Dan Wan

Methionine restriction on lipid metabolism and its possible mechanisms

Methionine restriction (MR) exerts many beneficial effects, such as increasing longevity, decreasing oxidative damage and alleviating inflammatory responses. Much attention has been recently focused on the effects of MR on metabolic health, especially lipid metabolism, since the increasing incidence of obesity, insulin resistance and type 2 diabetes causes a worldwide health problem. In general, MR is considered to increase de novo lipogenesis, lipolysis and fatty acid oxidation, with a result of reduced fat accumulation. However, different responses in lipid metabolism between adipose tissue and liver are declared. Therefore, in this review, we will focus on the changes of lipid metabolism responses to dietary MR. Moreover, the comparison of alterations of fat metabolism responses to dietary MR between adipose tissue and liver, and the comparison of changes between rodents and pigs is made to illustrate the tissue- and species-specific responses. In addition, the possible mechanisms that might be engaged in the regulation of MR diet on lipid metabolism are also discussed.

Toxicological evaluation of ferrous N-carbamylglycinate chelate: Acute, Sub-acute toxicity and mutagenicity.

Iron is an essential trace element that is vital important in various biological process. A deficiency in iron could induce public health problem e.g. anaemia, while an overload could induce ROS production, lipid peroxidation and DNA bases modifications. In the present study, a new iron fortifier was synthesized, and its acute/sub-acute toxicity was investigated. According to the improved Karbers method, the median lethal dose (LD50) of the ferrous N-carbamylglycinate in SD rat was 3.02 g/kg and the 95% confidence intervals were between 2.78 and 3.31 g/kg. No biologically significant or test substance-related differences were observed in body weights, feed consumption, clinical signs, organ weights, histopathology, ophthalmology, hematology, and clinical chemistry parameters in any of the treatment groups of ferrous N-carbamylglycinate at target concentrations corresponding to 150, 300, and 600 mg/kg/day for 28 days. The no observed adverse effect level (NOAEL) for ferrous N-carbamylglycinate was at least 600 mg/kg b.w. day in rats. In addition, no evidence of mutagenicity was found, either in vitro in bacterial reverse mutation assay or in vivo in mice bone marrow micronucleus assay and sperm shape abnormality assay. On the basis of our findings, we conclude that ferrous N-carbamylglycinate is a low-toxic substance with no genotoxicity.

Acute and sub-acute oral toxicological evaluations and mutagenicity of N-carbamylglutamate (NCG).

N-carbamylglutamate (NCG) is a metabolically stable analog of N-acetylglutamate that activates carbamyl phosphate synthase-1, a key arginine synthesis enzyme in enterocytes. It is a promising feed additive in swine in China. In this study, we assessed the acute and sub-acute toxicity of NCG in Sprague-Dawley (SD) rats. All rats survived until they were killed at a scheduled time point. No adverse effects or mortality was observed following acute oral administration of 5000 mg/kg NCG to SD rats. No biologically significant or test substance-related differences were observed in body weights, feed consumption, clinical signs, a functional observational battery, organ weights, histopathology, ophthalmology, hematology, coagulation, and clinical chemistry parameters in any of the treatment groups in sub-acute doses of NCG at target concentrations corresponding to 500, 2000, and 3000 mg/kg/day for 28 days neither. In addition, no evidence of mutagenicity or genotoxicity was found, either in vitro in bacterial reverse mutation assay or in vivo in mice bone marrow micronucleus assay and sperm shape abnormality assay. On the basis of our findings, we conclude that NCG is a non-toxic substance with no genotoxicity.

Diurnal variations in iron concentrations and expression of genes involved in iron absorption and metabolism in pigs

Diurnal variations in serum iron levels have been well documented in clinical studies, and serum iron is an important diagnostic index for iron-deficiency anemia. However, the underlying mechanism of dynamic iron regulation in response to the circadian rhythm is still unclear. In this study, we investigated daily variations in iron status in the plasma and liver of pigs. The transcripts encoding key factors involved in iron uptake and homeostasis were evaluated. The results showed that iron levels in the plasma and liver exhibited diurnal rhythms. Diurnal variations were also observed in transcript levels of divalent metal transporter 1 (DMT1), membrane-associated ferric reductase 1 (DCYTB), and transferrin receptor (TfR) in the duodenum and jejunum, as well as hepcidin (HAMP) and TfR in the liver. Moreover, the results showed a network in which diurnal variations in systemic iron levels were tightly regulated by hepcidin and Tf/TfR via DCYTB and DMT1. These findings provide new insights into circadian iron homeostasis regulation. The diurnal variations in serum iron levels may also have pathophysiological implications for clinical diagnostics related to iron deficiency anemia in pigs.

