Role of one-carbon nutrient intake and diabetes during pregnancy in children's growth and neurodevelopment: A 2-year follow-up study of a prospective cohort.

BACKGROUND & AIMS: Both maternal metabolic dysregulation, e.g., gestational diabetes mellitus (GDM), and maternal supply of nutrients that participate in one-carbon (1C) metabolism, e.g., folate, choline, betaine, and vitamin B12, have been demonstrated to influence epigenetic modification such as DNA methylation, thereby exerting long-lasting impacts on growth and development of offspring. This study aimed to determine how maternal 1C nutrient intake was associated with DNA methylation and further, development of children, as well as whether maternal GDM status modified the association in a prospective cohort. METHODS: In this study, women with (n = 18) and without (n = 20) GDM were recruited at 25-33 weeks gestation. Detailed dietary intake data was collected by 3-day 24-h dietary recall and nutrient levels in maternal blood were also assessed at enrollment. The maternal-child dyads were invited to participate in a 2-year follow-up during which anthropometric measurement and the Bayley Scales of Infant and Toddler Development™ Screening Test (Third Edition) were conducted on children. The association between maternal 1C nutrients and children's developmental outcomes was analyzed with a generalized linear model controlling for maternal GDM status. RESULTS: We found that children born to mothers with GDM had lower scores in the language domain of the Bayley test (p = 0.049). Higher maternal food folate and choline intakes were associated with better language scores in children (p = 0.01 and 0.025, respectively). Higher maternal food folate intakes were also associated with better cognitive scores in children (p = 0.002). Higher 1C nutrient intakes during pregnancy were associated with lower body weight of children at 2 years of age (p < 0.05). However, global DNA methylation of children's buccal cells was not associated with any maternal 1C nutrients. CONCLUSIONS: In conclusion, higher 1C nutrient intake during pregnancy was associated with lower body weight and better neurodevelopmental outcomes of children. This may help overcome the lower language scores seen in GDM-affected children in this cohort. Studies in larger cohorts and with a longer follow-up duration are needed to further delineate the relationship between prenatal 1C nutrient exposure, especially in GDM-affected pregnancies, and offspring health outcomes.

Associations between nutrients in one-carbon metabolism and fetal DNA methylation in pregnancies with or without gestational diabetes mellitus.

BACKGROUND: Gestational diabetes mellitus (GDM), characterized by hyperglycemia that develops during pregnancy, increases the risk of fetal macrosomia, childhood obesity and cardiometabolic disorders later in life. This process has been attributed partly to DNA methylation modifications in growth and stress-related pathways. Nutrients involved with one-carbon metabolism (OCM), such as folate, choline, betaine, and vitamin B12, provide methyl groups for DNA methylation of these pathways. Therefore, this study aimed to determine whether maternal OCM nutrient intakes and levels modified fetal DNA methylation and in turn altered fetal growth patterns in pregnancies with and without GDM. RESULTS: In this prospective study at a single academic institution from September 2016 to June 2019, we recruited 76 pregnant women with and without GDM at 25-33 weeks gestational age and assessed their OCM nutrient intake by diet recalls and measured maternal blood OCM nutrient levels. We also collected placenta and cord blood samples at delivery to examine fetal tissue DNA methylation of the genes that modify fetal growth and stress response such as insulin-like growth factor 2 (IGF2) and corticotropin-releasing hormone (CRH). We analyzed the association between maternal OCM nutrients and fetal DNA methylation using a generalized linear mixed model. Our results demonstrated that maternal choline intake was positively correlated with cord blood CRH methylation levels in both GDM and non-GDM pregnancies (r = 0.13, p = 0.007). Further, the downstream stress hormone cortisol regulated by CRH was inversely associated with maternal choline intake (r = - 0.36, p = 0.021). Higher maternal betaine intake and serum folate levels were associated with lower cord blood and placental IGF2 DNA methylation (r = - 0.13, p = 0.049 and r = - 0.065, p = 0.034, respectively) in both GDM and non-GDM pregnancies. Further, there was an inverse association between maternal betaine intake and birthweight of infants (r = - 0.28, p = 0.015). CONCLUSIONS: In conclusion, we observed a complex interrelationship between maternal OCM nutrients and fetal DNA methylation levels regardless of GDM status, which may, epigenetically, program molecular pathways related to fetal growth and stress response.

