Hygiene of housing conditions and proinflammatory signals alter gene expressions in porcine adipose tissues and blood cells.

Adipose tissue is an organ with metabolic, endocrine and immune functions. In this tissue, the expressions of genes associated with several metabolic pathways, including lipid metabolism, have been shown to be affected by genetic selection for feed efficiency, an important trait to consider in livestock. We hypothesized that the stimulation of immune system caused by poor hygiene conditions of housing impacts the molecular and cellular features of adipose tissue and that the impact may differ between pigs that diverge in feed efficiency. At the age of 12 weeks, Large White pigs from two genetic lines divergent for residual feed intake (RFI) were housed in two contrasting hygiene conditions (good vs poor). After six weeks of exposure, pigs were slaughtered (n = 36). Samples of blood, subcutaneous (SCAT) and perirenal (PRAT) adipose tissues were collected for cell response and gene expression investigations. The decrease in the relative weight of PRAT was associated with a decline in mRNA levels of FASN, ME, LCN2 and TLR4 (P < 0.05) in pigs housed in poor conditions compared with pigs housed in good conditions for both RFI lines. In SCAT, the expressions of only two key genes (PPARG and TLR4) were significantly affected by the hygiene of housing conditions. Besides, the mRNA levels of both LCN2 and GPX3 were influenced by the RFI line (P < 0.05). Because we suspected an effect of poor hygiene at the cellular levels, we investigated the differentiation of stromal vascular cells isolated from SCAT in vitro in the absence or presence of a pro-inflammatory cytokine, Tumor Necrosis Factor-α (TNF-α). The ability of these cells to differentiate in the absence or presence of TNF-α did not differ among the four groups of animals (P > 0.05). We also investigated the expressions of genes involved in the immune response and lipid metabolism in whole blood cells cultured in the absence and presence of LPS. The hygiene conditions had no effect but, the relative expression of the GPX3 gene was higher (P < 0.001) in high RFI than in low RFI pigs while the expressions of IL-10 (P = 0.027), TGFß1 (P = 0.023) and ADIPOR2 (P = 0.05) genes were lower in high RFI than in low RFI pigs. Overall, the current study indicates that the hygiene of housing had similar effects on both RFI lines on the expression of genes in adipose tissues and on the features of SCAT adipose cells and whole blood cells in response to TNF-α and LPS. It further demonstrates that the number of genes with expression impacted by housing conditions was higher in PRAT than in SCAT. It suggests a depot-specific response of adipose tissue to the current challenge.

Improving maternal welfare during gestation has positive outcomes on neonatal survival and modulates offspring immune response in pigs.

Improving the housing of pregnant sows by giving them more space and access to deep straw had positive effects on their welfare, influenced their maternal behavior and improved the survival of their offspring. The present study aimed at determining whether these effects were actually due to environmental enrichment and whether the provision of straw pellets and wood can partly mimic the effects of straw bedding during gestation. Three graded levels of enrichment were used, that were, collective conventional pens on slatted floor (C, n = 26), the same pens with manipulable wood materials and distribution of straw pellets after the meals (CE, n = 30), and larger pens on deep straw litter (E, n = 27). Sows were then housed in identical farrowing crates from 105 days of gestation until weaning. Decreased stereotypies, blood neutrophils, and salivary cortisol, and increased behavioral investigation indicated that health and welfare of sows during gestation were improved in the E environment compared with the C environment. The CE sows responded as C or E sows depending on the trait. Piglet mortality rate in the first 12 h after birth was lower in E and CE litters than in C litters, but enrichment level during gestation had only small effects on lactating sow behavior and milk composition postpartum. On days 2 and 3 of lactation, E sows interrupted less often their nursing sequences than C and CE sows. On day 2, milk from both E and CE sows contained more minerals than that from C sows. In one-day-old piglets, the expression levels of genes encoding toll-like receptors (TLR2, TLR4) and cytokines (interleukin-1, -6 and -10) in whole blood after 20-h culture, were greater in E piglets than in CE or C piglets. In conclusion, housing sows in an enriched environment during gestation improved early neonatal survival, probably via moderate and cumulative positive effects on sow behavior, milk composition, and offspring innate immune response. The gradation in the effects observed in C, CE and E housing environment reinforced the hypothesis of a causal relationship between maternal environmental enrichment, sow welfare and postnatal piglet traits.

