Genome-wide cross-cancer analysis illustrates the critical role of bimodal miRNA in patient survival and drug responses to PI3K inhibitors.

Heterogeneity of cancer means many tumorigenic genes are only aberrantly expressed in a subset of patients and thus follow a bimodal distribution, having two modes of expression within a single population. Traditional statistical techniques that compare sample means between cancer patients and healthy controls fail to detect bimodally expressed genes. We utilize a mixture modeling approach to identify bimodal microRNA (miRNA) across cancers, find consistent sources of heterogeneity, and identify potential oncogenic miRNA that may be used to guide personalized therapies. Pathway analysis was conducted using target genes of the bimodal miRNA to identify potential functional implications in cancer. In vivo overexpression experiments were conducted to elucidate the clinical importance of bimodal miRNA in chemotherapy treatments. In nine types of cancer, tumors consistently displayed greater bimodality than normal tissue. Specifically, in liver and lung cancers, high expression of miR-105 and miR-767 was indicative of poor prognosis. Functional pathway analysis identified target genes of miR-105 and miR-767 enriched in the phosphoinositide-3-kinase (PI3K) pathway, and analysis of over 200 cancer drugs in vitro showed that drugs targeting the same pathway had greater efficacy in cell lines with high miR-105 and miR-767 levels. Overexpression of the two miRNA facilitated response to PI3K inhibitor treatment. We demonstrate that while cancer is marked by considerable genetic heterogeneity, there is between-cancer concordance regarding the particular miRNA that are more variable. Bimodal miRNA are ideal biomarkers that can be used to stratify patients for prognosis and drug response in certain types of cancer.

A maternal low-protein diet during gestation induces hepatic autophagy-related gene expression in a sex-specific manner in Sprague-Dawley rats.

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.

Maternal high-fat diet activates hepatic interleukin-4 in rat male offspring accompanied by increased eosinophil infiltration.

Interleukin-4 (IL-4) is activated as an immune response during infection or tissue injury. Epigenetic programming of maternal high-fat (HF) diet has long-term effects in the offspring. In the present study, we investigated the epigenetic regulation of IL-4 in a maternal HF diet model in the liver of adult offspring. Timed-pregnant Sprague-Dawley rats were fed either control (C) or HF diet throughout gestation and lactation. Offspring were placed on a control diet after weaning, generating C/C and HF/C groups. The liver was collected at 12 wk of age, followed by histological and molecular analysis to investigate the maternal programming effects on IL-4 by HF diet. Maternal HF diet significantly induced mRNA expression and protein level of IL-4 and promoted hypomethylation of Il4 compared with the control group. Methylation-selective PCR (MSP) confirmed that maternal HF diet increased RNA polymerase II, acetylation of histone H4, and dimethylation of histone 3 lysine 4 at the +6 kb region of Il4. Moreover, the rat eosinophil marker Siglec-F was increased and colocalized with IL-4 in the liver. In conclusion, our study indicated that IL-4 was increased in liver cells in response to maternal HF diet. This coincides with DNA hypomethylation in combination with chromatin remodeling at the +6 kb region of the 3' downstream region as well as an induced immune cell infiltration, especially eosinophil infiltration, in the liver of offspring.NEW & NOTEWORTHY The present study identifies that maternal high-fat-diet-induced IL-4 upregulation is associated with DNA hypomethylation at the +6 kb region of the 3' downstream region of the gene. Furthermore, our results confirm that the induced Il4 expression in the liver of male offspring corresponds to the induced immune cell, especially eosinophil, infiltration.

A Low Protein Diet during Gestation and Lactation Increases Hepatic Lipid Accumulation through Autophagy and Histone Deacetylase.