TCGA Expression Analyses of 10 Carcinoma Types Reveal Clinically Significant Racial Differences.

Epidemiological studies reveal disparities in cancer incidence and outcome rates between racial groups in the United States. In our study, we investigated molecular differences between racial groups in 10 carcinoma types. We used publicly available data from The Cancer Genome Atlas to identify patterns of differential gene expression in tumor samples obtained from 4112 White, Black/African American, and Asian patients. We identified race-dependent expression of numerous genes whose mRNA transcript levels were significantly correlated with patients' survival. Only a small subset of these genes was differentially expressed in multiple carcinomas, including genes involved in cell cycle progression such as CCNB1, CCNE1, CCNE2, and FOXM1. In contrast, most other genes, such as transcriptional factor ETS1 and apoptotic gene BAK1, were differentially expressed and clinically significant only in specific cancer types. Our analyses also revealed race-dependent, cancer-specific regulation of biological pathways. Importantly, homology-directed repair and ERBB4-mediated nuclear signaling were both upregulated in Black samples compared to White samples in four carcinoma types. This large-scale pan-cancer study refines our understanding of the cancer health disparity and can help inform the use of novel biomarkers in clinical settings and the future development of precision therapies.

Prenatal Choline Supplement in a Maternal Obesity Model Modulates Offspring Hepatic Lipidomes.

Maternal obesity during pregnancy adversely impacts offspring health, predisposing them to chronic metabolic diseases characterized by insulin resistance, dysregulated macronutrient metabolism, and lipid overload, such as metabolic-associated fatty liver disease (MAFLD). Choline is a semi-essential nutrient involved in lipid and one-carbon metabolism that is compromised during MAFLD progression. Here, we investigated under high-fat (HF) obesogenic feeding how maternal choline supplementation (CS) influenced the hepatic lipidome of mouse offspring. Our results demonstrate that maternal HF+CS increased relative abundance of a subclass of phospholipids called plasmalogens in the offspring liver at both embryonic day 17.5 and after 6 weeks of postnatal HF feeding. Consistent with the role of plasmalogens as sacrificial antioxidants, HF+CS embryos were presumably protected with lower oxidative stress. After postnatal HF feeding, the maternal HF+CS male offspring also had higher relative abundance of both sphingomyelin d42:2 and its side chain, nervonic acid (FA 24:1). Nervonic acid is exclusively metabolized in the peroxisome and is tied to plasmalogen synthesis. Altogether, this study demonstrates that under the influence of obesogenic diet, maternal CS modulates the fetal and postnatal hepatic lipidome of male offspring, favoring plasmalogen synthesis, an antioxidative response that may protect the mouse liver from damages due to HF feeding.

In silico study predicts a key role of RNA-binding domains 3 and 4 in nucleolin-miRNA interactions.

RNA binding proteins (RBPs) regulate many important cellular processes through their interactions with RNA molecules. RBPs are critical for posttranscriptional mechanisms keeping gene regulation in a fine equilibrium. Conversely, dysregulation of RBPs and RNA metabolism pathways is an established hallmark of tumorigenesis. Human nucleolin (NCL) is a multifunctional RBP that interacts with different types of RNA molecules, in part through its four RNA binding domains (RBDs). Particularly, NCL interacts directly with microRNAs (miRNAs) and is involved in their aberrant processing linked with many cancers, including breast cancer. Nonetheless, molecular details of the NCL-miRNA interaction remain obscure. In this study, we used an in silico approach to characterize how NCL targets miRNAs and whether this specificity is imposed by a definite RBD-interface. Here, we present structural models of NCL-RBDs and miRNAs, as well as predict scenarios of NCL-miRNA interactions generated using docking algorithms. Our study suggests a predominant role of NCL RBDs 3 and 4 (RBD3-4) in miRNA binding. We provide detailed analyses of specific motifs/residues at the NCL-substrate interface in both these RBDs and miRNAs. Finally, we propose that the evolutionary emergence of more than two RBDs in NCL in higher organisms coincides with its additional role/s in miRNA processing. Our study shows that RBD3-4 display sequence/structural determinants to specifically recognize miRNA precursor molecules. Moreover, the insights from this study can ultimately support the design of novel antineoplastic drugs aimed at regulating NCL-dependent biological pathways with a causal role in tumorigenesis.

