Search

This study investigates the mechanism by which maternal protein restriction induces hepatic autophagy-related gene expression in the offspring of rats. Pregnant Sprague-Dawley rats were fed either a control diet (C, 18 % energy from protein) or a low-protein diet (LP, 8·5 % energy from protein) during gestation, followed by the control diet during lactation and post-weaning. Liver tissue was collected from the offspring at postnatal day 38 and divided into four groups according to sex and maternal diet (F-C, F-LP, M-C and M-LP) for further analysis. Autophagy-related mRNA and protein levels were determined by real-time PCR and Western blotting, respectively. In addition, chromatin immunoprecipitation (ChIP) was performed to investigate the interactions between transcription factors and autophagy-related genes. Protein levels of p- eukaryotic translation initiation factor 2a and activating transcription factor 4 (ATF4) were increased only in the female offspring born to dams fed the LP diet. Correlatively, the mRNA expression of hepatic autophagy-related genes including Map1lc3b, P62/Sqstm1, Becn1, Atg3, Atg7 and Atg10 was significantly greater in the F-LP group than in the F-C group. Furthermore, ChIP results showed greater ATF4 and C/EBP homology protein (CHOP) binding at the regions of a set of autophagy-related genes in the F-LP group than in the F-C group. Our data demonstrated that a maternal LP diet transcriptionally programmed hepatic autophagy-related gene expression only in female rat offspring. This transcriptional programme involved the activation of the eIF2α/ATF4 pathway and intricate regulation by transcription factors ATF4 and CHOP.

The simultaneous introduction of wheel running (WR) and diet choice (high-carbohydrate chow vs. high-fat diet) results in sex-specific diet choice patterns in rats. WR induces a high-fat (HF) diet avoidance, and such avoidance persists in the majority of males, but not females, throughout a 2-wk period. Exercise is a physiological stressor that activates the hypothalamic-pituitary-adrenal (HPA) axis and stimulates glucocorticoid (GC) release, which can alter dietary preferences. Here, we examined the role of the HPA axis and GC signaling in mediating exercise-induced changes in diet preference and the associated neurobiological adaptations that may underlie sex differences in diet choice patterns. Experiment 1 revealed that adrenalectomy did not significantly alter the initiation and persistence of running-induced HF diet avoidance in male rats. Experiment 2 showed that acute WR resulted in greater neural activation than chronic WR in the medial prefrontal (mPFC) and insular cortices (IC) in male rats. Experiment 3 revealed sex differences in the molecular adaptation to exercise and diet preference. First, exercise increased gene expression of fkbp5 in the mPFC, IC, and hippocampus of WR females but had limited influence in males. Second, male and female WR rats that reversed or maintained HF diet avoidance showed distinct sex- and HF diet preference-dependent expression profiles of genes involved in cortical GC signaling (e.g., nr3c1, nr3c2, and src1). Taken together, our results suggest sex differences in region-specific neural adaptations may underlie sex differences in diet preference and the health benefits from exercise.

Both high-fat diets (HFD) and bisphenol A (BPA), an environmental endocrine disruptor, are prevalent in industrialized societies. Previous studies have detected separate effects of BPA and HFD; however, none have assessed possible interactive effects. Here, pregnant dams consumed 0, 40, or 400 µg BPA/kg/day and were fed either a control (CON; 15.8% kcal fat) or HFD (45% kcal fat) from gestational day 2 through parturition. The pups were individually dosed with BPA from postnatal days (P) 1–10, while the dams continued to consume one of the two diets. Maternal behavior increased with the HFD while the offspring’s periadolescent social play decreased with BPA, but no interactive effects were observed. Neither HFD nor BPA exposure changed performance on a social recognition task, and only BPA had an effect on the elevated plus maze. BPA increased several cytokines in the medial prefrontal cortex (mPFC) of P10 males but not females. Expression of several genes related to hormone synthesis and receptors, inflammation, oxidative stress, and apoptosis in the mPFC on P10 and P90 were altered due to BPA and/or HFD exposure with rare interactive effects. BPA resulted in an increase in the gene expression of Esr1 in the mPFC of females on both P10 and P90. Epigenetic analysis on P90 did not show a change in methylation or in the levels of pre-mRNA or microRNA. Thus, perinatal BPA and HFD have separate effects but rarely interact.