3D in vitro models of skeletal muscle: myopshere, myobundle and bioprinted muscle construct.

Typical two-dimensional (2D) culture models of skeletal muscle-derived cells cannot fully recapitulate the organization and function of living muscle tissues, restricting their usefulness in in-depth physiological studies. The development of functional 3D culture models offers a major opportunity to mimic the living tissues and to model muscle diseases. In this respect, this new type of in vitro model significantly increases our understanding of the involvement of the different cell types present in the formation of skeletal muscle and their interactions, as well as the modalities of response of a pathological muscle to new therapies. This second point could lead to the identification of effective treatments. Here, we report the significant progresses that have been made the last years to engineer muscle tissue-like structures, providing useful tools to investigate the behavior of resident cells. Specifically, we interest in the development of myopshere- and myobundle-based systems as well as the bioprinting constructs. The electrical/mechanical stimulation protocols and the co-culture systems developed to improve tissue maturation process and functionalities are presented. The formation of these biomimetic engineered muscle tissues represents a new platform to study skeletal muscle function and spatial organization in large number of physiological and pathological contexts.

Autophagy in farm animals: current knowledge and future challenges.

Autophagy (a process of cellular self-eating) is a conserved cellular degradative process that plays important roles in maintaining homeostasis and preventing nutritional, metabolic, and infection-mediated stresses. Surprisingly, little attention has been paid to the role of this cellular function in species of agronomical interest, and the details of how autophagy functions in the development of phenotypes of agricultural interest remain largely unexplored. Here, we first provide a brief description of the main mechanisms involved in autophagy, then review our current knowledge regarding autophagy in species of agronomical interest, with particular attention to physiological functions supporting livestock animal production, and finally assess the potential of translating the acquired knowledge to improve animal development, growth and health in the context of growing social, economic and environmental challenges for agriculture.Abbreviations: AKT: AKT serine/threonine kinase; AMPK: AMP-activated protein kinase; ASC: adipose-derived stem cells; ATG: autophagy-related; BECN1: beclin 1; BNIP3: BCL2 interacting protein 3; BVDV: bovine viral diarrhea virus; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CMA: chaperone-mediated autophagy; CTSB: cathepsin B; CTSD: cathepsin D; DAP: Death-Associated Protein; ER: endoplasmic reticulum; GFP: green fluorescent protein; Gln: Glutamine; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; IF: immunofluorescence; IVP: in vitro produced; LAMP2A: lysosomal associated membrane protein 2A; LMS: lysosomal membrane stability; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MDBK: Madin-Darby bovine kidney; MSC: mesenchymal stem cells; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; NBR1: NBR1 autophagy cargo receptor; NDV: Newcastle disease virus; NECTIN4: nectin cell adhesion molecule 4; NOD1: nucleotide-binding oligomerization domain 1; OCD: osteochondritis dissecans; OEC: oviduct epithelial cells; OPTN: optineurin; PI3K: phosphoinositide-3-kinase; PPRV: peste des petits ruminants virus; RHDV: rabbit hemorrhagic disease virus; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy.

Response of adult stem cell populations to a high-fat/high-fiber diet in skeletal muscle and adipose tissue of growing pigs divergently selected for feed efficiency.

PURPOSE: The control of body composition by genetics and dietary nutrients is of the upmost importance for both human and animal physiology. Adult stem cells (aSC) may represent a relevant level of tissue adaptation. The purpose of this study was to determine the impact of macronutrient composition on aSC populations isolated from adipose tissue or muscle in growing pigs. METHODS: Pigs from two lines divergently selected for feed efficiency were fed ad libitum either a high-fat/high-fiber (HF) diet or a low-fat/low-fiber (LF) diet (n = 6 per line and diet) from 74 to 132 days of age. Stroma vascular cells were isolated from adipose tissue and muscle and characterized with cell surface markers. RESULTS: In both lines, pigs fed the HF diet exhibited a reduced adiposity (P < 0.001) compared with pigs fed the LF diet. In the four groups, CD90 and PDGFRα markers were predominantly expressed in adipose cells, whereas CD90 and CD56 markers were highly expressed in muscle cells. In adipose tissue, the proportions of CD56+/PDGFRα + and of CD90+/PDGFRα + cells were lower (P < 0.05) in HF pigs than in LF pigs. On the opposite, in muscle, these proportions were higher (P < 0.001) in HF pigs. CONCLUSION: This study indicates that dietary nutrients affected the relative proportions of CD56+/PDGFRα + cells with opposite effects between muscle and adipose tissue. These cell populations exhibiting adipogenic potential in adipose tissue and myogenic potential in muscle may be a target to modulate body composition.