Maternal Choline Supplementation and High-Fat Feeding Interact to Influence DNA Methylation in Offspring in a Time-Specific Manner.

Maternal methyl donor supplementation during pregnancy has demonstrated lasting influence on offspring DNA methylation. However, it is unknown whether an adverse postnatal environment, such as high-fat (HF) feeding, overrides the influence of prenatal methyl donor supplementation on offspring epigenome. In this study, we examined whether maternal supplementation of choline (CS), a methyl donor, interacts with prenatal and postnatal HF feeding to alter global and site-specific DNA methylation in offspring. We fed wild-type C57BL/6J mouse dams a HF diet with or without CS throughout gestation. After weaning, the offspring were exposed to HF feeding for 6 weeks resembling a continued obesogenic environment. Our results suggest that maternal CS under the HF condition (HFCS) increased global DNA methylation and DNA methyltransferase 1 (Dnmt1) expression in both fetal liver and brain. However, during the postnatal period, HFCS offspring demonstrated lower global DNA methylation and Dnmt1 expression was unaltered in both the liver and visceral adipose tissue. Site-specific DNA methylation analysis during both fetal and postnatal periods demonstrated that HFCS offspring had higher methylation of CpGs in the promoter of Srebf1, a key mediator of de novo lipogenesis. In conclusion, the influence of maternal CS on offspring DNA methylation is specific to HF feeding status during prenatal and postnatal periods. Without continued CS during the postnatal period, global DNA methylation enhanced by prenatal CS in the offspring was overridden by postnatal HF feeding.

Intrahepatic microbes govern liver immunity by programming NKT cells.

The gut microbiome shapes local and systemic immunity. The liver is presumed to be a protected sterile site. As such, a hepatic microbiome has not been examined. Here, we showed a liver microbiome in mice and humans that is distinct from that of the gut and is enriched in Proteobacteria. It undergoes dynamic alterations with age and is influenced by the environment and host physiology. Fecal microbial transfer experiments revealed that the liver microbiome is populated from the gut in a highly selective manner. Hepatic immunity is dependent on the microbiome, specifically the bacteroidetes species. Targeting bacteroidetes with oral antibiotics reduced hepatic immune cells by approximately 90%, prevented antigen-presenting cell (APC) maturation, and mitigated adaptive immunity. Mechanistically, our findings are consistent with presentation of bacteroidetes-derived glycosphingolipids to NKT cells promoting CCL5 signaling, which drives hepatic leukocyte expansion and activation, among other possible host-microbe interactions. Collectively, we reveal a microbial/glycosphingolipid/NKT/CCL5 axis that underlies hepatic immunity.

Fungi, host immune response, and tumorigenesis.

Advances in -omics analyses have tremendously enhanced our understanding of the role of the microbiome in human health and disease. Most research is focused on the bacteriome, but scientists have now realized the significance of the virome and microbial dysbiosis as well, particularly in noninfectious diseases such as cancer. In this review, we summarize the role of mycobiome in tumorigenesis, with a dismal prognosis, and attention to pancreatic ductal adenocarcinoma (PDAC). We also discuss bacterial and mycobial interactions to the host's immune response that is prevalently responsible for resistance to cancer therapy, including immunotherapy. We reported that the Malassezia species associated with scalp and skin infections, colonize in human PDAC tumors and accelerate tumorigenesis via activating the C3 complement-mannose-binding lectin (MBL) pathway. PDAC tumors thrive in an immunosuppressive microenvironment with desmoplastic stroma and a dysbiotic microbiome. Host-microbiome interactions in the tumor milieu pose a significant threat in driving the indolent immune behavior of the tumor. Microbial intervention in multimodal cancer therapy is a promising novel approach to modify an immunotolerant ("cold") tumor microenvironment to an immunocompetent ("hot") milieu that is effective in eliminating tumorigenesis.