Bladder cancer has high morbidity and mortality rates among the male genitourinary system tumor types. MicroRNA‑218 (miR‑218) is associated with the development of a variety of cancer types, including bladder cancer. Rab6c is a member of the Rab family and is involved in drug resistance in MCF7 cells. The aim of the present study was to clarify the relationship between Rab6c and miR‑218 in bladder cancer cell lines. In this study, the expression levels of miR‑218 and Rab6c were evaluated via reverse transcription‑quantitative PCR and western blotting, respectively. The association between Rab6c and miR‑218 was recognized via TargetScan analysis and dual luciferase reporter gene detection. Cell proliferation was analyzed using Cell Counting Kit‑8 and colony formation assays, and the invasive ability was measured via Transwell assays. Rab6c was highly expressed in bladder cancer, while miR‑218 had abnormally low expression in bladder cancer. In addition, there was a mutual regulation between Rab6c and miR‑218 in bladder cancer. It was found that overexpression of Rab6c significantly enhanced the proliferation, colony formation and invasion of T24 and EJ cells. Furthermore, miR‑218 overexpression blocked the promoting effects of Rab6c on the malignant behavior of bladder cancer cells. Thus, Rab6c promotes the proliferation and invasion of bladder cancer cells, while miR‑218 has the opposite effect, which may provide a novel insight for the treatment of bladder cancer.

OBJECTIVES: Previously we found that intestinal-epithelial-cell-specific-hnRNPI knockout (KO) upregulates pro-inflammatory cytokines expression in colon, suggesting a potential link between hnRNPI KO and colonic immunity. Moreover, high protein diets were found to inhibit the inflammatory responses in KO mice, mechanism remains unclear. Hence, this current study is aimed to profile cell types in colon of hnRNPI wildtype (WT) and KO mice, characterize differences on cellular population, and identify cell types mostly responsible for the cytokine expression. METHODS: In this study, whole colon tissues from WT and KO mice (n = 3) were collected for scRNA-seq. Single cells were filtered by mitochondria percentage and UMI counts. 31,798 cells were used for profiling. Uniform manifold approximation and projection (UMAP) was adopted for dimension reduction and graph clustering, proportion of each cell subtype was normalized to cell density. Gene expression of specific pro-inflammatory markers were calculated by Loupe Browser (10X Genomics). RESULTS: A total of 25 clusters were sorted to the following cell types: B-cell, endothelial, epithelial, erythrocytes, fibroblast, innate lymphoid cell (ILC), mast, monocytes, myeloid and T-cell. Comparing to WT, proportion of tuft cells is increased (P = 0.08) in KO. As for immune cells, total T-cell (P = 0.1), especially non-cytotoxic T-cell (P

Perinatal exposure to endocrine disrupting chemicals negatively impacts health, but the mechanism by which such toxicants damage long-term reproductive and metabolic function is unknown. Lipid metabolism plays a pivotal role in steroid hormone synthesis as well as energy utilization and storage; thus, aberrant lipid regulation may contribute to phthalate-driven health impairments. In order to test this hypothesis, we specifically examined epigenetic disruptions in lipid metabolism pathways after perinatal phthalate exposure. During gestation and lactation, pregnant Long-Evans rat dams were fed environmentally relevant doses of phthalate mixture: 0 (CON), 200 (LO), or 1000 (HI) µg/kg body weight/day. On PND90, male offspring in the LO and HI groups had higher body weights than CON rats. Gene expression of lipid metabolism pathways was altered in testis and adipose tissue of males exposed to the HI phthalate dosage. Specifically, Srebf1 was downregulated in testis and Srebf2 was upregulated in adipose tissue. In testis of HI rats, DNA methylation was increased at two loci and reduced at one other site surrounding Srebf1 transcription start site. In adipose tissue of HI rats, we observed increased DNA methylation at one region within the first intron of Srebf2. Computational analysis revealed several potential transcriptional regulator binding sites, suggesting functional relevance of the identified differentially methylated CpGs. Overall, we show that perinatal phthalate exposure affects lipid metabolism gene expression in a tissue-specific manner possibly through altering DNA methylation of Srebf1 and Srebf2.

Perinatal exposure to endocrine disrupting chemicals negatively impacts health, but the mechanism by which such toxicants damage long-term reproductive and metabolic function is unknown. Lipid metabolism plays a pivotal role in steroid hormone synthesis as well as energy utilization and storage; thus, aberrant lipid regulation may contribute to phthalate-driven health impairments. In order to test this hypothesis, we specifically examined epigenetic disruptions in lipid metabolism pathways after perinatal phthalate exposure. During gestation and lactation, pregnant Long–Evans rat dams were fed environmentally relevant doses of phthalate mixture: 0 (CON), 200 (LO), or 1000 (HI) µg/kg body weight/day. On PND90, male offspring in the LO and HI groups had higher body weights than CON rats. Gene expression of lipid metabolism pathways was altered in testis and adipose tissue of males exposed to the HI phthalate dosage. Specifically, Srebf1 was downregulated in testis and Srebf2 was upregulated in adipose tissue. In testis of HI rats, DNA methylation was increased at two loci and reduced at one other site surrounding Srebf1 transcription start site. In adipose tissue of HI rats, we observed increased DNA methylation at one region within the first intron of Srebf2. Computational analysis revealed several potential transcriptional regulator binding sites, suggesting functional relevance of the identified differentially methylated CpGs. Overall, we show that perinatal phthalate exposure affects lipid metabolism gene expression in a tissue-specific manner possibly through altering DNA methylation of Srebf1 and Srebf2.

