Zinc lactate alleviates oxidative stress by modulating crosstalk between constitutive androstane receptor signaling pathway and gut microbiota profile in weaned piglets.

This study aimed to determine the regulatory mechanism of dietary zinc lactate (ZL) supplementation on intestinal oxidative stress damage in a paraquat (PQ)-induced piglet model. Twenty-eight piglets (mean body weight 9.51 ± 0.23 kg) weaned at 28 d of age were randomly divided into control, ZL, PQ, and ZL + PQ groups (n = 7 in each group). The ZL-supplemented diet had little effect on growth performance under normal physiological conditions. However, under PQ challenge, ZL supplementation significantly improved average daily gain (P < 0.05) and reduced the frequency of diarrhea. ZL improved intestinal morphology and ultrastructure by significantly increasing the expression level of the jejunal tight junction protein, zonula occludens-1 (ZO-1) (P < 0.05), and intestinal zinc transport and absorption in PQ-induced piglets, which reduced intestinal permeability. ZL supplementation also enhanced the expression of antioxidant and anti-inflammatory factor-related genes and decreased inflammatory cytokine expression and secretion in PQ-induced piglets. Furthermore, ZL treatment significantly inhibited the activation of constitutive androstane receptor (CAR) signaling (P < 0.01) in PQ-induced piglets and altered the structure of the gut microbiota, especially by significantly increasing the abundance of beneficial gut microbes, including UCG_002, Ruminococcus, Rikenellaceae_RC9_gut_group, Christensenellaceae_R_7_group, Treponema, unclassified_Christensenellaceae, and unclassified_Erysipelotrichaceae (P < 0.05). These data reveal that pre-administration of ZL to piglets can suppress intestinal oxidative stress by improving antioxidant and anti-inflammatory capacity and regulating the crosstalk between CAR signaling and gut microbiota.

Crosstalk between trace elements and T-cell immunity during early-life health in pigs.

With gradual ban on the use of antibiotics, the deficiency and excessive use of trace elements in intestinal health is gaining attention. In mammals, trace elements are essential for the development of the immune system, specifically T-cell proliferation, and differentiation. However, there remain significant gaps in our understanding of the effects of certain trace elements on T-cell immune phenotypes and functions in pigs. In this review, we summarize the specificity, development, subpopulations, and responses to pathogens of porcine T cells and the effects of functional trace elements (e.g., iron, copper, zinc, and selenium) on intestinal T-cell immunity during early-life health in pigs. Furthermore, we discuss the current trends of research on the crosstalk mechanisms between trace elements and T-cell immunity. The present review expands our knowledge of the association between trace elements and T-cell immunity and provides an opportunity to utilize the metabolism of trace elements as a target to treat various diseases.

Serine Administration Improves Selenium Status, Oxidative Stress, and Mitochondrial Function in Longissimus Dorsi Muscle of Piglets with Intrauterine Growth Retardation.

Intrauterine growth retardation (IUGR) causes oxidative stress in the skeletal muscle. Serine and selenoproteins are involved in anti-oxidative processes; however, whether IUGR affects selenium status and whether serine has beneficial effects remain elusive. Here, we investigated the effects of serine administration on selenium nutritional status and oxidative stress in the longissimus dorsi muscle of piglets with IUGR. Six newborn Min piglets having normal birth weight were administered saline, and 12 IUGR piglets were either administered saline or 0.8% serine. The results showed a lower selenium content in skeletal muscle in IUGR piglets, which was restored after serine administration. IUGR piglets showed a disturbed expression of genes encoding selenoproteins, with decreased expression of GPX2, GPX4, TXNRD1, and TXNRD3 and increased expression of DIO1, DIO2, SELF, SELM, SELP, and SELW. Notably, serine administration restored the expression levels of these genes. In accordance with the changes in gene expression, the activity of GPX, TXNRD, and DIO and the content of GSH and SELP were also altered, whereas serine administration restored their contents and activities. Moreover, we observed severe oxidative stress in the skeletal muscle of IUGR piglets, as indicated by decreased GSH content and increased MDA and PC content, whereas serine administration alleviated these changes. In conclusion, our results indicate that IUGR piglets showed a disturbed expression of genes encoding selenoproteins, accompanied by severe oxidative stress. Serine administration can improve selenium status, oxidative stress, and mitochondrial function in the longissimus dorsi muscle of piglets with IUGR. These results suggest that serine could potentially be used in the treatment of IUGR in piglets.