Nucleolin regulates 14-3-3ζ mRNA and promotes cofilin phosphorylation to induce tunneling nanotube formation.

Tunneling nanotubes (TNTs) mediate intercellular communication between animal cells in health and disease, but the mechanisms of their biogenesis and function are poorly understood. Here we report that the RNA-binding protein (RBP) nucleolin, which interacts with the known TNT-inducing protein MSec, is essential for TNT formation in mammalian cells. Nucleolin, through its RNA-binding domains (RBDs), binds to and maintains the cytosolic levels of 14-3-3ζ mRNA, and is, therefore, required for TNT formation. A specific region of the 3'-untranslated region (UTR) of the 14-3-3ζ mRNA is likely to be involved in its regulation by nucleolin. Functional complementation experiments suggest that nucleolin and 14-3-3ζ form a linear signaling axis that promotes the phosphorylation and inactivation of the F-actin depolymerization factor cofilin to induce TNT formation. MSec also similarly inactivates cofilin, but potentiates TNT formation independent of the nucleolin-14-3-3ζ axis, despite biochemically interacting with both proteins. We show that 14-3-3ζ and nucleolin are required for the formation of TNTs between primary mouse neurons and astrocytes and in multiple other mammalian cell types. We also report that the Caenorhabditis elegans orthologs of 14-3-3ζ and MSec regulate the size and architecture of the TNT-like cellular protrusions of the distal tip cell (DTC), the germline stem cell niche in the gonad. Our study demonstrates a novel and potentially conserved mRNA-guided mechanism of TNT formation through the maintenance of cellular 14-3-3ζ mRNA levels by the RBP nucleolin.

Targeting Piezo1 unleashes innate immunity against cancer and infectious disease.

Piezo1 is a mechanosensitive ion channel that has gained recognition for its role in regulating diverse physiological processes. However, the influence of Piezo1 in inflammatory disease, including infection and tumor immunity, is not well studied. We postulated that Piezo1 links physical forces to immune regulation in myeloid cells. We found signal transduction via Piezo1 in myeloid cells and established this channel as the primary sensor of mechanical stress in these cells. Global inhibition of Piezo1 with a peptide inhibitor was protective against both cancer and septic shock and resulted in a diminution in suppressive myeloid cells. Moreover, deletion of Piezo1 in myeloid cells protected against cancer and increased survival in polymicrobial sepsis. Mechanistically, we show that mechanical stimulation promotes Piezo1-dependent myeloid cell expansion by suppressing the retinoblastoma gene Rb1 We further show that Piezo1-mediated silencing of Rb1 is regulated via up-regulation of histone deacetylase 2. Collectively, our work uncovers Piezo1 as a targetable immune checkpoint that drives immunosuppressive myelopoiesis in cancer and infectious disease.

Prenatal Choline Supplementation during High-Fat Feeding Improves Long-Term Blood Glucose Control in Male Mouse Offspring.

