A male pheromone that improves the quality of the oogenic germline.

Pheromones exchanged by conspecifics are a major class of chemical signals that can alter behavior, physiology, and development. In particular, males and females communicate with potential mating partners via sex pheromones to promote reproductive success. Physiological and developmental mechanisms by which pheromones facilitate progeny production remain largely enigmatic. Here, we describe how a Caenorhabditis elegans male pheromone, ascr#10, improves the oogenic germline. Before most signs of aging become evident, C. elegans hermaphrodites start producing lower-quality gametes characterized by abnormal morphology, increased rates of chromosomal nondisjunction, and higher penetrance of deleterious alleles. We show that exposure to the male pheromone substantially ameliorates these defects and reduces embryonic lethality. ascr#10 stimulates proliferation of germline precursor cells in adult hermaphrodites. Coupled to the greater precursor supply is increased physiological germline cell death, which is required to improve oocyte quality in older mothers. The hermaphrodite germline is sensitive to the pheromone only during a time window, comparable in duration to a larval stage, in early adulthood. During this period, prereproductive adults assess the suitability of the environment for reproduction. Our results identify developmental events that occur in the oogenic germline in response to a male pheromone. They also suggest that the opposite effects of the pheromone on gamete quality and maternal longevity arise from competition over resource allocation between soma and the germline.

Serotonergic signaling plays a deeply conserved role in improving oocyte quality.

Declining germline quality is a major cause of reproductive senescence. Potential remedies could be found by studying regulatory pathways that promote germline quality. Several lines of evidence, including a C. elegans male pheromone ascr#10 that counteracts the effects of germline aging in hermaphrodites, suggest that the nervous system plays an important role in regulating germline quality. Inspired by the fact that serotonin mediates ascr#10 signaling, here we show that serotonin reuptake inhibitors recapitulate the effects of ascr#10 on the germline and promote healthy oocyte aging in C. elegans. Surprisingly, we found that pharmacological increase of serotonin signaling stimulates several developmental processes in D. melanogaster, including improved oocyte quality, although underlying mechanisms appear to be different between worms and flies. Our results reveal a plausibly conserved role for serotonin in maintaining germline quality and identify a class of therapeutic interventions using available compounds that could efficiently forestall reproductive aging.

A Caenorhabditis elegans Male Pheromone Feminizes Germline Gene Expression in Hermaphrodites and Imposes Life-History Costs.

Sex pheromones not only improve the reproductive success of the recipients, but also impose costs, such as a reduced life span. The underlying mechanisms largely remain to be elucidated. Here, we show that even a brief exposure to physiological amounts of the dominant Caenorhabditis elegans male pheromone, ascr#10, alters the expression of thousands of genes in hermaphrodites. The most dramatic effect on the transcriptome is the upregulation of genes expressed during oogenesis and the downregulation of genes associated with male gametogenesis. This result reveals a way in which social signals help to resolve the inherent conflict between spermatogenesis and oogenesis in a simultaneous hermaphrodite, presumably to optimally align reproductive function with the presence of potential mating partners. We also found that exposure to ascr#10 increased the risk of persistent intestinal infections in hermaphrodites due to pathological pharyngeal hypertrophy. Thus, our study reveals ways in which the male pheromone can not only have beneficial effects on the recipients' reproduction, but also cause harmful consequences that reduce life span.

The serotonin circuit that coordinates germline proliferation and egg laying with other reproductive functions in Caenorhabditis elegans.

Behaviour and physiology are altered in reproducing animals, but neuronal circuits that regulate these changes remain largely unknown. Insights into mechanisms that regulate and possibly coordinate reproduction-related traits could be gleaned from the study of sex pheromones that can improve the reproductive success of potential mating partners. In Caenorhabditis elegans, the prominent male pheromone, ascr#10, modifies reproductive behaviour and several aspects of reproductive physiology in hermaphrodite recipients, including improving oocyte quality. Here we show that a circuit that contains serotonin-producing and serotonin-uptaking neurons plays a key role in mediating effects of ascr#10 on germline development and egg laying behaviour. We also demonstrate that increased serotonin signalling promotes proliferation of germline progenitors in adult hermaphrodites. Our results establish a role for serotonin in maintaining germline quality and highlight a simple neuronal circuit that acts as a linchpin that couples food intake, mating behaviour, reproductive output, and germline renewal and provisioning.

The roles of TGFß and serotonin signaling in regulating proliferation of oocyte precursors and germline aging.

