Concentration-dependent change in hypothalamic neuronal transcriptome by the dietary fatty acids: oleic and palmitic acids.

Prenatal high-fat diet exposure increases hypothalamic neurogenesis events in embryos and programs offspring to be obesity-prone. The molecular mechanism involved in these dietary effects of neurogenesis is unknown. This study investigated the effects of oleic and palmitic acids, which are abundant in a high-fat diet, on the hypothalamic neuronal transcriptome and how these changes impact neurogenesis events. The results show the differential effects of low and high concentrations of oleic or palmitic acid treatment on differential gene transcription. Gene ontology analysis uncovered significant gene enrichment in several cellular pathways involved in gene regulation and protein production, particularly with proliferation, migration, and cell survival. The enriched signaling pathways include Wnt, integrin, PDGF, and apoptosis, in addition to endocrine function signaling pathways CCKR and GnRH. Further examination of proliferation and migration shows low concentrations of oleic acid to stimulate proliferation and high concentrations of both oleic and palmitic acid to stimulate apoptosis. Oleic acid also reduces hypothalamic neuronal migration, with little effect from palmitic acid. The results show the two most abundant fatty acids in a high-fat diet impact hypothalamic neuronal proliferation and migration. The results also uncovered potential signaling pathways affected by oleic and palmitic acid and suggest one mechanism of prenatal high-fat diet-induced neurogenesis events may be through these two abundant fatty acids.

Behavioral Feeding Circuit: Dietary Fat-Induced Effects of Inflammatory Mediators in the Hypothalamus.

Excessive dietary fat intake has extensive impacts on several physiological systems and can lead to metabolic and nonmetabolic disease. In animal models of ingestion, exposure to a high fat diet during pregnancy predisposes offspring to increase intake of dietary fat and causes increase in weight gain that can lead to obesity, and without intervention, these physiological and behavioral consequences can persist for several generations. The hypothalamus is a region of the brain that responds to physiological hunger and fullness and contains orexigenic neuropeptide systems that have long been associated with dietary fat intake. The past fifteen years of research show that prenatal exposure to a high fat diet increases neurogenesis of these neuropeptide systems in offspring brain and are correlated to behavioral changes that induce a pro-consummatory and obesogenic phenotype. Current research has uncovered several potential molecular mechanisms by which excessive dietary fat alters the hypothalamus and involve dietary fatty acids, the immune system, gut microbiota, and transcriptional and epigenetic changes. This review will examine the current knowledge of dietary fat-associated changes in the hypothalamus and the potential pathways involved in modifying the development of orexigenic peptide neurons that lead to changes in ingestive behavior, with a special emphasis on inflammation by chemokines.

Involvement of the CXCL12 System in the Stimulatory Effects of Prenatal Exposure to High-Fat Diet on Hypothalamic Orexigenic Peptides and Behavior in Offspring.

Exposure to a high fat diet (HFD) during gestation stimulates neurogenesis and expression of hypothalamic orexigenic neuropeptides that affect consummatory and emotional behaviors. With recent studies showing a HFD to increase inflammation, this report investigated the neuroinflammatory chemokine, CXCL12, and compared the effects of prenatal CXCL12 injection to those of prenatal HFD exposure, first, by testing whether the HFD affects circulating CXCL12 in the dam and the CXCL12 system in the offspring brain, and then by examining whether prenatal exposure to CXCL12 itself mimics the effects of a HFD on hypothalamic neuropeptides and emotional behaviors. Our results showed that prenatal exposure to a HFD significantly increased circulating levels of CXCL12 in the dam, and that daily injections of CXCL12 induced a similar increase in CXCL12 levels as the HFD. In addition, prenatal HFD exposure significantly increased the expression of CXCL12 and its receptors, CXCR4 and CXCR7, in the hypothalamic paraventricular nucleus (PVN) of the offspring. Finally, the results revealed strong similarities in the effects of prenatal HFD and CXCL12 administration, which both stimulated neurogenesis and enkephalin (ENK) expression in the PVN, while having inconsistent or no effect in other regions of the hypothalamus, and also increased anxiety as measured by several behavioral tests. These results focus attention specifically on the CXCL12 chemokine system in the PVN of the offspring as being possibly involved in the stimulatory effects of prenatal HFD exposure on ENK-expressing neurons in the PVN and their associated changes in emotional behavior.

