Mono-(2-ethylhexyl) phthalate induces trophoblast hypoxia and mitochondrial dysfunction through HIF-1α-miR-210-3p axis in HTR-8/SVneo cell line.

The exposure to the ubiquitous phthalate metabolite mono-(2-ethylhexyl) phthalate (MEHP) is connected to dysregulated trophoblast function and placenta health; however, the underlying mechanisms preluding this scenario remain to be elucidated. In this study, we explored the hypoxemic effects of MEHP on a human placental first-trimester trophoblast cell line (HTR-8/Svneo). MEHP-treated trophoblast cells displayed significantly increased levels of oxidative stress and hypoxia-inducible factor-1 alpha (HIF-1α) attributed by the induction of hypoxia. Further, HIF-1α exhibited higher DNA binding activity and upregulated gene expression of its downstream target vascular endothelial growth factor A (VEGFA). The hypoxia-induced microRNA miR-210-3p was also significantly increased upon MEHP treatment followed by disrupted mitochondrial ATP generation and membrane potential. This was identified to possibly be facilitated by lowered mitochondrial DNA copy number and inhibited expression of electron transport chain subunits, such as mitochondrial complex-IV. These results suggest potential adverse effects of MEHP exposure in a trophoblast cell line mediated by HIF-1α and the epigenetic modulator miR-210-3p. Chronic placental hypoxia and oxidative stress have long been implicated in the pathogenesis of pregnancy complications such as preeclampsia. As we've revealed genetic and epigenetic factors underscoring a potential mechanism induced by MEHP, this brings to light another significant implication of phthalate exposure on maternal and fetal health.

LncRNA DLEU2 regulates sirtuins and mitochondrial respiratory chain complex IV: a novel pathway in obesity and offspring's health.

BACKGROUND: Long non-coding RNAs (lncRNAs) have emerged as a rapidly expanding area of interest in chronic diseases. They are mostly unknown for roles in metabolic regulation. Sirtuins, an epigenetic modulator class, regulate metabolic pathways. However, how sirtuins are regulated via lncRNA is unknown. We hypothesized that a high-fat high-fructose diet (HFD-HF) during pregnancy would increase the risk for obesity via lncRNA-Sirtuin pathways. METHODS: Female C57Bl/6 mice (F0) were fed either chow diet (CD) or HFD-HF for 6 weeks till birth. The pups (F1) were fed either CD or HFD-HF for 20 weeks. Expression of Dleu2, sirtuins, mitochondrial respiratory complexes, and oxidative stress were investigated in the F1 livers. Fasting blood glucose, insulin sensitivity, glucose tolerance, body and tissues weight were measured. A mechanistic interaction was then carried out using a DLEU2 knockdown experiment in the HepG2 cell. RESULTS: Dleu2 and sirtuins were both significantly decreased in the livers of HFD-HF fed male F1 whose mothers were either fed CD or HFD-HF during reproductive and pregnancy windows. Confirming this connection, upon silencing DLEU2, transcription levels of SIRT1 through 6 and translational levels of SIRT1, 3, 5, and 6 were significantly downregulated. Knockdown of DLEU2 significantly decreased the protein level of cytochrome-c oxidase (complex IV, MTCO1) without altering other mitochondrial complexes, decreased mitochondrial membrane potential, decreased ATP, and increased reactive oxygen species. Interestingly, in F1 livers, the protein level of MTCO1 was also significantly decreased under an HFD-HF diet or even under chow diet if the mother was exposed to HFD-HF. CONCLUSION: Our findings reveal for the first time that one lncRNA can regulate sirtuins and a specific mitochondrial complex. Furthermore, diet or maternal diet can modulate Dleu2 and its downstream regulators in offspring, suggesting a potential role of DLEU2 in metabolic disorders over one or more generations.

Pyrroloquinoline quinone attenuated benzyl butyl phthalate induced metabolic aberration and a hepatic metabolomic analysis.

