Dingan Pi

Dietary supplementation of aspartate enhances intestinal integrity and energy status in weanling piglets after lipopolysaccharide challenge

The intestine has a high requirement for ATP to support its integrity, function and health, and thus, energy deficits in the intestinal mucosa may play a critical role in intestinal injury. Aspartate (Asp) is one of the major sources of ATP in mammalian enterocytes via mitochondrial oxidation. We hypothesized that dietary supplementation of Asp could attenuate lipopolysaccharide (LPS)-induced intestinal damage via modulation of intestinal energy status. Twenty-four weanling piglets were allotted to one of four treatments: (1) nonchallenged control, (2) LPS-challenged control, (3) LPS+0.5% Asp treatment, and (4) LPS+1.0% Asp treatment. On day 19, pigs were injected with saline or LPS. At 24 h postinjection, pigs were killed and intestinal samples were obtained. Asp attenuated LPS-induced intestinal damage indicated by greater villus height and villus height/crypt depth ratio as well as higher RNA/DNA and protein/DNA ratios. Asp improved intestinal function indicated by increased intestinal mucosal disaccharidase activities. Asp also improved intestinal energy status indicated by increased ATP, ADP and total adenine nucleotide contents, adenylate energy charge and decreased AMP/ATP ratio. In addition, Asp increased the activities of tricarboxylic acid cycle key enzymes including citrate synthase, isocitrate dehydrogenase and alpha-oxoglutarate dehydrogenase complex. Moreover, Asp down-regulated mRNA expression of intestinal AMP-activated protein kinase α1 (AMPKα1), AMPKα2, silent information regulator 1 (SIRT1) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC1α) and decreased intestinal AMPKα phosphorylation. These results indicate that Asp may alleviate LPS-induced intestinal damage and improve intestinal energy status.

Aspartate alleviates liver injury and regulates mRNA expressions of TLR4 and NOD signaling-related genes in weaned pigs after lipopolysaccharide challenge

Pro-inflammatory cytokines play a critical role in many models of liver injury. In addition, aspartate (Asp) plays an important role in many biological and physiological processes including liver physiology. We hypothesized that Asp could alleviate lipopolysaccharide (LPS)-induced liver injury. Forty-eight weanling pigs were assigned to four treatments including: (1) non-challenged control; (2) LPS challenged control; (3) LPS+0.5% Asp; (4) LPS+1.0% Asp. After 20-d feeding with control (0% Asp), 0.5% or 1.0% Asp supplemented diets, pigs were injected with saline or LPS. At 4 (early phase) and 24 h (late phase) post-injection, blood and liver samples were obtained. Asp attenuated liver injury indicated by reduced serum aspartate aminotransferase activity and increased ratio of serum alanine aminotransferase and aspartate aminotransferase at 24 h, and less severe histological liver damage induced by LPS challenge at 4 or 24 h. In addition, Asp supplementation to LPS challenged pigs decreased mRNA expressions of tumor necrosis factor (TNF)-α and cyclooxygenase-2 linearly and quadratically at 4 h, and increased mRNA expressions of these pro-inflammatory mediators linearly and quadratically at 24 h. Finally, Asp decreased mRNA expression of toll-like receptor 4 (TLR4) signaling related genes (TLR4, myeloid differentiation factor 88, IL-1 receptor-associated kinase 1, TNF-α receptor-associated factor (6), nucleotide-binding oligomerization domain protein (NOD) signaling related genes (NOD1, NOD2 and receptor-interacting serine/threonine-protein kinase 2) and nuclear factor-κB p65 linearly or quadratically at 4 h. However, Asp increased mRNA expressions of these signaling molecules linearly or quadratically at 24 h. These results indicate that, at early and late phases of LPS challenge, Asp exerts opposite regulatory effects on mRNA expression of hepatic pro-inflammatory cytokines and TLR4 and NOD signalling related genes, and improves liver integrity.

Asparagine attenuates intestinal injury, improves energy status and inhibits AMP-activated protein kinase signalling pathways in weaned piglets challenged with Escherichia coli lipopolysaccharide

