Bovine mammary epithelial cells can grow and express milk protein synthesis genes at reduced fetal bovine serum concentration.

Milk proteins produced by lactating cells isolated from bovine mammary tissue can offer a sustainable solution to the high protein demand of a global growing population. Serum is commonly added to culture systems to provide compounds necessary for optimal growth and function of the cells. However, in a cellular agricultural context, its usage is desired to be decreased. This study aims at examining the minimum level of fetal bovine serum (FBS) required for the growth and functionality of bovine mammary epithelial cells (MECs). The cells were isolated from dairy cows in early and mid-lactation and cultured in reduced concentrations of FBS (10%, 5%, 1.25%, and 0%). Real-time cell analysis showed a significant effect of lactation stage on growth rate and 5% FBS resulted in similar growth rate as 10% while 0% resulted in the lowest. The effect of reducing FBS on cell functionality was examined by studying the expressions of selected marker genes involved in milk protein and fat synthesis, following differentiation. The gene expressions were not affected by the level of FBS. A reduction of FBS in the culture system of MEC, at least down to 5%, does not assert any negative effect on the growth and expression levels of studied genes. As the first attempt in developing an in-vitro model for milk component production using MEC, our results demonstrate the potential of MEC to endure FBS-reduced conditions.

Coupling in vitro food digestion with in vitro epithelial absorption; recommendations for biocompatibility.

As food transits the gastrointestinal tract, food structures are disrupted and nutrients are absorbed across the gut barrier. In the past decade, great efforts have focused on the creation of a consensus gastrointestinal digestion protocol (i.e., INFOGEST method) to mimic digestion in the upper gut. However, to better determine the fate of food components, it is also critical to mimic food absorption in vitro. This is usually performed by treating polarized epithelial cells (i.e., differentiated Caco-2 monolayers) with food digesta. This food digesta contains digestive enzymes and bile salts, and if following the INFOGEST protocol, at concentrations that although physiologically relevant are harmful to cells. The lack of a harmonized protocol on how to prepare the food digesta samples for downstream Caco-2 studies creates challenges in comparing inter laboratory results. This article aims to critically review the current detoxification practices, highlight potential routes and their limitations, and recommend common approaches to ensure food digesta is biocompatible with Caco-2 monolayers. Our ultimate aim is to agree a harmonized consensus protocol or framework for in vitro studies focused on the absorption of food components across the intestinal barrier.

Curcumin and quercetin modify warfarin-induced regulation of porcine CYP1A2 and CYP3A expression and activity in vitro.

The anticoagulant drug warfarin is used treat atrial fibrillation. Several cases of drug-drug and drug-food interactions have been reported for warfarin.The aim of this study was to investigate the interaction between simultaneous administration of warfarin with the two ubiquitous flavonoids quercetin and curcumin.Using porcine primary hepatocytes we demonstrated that warfarin treatment increased the mRNA and protein expression of CYP3A(29), while no changes in CYP1A2 were observed. Co-treatment with quercetin and/or curcumin decreased the warfarin-induced CYP3A protein expression. Moreover, when quercetin and curcumin were co-administrated to warfarin-exposed hepatocytes the protein expression of CYP1A2 was decreased. In hepatic microsomes, curcumin inhibited the activity of both CYP1A2 and CYP3A, while warfarin had no effect. Both quercetin and curcumin decreased the CYP1A2 and CYP3A activity when co-administrated with warfarin.The results clearly demonstrated that quercetin and curcumin can cause food-drug interactions with warfarin, and that the cocktail effect of exposure to more compounds than one can further enhance these interactions.

Bovine Satellite Cells Isolated after 2 and 5 Days of Tissue Storage Maintain the Proliferative and Myogenic Capacity Needed for Cultured Meat Production.