Substance P in the anterior thalamic paraventricular nucleus: promotion of ethanol drinking in response to orexin from the hypothalamus.

The paraventricular nucleus of the thalamus (PVT) appears to participate in drug addiction. Recent evidence in rats shows that ethanol drinking is increased by orexin/hypocretin (OX) afferents from the hypothalamus, acting specifically in the anterior (aPVT) rather than posterior (pPVT) PVT subregion. The present study sought to identify neuropeptides transcribed within the PVT, which themselves might contribute to ethanol drinking and possibly mediate the actions of OX. We discovered that substance P (SP) in the aPVT can stimulate intermittent-access ethanol drinking, similar to OX, and that SP receptor [neurokinin 1 receptor/tachykinin receptor 1 (NK1R)] antagonists in this subregion reduce ethanol drinking. As with OX, this effect is site specific, with SP in the pPVT or dorsal third ventricle having no effect on ethanol drinking, and it is behaviorally specific, with SP in the aPVT reducing the drinking of sucrose and stimulating it in the pPVT. A close relationship between SP and OX was demonstrated by a stimulatory effect of local OX injection on SP mRNA and peptide levels, specifically in the aPVT but not pPVT, and a stimulatory effect of OX on SP expression in isolated thalamic neurons, reflecting postsynaptic actions. A functional relationship between OX and SP in the aPVT is suggested by our additional finding that ethanol drinking induced by OX is blocked by a local NK1R antagonist administered at a sub-threshold dose. These results, suggesting that SP in the aPVT mediates the stimulatory effect of OX on ethanol drinking, identify a new role for SP in the control of this behavior.

Consumption of Substances of Abuse during Pregnancy Increases Consumption in Offspring: Possible Underlying Mechanisms.

Correlative human observational studies on substances of abuse have been highly dependent on the use of rodent models to determine the neuronal and molecular mechanisms that control behavioral outcomes. This is particularly true for gestational exposure to non-illicit substances of abuse, such as excessive dietary fat, ethanol, and nicotine, which are commonly consumed in our society. Exposure to these substances during the prenatal period has been shown in offspring to increase their intake of these substances, induce other behavioral changes, and affect neurochemical systems in several brain areas that are known to control behavior. More importantly, emerging studies are linking the function of the immune system to these neurochemicals and ingestion of these abused substances. This review article will summarize the prenatal rodent models used to study developmental changes in offspring caused by prenatal exposure to dietary fat, ethanol, or nicotine. We will discuss the various techniques used for the administration of these substances into rodents and summarize the published outcomes induced by prenatal exposure to these substances. Finally, this review will cover some of the recent evidence for the role of immune factors in causing these behavioral and neuronal changes.

Relationship of the Chemokine, CXCL12, to Effects of Dietary Fat on Feeding-Related Behaviors and Hypothalamic Neuropeptide Systems.

The intake of a high fat diet (HFD), in addition to stimulating orexigenic neuropeptides in the hypothalamus while promoting overeating and reducing locomotor behavior, is known to increase inflammatory mediators that modulate neuronal systems in the brain. To understand the involvement of chemokines in the effects of a HFD, we examined in rats whether HFD intake affects a specific chemokine, CXCL12, and its receptors, CXCR4 and CXCR7, in the hypothalamus together with the neuropeptides and whether CXCL12 itself acts similarly to a HFD in stimulating the neuropeptides and altering ingestion and locomotor behavior. Compared to low-fat chow, a HFD for 5 days significantly increased the expression of CXCL12 and its receptors, in both the paraventricular nucleus (PVN) where the neuropeptides enkephalin (ENK) and galanin were also stimulated and the perifornical lateral hypothalamus (PFLH) where orexin (OX) and melanin-concentrating hormone (MCH) were increased. In contrast, the HFD had no impact on expression of CXCL12 or its receptors in the arcuate nucleus (ARC) where the carbohydrate-related peptide, neuropeptide Y (NPY), was suppressed. Analysis of protein levels revealed a similar stimulatory effect of a HFD on CXCL12 levels in the PVN and PFLH, as well as in blood, and an increase in the number of CXCR4-positive cells in the PVN. In the ARC, in contrast, levels of CXCL12 and number of CXCR4-positive cells were too low to measure. When centrally administered, CXCL12 was found to have similar effects to a HFD. Injection of CXCL12 into the third cerebral ventricle immediately anterior to the hypothalamus significantly stimulated the ingestion of a HFD, reduced novelty-induced locomotor activity, and increased expression of ENK in the PVN where the CXCR4 receptors were dense. It had no impact, however, on NPY in the ARC or on OX and MCH in the PFLH where the CXCR4 receptors were not detected. These results, showing CXCL12 in the hypothalamus to be stimulated by a HFD and to mimic the effects of the HFD where its receptors are located, suggest that this chemokine system may have a role in mediating both the neuronal and behavioral effects induced by a fat-rich diet.