Maternal obesity increases the risk of metabolic dysregulation in rodent offspring, especially when offspring are exposed to a high-fat (HF), obesogenic diet later in life. We previously demonstrated that maternal choline supplementation (MCS) in HF-fed mouse dams during gestation prevents fetal overgrowth and excess adiposity. In this study, we examined the long-term metabolic influence of MCS. C57BL/6J mice were fed a HF diet with or without choline supplementation prior to and during gestation. After weaning, their pups were exposed to either a HF or control diet for 6 weeks before measurements. Prenatal and post-weaning dietary treatments led to sexually dimorphic responses. In male offspring, while post-weaning HF led to impaired fasting glucose and worse glucose tolerance (p < 0.05), MCS in HF dams (HFCS) attenuated these changes. HFCS (versus maternal normal fat control) appeared to improve metabolic functioning of visceral adipose tissue during post-weaning HF feeding, preventing the elevation in leptin and increasing (p < 0.05) mRNA expression of insulin receptor substrate 1 (Irs1) that promotes peripheral insulin signaling in male offspring. In contrast, MCS had minimal effects on metabolic outcomes of female offspring. In conclusion, MCS during HF feeding in mice improves long-term blood glucose homeostasis in male offspring when they are faced with a postnatal obesogenic environment.

The fungal mycobiome promotes pancreatic oncogenesis via activation of MBL.

Bacterial dysbiosis accompanies carcinogenesis in malignancies such as colon and liver cancer, and has recently been implicated in the pathogenesis of pancreatic ductal adenocarcinoma (PDA)1. However, the mycobiome has not been clearly implicated in tumorigenesis. Here we show that fungi migrate from the gut lumen to the pancreas, and that this is implicated in the pathogenesis of PDA. PDA tumours in humans and mouse models of this cancer displayed an increase in fungi of about 3,000-fold compared to normal pancreatic tissue. The composition of the mycobiome of PDA tumours was distinct from that of the gut or normal pancreas on the basis of alpha- and beta-diversity indices. Specifically, the fungal community that infiltrated PDA tumours was markedly enriched for Malassezia spp. in both mice and humans. Ablation of the mycobiome was protective against tumour growth in slowly progressive and invasive models of PDA, and repopulation with a Malassezia species-but not species in the genera Candida, Saccharomyces or Aspergillus-accelerated oncogenesis. We also discovered that ligation of mannose-binding lectin (MBL), which binds to glycans of the fungal wall to activate the complement cascade, was required for oncogenic progression, whereas deletion of MBL or C3 in the extratumoral compartment-or knockdown of C3aR in tumour cells-were both protective against tumour growth. In addition, reprogramming of the mycobiome did not alter the progression of PDA in Mbl- (also known as Mbl2) or C3-deficient mice. Collectively, our work shows that pathogenic fungi promote PDA by driving the complement cascade through the activation of MBL.

Maternal Choline and Betaine Supplementation Modifies the Placental Response to Hyperglycemia in Mice and Human Trophoblasts.

Gestational diabetes mellitus (GDM) is characterized by excessive placental fat and glucose transport, resulting in fetal overgrowth. Earlier we demonstrated that maternal choline supplementation normalizes fetal growth in GDM mice at mid-gestation. In this study, we further assess how choline and its oxidation product betaine influence determinants of placental nutrient transport in GDM mice and human trophoblasts. C57BL/6J mice were fed a high-fat (HF) diet 4 weeks prior to and during pregnancy to induce GDM or fed a control normal fat (NF) diet. The HF mice also received 25 mM choline, 85 mM betaine, or control drinking water. We observed that GDM mice had an expanded placental junctional zone with an increased area of glycogen cells, while the thickness of the placental labyrinth zone was decreased at E17.5 compared to NF control mice (p < 0.05). Choline and betaine supplementation alleviated these morphological changes in GDM placentas. In parallel, both choline and betaine supplementation significantly reduced glucose accretion (p < 0.05) in in vitro assays where the human choriocarcinoma BeWo cells were cultured in high (35.5 mM) or normal (5.5 mM) glucose conditions. Expression of angiogenic genes was minimally altered by choline or betaine supplementation in either model. In conclusion, both choline and betaine modified some but not all determinants of placental transport in response to hyperglycemia in mouse and in vitro human cell line models.

Maternal betaine supplementation affects fetal growth and lipid metabolism of high-fat fed mice in a temporal-specific manner.

