Npy transcription is regulated by noncanonical STAT3 signaling in hypothalamic neurons: Implication with lipotoxicity and obesity.

Neuropeptide Y (Npy) is an abundant neuropeptide expressed in the central and peripheral nervous systems. NPY-secreting neurons in the hypothalamic arcuate nucleus regulate energy homeostasis, and Npy mRNA expression is regulated by peripheral nutrient and hormonal signals like leptin, interleukin-6 (IL-6), and fatty acids. This study demonstrates that IL-6, which phosphorylates tyrosine 705 (Y705) of STAT3, decreased Npy mRNA in arcuate immortalized hypothalamic neurons. In parallel, inhibitors of STAT3-Y705 phosphorylation, stattic and cucurbitacin I, robustly upregulated Npy mRNA. Chromatin-immunoprecipitation showed high baseline total STAT3 binding to multiple regulatory regions of the Npy gene, which are decreased by IL-6 exposure. The STAT3-Npy interaction was further examined in obesity-related pathologies. Notably, in four different hypothalamic neuronal models where palmitate potently stimulated Npy mRNA, Socs3, a specific STAT3 activity marker, was downregulated and was negatively correlated with Npy mRNA levels (R2 = 0.40, p < 0.001), suggesting that disrupted STAT3 signaling is involved in lipotoxicity-mediated dysregulation of Npy. Finally, human NPY SNPs that map to human obesity or body mass index were investigated for potential STAT3 binding sites. Although none of the SNPs were linked to direct STAT3 binding, analysis show that rs17149106 (-602 G > T) is located on an upstream enhancer element of NPY, where the variant is predicted to disrupt validated binding of KLF4, a known inhibitory cofactor of STAT3 and downstream effector of leptin signaling. Collectively, this study demonstrates that STAT3 signaling negatively regulates Npy transcription, and that disruption of this interaction may contribute to metabolic disorders.

Phenylbutyric acid robustly increases Npy mRNA expression in hypothalamic neurons by increasing H3K9/14 acetylation at the Npy promoter.

Phenylbutyric acid (PBA) is a commonly used inhibitor of endoplasmic reticulum stress, as well as a histone deacetylase (HDAC) inhibitor, that increases hypothalamic expression of orexigenic neuropeptide Y (Npy). Elucidation of the dose-response relationship and mechanism of action of PBA may position this compound as a potential therapeutic for eating disorders where Npy is dysregulated, such as anorexia nervosa. The hypothalamic neuronal model mHypoE-41 was exposed to PBA (5 µM-5 mM) to assess the maximal Npy upregulation. Transcription factors and histone acetylation-related genes were assessed by qRT-PCR, as well as the involvement estrogen receptors (ER) using siRNA knockdown. Changes in global and Npy promoter-specific H3K9/14 acetylation were detected using western analysis and chromatin immunoprecipitation. Treatment with 5 mM PBA led to a 10-fold and 206-fold increase in Npy mRNA at 4 and 16 h, respectively, as well as increased NPY secretion. This induction was not observed with another orexigenic neuropeptide Agrp. PBA significantly increased the expression of Foxo1, Socs3 and Atf3 and the ERs Esr1 and Esr2 mRNA, but the PBA-mediated induction of Npy was not dependent on ERα or ERß. PBA induced histone H3K9/14 acetylation at 3 distinct Npy promoter regions, suggesting increased Npy transcriptional activation due to a more open chromatin structure. We also report changes in Hdac mRNAs by PBA and the fatty acid palmitate, highlighting the importance of epigenetic regulation in Npy transcription. Overall, we conclude that PBA has strong orexigenic potential and can robustly and specifically induce Npy in hypothalamic neurons through a mechanism likely involving histone H3 acetylation.

Oleate restores altered autophagic flux to rescue palmitate lipotoxicity in hypothalamic neurons.