The present study examined the mechanism of a low protein (LP) diet on hepatic lipid metabolism during gestation and lactation. Timed-pregnant Sprague-Dawley rats were fed a control or an LP diet during gestation and lactation. LP dams had increased hepatic triglyceride accumulation and significantly higher aspartate/alanine transaminase ratio, accompanied by a decrease in circulating very low-density/low-density lipoprotein ratio. LC3B (Microtubule Associated Protein 1 Light Chain 3 Beta) expression was stimulated in LP dams along with increased histone acetylation. LP diet-induced co-localization of the LC3 binding motif-interacting proteins APOB or MTTP with LC3B, suggesting autophagic degradation. HDAC3 is found necessary to prevent lipid accumulation in response to amino acid deprivation in HepG2 cells. LC3B-mediated APOB protein degradation is related to increases in lipid accumulation. Conclusion: HDAC3 regulated LC3B-induced lipid accumulation potentially through autophagic degradation of APOB and MTTP in response to amino acid limitation caused by a low protein diet.

Role of glucocorticoid signaling in exercise-associated changes in high-fat diet preference in rats.

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.

Effects of obesity and exercise on colon cancer induction and hematopoiesis in mice.

Obesity-induced inflammation is associated with increased risk for colorectal cancer (CRC). The role of diet and exercise in modulating increased CRC risk in obesity and the potential role of altered hematopoiesis as a contributor to these effects remain unknown. The purpose of this study was to examine how weight loss induced during CRC induction with or without exercise alters CRC initiation and its relationship to altered hematopoiesis. Mice consumed either a control (CON) or a high-fat diet to induce obesity. All mice were then placed on the control diet during CRC induction with azoxymethane (AOM). Following AOM injection, mice originally on the high-fat diet were randomized into sedentary (HF-SED) or exercise trained (HF-EX) conditions. At euthanasia, body weight and fat mass were similar among all three groups ( P < 0.05). Compared with CON and HF-EX, HF-SED developed increased content of preneoplastic lesions ( P < 0.05), and HF-SED had significantly increased markers of colon inflammation compared with CON. Compared with both CON and HF-EX, HF-SED had decreased content of short-term hematopoietic stem cells and increased content of common myeloid progenitor cells (both P < 0.05). Similarly, HF-SED had increased bone marrow adiposity compared with CON and HF-EX ( P < 0.05), and proteomics analysis revealed an increased marker of bone marrow inflammation in HF-SED compared with CON and HF-EX. Our results suggest that the early removal of a high-fat diet reduces CRC incidence when combined with an exercise training intervention. This reduction in risk was related to lower colon inflammation with anti-inflammatory changes in hematopoiesis induced by exercise.

Perinatal High-Fat Diet and Bisphenol A: Effects on Behavior and Gene Expression in the Medial Prefrontal Cortex.

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.

Methionine Supply During Late-Gestation Triggers Offspring Sex-Specific Divergent Changes in Metabolic and Epigenetic Signatures in Bovine Placenta.

Background: Nonruminant male and female offspring respond differently to gestational nutrition, with placenta contributing to the underlying mechanisms. However, similar data are lacking in large ruminants. Objectives: The aim of this study was to investigate the impact of methionine supply during late-gestation on metabolism and DNA methylation in placenta from cows carrying male or female calves. Methods: During the last 28 d of pregnancy, cows were individually fed a control diet (CON) or the control diet plus rumen-protected d,l-methionine (MET; 0.9 g/kg dry matter intake). Placentomes collected at term were classified according to cow dietary treatment and offspring sex as follows: Male CON (n = 7), Male MET (n = 7), Female CON (n = 8), and Female MET (n = 8). Calf growth was measured until 9 wk of age. Targeted metabolomics, RT-PCR, global DNA methylation, and activity of selected enzymes in one-carbon metabolism and transsulfuration pathways were performed. Statistics were conducted via ANOVA using MIXED models. Results: At birth, Male MET calves were heavier than Male CON calves (7.6%, P = 0.02), but body mass was similar at 9 wk of age. In contrast, compared with Female CON, Female MET calves had greater body mass at 9 wk of age (6.3%, P = 0.03). Compared with Male CON, placenta from Male MET calves had greater concentrations of tricarboxylic acid (TCA) cycle and transsulfuration intermediates (23-100%, P < 0.05), along with greater 5-methyltetrahydrofolatehomocysteine methyltransferase activity (67%, P = 0.03). Compared with Female CON, placenta from Female MET calves had greater concentrations of one-carbon metabolism intermediates (13-52%, P < 0.05). DNA methyltransferase 3A (DNMT3A) was upregulated (43%, P < 0.01) in placenta from Female MET compared with Female CON calves. Global DNA methylation was lower in placenta from Female MET compared with Female CON calves (45%, P = 0.06). Conclusions: Methionine supply affects placental metabolism, DNA methylation, and body mass of the calf in a sex-specific manner, underscoring its importance as dietary methyl-donor for pregnant cows.