BACKGROUND/OBJECTIVES: Maternal obesity increases the risk of gestational diabetes mellitus (GDM), which results in fetal overgrowth and long-lasting metabolic dysfunctioning in the offspring. Previous studies show that maternal choline supplementation normalizes fetal growth and adiposity of progeny from obese mice. This study examines whether supplementation of betaine, a choline derivative, has positive effects on fetal metabolic outcomes in mouse progeny exposed to maternal obesity and GDM. METHODS: C57BL/6J mice were fed either a high-fat (HF) diet or a control (normal-fat, NF) diet and received either 1% betaine (BS) or control untreated (BC) drinking water 4-6 weeks before timed-mating and throughout gestation. Maternal, placental, and fetal samples were collected for metabolite and gene-expression assays. RESULTS: At E12.5, BS prevented fetal and placental overgrowth and downregulated glucose and fatty acid transporters (Glut1 and Fatp1) and the growth-promoting insulin-like growth factor 2 (Igf2) and its receptor Igf1r in the placenta of HF, glucose-intolerant dams (P < 0.05). However, these effects disappeared at E17.5. At E17.5, BS reduced fetal adiposity and prevented liver triglyceride overaccumulation in HF versus NF fetuses (P < 0.05). BS fetal livers had enhanced mRNA expression of microsomal triglyceride transfer protein (Mttp) (P < 0.01), which promotes VLDL synthesis and secretion. Although we previously reported that maternal choline supplementation downregulated mRNA expression of genes involved in de novo lipogenesis in fetal livers, such alterations were not observed with BS, suggesting differential effects of betaine and choline on fetal gene expression. CONCLUSION: We propose a temporal-specific mechanism by which maternal BS influences fetal growth and lipid metabolic outcomes of HF mice during prenatal development.

The Pancreatic Cancer Microbiome Promotes Oncogenesis by Induction of Innate and Adaptive Immune Suppression.

We found that the cancerous pancreas harbors a markedly more abundant microbiome compared with normal pancreas in both mice and humans, and select bacteria are differentially increased in the tumorous pancreas compared with gut. Ablation of the microbiome protects against preinvasive and invasive pancreatic ductal adenocarcinoma (PDA), whereas transfer of bacteria from PDA-bearing hosts, but not controls, reverses tumor protection. Bacterial ablation was associated with immunogenic reprogramming of the PDA tumor microenvironment, including a reduction in myeloid-derived suppressor cells and an increase in M1 macrophage differentiation, promoting TH1 differentiation of CD4+ T cells and CD8+ T-cell activation. Bacterial ablation also enabled efficacy for checkpoint-targeted immunotherapy by upregulating PD-1 expression. Mechanistically, the PDA microbiome generated a tolerogenic immune program by differentially activating select Toll-like receptors in monocytic cells. These data suggest that endogenous microbiota promote the crippling immune-suppression characteristic of PDA and that the microbiome has potential as a therapeutic target in the modulation of disease progression.Significance: We found that a distinct and abundant microbiome drives suppressive monocytic cellular differentiation in pancreatic cancer via selective Toll-like receptor ligation leading to T-cell anergy. Targeting the microbiome protects against oncogenesis, reverses intratumoral immune tolerance, and enables efficacy for checkpoint-based immunotherapy. These data have implications for understanding immune suppression in pancreatic cancer and its reversal in the clinic. Cancer Discov; 8(4); 403-16. ©2018 AACR.See related commentary by Riquelme et al., p. 386This article is highlighted in the In This Issue feature, p. 371.

Nucleolin phosphorylation regulates PARN deadenylase activity during cellular stress response.