Regulation of the orexigenic neuropeptide, enkephalin, by PPARδ and fatty acids in neurons of the hypothalamus and forebrain.

Ingestion of a high-fat diet composed mainly of the saturated fatty acid, palmitic (PA), and the unsaturated fatty acid, oleic (OA), stimulates transcription in the brain of the opioid neuropeptide, enkephalin (ENK), which promotes intake of substances of abuse. To understand possible underlying mechanisms, this study examined the nuclear receptors, peroxisome proliferator-activated receptors (PPARs), and tested in hypothalamic and forebrain neurons from rat embryos whether PPARs regulate endogenous ENK and the fatty acids themselves affect these PPARs and ENK. The first set of experiments demonstrated that knocking down PPARδ, but not PPARα or PPARγ, increased ENK transcription, activation of PPARδ by an agonist decreased ENK levels, and PPARδ neurons coexpressed ENK, suggesting that PPARδ negatively regulates ENK. In the second set of experiments, PA treatment of hypothalamic and forebrain neurons had no effect on PPARδ protein while stimulating ENK mRNA and protein, whereas OA increased both mRNA and protein levels of PPARδ in forebrain neurons while having no effect on ENK mRNA and increasing ENK levels. These findings show that PA has a strong, stimulatory effect on ENK and weak effect on PPARδ protein, whereas OA has a strong stimulatory effect on PPARδ and weak effect on ENK, consistent with the inhibitory effect of PPARδ on ENK. They suggest a function for PPARδ, perhaps protective in nature, in embryonic neurons exposed to fatty acids from a fat-rich diet and provide evidence for a mechanism contributing to differential effects of saturated and monounsaturated fatty acids on neurochemical systems involved in consummatory behavior. Our findings show that PPARδ in forebrain and hypothalamic neurons negatively regulates enkephalin (ENK), a peptide known to promote ingestive behavior. This inverse relationship is consistent with our additional findings, that a saturated (palmitic; PA) compared to a monounsaturated fatty acid (oleic; OA) has a strong stimulatory effect on ENK and weak effect on PPARδ. These results suggest that PPARδ protects against the neuronal effects of fatty acids, which differentially affect neurochemical systems involved in ingestive behavior.

Stimulatory role of the chemokine CCL2 in the migration and peptide expression of embryonic hypothalamic neurons.

Neuroinflammation is a feedback mechanism against infection, with recent studies suggesting a neuromodulatory role. The chemokine, (C-C motif) ligand 2 (CCL2), and its receptor, (C-C motif) receptor type 2 (CCR2), affect neuromodulation and migration in response to damage. Although CCL2 co-localizes with neuropeptides in the hypothalamus that control voluntary behavior, the function of CCL2/CCR2 is unknown. This led us to consider the possibility that CCL2 acting through CCR2, under natural conditions, may affect the migration and peptide levels of hypothalamic neurons that control voluntary behavior. This study used primary embryonic hypothalamic neurons to examine the effect of CCL2 on migratory behavior and on levels of the peptides, enkephalin (ENK) and galanin. Treatment with CCL2 led to a significant, dose-dependent increase in the number of migrated neurons and an increase in the velocity and distance traveled. CCL2 also significantly increased the number of ENK-expressing and CCR2/ENK co-expressing neurons and the percentage of neurons that contain higher levels of ENK. Lastly, CCL2 produced a dose-dependent increase in expression of ENK and galanin. These results provide evidence for a stimulatory effect of CCL2 on embryonic hypothalamic neurons involving changes in migratory behavior, expression, and synthesis of neuropeptides that function in controlling behavior. Our results demonstrate that the chemokine, CCL2, functions through its receptor, CCR2, to stimulate the migration and expression of the orexigenic peptides, enkephalin (ENK) and galanin (GAL), in developing embryonic hypothalamic neurons that are important for controlling ingestive behavior. This evidence reveals broad effects of CCL2 in the developing hypothalamus, showing this chemokine system to be tightly linked to the hypothalamic peptide neurons.