Sterile α motif and HD domain-containing protein 1 (SAMHD1) is a novel intrinsic restriction factor that inhibits the replication of certain retroviruses and DNA viruses through its deoxynucleoside triphosphate triphosphohydrolase activity. A previous study by our group showed that SAMHD1 restrained hepatitis B virus replication and interferon (IFN)‑α induced SAMHD1 expression in liver cells. However the mechanisms of SAMHD1 upregulation by IFN‑α in liver cells have remained elusive. The present study demonstrated that IFN‑α treatment increased SAMHD1 mRNA levels in SMMC‑7721 cells in a time‑dependent manner. Knockdown of STAT1 inhibited the induction of SAMHD1 expression by IFN‑α in SMMC‑7721 cells. STAT2 silencing also suppressed the induction of SAMHD1 expression by IFN‑α in SMMC‑7721 cells. Furthermore, the induction of SAMHD1 expression in SMMC‑7721 cells by IFN‑α was found to be dependent on IFN‑regulatory factor 9 (IRF9). In conclusion, these results suggested that the interferon‑stimulated gene factor 3 complex, which consists of STAT1, STAT2 and IRF9, is required for the induction of SAMHD1 expression by IFN-α in SMMC-7721 cells.

The aim of the present study was to investigate the mechanistic basis of protein deficiency during pregnancy in mother that is transduced to offspring. To this end, timed-pregnant Sprague–Dawley rats were fed either a control (20 % of energy from protein) or low-protein (LP, 8 % of energy from protein) diet during gestation. Tissues were collected after delivery from rat dams, and skeletal muscle was collected at postnatal day 38 from the offspring. Quantitative RT-PCR and Western blot analyses were performed to determine mRNA and protein levels. Histological analysis was performed to evaluate myofibre size. LP dams gained significantly less weight during pregnancy, developed muscle atrophy, and had significantly lower circulating threonine and histidine levels than control dams. The mRNA expression of the well-known amino acid response (AAR) pathway-related target genes was increased only in the skeletal muscle of LP dams, as well as the protein expression levels of activating transcription factor 4 (ATF4) and phosphorylated eukaryotic translation initiation factor 2α (p-eIF2α). The mRNA expression of autophagy-related genes was significantly increased in the skeletal muscle of LP dams. Moreover, the mRNA expression of genes involved in both AAR and autophagy pathways remained elevated and was memorised in the muscle of LP offspring that consumed a post-weaning control diet. Additionally, the LP diet increased an autophagy marker, microtubule-associated proteins 1A/1B light chain 3B (LC3B) protein expression in the skeletal muscle of rat dams, consistent with the initiation of autophagy. The LP diet further increased ATF4 binding at the predicted regions of AAR and autophagy pathway-related genes. Increased binding of ATF4 unveils the crucial role of ATF4 in the activation of autophagy in response to protein restriction. Our data suggest that molecular changes in maternal muscle are memorised in the offspring long after gestational protein restriction, reinforcing the role of maternal signalling in programming offspring health.

cope Soy flour diet (MS) prevented isoflavones from stimulating MCF-7 tumor growth in athymic nude mice, indicating that other bioactive compounds in soy can negate the estrogenic properties of isoflavones. The underlying signal transduction pathways to explain the protective effects of soy flour consumption were studied here. Methods and results Ovariectomized athymic nude mice inoculated with MCF-7 human breast cancer cells were fed either Soy flour diet (MS) or purified isoflavone mix diet (MI), both with equivalent amounts of genistein. Positive controls received estradiol pellets and negative controls received sham pellets. GeneChip Human Genome U133 Plus 2.0 Array platform was used to evaluate gene expressions, and results were analyzed using bioinformatics approaches. Tumors in MS-fed mice exhibited higher expression of tumor growth suppressing genes ATP2A3 and BLNK and lower expression of oncogene MYC. Tumors in MI-fed mice expressed a higher level of oncogene MYB and a lower level of MHC-I and MHC-II, allowing tumor cells to escape immunosurveillance. MS-induced gene expression alterations were predictive of prolonged survival among estrogen-receptor-positive breast cancer patients, whilst MI-induced gene changes were predictive of shortened survival. Conclusion Our findings suggest that dietary soy flour affects gene expression differently than purified isoflavones, which may explain why soy foods prevent isoflavones-induced stimulation of MCF-7 tumor growth in athymic nude mice.