Improvement of Ulcerative Colitis by Aspartate via RIPK Pathway Modulation and Gut Microbiota Composition in Mice.

The intestine requires a great deal of energy to maintain its health and function; thus, energy deficits in the intestinal mucosa may lead to intestinal damage. Aspartate (Asp) is an essential energy source in the intestinal mucosa and plays a vital part in gut health. In the current study, we hypothesized that dietary supplementation of Asp could alleviate DSS-induced colitis via improvement in the colonic morphology, oxidative stress, cell apoptosis, and microbiota composition in a mouse model of dextran. Asp administration decreased the disease activity index, apoptosis, myeloperoxidase, eosinophil peroxidase, and proinflammatory cytokine (IL-1ß and TNF-α) concentrations in the colonic tissue, but improved the body weight, average daily food intake, colonic morphology, and antioxidant-related gene (GPX1 and GPX4) expression in DSS-treated mice. Expression levels of RIPK1 and RIPK3 were increased in the colon following Asp administration in the DSS-induced mice, whereas the MLKL protein expression was decreased. 16S rRNA sequencing showed that Asp treatment increased the abundance of Lactobacillus and Alistipes at the gene level, and Bacteroidetes at the phylum level, but decreased the abundance of Actinobacteria and Verrucomicrobia at the phylum level. Asp may positively regulate the recovery of DSS-induced damage by improving the immunity and antioxidative capacity, regulating RIPK signaling and modulating the gut microbiota composition.

Glutathione Protects against Paraquat-Induced Oxidative Stress by Regulating Intestinal Barrier, Antioxidant Capacity, and CAR Signaling Pathway in Weaned Piglets.

Endogenous glutathione (GSH) effectively regulates redox homeostasis in the body. This study aimed to investigate the regulatory mechanism of different dietary levels of GSH supplementation on the intestinal barrier and antioxidant function in a paraquat-induced stress-weaned piglet model. Our results showed that dietary 0.06% GSH supplementation improved the growth performance of weaned piglets under normal and stressful conditions to some degree and decreased the diarrhea rate throughout. Exogenous GSH improved paraquat-induced changes in intestinal morphology, organelle, and permeability and reduced intestinal epithelial cell apoptosis. Moreover, GSH treatment alleviated intestinal oxidative stress damage by upregulating antioxidant (GPX4, CnZnSOD, GCLC, and GCLM) and anti-inflammatory (IL-10) gene expression and downregulating inflammatory cytokines (IFN-γ and IL-12) gene expression. Furthermore, GSH significantly reduced the expression levels of constitutive androstane receptor (CAR), RXRα, HSP90, PP2Ac, CYP2B22, and CYP3A29, and increased the expression levels of GSTA1 and GSTA2 in the jejunum and ileum of paraquat-induced piglets. We conclude that exogenous GSH protects against oxidative stress damage by regulating the intestinal barrier, antioxidant capacity, and CAR signaling pathway.

Serine Supplementation in the Diets of Late Gestating and Lactating Sows Improves Selenium Nutritional Status in Sows and Their Offspring.

Serine can regulate selenoprotein expression, and dietary serine is correlated with the contents of plasma selenoprotein P (Sepp1) and milk selenium (Se) in lactating mothers. Based on this, we investigated the effects of serine supplementation in the diets of late gestating and lactating sows on Sepp1 and Se contents in sows and their offspring. A total of 72 sows were assigned to four groups. During the experiment, sows were fed either a basal diet or basal diets supplemented with three different levels of serine. The results showed that maternal dietary serine had no effect on the Se content in the serum of sows and their offspring, whereas it significantly increased the Se content in the liver of piglets at the age of 21 days. Maternal dietary serine significantly increased Sepp1 content, either in the serum of sows or that in their offspring at the ages of 3 days, 7 days, and 21 days. Additionally, maternal dietary serine significantly increased litter weight and the average body weight of piglets at the age of 11 days. Notably, a positive correlation was found between the average body weight of piglets at the age of 11 days and serum Sepp1 content in piglets, at the age of either 3 days or 7 days. In conclusion, maternal dietary serine supplementation could improve Se nutritional status in sows and their offspring. These beneficial changes may contribute to the higher body weight of the offspring.