Accumulation of excess lipids in non-adipose tissues, such as the hypothalamus, is termed lipotoxicity and causative of free fatty acid-mediated pathology in metabolic disease. This study aimed to elucidate the molecular mechanisms behind oleate (OA)- and palmitate (PA)-mediated changes in hypothalamic neurons. Using the well-characterized hypothalamic neuronal cell model, mHypoE-46, we assessed gene changes through qRT-PCR, cell death with quantitative imaging, PA metabolism using stable isotope labeling, and cellular mechanisms using pharmacological modulation of lipid metabolism and autophagic flux. Palmitate (PA) disrupts gene expression, including Npy, Grp78, and Il-6 mRNA in mHypoE-46 hypothalamic neurons. Blocking PA metabolism using triacsin-C prevented the increase of these genes, implying that these changes depend on PA intracellular metabolism. Co-incubation with oleate (OA) is also potently protective and prevents cell death induced by increasing concentrations of PA. However, OA does not decrease U-13C-PA incorporation into diacylglycerol and phospholipids. Remarkably, OA can reverse PA toxicity even after significant PA metabolism and cellular impairment. OA can restore PA-mediated impairment of autophagy to prevent or reverse the accumulation of PA metabolites through lysosomal degradation, and not through other reported mechanisms. The autophagic flux inhibitor chloroquine (CQ) mimics PA toxicity by upregulating autophagy-related genes, Npy, Grp78, and Il-6, an effect partially reversed by OA. CQ also prevented the OA defense against PA toxicity, whereas the autophagy inducer rapamycin provided some protection. Thus, PA impairment of autophagic flux significantly contributes to its lipotoxicity, and OA-mediated protection requires functional autophagy. Overall, our results suggest that impairment of autophagy contributes to hypothalamic lipotoxicity.

Hypothalamic miR-1983 Targets Insulin Receptor ß and the Insulin-mediated miR-1983 Increase Is Blocked by Metformin.

MicroRNAs (miRNAs) expressed in the hypothalamus are capable of regulating energy balance and peripheral metabolism by inhibiting translation of target messenger RNAs (mRNAs). Hypothalamic insulin resistance is known to precede that in the periphery, thus a critical unanswered question is whether central insulin resistance creates a specific hypothalamic miRNA signature that can be identified and targeted. Here we show that miR-1983, a unique miRNA, is upregulated in vitro in 2 insulin-resistant immortalized hypothalamic neuronal neuropeptide Y-expressing models, and in vivo in hyperinsulinemic mice, with a concomitant decrease of insulin receptor ß subunit protein, a target of miR-1983. Importantly, we demonstrate that miR-1983 is detectable in human blood serum and that its levels significantly correlate with blood insulin and the homeostatic model assessment of insulin resistance. Levels of miR-1983 are normalized with metformin exposure in mouse hypothalamic neuronal cell culture. Our findings provide evidence for miR-1983 as a unique biomarker of cellular insulin resistance, and a potential therapeutic target for prevention of human metabolic disease.

Mechanisms Driving Palmitate-Mediated Neuronal Dysregulation in the Hypothalamus.

The hypothalamus maintains whole-body homeostasis by integrating information from circulating hormones, nutrients and signaling molecules. Distinct neuronal subpopulations that express and secrete unique neuropeptides execute the individual functions of the hypothalamus, including, but not limited to, the regulation of energy homeostasis, reproduction and circadian rhythms. Alterations at the hypothalamic level can lead to a myriad of diseases, such as type 2 diabetes mellitus, obesity, and infertility. The excessive consumption of saturated fatty acids can induce neuroinflammation, endoplasmic reticulum stress, and resistance to peripheral signals, ultimately leading to hyperphagia, obesity, impaired reproductive function and disturbed circadian rhythms. This review focuses on the how the changes in the underlying molecular mechanisms caused by palmitate exposure, the most commonly consumed saturated fatty acid, and the potential involvement of microRNAs, a class of non-coding RNA molecules that regulate gene expression post-transcriptionally, can result in detrimental alterations in protein expression and content. Studying the involvement of microRNAs in hypothalamic function holds immense potential, as these molecular markers are quickly proving to be valuable tools in the diagnosis and treatment of metabolic disease.

Bisphenol A Induces Agrp Gene Expression in Hypothalamic Neurons through a Mechanism Involving ATF3.