The decline of oocyte quality in aging but otherwise relatively healthy individuals compels a search for underlying mechanisms. Building upon a finding that exposure to male pheromone ascr#10 improves oocyte quality in C. elegans, we uncovered a regulatory cascade that promotes proliferation of oocyte precursors in adults and regulates oocyte quality. We found that the male pheromone promotes proliferation of oocyte precursors by upregulating LAG-2, a ligand of the Notch-like pathway in the germline stem cell niche. LAG-2 is upregulated by a TGFß-like ligand DAF-7 revealing similarity of regulatory mechanisms that promote germline proliferation in adults and larvae. A serotonin circuit that also regulates food search and consumption upregulates DAF-7 specifically in adults. The serotonin/DAF-7 signaling promotes germline expansion to compensate for oocyte expenditure which is increased by the male pheromone. Finally, we show that the earliest events in reproductive aging may be due to declining expression of LAG-2 and DAF-7. Our findings highlight neuronal signals that promote germline proliferation in response to the environment and argue that deteriorating oocyte quality may be due to reduced neuronal expression of key germline regulators.

A C. elegans male pheromone feminizes germline gene expression in hermaphrodites and imposes life-history costs.

Sex pheromones improve reproductive success, but also impose costs. Here we show that even brief exposure to physiological amounts of the dominant C. elegans male pheromone, ascr#10, alters the expression of thousands of genes in hermaphrodites. The most dramatic effect on the transcriptome was the upregulation of genes expressed during oogenesis and downregulation of genes associated with male gametogenesis. Among the detrimental effects of ascr#10 on hermaphrodites is the increased risk of persistent infections caused by pathological pharyngeal hypertrophy. Our results reveal a way in which social signals help to resolve the inherent conflict between spermatogenesis and oogenesis in a simultaneous hermaphrodite, presumably to optimally align reproductive function to the presence of potential mating partners. They also show that the beneficial effects of the pheromone are accompanied by harmful consequences that reduce lifespan.

The E2 Marie Kondo and the CTLH E3 ligase clear deposited RNA binding proteins during the maternal-to-zygotic transition.

The maternal-to-zygotic transition (MZT) is a conserved step in animal development, where control is passed from the maternal to the zygotic genome. Although the MZT is typically considered from its impact on the transcriptome, we previously found that three maternally deposited Drosophila RNA-binding proteins (ME31B, Trailer Hitch [TRAL], and Cup) are also cleared during the MZT by unknown mechanisms. Here, we show that these proteins are degraded by the ubiquitin-proteasome system. Marie Kondo, an E2 conjugating enzyme, and the E3 CTLH ligase are required for the destruction of ME31B, TRAL, and Cup. Structure modeling of the Drosophila CTLH complex suggests that substrate recognition is different than orthologous complexes. Despite occurring hours earlier, egg activation mediates clearance of these proteins through the Pan Gu kinase, which stimulates translation of Kdo mRNA. Clearance of the maternal protein dowry thus appears to be a coordinated, but as-yet underappreciated, aspect of the MZT.

Bestselling author and organizing consultant Marie Kondo has helped people around the world declutter their homes by getting rid of physical items that do not bring them joy. Keeping the crowded environment inside a living cell organized also requires work and involves removing molecules that are no longer needed. A fertilized egg cell, for example, contains molecules from the mother that regulate the initial stages as it develops into an embryo. Later on, the embryo takes control of its own development by destroying these inherited molecules and switches to making its own instead. This process is called the maternal-to-zygotic transition. The molecules passed from the mother to the egg cell include proteins and messenger RNAs (molecules that include the coded instructions to make new proteins). Previous research has begun to reveal how the embryo destroys the mRNAs it inherits from its mother and how it starts to make its own. Yet almost nothing is known about how an embryo gets rid of its mother's proteins. To address this question, Zavortink, Rutt, Dzitoyeva et al. used an approach known as an RNA interference screen to identify factors required to destroy three maternal proteins in fruit fly embryos. The experiments helped identify one enzyme that worked together with another larger enzyme complex to destroy the maternal proteins. This enzyme belongs to a class of enzymes known as ubiquitin-conjugating enzymes (or E2 enzymes) and it was given the name "Kdo", short for "Marie Kondo". Further experiments showed that the mRNAs that code for the Kdo enzyme were present in unfertilized eggs, but in a repressed state that prevented the eggs from making the enzyme. Once an egg started to develop into an embryo, these mRNAs became active and the embryo started to make Kdo enzymes. This led to the three maternal proteins being destroyed during the maternal-to-zygotic transition. These findings reveal a new pathway that regulates the destruction of maternal proteins as the embryo develops. The next challenge will be identifying other maternal proteins that do not "spark joy" and understanding the role their destruction plays in the earliest events of embryonic development.