Aspartate Metabolism Facilitates IL-1ß Production in Inflammatory Macrophages.

Increasing evidence support that cellular amino acid metabolism shapes the fate of immune cells; however, whether aspartate metabolism dictates macrophage function is still enigmatic. Here, we found that the metabolites in aspartate metabolism are depleted in lipopolysaccharide (LPS) plus interferon gamma (IFN-γ)-stimulated macrophages. Aspartate promotes interleukin-1ß (IL-1ß) secretion in M1 macrophages. Mechanistically, aspartate boosts the activation of hypoxia-inducible factor-1α (HIF-1α) and inflammasome and increases the levels of metabolites in aspartate metabolism, such as asparagine. Interestingly, asparagine also accelerates the activation of cellular signaling pathways and promotes the production of inflammatory cytokines from macrophages. Moreover, aspartate supplementation augments the macrophage-mediated inflammatory responses in mice and piglets. These results uncover a previously uncharacterized role for aspartate metabolism in directing M1 macrophage polarization.

Dietary Serine Supplementation Regulates Selenoprotein Transcription and Selenoenzyme Activity in Pigs.

The synthesis of selenocysteine and its incorporation into selenoproteins require serine during the action of seryl-tRNA synthetase. In view of this, we conducted this study to explore the effects of dietary serine supplementation on selenoprotein transcription and selenoenzyme activity in pigs. A total of 35 crossbred barrows (28 days old) were randomly assigned to five treatment groups. During the 42-day growth experiment, pigs were fed either a basal diet with no supplemented serine or diets supplemented with 0.25%, 0.5%, 0.75%, or 1% serine. The results showed that serine supplementation had no effect on the selenium content in the serum, skeletal muscle, and kidney of pigs. However, dietary supplementation with 0.5% serine significantly increased the selenium content in the liver. Diets supplemented with different levels of serine significantly increased the gene expression of glutathione peroxidase 1 (Gpx1), Gpx2, thioredoxin reductase 1 (Txnrd1), Txnrd2, and selenoprotein P (Sepp1) in the skeletal muscle and liver of pigs. Moreover, pigs supplemented with 0.5% serine had the highest selenoprotein P concentration and glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) activities in the skeletal muscle, which were significantly higher than those in the control pigs. Additionally, pigs supplemented with 0.25% serine had the highest GPx and TrxR activities in the liver, which were significantly higher than those in the control pigs. In conclusion, dietary serine supplementation could improve selenoprotein transcription and selenoenzyme activity in pigs, with the appropriate concentrations of serine to be included in the diet being 0.25% or 0.5%.

The Associated Regulatory Mechanisms of Zinc Lactate in Redox Balance and Mitochondrial Function of Intestinal Porcine Epithelial Cells.

Zinc lactate (ZnLA) is a new organic zinc salt which has antioxidant properties in mammals and can improve intestinal function. This study explored the effects of ZnLA and ZnSO4 on cell proliferation, Zn transport, antioxidant capacity, mitochondrial function, and their underlying molecular mechanisms in intestinal porcine epithelial cells (IPEC-J2). The results showed that addition of ZnLA promoted cell proliferation, inhibited cell apoptosis and IL-6 secretion, and upregulated the mRNA expression and concentration of MT-2B, ZNT-1, and CRIP, as well as affected the gene expression and activity of oxidation or antioxidant enzymes (e.g., CuZnSOD, CAT, and Gpx1, GSH-PX, LDH, and MDA), compared to ZnSO4 or control. Compared with the control, ZnLA treatment had no significant effect on mitochondrial membrane potential, whereas it markedly increased the mitochondrial basal OCR, nonmitochondrial respiratory capacity, and mitochondrial proton leakage and reduced spare respiratory capacity and mitochondrial reactive oxygen (ROS) production in IPEC-J2 cells. Furthermore, ZnLA treatment increased the protein expression of Nrf2 and phosphorylated AMPK, but reduced Keap1 and p62 protein expression and autophagy-related genes LC3B-1 and Beclin mRNA abundance. Under H2O2-induced oxidative stress conditions, ZnLA supplementation markedly reduced cell apoptosis and mitochondrial ROS levels in IPEC-J2 cells. Moreover, ZnLA administration increased the protein expression of Nrf2 and decreased the protein expression of caspase-3, Keap1, and p62 in H2O2-induced IPEC-J2 cells. In addition, when the activity of AMPK was inhibited by Compound C, ZnLA supplementation did not increase the protein expression of nuclear Nrf2, but when Compound C was removed, the activities of AMPK and Nfr2 were both increased by ZnLA treatment. Our results indicated that ZnLA could improve the antioxidant capacity and mitochondrial function in IPEC-J2 cells by activating the AMPK-Nrf2-p62 pathway under normal or oxidative stress conditions. Our novel finding also suggested that ZnLA, as a new feed additive for piglets, has the potential to be an alternative for ZnSO4.