The intestine requires a high amount of energy to maintain its health and function; thus, energy deficits in intestinal mucosa may lead to intestinal damage. Asparagine (Asn) is a precursor for many other amino acids such as aspartate, glutamine and glutamate, which can be used to supply energy to enterocytes. In the present study, we hypothesise that dietary supplementation of Asn could alleviate bacterial lipopolysaccharide (LPS)-induced intestinal injury via improvement of intestinal energy status. A total of twenty-four weaned piglets were assigned to one of four treatments: (1) non-challenged control; (2) LPS+0 % Asn; (3) LPS+0·5 % Asn; (4) LPS+1·0 % Asn. On day 19, piglets were injected with LPS or saline. At 24 h post-injection, piglets were slaughtered and intestinal samples were collected. Asn supplementation improved intestinal morphology, indicated by higher villus height and villus height:crypt depth ratio, and lower crypt depth. Asn supplementation also increased the ratios of RNA:DNA and protein:DNA as well as disaccharidase activities in intestinal mucosa. In addition, Asn supplementation attenuated bacterial LPS-induced intestinal energy deficits, indicated by increased ATP and adenylate energy charge levels, and decreased AMP:ATP ratio. Moreover, Asn administration increased the activities of key enzymes involved in the tricarboxylic acid cycle, including citrate synthase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase complex. Finally, Asn administration decreased the mRNA abundance of intestinal AMP-activated protein kinase-α1 (AMPKα1), AMPKα2, silent information regulator 1 (SIRT1) and PPARγ coactivator-1α (PGC1α), and reduced intestinal AMPKα phosphorylation. Collectively, these results indicate that Asn supplementation alleviates bacterial LPS-induced intestinal injury by modulating the AMPK signalling pathway and improving energy status.

Asparagine improves intestinal integrity, inhibits TLR4 and NOD signaling, and differently regulates p38 and ERK1/2 signaling in weanling piglets after LPS challenge

Asparagine (Asn), an activator of ornithine decarboxylase (ODC), stimulates cell proliferation in intestinal epithelial cells. We hypothesized that Asn can mitigate LPS-induced injury of intestinal structure and barrier function by regulating inflammatory signaling pathways. We executed the following experiment using weanling pigs for each of the groups: (1) non-challenged control; (2) LPS-challenged control; (3) LPS + 0.5% Asn; (4) LPS + 1.0% Asn. After 21-d feeding, pigs received an i.p. injection of either saline or LPS. Four h after injection, the mid-jejunum and mid-ileum samples were collected. We found that Asn restored ODC expression that was decreased by LPS treatment. Asn also restored intestinal morphology and barrier function that were impaired by LPS treatment. In addition, Asn down-regulated intestinal caspase-3 protein expression and TNF-α concentration, and decreased the mRNA expression of intestinal TLR4, TLR4 downstream signals (myeloid differentiation factor 88, IL-1 receptor-associated kinase 1 and TNF-α receptor-associated factor 6 and NOD1, NOD2 and their adaptor molecule (receptor-interacting serine/threonine-protein kinase 2). Moreover, Asn decreased p38 phosphorylation but increased ERK1/2 phosphorylation. Our results suggest that Asn improves intestinal integrity during an inflammatory insult, which appears to be related to the decrease of intestinal pro-inflammatory cytokine (via TLR4, NODs and p38) and of enterocyte apoptosis (via p38 and ERK1/2).

Fish Oil Alleviates Activation of the Hypothalamic-Pituitary-Adrenal Axis Associated with Inhibition of TLR4 and NOD Signaling Pathways in Weaned Piglets after a Lipopolysaccharide Challenge

Long-chain n-3 (ω-3) polyunsaturated fatty acids exert beneficial effects in neuroendocrine dysfunctions in animal models and clinical trials. However, the mechanism(s) underlying the beneficial effects remains to be elucidated. We hypothesized that dietary treatment with fish oil (FO) could mitigate LPS-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis through inhibition of Toll-like receptor 4 and nucleotide-binding oligomerization domain protein signaling pathways. Twenty-four weaned pigs were used in a 2 × 2 factorial design, and the main factors consisted of diet (5% corn oil vs. 5% FO) and immunological challenge (saline vs. LPS). After 21 d of dietary treatment with 5% corn oil or FO diets, pigs were treated with saline or LPS. Blood samples were collected at 0 (preinjection), 2, and 4 h postinjection, and then pigs were humanely killed by intravenous injection of 40 mg/kg body weight sodium pentobarbital for tissue sample collection. FO led to enrichment of eicosapentaenoic acid and docosahexaenoic acid and total n-3 polyunsaturated fatty acids in hypothalamus, pituitary gland, adrenal gland, spleen, and thymus. FO decreased plasma adrenocorticotrophin and cortisol concentrations as well as mRNA expressions of hypothalamic corticotropin releasing hormone and pituitary proopiomelanocortin. FO also reduced mRNA expression of tumor necrosis factor-α in hypothalamus, adrenal gland, spleen, and thymus, and of cyclooxygenase 2 in hypothalamus. Moreover, FO downregulated the mRNA expressions of Toll-like receptor 4 (TLR4) and its downstream molecules, including cluster differentiation factor 14, myeloid differentiation factor 2, myeloid differentiation factor 88, interleukin-1 receptor-associated kinase 1, tumor necrosis factor-α receptor-associated factor 6, and nuclear factor kappa-light-chain-enhancer of activated B cells p65, and also decreased the mRNA expressions of nucleotide-binding oligomerization domain 1, nucleotide-binding oligomerization domain 2, and their adaptor molecule receptor-interacting serine/threonine-protein kinase 2. These results suggested that FO attenuates the activation of the HPA axis induced by LPS challenge. The beneficial effects of FO on the HPA axis may be associated with decreasing the production of brain or peripheral proinflammatory cytokines through inhibition of TLR4 and nucleotide-binding oligomerization domain protein signaling pathways.