Prenatal exposure to dietary fat induces changes in the transcriptional factors, TEF and YAP, which may stimulate differentiation of peptide neurons in rat hypothalamus.

Gestational exposure to a high-fat diet (HFD) stimulates the differentiation of orexigenic peptide-expressing neurons in the hypothalamus of offspring. To examine possible mechanisms that mediate this phenomenon, this study investigated the transcriptional factor, transcription enhancer factor-1 (TEF), and co-activator, Yes-associated protein (YAP), which when inactivated stimulate neuronal differentiation. In rat embryos and postnatal offspring prenatally exposed to a HFD compared to chow, changes in hypothalamic TEF and YAP and their relationship to the orexigenic peptide, enkephalin (ENK), were measured. The HFD offspring at postnatal day 15 (P15) exhibited in the hypothalamic paraventricular nucleus a significant reduction in YAP mRNA and protein, and increased levels of inactive and total TEF protein, with no change in mRNA. Similarly, HFD-exposed embryos at embryonic day 19 (E19) showed in whole hypothalamus significantly decreased levels of YAP mRNA and protein and TEF mRNA, and increased levels of inactive TEF protein, suggesting that HFD inactivates TEF and YAP. This was accompanied by increased density and fluorescence intensity of ENK neurons. A close relationship between TEF and ENK was suggested by the finding that TEF co-localizes with this peptide in hypothalamic neurons and HFD reduced the density of TEF/ENK co-labeled neurons, even while the number and fluorescence intensity of single-labeled TEF neurons were increased. Increased YAP inactivity by HFD was further evidenced by a decrease in number and fluorescence intensity of YAP-containing neurons, although the density of YAP/ENK co-labeled neurons was unaltered. Genetic knockdown of TEF or YAP stimulated ENK expression in hypothalamic neurons, supporting a close relationship between these transcription factors and neuropeptide. These findings suggest that prenatal HFD exposure inactivates both hypothalamic TEF and YAP, by either decreasing their levels or increasing their inactive form, and that this contributes to the stimulatory effect of HFD on ENK expression and possibly the differentiation of ENK-expressing neurons.

Developmental changes in embryonic hypothalamic neurons during prenatal fat exposure.

Maternal consumption of a fat-rich diet during pregnancy, which causes later overeating and weight gain in offspring, has been shown to stimulate neurogenesis and increase hypothalamic expression of orexigenic neuropeptides in these postnatal offspring. The studies here, using an in vitro model that mimics in vivo characteristics after prenatal high-fat diet (HFD) exposure, investigate whether these same peptide changes occur in embryos and if they are specific to neurons. Isolated hypothalamic neurons were compared with whole hypothalamus from embryonic day 19 (E19) embryos that were prenatally exposed to HFD and were both found to show similar increases in mRNA expression of enkephalin (ENK) and neuropeptide Y (NPY) compared with that of chow-exposed embryos, with no change in melanin-concentrating hormone, orexin, or galanin. Further examination using immunofluorescence cytochemistry revealed an increase in the number of cells expressing ENK and NPY. By plotting the fluorescence intensity of each cell as a probability density function, three different populations of neurons with low, medium, or high levels of ENK or NPY were found in both HFD and chow groups. The prenatal HFD shifted the density of neurons from the population containing low peptide levels to the population containing high peptide levels. This study indicates that neuronal culture is a useful in vitro system for studying diet effects on neuronal development and shows that prenatal HFD exposure alters the population of hypothalamic neurons containing ENK and NPY in the embryo. These changes may contribute to the increase in HFD intake and body weight observed in offspring.

Mechanisms of modal activation of GluA3 receptors.

