mTOR signaling pathway regulation HIF-1 α effects on LPS induced intestinal mucosal epithelial model damage.

BACKGROUND: Sepsis-induced small-intestinal injury is associated with increased morbidity and mortality. Our previous study and other papers have shown that HIF-1α has a protective effect on intestinal mucosal injury in septic rats. The purpose of this study is to further verify the protective effect of HIF-1α on intestinal mucosa and its molecular mechanism in vitro experiments. METHODS: Caco-2 cells were selected and experiment was divided into 2 parts. Part I: HIF-1α activator and inhibitor were used to treat lipopolysacchrides (LPS)-stimulated Caco-2 cells respectively, to explore the effect of HIF-1α on LPS induced Caco-2 cell epithelial model; Part II: mTOR activator or inhibitor combined with or without HIF-1α activator, inhibitor to treat LPS-stimulated Caco-2 cells respectively, and then the molecular mechanism of HIF-1α reducing LPS induced Caco-2 cell epithelial model damage was detected. RESULTS: The results showed that HIF-1α activator decreased the permeability and up regulated tight junction (TJ) expression, while HIF-1α inhibitor had the opposite effect with the HIF-1α activator. mTOR activation increased, while mTOR inhibition decreased HIF-1α protein and expression of its downstream target molecules, which can be attenuated by HIF-1α activator or inhibitor. CONCLUSION: This study once again confirmed that HIF-1α alleviates LPS-induced mucosal epithelial model damage through P70S6K signalling pathway. It is of great value to explore whether HIF-2α plays crucial roles in the regulation of mucosal epithelial model functions in the future.

Exploring the effects of lysozyme dietary supplementation on laying hens: performance, egg quality, and immune response.

An experiment was conducted to evaluate the dietary supplementation with lysozyme's impacts on laying performance, egg quality, biochemical analysis, body immunity, and intestinal morphology. A total of 720 Jingfen No. 1 laying hens (53 weeks old) were randomly assigned into five groups, with six replicates in each group and 24 hens per replicate. The basal diet was administered to the laying hens in the control group, and it was supplemented with 100, 200, 300, or 400 mg/kg of lysozyme (purity of 10% and an enzyme activity of 3,110 U/mg) for other groups. The preliminary observation of the laying rate lasted for 4 weeks, and the experimental period lasted for 8 weeks. The findings demonstrated that lysozyme might enhance production performance by lowering the rate of sand-shelled eggs (P < 0.05), particularly 200 and 300 mg/kg compared with the control group. Lysozyme did not show any negative effect on egg quality or the health of laying hens (P > 0.05). Lysozyme administration in the diet could improve intestinal morphology, immune efficiency, and nutritional digestibility in laying hens when compared with the control group (P < 0.05). These observations showed that lysozyme is safe to use as a feed supplement for the production of laying hens. Dietary supplementation with 200 to 300 mg/kg lysozyme should be suggested to farmers as a proper level of feed additive in laying hens breeding.

Lactiplantibacillus plantarum L47 and inulin alleviate enterotoxigenic Escherichia coli induced ileal inflammation in piglets by upregulating the levels of α-linolenic acid and 12,13-epoxyoctadecenoic acid.

Alternatives to antibiotics for preventing bacteria-induced inflammation in early-weaned farm animals are sorely needed. Our previous study showed that Lactiplantibacillus plantarum L47 and inulin could alleviate dextran sulfate sodium (DSS)-induced colitis in mice. To explore the protective effects of L. plantarum L47 and inulin on the ileal inflammatory response in weaned piglets challenged with enterotoxigenic Escherichia coli (ETEC), 28 weaned piglets were assigned into four groups, namely, CON group-orally given 10 mL/d phosphate buffer saline (PBS), LI47 group-orally given a mixture of 10 mL/d L. plantarum L47 and inulin, ECON group-orally given 10 mL/d PBS and challenged by ETEC, and ELI47 group-orally given 10 mL/d L. plantarum L47 and inulin mixture and challenged by ETEC. The results demonstrated that the combination of L. plantarum L47 and inulin reduced inflammatory responses and relieved the inflammatory damage caused by ETEC, including ileal morphological damage, reduced protein expression of ileal tight junction, decreased antioxidant capacity, and decreased anti-inflammatory factors. Transcriptome analysis revealed that L. plantarum L47 and inulin up-regulated the gene expression of phospholipase A2 group IIA (PLA2G2A) (P < 0.05) as well as affected alpha-linolenic acid (ALA) metabolism and linoleic acid metabolism. Moreover, L. plantarum L47 and inulin increased the levels of ALA (P < 0.05), lipoteichoic acid (LTA) (P < 0.05), and 12,13-epoxyoctadecenoic acid (12,13-EpOME) (P < 0.05) and the protein expression of Toll-like receptor 2 (TLR2) (P = 0.05) in the ileal mucosa. In conclusion, L. plantarum L47 and inulin together alleviated ETEC-induced ileal inflammation in piglets by up-regulating the levels of ALA and 12,13-EpOME via the LTA/TLR2/PLA2G2A pathway.