Fish oil enhances intestinal barrier function and inhibits corticotropin-releasing hormone/corticotropin-releasing hormone receptor 1 signalling pathway in weaned pigs after lipopolysaccharide challenge.

Stress induces injury in intestinal barrier function in piglets. Long-chain n-3 PUFA have been shown to exhibit potential immunomodulatory and barrier protective effects in animal models and clinical trials. In addition, corticotropin-releasing hormone (CRH)/CRH receptor (CRHR) signalling pathways play an important role in stress-induced alterations of intestinal barrier function. We hypothesised that fish oil could affect intestinal barrier function and CRH/CRHR signalling pathways. In total, thirty-two weaned pigs were allocated to one of four treatments. The experiment consisted of a 2×2 factorial design, and the main factors included immunological challenge (saline or lipopolysaccharide (LPS)) and diet (5 % maize oil or 5 % fish oil). On d 19 of the trial, piglets were treated with saline or LPS. At 4 h after injection, all pigs were killed, and the mesenteric lymph nodes (MLN), liver, spleen and intestinal samples were collected. Fish oil decreased bacterial translocation incidence and the number of translocated micro-organisms in the MLN. Fish oil increased intestinal claudin-1 protein relative concentration and villus height, as well as improved the intestinal morphology. In addition, fish oil supplementation increased intestinal intraepithelial lymphocyte number and prevented elevations in intestinal mast cell and neutrophil numbers induced by LPS challenge. Moreover, fish oil tended to decrease the mRNA expression of intestinal CRHR1, CRH and glucocorticoid receptors. These results suggest that fish oil supplementation improves intestinal barrier function and inhibits CRH/CRHR1 signalling pathway and mast cell tissue density.

Asparagine attenuates hepatic injury caused by lipopolysaccharide in weaned piglets associated with modulation of Toll-like receptor 4 and nucleotide-binding oligomerisation domain protein signalling and their negative regulators.

Pro-inflammatory cytokines play a key role in many models of hepatic damage. In addition, asparagine (Asn) plays an important role in immune function. We aimed to investigate whether Asn could attenuate lipopolysaccharide (LPS)-induced liver damage. Forty-eight castrated barrows were allotted to four groups including: (1) non-challenged control; (2) LPS-challenged control; (3) LPS + 0.5% Asn; and (4) LPS + 1.0% Asn. After 19 d feeding with control, 0.5 or 1.0% Asn diets, pigs were injected with LPS or saline. Blood and liver samples were obtained at 4 h (early stage) and 24 h (late stage) post-injection. Asn alleviated liver injury, indicated by reduced serum aspartate aminotransferase and alkaline phosphatase activities linearly and quadratically; it increased claudin-1 protein expression linearly and quadratically at 24 h, and less severe liver morphological impairment at 4 or 24 h. In addition, Asn decreased mRNA expression of TNF-α and heat shock protein 70 (HSP70) linearly and quadratically at 4 h; it increased TNF-α mRNA expression, and HSP70 protein expression linearly and quadratically at 24 h. Moreover, Asn increased inducible NO synthase activity linearly and quadratically. Finally, Asn down-regulated the mRNA expression of Toll-like receptor 4 (TLR4) signalling molecules (TLR4, IL-1 receptor-associated kinase 1 (IRAK1), TNF-α receptor-associated factor 6), nucleotide-binding oligomerisation domain protein (NOD) signalling molecules (NOD1, NOD2 and their adaptor molecule receptor-interacting serine/threonine-protein kinase 2 (RIPK2)), and NF-κB p65 linearly or quadratically at 4 h. Oppositely, Asn up-regulated mRNA expressions of TLR4 and NOD signalling molecules (TLR4, myeloid differentiation factor 88, IRAK1, NOD2 and RIPK2), and their negative regulators (radioprotective 105, single Ig IL-1R-related molecule, Erbb2 interacting protein and centaurin β1) linearly or quadratically at 24 h. These results indicate that, in early and late stages of LPS challenge, Asn improves liver integrity and exerts different regulatory effects on mRNA expression of TLR4 and NOD signalling molecules.