Calorie-dense high-fat diets (HF) are associated with detrimental health outcomes, including obesity, cardiovascular disease, and diabetes. Both pre- and post-natal HF diets have been hypothesized to negatively impact long-term metabolic health via epigenetic mechanisms. To understand how the timing of HF diet intake impacts DNA methylation and metabolism, male Sprague&ndash, Dawley rats were exposed to either maternal HF (MHF) or post-weaning HF diet (PHF). At post-natal week 12, PHF rats had similar body weights but greater hepatic lipid accumulation compared to the MHF rats. Genome-wide DNA methylation was evaluated, and analysis revealed 1744 differentially methylation regions (DMRs) between the groups with the majority of the DMR located outside of gene-coding regions. Within differentially methylated genes (DMGs), intragenic DNA methylation closer to the transcription start site was associated with lower gene expression, whereas DNA methylation further downstream was positively correlated with gene expression. The insulin and phosphatidylinositol (PI) signaling pathways were enriched with 25 DMRs that were associated with 20 DMGs, including PI3 kinase (Pi3k), pyruvate kinase (Pklr), and phosphodiesterase 3 (Pde3). Together, these results suggest that the timing of HF diet intake determines DNA methylation and gene expression patterns in hepatic metabolic pathways that target specific genomic contexts.

Standard methods for detecting cancer-associated genes rely on comparison of sample means between cancer patients and healthy controls. While such methods have successfully identified several oncogenes and tumor suppressor genes, they neglect to account for heterogeneity within the cancer population. Genetic mutations, translocations, and amplifications are often inconsistent across tumors, and instead they often affect smaller subsets of patients. This concept gives rise to the idea of bimodally expressed genes, or genes that display two modes of expression within one population. Analysis of bimodal gene expression has been explored via a variety of techniques including test statistics and clustering. In this review, we summarize the methodologies used to quantify bimodal gene expression and address the utility of these genes in patient stratification and specialized therapeutics in breast and lung cancer. Finally we discuss the limitations and future directions for bimodal genes in the era of high-throughput sequencing and personalized medicine. Keywords: Bimodality, Mixture-model, Clustering, Unsupervised learning, Prognosis, Personalized medicine

DNA methylation is a vital modification process in the control of genetic information, which contributes to the epigenetics by regulating gene expression without changing the DNA sequence. Abnormal DNA methylation—both hypomethylation and hypermethylation—has been associated with improper gene expression, leading to several disorders. Two types of risk factors can alter the epigenetic regulation of methylation pathways: genetic factors and modifiable factors. Nutrition is one of the strongest modifiable factors, which plays a direct role in DNA methylation pathways. Large numbers of studies have investigated the effects of nutrition on DNA methylation pathways, but relatively few have focused on the biochemical mechanisms. Understanding the biological mechanisms is essential for clarifying how nutrients function in epigenetics. It is believed that nutrition affects the epigenetic regulations of DNA methylation in several possible epigenetic pathways: mainly, by altering the substrates and cofactors that are necessary for proper DNA methylation; additionally, by changing the activity of enzymes regulating the one-carbon cycle; and, lastly, through there being an epigenetic role in several possible mechanisms related to DNA demethylation activity. The aim of this article is to review the potential underlying biochemical mechanisms that are related to diet modifications in DNA methylation and demethylation.

4-Coumarate:CoA ligase (4CL) is a key enzyme in the phenylpropanoid synthesis pathway. The Pto4CL2 promoter was cloned from Populus tomentosa Carr. and fused to the reporter gene encoding β-glucuronidase (GUS); the complex expression patterns directed by the Pto4CL2 promoter were then characterized in Nicotiana tabacum Xanthi by histochemical assays. The promoter 5'-deletion and histochemical assay conducted on transformants indicated that the -317 to -292 nt region supports Pto4CL2 expression in the epidermis and petals and the deletion of the -266 to -252 nt region resulted in the loss of tissue specificity and a dramatic reduction in GUS activity. Furthermore, electrophoretic mobility shift assays testified that an adenine and cytosine-rich element (-264 to -255 nt) and an abscisic acid-responsive element (-242 to -235 nt) in the Pto4CL2 promoter would have functions for the complex expression profiling and efficient basal expression, respectively. These results further clarify the mode of the regulatory expression of class II 4CL promoters in higher plants.