Maternal serine supply from late pregnancy to lactation improves offspring performance through modulation of metabolic pathways.

Maternal dietary serine affects free amino acid content in milk and the antioxidant ability of progeny. However, whether maternal dietary serine has any effects on offspring performance in pigs and related metabolic consequences remains unknown. This study was conducted to investigate the effects of different levels of maternal dietary serine from late pregnancy to lactation on sow reproductive performance and offspring performance, and on the metabolome of milk and the serum of sows and their offspring. The results showed that sows fed a diet supplemented with 25% serine of the basal diet (l-Ser) had a higher litter weight, and higher average piglet weight at birth and aged 21 days when compared with sows fed the basal diet (CON). We found a large number of metabolites in both colostrum and milk that differed significantly between sows in the CON and l-Ser groups. Additionally, twenty metabolites differed in the serum of piglets aged 21 days between the CON and l-Ser groups. Most of these metabolites are involved in purine and pyrimidine metabolism, glutathione and taurine metabolism, as well as phospholipid and sphingolipid metabolism, which may contribute to the growth-promoting effects of serine on offspring. Our results imply that maternal serine has the potential to improve offspring outcomes.

Antioxidant and Anti-Inflammatory Effects of Different Zinc Sources on Diquat-Induced Oxidant Stress in a Piglet Model.

Zinc (Zn) plays a crucial role in reducing oxidative stress and diarrhea in postweanling piglets. This study is aimed at comparing the effects of zinc chelate of 2-hydroxy-4 methyl-thio butanoic acid (HMZn) and ZnSO4 on the oxidative stress in weaned piglets. A total of 32 piglets were randomly divided into 4 treatments: CON: basal diet+80 mg/kg Zn as ZnSO4; DIQ: basal diet+80 mg/kg Zn as ZnSO4; HMZn: basal diet+200 mg/kg Zn as HMZn; and ZnSO4: basal diet+200 mg/kg Zn as ZnSO4. On day 15, the DIQ, HMZn, and ZnSO4 groups were injected intraperitoneally with diquat except for the CON group. The trial lasted 21 days. The results showed that zinc sources did not influence the growth performance during the first 14 days. But HMZn increased activities of superoxide dismutase (SOD), glutathione peroxidase (GPX), and total antioxidant capacity (T-AOC) in serum (P < 0.05). After diquat injection, the fecal score was decreased in the HMZn group. Both HMZn and ZnSO4 increased the activities of GPX and T-AOC in serum and the relative mRNA expressions of hepatic and renal Nrf2, SOD1, and GPX compared with the DIQ group (P < 0.05). Moreover, the relative mRNA expression of inflammatory factors in the small intestine, liver, and kidney was downregulated; the phosphorylation of NF-κB protein was inhibited in the HMZn group compared with the DIQ and ZnSO4 groups (P < 0.05). In general, HMZn showed notable advantage over ZnSO4 in reducing diarrhea and improving antioxidant and anti-inflammatory ability in piglets challenged with diquat.

Glutamate and aspartate alleviate testicular/epididymal oxidative stress by supporting antioxidant enzymes and immune defense systems in boars.