Effect of the flavonoid baicalin on the proliferative capacity of bovine mammary cells and their ability to regulate oxidative stress.

BACKGROUND: High-yielding dairy cows are prone to oxidative stress due to the high metabolic needs of homeostasis and milk production. Oxidative stress and inflammation are tightly linked; therefore, anti-inflammatory and/or natural antioxidant compounds may help improve mammary cell health. Baicalin, one of the major flavonoids in Scutellaria baicalensis, has natural antioxidant and anti-inflammatory properties in various cell types, but its effects on bovine mammary epithelial cells (BMECs) have not been investigated. METHODS: Explants from bovine mammary glands were collected by biopsy at the peak of lactation (approximately 60 days after the start of lactation) (n = three animals) to isolate BMECs corresponding to mature secretory cells. Cell viability, apoptosis, proliferative capacity and reactive oxygen species (ROS) production by BMECs were measured after increasing doses of baicalin were added to the culture media in the absence or presence of H2O2, which was used as an in vitro model of oxidative stress. RESULTS: Low doses of baicalin (1-10 µg/mL) had no or only slightly positive effects on the proliferation and viability of BMECs, whereas higher doses (100 or 200 µg/mL) markedly decreased BMEC proliferation. Baicalin decreased apoptosis rate at low concentrations (10 µg/mL) but increased apoptosis at higher doses. ROS production was decreased in BMECs treated with increasing doses of baicalin compared with untreated cells, and this decreased production was associated with increased intracellular concentrations of catalase and NRF-2. Irrespective of the dose, baicalin pretreatment attenuated H2O2-induced ROS production. DISCUSSION: These results indicate that baicalin exerts protective antioxidant effects on bovine mammary cells. This finding suggests that baicalin could be used to prevent oxidative metabolic disorders in dairy cows.

Improving pork quality traits by a short-term dietary hydroxy methionine supplementation at levels above growth requirements in finisher pigs.

The effects of dietary methionine (Met) supplies above growth requirements on tissue biology and pork quality were studied. At 70 kg, 45 crossbred pigs were fed a control (CONT) diet adequate in Met (0.22% Met) up to 105 kg. For the last 14 days before slaughter, pigs were fed with the CONT diet or with diets where the Met level was increased to Met3 (0.66% Met) or Met5 (1.10% Met). Growth performance and carcass composition did not change with the treatment. Pigs fed the Met5 treatment displayed lower TBARS and higher glutathione levels (P ≤ .05), along with higher ultimate pH (P < .01) and lower drip, lightness and hue (P ≤ .10) in the longissimus muscle, compared to the CONT and Met3 pigs. Extra-dietary Met improved ham's technological quality in the Met3 and Met5 groups (P ≤ .05). Thus, dietary Met supplementation improves pork quality without impairing growth or carcass traits.

Methionine and cysteine deficiencies altered proliferation rate and time-course differentiation of porcine preadipose cells.

Methionine (Met) is an essential sulfur amino acid (AA) limiting growth and is the precursor of cysteine (Cys), the rate-limiting factor in the synthesis of glutathione, and the main intracellular non-enzymatic antioxidant. This study aimed at determining the effects of limited supplies in Met and(or) Cys in early aspects of adipose tissue development and oxidative stress in differentiated adipocytes. Incremental reductions in Met (70, 40, and 0 µM) were compared with Met 100 µM (control dose) in porcine preadipocytes cultured in media without or with Cys (250 µM). In Cys-deprived media, both the absence (0 µM) and the lowest dose of Met (40 µM) reduced preadipocyte proliferation. Adding Cys in media only partly compensated for this decrease. On the opposite, mild Met deficiency (70 µM) did not alter preadipocyte proliferation in media without or with Cys. Strong Met deficiency (40 µM) also reduced differentiation and lipid accumulation into preadipose cells. Mild Met deficiency also reduced preadipocyte differentiation when Cys was present in the culture media, whereas in Cys-deprived media, percent of differentiated cell was similar and intracellular lipid content was slightly higher at Met 70 µM than at Met 100 µM. Finally, incremental reductions in Met in media with or without Cys lowered reactive oxygen species (ROS) production by differentiated cells. These results demonstrate the strong dependency of porcine adipogenesis to sulfur AA supplies. Strong Met deficiency decreases both proliferation and differentiation, whereas mild deficiency only alters differentiation.

