This study was based on our previously developed double-layered enterohepatic co-culture system, composed of nontumorigenic porcine intestinal epithelial cell line (IPEC-J2) and primary culture of porcine hepatocytes. The anti-inflammatory effect of spent culture supernatant of Lactobacillus plantarum 2142 (Lp2142; 13.3%) and sodium n-butyrate (2 mM) was tested on IPEC-J2 and hepatocyte monocultures as well as on the gut-liver co-culture. To mimic inflammation, lipopolysaccharide (LPS; 1 and 10 μg/mL) was applied. Production of IL-8 and IL-6 was measured as a marker of inflammatory responses. The paracellular permeability of the intestinal epithelium was also monitored by fluoresceinisothiocyanate-labeled dextran 4 assay. Significant increase of IL-8 concentration was observed in the IPEC-J2 monoculture (P < 0.01) while the level of IL-6 was not changed following LPS treatment. Concentration of IL-8 and IL-6 was grown significantly in hepatocyte monocultures (P < 0.05 and P < 0.001) as well as in the co-culture after 10 μg/mL LPS treatment (P < 0.001 and P < 0.001). One microgram per milliliter LPS caused elevated IL-8 level in the co-culture (P < 0.001) and in the hepatocyte monoculture (P < 0.01), while it caused increased IL-6 level only in the hepatocytes (P < 0.001). Production of IL-8 was significantly decreased by butyrate in case of 1 μg/mL as well as 10 μg/mL LPS exposure in the co-culture (P < 0.001). Application of butyrate also reduced IL-6 level in the co-culture after 10 μg/mL LPS treatment (P < 0.01). Lactobacillus plantarum 2142 decreased IL-8 level after incubation with 1 μg/mL LPS (P < 0.001), while in case of 10 μg/mL LPS treatment only a marginal lowering in IL-8 (P = 0.064) release was measured. The IL-6 concentration was significantly reduced (P < 0.01 in case of 1 μg/mL LPS treatment) by Lp2142 in the co-culture. Contrarily, the elevated IL-8 and IL-6 level of hepatocytes has not been reduced in case of either butyrate or Lp2142 addition. The enterohepatic co-culture model offers a possibility for fast and reliable screening of new candidates against enteric inflammation, which are of special interest in porcine medicine and health management. According to our results, Lp2142 and butyrate both seem to be effective as anti-inflammatory agents in LPS-triggered inflammatory response, tested in the gut-liver co-culture model.

No abstract

As Kupffer cells are highly involved in the regulation of hepatic inflammatory response, the main goal of this study was to improve and to characterize a hepatocyte-Kupffer cell co-culture of pig origin for modelling endotoxin-induced hepatic inflammation and for testing the efficacy of potential anti-inflammatory substances. This monolayer co-culture was prepared from primary isolated swine hepatocytes and Kupffer cells in the ratio of 6:1 and 2:1, mimicking different states of liver inflammation. The prepared cell cultures were characterized by immunohistochemical CD-68 detection. Lipopolysaccharide (LPS) challenge of both co-cultures resulted in elevated interleukin-8 (IL-8) and that of 6:1 co-cultures in increased IL-6 production with a higher extent than on hepatocyte monocultures, justifying the key role of Kupffer cells in pro-inflammatory cytokine production. LPS-induced IL-8 production was successfully attenuated by concomitant application of both sodium butyrate and terpinen-4-ol on hepatocyte monocultures, but not on co-cultures, demonstrating the importance of the presence of Kupffer cells in cell cultures as inflammatory models. Based on these initial data, the applied porcine primary hepatocyte-Kupffer cell co-culture is suggested to be a proper tool for in vitro investigations on liver physiology and hepatic inflammation in pigs and can be used as a useful model mimicking in vivo conditions in veterinary research.

The short-chain fatty acid butyrate, either in unprotected or protected form, is widely applied as a growth-promoting feed additive in poultry nutrition; however, its possible effects on the carcass composition of broilers have not been fully elucidated. Further, lowering dietary crude protein (CP) levels is an important issue in poultry farming, contributing to ecologically beneficial lower nitrogen excretion. The main aims of this study were to test how unprotected and protected forms of butyrate and decreased dietary CP content with essential amino acid (lysine, methionine, threonine, tryptophan) supplementation (“LP-EAA” diet) affect carcass parameters and the chemical composition of muscles in broilers. Ross 308 chickens were randomized to seven groups (n=10/group) receiving adequate CP-containing (normal protein, “NP”) or LP-EAA diets, both supplemented with or without unprotected sodium butyrate, and NP diets with different forms of protected sodium butyrate. Carcass traits were measured, and the chemical composition of pectoral and femoral muscles was analyzed at the age of 6 weeks. Carcass weight was significantly increased by the LP-EAA diet and all protected butyrate types tested, while the relative breast meat yield was significantly higher in LP-EAA than NP groups and in both unprotected and protected butyrate-supplemented chickens compared to controls. The protein content of the femoral muscle was significantly decreased, but its lipid content was significantly elevated by the LP-EAA diet and by all types of butyrate addition. However, no changes were detected in the chemical composition of pectoral muscle. In conclusion, breast meat production can be effectively stimulated by dietary factors, such as by reducing dietary CP content with essential amino acid supplementation and by applying butyrate as a feed additive, while its chemical composition remains unchanged, in contrast to the femoral muscle. The aforementioned nutritional strategies seem to be the proper tools to increase carcass yield and to alter meat composition of broilers, contributing to more efficient poultry meat production.