Several potential oxidative agents have damaging effects on mammalian reproductive systems. This study was conducted to investigate the effects of glutamate (Glu) and aspartate (Asp) supplementation on antioxidant enzymes and immune defense systems in the outer scrotum of boars injected with H2O2. A total of 24 healthy boars were randomly divided into 4 treatment groups: control (basal diet, saline-treated), H2O2 (basal diet, H2O2-challenged outer scrotum (1 mL kg-1 BW)), Glu (basal diet +2% Glu, H2O2-challenged), and Asp (basal diet+2% Asp, H2O2-challenged). Our results showed that both Glu and Asp supplementation improved testicular morphology and decreased the genital index in the H2O2-treated boars. Glu and Asp administration increased the antioxidant enzyme activities and affected the testicular inflammatory cytokine secretion but had no effect on sex hormone levels. Furthermore, the mRNA expression of CAT, CuZnSOD, and GPx4 was altered in the testes and epididymis of boars treated with Asp and Glu. Glu and Asp supplementation also modulated the expression of TGF-ß1, IL-10, TNF-α, IL-6 and IL-1ß in the testis and epididymis. These results indicate that dietary Glu and Asp supplementation might enhance antioxidant capacity and regulate the secretion and expression of inflammatory cytokines to protect the testes and epididymis of boars against oxidative stress.

Prevention of Oxidative Stress by α-Ketoglutarate via Activation of CAR Signaling and Modulation of the Expression of Key Antioxidant-Associated Targets in Vivo and in Vitro.

α-Ketoglutarate (AKG) can act as an antioxidant both in vitro and in vivo. However, the mechanisms of the protective effects of AKG are still not well understood. We evaluated the effects of AKG supplementation on the regulation of the constitutive-androstane-receptor (CAR) pathway in porcine intestinal cells and piglets exposed to H2O2. Our data showed that AKG treatment significantly increased not only the intra- and extracellular levels of AKG (26.9 ± 1.31 µmol/g protein, 1064.4 ± 39.80 µmol/L medium) but also those of Asp (29.3 ± 0.21 µmol/g, 4.20 ± 0.11 µmol/L), Gln (24.82 ± 1.50 µmol/g, 1087.80 ± 16.10 µmol/L), and Glu (91.90 ± 3.6 µmol/g, 19.76 ± 1.00 µmol/L). There was approximately a 4-fold increase in α-ketoglutarate dehydrogenase mRNA levels in enterocytes and a simultaneous reduction in ROS levels ( P < 0.05). Moreover, AKG treatment increased the activities of the antioxidant enzymes and the efficiency of cellular respiration ( P < 0.05). AKG also regulated the mRNA levels of the target genes involved in antioxidant responses and xenobiotic detoxification in enterocytes. Increases in the protein levels of SOD1, SOD2, CAR, RXRα, and UCP2 and marked reductions in the expression levels of Nrf2 and Keap1 proteins ( P < 0.05) were observed after AKG administration in the H2O2-induced piglets. Our results indicated that AKG may protect against oxidative stress by activating CAR signaling and modulating the expression of key antioxidant-related targets, which improves cellular respiration and antioxidant capacity. The in vivo and in vitro effects of AKG suggest that it may prove to be useful in the reduction of oxidative stress in animal and human trials and subsequent prevention of gastrointestinal pathologies.

Effect of dietary α-ketoglutarate and allicin supplementation on the composition and diversity of the cecal microbial community in growing pigs.

BACKGROUND: The search for substitutes for antibiotics has recently become urgent. In our previous work, dietary α-ketoglutarate (AKG) combined with allicin improved growth performance and enhanced immunity in growing pigs, whereas the effects on them of intestinal microbiota were unclear. Here, we further investigate the effects of dietary AKG and allicin supplementation on the composition and diversity of intestinal microbiota in growing pigs. RESULTS: Treatment with a combination of AKG and allicin enhanced cecal bacteria richness and diversity, as evidenced by changes in Chao 1, ACE, Shannon, and Simpson values when compared to the control group and antibiotics group. At the phylum level, Bacteroidetes and Firmicutes were the two most abundant phyla. Treatment with a combination of AKG and allicin increased the numbers of Firmicutes and reduced the numbers of Bacteroidetes. Prevotella was the most abundant genus; it was increased by treatment with a combination of AKG and allicin. Furthermore, compared with the antibiotic group, the level of acetate was increased in the AKG group with or without allicin. Treatment with a combination of AKG and allicin increased the levels of cecal butyrate and total volatile fatty acids (VFA) when compared with the control group in growing pigs. CONCLUSION: Dietary 1.0% AKG combined with 0.5% allicin improved cecal microbial composition and diversity, which might further promote VFA metabolism in growing pigs. © 2018 Society of Chemical Industry.