AMPA receptors are the major excitatory neurotransmitter receptors in the central nervous system and are involved in numerous neurological disorders. An agonist-binding site is present in each of four subunits that form a functional channel. Binding consists of three steps: docking of agonist to the bilobed ligand binding domain (LBD), closure of the LBD, and increased stability of the closed-lobe conformation through interlobe hydrogen bonding. We describe GluA3 single channel currents activated by nitrowillardiine (NO(2)W) and chlorowillardiine (ClW) in the presence of cyclothiazide, in conjunction with crystal structures of GluA2 and GluA3 LBDs bound to fluorowillardiine (FW), ClW, and NO(2)W. When bound to NO(2)W or ClW, the GluA3 channel opens to three conductance levels with comparable open probabilities and displays modal behavior similar to that obtained with glutamate and FW as agonists (Poon et al., 2010). At lower concentrations, ClW evoked an alternate kinetic behavior, consisting of high open probability in lower conductance states. The structure of ClW bound to GluA3 LBD exhibits a unique partially open hydrogen bonding structure that may be associated with these alternative kinetics. NO(2)W evoked longer open times than seen for other agonists in high and very high modes. The structure ofGluA2 LBD bound to NO(2)W exhibits fully closed lobes with additional interlobe interactions mediated by the nitro group. Beyond differences in efficacy between full and partial agonists, the complexities of the single channel behavior of AMPA receptors may also be associated with small interactions that modify the stability of various degrees of closure.

Characterizing single-channel behavior of GluA3 receptors.

AMPA receptors play a major role in excitatory neurotransmission in the CNS and are involved in numerous neurological disorders. Agonists bind to each of four bilobed LBDs of this tetrameric receptor, and upon binding, the lobes close to envelope the agonist, leading to channel activation. However, AMPA receptors exhibit complex activation kinetics, the mechanism of which has not yet been determined. We report here single-channel studies of a homomeric AMPA receptor (GluA3) activated by the full agonist, glutamate, and a partial agonist, fluorowillardiine. Both agonists activate the channel to the same three open conductance levels but with different open probabilities in each level. The closed probability (P(c)) varied within records, particularly at low agonist concentrations. By sorting discrete segments of the record according to P(c) using the X-means algorithm, we defined five modes of activity. The kinetic behavior could then be analyzed for both agonists over a range of agonist concentrations with a relatively simple model (three closed states and two open states for each open conductance level). The structural mechanism underlying the modal behavior is not clear; however, it occurs on a timescale consistent with hydrogen bonding across the lobe interface in the LBD.

Distinct modulations of human capsaicin receptor by protons and magnesium through different domains.

The capsaicin receptor (TRPV1) is a nonselective cation channel that integrates multiple painful stimuli, including capsaicin, protons, and heat. Protons facilitate the capsaicin- and heat-induced currents by decreasing thermal threshold or increasing agonist potency for TRPV1 activation (Tominaga, M., Caterina, M. J., Malmberg, A. B., Rosen, T. A., Gilbert, H., Skinner, K., Raumann, B. E., Basbaum, A. I., and Julius, D. (1998) Neuron 21, 531-543). In the presence of saturating capsaicin, rat TRPV1 (rTRPV1) reaches full activation, with no further stimulation by protons. Human TRPV1 (hTRPV1), a species ortholog with high homology to rTRPV1, is potentiated by extracellular protons and magnesium, even at saturating capsaicin. We investigated the structural basis for protons and magnesium modulation of fully capsaicin-bound human receptors. By analysis of chimeric channels between hTRPV1 and rTRPV1, we found that transmembrane domain 1-4 (TM1-4) of TRPV1 determines whether protons can further open the fully capsaicin-bound receptors. Mutational analysis identified a titratable glutamate residue (Glu-536) in the linker between TM3 and TM4 critical for further stimulation of fully liganded hTRPV1. In contrast, hTRPV1 TM5-6 is required for magnesium augmentation of capsaicin efficacy. Our results demonstrate that capsaicin efficacy of hTRPV1 correlates with the extracellular ion milieu and unravel the relevant structural basis of modulation by protons and magnesium.

Miscoding properties of 6alpha- and 6beta-diastereoisomers of the N(2)-(estradiol-6-yl)-2'-deoxyguanosine DNA adduct by Y-family human DNA polymerases.