Benzyl butyl phthalate (BBP) has recently been implicated as an obesogen. Our recent study demonstrated that BBP can exacerbate high fat diet (HFD) induced diabesity in male mice. Here, we explored if pyrroloquinoline quinone (PQQ), a natural antioxidant andphytochemical, can attenuate metabolic aberrations induced by HFD or HFD-BBPcombination. C57Bl/6 male and female mice were fed either a chow diet (CD) or HFD with or without BBP (3 mg/kg body weight/day)and/or PQQ (20 mg/kg/day)for 16 weeks. The mice's body and tissue weight, fasting blood glucose, glucose and insulin tolerance test, and liver metabolites level weremeasured. In HFD-fed male mice, PQQ significantly attenuated the increased body weight, liver weight, fasting blood glucose, and insulin intolerance under BBP exposure.Even though female mice did show some reversal of metabolic characteristics by PQQ, the response was not similar nor consistent with the male population. Amongthe 14 hepatic metabolites that were significantly altered by HFD compared to CD, only three major metabolites (acetyl-L-carnitine, DL-stachytine, and propionylcarnitine) were decreased. These three were shown to have more reduction under BBP exposure in the presence of HFD whereas with addition of PQQ, these metabolites were restored. Pathway analysis and literature search revealed that these metabolites were negatively associated with obesity and were involved in several pathways including beta-oxidation, oxidative stress, and mitochondrial function. Overall,this finding indicated the potential use of PQQ to restore thewide range of aberrant metabolic effectinduced by an obesogen in the presence of a western diet.

Butyl Benzyl Phthalate Promotes Adipogenesis in 3T3-L1 Cells via the miRNA-34a-5p Signaling Pathway in the Absence of Exogenous Adipogenic Stimuli.

Phthalates, a plasticizer group, are used extensively in many of the products we use every day. Public health concerns are growing as recent studies have implicated butyl benzyl phthalate (BBP) as an obesogen. However, BBP-induced epigenetic regulation during adipogenesis is still unknown. We investigated if BBP altered miR-34a-5p, a key miRNA involved in obesity, and regulated its downstream pathway. Differentiating 3T3-L1 cells were exposed to various doses of BBP without exogenous adipogenic stimuli, tested for adipogenesis markers (PPARγ and aP2), and stained for lipid accumulation with Oil Red O staining. We then measured the expression of miR-34a-5p and its target genes, Nampt and Sirt1, along with another significant epigenetic modulator, Sirt3. Furthermore, using antagomiR, we examined whether miR-34a-5p knockdown decreased adipogenesis. BBP exposure resulted in augmented expression levels of miR-34a-5p with an associated increase in adipogenesis. BBP significantly decreased the Nampt, Sirt1, and Sirt3 gene expression levels. However, a decrease in the protein expression was observed only for Nampt, indicating that miR-34a-5p under BBP exposure may regulate Sirt1/Sirt3 only at the transcriptional level. Interestingly, in the presence of BBP, knockdown of miR-34a-5p decreased adipogenesis in the differentiating 3T3-L1 cells. Furthermore, miR-34a-5p knockdown increased the Nampt protein expression levels as well as NAD+ levels, indicating that miR-34a-5p regulates Nampt during BBP exposure. Additionally, the NAD+-dependent sirtuin activity decreased in BBP-treated cells and increased in miR-34a-5p knockdown cells with BBP treatment. BBP exposure demonstrated the involvement of epigenetic regulation by altering the expression patterns of miR-34a-5p and its target Nampt, which may perturb the energy homeostasis of the differentiating adipocytes by altering NAD+ levels and sirtuin activity, resulting in increased adipogenesis.

Effect of Chronic Western Diets on Non-Alcoholic Fatty Liver of Male Mice Modifying the PPAR-γ Pathway via miR-27b-5p Regulation.

Western diets contribute to metabolic diseases. However, the effects of various diets and epigenetic mechanisms are mostly unknown. Here, six week-old C57BL/6J male and female mice were fed with a low-fat diet (LFD), high-fat diet (HFD), and high-fat high-fructose diet (HFD-HF) for 20 weeks. We determined that HFD-HF or HFD mice experienced significant metabolic dysregulation compared to the LFD. HFD-HF and HFD-fed male mice showed significantly increased body weight, liver size, and fasting glucose levels with downregulated PPARγ, SCD1, and FAS protein expression. In contrast, female mice were less affected by HFD and HFD-HF. As miR-27b contains a seed sequence in PPARγ, it was discovered that these changes are accompanied by male-specific upregulation of miR-27b-5p, which is even more pronounced in the HFD-HF group (p < 0.01 vs. LFD) compared to the HFD group (p < 0.05 vs. LFD). Other miR-27 subtypes were increased but not significantly. HFD-HF showed insignificant changes in fibrosis markers when compared to LFD. Interestingly, fat ballooning in hepatocytes was increased in HFD-fed mice compared to HFD-HF fed mice, however, the HFD-HF liver showed an increase in the number of small cells. Here, we concluded that chronic Western diet-composition administered for 20 weeks may surpass the non-alcoholic fatty liver (NAFL) stage but may be at an intermediate stage between fatty liver and fibrosis via miR-27b-5p-induced PPARγ downregulation.