5-Lipoxygenase DNA methylation and mRNA content in the brain and heart of young and old mice.

The expression of 5-lipoxygenase (5-LOX) is affected by aging and regulated by epigenetic mechanisms including DNA methylation. We used methylation-sensitive restriction endonucleases (AciI, BstUI, HpaII, and HinP1I) to assess 5-LOX DNA methylation in brain and heart tissue samples from young (2 months) and old (22 months) mice. We also measured mRNA content for 5-LOX and the DNA methyltransferases DNMT1 and DNMT3a. In young mice, the 5-LOX mRNA content was significantly greater in the heart compared to the brain; 5-LOX DNA methylation was lower, except in the AciI assay in which it was higher in the heart. Aging decreased 5-LOX mRNA content in the heart and increased it in the brain. Aging also increased 5-LOX DNA methylation and this effect was site- (i.e., enzyme) and tissue-specific. Generally, DNMT1 and DNMT3a mRNA content was lower in the brain regions compared to the heart; the only effect of aging was observed in the mRNA content of DNMT3a, which was decreased in the heart of old mice. These results indicate a complex tissue-specific and aging-dependent interplay between the DNA methylation system and 5-LOX mRNA content. Interpretation of this data must take into account that the tissue samples contained a mixture of various cell types.

The lncRNA BDNF-AS is an epigenetic regulator in the human amygdala in early onset alcohol use disorders.

Adolescent alcohol drinking is known to contribute to the development and severity of alcohol use disorders (AUDs) later in adulthood. Recent studies have shown that long non-coding RNAs (lncRNAs) are critical for brain development and synaptic plasticity. One such lncRNA is natural occurring brain-derived neurotrophic factor antisense (BDNF-AS) that has been shown to regulate BDNF expression. The role of BDNF-AS lncRNA in the molecular mechanisms of AUD is unknown. Here, we evaluated the expression and functional role of BDNF-AS in postmortem amygdala of either early onset or late onset alcoholics (individuals who began drinking before or after 21 years of age, respectively) and age-matched control subjects. BDNF-AS expression is increased in early onset but not in late onset AUD amygdala and appears to be regulated epitranscriptomically via decreased N6-methyladenosine on BDNF-AS. Upregulation of BDNF-AS is associated with a significant decrease in BDNF expression and increased recruitment of EZH2, which deposits repressive H3K27 trimethylation (H3K27me3) at regulatory regions in the BDNF gene in the early onset AUD group. Drinking during adolescence also contributed to significant decreases in activity-regulated cytoskeleton-associated protein (ARC) expression which also appeared to be mediated by increased EZH2 deposition of repressive H3K27me3 at the ARC synaptic activity response element. These results suggest an important role for BDNF-AS in the regulation of synaptic plasticity via epigenetic reprogramming in the amygdala of AUD subjects who began drinking during adolescence.

Melatonin signaling in mouse cerebellar granule cells with variable native MT1 and MT2 melatonin receptors.

Although G protein-coupled MT1 and MT2 melatonin receptors are expressed in neurons of the mammalian brain including in humans, relatively little is known about the influence of native MT1 and MT2 melatonin receptors on neuronal melatonin signaling. Whereas human cerebellar granule cells (CGC) express only MT1 receptors, mouse CGC express both MT1 and MT2. To study the effects of altered neuronal MT1/MT2 receptors, we used CGC cultures prepared from immature cerebella of wild-type mice (MT1/MT2 CGC) and MT1- and MT2-knockout mice (MT2 and MT1 CGC, respectively). Here we report that in MT1/MT2 cultures, physiological (low nanomolar) concentrations of melatonin decrease the activity (phosphorylation) of extracellular-signal-regulated kinase (ERK) whereas a micromolar concentration was ineffective. Both MT1 and MT2 deficiencies transformed the melatonin inhibition of ERK into melatonin-induced ERK activation. In MT1/MT2 CGC, 1 nM melatonin inhibited serine/threonine kinase Akt, whereas in MT1 and MT2 CGC, this concentration was ineffective. Under these conditions, both MT1 and MT2 deficiencies prevented melatonin from inhibiting forskolin-stimulated cAMP levels and cFos immunoreactivity. We demonstrated that selective removal of native neuronal MT1 and MT2 receptors has a profound effect on the intracellular actions of low/physiological concentrations of melatonin. Since the expression of MT1 and MT2 receptors is cell-type-specific and species-dependent, we postulate that the pattern of expression of neuronal melatonin receptor types in different brain areas and cells could determine the capabilities of endogenous melatonin in regulating neuronal functioning.