BACKGROUND: Bisphenol A (BPA) is a ubiquitous endocrine disrupting chemical and obesogen. Although limited evidence exists of the effects of BPA on hypothalamic agouti-related peptide (AgRP) levels, the mechanisms underlying these effects remain unknown. Given that AgRP is a potent orexigenic neuropeptide, determining the mechanism by which BPA increases AgRP is critical to preventing the progression to metabolic disease. METHODS: Using quantitative reverse transcriptase polymerase chain reaction, we investigated the response of Agrp-expressing mouse hypothalamic cell lines to BPA treatment. The percentage of total BPA entering hypothalamic cells in culture was quantified using an enzyme-linked immunosorbent assay. In order to identify the mechanism underlying BPA-mediated changes in Agrp, siRNA knockdown of transcription factors, FOXO1, CHOP, ATF3, ATF4, ATF6, and small-molecule inhibitors of endoplasmic reticulum stress, JNK or MEK/ERK were used. RESULTS: BPA increased mRNA levels of Agrp in six hypothalamic cell lines (mHypoA-59, mHypoE-41, mHypoA-2/12, mHypoE-46, mHypoE-44, and mHypoE-42). Interestingly, only 18% of the total BPA in the culture medium entered the cells after 24 h, suggesting that the exposure concentration is much lower than the treatment concentration. BPA increased pre-Agrp mRNA levels, indicating increased Agrp transcription. Knockdown of the transcription factor ATF3 prevented BPA-mediated increase in Agrp, pre-Agrp, and in part Npy mRNA levels. However, chemical chaperone, sodium phenylbutyrate, JNK inhibitor, SP600125, or the MEK/ERK inhibitor PD0352901 did not block BPA-induced Agrp upregulation. CONCLUSION: Overall, these results indicate that hypothalamic Agrp is susceptible to dysregulation by BPA and implicate ATF3 as a common mediator of the orexigenic effects of BPA in hypothalamic neurons.

BPA Differentially Regulates NPY Expression in Hypothalamic Neurons Through a Mechanism Involving Oxidative Stress.

Bisphenol A (BPA), a ubiquitous endocrine-disrupting chemical, interferes with reproduction and is also considered an obesogen. The neuropeptide Y (NPY) neurons of the hypothalamus control both food intake and reproduction and have emerged as potential targets of BPA. These functionally diverse subpopulations of NPY neurons are differentially regulated by peripheral signals, such as estrogen and leptin. Whether BPA also differentially alters Npy expression in subpopulations of NPY neurons, contributing to BPA-induced endocrine dysfunction is unclear. We investigated the response of 6 immortalized hypothalamic NPY-expressing cell lines to BPA treatment. BPA upregulated Npy mRNA expression in 4 cell lines (mHypoA-59, mHypoE-41, mHypoA-2/12, mHypoE-42), and downregulated Npy in 2 lines (mHypoE-46, mHypoE-44). This differential expression of Npy occurred concurrently with differential expression of estrogen receptor mRNA levels. Inhibition of G-protein coupled estrogen receptor GPR30 or estrogen receptor ß prevented the BPA-mediated decrease in Npy, whereas inhibition of energy sensor 5' adenosine monophosphate-activated protein kinase (AMPK) with compound C prevented BPA-induced increase in Npy. BPA also altered neuroinflammatory and oxidative stress markers in both mHypoA-59 and mHypoE-46 cell lines despite the differential regulation of Npy. Remarkably, treatment with BPA in an antioxidant-rich media, Neurobasal A (NBA), or with reactive oxygen species scavenger tauroursodeoxycholic acid mitigated the BPA-induced increase and decrease in Npy. Furthermore, 2 antioxidant species from NBA-N-acetylcysteine and vitamin B6-diminished the induction of Npy in the mHypoA-59 cells, demonstrating these supplements can counteract BPA-induced dysregulation in certain subpopulations. Overall, these results illustrate the differential regulation of Npy by BPA in neuronal subpopulations, and point to oxidative stress as a pathway that can be targeted to block BPA-induced Npy dysregulation in hypothalamic neurons.

Analysis of Western diet, palmitate and BMAL1 regulation of neuropeptide Y expression in the murine hypothalamus and BMAL1 knockout cell models.

