DNA methylation of leptin and adiponectin promoters in children is reduced by the combined presence of obesity and insulin resistance.

OBJECTIVE: Epigenetic alterations have been suggested to be associated with obesity and related metabolic disorders. Here we examined the correlation between obesity and insulin resistance with the methylation frequency of the leptin (LEP) and adiponectin (ADIPOQ) promoters in obese adolescents with the aim to identify epigenetic markers that might be used as tools to predict and follow up the physiological alterations associated with the development of the metabolic syndrome. SUBJECTS: One hundred and six adolescents were recruited and classified according to body mass index and homeostasis model of assessment-insulin resistance index. The circulating concentrations of leptin, adiponectin and of several metabolic markers of obesity and insulin resistance were determined by standard methods. The methylation frequency of the LEP and ADIPOQ promoters was determined by methylation-specific PCR (MS-PCR) in DNA obtained from peripheral blood samples. RESULTS: Obese adolescents without insulin resistance showed higher and lower circulating levels of, respectively, leptin and adiponectin along with increased plasmatic concentrations of insulin and triglycerides. They also exhibited the same methylation frequency than lean subjects of the CpG sites located at -51 and -31 nt relative to the transcription start site of the LEP gene. However, the methylation frequency of these nucleotides dropped markedly in obese adolescents with insulin resistance. We found the same inverse relationship between the combined presence of obesity and insulin resistance and the methylation frequency of the CpG site located at -283 nt relative to the start site of the ADIPOQ promoter. CONCLUSIONS: These observations sustain the hypothesis that epigenetic modifications might underpin the development of obesity and related metabolic disorders. They also validate the use of blood leukocytes and MS-PCR as a reliable and affordable methodology for the identification of epigenetic modifications that could be used as molecular markers to predict and follow up the physiological changes associated with obesity and insulin resistance.

Perinatal nutrient restriction induces long-lasting alterations in the circadian expression pattern of genes regulating food intake and energy metabolism.

OBJECTIVE: Several lines of evidence indicate that nutrient restriction during perinatal development sensitizes the offspring to the development of obesity, insulin resistance and cardiovascular disease in adulthood via the programming of hyperphagia and reduced energy expenditure. Given the link between the circadian clock and energy metabolism, and the resetting action of food on the circadian clock, in this study, we have investigated whether perinatal undernutrition affects the circadian expression rhythms of genes regulating food intake in the hypothalamus and energy metabolism in the liver. DESIGN: Pregnant Sprague-Dawley rats were fed ad libitum either a control (20% protein) or a low-protein (8% protein) diet throughout pregnancy and lactation. At weaning, pups received a standard diet and at 17 and 35 days of age, their daily patterns of gene expression were analyzed by real-time quantitative PCR experiments. RESULTS: 17-day-old pups exposed to perinatal undernutrition exhibited significant alterations in the circadian expression profile of the transcripts encoding diverse genes regulating food intake, the metabolic enzymes fatty acid synthase and glucokinase as well as the clock genes BMAL1 and Period1. These effects persisted after weaning, were associated with hyperphagia and mirrored the results of the behavioral analysis of feeding. Thus, perinatally undernourished rats exhibited an increased hypothalamic expression of the orexigenic peptides agouti-related protein and neuropeptide Y. Conversely, the mRNA levels of the anorexigenic peptides pro-opiomelanocortin and cocaine and amphetamine-related transcripts were decreased. CONCLUSION: These observations indicate that the circadian clock undergoes nutritional programming. The programming of the circadian clock may contribute to the alterations in feeding and energy metabolism associated with malnutrition in early life, which might promote the development of metabolic disorders in adulthood.

Dystrophin and Dp71, two products of the DMD gene, show a different pattern of expression during embryonic development in zebrafish.

Dystrophin, the protein defective in Duchenne muscular dystrophy (DMD), plays a critical role in the formation and maintenance of the neuromuscular junction. In addition to dystrophin, activation of internal promoters of the DMD gene leads to the production of several short products. Among these, Dp71, which consists of the C-terminal domain of dystrophin, is the most abundant product of the gene in non-muscle tissues and brain. In this report, we compare the temporal and regional expression patterns of dystrophin and Dp71 at different stages of embryonic development and during retinal differentiation in zebrafish. The Dp71 transcripts are the earliest to be expressed at 9-10 h post-fertilization (hpf) in the axial mesoderm. As development proceeds, intense Dp71 staining is observed in the notochord, the developing brain, the marginal regions of the somites and the eye primordium. At the completion of retinal differentiation, Dp71 is expressed in the ganglion and inner nuclear layers. Transcripts encoding dystrophin have a slightly later onset of expression, 13-14 hpf, and remain restricted to the transverse myosepta through all the developmental stages examined. The complementary patterns of expression of dystrophin and Dp71 suggest that these two proteins exert different functions during embryonic development in zebrafish.