Stimulatory effects of a melatonin receptor agonist, ramelteon, on BDNF in mouse cerebellar granule cells.

Melatonin receptor activation has been linked to the regulation of neurotrophic factors, including the brain-derived neurotrophic factor (BDNF). To further characterize the effects of melatonin receptor stimulation on neuronal BDNF, we used a clinically available novel agonist for MT1 and MT2 melatonin receptors, ramelteon. Primary cultures of cerebellar granule cells (CGC) have been established as an in vitro model for studying neuronal BDNF. We took advantage of the availability of MT1- and MT2-deficient (knockout; KO) mice to employ primary CGC prepared from wild type (WT), MT1 KO, and MT2 KO mice. We investigated the effects of ramelteon on BDNF protein and mRNA content. Administered in a low nanomolar range, ramelteon increased BDNF protein content in all three types of mouse CGC. This ramelteon-triggered BDNF protein elevation was not preceded by a BDNF mRNA increase. However, it was prevented by treatment of cultures with a protein synthesis inhibitor cycloheximide. These results demonstrated that the MT1/MT2 melatonin receptor agonist ramelteon is capable of increasing BDNF protein in neurons expressing either of the two melatonin receptor types and that this action of ramelteon involves translational mechanisms. Further research is needed to explore the putative influence of ramelteon on BDNF-associated neuroplasticity.

Techniques: fruit flies as models for neuropharmacological research.

An unlikely animal model is gaining popularity in neuropharmacological research: the 2-mm fruit fly (Drosophila melanogaster). Drugs have been administered to adult flies in their food and, more recently, via gasses and injections. Pharmacological tools have introduced behavioral alterations in Drosophila reminiscent of human behavior, rescued flies from gene-alteration-triggered neuropathologies, and triggered gene silencing. Combined, these methods hold promise for significant neuropharmacological advancement.

Drosophila GABA(B) receptors are involved in behavioral effects of gamma-hydroxybutyric acid (GHB).

Gamma-hydroxybutyric acid (GHB) can be synthesized in the brain but is also a known drug of abuse. Although putative GHB receptors have been cloned, it has been proposed that, similar to the behavior-impairing effects of ethanol, the in vivo effects of pharmacological GHB may involve metabotropic gamma-aminobutyric acid (GABA) GABA(B) receptors. We developed a fruitfly (Drosophila melanogater) model to investigate the role of these receptors in the behavioral effects of exogenous GHB. Injecting GHB into male flies produced a dose-dependent motor impairment (measured with a computer-assisted automated system), which was greater in ethanol-sensitive cheapdate mutants than in wild-type flies. These effects of pharmacological concentrations of GHB require the presence and activation of GABA(B) receptors. The evidence for this was obtained by pharmacological antagonism of GABA(B) receptors with CGP54626 and by RNA interference (RNAi)-induced knockdown of the GABA(B(1)) receptor subtype. Both procedures inhibited the behavioral effects of GHB. GHB pretreatment diminished the behavioral response to subsequent GHB injections; i.e., it triggered GHB tolerance, but did not produce ethanol tolerance. On the other hand, ethanol pretreatment produced both ethanol and GHB tolerance. It appears that in spite of many similarities between ethanol and GHB, the primary sites of their action may differ and that recently cloned putative GHB receptors may participate in actions of GHB that are not mediated by GABA(B) receptors. These receptors do not have a Drosophila orthologue. Whether Drosophila express a different GHB receptor should be explored.

Developmental role of GABAB(1) receptors in Drosophila.

Previously, an RNA interference (RNAi) knockdown of GABAB(1) subunit in adult Drosophila was used for behavioral studies. Here we report on developmental deficits caused by embryonic Drosophila GABAB(1) RNAi and drug antagonism. Injecting embryos with CGP54626 (a GABAB receptor antagonist) reduced hatching and caused lethality. Similar effects were produced by injecting embryos with GABAB(1) double-stranded RNA (RNAi). The surviving GABAB(1) RNAi larvae were significantly smaller than controls and showed a peculiar phenotype; their tracheae were folded. Our results suggest that GABAB receptors are required for normal development and that the Drosophila model could be used to investigate the participating molecular mechanisms.

