Caspase-1-dependent mechanism mediating the harmful impacts of the quorum-sensing molecule N-(3-oxo-dodecanoyl)-l-homoserine lactone on the intestinal cells.

N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL), a quorum-sensing (QS) molecule produced by Gram-negative bacteria in the gastrointestinal tract, adversly impacts host cells. Our previous study demonstrated that 3-oxo-C12-HSL induced a decrease in cell viability via cell apoptosis and eventually disrupted mucin synthesis from LS174T goblet cells. However, the molecular mechanism underlying cell apoptosis and whether pyroptosis was involved in this process are still unknown. In this study, we emphasized on the caspases signal pathway and sterile inflammation to reveal the harmful effects of 3-oxo-C12-HSL on LS174T goblet cells. Our data showed that 3-oxo-C12-HSL is a major inducer of oxidative stress indicated by a high level of intracellular reactive oxygen species (ROS). However, TQ416, an inhibitor of paraoxonase 2, can effectively block oxidative stress. A higher ROS level is the trigger for activating the caspase-1 and 3 cascade signal pathways. Blockade of ROS synthesis and caspase-1 and 3 cascades can obviously rescue the viability of LS174T cells after 3-oxo-C12-HSL treatment. We also found that paralleled with a higher level of ROS and caspases activation, an abnormal expression of proinflammatory cytokines was induced by 3-oxo-C12-HSL treatment; however, the blockage of TLRs-NF-κB pathway cannot restore cell viability and secretary function. These data collectively indicate that 3-oxo-C12-HSL exposure induces damages to cell viability and secretary function of LS174T goblet cells, which is mediated by oxidative stress, cell apoptosis, and sterile inflammation. Overall, the data in this study will provide a better understanding of the harmful impacts of some QS molecules on host cells and their underlying mechanism.

Corticosterone-Induced Lipogenesis Activation and Lipophagy Inhibition in Chicken Liver Are Alleviated by Maternal Betaine Supplementation.

Background: We have shown previously that in ovo betaine injection can prevent nonalcoholic fatty liver induced by glucocorticoid exposure in chickens; yet it remains unknown whether feeding betaine to laying hens may exert similar effects in their progeny. Objective: In this study, we fed laying hens a betaine-supplemented diet, and the progeny were later exposed chronically to corticosterone (CORT) to test hepatoprotective effects and further elucidate underlying mechanisms. Methods: Rugao yellow-feathered laying hens (n = 120) were fed a basal (control, C) diet or a 0.5% betaine-supplemented (B) diet for 28 d before their eggs were collected for incubation. At 49 d of age, male chickens selected from each group were daily injected subcutaneously with solvent (15% ethanol; vehicle, VEH) or CORT (4.0 mg/kg body mass) for 7 d to establish a fatty liver model. Chickens in the 4 groups (C-VEH, C-CORT, B-VEH, and B-CORT) were killed at day 57. Plasma and hepatic triglyceride (TG) concentrations, as well as the hepatic expression of genes involved in lipogenesis and lipophagy, were determined. Results: CORT induced a 1.6-fold increase in the plasma TG concentration (P < 0.05) and a 1.8-fold increment in the hepatic TG concentration (P < 0.05), associated with activation of lipogenic genes (70-780%). In contrast, lipophagy and mitochondrial ß-oxidation genes were inhibited by 30-60% (P < 0.05) in CORT-treated chickens. These CORT-induced changes were completely normalized by maternal betaine supplementation or were partially normalized to intermediate values that were significantly different from those in the C-VEH and C-CORT groups. These effects were accompanied by modifications in CpG methylation and glucocorticoid receptor binding to the promoters of major lipogenic and lipophagic genes (P < 0.05). Conclusions: These results indicate that maternal betaine supplementation protects male juvenile chickens from CORT-induced TG accumulation in the liver via epigenetic modulation of lipogenic and lipophagic genes.

Prenatal betaine exposure alleviates corticosterone-induced inhibition of CYP27A1 expression in the liver of juvenile chickens associated with its promoter DNA methylation.