Nucleolin (NCL) is an abundant stress-responsive, RNA-binding phosphoprotein that controls gene expression by regulating either mRNA stability and/or translation. NCL binds to the AU-rich element (ARE) in the 3'UTR of target mRNAs, mediates miRNA functions in the nearby target sequences, and regulates mRNA deadenylation. However, the mechanism by which NCL phosphorylation affects these functions and the identity of the deadenylase involved, remain largely unexplored. Earlier we demonstrated that NCL phosphorylation is vital for cell cycle progression and proliferation, whereas phosphorylation-deficient NCL at six consensus CK2 sites confers dominant-negative effect on proliferation by increasing p53 expression, possibly mimicking cellular DNA damage conditions. In this study, we show that NCL phosphorylation at those CK2 consensus sites in the N-terminus is necessary to induce deadenylation upon oncogenic stimuli and UV stress. NCL-WT, but not hypophosphorylated NCL-6/S*A, activates poly (A)-specific ribonuclease (PARN) deadenylase activity. We further demonstrate that NCL interacts directly with PARN, and under non-stress conditions also forms (a) complex (es) with factors that regulate deadenylation, such as p53 and the ARE-binding protein HuR. Upon UV stress, the interaction of hypophosphorylated NCL-6/S*A with these proteins is favored. As an RNA-binding protein, NCL interacts with PARN deadenylase substrates such as TP53 and BCL2 mRNAs, playing a role in their downregulation under non-stress conditions. For the first time, we show that NCL phosphorylation offers specificity to its protein-protein, protein-RNA interactions, resulting in the PARN deadenylase regulation, and hence gene expression, during cellular stress responses.

Choline prevents fetal overgrowth and normalizes placental fatty acid and glucose metabolism in a mouse model of maternal obesity.

Maternal obesity increases placental transport of macronutrients, resulting in fetal overgrowth and obesity later in life. Choline participates in fatty acid metabolism, serves as a methyl donor and influences growth signaling, which may modify placental macronutrient homeostasis and affect fetal growth. Using a mouse model of maternal obesity, we assessed the effect of maternal choline supplementation on preventing fetal overgrowth and restoring placental macronutrient homeostasis. C57BL/6J mice were fed either a high-fat (HF, 60% kcal from fat) diet or a normal (NF, 10% kcal from fat) diet with a drinking supply of either 25 mM choline chloride or control purified water, respectively, beginning 4 weeks prior to mating until gestational day 12.5. Fetal and placental weight, metabolites and gene expression were measured. HF feeding significantly (P<.05) increased placental and fetal weight in the HF-control (HFCO) versus NF-control (NFCO) animals, whereas the HF choline-supplemented (HFCS) group effectively normalized placental and fetal weight to the levels of the NFCO group. Compared to HFCO, the HFCS group had lower (P<.05) glucose transporter 1 and fatty acid transport protein 1 expression as well as lower accumulation of glycogen in the placenta. The HFCS group also had lower (P<.05) placental 4E-binding protein 1 and ribosomal protein s6 phosphorylation, which are indicators of mechanistic target of rapamycin complex 1 activation favoring macronutrient anabolism. In summary, our results suggest that maternal choline supplementation prevented fetal overgrowth in obese mice at midgestation and improved biomarkers of placental macronutrient homeostasis.

Choline Supplementation Normalizes Fetal Adiposity and Reduces Lipogenic Gene Expression in a Mouse Model of Maternal Obesity.

Maternal obesity increases fetal adiposity which may adversely affect metabolic health of the offspring. Choline regulates lipid metabolism and thus may influence adiposity. This study investigates the effect of maternal choline supplementation on fetal adiposity in a mouse model of maternal obesity. C57BL/6J mice were fed either a high-fat (HF) diet or a control (NF) diet and received either 25 mM choline supplemented (CS) or control untreated (CO) drinking water for 6 weeks before timed-mating and throughout gestation. At embryonic day 17.5, HF feeding led to higher (p < 0.05) percent total body fat in fetuses from the HFCO group, while the choline supplemented HFCS group did not show significant difference versus the NFCO group. Similarly, HF feeding led to higher (p < 0.05) hepatic triglyceride accumulation in the HFCO but not the HFCS fetuses. mRNA levels of lipogenic genes such as Acc1, Fads1, and Elovl5, as well as the transcription factor Srebp1c that favors lipogenesis were downregulated (p < 0.05) by maternal choline supplementation in the HFCS group, which may serve as a mechanism to reduce fat accumulation in the fetal liver during maternal HF feeding. In summary, maternal choline supplementation improves indices of fetal adiposity in obese dams at late gestation.