A moderate physiological dose of benzyl butyl phthalate exacerbates the high fat diet-induced diabesity in male mice.

Exposure to endocrine disrupting chemicals (EDCs) used in plastic manufacturing processes may be contributing to the current increase in metabolic disorders. Here, we determined that benzyl butyl phthalate (BBP), a common EDC and food packaging plasticizer, mixed into chow diet (CD) and high fat diets (HFD) at varying concentrations (4 µg/kg body weight (bw)/day, 169 µg/kg bw/day, 3 mg/kg bw/day, 50 mg/kg bw/day) produced a number of detrimental and sex-specific metabolic effects in C57BL/6 male and female mice after 16 weeks. Male mice exposed to moderate (3 mg/kg bw/day) concentrations of BBP in an HFD were especially affected, with significant increases in body weight due to significant increases in weight of liver and adipose tissue. Other doses did not show any significant changes when compared to only CD or HFD alone. HFD in the presence of 3 mg/kg bw/day BBP showed significant increases in fasting blood glucose, glucose intolerance, and insulin intolerance when compared to HFD alone. Furthermore, this group significantly alters transcriptional regulators involved in hepatic lipid synthesis and its downstream pathway. Interestingly, most of the BBP doses had no phenotypic effect when mixed with CD and compared to CD alone. The female mice did not show a similar response as the male population even though they consumed a similar amount of food. Overall, these data establish a dose which can be used for a BBP-induced metabolic research model and suggest that a moderate dosage level of EDC exposure can contribute to widely ranging metabolic effects.

Mono-(2-ethylhexyl) Phthalate Increases Oxidative Stress Responsive miRNAs in First Trimester Placental Cell Line HTR8/SVneo.

Phthalates, an endocrine disruptor group, cause oxidative stress (OS) in the placenta. However, no studies have reported OS-related miRNAs induced by phthalates. In the present study, we demonstrate that mono-(2-ethylhexyl) phthalate (MEHP) induces OS responsive miR-17-5p, miR-155-5p, and miR-126-3p in HTR8/SVneo in a dose- and time-dependent manner. Furthermore, MEHP altered the expression of phosphoinositide-3-kinase regulatory subunit 1α, phosphatase and tensin homolog, CDKN2A interacting protein, superoxide dismutase 2, and 3ß-hydroxysterol-D24 reductase, which are involved in OS and predicted to be regulated by these miRNAs. Our results suggest that placental exposure to MEHP may result in aberrant miRNA expression leading to pregnancy complications.

Mono-(2-ethylhexyl) phthalate induces apoptosis through miR-16 in human first trimester placental cell line HTR-8/SVneo.

Phthalates have been linked to adverse pregnancy complications. Mono-(2-ethylhexyl) phthalate, an active metabolite of di-(2-ethylhexyl) phthalate and an endocrine disruptor, has been shown to induce apoptosis in various cell types including placental cells. However, the mechanism of action of MEHP induced apoptosis is still unknown. We hypothesized that apoptosis may be mediated in part through altered microRNA(s) in placenta under MEHP exposure. In the present study, we report that MEHP increases miR-16 expression in a time- and dose-dependent manner (p<0.05), while inducing apoptosis in HTR-8/SVneo. Cells treated with MEHP showed a dose-dependent increase in cytotoxicity and reactive oxygen species along with decreased cell viability. Consistent with significant increase in apoptosis analyzed by flow cytometry, we detected decreased anti-apoptotic BCL-2 at transcriptional and translational levels with MEHP (p<0.05). Knockdown of miR-16 did not decrease the BCL-2/BAX protein expression ratio in the presence of MEHP when compared to negative control demonstrating that MEHP induces apoptosis directly through miR-16. In conclusion, our study demonstrates for the first time that MEHP induces miR-16, which in turn, alters BCL-2/BAX ratio leading to increased apoptosis. This study provides a novel insight into MEHP induced epigenetic regulation in placental apoptosis which may lead to pregnancy complications.