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.

Serine alleviates oxidative stress via supporting glutathione synthesis and methionine cycle in mice.

SCOPE: Serine lies at the central node linking biosynthetic flux from glycolysis to glutathione synthesis and one-carbon metabolic cycle which are closely related to antioxidant capacity. The present study was conducted to determine the effects of serine supplementation on oxidative stress and its relative mechanisms. METHODS AND RESULTS: Diquat treatment was performed to induce oxidative stress in mice and primary hepatocytes. The results showed that hepatic glutathione anti-oxidant systems were impaired and reactive oxygen species and homocysteine were increased in diquat-induced mice and hepatocytes, while such disadvantageous changes were diminished by serine supplementation both in vivo and in vitro. However, when cystathionine ß-synthase expression was inhibited by interference RNA in hepatocytes, the effects of serine supplementation on the improvement of glutathione synthesis and the alleviation of oxidative stress were diminished. Moreover, when hepatocytes were treated with cycloleucine, an inhibitor of methionine adenosyltransferase, the effects of serine supplementation on the improvement of methionine cycle and the alleviation of DNA hypomethylation and oxidative stress were also diminished. CONCLUSION: Our results indicated that serine supplementation alleviated oxidative stress via supporting glutathione synthesis and methionine cycle, mostly by condensing with homocysteine to synthesize cysteine and providing one-carbon units for homocysteine remethylation.

AMPK/α-Ketoglutarate Axis Regulates Intestinal Water and Ion Homeostasis in Young Pigs.

Water and ion absorption via sensitive aquaporins (AQPs) and ion channels is of critical importance in intestinal health. However, whether α-ketoglutarate (AKG) could improve intestinal water and ion homeostasis in lipopolysaccharide (LPS)-challenged piglets and whether the AMP-activated protein kinase (AMPK) pathway is involved remains largely unknown. This study was conducted to investigate the effect of dietary AKG supplementation on the small intestinal water and ion homeostasis through modulating the AMPK pathway in a piglet diarrhea model. A total of 32 weaned piglets were used in a 2 × 2 factorial design; the major factors were diet (basal diet or 1% AKG diet) and challenge (Escherichia coli LPS or saline). The results showed that LPS challenge increased the diarrhea index and affected the concentrations of serum Na+, K+, Cl-, glucose, and AKG and its metabolites in piglets fed the basal or AKG diet. However, the addition of AKG attenuated diarrhea incidence and reversed these serum parameter concentrations. Most AQPs (e.g., AQP1, AQP3, AQP4, AQP5, AQP8, AQP10, and AQP11) and ion transporters (NHE3, ENaC, and DRA/PAT1) were widely distributed in the duodenum and jejunum of piglets. We also found that AKG up-regulated the expression of intestinal epithelial AQPs while inhibiting the expression of ion transporters. LPS challenge decreased (P < 0.05) the gene and protein expression of the AMPK pathway (AMPKα1, AMPKα2, SIRT1, PGC-1α, ACC, and TORC2) in the jejunum and ileum. Notably, AKG supplementation enhanced the abundance of these proteins in the LPS-challenged piglets. Collectively, AKG plays an important role in increasing water and ion homeostasis through modulating the AMPK pathway. Our novel finding has important implications for the prevention and treatment of gut dysfunction in neonates.

Effects of Alpha-Ketoglutarate on Glutamine Metabolism in Piglet Enterocytes in Vivo and in Vitro.