Cultured meat is an emerging alternative food technology which aims to deliver a more ethical, sustainable, and healthy muscle-tissue-derived food item compared to conventional meat. As start-up companies are rapidly forming and accelerating this technology, many aspects of this multi-faceted science have still not been investigated in academia. In this study, we investigated if bovine satellite cells with the ability to proliferate and undergo myogenic differentiation could be isolated after extended tissue storage, for the purpose of increasing the practicality for cultured meat production. Proliferation of bovine satellite cells isolated on the day of arrival or after 2 and 5 days of tissue storage were analyzed by metabolic and DNA-based assays, while their myogenic characteristics were investigated using RT-qPCR and immunofluorescence. Extended tissue storage up to 5 days did not negatively affect proliferation nor the ability to undergo fusion and create myosin heavy chain-positive myotubes. The expression patterns of myogenic and muscle-specific genes were also not affected after tissue storage. In fact, the data indicated a positive trend in terms of myogenic potential after tissue storage, although it was non-significant. These results suggest that the timeframe of which viable myogenic satellite cells can be isolated and used for cultured meat production can be greatly extended by proper tissue storage.

Porcine cytochrome P450 3A: current status on expression and regulation.

The cytochrome P450s (CYPs) constitute a family of enzymes maintaining vital functions in the body and are mostly recognized for their significant role in detoxification. Of the CYP subfamilies, CYP3A, is one of the most active in the clearance of drugs and other xenobiotics. During the last decades, much focus has been on exploring different models for human CYP3A regulation, expression and activity. In that respect, the growing knowledge of the porcine CYP3As is of great interest. Although many aspects of porcine CYP3A regulation and activity are still unknown, the current literature provides a basic understanding of the porcine CYP3As that can be used e.g., when translating results from studies done in the porcine model into human settings. In this review, the current knowledge about porcine CYP3A expression, regulation, activity and metabolic significance are highlighted. Future research needs are also identified.

Correction to: Effects of multi-component mixtures from sewage treatment plant effluent on common carp (Cyprinus carpio) under fully realistic condition.

The original version of this Article unfortunately contained an error. The authors' given and family names were transposed erroneously. It has been corrected now in this Erratum.

Effects of Multi-Component Mixtures from Sewage Treatment Plant Effluent on Common Carp (Cyprinus carpio) under Fully Realistic Condition.

This study characterized changes in biomarker responses in common carp (Cyprinus carpio) upon exposure to effluent water discharged from a sewage treatment plant (STP) under real conditions. Fish were exposed to contamination in Cezarka pond, which receives all of its water input from the STP in the town of Vodnany, Czech Republic. Five sampling events were performed at day 0, 30, 90, 180, and 360 starting in April 2015. In total, 62 pharmaceutical and personal care products (PPCPs) were detected in the polar organic chemical integrative sampler. Compared to a control pond, the total concentration of PPCPs was 45, 16, 7, and 7 times higher in Cezarka pond at day 30, 90, 180, and 360, respectively. The result of oxidative stress and antioxidant enzyme biomarkers indicated alterations in the liver and intestine tissues of fish from Cezarka pond at day 30 and 360, respectively. High plasma vitellogenin levels were observed in both exposed females (180 and 360 days) and males (360 days) compared with their respective controls. However, only exposed female fish had higher vitellogenin mRNA expression than the control fish in these periods. Exposed female fish showed irregular structure of the ovary with scattered oocytes, which further developed to a vitellogenic stage at day 360. Low white blood cell levels were indicated in all exposed fish. Despite numerous alterations in exposed fish, favorable ecological conditions including high availability of food resulted in a better overall condition of the exposed fish after 1 year of exposure compared to the controls.

Induction of cytochrome P450 mRNA in porcine primary hepatocytes cultured under serum free conditions: Comparison of freshly isolated cells and cryopreserved.

Primary hepatocytes are widely used in the study of dynamic events like regulation of gene expression, as they are superior to most cell-lines. However, the culturing of the hepatocytes often results in loss of phenotype, e.g. the expression of the cytochrome p450s (CYP). The present study investigated the impact of serum in the culture medium of porcine primary hepatocytes (PPH) on markers of dedifferentiation as well as the impact on CYP induction. The effects were studied in both freshly isolated primary hepatocytes as well as cryopreserved. The exclusion of serum in the culturing media were not introducing significant dedifferentiation as judged by the gene expression of α-fetoprotein, albumin, glucose-6-phosphatase and the constitutive expression of selected transcription factors and CYP. The induction of CYP2B22 and CYP3A29 by phenobarbital and rifampicin, were greater in hepatocytes cultured without serum. The same were not observed for TCDD induced CYP1A2 expression. In conclusion, PPH cultured under serum free conditions results in little or no dedifferentiation, while being more responsive to known CYP inducers. Hence, it can be suggested that PPH cultured under serum free conditions provides a reliable hepatocyte model to investigate CYP gene regulation.