Duodenal infusion of soy protein hydrolysate activates cAMP signaling and hypothalamic neurotransmitter synthesis in pigs.

Neurotransmitters in the brain are important for cognition and memory. As bioactive substrates, whether increased soy protein levels in pigs can promote hypothalamic neurotransmitter synthesis remains unclear. The effect of increased soy protein hydrolysate (SPH) levels in the small intestine of pigs on neurotransmitter precursor supply, hypothalamic neurotransmitter synthesis and underlying molecular processes was investigated by using sixteen pigs (35.2 ± 0.3 kg) infused either with SPH (70 g day-1) or sterile saline (control) twice daily for 15 days via a duodenal fistula. It demonstrated that SPH infusion increased the expression of the neutral amino acid transporter B0AT1 in the jejunal mucosa, the serum tyrosine/large neutral amino acid ratio, the concentrations of serum tyrosine, hypothalamic tyrosine, dopamine and brain-derived neurotrophic factor (BDNF) (P < 0.05). It also increased the jejunal and serum choline, hypothalamic choline and acetylcholine levels (P < 0.05). Hypothalamic transcriptome revealed that differential genes were significantly enriched in the cholinergic synapse, dopaminergic synapse and cyclic adenosine monophosphate (cAMP) signalling pathways, and that the expression of key enzyme genes in the synthesis of acetylcholine and dopamine and dopamine receptors 1 (DRD1) was upregulated by SPH (P < 0.05). Western blotting showed that SPH infusion activated the hypothalamic cAMP signalling pathways. Overall, SPH infusion promoted the synthesis of hypothalamic dopamine and acetylcholine, and the synthesised dopamine promoted BDNF production most likely through the activation of the cAMP signalling pathways by the DRD1.

Effects of the increased protein level in small intestine on the colonic microbiota, inflammation and barrier function in growing pigs.

BACKGROUND: An increased level of the dietary protein alters the colonic microbial community and metabolic profile of pigs, but it remains unclear whether this leads to colonic inflammation and impairs barrier function in growing pigs. RESULTS: Sixteen pigs (35.2 ± 0.3 kg) were infused with sterile saline (control) or soy protein hydrolysate (SPH) (70 g/day) through a duodenal fistula twice daily during a 15-day experimental period. The SPH treatment did not affect their average daily feed intake and daily weight gain (P > 0.05), but reduced colon index and length (P < 0.05). Illumina MiSeq sequencing revealed that species richness was increased following SPH intervention (P < 0.05). Furthermore, SPH reduced the abundance of butyrate- and propionate-producing bacteria-such as Lachnospiraceae NK4A136 group, Lachnospiraceae_uncultured, Coprococcus 3, Lachnospiraceae UCG-002, and Anaerovibrio-and increased the abundance of potentially pathogenic bacteria and protein-fermenting bacteria, such as Escherichia-Shigella, Dialister, Veillonella, Prevotella, Candidatus Saccharimonas, Erysipelotrichaceae UCG-006, Prevotellaceae_uncultured, and Prevotellaceae UCG-003 (P < 0.05). In addition, a lower content of total short-chain fatty acids, propionate, and butyrate and a higher concentration of cadaverine, putrescine, total biogenic amines, ammonia, and isovalerate were observed following SPH infusion (P < 0.05). Further analysis revealed that SPH increased the concentration of tumour necrosis factor-α, interleukin (IL)-1ß, IL-6, and IL-8 in the colonic mucosa (P < 0.05). Interestingly, SPH intervention increased the expression of occludin, zonula occludens (ZO)-1, and claudin-1 in colonic mucosa (P < 0.05). Correlation analysis showed that different genera were significantly related to the production of metabolites and the concentrations of pro-inflammatory cytokines. CONCLUSION: An increased soy protein level in the small intestine altered the colonic microbial composition and metabolic profile, which resulted in the secretion of colonic proinflammatory cytokines and the increased expression of tight junction proteins.