Molecular cloning and characterization of dystrophin and Dp71, two products of the Duchenne Muscular Dystrophy gene, in zebrafish.

Dystrophin, the protein responsible for Duchenne Muscular Dystrophy (DMD), plays a critical role in the maintenance of the muscle membrane integrity. There are several forms of dystrophin derived from the DMD gene by alternative promoter usage. In addition to full-length dystrophin (Dp427), four shorter transcripts have been identified: Dp260, Dp140, Dp116 and Dp71. The functional role played by the different products of the DMD gene is not yet determined. To get insight into the function of dystrophin and related products, we have investigated the presence of dystrophin in zebrafish. This choice takes advantage of large-scale mutagenesis screens in zebrafish, which have led to the identification of several mutants with motility defects. The identification and characterization of the genes affected by these mutations is likely to provide relevant information for the understanding of the molecular mechanisms of muscle development and function. Two cDNA clones encoding the homologues of dystrophin and Dp71 in zebrafish were identified and characterized. Both transcripts exhibit a high degree of sequence homology with the dystrophin and Dp71 proteins described in higher vertebrates. In addition, three alternative spliced transcripts that occur at the C-terminal end of the zebrafish DMD gene have been identified. These transcripts exhibit different patterns of tissue expression. We have also determined the chromosomal localization of dystrophin on the radiation hybrid map of the zebrafish genome. Our results indicate that the dystrophin gene is localized to linkage group one. Altogether, these results give new insights on the physiological role played by dystrophin and related proteins, and provide new tools for the identification of mutated genes associated with muscle defects in zebrafish.

Effect of chronic antidepressant treatment on 5-HT1B presynaptic heteroreceptors inhibiting acetylcholine release.

The effect of long term treatment with two tricyclic antidepressants on the sensitivity of 5-HT1B presynaptic heteroreceptors inhibiting acetylcholine (ACh) release was investigated. Groups of male rats received during 14 days either saline, citalopram (20 mg/kg), a serotonin (5-HT) uptake blocker, or tianeptine (2 x 10 mg/kg), an antidepressant that enhances 5-HT uptake. The efficacy of the 5-HT1B selective agonist 7-trifluoromethyl-4-(4-1-piperazinyl)-pyrrolo[1,2-a]quinoxaline (CGS 12066B) in reducing K(+)-evoked [3H]acetylcholine release from hippocampal synaptosomes was determined 24 hr after the last administration. The chronic treatment with citalopram or tianeptine modified neither the basal nor the K(+)-evoked release of [3H]acetylcholine. In contrast, these treatments significantly reduced the efficacy of CGS 12066B to inhibit the release of [3H]acetylcholine induced by K+ depolarization. These data suggest that chronic antidepressant treatment desensitizes 5-HT1B presynaptic heteroreceptors through a mechanism which seems to be independent of the synaptic availability of 5-HT.

Tianeptine stimulates uptake of 5-hydroxytryptamine in vivo in the rat brain.

The neurochemical effects of the atypical tricyclic antidepressant, tianeptine, were further assessed on central serotoninergic and dopaminergic systems in the rat. Acute treatment with tianeptine (10 mg/kg i.p.) significantly enhanced the levels of metabolites of 5-HT and DA, 5-hydroxyindole acetic acid and dihydroxyphenylacetic acid respectively, in the brain stem, striatum and cerebral cortex. These effects could be prevented by the administration of drugs acting selectively (or preferentially) on serotoninergic systems such as d,l-fenfluramine and 4-methyl-alpha-ethyl-metatyramine (H75/12), suggesting that the increased metabolism of DA was secondary to a modification of serotoninergic systems in tianeptine-treated rats. In contrast to that found with inhibitors of the uptake of 5-HT, treatment with tianeptine markedly enhanced depletion of 5-HT due to administration of H75/12. However, depletion of DA induced by H75/12, was not altered by tianeptine. In vitro measurement of the uptake of [3H]5-HT also confirmed that tianeptine exerted opposite effects to those of classical tricyclic antidepressants, since the in vivo administration of tianeptine (2 x 10 mg/kg i.p.) induced a significant increase in the uptake of [3H]5-HT in cortical synaptosomes. The fact that both inhibitors of the uptake of 5-HT and tianeptine which, in contrast, enhanced the in vivo uptake of 5-HT, are potent antidepressants, challenges the current hypothesis on the central mechanisms of action of these drugs.