Mammary Epithelial Cell Hierarchy in the Dairy Cow Throughout Lactation.

The plasticity of the mammary gland relies on adult mammary stem cells (MaSCs) and their progenitors, which give rise to various populations of mammary epithelial cells (MECs). To face global challenges, an in-depth characterization of milk-producing animal mammary gland plasticity is required, to select more sustainable and robust dairy cows. The identification and characterization of MaSC and their progenitors will also provide innovative tools in veterinary/human medicine regarding mammary tissue damage (carcinogenesis, bacterial infections). This study aimed to determine the dynamics of mammary cell populations throughout a lactation cycle. Using mammary biopsies from primiparous lactating dairy cows at 30, 90, 150, and 250 days of lactation, we phenotyped cell populations by flow cytometry. To investigate cell lineages, we used specific cell-surface markers, including CD49f, CD24, EpCAM (epithelial cell adhesion molecule), and CD10. Two cell populations linked to milk production were identified: CD49f(+)/EpCAM(-) (y = 0.88x + 4.42, R(2) = 0.36, P < 0.05) and CD49f(-)/EpCAM(-) (y = -1.15x + 92.44, R(2) = 0.51, P < 0.05) cells. Combining immunostaining analysis, flow cytometry, daily milk production data, and statistical approaches, we defined a stem cell population (CD24(+)/CD49f(+)) and four progenitor cell populations that include bipotent luminal progenitors (CD24(-)/CD49f(+)), lumino-alveolar progenitors (CD24(-)/EpCAM(+)), myoepithelial progenitors (CD24(+)/CD10(-)), and lumino-ductal progenitors (CD49f(-)/EpCAM(+)). Interestingly, we found that the bipotent luminal progenitors (CD24(-)/CD49f(+)) decreased significantly (P < 0.05) during lactation. This study provides the first results of mammary cell lineage, allowing insight into mammary cell plasticity during lactation.

Phenotypic and functional characterization of two bovine mammary epithelial cell lines in 2D and 3D models.

Immortalized bovine mammary epithelial cells (BME-UV1) and immortalized bovine mammary alveolar cells (MAC-T) have been extensively used as in vitro cell models to understand milk production in dairy cows. Precise knowledge about their phenotype and performance remains, however, unknown. This study aims to characterize MAC-T and BME-UV1 profiles when cultured in two-dimensional adherent, three-dimensional adherent (Matrigel), and three-dimensional no adherent [ultralow attachment (ULA)] supports. MAC-T and BME-UV1 were compared according to their proliferation capacities and to specific cell surface markers CD24, CD326 [epithelial cell adhesion molecule (EpCAM)], CD10, and integrin CD49f (α-6). Cytokeratin (CK14 and CK19), signal transducer and activator of transcription 5, and other proteins (occludin and cadherin-1) were analyzed. BME-UV1 in ULA support expressed higher CD49f marker. A different intensity of CD49 staining allowed the discrimination between the two cell lines in adherent condition. CD10, EpCAM, and CK19 expressions show that BME-UV1 cells have luminal capacity, while MAC-T has a myoepithelial profile with a high expression of CK14. BME-UV1 cells possess a closer committed progenitor profile due to their higher expression in aldehyde dehydrogenase and EpCAM. We observed that BME-UV1 cells have a better capacity to form spherical structures, mammospheres, in Matrigel than MAC-T, which was confirmed by the higher mammosphere area. In the ULA condition, BME-UV1 proliferated over the 6 days of culture. Taken together, our results clearly confirm the BME-UV1 luminal profile and MAC-T ductal/myoepithelial-like phenotype.

Flow Cytometry Approach to Quantify the Viability of Milk Somatic Cell Counts after Various Physico-Chemical Treatments.

