Prenatal Exposure to Dietary Fat Induces Changes in the Transcriptional Factors,TEF and YAP, Which May Stimulate Differentiation of Peptide Neurons in Rat Hypothalamus

Which May Stimulate Differentiation of Peptide Neurons in Rat Hypothalamus. PLoS ONE 8(10): e77668. doi:10.1371/journal.pone. 0077668 Prenatal Exposure to Dietary Fat Induces Changes in the Transcriptional Factors,TEF and YAP, Which May Stimulate Differentiation of Peptide Neurons in Rat Hypothalamus Kinning Poon 0 Sushma Mandava 0 Karen Chen 0 Jessica R. Barson 0 Sylvie Buschlen 0 Sarah F. Leibowitz 0 Thierry Alquier, CRCHUM-Montreal Diabetes Research Center, Canada 0 The Rockefeller University, Laboratory of Behavioral Neurobiology , New York, New York , United States of America 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. - Funding: This work was supported by the NIH Grant 5R01DA021518, and by NARSAD grant CEN5401363. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Obesity is a growing epidemic, with the latest National Health and Nutrition Survey finding that 36% of adults and 17% of adolescents and children are obese [1]. Clinical and animal studies have attributed this rise, in part, to fetal programming produced by maternal obesity and overconsumption of a fatrich diet, which in the offspring increases preference for a highfat diet (HFD) and risk for higher weight gain and metabolic disorders [2-6]. The hypothalamus, which is an important part food intake and energy expenditure, has been shown to be markedly affected by prenatal exposure to a HFD. Maternal ingestion of a HFD stimulates neurogenesis in early embryonic hypothalamus and increases the number of neurons that express neuropeptides known to stimulate ingestive behavior [4,7]. Further, prenatal HFD exposure increases the peptideexpressing neuroprogenitor cell population in embryos, which later differentiate into functional peptide neurons [8,9]. The cellular factors involved in this stimulatory effect on the differentiation and expression of peptide neurons are unknown of the brain that controls energy homeostasis by regulating but are likely to involve transcriptional regulators of neuronal differentiation, many of which, with the exception of c-Fos and CREB [10-12], have never been studied in relation to the orexigenic peptides in the hypothalamus. The transcription enhancer factor-1 (TEF) is a transcriptional factor that partners predominantly with the transcriptional coactivator, Yes-associated protein (YAP) [13,14], and is very important for regulating organ growth during development, with genetic deletion of either TEF or YAP found to be fatal to the developing embryo [15,16]. The association of the active forms of TEF and YAP is found to induce transcriptional activation of cell-cycle related elements that affect neuronal differentiation and proliferation [17-19]. Phosphorylation of serine residues of these two transcriptional factors causes them to sequester in the cytoplasm and become inactive [20-23]. A suppression of TEF and YAP activity, either by decreasing levels or increasing the inactive forms of these proteins, activates neuronal differentiation while inhibiting proliferation, whereas excess TEF and YAP causes an expansion of neuroprogenitor cells by inhibiting differentiation and increasing proliferation [17,19,24]. Furthermore, genetic profiling of altered TEF or YAP activity has revealed changes in neuropeptide and neurotransmitter expression [25], hinting at the possibility that these two factors may be involved in the differentiation of hypothalamic neurons that express the orexigenic peptides and in the stimulatory effect produced by prenatal exposure to a HFD. Recent studies focus attention on the orexigenic peptide, enkephalin (ENK), in terms of its close relationship to dietary fat in both adults and embryos. In adult rats, this opioid peptide stimulates the intake of a HFD when injected into the hypothalamic paraventricular nucleus (PVN) [26-28], and its levels are endogenously increased in the PVN of animals consuming or prone to overeating the HFD [29]. Also, exposure to a HFD during gestation is found to increase in the PVN not only the expression of ENK but also the number of ENK neurons in the offspring, as demonstrated both in postnatal day 15 (P15) offspring and embryonic day 19 (E19) embryos [4,9], an effect that is sustained into adulthood. As the hypothalamus forms early in development and undergoes restructuring at E19 [30,31] while becoming fully formed by P15, it is interesting that the expression of both TEF and YAP is also at its peak during the embryonic developmental period [32], while decreasing postnatally into adulthood [33]. This temporal overlap of neuronal differentiation with TEF and YAP expression suggests that changes in TEF and YAP activity may be contributing to the increase in differentiated ENK neurons and ENK expression that occurs in th (...truncated)