Are epigenetic drugs for diabetes and obesity at our door step?

Recent interest in epigenetics has focused on small molecules aimed at modifying disease-specific gene expression, including diabetes and obesity. Several major classes of epigenetic modifier include drugs already in the marketplace as well as several in various stages of study. These classes include histone deacetylase inhibitors (HDACi), histone acetyltransferase inhibitors (HATi), protein arginine methyltransferase inhibitors (PRMTis), DNA methyltransferase inhibitors (DNMTis), histone demethylating inhibitors (HDMis), and sirtuin-activating compounds (STACs). In this review, we discuss drugs with epigenetic properties that have been identified as potential therapeutic agents in the treatment of diabetes and obesity, including those currently in clinical trials.

Pyrroloquinoline quinone increases the expression and activity of Sirt1 and -3 genes in HepG2 cells.

Sirtuin (Sirt) 1 and Sirt 3 are nicotinamide adenine dinucleotide ((+))-dependent protein deacetylases that are important to a number of mitochondrial-related functions; thus, identification of sirtuin activators is important. Herein, we hypothesize that pyrroloquinoline quinone (PQQ) can act as a Sirt1/Sirt3 activator. In HepG2 cell cultures, PQQ increased the expression of Sirt1 and Sirt3 gene, protein, and activity levels (P < .05). We also observed a significant increase in nicotinamide phosphoribosyltransferase gene expression (as early as 18 hours) and increased NAD(+) activity at 24 hours. In addition, targets of Sirt1 and Sirt3 (peroxisome proliferator-activated receptor γ coactivator 1α, nuclear respiratory factor 1 and 2, and mitochondrial transcription factor A) were increased at 48 hours. This is the first report that demonstrates PQQ as an activator of Sirt1 and Sirt3 expression and activity, making it an attractive therapeutic agent for the treatment of metabolic diseases and for healthy aging. Based on our study and the available data in vivo, PQQ has the potential to serve as a therapeutic nutraceutical, when enhancing mitochondrial function.

The adipogenic transcriptional cofactor ZNF638 interacts with splicing regulators and influences alternative splicing.

Increasing evidence indicates that transcription and alternative splicing are coordinated processes; however, our knowledge of specific factors implicated in both functions during the process of adipocyte differentiation is limited. We have previously demonstrated that the zinc finger protein ZNF638 plays a role as a transcriptional coregulator of adipocyte differentiation via induction of PPARγ in cooperation with CCAAT/enhancer binding proteins (C/EBPs). Here we provide new evidence that ZNF638 is localized in nuclear bodies enriched with splicing factors, and through biochemical purification of ZNF638's interacting proteins in adipocytes and mass spectrometry analysis, we show that ZNF638 interacts with splicing regulators. Functional analysis of the effects of ectopic ZNF638 expression on a minigene reporter demonstrated that ZNF638 is sufficient to promote alternative splicing, a function enhanced through its recruitment to the minigene promoter at C/EBP responsive elements via C/EBP proteins. Structure-function analysis revealed that the arginine/serine-rich motif and the C-terminal zinc finger domain required for speckle localization are necessary for the adipocyte differentiation function of ZNF638 and for the regulation of the levels of alternatively spliced isoforms of lipin1 and nuclear receptor co-repressor 1. Overall, our data demonstrate that ZNF638 participates in splicing decisions and that it may control adipogenesis through regulation of the relative amounts of differentiation-specific isoforms.

Thyroid hormone analogues: where do we stand in 2013?

Thyroid hormones (THs) are important in the development and maintenance of lipid and energy homeostasis. THs act through two closely related TH receptors (TRs α and ß), which are conditional transcription factors. Recently, TH analogues or thyromimetics with varying degrees of TR subtype and liver uptake selectivity have been developed. These compounds exert beneficial effects of TH excess states without many undesirable TR-dependent side effects. Several selective TR modulators (STRMs) showed exceptionally promising results in lowering serum cholesterol in preclinical animal models and human clinical studies. Moreover, some first generation STRMs elicit other potentially beneficial effects on obesity, glucose metabolism, and nonalcoholic fatty liver disease (NAFLD). While it was initially thought that STRMs would be an effective long-term therapy to combat elevated cholesterol, possibly in conjunction with another cholesterol-lowering therapy, the statins, three major first generation STRMs failed to progress beyond early phase III human trials. The aim of this review is to discuss how STRMs work, their actions in preclinical animal models and human clinical trials, why they did not progress beyond clinical trials as cholesterol-lowering therapeutics, whether selective TR modulation continues to hold promise for dyslipidemias, and whether members of this drug class could be applied to the treatment of other aspects of metabolic syndrome and human genetic disease.