Western diets that are high in saturated fat and sugar disrupt circadian rhythms, induce weight gain, and lead to metabolic diseases including obesity. However, the mechanistic link between altered circadian rhythms and energy homeostasis remains poorly understood. In C57BL/6J mice, consuming a Western diet for 16 weeks significantly reduced food intake (at zeitgeber 12-16), in association with decreases in hypothalamic expression of the orexigenic neuropeptides, neuropeptide Y (Npy) and agouti-related peptide (AgRP). To examine the acute effects of the most prevalent saturated fatty acid in a Western diet, palmitate, and the role of the core clock gene, Bmal1, in the regulation of hypothalamic feeding neuropeptides, we used heterogeneous and clonal BMAL1 knockout (KO) immortalized hypothalamic cell lines, expressing specific neuropeptides, derived from male (M) and female (F) mice. Both mHypoA-BMAL1-KO/F and mHypoA-BMAL1-KO/M cells demonstrated a loss of circadian rhythmicity in expression of the clock gene, Per2, as compared to wild-type (control) cultures. Loss of BMAL1 also altered the time-dependent expression of Npy and proopiomelanocortin, and disrupted AgRP rhythmicity. Furthermore, palmitate increased BMAL1 binding to the Npy promotor region, and palmitate treatment (50 µM for 24 h) stimulated Npy expression in a BMAL1-dependent manner in both heterogeneous and clonal NPY-expressing female-derived cell models. The results of this study demonstrate that circadian expression of Bmal1 serves as a mechanistic link between Western diet- and palmitate-induced disruptions of the normal rhythmic patterns in hypothalamic feeding-related neuropeptides.

Palmitate and Nitric Oxide Regulate the Expression of Spexin and Galanin Receptors 2 and 3 in Hypothalamic Neurons.

Spexin (SPX) is a novel satiety factor that putatively binds the galanin receptors R2 and R3 (GalR2/R3). SPX reduces body weight, and circulating SPX is decreased in obesity. It is unknown how SPX and its receptors are regulated in the hypothalamus, critical for energy homeostasis. We therefore examined the regulation of hypothalamic Spx, GalR2 and GalR3 gene expression in mouse primary and immortalized hypothalamic neurons. We report that Spx, GalR2 and GalR3 mRNA levels were regulated by acute treatments of palmitate, a dietary saturated fatty acid, as well as the nitric oxide (NO) donor sodium nitroprusside (SNP), but through a pathway independent of cyclic GMP and protein kinase G. Additionally, the palmitate- and NO-mediated induction of Spx and galanin receptors was blocked with the PKC inhibitor k252c. Furthermore, palmitate induced mRNA levels of endoplasmic reticulum (ER) stress markers, including Chop, Grp78 and Bax/Bcl2, as well as C/ebp-ß, whereas SNP induced Bax/Bcl2 and C/ebp-ß. Transcriptional changes in Spx, GalR2, GalR3, C/ebp-ß and ER stress marker mRNAs were blocked by pre-treatment with at least one of the chemical chaperones PBA or TUDCA. We also describe the presence of OCT-1 and C/EBP-ß response elements in the 5' regulatory region of Spx and demonstrate that SNP increases binding of C/EBP-ß to this region, but not Oct-1 mRNA nor OCT-1 binding. Our findings suggest an acute modulation of anorexigenic SPX signaling by palmitate and NO. Furthermore, ER stress and C/EBP-ß appear to mediate the changes in Spx, GalR2 and GalR3 in hypothalamic neurons.

Regulation of Gpr173 expression, a putative phoenixin receptor, by saturated fatty acid palmitate and endocrine-disrupting chemical bisphenol A through a p38-mediated mechanism in immortalized hypothalamic neurons.

GPR173 is a highly conserved G protein coupled receptor associated with the hypothalamic-pituitary-gonadal reproductive axis. It is expressed in the brain and ovaries, however considerable knowledge about its function remains unknown. One putative ligand for this receptor is phoenixin (PNX), a newly identified reproductive peptide involved in hypothalamic coordination of the estrous cycle. In order to characterize GPR173, it is vital to determine how Gpr173 is regulated in the hypothalamus. Since the hypothalamus senses compounds from the blood, such as nutrients and chemicals, we examined the effect of palmitate, a saturated fatty acid, and bisphenol A (BPA), an endocrine disrupting chemical, on Gpr173 gene expression. Immortalized hypothalamic neurons were treated with palmitate or BPA for 2-24 h and Gpr173 mRNA levels were assessed with RT-qPCR. Palmitate and BPA both reduced Gpr173 mRNA levels, in part through the mitogen-activated protein kinase (MAPK), p38. Pre-treatment with palmitate for 24 h blocked the PNX-induction of phosphorylated cAMP response element-binding protein (CREB) levels. In conclusion, nutrition levels and environmental chemicals may influence reproductive function through modulation of Gpr173 expression, which may prove to be a future therapeutic target in reproductive health.