In vitro differentiation of progenitor cells isolated from juvenile pig hearts - expression of relevant gene and protein markers.

OBJECTIVES: Heart failure is a significant cause of mortality worldwide, and most current therapies treat only its symptoms. The results of cardiac stem cell research suggest a promising treatment option for heart failure, but there is currently an unmet demand for better research models. We have therefore, for the first time, isolated, expanded and differentiated progenitor cells obtained from juvenile pig hearts to use as a platform for cardiac stem cell research. DESIGN: Progenitor cells were isolated from the left ventricles of porcine hearts using collagenase enzymatic digestion and Percoll®-gradient centrifugation. Cells were proliferated in Matrigel®-coated wells. Cell differentiation was initiated by applying 5-azacytidine and subsequently controlled by modifying the serum concentration. Western blotting and qPCR were used to determine protein and gene expression, respectively. RESULTS: Cardiac-specific genes, from the following proteins: troponin I-3, and myosin-heavy-chain 7 were stably expressed during proliferation and differentiation. Connexin-43 was upregulated and Actinin alfa 2 was downregulated during differentiation. The immature-cardiomyocyte marker GATA binding protein 4 was stably expressed but with a decrease in expression at day 4 of differentiation. Smooth muscle actin decreased in expression and Von Willebrand factor were stably expressed during differentiation. Smooth muscle protein expression was documented but no expression of cardiac-specific proteins after differentiation was found. CONCLUSION: The isolated progenitor cells had key cardiac-lineage gene expression characteristics but they did not express cardiac-specific proteins. Smooth muscle protein was expressed confirming commitment to the smooth muscle lineage.

Co-treatment with indole-3-carbinol and resveratrol modify porcine CYP1A and CYP3A activities and expression.

1. Humans and animals are commonly exposed to indole-3-carbinol (I3C) and resveratrol (RES) via food or beverages. Moreover, these compounds have been demonstrated to potentially cause food-drug interactions. However, information about their combined effects is limited. Therefore, we investigated the effects of I3C and RES, both as single compounds and in combination, on cytochrome P450 1A and 3A activity and gene expression. 2. Using porcine microsomes, we demonstrated that RES caused non-competitive inhibition of CYP1A activity and un-competitive inhibition of CYP3A activity. Compared to the effect of single compounds, co-treatment with I3C and RES increased a degree of inhibition of CYP1A activity. 3. In porcine primary hepatocytes, treatment with I3C and RES resulted in induction of CYP1A1, CYP1A2 and CYP3A29 mRNA expression. 4. In conclusion, we demonstrated that both RES and I3C could cause food-drug interactions and that the combined effect could be more potent in doing so.

Tissue-specific regulation of CYP3A by hydrolysable tannins in male pigs.

1. Little is known about the activities and regulation of cytochrome P4503A (CYP3A) enzymes in porcine colon in response to specific feeding components. 2. We added hydrolyzable tannins to the diet of fattening boars and studied its effect on the expression of hepatic and intestinal CYP3A. 3. In total, 51 Landrace × Large White boars were assigned to the following treatment groups: control (without the addition of hydrolysable tannins), T1 (diet-containing 1% hydrolysable tannin extract), T2 (diet-containing 2% hydrolysable tannin extract) and T3 (diet-containing 3% hydrolysable tannin extract). CYP3A expression and activity were measured in microsomes prepared from liver and colon tissue. 4. CYP3A protein expression and activity were increased in the colon of pigs fed 2% and 3% tannins, while no changes were observed with lower tannin concentrations, or in the liver of any treatment groups. Also, it was demonstrated that colon mucosa possess CYP3A activity similar to that measured in the liver. 5. The present results provide the first evidence that tannin supplementation can modulate CYP3A in porcine colon mucosa in vivo. The physiological significance of this finding for the health status of the individual animal needs further investigation.

