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.

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.

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.

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.

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.

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.

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.

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.

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.

Primary hepatocytes isolated from human and porcine donors display similar patterns of cytochrome p450 expression following exposure to prototypical activators of AhR, CAR and PXR.

The hepatic cytochrome p450's (CYP) are of major importance for the metabolism of xenobiotics and knowledge about their regulation is crucial. This knowledge often originates from cell models; primary human hepatocytes (PHH) being the gold standard. However, due to limited availability of high-quality human donor organs, basic knowledge on alternative models are needed. Primary porcine hepatocytes (PPH) have been suggested as an alternative to PHH. Unfortunately, data comparing the response in gene-transcription to standard CYP inducers between PHH and PPH are missing. In the present study we, cultured PHH and PPH under the same conditions, treated them with standard inducers of the CYP1-3 and determined the response in gene and protein expression. The results demonstrated that in both species TCDD and omeprazole caused an increase in CYP1A/B expression. In PPH, CITCO increased the content of CYP1A/B. For the CYP2B/C/D's, phenobarbital and rifampicin caused increases in expression. For the CYP2D's, TCDD and omeprazole caused increased gene expression in PPH, which were not the case for PHH. Both phenobarbital, rifampicin and omeprazole increased CYP3A expression in PHH and PPH. Moreover, TCDD increased the gene expression of CYP3A in PPH; this was not the case for PHH. Multivariate data analysis found no difference in gene expression between PHH and PPH for phenobarbital, rifampicin and CITCO. However, differential clustering was observed for TCDD and omeprazole. In conclusion, despite model specificity, there are a high number of similar responses, and experiments investigating mRNA regulation made in PPH permits for a reliable translation into human setting.

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.

Tissue-specific expression and activity of cytochrome P450 1A and 3A in rainbow trout (Oncorhynchus mykiss).

Piscine cytochrome P450 (CYP) enzymes play an important role in the metabolism of xenobiotics. Xenobiotics often act as inducers of CYP1A1 and CYP3A expression and activity in fish. We compared constitutive mRNA expression of CYP1A1, CYP3A27, and CYP3A45 and catalytic activity of CYP1A (7-ethoxyresorufin-O-deethylation, EROD) and CYP3A-like (benzyloxy-4-trifluoromethylcoumarin-O-debenzyloxylation, BFCOD) enzymes in the following six rainbow trout tissues: liver, gill, heart, brain, intestine, and gonad. mRNA expression and activity were present in all investigated tissues. The CYP1A1 mRNA expression was higher in the liver, gill, heart, and brain compared to gonad and intestine. The intestine was the main site of CYP3A27 and CYP3A45 expression. The highest EROD and BFCOD activity was observed in liver tissue followed in descending order by heart, brain, gill, intestine, and gonad. Such differences might be related to the role of CYP physiological functions in the specific tissue. Rainbow trout exposure to 50 mg/kg of ß-naphthoflavone for 48 h resulted in a 7.5- and 5.9-fold increase in liver EROD and BFCOD activity, respectively. In vitro EROD activity inhibition with ellipticine showed tissue-specific inhibition, while ketoconazole decreased BFCOD activity by 50-98 % in all tissues. Further studies are needed to identify all CYP isoforms that are responsible for these activities and modes of regulation.

Brain foods - the role of diet in brain performance and health.

The performance of the human brain is based on an interplay between the inherited genotype and external environmental factors, including diet. Food and nutrition, essential in maintenance of brain performance, also aid in prevention and treatment of mental disorders. Both the overall composition of the human diet and specific dietary components have been shown to have an impact on brain function in various experimental models and epidemiological studies. This narrative review provides an overview of the role of diet in 5 key areas of brain function related to mental health and performance, including: (1) brain development, (2) signaling networks and neurotransmitters in the brain, (3) cognition and memory, (4) the balance between protein formation and degradation, and (5) deteriorative effects due to chronic inflammatory processes. Finally, the role of diet in epigenetic regulation of brain physiology is discussed.

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.

Background Diet Influences TMAO Concentrations Associated with Red Meat Intake without Influencing Apparent Hepatic TMAO-Related Activity in a Porcine Model.

Red meat has been associated with an increased cardiovascular disease (CVD) risk, possibly through gut microbial-derived trimethylamine-N-oxide (TMAO). However, previous reports are conflicting, and influences from the background diet may modulate the impact of meat consumption. This study investigated the effect of red and white meat intake combined with two different background diets on urinary TMAO concentration and its association with the colon microbiome in addition to apparent hepatic TMAO-related activity. For 4 weeks, 32 pigs were fed chicken or red and processed meat combined with a prudent or western background diet. 1H NMR-based metabolomics analysis was conducted on urine samples and hepatic mRNA expression of TMAO-related genes determined. Lower urinary TMAO concentrations were observed after intake of red and processed meat when consumed with a prudent compared to a western background diet. In addition, correlation analyses between urinary TMAO concentrations and relative abundance of colon bacterial groups suggested an association between TMAO and specific bacterial taxa. Diet did not affect the hepatic mRNA expression of genes related to TMAO formation. The results suggest that meat-induced TMAO formation is regulated by mechanisms other than alterations at the hepatic gene expression level, possibly involving modulations of the gut microbiota.