Placental efficiency is a predictor of fetal growth and development, which is also controlled by maternal gestational health and diet. The present study investigated the effects of a gestational low-protein diet on offspring growth capacity as well as the diet’s contribution to altered expression of placental genes associated with the mammalian amino acid response (AAR) pathway. To assess these outcomes, timed-pregnant Sprague Dawley rats were fed a control (C) diet with 18% protein or a low-protein (LP) diet with 9% protein throughout gestation (Expt. 1) or throughout gestation and lactation (Expt. 2). Placentas were collected during natural delivery and quantitative RT-PCR and Western-blot analyses were performed to determine placental mRNA and protein levels. By the end of the lactation period, offspring of dams fed the LP diet had stunted growth in both experiments. mRNA expressionof target genes in the AAR pathway, such as activating transcription factor-3 (Atf3), asparagine synthetase (Asns), and Sodium-dependent neutral amino acid transporter-2 (Snat2), was greater in placentas of rats fed the LP diet compared with controls, as were placental ATF4 and p-eIF2a protein levels. The increase in mRNA expression of AAR pathway-associated genes was correlated with the stunting of offspring growth (Atf3: R 2 = 0.32, P = 0.086;Asns: R 2 = 0.44, P , 0.05; Snat2: R 2 = 0.33, P = 0.084). Our study showed that the mammalian AAR pathway in placenta is upregulated by a maternal low-protein diet and this activation may act as a cue for the fetus to develop an adaptive response suited to their predicted postnatal environment, i.e. a more favorable phenotype for their survival. J. Nutr. 140: 2116‐2120, 2010.

Biological responses to environmental stress, including nutrient limitation are mediated in part by epigenetic modifications including DNA methylation. Insulin-like growth factor II (Igf2) and H19 are subject to epigenetic modifications leading to genomic imprinting. The present study was designed to test the effect of maternal low protein diet on the Igf2/H19 locus in offspring. Pregnant Sprague-Dawley rats were fed diets containing 180 g/kg casein (control) or 90 g/kg (LP) casein with either 1 mg/kg (LP) or 3 mg/kg folic acid (LPF). LP diet increased Igf2 and H19 gene expression in the liver of day 0 male offspring and the addition of folic acid reduced the mRNA level in LPF rats to that of the control group. DNA methylation in Imprinting Control Region (ICR) of Igf2/H19 locus increased significantly following maternal LP diet but rats fed the LPF diet did not exhibit the hypermethylation. The Differential Methylation Region 2 (DMR2) did not show any change in methylation in either LP or LPF rats. The expression of Dnmt1 and Dnmt3a, the members of DNA methyltransferase family, and methyl CpG-binding domain 2 (Mbd2) was significantly increased following the maternal LP diet but did not differ between the control and LPF group. There is a strong correlation between methylation of ICR with the expression of Igf2 and H19. These results suggested that maternal exposure to a low protein diet and folic acid during gestation alters gene expression of Igf2 and H19 in the liver by regulating the DNA methylation of these genes. The DNA methyltransferase machinery may be involved into the programming of imprinted genes through the imprinted control region.

Promoters that have been widely used for both basic research and biotechnological application in plants are generally unidirectional. Here we describe a strategy to bi-directionalize the vascular-specific expression grp1.8 promoter (here named GRPp) and 4CL1 promoter (here named 4CL1p) so that one promoter can direct the vascular-specific expression of two genes, one on each end of the promoter. The minimal promoter (35Smini or GRP mini), when fused at the 5' end of the specific expression promoter (GRPp or 4CL1p) to form bi-directional promoter (35Smini-GRPp, 35Smini-4CL1p or GRPmini-GRPp), was able to direct expression of the glucuronidase (gus) and green fluorescent protein (gfp) gene in all independent transgenic tobacco lines. Stable expression of gusA and gfp genes in transgenic plants was analyzed by histochemical staining for GUS and fluorescence microscopic observation under UV for GFP in transgenic plants. The remarkable transcript levels of GFP and GUS were detected by real-time PCR in independent transgenic tobacco lines. Their vascular-specific bi-directional promoters should be used to vascular-specific expression several functional genes in transgenic plants simultaneously.