Comparison of secreted miRNAs and proteins during proliferation and differentiation of bovine satellite cells in culture implies potential roles in regulating myogenesis.

Cultivated meat is an emerging new technology to produce sustainable meat for the future. The common approach for cultivated meat, is the isolation of satellite cells from donor animals, followed by in vitro proliferation and differentiation into primitive muscle fibers. The transformation of satellite cells into myofibers is tightly orchestrated by intra-cellular signaling, while the inter-cellular signaling is less well understood. Thus, the current study was conducted to map the secretion of potential signaling molecules (MicroRNAs and proteins) during proliferation and differentiation. Primary cultures of satellite cells were grown to 50% and 80% confluence, representing the proliferative phase or serum-starved for 1 and 3 days to induce differentiation. Post incubation in FBS-free media, the media were collected and analyzed for miRNA and protein content using gene-arrays and LC-MS/MS, respectively. When comparing the miRNA secretome at 50% and 80% confluence, we observed four differentially expressed miRNA, while only five were differentially expressed when comparing Day 1 to Day 3. A subsequent in silico analysis suggested that pathways of importance for myogenesis, e.g., MAPK and AMPK signaling, could be regulated by the secreted miRNAs. In addition, >300 proteins were secreted, including insulin-like growth factor 1 binding proteins 2, 3, 4, 5 and 6. In conclusion, this study demonstrated differential secretion of several miRNAs and proteins during both proliferation and differentiation of bovine satellite cells in vitro.

Regulation of 3ß-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase: a review.

This review focuses on the expression and regulation of 3ß-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3ß-HSD), with emphasis on the porcine version. 3ß-HSD is often associated with steroidogenesis, but its function in the metabolism of both steroids and xenobiotics is more obscure. Based on currently available literature covering humans, rodents and pigs, this review provides an overview of the present knowledge concerning the regulatory mechanisms for 3ß-HSD at all omic levels. The HSD isoenzymes are essential in steroid hormone metabolism, both in the synthesis and degradation of steroids. They display tissue-specific expression and factors influencing their activity, which therefore indicates their tissue-specific responses. 3ß-HSD is involved in the synthesis of a number of natural steroid hormones, including progesterone and testosterone, and the hepatic degradation of the pheromone androstenone. In general, a number of signaling and regulatory pathways have been demonstrated to influence 3ß-HSD transcription and activity, e.g., JAK-STAT, LH/hCG, ERα, AR, SF-1 and PPARα. The expression and enzymic activity of 3ß-HSD are also influenced by external factors, such as dietary composition. Much of the research conducted on porcine 3ß-HSD is motivated by its importance for the occurrence of the boar taint phenomenon that results from high concentrations of steroids such as androstenone. This topic is also examined in this review.

Hepatic PGC-1α has minor regulatory effect on the liver transcriptome and metabolome during high fat high fructose diet and exercise training.

The prevalence of non-alcoholic fatty liver diseases (NAFLD) has reached epidemic levels during recent years and a major driver of NAFLD are diets high in fat and fructose. A common practice in the treatment of NAFLD are life-style interventions including for example increased physical activity. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) has been shown to be central in mediating the beneficial effects of exercise training by regulating the expression of key metabolic genes. However, the significance of hepatic PGC-1α for high fat high fructose (HFFD) induced changes in gene expression and metabolites associated with NAFLD has not been elucidated. Therefore the aim of the present study was to investigate the effect of hepatic PGC-1α on HFFD and exercise-induced changes in the hepatic transcriptome and metabolome in mice. Using gene-arrays and 1H NMR spectroscopy, the liver transcriptome and metabolome of liver-specific PGC-1α knock-out mice receiving either standard chow, HFFD or HFFD + exercise (HFFD + Ex) were determined. In total 122 genes were identified as differently expressed in mice receiving HFFD for 13 weeks compared to chow, while the loss of hepatic PGC-1α only had very minor effects on the transcriptome. The same was observed for the liver metabolome. The effect of 4 weeks exercise training in combination with 13 weeks of HFFD, had small effects on the transcriptome and metabolome compared to HFFD alone. Together our results highlight a minor regulatory effect of hepatic PGC-1α on the liver transcriptome during high fat high fructose diet and exercise training.