Dietary Moringa oleifera leaf powder improves jejunal permeability and digestive function by modulating the microbiota composition and mucosal immunity in heat stressed rabbits.

Heat stress (HS) has detrimental effects on intestinal health by altering digestive and immune responses in animals. Dietary Moringa oleifera leaf powder (MOLP) has been implicated in ameliorating the impact of HS, but its effects in terms of intestinal function improvement under HS remain poorly characterized. Therefore, the current study investigated the impact of HS and MOLP supplementation on tight junction barriers, intestinal microbiota (jejunal digesta), and differentially expressed genes (DEGs) in jejunal mucosa of heat-stressed rabbits by using the next-generation sequencing techniques. A total of 21 male New Zealand White rabbits (32 weeks old mean body weight of 3318 ± 171 g) were divided into three groups (n = 7/group) as control (CON, 25 °C), heat stress (HS, 35 °C for 7 h daily), and HS with MOLP supplementation (HSM, 35 °C for 7 h daily) gavage at 200 mg/kg body weight per day for 4 weeks. The results indicated that MOLP supplementation increased mRNA expression of tight junction proteins and glutathione transferase activity, while the malonaldehyde concentration was decreased in the jejunal mucosa compared to HS group (P < 0.05). Furthermore, MOLP decreased the concentrations of lipopolysaccharide, pro-inflammatory cytokines, and myeloperoxidase compared with HS group (P < 0.05). Intestinal microbiota analysis revealed that at phyla level, the relative abundance of Bacteroidetes was higher in HSM group compared to CON and HS groups. MOLP supplementation also resulted in higher abundance of putatively health-associated genera such as Christensenellaceae R-7 gut group, Ruminococcaceae NK4A214 group, Ruminococcus 2, Lachnospiraceae NK4A136 group, and Lachnospiraceae unclassified along with higher butyrate levels in HSM group as compared to HS group. The analysis of DEGs revealed that MOLP reversed inflammatory response by downregulation of genes, such as TNFRSF13C, LBP, and COX2 in enriched KEGG pathway of NF-kß pathway. MOLP supplementation also significantly upregulated the expression of genes in protein digestion and absorption pathway, including PRSS2, LOC100349163, CPA1, CPB1, SLC9A3, SLC1A1, and SLC7A9 in HSM group. Three genes of fibrillar collagens, i.e., COL3A1, COL5A3, and COL12A1 in protein digestion were also down-regulated in HSM group. In conclusion, MOLP supplementation could improve jejunal permeability and digestive function, positively modulate microbiota composition and mucosal immunity in heat-stressed rabbits.

Determination of the Effects of Duodenal Infusion Soy Protein Hydrolysate on Hepatic Glucose and Lipid Metabolism in Pigs Through Multi-Omics Analysis.

High animal protein intake increases hepatic lipid deposition and the risk of diabetes. However, the effects of high plant protein (HPP) intake on glycaemic responses and hepatic lipid metabolism in healthy people, as well as the underlying mechanisms, remain unclear. The current study explored the metabolomic and transcriptomic responses in the livers of pigs to assess the effects of HPP intake on host glucose and lipid metabolism. Sixteen pigs were infused with sterile saline or soy protein hydrolysate (SPH; 70 g/day) through a duodenal fistula twice daily during a 15 days experimental period. Hepatic metabolomic and transcriptomic analyses were performed, and the serum and hepatic biochemical parameters were measured. The results revealed that SPH infusion decreased serum glucose, hepatic triglyceride (TG), total cholesterol and low-density lipoprotein cholesterol levels, while it increased serum urea and eight hepatic amino acid levels (P < 0.05). Hepatic metabolomics displayed that SPH treatment produced seven different metabolites, four of which were related to lipid metabolism and one was related to glucose metabolism. In particular, lower (P < 0.05) glycocholic acid and glucose 1-phosphate levels and higher (P < 0.05) phosphatidylethanolamine (PE), arachidonic acid, prostaglandin F2α, l-carnitine and indole-3 acetic acid levels were observed following SPH infusion. A further metabolic pathway enrichment analysis found that these differential metabolites were mainly enriched in pathways related to lipid and glucose metabolism. Hepatic transcriptomics also demonstrated that multiple genes related to glucose and lipid metabolism were affected by SPH (P < 0.05). Together, SPH infusion reduced the hepatic TG levels by accelerating fatty acid ß-oxidation and inhibiting TG synthesis. In addition, SPH infusion reduced the serum glucose levels by promoting hepatic glucose uptake and glycolysis. This study's result demonstrated that HPP intake regulated glycaemic responses and hepatic lipid metabolism in pigs without increasing the risk of hepatic lipid deposition and hyperglycaemia.