Induced expression of nucleolin phosphorylation-deficient mutant confers dominant-negative effect on cell proliferation.

Nucleolin (NCL) is a major nucleolar phosphoprotein that has pleiotropic effects on cell proliferation and is elevated in a variety of tumors. NCL is highly phosphorylated at the N-terminus by two major kinases: interphase casein kinase 2 (CK2) and mitotic cyclin-dependent kinase 1 (CDK1). Earlier we demonstrated that a NCL-mutant that is partly defective in undergoing phosphorylation by CK2 inhibits chromosomal replication through its interactions with Replication Protein A, mimicking the cellular response to DNA damage. We further delineated that the N-terminus of NCL associates with Hdm2, the most common E3 ubiquitin ligase of p53. We reported that NCL antagonizes Hdm2 to stabilize p53 and stimulates p53 transcriptional activity. Although NCL-phosphorylation by CK2 and ribosomal DNA transcription are closely coordinated during interphase, the role of NCL phosphorylation in regulating cell proliferation remains unexplored. We have therefore engineered unique human cells that specifically induce expression of NCL-wild type (WT) or a phosphorylation-deficient NCL-mutant, 6/S*A where all the six CK2 consensus serine sites residing in the N-terminus NCL were mutated to alanine. Here we show that this NCL-mutant is defective in undergoing phosphorylation by CK2. We also demonstrate that NCL-phosphorylation by CK2 is required through the S-phase progression in cell cycle and hence proliferation. Induced expression of NCL with mutated CK2 phosphorylation sites stabilizes p53, results in higher expression of Bcl2 (B-cell lymphoma 2) homology 3 (BH3)-only apoptotic markers and causes a dominant-negative effect on cell viability. Our unique cellular system thus provides the first evidential support to delineate phospho-specific functions of NCL on cell proliferation.

Metformin targets c-MYC oncogene to prevent prostate cancer.

UNLABELLED: Prostate cancer (PCa) is the second leading cause of cancer-related death in American men and many PCa patients develop skeletal metastasis. Current treatment modalities for metastatic PCa are mostly palliative with poor prognosis. Epidemiological studies indicated that patients receiving the diabetic drug metformin have lower PCa risk and better prognosis, suggesting that metformin may have antineoplastic effects. The mechanism by which metformin acts as chemopreventive agent to impede PCa initiation and progression is unknown. The amplification of c-MYC oncogene plays a key role in early prostate epithelia cell transformation and PCa growth. The purpose of this study is to investigate the effect of metformin on c-myc expression and PCa progression. Our results demonstrated that (i) in Hi-Myc mice that display murine prostate neoplasia and highly resemble the progression of human prostate tumors, metformin attenuated the development of prostate intraepithelial neoplasia (PIN, the precancerous lesion of prostate) and PCa lesions. (ii) Metformin reduced c-myc protein levels in vivo and in vitro. In Myc-CaP mouse PCa cells, metformin decreased c-myc protein levels by at least 50%. (iii) Metformin selectively inhibited the growth of PCa cells by stimulating cell cycle arrest and apoptosis without affecting the growth of normal prostatic epithelial cells (RWPE-1). (iv) Reduced PIN formation by metformin was associated with reduced levels of androgen receptor and proliferation marker Ki-67 in Hi-Myc mouse prostate glands. Our novel findings suggest that by downregulating c-myc, metformin can act as a chemopreventive agent to restrict prostatic neoplasia initiation and transformation. SUMMARY: Metformin, an old antidiabetes drug, may inhibit prostate intraepithelial neoplasia transforming to cancer lesion via reducing c-MYC, an 'old' overexpressed oncogene. This study explores chemopreventive efficacy of metformin in prostate cancer and its link to cMYC in vitro and in vivo.