A simple and robust serum-free media for the proliferation of muscle cells.

Cultivated meat production requires an efficient, robust and highly optimized serum-free cell culture media for the needed upscaling of muscle cell expansion. Existing formulations of serum-free media are complex, expensive and have not been optimized for muscle cells. Thus, we undertook this work to develop a simple and robust serum-free media for the proliferation of bovine satellite cells (SCs) through Design of Experiment (DOE) and Response Surface Methodology (RSM) using precise and high-throughput image-based cytometry. Proliferative attributes were investigated with transcriptomics and long-term performance was validated using multiple live assays. Here we formulated a media based on three highly optimized components; FGF2 (2 ng/mL), fetuin (600 µg/mL) and BSA (75 µg/mL) which together with an insulin-transferrin-selenium (1x) supplement, sustained the proliferation of bovine SCs, porcine SCs and murine C2C12 muscle cells. Remarkably, cells cultured in our media named Tri-basal 2.0+ performed better than cell cultured in 10% FBS, with respect to proliferation. Hence, the optimized Tri-basal 2.0+ enhanced serum-free cell attachment and long-term proliferation, providing an alternative solution to the use of FBS in the production of cultivated meat.

Inulin Supplementation Modulates the Hepatic Transcriptome, Metabolome, and Ferritin Content in Ovariectomized Rats.

SCOPE: Liver is an important metabolic organ regulating whole-body homeostasis. This study aims to investigate how prebiotic-induced changes in the metabolic activity of the gut microbiome (GM) and dietary calcium depletion modulates the hepatic metabolome and transcriptome. METHODS AND RESULTS: The serum metabolome, liver metabolome, and transcriptome are determined on samples from ovariectomized (OVX) rats fed a control diet (Control, n = 7), a control diet supplemented with 5% w/w inulin (Inulin, n = 7), or a calcium-deficient diet (CaDef, n = 7). Inulin fortification is associated with higher serum concentrations of acetate, 3-hydroxybutyrate, and reduced concentration of dimethyl sulfone, revealing that changes in the metabolic activity of the GM are reflected in circulating metabolites. Metabolomics also reveal that the inulin-fortified diet results in lower concentrations of hepatic glutamate, serine, and hypoxanthine while transcriptomics reveal accompanying effects on the hepatic expression of ferric iron binding-related genes. Inulin fortification also induces effects on the hepatic expression of genes involved in olfactory transduction, suggesting that prebiotics regulate liver function through yet unidentified mechanisms involving olfactory receptors. CONCLUSION: Inulin ingestion impacts hepatic gene expression and is associated with an upregulation of ferritin synthesis-related genes and liver ferritin content.

Expression of hepatic 3ß-hydroxysteroid dehydrogenase and sulfotransferase 2A1 in entire and castrated male pigs.

The present study investigated the effect of surgical (SC) and immunological castration on the steroid metabolizing enzymes 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and sulfotransferase 2A1 (SULT2A1) in male pigs. Thirty-two male pigs were divided in four groups; in one group the pigs were SC before the age of 7 days, two groups were injected with Improvac(®) a vaccine against gonadotropin releasing hormone (immunological castration), while the pigs in the last group remained entire males (EMs). Immunological castration was in one group performed by vaccine injection at ages 11 and 14 weeks, while the other group received injections at ages 17 and 21 weeks. Plasma, adipose and liver tissue were collected at the time of slaughter. Plasma was analyzed for concentrations of testosterone and oestradiol. The adipose tissue was analyzed for the concentration of androstenone, while the liver tissue was analyzed for mRNA and protein expression of 3ß-HSD and SULT2A1. Independent of method, all castrated pigs showed greater mRNA and protein expression of 3ß-HSD and lower levels of all steroids in plasma compared with EMs. Moreover, there was a strong correlation between mRNA and protein expression of 3ß-HSD and steroid levels. The same was not valid for expression of SULT2A1. It is concluded that steroid levels can increase expression of the steroid metabolizing enzyme 3ß-HSD and thereby influence steroid metabolism, e.g. of androstenone.