Liver transcriptome of rabbits supplemented with oral Moringa oleifera leaf powder under heat stress is associated with modulation of lipid metabolism and up-regulation of genes for thermo-tolerance, antioxidation, and immunity.

Heat stress resulting from global warming is a serious threat to livestock and humans and can cause impaired metabolism, dysregulated immune functions, and even death. Liver transcriptome of the heat-stressed rabbits supplemented with oral Moringa oleifera leaf powder (MOLP) was performed with the hypothesis that antioxidative properties of MOLP might help to maintain homeostasis under heat stress environment. A total of 21 rabbits were divided into 3 groups (n = 7/group); control (CON, 25°C), heat stress (HS, 35°C for 7 hours daily), and HS supplemented with MOLP (HSM, 35°C) at 200 mg/kg body weight daily for 28 days. Serum analysis indicated that dietary MOLP (HSM) reduced glucose, total cholesterol, triglycerides, and low-density lipoprotein cholesterol contents compared with the HS group (P < .05). The HS group showed increased mRNA expression of interleukin (IL)-1α and IL-1ß (P < .05), whereas enhanced expression of Nrf2 was observed in HSM compared with the HS group indicates antioxidative capacity of MOLP. Up-regulated genes PCK1 and ANGPTL4 as indicated by transcriptome analysis can explain increased serum glucose and lipid levels in the HS group. Up-regulation of antiapoptotic gene BCL2A1 by MOLP may suggest protection from heat stress induced apoptosis. In HSM, the up-regulated IL-6 family genes dictate their importance for immune and survival response, whereas genes PIK3R5 and TLR-2 are vital in thermo-tolerance. Further, identification of marker genes elucidates stress regulation response. In conclusion, findings of current study reveal beneficial aspects of dietary MOLP on liver function in heat-stressed rabbits.

Soybean protein hydrolysate stimulated cholecystokinin secretion and inhibited feed intake through calcium-sensing receptors and intracellular calcium signalling in pigs.

Although soybean protein is the major component in livestock feeds, its effect on pigs' appetites is largely unknown. Recently, the importance of gut nutrient-sensing for appetite modulation by regulating anorectic gut hormone release has been recognised. This study investigates the roles of soybean proteins in appetite regulation, anorectic gut hormone secretion, and underlying mechanisms. The duodenal-cannulated piglets were used to evaluate the effects of soybean protein hydrolysate (SPH) on feed intake and anorectic hormone release, including cholecystokinin (CCK), peptide YY (PYY), glucagon-like peptide 1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP) in the hepatic vein by infusing SPH. Identifying which nutrient-sensing receptor in pig duodenum response to SPH stimulation for gut hormone release was conducted. Using its antagonist, the role of the identified receptor in feed intake and anorectic hormone release was also investigated. Combination with an ex vivo perfusion system, the possible mechanism by which SPH exerts the effects in porcine duodenum was further illustrated. Results in vivo showed that intraduodenal infusion of SPH inhibited short-term feed intake in pigs and promoted CCK, PYY, and GIP secretion in the hepatic vein. SPH also increased duodenum calcium-sensing receptor (CaSR) expression. Pre-treated with CaSR antagonist NPS 2143, the feed intake of pigs tended to be attenuated by SPH (P = 0.09), and CCK release was also suppressed (P < 0.05), indicating that CaSR was involved in SPH-stimulated CCK release and inhibited feed intake in pigs. The ex vivo perfused duodenum tissues revealed that SPH-triggered CCK secretion was likeliest due to the activation of the intracellular Ca2+/TRPM5 pathway. Overall, this study's result illustrates that the diet soybean protein might decrease appetite in pigs by triggering duodenum CCK secretion by activating CaSR and the intracellular Ca2+/TRPM5 pathway.

Oral administration of Moringa oleifera leaf powder relieves oxidative stress, modulates mucosal immune response and cecal microbiota after exposure to heat stress in New Zealand White rabbits.