Mouse 5-HT2B receptor-mediated serotonin trophic functions.

5-HT2B receptors, in addition to phospholipase C stimulation, are able to trigger activation of the proto-oncogene product p21ras. During mouse embryogenesis, a peak of 5-HT2B receptor expression is detected at the neurulation stage; we localized the 5-HT2B expression in neural crest cells, heart myocardium, and somites. The requirement for functional 5-HT2B receptors shortly after gastrulation, is supported by culture of embryos exposed to 5-HT2B-high affinity antagonist such as ritanserin, which induces morphological defects in the cephalic region, heart and neural tube. Functional 5-HT2B receptors are also expressed during the serotonergic differentiation of the mouse F9 teratocarcinoma-derived clonal cell line 1C11. Upon 2 days of induction by cAMP, 5-HT2B receptors become functional, and on day 4, the appearance of 5-HT2A receptors coincides with the onset of active serotonin transporter by these cells. Active serotonin uptake is modulated by serotonin suggesting autoreceptor functions for 5-HT2B receptors.

Preferential expression of 5-HT1D over 5-HT1B receptors during early embryogenesis.

The cloning and pharmacological characterization of mouse 5-HT1D receptors as well as the comparative analysis of its embryonic expression vs that of 5-HT1B receptors are reported. High densities of both 5-HT1D receptors mRNA and specific 5-HT1D binding sites were detected at 8, 9.5, 10.5 and 13.5 days of prenatal development. In contrast, no specific 5-HT1B binding sites could be detected until 13.5 days of development, when they were present at lower levels than 5-HT1D receptors. This differs markedly from the situation in the adult brain, in which 5-HT1B receptors are present at a much higher density than the 5-HT1D subtype. These data suggest the involvement of 5-HT1D receptors in the mitogenic and proliferative effects of serotonin during early embryonic development.

Antagonism by citalopram and tianeptine of presynaptic 5-HT1B heteroreceptors inhibiting acetylcholine release.

The interactions of citalopram and tianeptine, two antidepressants having opposite effects on serotonin (5-HT) uptake, with 5-HT1B presynaptic heteroreceptors located on cholinergic terminals were investigated. In rat hippocampal synaptosomes, citalopram (0.01 or 0.1 microM) or tianeptine (0.01-10 microM) did not modify the basal or the K(+)-evoked release of [3H]acetylcholine. Only at the concentration of 100 microM did tianeptine significantly decrease (-18%) the K(+)-evoked release of [3H]acetylcholine without affecting the spontaneous outflow of radioactivity. The inhibitory effect of 7-trifluoromethyl-4-(4-1-piperazinyl)-pyrrolo[1,2-a]quinoxaline (CGS 12066B), a 5-HT1B receptor agonist, on the stimulation-induced release of [3H]acetylcholine was reduced in a concentration-dependent manner by citalopram and tianeptine. Both drugs completely reversed the inhibitory effects of CGS 12066B at concentrations that did not modify by themselves the release of [3H]acetylcholine. In contrast, tianeptine, up to a concentration of 1 microM, failed to antagonise the inhibitory effect of the muscarinic receptor agonist carbachol on K(+)-evoked [3H]acetylcholine release. Finally, the administration of tianeptine ex vivo (10 or 20 mg/kg) modified neither the depolarisation-induced release of [3H]acetylcholine nor the inhibitory effect of CGS 12066B on this presynaptic process. These findings further confirm that antidepressants interact in vitro with presynaptic 5-HT1B heteroreceptors.

Characterization of galanin and 5-HT1A receptor coupling to adenylyl cyclase in discrete regions of the rat brain.

We have studied the coupling of galanin and 5-HT1A receptors with adenylyl cyclase in the hypothalamus, the entorhinal cortex and the hippocampus of the rat brain. Furthermore, we have evaluated the effects of simultaneous activation of galanin and 5-HT1A receptors on adenylyl cyclase activity. Galanin-(1-29) and galanin-(1-15) showed a dose-dependent inhibitory effect on forskolin-stimulated adenylyl cyclase activity in the hypothalamus and entorhinal cortex. No clear effects were observed in the hippocampus. Neither galanin-(1-29) nor galanin-(1-15) had any effect on the basal activity of adenylyl cyclase in these regions. The selective 5-HT1A receptor agonist 8-OH-2-(di-n-propylamino)-tetralin (8-OH-DPAT) induced a dose-dependent inhibition of forskolin stimulated adenylyl cyclase activity in the hippocampus and the entorhinal cortex. 5-HT induced an inhibition in the hypothalamus. In all regions the effects could be fully counteracted by methiothepin. 5-HT was shown to stimulate the basal activity of adenylyl cyclase in the hippocampus and the entorhinal cortex. The effects could be counteracted by methiothepin. When galanin-(1-29) and 5-HT/8-OH-DPAT were incubated simultaneously additive inhibitory effects, but no synergistic interactions, could be observed on the stimulated adenylyl cyclase activity. In conclusion, galanin and 5-HT1A receptors seem to be linked to different independent pools of G proteins, indicating that the previously demonstrated intramembrane interactions between galanin and 5-HT1A receptors involve a mechanism not directly related to adenylyl cyclase.