Differentially expressed marker genes and glycogen levels in pectoralis major of Ross308 broilers with wooden breast syndrome indicates stress, inflammation and hypoxic conditions.

The occurrence of wooden breast (WB) in broiler production is increasing, but onset of its development is only described in part. In this study, we determined the regulation of marker genes related to oxidative stress in Ross308 broilers categorized as no-, mild- or severe-WB, on days 21 and 30 of production. The biochemical parameters, lactate dehydrogenase and pro- and macro-glycogen, were also determined. On day 21, breast meat from birds affected severely by WB had increased mRNA abundances of heat-shock protein 70, heme-oxygenase 1, cyclooxygenase 2, tumor necrosis factor 1, and hypoxia inducible factors as well as higher pH and lower dry matter contents. On day 30, breast meat from both mild and severely affected birds had increased mRNA for heme oxygenase 1, lactate dehydrogenase, and hypoxia inducible factor. Moreover, pro- and micro-glycogen, as well as the total pool of glycogen, were decreased compared with the non-WB birds. In conclusion, this study indicates oxidative stress, inflammation and hypoxic conditions in WB.

Hepatic PGC-1α is not essential for fasting-induced cytochrome p450 regulation in mouse liver.

Fasting has been shown to regulate the expression of the cytochrome p450 (CYP) enzyme system in the liver. However, the exact mechanism behind the fasting-induced regulation of the CYP's remains unknown. In the present study we tested the hypothesis that the peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), which is a key-regulator of energy metabolism, is responsible for the fasting-induced regulation of the CYP's. Lox/lox and liver specific PGC-1α (LKO) mice of both sexes, fasted for 18 h and the content of the CYP's as well as the hepatic metabolome was assessed. Fasting increased the mRNA content of Cyp2a4, Cyp2e1, Cyp3a11 and Cyp4a10. The fasting-induced response in Cyp4a10 mRNA content was different between lox/lox and LKO mice, while the absence of PGC-1α had no effect on the fasting-induced response for the other Cyp's. Moreover, the fasting-induced response in mRNA content of Sirtinus 1 and Perilipin 2 was different between lox/lox and LKO mice. Only the CYP1A isoform showed a fasting-induced response at the protein level. Absence of hepatic PGC-1α had no effect on the apparent metabolome, where fasting vs fed was the only discriminate in the following multivariate analysis. In conclusion, hepatic PGC-1α is not essential for the fasting-induced regulation of hepatic CYP's.

Sex dictates the constitutive expression of hepatic cytochrome P450 isoforms in Göttingen minipigs.

The cytochrome P450 enzyme (CYP) family includes key enzymes for the metabolism of drugs and xenobiotics. Several animal models have been used to determine the metabolite profile of specific drugs. Among these are porcine microsomes prepared from Göttingen minipigs. However, CYP expression profile in microsomes from this pig breed is unknown. In the present study, we determined the mRNA and protein profiles of a comprehensive selection of CYPs in microsomes prepared from male and female Göttingen minipigs. Using RT-PCR, western blotting and mass spectroscopy, we found that the expression levels of CYP1A, CYP2A and CYP2E1 were significantly higher in females than males. Moreover, some of the transcription factors controlling CYP transcription also showed a sex-dependent expression pattern. Conversely, expression of CYP2B, CYP2D and CYP3A was comparable between sexes. The overall CYP expression distribution showed high similarity with what previously been reported in humans. In conclusion, our results suggest that Göttingen minipigs are a reliable model for studying CYPs.

Supplementation of sows with L-Arginine during gestating and lactation affects muscle traits of offspring related with postnatal growth and meat quality: From conception to consumption.

This study investigated the effect of dietary inclusion of 25 g/day of L-Arginine (n = 7) or iso­nitrogenous amounts of alanine (n = 6) from d 30 of gestation to d 28 of lactation of sows on performance, muscle traits and meat quality in offspring. From each litter, heaviest and smallest littermate of both sexes were reared from d 28 and slaughtered at d 140 in accordance with a 23factorial design. A response to L-Arginine were obtained on small females where L-Arginine increased birth weight, however this effect disappeared at weaning. L-Arginine increased daily gain by 7% and increased the cross-sectional area of the M. semitendinosus in small females by 14%, suggesting an increased lean ratio. Mechanistic studies showed firstly, that small female littermates had increased number of muscle fibres (myogenesis) after L-Arginine treatment (11%) and secondly increased total DNA (12%) as a consequence of satellite cell proliferation. Traits describing tenderness seem to be affected by L-Arginine but further studies are needed.