Regulation of adipocyte differentiation by the zinc finger protein ZNF638.

Zinc finger proteins constitute the largest family of transcription regulators in eukaryotes. These factors are involved in diverse processes in many tissues, including development and differentiation. We report here the characterization of the zinc finger protein ZNF638 as a novel regulator of adipogenesis. ZNF638 is induced early during adipocyte differentiation. Ectopic expression of ZNF638 increases adipogenesis in vitro, whereas its knockdown inhibits differentiation and decreases the expression of adipocyte-specific genes. ZNF638 physically interacts and transcriptionally cooperates with CCAAT/enhancer-binding protein (C/EBP) ß and C/EBPδ. This interaction leads to the expression of peroxisome proliferator-activated receptor γ, which is the key regulator of adipocyte differentiation. In summary, ZNF638 is a novel and early regulator of adipogenesis that works as a transcription cofactor of C/EBPs.

Serum- and glucocorticoid-inducible kinase 1 (SGK1) regulates adipocyte differentiation via forkhead box O1.

The serum and glucocorticoid-inducible kinase 1 (SGK1) is an inducible kinase the physiological function of which has been characterized primarily in the kidney. Here we show that SGK1 is expressed in white adipose tissue and that its levels are induced in the conversion of preadipocytes into fat cells. Adipocyte differentiation is significantly diminished via small interfering RNA inhibition of endogenous SGK1 expression, whereas ectopic expression of SGK1 in mesenchymal precursor cells promotes adipogenesis. The SGK1-mediated phenotypic effects on differentiation parallel changes in the mRNA levels for critical regulators and markers of adipogenesis, such as peroxisome proliferator-activated receptor gamma, CCAAT enhancer binding protein alpha, and fatty acid binding protein aP2. We demonstrate that SGK1 affects differentiation by direct phosphorylation of Foxo1, thereby changing its cellular localization from the nucleus to the cytosol. In addition we show that SGK1-/- cells are unable to relocalize Foxo1 to the cytosol in response to dexamethasone. Together these results show that SGK1 influences adipocyte differentiation by regulating Foxo1 phosphorylation and reveal a potentially important function for this kinase in the control of fat mass and function.

Sequence determinants for the reaction specificity of murine (12R)-lipoxygenase: targeted substrate modification and site-directed mutagenesis.

Mammalian lipoxygenases (LOXs) are categorized with respect to their positional specificity of arachidonic acid oxygenation. Site-directed mutagenesis identified sequence determinants for the positional specificity of these enzymes, and a critical amino acid for the stereoselectivity was recently discovered. To search for sequence determinants of murine (12R)-LOX, we carried out multiple amino acid sequence alignments and found that Phe(390), Gly(441), Ala(455), and Val(631) align with previously identified positional determinants of S-LOX isoforms. Multiple site-directed mutagenesis studies on Phe(390) and Ala(455) did not induce specific alterations in the reaction specificity, but yielded enzyme species with reduced specific activities and stereo random product patterns. Mutation of Gly(441) to Ala, which caused drastic alterations in the reaction specificity of other LOX isoforms, failed to induce major alterations in the positional specificity of mouse (12R)-LOX, but markedly modified the enantioselectivity of the enzyme. When Val(631), which aligns with the positional determinant Ile(593) of rabbit 15-LOX, was mutated to a less space-filling residue (Ala or Gly), we obtained an enzyme species with augmented catalytic activity and specifically altered reaction characteristics (major formation of chiral (11R)-hydroxyeicosatetraenoic acid methyl ester). The importance of Val(631) for the stereo control of murine (12R)-LOX was confirmed with other substrates such as methyl linoleate and 20-hydroxyeicosatetraenoic acid methyl ester. These data identify Val(631) as the major sequence determinant for the specificity of murine (12R)-LOX. Furthermore, we conclude that substrate fatty acids may adopt different catalytically productive arrangements at the active site of murine (12R)-LOX and that each of these arrangements may lead to the formation of chiral oxygenation products.