Sterol 27-hydroxylase (CYP27A1) plays an important role in cholesterol homeostasis by degrading cholesterol to bile acids. Betaine can alleviate high-fat diet-induced hepatic cholesterol accumulation and maternal betaine treatment programs the hepatic expression of CYP27A1 in offspring. Excessive corticosterone (CORT) exposure causes hepatic cholesterol deposition in chickens, yet it remains unknown whether prenatal betaine modulates CORT-induced cholesterol accumulation in chicken liver later in life and whether it involves epigenetic gene regulation of CYP27A1. In this study, fertilized eggs were injected with saline or betaine at 2.5mg/egg before incubation, and the hatchlings were raised under the same condition till 56days of age followed by 7days of subcutaneous CORT injection. Plasma concentrations of total cholesterol (Tch), HDL- and LDL-cholesterol were significantly increased (P<0.05), after CORT challenge, in both control and betaine groups. However, prenatal betaine exposure prevented CORT-induced increase (P<0.05) in hepatic Tch content. Hepatic expression of cholesterol biosynthesis genes and ACAT1 protein that esterifies cholesterol for storage, were activated in both control and betaine groups upon CORT challenge. However, betaine-treated chickens were protected from CORT-induced repression (P<0.05) in LXR and CYP27A1 expression in the liver. CORT-induced down-regulation of LXR and CYP27A1 coincided with significantly increased (P<0.05) CpG methylation on their promoters, which was significantly ameliorated in betaine-treated chickens. These results suggest that in ovo betaine injection alleviates CORT-induced hepatic cholesterol deposition most probably through epigenetic regulation of CYP27A1 and LXR genes in juvenile chickens.

Maternal low-protein diet affects myostatin signaling and protein synthesis in skeletal muscle of offspring piglets at weaning stage.

PURPOSE: We tested the hypothesis that maternal low-protein (LP) diet during gestation and lactation can program myostatin (MSTN) signaling and protein synthesis in skeletal muscle of offspring at weaning stage (35 days). METHODS: Fourteen Meishan sows were fed either LP or standard-protein diets throughout gestation and lactation, male offspring piglets were killed at weaning stage and longissimus dorsi (LD) muscles were taken. The cross-sectional areas (CSA) of LD muscles were measured by hematoxylin and eosin staining. The levels of free amino acids in plasma were measured by amino acid auto-analyzer. Proteins and mRNA were determined by Western blot and RT-qPCR, respectively. RESULTS: Body weight, LD muscle weight and the myofiber CSA were significantly decreased (P < 0.05) in LP piglets; meanwhile, the concentration of branched-chain amino acids was also significantly decreased (P < 0.001). MSTN protein content tended to be higher (P = 0.098) in LP piglets, while the expression of MSTN receptors, activin type II receptor-beta and transforming growth factor type-beta type I receptor kinase, was significantly up-regulated (P < 0.05). Furthermore, p38 mitogen-activated protein kinase, the downstream signaling factor of MSTN, was also enhanced significantly (P < 0.05). In addition, key factors of translation initiation, phosphorylated eukaryotic initiation factor 4E and the 70 kDa ribosomal protein S6 kinase, were significantly decreased (P < 0.05) in LP piglets. CONCLUSIONS: Our results suggest that maternal LP diet during gestation and lactation affects MSTN signaling and protein synthesis in skeletal muscle of offspring at weaning stage.

Dietary betaine supplementation to gestational sows enhances hippocampal IGF2 expression in newborn piglets with modified DNA methylation of the differentially methylated regions.

PURPOSE: The adequate supply of methyl donors is critical for the normal development of brain. The purpose of the present study was to investigate the effects of maternal betaine supplementation on hippocampal gene expression in neonatal piglets and to explore the possible mechanisms. METHODS: Gestational sows were fed control or betaine-supplemented (3 g/kg) diets throughout the pregnancy. Immediately after birth, male piglets were killed, and the hippocampus was dissected for analyses. The mRNA abundance was determined by reverse transcription real-time polymerase chain reaction. Protein content was measured by Western blot, and DNA methylation was detected by methylated DNA immunoprecipitation assay. RESULTS: Prenatal betaine supplementation did not alter the body weight or the hippocampus weight, but increased the hippocampal DNA content as well as the mRNA expression of proliferation-related genes. Prenatal betaine supplementation increased serum level of methionine (P < 0.05) and up-regulated (P < 0.05) the mRNA and protein expression of betaine-homocysteine methyltransferase, glycine N-methyltransferase and DNA methyltransferase 1 in the neonatal hippocampus. Hippocampal expression of insulin growth factor II (IGF2) and its receptors IGF1R and IGF2R were all significantly up-regulated (P < 0.05) in betaine-treated group, together with a significant activation (P < 0.01) of the downstream extracellular signal-regulated kinase 1/2. Moreover, the differentially methylated region (DMR) 1 and 2 on IGF2 locus was found to be hypermethylated (P < 0.05) in the hippocampus of betaine-treated piglets. CONCLUSIONS: These results indicate that maternal betaine supplementation enhances betaine/methionine metabolism and DNA methyltransferase expression, causes hypermethylation of DMR on IGF2 gene, which was associated with augmented expression of IGF2 and cell proliferation/anti-apoptotic markers in the hippocampus of neonatal piglets.