Developmental bisphenol A (BPA) exposure increases adulthood hepatic steatosis with reduced mitochondrial function. To investigate the potential epigenetic mechanisms behind developmental BPA-induced hepatic steatosis, pregnant Sprague-Dawley rats were dosed with vehicle (oil) or BPA (100μg/kg/day) from gestational day 6 until postnatal day (PND) 21. After weaning, offspring were either challenged with a high-fat (HF; 45% fat) or remained on a control (C) diet until PND110. From PND60 to 90, both BPA and HF diet increased the fat/lean ratio in males only, and the combination of BPA and HF diet appeared to cause the highest ratio. On PND110, Oil-HF, BPA-C, and BPA-HF males had higher hepatic lipid accumulation than Oil-C, with microvesicular steatosis being marked in the BPA-HF group. Furthermore, on PND1, BPA increased and modified hepatic triglyceride (TG) and free fatty acid (FFA) compositions in males only. In PND1 males, BPA increased hepatic expression of FFA uptake gene Fat/Cd36, and decreased the expression of TG synthesis- and β-oxidation-related genes (Dgat, Agpat6, Cebpα, Cebpβ, Pck1, Acox1, Cpt1a, Cybb). BPA altered DNA methylation and histone marks (H3Ac, H4Ac, H3Me2K4, H3Me3K36), and decreased the binding of several transcription factors (Pol II, C/EBPβ, SREBP1) within the male Cpt1a gene, the key β-oxidation enzyme. In PND1 females, BPA only increased the expression of genes involved in FFA uptake and TG synthesis (Lpl, Fasn, and Dgat). These data suggest that developmental BPA exposure alters and reprograms hepatic β-oxidation capacity in males, potentially through the epigenetic regulation of genes, and further alters the response to a HF diet.

A high-fat (HF) diet is associated with progression of liver diseases. To illustrate genome-wide landscape of DNA methylation in liver of rats fed either a control or HF diet, two enrichment-based methods, namely methyl-DNA immunoprecipitation assay with high-throughput sequencing (MeDIP-seq) and methylation-sensitive restriction enzyme sequencing (MRE-seq), were performed in our study. Rats fed with the HF diet exhibited an increased body weight and liver fat accumulation compared with that of the control group when they were 12 wk of age. Genome-wide analysis of differentially methylated regions (DMRs) showed that 12,494 DMRs induced by HF diet were hypomethylated and 6,404 were hypermethylated. DMRs with gene annotations [differentially methylated genes (DMGs)] were further analyzed to show gene-specific methylation profile. There were 88, 2,680, and 95 hypomethylated DMGs identified with changes in DNA methylation in the promoter, intragenic and downstream regions, respectively, compared with fewer hypermethylated DMGs (45, 1,623, and 50 in the respective regions). Some of these genes also contained an ACGT cis-acting motif whose DNA methylation status may affect gene expression. Pathway analysis showed that these DMGs were involved in critical hepatic signaling networks related to hepatic development. Therefore, HF diet had global impacts on DNA methylation profile in the liver of rats, leading to differential expression of genes in hepatic pathways that may involve in functional changes in liver development.

▪ Abstract The amino acid response (AAR) pathway in mammalian cells is designed to detect and respond to amino acid deficiency. Limiting any essential amino acid initiates this signaling cascade, which leads to increased translation of a “master regulator,” activating transcription factor (ATF) 4, and ultimately, to regulation of many steps along the pathway of DNA to RNA to protein. These regulated events include chromatin remodeling, RNA splicing, nuclear RNA export, mRNA stabilization, and translational control. Proteins that are increased in their expression as targets of the AAR pathway include membrane transporters, transcription factors from the basic region/leucine zipper (bZIP) superfamily, growth factors, and metabolic enzymes. Significant progress has been achieved in understanding the molecular mechanisms by which amino acids control the synthesis and turnover of mRNA and protein. Beyond gaining additional knowledge of these important regulatory pathways, further characterization of how these processes contribute to the pathology of various disease states represents an interesting aspect of future research in molecular nutrition.

The objective of this study was to evaluate the effect of dietary protein intake and stage of development on chicken intestinal peptide transporter (cPepT1) mRNA abundance. Chicks were sampled at embryonic d 16 (e 16) until the day of hatch (d 0). After hatch, mixed sex Cobb chicks were randomly assigned to diets containing 12, 18, or 24% crude protein (CP). Intakes of the 18 and 24% CP-fed birds were restricted to that consumed by the 12% CP-fed birds. Chickens were sampled on d 0, 1, 3, 5, 7, 10, 14, 21, 28, and 35. The experiment was repeated with the addition of a 4th group with free access to the 24% CP diet. PepT1 mRNA abundance in the duodenum, jejunum, and ileum was quantified by Northern blot. PepT1 mRNA was barely detectable between e 16 and 20 but increased 14- to 50-fold by d 0 (P < 0.001). In chickens fed 12% CP, cPepT1 mRNA abundance decreased throughout the experiment, whereas those fed the restricted 18 or 24% CP diets showed an increase in cPepT1 mRNA abundance (protein x time interaction, P < 0.01). In chicks with free access to the 24% CP diet, cPepT1 mRNA decreased until d 14 but returned to an intermediate level at d 35 (protein x time interaction, P < 0.01). The relative abundance of cPepT1 mRNA varied among intestinal segments with greater abundance occurring in the duodenal or jejunal sections (P < 0.05). Our results indicate that expression of cPepT1 mRNA is regulated by both dietary protein intake and developmental stage.