BACKGROUND: Heat stress (HS) disrupts the gut barrier allowing the uptake of lipopolysaccharide (LPS) and leads to an inflammatory response and changes in gut microbiota composition. Moringa oleifera leaf powder (MOLP) has been proposed to combat HS, yet its alleviate role is currently under investigation. The current study investigated the effects of chronic HS and MOLP supplementation on changes in redox status and immune response of cecal mucosa along with alteration in cecal microbiota. METHODS: A total of 21 young New Zealand White (NZW) rabbits (male) about 32 weeks old (mean body weight of 3318 ± 171 g) reared on a commercial pelleted diet were employed; divided into three groups (n = 7): control (CON, 25 °C), heat stress (HS, 35 °C for 7 h daily), and HS supplemented orally with MOLP (HSM, 35 °C) at 200 mg/kg body weight per day for 4 weeks. RESULTS: The results demonstrated that MOLP supplementation increased organ index of cecal tissue compared with the HS group (P > 0.05). Levels of malonaldehyde (MDA) and activity of superoxide dismutase (SOD) as well as lactate dehydrogenase (LDH) were reduced in the cecal mucosa of the HSM group compared with the HS group. MOLP downregulated the contents of cecal mucosa LPS, several inflammatory markers (TNF-α/IL-1α/IL-1ß), and myeloperoxidase (MPO) in the HSM group (P < 0.05). Secretory immunoglobulin A (SIgA) was increased in the HSM group compared with the HS group (P < 0.05). The transcriptome of cecal mucosa showed that MOLP reduced gene expression relative to several immune factors, including IL-10, IFNG, and RLA, whereas both HS and MOLP increased the gene expression of fat digestion and absorption pathway, including APOA1, FABP1, FABP2, MTTP, and LOC100344166, compared to the CON group (P < 0.001). At the phylum level, the relative abundance of Proteobacteria was increased by HS, while Actinobacteria was significantly increased by HSM compared to other groups (P < 0.05). At genus level, Papillibacter was higher in abundance in HSM groups compared to CON and HS groups (P < 0.05). Higher butyrate concentrations were observed in the HSM group than HS and CON groups (P < 0.05). CONCLUSION: In conclusion, HS in growing rabbits resulted in alteration of cecal microbiota at phyla level as well as increased oxidative stress and expression of mucosal inflammatory genes. Whereas, oral MOLP supplementation elevated the relative weight of cecum, affected their immunological and cecal micro-ecosystem function by improving antioxidant status and down-regulating mucosal tissue inflammatory response.

Supplementation of Moringa oleifera leaf powder orally improved productive performance by enhancing the intestinal health in rabbits under chronic heat stress.

Heat stress jeopardizes animal's growth and health mainly through induction of oxidative stress and inflammation. The current study investigated the effects of Moringa oleifera leaf powder (MOLP) supplementation on productive performance and intestinal health of rabbits under chronic heat stress (HS). Young New Zealand White rabbits (male) at the age of 32 weeks (n = 21, mean body weight of 3318 ± 171 g) for four weeks' period were reared on commercial pelleted diet and divided into three groups: control (CON, 25 °C), HS (35 ± 1 °C) and HS (35 ± 1 °C) with MOLP (HSM) supplemented orally (200 mg/kg body weight). The results demonstrated that rabbits in the HSM group had reduced rectal temperature, respiration rate and improved FCR due to improved daily gain and better crude fiber (NDF) digestibility (P < 0.05) compared with HS group. MOLP improved intestinal integrity and function as indicated by lower serum diamine oxidase level and increased jejunal weight, length, villus height and ratio of villus height to crypt depth than heat-stressed rabbits. MOLP reversed the increased levels of serum cortisol, metabolic indicators i.e. glucose, insulin, and reduced concentrations of serum triiodothyronine. MOLP supplementation also significantly down-regulated the mRNA expression of tumor necrosis factor alpha (α), heat shock protein A2, glutathione peroxidase-1, interleukin (IL)-1α and increased the expression of IL-6. In conclusion, MOLP supplementation could enhance intestinal health along with production and metabolic indicators by alleviating the oxidative stress and inflammatory response in small intestine of hyper-thermic rabbits.

l-Glutamate stimulates cholecystokinin secretion via the T1R1/T1R3 mediated PLC/TRPM5 transduction pathway.