Time-dependent regulation of hepatic cytochrome P450 mRNA in male liver-specific PGC-1α knockout mice.

The circadian rhythm has profound effect on the body, exerting effects on diverse events like sleep-wake patterns, eating behavior and hepatic detoxification. The cytochrome p450 s (Cyps) is the main group of enzymes responsible for detoxification. However, the underlying mechanisms behind circadian regulation of the Cyps are currently not fully clarified. Therefore, the aim of the present study was to investigate the requirement of hepatic peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) for the circadian regulation of the hepatic expression of Cyp1-4 using liver-specific PGC-1α knockout (LKO) mice and littermate controls. The circadian regulator genes Bmal1 and Clock displayed decreased mRNA content at zeitgeber time (ZT) 12, compared to ZT-2 and the mRNA content of Cyp2a4 and Cyp2e1 was higher at ZT-12 than at ZT-2. Moreover, the increase in Cyp2e1 mRNA content was not observed in the PGC-1α LKO mice and hepatic PGC-1α deficiency tended to blunt the rhythmic expression of Clock and Bmal1. However, no circadian regulation was evident at the protein level for the investigated Cyps except for a change in Cyp2e1 protein content in the LKO mice. Of the measured transcription factors, only, the mRNA content of peroxisome proliferator-activated receptor α, showed rhythmic expression. To further analyze the difference between the control and LKO mice, principal component analysis were executed on the mRNA data. This demonstrated a clear separation of the experimental groups with respect to ZT and genotype. Our finding provides novel insight into the role of hepatic PGC-1α for basic and circadian expression of Cyps in mouse liver. This is important for our understanding of the molecular events behind circadian Cyp regulation and hence circadian regulation of hepatic detoxification capacity.

Effects of Calcium Source, Inulin, and Lactose on Gut-Bone Associations in an Ovarierectomized Rat Model.

SCOPE: Osteoporosis poses a health challenge especially for postmenopausal women. This study aims to explore nutritional strategies to counteract bone demineralization in ovarierectomized (OVX) rats. METHODS AND RESULTS: OVX rats (n = 49) are fed with one of six different diets, where two different calcium sources (dairy calcium or calcium carbonate) are provided alone or in combination with either inulin (5%) or lactose (0.5%). In addition, a calcium-deficient diet is included. Calcium supplementation increases intestinal concentrations of short-chain fatty acids (SCFAs) and the abundance of fecal Acinetobacter and Propionibacterium. Accompanied with these effects, rats fed with calcium-fortified diets have higher bone mineral density, bone mineral content and femur mechanical strength, lower serum levels of bone markers, and lower expression of calcium absorption-related genes (transient receptor potential vanilloid type 6 (TRPV6), calcium-binding protein (CaBP) compared with control. Inulin supplementation results in a markedly increased production of intestinal SCFAs, a decreased intestinal pH, an increased abundance of Allobaculum and Bifidobacterium, and an increased expression of Trpv6. Inulin and lactose show beneficial effects on spine bone. CONCLUSION: Calcium modulates gut microbiome composition and function. A pronounced effect of inulin on metabolic activity in the gastrointestinal tract is evident, and lactose supplementation decreases jejunal pH that might be associated with slightly enhanced bone mineralization.

Recombinant production of growth factors for application in cell culture.

Culturing eukaryotic cells has widespread applications in research and industry, including the emerging field of cell-cultured meat production colloquially referred to as "cellular agriculture". These applications are often restricted by the high cost of growth medium necessary for cell growth. Mitogenic protein growth factors (GFs) are essential components of growth medium and account for upwards of 90% of the total costs. Here, we present a set of expression constructs and a simplified protocol for recombinant production of functionally active GFs, including FGF2, IGF1, PDGF-BB, and TGF-ß1 in Escherichia coli. Using this E. coli expression system, we produced soluble GF orthologs from species including bovine, chicken, and salmon. Bioactivity analysis revealed orthologs with improved performance compared to commercially available alternatives. We estimated that the production cost of GFs using our methodology will significantly reduce the cost of cell culture medium, facilitating low-cost protocols tailored for cultured meat production and tissue engineering.