Flow cytometry has been used as a routine method to count somatic cells in milk, and to ascertain udder health and milk quality. However, few studies investigate the viability of somatic cells and even fewer at a subpopulation level to follow up how the cells can resist to various stresses that can be encountered during technological processes. To address this issue, a flow cytometry approach was used to simultaneously identify cell types of bovine milk using cell-specific antibodies and to measure the cell viability among the identified subpopulations by using a live/dead cell viability kit. Confirmation of the cell viability was performed by using conventional microscopy. Different physico-chemical treatments were carried out on standardized cell samples, such as heat treatment, various centrifugation rates and storage in milk or in PBS pH 7.4 for three days. Cytometry gating strategy was developed by using blood cell samples stored at 4°C in PBS and milk cell samples heat-treated at 80°C for 30 min as a control for the maximum (95.9%) and minimum (0.7%) values of cell viability respectively. Cell viability in the initial samples was 39.5% for all cells and varied for each cell population from 26.7% for PMNs, to 32.6% for macrophages, and 58.3% for lymphocytes. Regarding the physico-chemical treatments applied, somatic cells did not sustain heat treatment at 60°C and 80°C in contrast to changes in centrifugation rates, for which only the higher level, i.e. 5000×g led to a cell viability decrease, down to 9.4%, but no significant changes within the cell subpopulation distribution were observed. Finally, the somatic cells were better preserved in milk after 72h storage, in particular PMNs, that maintained a viability of 34.0 ± 2.9% compared to 4.9±1.9% in PBS, while there was almost no changes for macrophages (41.7 ± 5.7% in milk vs 31.2 ± 2.4% in PBS) and lymphocytes (25.3 ± 3.0% in milk vs 11.4 ± 3.1% in PBS). This study provides a new array to better understand milk cell biology and to establish the relationship between the cell viability and the release of their endogenous enzymes in dairy matrix.

A Methionine Deficient Diet Enhances Adipose Tissue Lipid Metabolism and Alters Anti-Oxidant Pathways in Young Growing Pigs.

Methionine is a rate-limiting amino-acid for protein synthesis but non-proteinogenic roles on lipid metabolism and oxidative stress have been demonstrated. Contrary to rodents where a dietary methionine deficiency led to a lower adiposity, an increased lipid accretion rate has been reported in growing pigs fed a methionine deficient diet. This study aimed to clarify the effects of a dietary methionine deficiency on different aspects of tissue lipid metabolism and anti-oxidant pathways in young pigs. Post-weaned pigs (9.8 kg initial body weight) were restrictively-fed diets providing either an adequate (CTRL) or a deficient methionine supply (MD) during 10 days (n=6 per group). At the end of the feeding trial, pigs fed the MD diet had higher lipid content in subcutaneous adipose tissue. Expression levels of genes involved in glucose uptake, lipogenesis but also lipolysis, and activities of NADPH enzyme suppliers were generally higher in subcutaneous and perirenal adipose tissues of MD pigs, suggesting an increased lipid turnover in those pigs. Activities of the anti-oxidant enzymes superoxide dismutase, catalase and glutathione reductase were increased in adipose tissues and muscle of MD pigs. Expression level and activity of the glutathione peroxidase were also higher in liver of MD pigs, but hepatic contents in the reduced and oxidized forms of glutathione and glutathione reductase activity were lower compared with control pigs. In plasma, superoxide dismutase activity was higher but total anti-oxidant power was lower in MD pigs. These results show that a dietary methionine deficiency resulted in increased levels of lipogenesis and lipolytic indicators in porcine adipose tissues. Decreased glutathione content in the liver and coordinated increase of enzymatic antioxidant activities in adipose tissues altered the cellular redox status of young pigs fed a methionine-deficient diet. These findings illustrate that a rapidly growing animal differently adapts tissue metabolisms when facing an insufficient methionine supply.

Age-related changes in the features of porcine adult stem cells isolated from adipose tissue and skeletal muscle.