BACKGROUND: It is known that cholecystokinin (CCK) plays an essential role in reducing food intake and driving weight loss. Previous studies demonstrated that amino acids were capable of triggering CCK release through G protein-coupled receptors, but the sensing mechanism remains obscure, especially the intracellular signaling pathway. RESULTS: l-Glu, rather than its d-isomer, robustly stimulated CCK secretion in a porcine duodenal model, and the secretory response was augmented by incubation with the allosteric ligand of T1R1, while T1R3 antagonist attenuated it. Upon inhibiting phospholipase C (PLC) or transient receptor potential M5 (TRPM5) activity, l-Glu failed to increase CCK release. Oral administration of monosodium glutamate in rats also suppressed food intake and increased plasma CCK levels, accompanied by elevated expression of T1R1, PLCß2 and TRPM5 in the duodenum. CONCLUSION: These data demonstrated that l-Glu stimulated CCK secretion through the activation of T1R1/T1R3 in a PLC/TRPM5-dependent manner. © 2020 Society of Chemical Industry.

L-phenylalanine Increased Gut Hormone Secretion through Calcium-Sensing Receptor in the Porcine Duodenum.

Luminal amino acids have a pivotal role in gut hormone secretion, and thereby modulate food intake and energy metabolism. However, the mechanisms by which amino acids exert this effect remains unknown. The purpose of this research was to investigate the response of L-phenylalanine (L-Phe) to gut hormone secretion and its underlying mechanisms by perfusing the pig duodenum. Eighty mM L-Phe and extracellular Ca2+ stimulated cholecystokinin (CCK) and glucose-dependent insulinotropic peptide (GIP) release, and upregulated the mRNA expression of the calcium-sensing receptor (CaSR), CCK, and GIP. Western blotting results showed that L-Phe also elevated the protein levels of CaSR, the inositol 1,4,5-triphosphate receptor (IP3R), and protein kinase C (PKC). However, the CaSR inhibitor NPS 2143 reduced the mRNA expression of CaSR, CCK, and GIP, and the secretion of CCK and GIP, as well as the protein level of CaSR, IP3R, and PKC. These results indicated that Phe stimulated gut secretion through a CaSR-mediated pathway and its downstream signaling molecules, PKC and IP3R.

Effects of low-protein diet on the intestinal morphology, digestive enzyme activity, blood urea nitrogen, and gut microbiota and metabolites in weaned pigs.

This study investigated the effects of low-protein diet supplemented with Lysine (Lys), Methionine (Met), Threonine (Thr), and Tryptophan (Trp) on small intestine morphology, enzyme activity, blood urea nitrogen, and gut microbiota and metabolites in weaned piglets. Eighteen weaned pigs weighing an average of 9.57 kg received one of three treatments: a normal protein diet with 20% crude protein (CP, diet [NP]), a moderately reduced protein diet with 17% CP (MP), or a low-protein diet with 14% CP (LP). All three diets were supplemented with Lys, Met, Thr and Trp to meet essential amino acid requirements for post-weaned piglets according to the NRC (2012). Following a 45 d study period, piglets on the LP and MP diets demonstrated atrophic small intestinal morphology, with decreased villus heights and lower ratios of villus height to crypt depth (p < 0.05); pepsin activity in the stomach was also reduced in these two groups (p < 0.05). Increased plasma cholesterol and decreased blood urea nitrogen presented in the MP and LP groups compared with the NP group (p < 0.05). Overall, gastrointestinal hormones were not affected by dietary protein levels with the exception of reduced somatostatin levels in the MP and LP groups. Jejunum and colon microbiota were not affected at either the phyla or genera level in any of the diets. Colonic ammonia nitrogen concentration was reduced in MP and LP groups. Dietary protein level had no effect on short chain fatty acids or biogenic amines. Our data suggest that reducing dietary protein levels by 3% (MP) or 6% (LP) in weaned pigs has the potential to decrease nitrogen emissions and impaired digestive capacity. Therefore, dietary protein level cannot be reduced by more than 3% in consideration of maladaptive changes to small intestinal morphology and pepsin activity in weaned piglets.

Effects of Long-Term Dietary Protein Restriction on Intestinal Morphology, Digestive Enzymes, Gut Hormones, and Colonic Microbiota in Pigs.

Using protein-restriction diets becomes a potential strategy to save the dietary protein resources. However, the mechanism of low-protein diets influencing pigs' growth performance is still controversial. This study aimed to investigate the effect of protein-restriction diets on gastrointestinal physiology and gut microbiota in pigs. Eighteen weaned piglets were randomly allocated to three groups with different dietary protein levels. After a 16-week trial, the results showed that feeding a low-protein diet to pigs impaired the epithelial morphology of duodenum and jejunum (p < 0.05) and reduced the concentration of many plasma hormones (p < 0.05), such as ghrelin, somatostatin, glucose-dependent insulin-tropic polypeptide, leptin, and gastrin. The relative abundance of Streptococcus and Lactobacillus in colon and microbiota metabolites was also decreased by extreme protein-restriction diets (p < 0.05). These findings suggested that long-term ingestion of a protein-restricted diet could impair intestinal morphology, suppress gut hormone secretion, and change the microbial community and fermentation metabolites in pigs, while the moderately low-protein diet had a minimal effect on gut function and did not impair growth performance.