Effects of stress on the functional properties of pre- and postsynaptic 5-HT1B receptors in the rat brain.

Numerous studies have clearly shown that the turnover and release of serotonin (5-hydroxytryptamine, 5-HT) are increased under acute stressful conditions. Inasmuch as this latter process is under the control of a feedback mechanism involving the stimulation of presynaptic 5-HT1B autoreceptors, we have investigated the possible effects of acute restraint (40 min) on the functional properties of 5-HT1B receptors. The efficacy of the selective 5-HT1B receptor agonist 3-[1,2,5,6-tetrahydropyrid-4-yl]pyrrolo-[3,2-b]pyrid-5-one (CP-93,129) in inhibiting in vitro the K+-evoked release of [3H]5-HT, was significantly reduced in stressed rats as compared to naive animals. Similarly, the responsiveness of 5-HT1B receptors inhibiting the release of [3H]acetylcholine (presynaptic 5-HT1B heteroreceptors), was reduced by restraint. These effects were observed in the hippocampus, but using the inhibitory effect of CP-93,129 on forskolin-stimulated adenylyl cyclase activity as an index of 5-HT1B receptor function, it could be shown that the 5-HT1B receptors located in the substantia nigra are also desensitized by stress. The number as well as the apparent affinity constant of 5-HT1B binding sites labelled by [125I]iodocyanopindolol, as measured by quantitative autoradiography and membrane binding, were similar in naive and restraint-stressed rats suggesting that the stress-induced desensitization of 5-HT1B receptors is not due to a reduced number of 5-HT1B binding sites. As stress is thought to be a causal factor for the etiology of anxiety and depression, these results support the potential involvement of 5-HT1B receptor dysfunction in the development of these neurological disorders.

Sub-chronic cold stress reduces 5-HT1A receptor responsiveness in the old but not in the young rat.

The inhibitory effect of the prototypical 5-hydroxytryptamine (5-HT)1A receptor agonist 8-hydroxy-2-(di-n-propyl amino)tetraline (8-OH-DPAT) on forskolin-stimulated adenylyl cyclase activity, has been examined as an index of the functional activity of 5-HT1A receptors in the hippocampus of young (3 months) and old (18 months) rats exposed during 24 h or 5 days to cold. In both young and old rats exposed to cold stress during 24 h, there was a reduction in the potency (EC50) and/or the maximal inhibitory effect (Emax) of 8-OH-DPAT in reducing forskolin-induced cAMP accumulation. The properties of the hippocampal 5-HT1A sites labelled by [3H]8-OH-DPAT were not affected by these stressful conditions. Moreover, while the sensitivity of 5-HT1A receptors to 8-OH-DPAT in young rats returned to control values after 5 days of cold exposure, old rats still exhibited a significant desensitization of 5-HT1A receptors as compared to naive animals. These results point out the capacity of young but not of old rats to adapt to the aversive effects of a subchronic stressor.

Stress-induced 5-HT1A receptor desensitization: protective effects of Ginkgo biloba extract (EGb 761).

The effects of sub-chronic cold stress on the functioning of hippocampal 5-HT1A receptors in old isolated rats and the possible protective effects of Ginkgo biloba extract (EGb 761) were investigated. Cold exposure during five days, produced a significant reduction of the inhibitory effect of 8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT) on forskolin-stimulated adenylyl cyclase activity. In contrast, neither the affinity nor the density of hippocampal [3H]8-OH-DPAT binding sites were affected indicating that the reduced sensitivity of 5-HT1A receptors induced by stress is probably due to a modification of their coupling mechanisms to adenylyl cyclase. The stress-induced desensitization of 5-HT1A receptors was prevented by the administration of EGb 761 (50 mg/kg per os/14 days). These results clearly indicate that 5-HT1A receptors are desensitized by stress and point out the reduced capacity of old rats to cope with the adverse effects of a chronic stressor. EGb 761 appears to restore the age-related decreased capacity to adapt to a chronic stressor.