Maternal low-protein diet induces gender-dependent changes in epigenetic regulation of the glucose-6-phosphatase gene in newborn piglet liver.

Glucose-6-phosphatase (G6PC) plays an important role in glucose homeostasis because it catalyzes the final steps of gluconeogenesis and glycogenolysis. Maternal malnutrition during pregnancy affects G6PC activity, yet it is unknown whether epigenetic regulations of the G6PC gene are also affected. In this study, we fed primiparous, purebred Meishan sows either standard-protein (SP; 12% crude protein) or low-protein (LP; 6% crude protein) diets throughout gestation and analyzed hepatic G6PC expression in both male and female newborn piglets. The epigenetic regulation of G6PC, including DNA methylation, histone modifications, and micro RNA (miRNA), was determined to reveal potential mechanisms. Male, but not female, LP piglets had a significantly lower serum glucose concentration and greater hepatic G6PC mRNA expression and enzyme activity. Also, in LP males, glucocorticoid receptor binding to the G6PC promoter was lower compared with SP males, which was accompanied by hypomethylation of the G6PC promoter. Modifications in histones also were gender dependent; LP males had less histone H3 and histone H3 lysine 9 trimethylation and more histone H3 acetylation and histone H3 lysine 4 trimethylation on the G6PC promoter compared with the SP males, whereas LP females had more H3 and greater H3 methylation compared with their SP counterparts. Moreover, two miRNA, ssc-miR-339-5p and ssc-miR-532-3p, targeting the G6PC 3' untranslated region were significantly upregulated by the LP diet only in females. These results suggest that a maternal LP diet during pregnancy causes hepatic activation of G6PC gene expression in male piglets, which possibly contributes to adult-onset hyperglycemia.

Glucocorticoid receptors involved in melatonin inhibiting cell apoptosis and NLRP3 inflammasome activation caused by bacterial toxin pyocyanin in colon.

The immunoinhibitory effect of glucocorticoid and immunoenhancing attributes of melatonin (MEL) are well known, however, the involvement of glucocorticoid receptor (GR) in melatonin modulation of bacterial toxins caused-inflammation has not been studied in colon. Pyocyanin (PCN), a toxin released by Pseudomonas aeruginosa, can destroy cells through generating superoxide products and inflammatory response. Here we report that PCN treatment elevated the generation of reactive oxygen species (ROS), which further lead to mitochondrial swelling and caspase cascades activation both in vivo and in vitro. However, MEL treatment alleviated the oxidative stress caused by PCN on cells through scavenging ROS and restoring the expression of antioxidant enzyme so that to effectively alleviate the apoptosis. Large amounts of ROS can activate the NLRP3 signaling pathway, so MEL inhibited PCN induced NLRP3 inflammasome activation and inflammatory cytokines (IL-1ß, IL-8, and TNF-α) secretion. In order to further investigate the molecular mechanism, goblet cells were exposed to MEL and PCN in the presence of luzindole and RU486, inhibitors of MEL receptors and GR respectively. It was found that PCN significantly inhibited the expression level of GR, and MEL effectively alleviated the inhibition phenomenon. Moreover, we found that MEL mainly upregulated the expression of GR to achieve its anti-inflammatory and anti-apoptotic functions rather than through its own receptor (MT2) in colon goblet cells. Therefore, MEL can reverse the inhibitory effects of PCN on GR/p-GR expression to present its anti-oxidative and anti-apoptotic function.

Chronic corticosterone exposure induces liver inflammation and fibrosis in association with m6A-linked post-transcriptional suppression of heat shock proteins in chicken.