The aim of the present study was to investigate the effects of butyrate as a feed supplement on the expression of insulin signalling proteins as potent regulators of metabolism and growth in Ross 308 broiler chickens fed maize-or wheatbased diets. Both diets were supplemented with non-protected butyrate (1.5 and 3.0 g/kg of diet, respectively) or with protected butyrate (0.2 g/kg of diet); the diet of the control groups was prepared without any additives (control). On day 42 of life, systemic blood samples were drawn for analyses of glucose and insulin concentrations, and tissue samples (liver, gastrocnemius muscle and subcutaneous adipose tissue) were taken for Western blotting examinations. The expression of key insulin signalling proteins (IRβ, PKCζ and mTOR) was assessed by semiquantitative Western blotting from the tissues mentioned. The type of diet had a remarkable influence on the insulin homeostasis of chickens. The wheat-based diet significantly increased IRβ and mTOR expression in the liver as well as mTOR and PKCζ expression in the adipose tissue when compared to animals kept on a maize-based diet. IRβ expression in the liver was stimulated by the lower dose of non-protected butyrate as well, suggesting the potential of butyrate as a feed additive to affect insulin sensitivity. Based on the results obtained, the present study shows new aspects of nutritional factors by comparing the special effects of butyrate as a feed additive and those of the cereal type, presumably in association with dietary non-starch polysaccharide-(NSP-) driven enteric shortchain fatty acid release including butyrate, influencing insulin homeostasis in chickens. As the tissues of chickens have physiologically lower insulin sensitivity compared to mammals, diet-associated induction of the insulin signalling pathway can be of special importance in improving growth and metabolic health.

This study investigates the metabolic effects of maize- or wheat-based diets with normal (NP) and lowered (LP) dietary crude protein level [the latter supplemented with limiting amino acids and sodium (n-)butyrate at 1.5 g/kg diet] at different phases of broiler fattening. Blood samples of Ross 308 broilers were tested at the age of 1, 3 and 6 weeks. Total protein (TP) concentration increased in wheat-based and decreased in LP groups in week 3, while butyrate reduced albumin/TP ratio in week 1. Uric acid level was elevated by wheat-based diet in week 1 and by wheat-based diet and butyrate in week 3, but decreased in LP groups in weeks 3 and 6. Aspartate aminotransferase activity was increased by wheat-based diet in week 3, and creatine kinase activity was intensified by LP in weeks 3 and 6. Blood glucose level decreased in wheat-based groups in week 3; however, triglyceride concentration was augmented in the same groups in week 3. No change of glucagon-like peptide 1, glucose-dependent insulinotropic polypeptide and insulin concentration was observed. In conclusion, an age-dependent responsiveness of broilers to dietary factors was found, dietary cereal type was a potent modulator of metabolism, and a low crude protein diet supplemented with limiting amino acids might have a beneficial impact on the growth of chickens.

The expression of hepatic drug-metabolizing cytochrome P450 (CYP) enzymes may be affected by several nutrition-derived compounds, such as by the commonly applied feed additive butyrate, possibly leading to feed-drug interactions. The aim of this study was to provide some evidence if butyrate can alter the activity of hepatic CYPs in chickens exposed to CYP-inducing xenobiotics, monitoring for the first time the possibility of such interaction. Ross 308 chickens in the grower phase were treated with daily intracoelomal phenobarbital (PB) injection (80 mg/kg BW), applied as a non-specific CYP-inducer, simultaneously with two different doses of intra-ingluvial sodium butyrate boluses (0.25 and 1.25 g/kg BW) for 5 days. Activity of CYP2H and CYP3A subfamilies was assessed by specific enzyme assays from isolated liver microsomes. According to our results, the lower dose of orally administered butyrate significantly attenuated the PB-triggered elevation of both hepatic CYP2H and CYP3A activities, which might be in association with the partly common signalling pathways of butyrate and CYP-inducing drugs, such as that of PB. Based on these data, butyrate may take part in pharmacoepigenetic interactions with simultaneously applied drugs or other CYP-inducing xenobiotics, with possible consequences for food safety and pharmacotherapy. Butyrate was found to be capable to maintain physiological CYP activity by attenuating CYP induction, underlining the safety of butyrate application in poultry nutrition.

The pancreatic secretion of insulin, a key endocrine regulator of metabolism and growth, can be greatly influenced by the gut-derived incretin hormones, namely by GIP (Glucose-dependent Insulinotropic Peptide) and GLP-1 (Glucagon-like Peptide 1). As insulin is a major stimulator of growth, affecting its producion may be of special importance in food-producing livestock. The aim of the present study was to investigate novel ways of modulating incretin and insulin homeostasis in chickens and rabbits by nutrition, e.g. by oral butyrate application, also studying the mechanisms of incretin action in both species as a comparative approach. Acute oral butyrate challenge significantly decreased plasma GIP levels by approx. 40% in both species: significant interactions of butyrate exposure and incubation time were found in both chickens (P = 0.038 and P = 0.034 at 30 and 60 min following butyrate ingestion [1.25 g/kg BW], respectively) and rabbits (P = 0.036 and P = 0.039 at 30 and 60 min after butyrate ingestion [0.25 g/kg BW], respectively), while plasma GLP-1, insulin and glucose concentrations remained unaffected by butyrate in both species over time. These results are in contrast to butyrate’s stimulating effect on both incretin and insulin secretion in mice, indicating specific, species-dependent differences even among mammalian species. Further, based on the analyzed correlations between the measured endocrine parameters (regardless of the butyrate exposure), it can be assumed that incretins may regulate pancreatic insulin release in rabbits on a partly different way compared to mice, humans and chickens.