Treatment with estrogen increases the risk of breast, ovary, and endometrial cancers in women. DNA damage induced by estrogen is thought to be involved in estrogen carcinogenesis. In fact, Y-family human DNA polymerases (pol) eta and kappa, which are highly expressed in the reproductive organs, miscode model estrogen-derived DNA adducts during DNA synthesis. Since the estrogen-DNA adducts are a mixture of 6alpha- and 6beta-diastereoisomers of dG-N(2)-6-estrogen or dA-N(6)-6-estrogen, the stereochemistry of each isomeric adduct on translesion synthesis catalyzed by DNA pols has not been investigated. We have recently established a phosphoramidite chemical procedure to insert 6alpha- or 6beta-isomeric N(2)-(estradiol-6-yl)-2'-deoxyguanosine (dG-N(2)-6-E(2)) into oligodeoxynucleotides. Using such site-specific modified oligomer as a template, the specificity and frequency of miscoding by dG-N(2)-6alpha-E(2) or dG-N(2)-6beta-E(2) were explored using pol eta and a truncated form of pol kappa (pol kappaDeltaC). Translesion synthesis catalyzed by pol eta bypassed both the 6alpha- and 6beta-isomers of dG-N(2)-6-E(2), with a weak blockage at the adduct site, while translesion synthesis catalyzed by pol kappaDeltaC readily bypassed both isomeric adducts. Quantitative analysis of base substitutions and deletions occurring at the adduct site showed that pol kappaDeltaC was more efficient than pol eta by incorporating dCMP opposite both 6alpha- and 6beta-isomeric dG-N(2)-6-E(2) adducts. The miscoding events occurred more frequently with pol eta, but not with pol kappaDeltaC. Pol eta promoted incorporation of dAMP and dTMP at both the 6alpha- and 6beta-isomeric adducts, generating G --> T transversions and G --> A transitions. One- and two-base deletions were also formed. The 6alpha-isomeric adduct promoted slightly lower frequency of dCMP incorporation and higher frequency of dTMP incorporation and one-base deletions, compared with the 6beta-isomeric adduct. These observations were supported by steady-state kinetic studies. Taken together, the miscoding property of the 6alpha-isomeric dG-N(2)-6-E(2) is likely to be similar to that of the 6beta-isomeric adduct.

Translesion synthesis past estrogen-derived DNA adducts by human DNA polymerases eta and kappa.

Newly discovered human DNA polymerase (pol) eta and kappa are highly expressed in the reproductive organs, such as testis, ovary, and uterus, where steroid hormones are produced. Because treatment with estrogen increases the risk of developing breast, ovary, and endometrial cancers, miscoding events occurring at model estrogen-derived DNA adducts were explored using pol eta and a truncated form of human pol kappa (pol kappaDeltaC). These enzymes bypassed N(2)-[3-methoxyestra-1,3,5(10)-trien-6-yl]-2'-deoxyguanosine (dG-N(2)-3MeE) and N(6)-[3-methoxyestra-1,3,5(10)-trien-6-yl]-2'-deoxyadenosine (dA-N(6)-3MeE), which were embedded in site-specifically modified oligodeoxynucleotide templates. Quantitative analysis of base substitutions and deletions occurring at the lesion site showed that pol kappaDeltaC was more efficient at incorporating dCMP opposite the dG-N(2)-3MeE lesion than pol eta. Surprisingly, the frequency of translesion synthesis beyond the dC*dG-N(2)-3MeE pair was 13% of the normal dC*dG pair and was 4 and 6 orders of magnitude higher than that of dC*(+)-trans-dG-N(2)-benzo[a]pyrene and dC*dG-C8-acetylaminofluorene pairs, respectively, suggesting that dG-N(2)-3MeE is a natural substrate for pol kappa. In contrast, the bypass frequency beyond the dT*dA-N(6)-3MeE pair was 7 orders of magnitude less than that for the normal dT*dA pair. dA-N(6)-3MeE is a more miscoding lesion than dG-N(2)-3MeE. Pol eta promoted incorporation of dAMP and dCMP at the dA-N(6)-3MeE lesion, while with pol kappaDeltaC, deletions were more frequently observed, along with incorporation of dAMP and dCMP opposite the lesion. These observations were also supported by steady-state kinetic studies. When taken together, the properties of pol eta and kappa are consistent with the mutagenic events attributed to estrogen-derived DNA adducts.