Our previous study had shown that chronic corticosterone (CORT) exposure causes excessive fat deposition in chicken liver, yet it remains unknown whether it is associated with inflammation and fibrosis. In general, heat shock proteins (HSPs) are activated in response to acute stress to play a cytoprotective role, and this activation is associated with m6A-mediated post-transcriptional regulation. However, changes of HSPs and the m6A methylation on their mRNAs in response to chronic CORT treatment in chicken liver have not been reported. In this study, chronic CORT exposure induced inflammation and fibrosis in chicken liver, associated with significantly modulated expression of HSPs that was significantly upregulated at mRNA level yet downregulated at protein level. Concurrently, m6A methyltransferases METTL3 content was upregulated together with the level of m6A methylation on HSPs transcripts. The m6A-seq analysis revealed 2-6 significantly (P < 0.05) hypermethylated m6A peaks in the mRNA of 4 different species of HSPs in CORT-treated chicken liver. HSP90B1 transcript had 6 differentially methylated m6A peaks among which peaks on exon 16 and exon 17 showed 3.14- and 4.72-fold of increase, respectively. Mutation of the 8 predicted m6A sites on exon 16 and exon 17 resulted in a significant (P < 0.05) increase in eGFP-fused content of HSP90B1 exon 16 and exon 17 fragment in 293 T cells, indicating a possible role of m6A in post-transcriptional regulation of HSPs. In conclusion, chronic CORT exposure induces inflammation and fibrosis in chicken liver along with an increase in the levels and m6A methylation of several HSPs mRNAs; HSPs levels were however reduced under the indicated conditions. Results presented suggest that the reduction in HSPs levels may be associated with m6A methylation in CORT-exposed chickens.

Fetal betaine exposure modulates hypothalamic expression of cholesterol metabolic genes in offspring cockerels with modification of promoter DNA methylation.

In documents, maternal betaine modulates hypothalamic cholesterol metabolism in chicken posthatchings, but it remains unclear whether this effect can be passed on by generations. In present study, eggs were injected with saline or betaine at 2.5 mg/egg, and the hatchlings (F1) were raised under the same condition until sexual maturation. Both the control group and the betaine group used artificial insemination to collect sperm from their cockerels. Fertilized eggs were incubated, and the hatchlings of the following generation (F2) were raised up to 64 D of age. F2 cockerels in betaine group showed significantly (P < 0.05) lower body weight, which was associated with significantly decreased (P < 0.05) hypothalamic content of total cholesterol and cholesterol ester. Concordantly, hypothalamic expression of cholesterol biosynthetic genes, SREBP2 and HMGCR, were significantly downregulated (P < 0.05), together with cholesterol conversion-related and excretion-related genes, CYP46A1 and ABCA1. These changes coincided with a significant downregulation in mRNA expression of regulatory neuropeptides including brain-derived neurotrophic factor, neuropeptide Y, and corticotropin-releasing hormone. Moreover, genes involved in methyl transfer cycle were also modified. Betaine homocysteine methyltransferase (P < 0.05) was downregulated, yet DNA methyltransferase1 tended to be upregulated (P = 0.06). S-adenosyl methionine/S-adenosylhomocysteine ratio was higher in the hypothalamus of betaine-treated F2 cockerels, which was associated with significantly modified CpG methylation on the promoter of those affected genes. These results suggested that betaine might regulate central cholesterol metabolism and hypothalamic expression of genes related to brain function by altering promoter DNA methylation in F2 cockerels.

Maternal betaine supplementation decreases hepatic cholesterol deposition in chicken offspring with epigenetic modulation of SREBP2 and CYP7A1 genes.

