane of a PEPcase-MDH(NADP)-Malic enzyme multienzymatic complex. We have been able to purify this complex. Its functional advantage has been investigated by comparing the kinetic properties of the constitutive enzymes in their free state with those in their complexed state.

Subject Areas: carotid arteries; emulsions; fatty acids; gene expression; heparin; hypothalamus; neurons; oxidation.; International audience; Variations in plasma fatty acid (FA) concentrations are detected by FA sensing neurons in specific brain areas such as the hypothalamus. These neurons play a physiological role in the control of food intake and the regulation of hepatic glucose production. Le Foll et al. previously showed in vitro that at least 50% of the FA sensing in ventromedial hypothalamic (VMH) neurons is attributable to the interaction of long chain FA with FA translocase/CD36 (CD36). The present work assessed whether in vivo effects of hypothalamic FA sensing might be partly mediated by CD36 or intracellular events such as acylCoA synthesis or β-oxidation. To that end, a catheter was implanted in the carotid artery toward the brain in male Wistar rats. After 1 wk recovery, animals were food-deprived for 5 h, then 10 min infusions of triglyceride emulsion, Intralipid +/- heparin (IL, ILH, respectively) or saline/heparin (SH) were carried out and food intake was assessed over the next 5 h. Experimental groups included: 1) Rats previously injected in ventromedian nucleus (VMN) with shRNA against CD36 or scrambled RNA; 2) Etomoxir (CPT1 inhibitor) or saline co-infused with ILH/SH; and 3) Triacsin C (acylCoA synthase inhibitor) or saline co-infused with ILH/SH. ILH significantly lowered food intake during refeeding compared to SH (p

Introduction L’hippocampe participe au controle de la prise alimentaire, du poids corporel et des depenses energetiques. De plus, il est le site majeur d’activite et d’expression de la lipoproteine lipase (LPL), responsable de l’hydrolyse des triglycerides en acides gras libres. Nous formons l’hypothese que l’activite LPL hippocampique, via une modulation du lipid sensing cerebral, pourrait jouer un role dans le controle de la balance energetique. Materiels et methodes Des souris Lpl lox/lox ont ete croisees avec des souris exprimant la Cre recombinase sous promoteur CamK2α; (expression majoritaire dans la couche CA1 de l’hippocampe), l’absence de recombinaison au niveau des organes peripheriques tels que le pancreas a ete verifiee. La prise alimentaire, le poids, la composition corporelle et les depenses energetiques ont ete caracterisees chez la descendance. L’homeostasie glucidique a ete exploree par des tests de tolerance au glucose oral (OGTT) associes a la mesure de la secretion d’insuline, et des tests de tolerance a l’insuline (ITT). Resultats Les souris heterozygotes pour la deletion du gene Lpl presentent une diminution significative de l’activite LPL hippocampique et developpent un surpoids transitoire sans hyperphagie, associe a une augmentation de masse grasse et a une diminution des depenses energetiques, par rapport aux controles. Ces souris sont egalement intolerantes au glucose et presentent un defaut de secretion d’insuline en reponse au glucose. Elles sont en revanche plus sensibles a l’insuline. Les souris homozygotes ne presentent quant a elles pas de phenotype ni de diminution de l’activite LPL hippocampique. Conclusion La deletion heterozygote du gene Lpl sous promoteur Camk2α; induit une intolerance au glucose associee a un defaut de secretion d’insuline. Ces resultats sont en faveur d’un role de l’activite LPL hippocampique dans la regulation nerveuse de l’homeostasie glucidique.

Polyphenol oxidase (PPO) is responsible for browning reactions in fruits and vegetables. In order to inhibit these reactions during processing, the use of chemical inhibitors including thiols such as L-cysteine has been proposed. The effect of this thiol on PPO activity was studied in order to establish if it reacts with quinone and/or directly inhibits the enzyme. The inhibition of palmito PPO (catecholase activity, with 4-methylcatechol as substrate) by L-cysteine was characterized spectrophotometrically. The effect of increasing cysteine concentration with respect to incubation time was measured by polarography and the kinetic parameters calculated. Kinetic analysis using spectrophotometric assays showed a lag period suggesting that there is no quinone accumulation in the reaction mixture. The increase of absorbance at 300 nm observed with u.v.—vis spectra indicated that a colourless compound (thiol—quinone complex) was produced. The plots of log remaining enzyme activity vs incubation time were characterized by two straight lines suggesting two possible inactivation models: (i) the deactivation of one enzyme that proceeds in two steps or (ii) the existence of two isoenzymes that behave differently. We have shown that the inactivation process of PPO by cysteine followed this model: N → X[C] → I where N represents one native form, X represents an intermediate form, the structure of which depends on the cysteine concentration [C], and where I is the completely inactive form of the enzyme. These results showed that there is a direct irreversible inhibition of PPO by cysteine according to a two-step model. The analytical approach illustrated by the present study of palmito PPO inhibition by L-cysteine may be applied to other investigations of mechanisms of enzyme inhibition.

An alternate method for enzyme study is proposed. Multidimensional statistical analysis applied on mid-infrared attenuated total reflectance spectra (Cadet et al. (1991) Appl. Spectrosc. 42, 166-172) collected during a kinetic allows a direct and fast quantification of the remaining substrate, as well as a one step enzymatic assay. Furthermore, the combination of these techniques may be used as a structural tool. The method applied to the study of beta-fructosidase is developed in this paper as an example. With appropriate calibration, the method may be extend to any enzyme.