To study peptide absorption in chickens, an intestinal peptide transporter cDNA (cPepT1) was isolated from a chicken duodenal cDNA library. The cDNA was 2914 bp long and encoded a protein of 714 amino acid residues with an estimated molecular size of 79.3 kDa and an isoelectric point of 7.48. cPepT1 protein is similar60% identical to PepT1 from rabbits, humans, mice, rats and sheep. Sixteen dipeptides, three tripeptides and four tetrapeptides that contained the essential amino acids Met, Lys and(or) Trp were used for functional analysis of cPepT1 in Xenopus oocytes and Chinese hamster ovary cells. For most di- and tripeptides tested, the substrate affinities were in the micromolar range, indicating that cPepT1 has high affinity for these peptides. Lys-Lys and Lys-Trp-Lys were exceptions, with substrate affinities in the millimolar range. Neither free amino acids nor tetrapeptides were transported by cPepT1. Northern blot analysis using a full-length cPepT1 cDNA as the probe demonstrated that cPepT1 is expressed strongly in the duodenum, jejunum and ileum, and at lower levels in kidney and ceca. The present study demonstrated for the first time the presence and functional characteristics of a peptide transport system from an avian species.

Cyclooxygenase-2 (COX-2) produces prostaglandins that participate in multiple physiological and pathological processes, including the activation of inflammatory responses. Induction of COX-2 gene expression has been reported to be correlated with DNA hypomethylation. This study characterized a novel enhancer located at an upstream region on the COX-2 gene and associated alterations of the DNA methylation patterns by high fat diet. Male Sprague-Dawley rats received a high fat diet at different life stages, including maternal (HF/C), post-weaning (C/HF), and lifelong (HF/HF). Liver was collected for analysis at 12 wks of age. Results showed that the high fat diet induced the expression of COX-2 mRNA in all three high fat groups when compared to the control group (C/C). Genome-wide methylated DNA immunoprecipitation (MeDIP) showed that DNA hypomethylation occurred at an upstream region of distal promoter of COX-2 gene in all three high fat groups. Site-specific hypomethylation of CpG in this region was furth...

We determined the primary structure, tissue distribution and in vitro functional characterization of a peptide transporter, oPepT1, from ovine intestine. Ovine PepT1 (oPepT1) cDNA was 2829-bp long, encoding a protein of 707 amino acid residues with an estimated molecular size of 78 kDa and an isoelectric point (pI) of 6.57. Transport function of oPepT1 was assessed by expressing oPepT1 in Xenopus oocytes using a two-electrode voltage-clamp technique. The transport process was electrogenic and pH dependent, but independent of Na+, Cl- and Ca2+. The oPepT1 displayed a broad substrate specificity for transport of neutral and charged dipeptides and tripeptides. All dipeptides and tripeptides examined evoked inward currents in a saturable manner, with an affinity constant (Kt) ranging from 27 micromol/L to 3.0 mmol/L. No responses were detected from tetrapeptides or free amino acids. Northern blot analysis demonstrated that oPepT1 was expressed in the small intestine, omasum and rumen, but was not expressed in liver and kidney. The presence of the peptide transporter in the forestomach at such levels could provide nutritionally important amino acid nitrogen to ruminants.

Amino acid response (AAR) pathway is activated when cells are deprived of amino acids. In the present study, using the human colon cancer cell line SW480, we observed that DKK1, an antagonist of the Wnt pathway, was significantly induced at the mRNA level after the removal of amino acids from the medium. Addition of the amino alcohol histidinol, which prevents the formation of histidinyl-tRNA(His), also increased DKK1 mRNA to a level similar to that observed when cells were deprived of all amino acids. Transcriptional activity and stability of DKK1 mRNA were both increased in the amino acid-deprived condition. The induction of DKK1 gene expression was confirmed by the increased immunofluorescent staining of the DKK1 protein in the amino acid deprived condition. Although chromatin immunoprecipitation assays showed increased RNA Polymerase II binding at the DKK1 promoter in amino acid-limited conditions, ATF4 binding to the promoter is absent. Luciferase reporter assays did not detect any functional AARE within the DKK1 gene structure. Knockdown of ATF4 by siRNA did not affect the increase of DKK1 mRNA during amino acid limitation. Inhibition of ERK phosphorylation abolished the induction of DKK1. Our study revealed that DKK1 is a novel target gene in the response to amino acid deficiency and that the expression of DKK1 is up-regulated through an ATF4-independent and an ERK-dependent pathway.