Maternal betaine was reported to regulate offspring hepatic cholesterol metabolism in mammals. However, it is unclear whether and how feeding betaine to laying hens affects hepatic cholesterol metabolism in offspring chickens. Rugao yellow-feathered laying hens (n = 120) were fed basal or 0.5% betaine-supplemented diet for 28 D before the eggs were collected for incubation. Maternal betaine significantly decreased the hepatic cholesterol content (P < 0.05) in offspring chickens. Accordingly, the cholesterol biosynthetic enzymes, sterol regulator element-binding protein 2 (SREBP2) and 3-hydroxy-3-methylglutaryl coenzyme A reductase, were decreased, while cholesterol-7alpha-hydroxylase (CYP7A1), which converts cholesterol to bile acids, was increased at both mRNA and protein levels in betaine-treated offspring chickens. Hepatic mRNA and protein expression of low-density lipoprotein receptor was significantly (P < 0.05) increased, while the mRNA abundance of cholesterol acyltransferase 1 (ACAT1) that mediates cholesterol esterification was significantly (P < 0.05) decreased in the betaine group. Meanwhile, hepatic protein contents of DNA methyltransferases 1 and betaine homocysteine methyltransferase were increased (P < 0.05), which was associated with modifications of CpG methylation on affected cholesterol metabolic genes. Furthermore, the level of CpG methylation on gene promoters was increased (P < 0.05) for sterol regulator element-binding protein 2 and abundance of cholesterol acyltransferase 1 yet decreased (P < 0.05) for cholesterol-7alpha-hydroxylase. These results indicate that maternal betaine supplementation significantly decreases hepatic cholesterol deposition through epigenetic regulation of cholesterol metabolic genes in offspring juvenile chickens.

Stress Response Simulated by Continuous Injection of ACTH Attenuates Lipopolysaccharide-Induced Inflammation in Porcine Adrenal Gland.

On modern farms, animals are at high risk of bacterial invasion due to environmental stress factors. The adrenal gland is the terminal organ of the stress response. The crosstalk between adrenal endocrine stress and innate immune response is critical for the maintenance of immune homeostasis during inflammation. Thus, it's important to explore whether stresses play a pivotal role in lipopolysaccharide (LPS)-induced inflammatory response in the porcine adrenal gland. Thirty-days-old Duroc × Landrace × Large White crossbred piglets (12 ± 0.5 kg) were randomly allocated into four groups in a 2 × 2 factorial arrangement of treatments, including ACTH pretreatment (with or without ACTH injection) and LPS challenge (with or without LPS injection). Each group consisted of six male piglets. The results showed that our LPS preparation alone induced mRNA expressions of IL-1ß, IL-6, TNF-α, IL-10, COX-2, TLR2, TLR4, and GR (P < 0.05). ACTH pretreatment downregulated the TLR2 mRNA and IL-6 protein level induced by our LPS preparation significantly (P < 0.05) by one-way ANOVA analysis. Treatment with LPS alone extremely significantly decreased ssc-miR-338 levels (P < 0.01). Interaction of ACTH × LPS was significant for cNOS level (P = 0.011) and ssc-miR-338 expression (P = 0.04) by two-way ANOVA analysis. The LPS treatment significantly downregulated cNOS levels (P < 0.01), which was significantly attenuated by ACTH pretreatment (P < 0.05). Lipopolysaccharide alone did not affect ssc-miR-146b expression levels compared to that in the vehicle group. However, ACTH pretreatment in combination with LPS significantly increased this micro-RNA expression (P < 0.05). TLRs 1-10 were all expressed in adrenal tissue. The LPS challenge alone induced remarkable compensatory mitochondrial damages at the ultrastructural level, which was alleviated by ACTH pretreatment. Accordingly, ACTH pretreatment was able to block LPS-induced secretion of local adrenal cortisol (P < 0.05). Taken together, our results demonstrate that ACTH pretreatment seems to attenuate LPS-induced mitochondria damage and inflammation that decreased cNOS activity in the adrenal gland and ultimately returned local adrenal cortisol to basal levels at 6 h post LPS injection.

White light emitting diode induces autophagy in hippocampal neuron cells through GSK-3-mediated GR and RORα pathways.

Autophagy plays an important role in cell survival under diverse stress conditions. Here, we show that white LED light exposure for 24 h significantly activated autophagy-related genes and increased autophagosome formation in hippocampal neural cells (HT-22). Concurrently, the rhythmic pattern of clock-related gene expression was disrupted, which was associated with augmented expression of SIRT1, AMPK and retinoid-related orphan receptor alpha (RORα). SR1001, a specific inhibitor of RORα, protected the cells from light-induced activation of autophagy. Moreover, light exposure increased glucocorticoid receptor (GR) phosphorylation and nuclear translocation. GR inhibitor RU486 prevented light-induced up-regulation of RORα and the activation of autophagy. These changes were associated with enhanced glycogen synthase kinase-3 (GSK-3) activity and its specific inhibitor CHIR-99021 significantly rescued light-induced autophagy and augmented GR, RORα and autophagy-related proteins. Furthermore, GSK-3 was identified as an upstream regulator of GR/RORα signaling as it was not affected by GR or RORα inhibitors. Taken together, our data demonstrate that GSK-3-mediated GR/RORα signaling pathway is involved in white LED light-induced autophagy in hippocampal neuron cells.