Computational methods to identify bimodal gene expression and facilitate personalized treatment in cancer patients.

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.

Early-life Programming of Type 2 Diabetes Mellitus: Understanding the Association between Epigenetics/Genetics and Environmental Factors.

Type 2 Diabetes Mellitus is an increasing public health problem that poses a severe social and economic burden affecting both developed and developing countries. Defects in insulin signaling itself are among the earliest indications that an individual is predisposed to the development of insulin resistance and subsequently Type 2 Diabetes Mellitus. To date, however, the underlying molecular mechanisms which result in resistance to the actions of insulin are poorly understood. Furthermore, it has been shown that maternal obesity is associated with an increased risk of obesity and insulin resistance in the offspring. However, the genetic and/or epigenetic modifications within insulin-sensitive tissues such as the liver and skeletal muscle, which contribute to the insulin-resistant phenotype, still remain unknown. More importantly, a lack of in-depth understanding of how the early life environment can have long-lasting effects on health and increased risk of Type 2 Diabetes Mellitus in adulthood poses a major limitation to such efforts. The focus of the current review is thus to discuss recent experimental and human evidence of an epigenetic component associated with components of nutritional programming of Type 2 Diabetes Mellitus, including altered feeding behavior, adipose tissue, and pancreatic beta-cell dysfunction, and transgenerational risk transmission.

Hepatic betaine-homocysteine methyltransferase and methionine synthase activity and intermediates of the methionine cycle are altered by choline supply during negative energy balance in Holstein cows.

Although choline requirements are unknown, enhanced postruminal supply may decrease liver triacylglycerol (TAG) storage and increase flux through the methionine cycle, helping cows during a negative energy balance (NEB). The objective was to investigate effects of postruminal choline supply during NEB on hepatic activity of betaine-homocysteine methyltransferase (BHMT), methionine synthase (MTR), methionine adenosyltransferase, transcription of enzymes, and metabolite concentrations in the methionine cycle. Ten primiparous rumen-cannulated Holstein cows (158 ± 24 d postpartum) were used in a replicated 5 × 5 Latin square design with 4-d treatment periods and 10 d of recovery (14 d/period). Treatments were unrestricted intake with abomasal infusion of water (A0), restricted intake (R; 60% of net energy for lactation requirements to induce NEB) with abomasal infusion of water (R0) or R plus abomasal infusion of 6.25, 12.5, or 25 g/d of choline ion. Liver tissue was collected on d 5 after the infusions ended, blood on d 1 to 5, and milk on d 1 to 4. Statistical contrasts were A0 versus R0 (CONT1) and tests of linear (L), quadratic (Q), and cubic (C) effects of choline dose. Plasma choline increased with R (CONT1) and choline (L). Although R decreased milk yield (CONT1), choline increased milk yield and liver phosphatidylcholine (PC), but decreased TAG (L). No differences were observed in plasma PC or very-low-density lipoprotein concentrations with R or choline. Activity and mRNA abundance of BHMT were greater with R (CONT1) and increased with choline (L). Although activity of MTR was lower with R (CONT1), it tended to increase with choline (L). No effect of R was detected for activity of methionine adenosyltransferase, but it changed cubically across dose of choline. Those responses were associated with linear increases in the concentrations of liver tissue (+13%) and plasma methionine concentrations. The mRNA abundance of CPT1A, SLC22A5, APOA5, and APOB, genes associated with fatty acid oxidation and lipoprotein metabolism, was upregulated by choline (Q). Overall, enhanced supply of choline during NEB increases hepatic activity of BHMT and MTR to regenerate methionine and PC, partly to help clear TAG. The relevance of these effects during the periparturient period merits further research.