Sensing of L-Arginine by Gut-Expressed Calcium Sensing Receptor Stimulates Gut Satiety Hormones Cholecystokinin and Glucose-Dependent Insulinotropic Peptide Secretion in Pig Model.

Nutrients regulate the secretion of gut satiety hormones, which is related to the modulation of food intake and blood glucose levels. Calcium-sensing receptor (CaSR) is involved in regulating gut hormone secretion in response to l-amino acids and multivalent cations. Rodents are often used to investigate the effect of nutrients on these hormonal release. However, results obtained using rodent models are difficult to be applied in humans, we used pigs as a model in this study because their physiology is similar to that of humans. In this study, we investigated whether l-Arginine (l-Arg) could induce gut hormones cholecystokinin (CCK) and glucose-dependent insulinotropic peptide (GIP) secretion in the porcine duodenum and if so, whether CaSR mediated l-Arg-regulated gut satiety hormone secretion. Our data showed that treatment with 20 and 50 mM l-Arg induced CCK and GIP secretion compared with 0 mM l-Arg. However, treatment with d-Arg (an inactive isomer) failed to elicit this response. The potency of l-Arg to induce CCK and GIP secretion was enhanced in the presence of extracellular Ca2+ and CaSR agonist cinacalcet. However, the effect of Arg on CCK and GIP secretion was attenuated by blocking CaSR and its downstream signaling molecules adenylate cyclase (AC) and phospholipase C (PLC). Taken all together, pig duodenum provides an appropriate model to explore the effects of l-Arg on the secretion of the satiety-related gut hormones CCK and GIP and the role of CaSR in this effect. Further investigations are needed to verify the effect of l-Arg on food intake and blood glucose in human study. PRACTICAL APPLICATION: l-Arginine is able to modulate cholecystokinin and glucose-dependent insulinotropic peptide secretion through the CaSR in pig model, which has a potential role in regulating food intake and blood glucose levels.

Phenylalanine and tryptophan stimulate gastrin and somatostatin secretion and H+-K+-ATPase activity in pigs through calcium-sensing receptor.

In rodents and humans, aromatic amino acids increase gut hormone secretion and H+-K+-ATPase activity by modulating calcium-sensing receptor (CaSR). However, the role of CaSR and its related signaling molecules in amino acid-induced gut hormone secretion in swine has not been investigated. Here, we examined whether a CaSR-dependent pathway modulated gastrin and somatostatin (SS) secretion and H+-K+-ATPase activity in pigs. Perfusion of pig stomach tissues in the presence of extracellular 80 mM l-phenylalanine (Phe) or 20 mM l-tryptophan (Trp) and a CaSR agonist cinacalcet triggered gastrin and SS secretion and H+-K+-ATPase activity (P < 0.05) and increased CaSR expression (P < 0.05). This effect of Phe and Trp was dependent on Ca2+ (P < 0.05) and was abolished after treatment with NPS 2143, an inhibitor of CaSR, and 2-aminoethyl diphenyl borinate, an inhibitor of CaSR downstream signaling molecule inositol 1,4,5-triphosphate receptor (IP3R). These findings indicate that Phe and Trp induce Ca2+-dependent gastrin and SS secretion and H+-K+-ATPase activity through CaSR and its downstream signaling molecule IP3R.

Calcium-sensing receptor-mediated L-tryptophan-induced secretion of cholecystokinin and glucose-dependent insulinotropic peptide in swine duodenum.

This study aimed to elucidate the effect of tryptophan (Trp) on gut hormone secretion as well as the roles of the calcium-sensing receptor (CaSR) and its downstream signaling pathway in gut hormone secretion by assessing swine duodenal perfusion in vitro. Swine duodenum was perfused with Krebs-Henseleit buffer as a basal solution. Various concentrations (0, 10, and 20 mM) of Trp were applied to investigate its effect on gut hormone secretion. A CaSR antagonist was used to detect the involvement of CaSR and its signal molecules. The 20 mM Trp concentration promoted the secretion of cholecystokinin (CCK) and glucose-dependent insulinotropic peptide (GIP), elevated the mRNA level of CaSR, and upregulated the protein levels of CaSR, protein kinase C (PKC), and inositol trisphosphate receptor (IP3R). However, NPS 2143, an inhibitor of CaSR, attenuated the CCK and GIP release, reduced the mRNA level of CaSR, and decreased the protein levels of CaSR, PKC, and IP3R with 20 mM Trp perfusion. The results indicate that CCK and GIP secretion can be induced by Trp in swine duodenum in vitro, and the effect is mediated by CaSR and its downstream signal molecules PKC and IP3R.