Brain-derived neurotrophic factor epigenetic modifications associated with schizophrenia-like phenotype induced by prenatal stress in mice.

BACKGROUND: Prenatal stress (PRS) is considered a risk factor for several neurodevelopmental disorders including schizophrenia (SZ). An animal model involving restraint stress of pregnant mice suggests that PRS induces epigenetic changes in specific GABAergic and glutamatergic genes likely to be implicated in SZ, including the gene for brain-derived neurotrophic factor (BDNF). METHODS: Studying adult offspring of pregnant mice subjected to PRS, we explored the long-term effects of PRS on behavior and on the expression of key chromatin remodeling factors including DNA methyltransferase 1, ten-eleven-translocation hydroxylases, methyl CpG binding protein 2, histone deacetylases, and histone methyltransferases and demethylase in the frontal cortex and hippocampus. We also measured the expression of BDNF. RESULTS: Adult PRS offspring demonstrate behavioral abnormalities suggestive of SZ and molecular changes similar to changes seen in postmortem brains of patients with SZ. This includes a significant increase in DNA methyltransferase 1 and ten-eleven-translocation hydroxylase 1 in the frontal cortex and hippocampus but not in cerebellum; no changes in histone deacetylases, histone methyltransferases and demethylases, or methyl CpG binding protein 2, and a significant decrease in Bdnf messenger RNA variants. The decrease of the corresponding Bdnf transcript level was accompanied by an enrichment of 5-methylcytosine and 5-hydroxymethylcytosine at Bdnf gene regulatory regions. In addition, the expression of Bdnf transcripts (IV and IX) correlated positively with social approach in both PRS mice and nonstressed mice. CONCLUSIONS: Because patients with psychosis and PRS mice show similar epigenetic signature, PRS mice may be a suitable model for understanding the behavioral and molecular epigenetic changes observed in patients with SZ.

Identification of a novel Drosophila gene, beltless, using injectable embryonic and adult RNA interference (RNAi).

BACKGROUND: RNA interference (RNAi) is a process triggered by a double-stranded RNA that leads to targeted down-regulation/silencing of gene expression and can be used for functional genomics; i.e. loss-of-function studies. Here we report on the use of RNAi in the identification of a developmentally important novel Drosophila (fruit fly) gene (corresponding to a putative gene CG5652/GM06434), that we named beltless based on an embryonic loss-of-function phenotype. RESULTS: Beltless mRNA is expressed in all developmental stages except in 0-6 h embryos. In situ RT-PCR localized beltless mRNA in the ventral cord and brain of late stage embryos and in the nervous system, ovaries, and the accessory glands of adult flies. RNAi was induced by injection of short (22 bp) beltless double-stranded RNAs into embryos or into adult flies. Embryonic RNAi altered cuticular phenotypes ranging from partially-formed to missing denticle belts (thus beltless) of the abdominal segments A2-A4. Embryonic beltless RNAi was lethal. Adult RNAi resulted in the shrinkage of the ovaries by half and reduced the number of eggs laid. We also examined Df(1)RK4 flies in which deletion removes 16 genes, including beltless. In some embryos, we observed cuticular abnormalities similar to our findings with beltless RNAi. After differentiating Df(1)RK4 embryos into those with visible denticle belts and those missing denticle belts, we assayed the presence of beltless mRNA; no beltless mRNA was detectable in embryos with missing denticle belts. CONCLUSIONS: We have identified a developmentally important novel Drosophila gene, beltless, which has been characterized in loss-of-function studies using RNA interference. The putative beltless protein shares homologies with the C. elegans nose resistant to fluoxetine (NRF) NRF-6 gene, as well as with several uncharacterized C. elegans and Drosophila melanogaster genes, some with prominent acyltransferase domains. Future studies should elucidate the role and mechanism of action of beltless during Drosophila development and in adults, including in the adult nervous system.

An automated assay of the behavioral effects of cocaine injections in adult Drosophila.

Introducing Drosophila models in neuropharmacological research helps to discover new mechanisms of drug action. In fruit flies, the pharmacobehavioral approach has been used to evaluate the effects of drugs of abuse including cocaine. Standard procedures of cocaine administration to flies employ drug vaporization whereas behavior is evaluated either by trained observers or by videotaping followed by analysis via a computer-operated tracking system. Because in mammalian studies cocaine is typically administered by injection, a procedure that ensures precise and timely dose delivery, we developed a method for injecting cocaine into adult Drosophila. To objectively measure the behavioral response of flies to cocaine injections, we adapted the standard Drosophila Activity Monitoring System (Trikinetics). We found that in wild-type Canton S flies, cocaine injections produce a dose-dependent increase in the number of hyperactivity bursts and that repeated injections of cocaine produce behavioral sensitization. Acute responses to cocaine were observed also in period null (per(0)) mutant flies, but in these flies, repeated injections of cocaine did not produce sensitization. In conclusion, we developed a method for accurately measuring the behavioral effects of cocaine in adult fruit flies that can be applied to studies of the mechanisms of behavioral sensitization.