Maternal and Post-weaning High-Fat Diets Produce Distinct DNA Methylation Patterns in Hepatic Metabolic Pathways within Specific Genomic Contexts.

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-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.

Molecular Mechanisms Underlying the Link between Diet and DNA Methylation.

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.

Placentome Nutrient Transporters and Mammalian Target of Rapamycin Signaling Proteins Are Altered by the Methionine Supply during Late Gestation in Dairy Cows and Are Associated with Newborn Birth Weight.

Background: To our knowledge, most research demonstrating a link between maternal nutrition and both fetal growth and offspring development after birth has been performed with nonruminants. Whether such relationships exist in large ruminants is largely unknown.Objective: We aimed to investigate whether increasing the methionine supply during late pregnancy would alter uteroplacental tissue nutrient transporters and mammalian target of rapamycin (mTOR) and their relation with newborn body weight.Methods: Multiparous Holstein cows were used in a randomized complete block design experiment. During the last 28 d of pregnancy, cows were fed a control diet or the control diet plus ethylcellulose rumen-protected methionine (0.9 g/kg dry matter intake) (Mepron; Evonik Nutrition & Care GmbH) to achieve a 2.8:1 ratio of lysine to methionine in the metabolizable protein reaching the small intestine. We collected placentome samples at parturition and used them to assess mRNA and protein expression and the phosphorylation status of mTOR pathway proteins.Results: Newborn body weight was greater in the methionine group than in the control group (44.1 kg and 41.8 kg, respectively; P ≤ 0.05). Increasing the methionine supply also resulted in greater feed intake (15.8 kg/d and 14.6 kg/d), plasma methionine (11.9 µM and 15.3 µM), and plasma insulin (1.16 µg/L and 0.81 µg/L) in cows during late pregnancy. As a result, mRNA expression of genes involved in neutral amino acid transport [solute carrier (SLC) family members SLC3A2, SLC7A5, SLC38A1, and SLC38A10], glucose transport [SLC2A1, SLC2A3, and SLC2A4], and the mTOR pathway [mechanistic target of rapamycin and ribosomal protein S6 kinase B1] were upregulated (P ≤ 0.07) in methionine-supplemented cows. Among 6 proteins in the mTOR pathway, increasing the methionine supply led to greater (P ≤ 0.09) protein expression of α serine-threonine kinase (AKT), phosphorylated (p)-AKT, p-eukaryotic elongation factor 2, and the p-mTOR:mTOR ratio.Conclusion: Supplemental methionine during late gestation increases feed intake and newborn body weight in dairy cows, and this effect may be mediated by alterations in the uteroplacental transport of nondispensable and dispensable amino acids and glucose at least in part through changes in gene transcription and mTOR signaling.

Compensatory induction of Fads1 gene expression in heterozygous Fads2-null mice and by diet with a high n-6/n-3 PUFA ratio.

In mammals, because they share a single synthetic pathway, n-6/n-3 ratios of dietary PUFAs impact tissue arachidonic acid (ARA) and DHA content. Likewise, SNPs in the human fatty acid desaturase (FADS) gene cluster impact tissue ARA and DHA. Here we tested the feasibility of using heterozygous Fads2-null-mice (HET) as an animal model of human FADS polymorphisms. WT and HET mice were fed diets with linoleate/α-linolenate ratios of 1:1, 7:1, and 44:1 at 7% of diet. In WT liver, ARA and DHA in phospholipids varied >2× among dietary groups, reflecting precursor ratios. Unexpectedly, ARA content was only <10% lower in HET than in WT livers, when fed the 44:1 diet, likely due to increased Fads1 mRNA in response to reduced Fads2 mRNA in HET. Consistent with the RNA data, C20:3n-6, which is elevated in minor FADS haplotypes in humans, was lower in HET than WT. Diet and genotype had little effect on brain PUFAs even though brain Fads2 mRNA was low in HET. No differences in cytokine mRNA were found among groups under unstimulated conditions. In conclusion, differential PUFA profiles between HET mice and human FADS SNPs suggest low expression of both FADS1 and 2 genes in human minor haplotypes.