Bisphenol A induces Pomc gene expression through neuroinflammatory and PPARγ nuclear receptor-mediated mechanisms in POMC-expressing hypothalamic neuronal models.

Endocrine disrupting chemicals, such as bisphenol A (BPA), have been linked to obesity. However, the direct effect of BPA on the hypothalamic pro-opiomelanocortin (POMC) neurons, which regulate energy homeostasis, remains unexplored. We define the effect of BPA on functionally characterized, POMC-expressing cell models, mHypoA-POMC/GFP-2 and mHypoE-43/5. Exposure to BPA significantly induced the mRNA levels of Pomc in both primary culture and the cell lines. Neuroinflammatory and steroid receptor mRNA levels were assessed to delineate the potential mechanisms, including inflammatory markers Nfκb, Il6 and Iκba, and steroid receptors Esr1, Esr2, Gpr30, Esrrg, and Pparg. Pre-treatment with anti-inflammatory compounds gonadotropin-releasing hormone, and PS1145, an IκB kinase inhibitor, abrogated the BPA-mediated Pomc induction. Furthermore, T0070907, a PPARγ antagonist, abolished Pomc induction, while the GPR30 antagonist G15 had no effect. These findings indicate that BPA may have direct effects on POMC neurons in the hypothalamus, utilizing neuroinflammatory mechanisms and through PPARγ nuclear receptors.

Bisphenol A Alters Bmal1, Per2, and Rev-Erba mRNA and Requires Bmal1 to Increase Neuropeptide Y Expression in Hypothalamic Neurons.

Bisphenol A (BPA), a ubiquitous environmental endocrine disruptor, is considered an obesogen. However, its role in the hypothalamic control of energy balance remains largely unexplored. Because disruption of the circadian clock is tightly associated with metabolic consequences, we explored how BPA affects the components of the molecular circadian clock in the feeding-related neurons of the hypothalamus. In immortalized POMC and NPY/AgRP-expressing hypothalamic cell lines and primary culture, we describe how BPA significantly alters mRNA expression of circadian clock genes Bmal1,Per2, and Rev-Erbα. Furthermore, we use newly generated Bmal1-knockout (KO) hypothalamic cell lines to link the BPA-induced neuropeptide dysregulation to the molecular clock. Specifically, BPA increased Npy, Agrp, and Pomc mRNA expression in wild type hypothalamic cells, whereas the increase in Npy, but not Agrp or Pomc, was abolished in cell lines lacking BMAL1. In line with this increase, BPA led to increased BMAL1 binding to the Npy promotor, potentially increasing Npy transcription. In conclusion, we show that BPA-mediated dysregulation of the circadian molecular clock is linked to the deleterious effects of BPA on neuropeptide expression. Furthermore, we describe hypothalamic Bmal1-KO cell lines to study the role of BMAL1 in hypothalamic responses to metabolic, hormonal, and environmental factors.

Nutrient-sensing mechanisms in hypothalamic cell models: neuropeptide regulation and neuroinflammation in male- and female-derived cell lines.

The hypothalamus is responsible for the control of many of our physiological responses, including energy homeostasis. Of interest, there are a number of instances of sexual dimorphism documented with regard to metabolic processes. This review will discuss the necessity of utilizing both male and female models when studying the mechanisms underlying energy homeostasis, particularly those originating at the level of the hypothalamus. Because obesity often results in central neuroinflammation, we describe markers that could be used to study differences between male and female models, both the whole organism and also at the cellular level. Our laboratory has generated a wide array of immortalized hypothalamic cell models, originating from male and female rodents that we suggest could be beneficial for these types of studies. It is imperative that both sexes are considered before any recommendations for therapeutic interventions are considered.