Serine prevented high-fat diet-induced oxidative stress by activating AMPK and epigenetically modulating the expression of glutathione synthesis-related genes

Serine deficiency has been observed in patients with nonalcoholic fatty liver disease (NAFLD). Whether serine supplementation has any beneficial effects on the prevention of NAFLD remains unknown. The present study was conducted to investigate the effects of serine supplementation on hepatic oxidative stress and steatosis and its related mechanisms. Forty male C57BL/6J mice (9week-old) were randomly assigned into four groups (n=10) and fed: i) a low-fat diet; ii) a low-fat diet supplemented with 1% (wt:vol) serine; iii) a high-fat (HF) diet; and iv) a HF diet supplemented with 1% serine, respectively. Palmitic acid (PA)-treated primary hepatocytes separated from adult mice were also used to study the effects of serine on oxidative stress. The results showed that serine supplementation increased glucose tolerance and insulin sensitivity, and protected mice from hepatic lipid accumulation, but did not significantly decreased HF diet-induced weight gain. In addition, serine supplementation protected glutathione (GSH) antioxidant system and prevented hypermethylation in the promoters of glutathione synthesis-related genes, while decreasing reactive oxygen species (ROS) in mice fed a HF diet. Moreover, we found that serine supplementation increased phosphorylation and S-glutathionylation of AMP-activated protein kinase α subunit (AMPKα), and decreased ROS, malondialdehyde and triglyceride contents in PA-treated primary hepatocytes. However, while AMPK activity or GSH synthesis was inhibited, the abovementioned effects of serine on PA-treated primary hepatocytes were not observed. Our results suggest that serine supplementation could prevent HF diet-induced oxidative stress and steatosis by epigenetically modulating the expression of glutathione synthesis-related genes and through AMPK activation.

Diurnal variations in polyunsaturated fatty acid contents and expression of genes involved in their de novo synthesis in pigs

The daily variations in circulating fatty acid (FA) contents and lipid metabolism have been well documented. However, whether long chain polyunsaturated FA (PUFA) contents and expression of genes involved in their de novo synthesis exhibit daily rhythms are yet unknown. We conducted the present study to investigate the daily variations in PUFA contents in plasma and liver of pigs. Moreover, diurnal expression of genes encode fatty acid desaturases and elongases, which are key enzymes catalyzed de novo synthesis of long chain PUFA, were also explored. The results showed that long chain PUFA contents in plasma and liver both exhibited diurnal rhythms. Diurnal variations were also observed in mRNA expression of FASD1 (Delta 5-desaturase), FASD2 (Delta 6-desaturase), ELOVL5 (fatty acid elongase 5) and ELOVL2 in liver, with an unexpectedly high level at night. Moreover, our results showed a similarity between the diurnal patterns of FASD1, FASD2, ELOVL2, ELOVL5 and Period 2. These results indicated a high activity of the desaturase-elongase pathway at night in pigs. These findings have important physiological and pathophysiological implications, since long chain PUFA are essential for cell function and closely involved in the development of metabolic syndrome.

Effects of dynamic feeding low and high methionine diets on egg quality traits in laying hens

&NA; This study was conducted to investigate effects of dynamic feeding low and high methionine (MET) diets on performance, egg quality traits, and serum metabolites in laying hens. A total of 180 laying hens (Brown Hy‐line, 41 wk old) were allocated into 3 equal groups with 6 replicates each. The average egg production (EP) of each group was around 87% during one‐week statistics before the formal experiment (P = 0.989). The control group (CON) received the control diet (contained 0.30% MET) at both 07:30 h and 15:30 hours. The low‐high group (LH) received a low MET diet (containing 0.27% MET) at 07:30 h and high MET diet (containing 0.33% MET) at 15:30 hours. The high‐low group (HL) received a high MET diet at 07:30 h and low MET diet at 15:30 hours. After 10 wk, blood samples were collected at 4‐hour intervals in a daily cycle initially starting at 07:30 h before feeding. Results showed that the MET intake of a d was not influenced by the treatments (P > 0.05), respectively. The EP of the LH and HL group increased by 2.28 and 2.45% when compared with the CON group (P > 0.05). The hens in the LH group had a lower albumen ratio and thicker eggshell thickness than both CON and HL groups. Egg yolk ratio of the LH group was higher than the HL group (P < 0.05). The serum total cholesterol (TC), total triglyceride (TG), total protein (TP), and calcium (Ca) of the LH group was significantly lower at 07:30 h than the CON and HL groups, and the serum TG of the HL group was lower at 07:30 h than the CON group (P < 0.05). Meanwhile, the LH group also had the lowest value of serum TP at 23:30 h and 03:30 h (P < 0.05). These results demonstrated that dynamic feeding low and high MET diets might alter the circadian variation of serum TC, TG, TP, and Ca, which is consistent with the change of component ratio of egg albumen and yolk and eggshell thickness.