Asparagine preserves intestinal barrier function from LPS-induced injury and regulates CRF/CRFR signaling pathway

Stress causes intestinal inflammation and barrier dysfunction. Corticotrophin-releasing factor (CRF)/CRF receptor (CRFR) signaling pathway has been shown to be important for stress-induced intestinal mucosal alteration. L-Asparagine (ASN) is a powerful stimulator of ornithine decarboxylase and cell proliferation in a variety of cell types, including colonic cells. In the present study, we investigated whether dietary ASN supplementation could alleviate the damage of intestinal barrier function caused by LPS through modulation of CRF/CRFR signaling pathway. Twenty-four weaned pigs were randomly divided into one of four treatments: (1) non-challenged control; (2) Escherichia coli LPS challenged control; (3) LPS + 0.5% ASN; (4) LPS + 1.0% ASN. LPS stress induced villous atrophy, intestinal morphology disruption and decreased claudin-1 expression. ASN supplementation increased intestinal claudin-1 protein expression and alleviated villous atrophy and intestinal morphology impairment caused by LPS stress. In addition, ASN supplementation increased the number of intestinal intraepithelial lymphocytes and reversed the elevations of intestinal mast cell number and neutrophil number induced by LPS stress. Moreover, ASN decreased the mRNA expression of intestinal CRF, glucocorticoid receptors and tryptase. These results indicate that ASN attenuates intestinal barrier dysfunction induced by LPS stress, and regulates CRF/CRFR1 signaling pathway and mast cell activation.

Aspartate inhibits LPS-induced MAFbx and MuRF1 expression in skeletal muscle in weaned pigs by regulating Akt, AMPKα and FOXO1

Infection and inflammation can result in the rapid loss of muscle mass and myofibrillar proteins (muscle atrophy). In addition, aspartate (Asp) is necessary for protein synthesis in mammalian cells. We hypothesized that Asp could attenuate LPS-induced muscle atrophy in a piglet model. Twenty-four weaning piglets were allotted to four treatments, including non-challenged control, LPS challenged control, LPS+0.5% Asp and LPS+1.0% Asp. On d 21, the piglets were injected with i.p. injection of LPS (100 ug/kg BM) or saline. At 4 h post-injection, blood, gastrocnemius and longissimus dorsi muscles samples were collected for biochemical and molecular analyses. Asp decreased the concentrations of cortisol and glucagon in plasma. In addition, Asp increased protein and RNA contents in muscles, and decreased mRNA expression of muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1). Moreover, Asp decreased phosphorylation of AMPKα but increased phosphorylation of Akt and Forkhead Box O (FOXO) 1 in the muscles. Our results indicate that Asp suppresses LPS-induced MAFbx and MuRF1 expression via activation of Akt signaling, and inhibition of AMPKα and FOXO1 signaling.

Asparagine reduces the mRNA expression of muscle atrophy markers via regulating protein kinase B (Akt), AMP-activated protein kinase α, toll-like receptor 4 and nucleotide-binding oligomerisation domain protein signalling in weaning piglets after lipopolysaccharide challenge.

Pro-inflammatory cytokines are critical in mechanisms of muscle atrophy. In addition, asparagine (Asn) is necessary for protein synthesis in mammalian cells. We hypothesised that Asn could attenuate lipopolysaccharide (LPS)-induced muscle atrophy in a piglet model. Piglets were allotted to four treatments (non-challenged control, LPS-challenged control, LPS+0·5 % Asn and LPS+1·0 % Asn). On day 21, the piglets were injected with LPS or saline. At 4 h post injection, piglet blood and muscle samples were collected. Asn increased protein and RNA content in muscles, and decreased mRNA expression of muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1). However, Asn had no effect on the protein abundance of MAFbx and MuRF1. In addition, Asn decreased muscle AMP-activated protein kinase (AMPK) α phosphorylation, but increased muscle protein kinase B (Akt) and Forkhead Box O (FOXO) 1 phosphorylation. Moreover, Asn decreased the concentrations of TNF-α, cortisol and glucagon in plasma, and TNF-α mRNA expression in muscles. Finally, Asn decreased mRNA abundance of muscle toll-like receptor (TLR) 4 and nucleotide-binding oligomerisation domain protein (NOD) signalling-related genes, and regulated their negative regulators. The beneficial effects of Asn on muscle atrophy may be associated with the following: (1) inhibiting muscle protein degradation via activating Akt and inactivating AMPKα and FOXO1; and (2) decreasing the expression of muscle pro-inflammatory cytokines via inhibiting TLR4 and NOD signalling pathways by modulation of their negative regulators.