Genistein exposure inhibits growth and alters steroidogenesis in adult mouse antral follicles.

Genistein is a naturally occurring isoflavone phytoestrogen commonly found in plant products such as soybeans, lentils, and chickpeas. Genistein, like other phytoestrogens, has the potential to mimic, enhance, or impair the estradiol biosynthesis pathway, thereby potentially altering ovarian follicle growth. Previous studies have inconsistently indicated that genistein exposure may alter granulosa cell proliferation and hormone production, but no studies have examined the effects of genistein on intact antral follicles. Thus, this study was designed to test the hypothesis that genistein exposure inhibits follicle growth and steroidogenesis in intact antral follicles. To test this hypothesis, antral follicles isolated from CD-1 mice were cultured with vehicle (dimethyl sulfoxide; DMSO) or genistein (6.0 and 36µM) for 18-96h. Every 24h, follicle diameters were measured to assess growth. At the end of each culture period, the media were pooled to measure hormone levels, and the cultured follicles were collected to measure expression of cell cycle regulators and steroidogenic enzymes. The results indicate that genistein (36µM) inhibits growth of mouse antral follicles. Additionally, genistein (6.0 and 36µM) increases progesterone, testosterone, and dehydroepiandrosterone (DHEA) levels, but decreases estrone and estradiol levels. The results also indicate that genistein alters the expression of steroidogenic enzymes at 24, 72 and 96h, and the expression of cell cycle regulators at 18h. These data indicate that genistein exposure inhibits antral follicle growth by inhibiting the cell cycle, alters sex steroid hormone levels, and dysregulates steroidogenic enzymes in cultured mouse antral follicles.

High-fat diet caused widespread epigenomic differences on hepatic methylome in rat.

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.

Developmental bisphenol A (BPA) exposure leads to sex-specific modification of hepatic gene expression and epigenome at birth that may exacerbate high-fat diet-induced hepatic steatosis.

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 ß-oxidation-related genes (Dgat, Agpat6, Cebpα, Cebpß, 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/EBPß, SREBP1) within the male Cpt1a gene, the key ß-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 ß-oxidation capacity in males, potentially through the epigenetic regulation of genes, and further alters the response to a HF diet.

Induction of autophagy through the activating transcription factor 4 (ATF4)-dependent amino acid response pathway in maternal skeletal muscle may function as the molecular memory in response to gestational protein restriction to alert offspring to maternal nutrition.

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.

Single-Cell RNA Sequencing (scRNA-seq) Identifies L1CAM as a Key Mediator between Epithelial Tuft Cell and Innate Lymphoid Cell in the Colon of Hnrnp I Knockout Mice.

(1) Background: Knockout (KO) of heterogeneous nuclear ribonucleoprotein I (Hnrnp I) in mouse intestinal epithelial cells (IECs) induced a severe inflammatory response in the colon, followed by hyperproliferation. This study aimed to investigate the epithelial lineage dynamics and cell-cell communications that underlie inflammation and colitis. (2) Methods: Single cells were isolated from the colons of wildtype (WT) and KO mice and used in scRNA-seq. Whole colons were collected for immunofluorescence staining and cytokine assays. (3) Results: from scRNA-seq, the number of DCLK1 + colonic tuft cells was significantly higher in the Hnrnp I KO mice compared to the WT mice. This was confirmed by immunofluorescent staining of DCLK1. The DCLK1 + colonic tuft cells in KO mice developed unique communications with lymphocytes via interactions between surface L1 cell adhesion molecule (L1CAM) and integrins. In the KO mice colons, a significantly elevated level of inflammatory cytokines IL4, IL6, and IL13 were observed, which marks type-2 immune responses directed by group 2 innate lymphoid cells (ILC2s). (4) Conclusions: This study demonstrates one critical cellular function of colonic tuft cells, which facilitates type-2 immune responses by communicating with ILC2s via the L1CAM-integrins interaction. This communication promotes pro-inflammatory signaling pathways in ILC2, leading to the increased secretion of inflammatory cytokines.