Differential developmental programming by early protein restriction of rat skeletal muscle according to its fibre-type composition.

AIMS: Differences in fibre-type composition of skeletal muscle have been associated with obesity and insulin resistance. As a poor nutrient environment early in life is a predisposing factor for the development of obesity and related metabolic diseases at adulthood, this study aimed at determining the long-term consequences of maternal undernutrition on the structural and metabolic properties of two skeletal muscles characterized by their different fibre-type composition and metabolic properties. METHODS: The fibre-type composition and enzymatic activities of hexokinase (HK), beta-hydroxyacyl-CoA dehydrogenase (ß-HAD) and citrate synthase (CS) were measured in soleus and extensor digitorum longus (EDL) muscles from adult rats born to dams fed a control (17% protein) or a low-protein [8% protein (PR)] diet throughout pregnancy and lactation. In addition, the expression levels of several genes regulating glycolysis, fatty acid oxidation and mitochondrial biogenesis were determined by real-time PCR. RESULTS: Protein rats exhibited enhanced density of type II fibres along with decreased rate of fatty acid oxidation and glycolysis in soleus but not EDL. Malnourished rats exhibited also a different gene expression profile in soleus and EDL. Altogether, these alterations correspond to a state of energy deficiency and are present in animals which do not show yet any sign of obesity or glucose intolerance. CONCLUSION: We conclude that maternal protein restriction alters in the long term the structural and enzymatic properties of offspring skeletal muscle in a fibre-type-dependent manner. These alterations might have a causative role in the development of obesity and related metabolic disorders later in life.

Unlimited access to low-energy diet causes acute malnutrition in dams and alters biometric and biochemical parameters in offspring.

Here we analyze the outcomes of unlimited access to a low-energy (LE) diet in dams and their offspring. At 3 weeks' gestation, pregnant Wistar rats were divided into two groups: (1) the control group received a normoenergetic diet; and (2) the experimental group received the LE diet. In dams, lactation outcomes, food intake, body weight, plasma IGF-1, prealbumin, transferrin and retinol-binding protein levels were evaluated; in offspring, biometric and biochemical parameters and food intake were evaluated. No differences were observed during pregnancy. However, after lactation, dams that received the LE diet demonstrated significant reductions in body weight (P<0.05), plasma IGF-1 (P=0.01), prealbumin and visceral fat (P<0.001). Pups born to dams that received the LE diet demonstrated reduced body length and weight at weaning (P<0.001) and were lighter than the control animals at the end of the experimental period. Pups also demonstrated reduced plasma, low-density lipoprotein (P=0.04), triglycerides (P=0.002) and glucose levels (P<0.05), and differences were noted in visceral fat. These results indicate that feeding dams with LE diet during the reproductive period induces acute malnutrition and impairs the growth and development of offspring, as well as certain metabolic parameters.

Nutrient restriction during early life reduces cell proliferation in the hippocampus at adulthood but does not impair the neuronal differentiation process of the new generated cells.

Maternal malnutrition results in learning deficits and predisposition to anxiety and depression in the offspring that extend into adulthood. At the cellular level, learning and memory rely on the production of new neurons in the dentate gyrus (DG) of the hippocampus, and hippocampal neurogenesis has been associated with the etiology and treatment of depression, but whether adult neurogenesis is affected by malnutrition during early life is not known. To investigate the effects of perinatal undernutrition on neurogenesis at adulthood, pregnant Sprague-Dawley rats were fed either ad libitum (C) or were undernourished by reducing their daily food intake by 50% in relation to the C group during gestation and lactation (FR/FR). At birth, one subset of control pups was cross-fostered to food-restricted dams to constitute a third group of animals that were undernourished during the lactation period only (AdLib/FR). At 90 days of age, pups were injected with bromodeoxyuridine (BrdU) and sacrificed 2 h, 1 week, or 3 weeks later. The number of BrdU-labeled cells in the DG was significantly reduced in the offspring of FR/FR dams in relation to controls at all the time points examined. However, the proportion of new cells exhibiting a neuronal phenotype was higher in FR/FR rats than in controls as revealed by the colabeling at 3 weeks of the BrdU-labeled cells with neuron-specific nuclear protein (NeuN). AdLib/FR animals exhibited also reduced BrdU labeling at 2 h and 1 week. Nevertheless, we found no significant differences at 3 weeks in either the number of BrdU-labeled cells or in the proportion of new neurons between controls and AdLib/FR rats. These results indicate that the decreased number of hippocampal neurons in perinatally undernourished rats is due to the deleterious effects of early nutrient restriction on cell proliferation but not on the neuronal differentiation process of the new generated cells.