Maternal betaine administration modulates hepatic type 1 iodothyronine deiodinase (Dio1) expression in chicken offspring through epigenetic modifications.

As a feed additive, betaine is widely used in livestock production for its ability to promote growth. Our previous studies had reported that maternal betaine supplementation altered hepatic metabolism in offspring. But it remains unknown whether and how maternal betaine modulates metabolism of thyroid hormones in the offspring chickens by epigenetic modification. In this study, one hundred and twenty Rugao yellow-feathered laying hens were randomly divided into two groups, and were fed basal diet with or without 0.5% betaine supplementation for 28 days. After that, all the hens were artificially inseminated and then four hundreds fertilized eggs were selected. After hatching, the newborn chicks were raised until 56 days old. Betaine fed female chicks showed significantly lower body weight and lower level of biologically active thyroid hormone in plasma compared to control group, which was associated with significantly decrease in expression of type 1 iodothyronine deiodinase (Dio1). Moreover, betaine also changed hepatic expression of betaine-homocysteine -S-methyltransferase (BHMT) and DNA methyltransferase 1 (DNMT1), which may contribute to hypermethylation of the Dio1 promoter. Interestingly, betaine treatments of hens caused none of these effects in male chicks except Dio1 expression. These results indicate that maternal betaine administration effects growth of offspring through differential modification of Dio1 gene methylation and expression in liver and this model of transgenerational effects may help elucidate the mechanisms of maternal effects arise in natural systems.

Spermidine-Activated Satellite Cells Are Associated with Hypoacetylation in ACVR2B and Smad3 Binding to Myogenic Genes in Mice.

Spermidine is an acetyltransferase inhibitor and a specific inducer of autophagy. Recently, spermidine is identified as a potential therapeutic agent for age-related muscle atrophy and inherited myopathies. However, the effect of spermidine on nonpathological skeletal muscle remains unclear. In this study, long-term spermidine administration in mice lowered the mean cross-sectional area of the gastrocnemius muscle and reduced the expression of myosin heavy chain isoforms in the muscle, which was associated with ubiquitination. Moreover, spermidine supplementation induced autophagy in satellite cells and enhanced satellite cell proliferation. ChIP assay revealed that spermidine repressed H3K56ac in the promoter of ACVR2B and lowered the binding affinity of Smad3 to the promoters of Myf5 and MyoD. Altogether, our results indicate that long-term administration of spermidine can activate satellite cells, as well as enhance autophagy, eventually resulting in muscle atrophy. In addition, H3K56ac and Smad3 emerged as key determinants of satellite cell activation.

Sodium Butyrate Ameliorates High-Fat-Diet-Induced Non-alcoholic Fatty Liver Disease through Peroxisome Proliferator-Activated Receptor α-Mediated Activation of ß Oxidation and Suppression of Inflammation.

Peroxisome proliferator-activated receptor α (PPARα) plays a protective role against non-alcoholic fatty liver disease (NAFLD). Sodium butyrate (NaB) has been shown to alleviate NAFLD, yet whether and how PPARα is involved in the action of NaB remains elusive. In this study, NaB administration alleviated high-fat-diet-induced NAFLD in adult rats, with a decrease of hepatic triglyceride content from 108.18 ± 5.77 to 81.34 ± 7.94 µg/mg ( p < 0.05), which was associated with a significant activation of PPARα. Nuclear factor κ-light-chain-enhancer of activated B cell (NF-κB)-mediated nucleotide-binding domain-like receptor protein 3 signaling and pro-inflammatory cytokine release were diminished by NaB treatment. NaB-induced PPARα upregulation coincided with a reduced protein content of histone deacetylase 1 and promoted histone H3 acetyl K9 (H3K9Ac) modification on the promoter of PPARα, whereas NaB-induced suppression of inflammation was linked to significantly increased PPARα binding with p-p65. NaB acts as a histone deacetylase inhibitor to upregulate PPARα expression with enhanced H3K9Ac modification on it promoter. NaB-induced PPARα activation stimulates fatty acid ß oxidation and inhibits NF-κB-mediated inflammation pathways via protein-protein interaction, thus contributing to amelioration of high-fat-diet-induced NAFLD in adult rats.