Progress in mitochondrial epigenetics.

Mitochondria, intracellular organelles with their own genome, have been shown capable of interacting with epigenetic mechanisms in at least four different ways. First, epigenetic mechanisms that regulate the expression of nuclear genome influence mitochondria by modulating the expression of nuclear-encoded mitochondrial genes. Second, a cell-specific mitochondrial DNA content (copy number) and mitochondrial activity determine the methylation pattern of nuclear genes. Third, mitochondrial DNA variants influence the nuclear gene expression patterns and the nuclear DNA (ncDNA) methylation levels. Fourth and most recent line of evidence indicates that mitochondrial DNA similar to ncDNA also is subject to epigenetic modifications, particularly by the 5-methylcytosine and 5-hydroxymethylcytosine marks. The latter interaction of mitochondria with epigenetics has been termed 'mitochondrial epigenetics'. Here we summarize recent developments in this particular area of epigenetic research. Furthermore, we propose the term 'mitoepigenetics' to include all four above-noted types of interactions between mitochondria and epigenetics, and we suggest a more restricted usage of the term 'mitochondrial epigenetics' for molecular events dealing solely with the intra-mitochondrial epigenetics and the modifications of mitochondrial genome.

Reduction of Cellular Lipid Content by a Knockdown of Drosophila PDP1 γ and Mammalian Hepatic Leukemia Factor.

In exploring the utility of double-stranded RNA (dsRNA) injections for silencing the PAR-domain protein 1 (Pdp1) gene in adult Drosophila, we noticed a dramatic loss of fat tissue lipids. To verify that our RNAi approach produced the expected Pdp1 knockdown, the abdominal fat tissues sections were stained with PDP1 antibodies. PDP1 protein immunostaining was absent in flies injected with dsRNA targeting a sequence common to all known Pdp1 isoforms. Subsequent experiments revealed that lipid staining is reduced in flies injected with dsRNA against Pdp1 γ (fat body specific) and not against Pdp1 ε (predominantly involved in circadian mechanisms). Drosophila PDP1 γ protein shows a high homology to mammalian thyrotroph embryonic factor (TEF), albumin D site-binding protein (DBP), and hepatic leukemia factor (HLF) transcription factors. In an in vitro model of drug- (olanzapine-) induced adiposity in mouse 3T3-L1 cells, the mRNA content of HLF but not TEF and DBP was increased by the drug treatment. A knockdown of the HLF mRNA by transfecting the cultures with HLF dsRNA significantly reduced their lipid content. Furthermore, the HLF RNAi prevented olanzapine from increasing the cell lipid content. These results suggest that the PDP1/HLF system may play a role in physiological and drug-influenced lipid regulation.

5-lipoxygenase-activating protein as a modulator of olanzapine-induced lipid accumulation in adipocyte.

Experiments were performed in 3T3-L1 preadipocytes differentiated in vitro into adipocytes. Cells were treated with olanzapine and a 5-lipoxygenase (5-LOX) activating protein (FLAP) inhibitor MK-886. Lipid content was measured using an Oil Red O assay; 5-LOX and FLAP mRNA content was measured using quantitative real-time PCR; the corresponding protein contents were measured using quantitative Western blot assay. Olanzapine did not affect the cell content of 5-LOX mRNA and protein; it decreased FLAP mRNA and protein content at day five but not 24 hours after olanzapine addition. In the absence of MK-886, low concentrations of olanzapine increased lipid content only slightly, whereas a 56% increase was induced by 50 µ M olanzapine. A 5-day cotreatment with 10 µ M MK-886 potentiated the lipid increasing action of low concentrations of olanzapine. In contrast, in the presence of 50 µ M olanzapine nanomolar and low micromolar concentrations of MK-886 reduced lipid content. These data suggest that FLAP system in adipocytes is affected by olanzapine and that it may modify how these cells respond to the second-generation antipsychotic drugs (SGADs). Clinical studies could evaluate whether the FLAP/5-LOX system could play a role in setting a variable individual susceptibility to the metabolic side effects of SGADs.