Alpha-ketoglutarate (AKG) plays a vital part in the tricarboxylic acid cycle and is a key intermediate in the oxidation of L-glutamine (Gln). The study was to evaluate effects of AKG on Gln metabolism in vivo and in vitro. A total of twenty-one piglets were weaned at 28 days with a mean body weight (BW) of 6.0 ± 0.2 kg, and randomly divided into 3 groups: corn soybean meal based diet (CON group); the basal diet with 1% alpha-ketoglutarate (AKG treatment group); and the basal diet with 1% L-glutamine (GLN treatment group). Intestinal porcine epithelial cells-1 (IPEC-1) were incubated to investigate effects of 0.5, 2, and 3 mM AKG addition on Gln metabolism. Our results showed that there were no differences (P > 0.05) among the 3 treatments in initial BW, final BW, and average daily feed intake. However, average daily gain (P = 0.013) and gain:feed (P = 0.041) of the AKG group were greater than those of the other two groups. In comparison with the CON group, the AKG and GLN groups exhibited an improvement in villus length, mucosal thickness, and crypt depth in the jejunum of piglets. Serum concentrations of Asp, Glu, Val, Ile, Tyr, Phe, Lys, and Arg in the piglets fed the 1% AKG or Gln diet were lower than those in the CON group. Compared with the CON group, the mRNA expression of jejunal and ileal amino acid (AA) transporters in the AKG and GLN groups were significantly increased (P < 0.05). Additionally, the in vitro study showed that the addition of 0.5, 2, and 3 mM AKG dose-dependently decreased (P < 0.05) the net utilization of Gln and formulation of ammonia, Glu, Ala, and Asp by IPEC-1. In conclusion, dietary AKG supplementation, as a replacement for Gln, could improve Gln metabolism in piglet enterocytes and enhance the utilization of AA.

Low-protein diets affect ileal amino acid digestibility and gene expression of digestive enzymes in growing and finishing pigs.

The objective of this study was to evaluate effects of dietary crude protein (CP) intake on ileal amino acid digestibilities and expression of genes for digestive enzymes in growing and finishing pigs. In Experiment 1, 18 growing pigs (average initial BW = 36.5 kg) were assigned randomly into one of three treatments (n = 6/treatment group) representing normal (18 % CP), low (15 % CP), and very low (12 % CP) protein intake. In Experiment 2, 18 finishing pigs (average initial BW = 62.3 kg) were allotted randomly into one of three treatments (n = 6/treatment group), representing normal (16 % CP), low (13 % CP) and very low (10 % CP) protein intake. In both experiments, diets with low and very low CP were supplemented with crystalline amino acids to achieve equal content of standardized ileal digestible Lys, Met, Thr, and Trp, and were provided to pigs ad libitum. Daily feed intake, BW, and feed/gain ratios were determined. At the end of each experiment, all pigs were slaughtered to collect pancreas, small-intestine samples, and terminal ileal chymes. Samples were used for determining expression of genes for digestive enzymes and ileal amino acid digestibilities. Growing pigs fed the 12 % CP and 15 % CP diets had lower final body weight (P < 0.01) and ADG (P < 0.0001) when compared with pigs fed the 18 % dietary CP diet. Growing pigs fed with the 12 % CP diet showed higher digestibilities for CP (P < 0.05), DM (P < 0.05), Lys (P < 0.0001), Met (P < 0.01), Cys (P < 0.01), Thr (P < 0.01), Trp (P < 0.05), Val (P < 0.05), Phe (P < 0.05), Ala (P < 0.05), Cys (P < 0.01), and Gly (P < 0.05) than those fed the 18 % CP diet. Finishing pigs fed the 16 % CP diet had a higher (P < 0.01) final body weight than those fed the 10 % CP diet. mRNA levels for digestive enzymes (trypsinogen, chymotrypsin B, and dipeptidases-II and III) differed among the three groups of pigs (P < 0.05), and no difference was noted in the genes expression between control group and lower CP group. These results indicated that a reduction of dietary CP by a six-percentage value limited the growth performance of growing-finishing pigs and that a low-protein diet supplemented with deficient amino acids could reduce the excretion of nitrogen into the environment without affecting weight gain.

The Physiological Basis and Nutritional Function of Alpha-ketoglutarate.

Alpha-Ketoglutarate (AKG) is a biological substance that plays important roles in cell metabolism and physiology. AKG is synthesized from glucose or oxaloacetate plus pyruvate. As an intermediate of the tricarboxylic acid cycle, AKG is essential for the oxidation of fatty acids, amino acids, and glucose. Extracellular AKG is a significant source of energy for cells of the gastrointestinal tract. As a precursor for the synthesis of glutamate and glutamine in multiple tissues (including liver, skeletal muscle, heart, brain, and white adipose tissue), AKG bridges carbohydrate and nitrogen metabolism for both conservation of amino acids and ammonia detoxification. Additionally, emerging evidence shows that AKG is a regulator of gene expression and cell signaling pathways (including the mammalian target of rapamycin and AMPactivated protein kinase). Thus, AKG is an attractive dietary supplement in animal and human nutrition to improve cellular energy status, immunity, and health.