Dietary betaine supplementation in hens modulates hypothalamic expression of cholesterol metabolic genes in F1 cockerels through modification of DNA methylation.

Betaine is widely used in animal nutrition to promote growth, development and methyl donor during methionine metabolism through nutritional reprogramming via regulation of gene expression. Prenatal betaine exposure is reported to modulate hypothalamic cholesterol metabolism in chickens, yet it remains unknown whether feeding hens with betaine-supplemented diet may affect hypothalamic cholesterol metabolism in F1 offspring. In this study, hens were fed with basal or betaine-supplemented (0.5%) for 30days, and the eggs were collected for incubation. The hatchlings were raised under the same condition up to 56days of age. Betaine-treated group showed significantly (P<0.05) higher plasma concentration of total cholesterol and HDL-cholesterol, together with increased hypothalamic content of total cholesterol and cholesterol ester. Concordantly, hypothalamic gene expression of SREBP2, HMGCR, and LDLR was significantly up regulated (P<0.05). Also, mRNA abundances of SREBP1, ACAT1 and APO-A1 were up-regulated, while that of CYP46A1 was significantly down-regulated (P<0.05). These changes coincided with a significant down-regulation of BDNF and CRH, and a significant up-regulation of NPY mRNA expression. Moreover, genes involved in methyl transfer cycle were also modulated. DNMT1 and BHMT were up-regulated (P<0.05) at both mRNA and protein levels, which was associated with significant modifications of CpG methylation on the promoter of SREBP-1, SREBP-2 and APO-A1 genes as detected by bisulfate sequencing. These results indicate that feeding betaine to hens modulates hypothalamic expression of genes involved in cholesterol metabolism and brain functions in F1 cockerels with modification of promoter DNA methylation.

White light emitting diode suppresses proliferation and induces apoptosis in hippocampal neuron cells through mitochondrial cytochrome c oxydase-mediated IGF-1 and TNF-α pathways.

Light emitting diode (LED) light has been tested to treat traumatic brain injury, neural degenerative diseases and psychiatric disorders. Previous studies indicate that blue LED light affects cell proliferation and apoptosis in photosensitive cells and cancer cells. In this study, we demonstrate that white LED light exposure impaired proliferation and induced apoptosis in HeLa and HT-22 hippocampal neural cells, but not C2C12 cells. Furthermore, the mechanisms underlying the effect of white LED light exposure on HT-22 cells were elucidated. In HeLa and HT-22 cells, white LED light activated mitochondrial cytochrome c oxidase (Cco), in association with enhanced ATP synthase activity and elevated intracellular ATP concentration. Also, reactive oxygen species (ROS) and nitric oxide (NO) production were increased, accompanied by higher calcium concentration and lower mitochondrial membrane potential. HT-22 cells exposed to white LED light for 24h showed reduced viability, with higher apoptotic rate and a cell cycle arrest at G0/G1 phase. Concurrently, the mRNA expression and the concentration of IGF-1 were decreased, while that of TNF-α were increased, in light-exposed cells, which was supported by the luciferase activity of both gene promoters. The down-stream mitogen-activated protein kinase (MAPK), AKT/mTOR pathways were inhibited, in association with an activation of apoptotic caspase 3. N-Acetylcysteine, a ROS scavenger, protected the cells from LED light-induced cellular damage, with rescued cell viability and restored mRNA expression of IGF-1 and TNF-α. Our data demonstrate that white LED light suppresses proliferation and induces apoptosis in hippocampal neuron cells through mitochondrial Cco/ROS-mediated IGF-1 and TNF-α pathways.

Dietary betaine supplementation increases adrenal expression of steroidogenic acute regulatory protein and yolk deposition of corticosterone in laying hens.