Progressive response of large intestinal bacterial community and fermentation to the stepwise decrease of dietary crude protein level in growing pigs.

The study aimed to determine the effects of reduction of dietary crude protein (CP) level with balanced essential amino acids (EAA) on intestinal bacteria and their metabolites of growing pigs. Forty pigs (initial BW 13.50 ± 0.50 kg, 45 ± 2 days of age) were randomly assigned to four dietary treatments containing CP levels at 20.00% (normal crude protein, NP); 17.16% (medium crude protein, MP); 15.30% (low crude protein, LP); and 13.90% (extremely low crude protein, ELP), respectively. Crystalline AAs were added to meet the EAA requirement of pigs. After 4-week feeding, eight pigs per treatment (n = 8) were randomly selected and slaughtered for sampling of ileal, cecal, and colonic digesta and mucosa. Pigs with moderately reduced CP level had increased bacterial diversity, with the Shannon diversity indices for the colon digesta in the LP group and mucosa in the MP and LP groups significantly (P < 0.05) higher than those in the NP and ELP groups. As the CP level reduces, the Bifidobacterium population were linearly decreased (P < 0.05) both in ileum, cecum, and colon, and the ELP group had the lowest Bifidobacterium population in the cecum and colon, with its value significantly lower than NP and MP groups (P < 0.05). However, the ELP group had the highest population of Escherichia coli in the colon, with its value significantly higher than the LP group (P < 0.05). For bacterial metabolites, as CP level decreased, total short-chain fatty acid (T-SCFA), acetate, and butyrate were linearly increased (linear, P < 0.05) in the ileum, while all SCFAs except formate in the cecum and T-SCFA and acetate in the colon, were linearly decreased (P < 0.05). Reducing CP level led to a linear decrease of microbial crude protein (MCP) in the ileum (P < 0.05) and ammonia in all intestine segments (P < 0.05). The spermidine in cecum and total amines, cadaverine, methylamine, and spermidine in colon were shown a quadratic change (P < 0.05) as dietary CP decreases, with the highest concentration in LP group. These findings suggest that moderate reduction of dietary CP level may benefit large intestinal bacterial community and its fermentation, which was negatively affected by extremely low CP diet.

[Feeding Lactobacillus plantarum and Lactobacillus casei increased microbial diversity and short chain fatty acids production in the gut-intestinal tract of weaning piglets].

Objective: The objective of the study was to study the effects of Lactobacillus plantarum and Lactobacillus casei on the changes in composition and quantity of microbial populations and the concentrations of short chain fatty acids in the gut digesta of piglets before and after weaning, to explore the mechanisms of the tested two strains to relieve weaning stress. Methods: Fifteen litters of piglets (Duroc×Yorkshire×Landrace) at the age of 7 days were randomly allocated to 3 groups (5 each), including the control group with an oral administration of saline, LP group with L. plantarum and and LC group, with L. casei. On day 21, 24 and 35, the piglets were slaughtered, and the ileum and colon digesta were analyzed for microbial populations and short chain fatty acids. Results: In the ileum and colon, the test strains significantly increased the microbial diversity (P<0.05), promoted the growth of Lactobacilli spp. and Bifidobacteria spp. after 2 weeks of post-weaning. The two test strains increased the concentration of acetate, propionate, butyrate and total short chain fatty acids both in ileum and colon before weaning, and the concentration of acetate, total short chain fatty acids after weaning. Correlation analysis showed that the decreased diarrhea rate before weaning in the LP and LC groups was significant associated with the increased short chain fatty acids concentration and total bacteria of ileum and colon, the increased height of the ileum villi. The improved average daily gain presented a correlation with the increased concentration of acetate and total short chain fatty acids of colon. Conclusion: The findings imply that the tested strains are contribute to increase the microbial diversity, the quantities of beneficial bacteria and the production of short chain fatty acids in the gut intestinal tract of weaning piglets.