Maternal high-fat (HF) diet feeding is associated with increased risk of developing metabolism-related diseases in adult offspring, including chronic liver disease. The present study tested the hypothesis that maternal HF diet leads to a decreased antioxidant defense capacity and causes cellular senescence in liver of adult offspring rats, which might increase risk of developing chronic liver disease. Timed-pregnant Sprague Dawley rats were fed a HF diet (45% of energy from fat) or a control (C) diet (16% of energy from fat) during gestation and lactation. The resulting offspring were fed a C diet after weaning to generate 2 offspring groups: C diet-fed offspring of dams fed C diet (C/C) and C diet-fed offspring of dams fed a HF diet (HF/C). At 12 wk of age, male rats were killed and samples were collected for analysis. Maternal HF diet significantly increased plasma TG and hepatic TBARS concentrations and the size of hepatic lipid droplets in offspring rats. The expression of antioxidant defense genes, such as glutathione peroxidase-1, Cu/Zn superoxide dismutase (Sod1), paraoxonase enzymes (Pon1, Pon2, and Pon3), were significantly lower in the liver of HF/C pups than in C/C pups. The expression of Inhibitor of cyclin dependent Kinase 4a (p16INK4a), a marker of cellular senescence, and cyclooxygenase-2 (Cox2), a proinflammatory marker, was significantly higher in the HF/C offspring group than in the C/C offspring group. Western-blot analysis shows that cyclin D1 and phosphorylated retinoblastoma protein were significantly lower in HF/C offspring than in C/C offspring. The results provide the first evidence to our knowledge that maternal HF diet might alter antioxidant defense capacity and program the p16INK4a-dependent cellular senescence in the liver of adult offspring.

In insulin resistance and type II diabetes, there is an elevation of hepatic gluconeogenesis, which contributes to hyperglycaemia. Studies in experimental animals have provided evidence that consumption of high fat (HF) diets by female rats programs the progeny for glucose intolerance in adulthood, but the mechanisms behind the in utero programming remain poorly understood. The present study analysed the effect of a maternal HF diet on fetal gluconeogenic gene expression and potential regulation mechanism related to histone modifications. Dams were fed either a Control (C, 16% kcal fat) or a high-fat (HF, 45% kcal fat) diet throughout gestation. Livers of the offspring were collected on gestational day 21 and analysed to determine the consequences of a maternal HF diet on molecular markers of fetal liver gluconeogenesis. We demonstrated that offspring of HF-fed dams were significantly heavier and had significantly higher blood glucose levels at the time of delivery than offspring of dams fed the C diet. While maternal gluconeogenesis and plasma glucose were not affected by the HF diet, offspring of HF-fed dams had significantly higher mRNA contents of gluconeogenic genes in addition to the elevated plasma glucose. In addition to increased transcription rate, a gestational HF diet resulted in modifications of the Pck1 histone code in livers of offspring. Our results demonstrate that in utero exposure to HF diet has the potential to program the gluconeogenic capacity of offspring through epigenetic modifications, which could potentially lead to excessive glucose production and altered insulin sensitivity in adulthood.

Neuropeptide S (NPS) is a newly identified neuromodulator located in the brainstem and regulates various biological functions by selectively activating the NPS receptors (NPSR). High level expression of NPSR mRNA in the olfactory cortex suggests that NPS-NPSR system might be involved in the regulation of olfactory function. The present study was undertaken to investigate the effects of intracerebroventricular (i.c.v.) injection of NPS or co-injection of NPSR antagonist on the olfactory behaviors, food intake, and c-Fos expression in olfactory cortex in mice. In addition, dual-immunofluorescence was employed to identify NPS-induced Fos immunereactive (-ir) neurons that also bear NPSR. NPS (0.1-1 nmol) i.c.v. injection significantly reduced the latency to find the buried food, and increased olfactory differentiation of different odors and the total sniffing time spent in olfactory habituation/dishabituation tasks. NPS facilitated olfactory ability most at the dose of 0.5 nmol, which could be blocked by co-injection of 40 nmol NPSR antagonist [D-Val(5)]NPS. NPS administration dose-dependently inhibited food intake in fasted mice. Ex-vivo c-Fos and NPSR immunohistochemistry in the olfactory cortex revealed that, as compared with vehicle-treated mice, NPS markedly enhanced c-Fos expression in the anterior olfactory nucleus (AON), piriform cortex (Pir), ventral tenia tecta (VTT), the anterior cortical amygdaloid nucleus (ACo) and lateral entorhinal cortex (LEnt). The percentage of Fos-ir neurons that also express NPSR were 88.5% and 98.1% in the AON and Pir, respectively. The present findings demonstrated that NPS, via selective activation of the neurons bearing NPSR in the olfactory cortex, facilitates olfactory function in mice.