Betaine, an important methyl donor, is known to execute epigenetic regulation of gene expression via nutritional reprogramming. Herein, we explore whether feeding a betaine-supplemented diet to laying hens would affect corticosteroid biosynthesis in the adrenal gland and corticosterone deposition in eggs, in correlation with the expression of methyl transfer enzymes and the promoter DNA methylation status of affected genes. Rugao yellow-feathered laying hens at 38 weeks of age were assigned to Control and Betaine groups, fed basal and betaine-supplemented diets, respectively, for four weeks. Betaine supplementation significantly increased (P < 0.05) the average laying rate, while the body weight and egg quality remained unchanged. Plasma concentrations of cholesterol and low-density lipoprotein-cholesterol were also higher (P < 0.05) in the Betaine group. Moreover, eggs in the Betaine group contained higher corticosterone in the yolk, which was associated with up-regulation of steroidogenesis genes in adrenal glands. Steroidogenic acute regulatory protein (StAR), the rate-limiting protein responsible for transporting cholesterol to the inner mitochondrial membrane, was significantly activated (P < 0.05), together with its transcription factors steroidogenic factor-1 (SF-1) and glucocorticoid receptor. Also, betaine supplementation significantly up-regulated (P < 0.05) the adrenal mRNA expression of adenosyl homocysteinase-like 1 and DNA methyltransferases1 and 3a. Bisulfite sequencing analysis revealed significant hypomethylation in several CpG sites within the promoter region of SF-1 gene in the adrenal gland. These results indicate that dietary supplementation of betaine in hens activates adrenal expression of StAR, possibly through epigenetic regulation of SF-1 gene.

Prenatal betaine exposure modulates hypothalamic expression of cholesterol metabolic genes in cockerels through modifications of DNA methylation.

Cholesterol is essential for neuronal development and brain function. Previously we reported that in ovo administration of betaine modulates hepatic cholesterol metabolism in the chicken, yet it remains unknown whether maternal betaine affects the cholesterol content and the expression of cholesterol metabolic genes in chicken hypothalamus. In this study, eggs were injected with saline or betaine at 2.5 mg/egg, and the hatchlings were raised under the same condition until 64 d of age. Maternal betaine significantly (P = 0.05) increased the body weight and suppressed aggressive behavior of 64-day-old cockerels, in association with significantly (P < 0.05) up-regulated expression of 5-HTR1A receptor in the hypothalamus. Concurrently, betaine in ovo significantly increased (P < 0.05) the hypothalamic content of total cholesterol and cholesterol ester, which coincided with significantly up-regulated (P < 0.05) hypothalamic expression of cholesterol biosynthetic genes, such as sterol-regulatory element binding protein 2 and 3-hydroxy-3-methyl-glutaryl-CoA reductase as well as acetyl-CoA cholesterol acyltransferase 1, which converts free cholesterol to cholesterol ester for storage. In contrast, low density lipoprotein receptor, which mediates the cholesterol uptake, was significantly down-regulated (P < 0.05). In ovo betaine administration significantly enhanced the expression of betaine-homocysteine methyltransferase and DNA methyltransferase 1 (P < 0.05), which was associated with alterations of CpG methylation on the promoter of modified cholesterol metabolic genes. These results indicate that maternal betaine modulates hypothalamic cholesterol metabolism in cockerels through modifying DNA methylation on the promoter of cholesterol metabolic genes.

In ovo injection of betaine alleviates corticosterone-induced fatty liver in chickens through epigenetic modifications.

Betaine alleviates high-fat diet-induced fatty liver and prenatal betaine programs offspring hepatic lipid metabolism. Excessive corticosterone (CORT) exposure causes fatty liver in chickens, yet it remains unknown whether and how prenatal betaine modulates the susceptibility of CORT-induced fatty liver later in life. In this study, fertilized eggs were injected with saline or betaine before incubation, and the hatchlings were raised at 8 weeks of age followed by 7 days of subcutaneous CORT injection. CORT-induced fatty liver was less severe in betaine-treated chickens, with significantly reduced oil-red staining and hepatic triglyceride content (P < 0.05). The protective effect of prenatal betaine was associated with significantly up-regulated expression of PPARα and CPT1α, as well as mitochondrial DNA (mtDNA)-encoded genes (P < 0.05). Moreover, betaine rescued CORT-induced alterations in methionine cycle genes, which coincided with modifications of CpG methylation on CPT1α gene promoter and mtDNA D-loop regions. Furthermore, the elevation of hepatic GR protein content after CORT treatment was significantly reduced (P < 0.05), while the reduction of GR binding to the control region of affected genes was significantly increased (P < 0.05), in betaine-treated chickens. These results indicate that in ovo betaine injection protects the juvenile chickens from CORT-induced fatty liver.