Intestinal microbiota in growing pigs: effects of stocking density

ABSTRACT This study aimed to assess oxidative stress and microbiota in pigs at different stocking densities. Markers for oxidative stress were investigated and gut bacteria identified. The pigs were randomly assigned to high-, medium- or low-density groups. The results showed that the superoxide dismutase (SOD) levels in the high- and low-density groups were lower than in the medium-density group. Glutathione peroxidase (GSH-Px) activity was significantly lower in the low-density group and highest in the medium-density group. We found no discrepancies between estimators of bacterial richness across the three groups. The jejunum and ileum were mainly occupied by Firmicutes and Proteobacteria of the classes Bacilli, Clostridia, Erysipelotrichia and Gammaproteobacteria. The ceacum was mainly occupied by Bacteroidetes and Firmicutes of the classes Bacteroidetes, Clostridia and Negativicutes. The results revealed that gut flora was not affected by stocking density. However, stocking density may influence oxidative stress.

Maternal dietary supplementation with ferrous N-carbamylglycinate chelate affects sow reproductive performance and iron status of neonatal piglets

Iron-deficiency anemia is a public health concern that frequently occurs in pregnant mammals and neonatal offspring. Ferrous N-carbamylglycinate chelate (Fe-CGly) is a newly designed iron fortifier with proven effects in iron-deficient rats and weanling piglets. However, the effects of this new compound on pregnant mammals are unknown. Therefore, this experiment was conducted to evaluate the effects of Fe-CGly on sow reproductive performance and iron status of both sows and neonatal piglets. A total of 40 large-white sows after second parity were randomly assigned to two groups (n=20). They were receiving a diet including 80 mg Fe/kg as FeSO4 or Fe-CGly, respectively, from day 85 of gestation to parturition. The serum (day 110 of pregnancy) and placentas of sows were sampled. Litter size, mean weight of live born piglets, birth (live) litter weight, number of live born piglets, and the number of still-born piglets, mummies, and weak-born piglets were recorded. Once delivered, eight litters were randomly selected from the 20 litters per treatment, and one new-born male piglet (1.503±0.142 kg) from each selected litter was slaughtered within 3 h after birth from the selected litters, without colostrum ingestion. The serum, longissimus muscle, liver and kidneys of the piglets were collected. The iron status of the serum samples and the messenger RNA level of iron-related genes in the placenta, liver and kidney were analyzed. The results showed that litter weight of live born piglets was higher (P=0.030) in the Fe-CGly group (19.86 kg) than in the FeSO4 group (17.34 kg). Fe-CGly significantly increased placental iron concentration (P<0.05) of sows. It also significantly increased iron saturation and reduced the total iron-binding capacity of piglets (P<0.05) at birth. However, the results revealed that supplementation of Fe-CGly in sows reduced liver and kidney iron concentration of neonatal piglets (P<0.05), indicating decreased iron storage. In addition, the concentration of iron in the colostrum was not significantly changed. Therefore, the present results suggested that replacement of maternal FeSO4 supplement with Fe-CGly in the late-gestating period for sows could improve litter birth weight, probably via enhanced iron transportation in the placenta.

Effects of Dietary Serine Supplementation on Intestinal Integrity, Inflammation and Oxidative Status in Early-Weaned Piglets

Background/Aims: Early weaning often causes gut dysfunction. Since serine alleviates oxidative stress and inflammatory response which are accompany with early weaning, we conducted the study to explore whether serine improves intestinal function in early-weaned piglets. Methods: Twenty-eight weaned piglets (aged 21 d) were fed either a basal diet or a basal diet plus 0.2% serine. We determined the effects of dietary serine supplementation on intestinal morphology by hematoxylin and eosin staining, expression of tight junction proteins (TJPs) by immunoblotting and immunofluorescence, expression of inflammatory cytokines and apoptosis markers by RT-qPCR and the level of antioxidant enzymes with ELISA kits in early-weaned piglets. Results: Serine supplementation increased daily body weight gain while decreasing diarrhea incidence. Both the jejunum and ileum of serine-supplemented piglets showed regularly arranged villi and microvilli. Moreover, dietary serine increased TJP expression, and alleviated apoptosis, inflammation, and oxidative stress in the intestine of early-weaned piglets. Conclusion: Our findings suggest that serine has the potential for use as a feed additive to prevent gut dysfunction caused by weaning.