A better understanding of the control of body fat distribution and muscle development is of the upmost importance for both human and animal physiology. This requires a better knowledge of the features and physiology of adult stem cells in adipose tissue and skeletal muscle. Thus the objective of the current study was to determine the type and proportion of these cells in growing and adult pigs. The different cell subsets of stromal vascular cells isolated from these tissues were characterized by flow cytometry using cell surface markers (CD11b, CD14, CD31, CD34, CD45, CD56, and CD90). Adipose and muscle cells were predominantly positive for the CD34, CD56, and CD90 markers. The proportion of positive cells changed with age especially in intermuscular adipose tissue and skeletal muscle where the percentage of CD90(+) cells markedly increased in adult animals. Further analysis using coimmunostaining indicates that eight populations with proportions ranging from 12 to 30% were identified in at least one tissue at 7 days of age, i.e., CD90(+)/CD34(+), CD90(+)/CD34(-), CD90(+)/CD56(+), CD90(+)/CD56(-), CD90(-)/CD56(+), CD56(+)/CD34(+), CD56(+)/CD34(-), and CD56(-)/CD34(+). Adipose tissues appeared to be a less heterogeneous tissue than skeletal muscle with two main populations (CD90(+)/CD34(-) and CD90(+)/CD56(-)) compared with five or more in muscle during the studied period. In culture, cells from adipose tissue and muscle differentiated into mature adipocytes in adipogenic medium. In myogenic conditions, only cells from muscle could form mature myofibers. Further studies are now needed to better understand the plasticity of those cell populations throughout life.

In vitro characterization of proliferation and differentiation of pig satellite cells.

Skeletal muscle contains various muscle fiber types exhibiting different contractile properties based on the myosin heavy chain (MyHC) isoform profile. Muscle fiber type composition is highly variable and influences growth performance and meat quality, but underlying mechanisms regulating fiber type composition remain poorly understood. The aim of the present work was to develop a model based on muscle satellite cell culture to further investigate the regulation of adult MyHC isoforms expression in pig skeletal muscle. Satellite cells were harvested from the mostly fast-twitch glycolytic longissimus (LM) and predominantly slow-twitch oxidative rhomboideus (RM) muscles of 6-week-old piglets. Satellite cells were allowed to proliferate up to 80% confluence, reached after 7 day of proliferation (D7), and then induced to differentiate. Kinetics of proliferation and differentiation were similar between muscles and more than 95% of the cells were myogenic (desmin positive) at D7 with a fusion index reaching 65 ± 9% after 4 day of differentiation. One-dimensional SDS polyacrylamide gel electrophoresis revealed that satellite cells from both muscles only expressed the embryonic and fetal MyHC isoforms in culture, without any of the adult MyHC isoforms that were expressed in vivo. Interestingly, triiodothyronine (T3) induced de novo expression of adult fast and α-cardiac MyHC in vitro making our culture system a valuable tool to study de novo expression of adult MyHC isoforms and its regulation by intrinsic and/or extrinsic factors.

Gender affects liver desaturase expression in a rat model of n-3 fatty acid repletion.

Dietary n-3 polyunsaturated fatty acids (PUFA) are major components of cell membranes and have beneficial effects on human health. Docosahexaenoic acid (DHA; 22:6n-3) is the most biologically important n-3 PUFA and can be synthesized from its dietary essential precursor, alpha-linolenic acid (ALA; 18:3n-3). Gender differences in the efficiency of DHA bioconversion have been reported, but underlying molecular mechanisms are unknown. We compared the capacity for DHA synthesis from ALA and the expression of related enzymes in the liver and cerebral cortex between male and female rats. Wistar rats, born with a low-DHA status, were supplied with a suboptimal amount of ALA from weaning to 8 weeks of age. Fatty acid composition was determined by gas chromatography, the mRNA expression of different genes involved in PUFA metabolism was determined by RT-PCR (low-density array) and the expression of proteins was determined by Western blot analysis. At 8 weeks, DHA content was higher (+20 to +40%) in each phospholipid class of female livers compared to male livers. The "Delta4," Delta5 and Delta6 desaturation indexes were 1.2-3 times higher in females than in males. The mRNA expression of Delta5- and Delta6-desaturase genes was 3.8 and 2.5 times greater, respectively, and the Delta5-desaturase protein was higher in female livers (+50%). No gender difference was observed in the cerebral cortex. We conclude that female rats replete their DHA status more readily than males, probably due to a higher expression of liver desaturases. Our results support the hypothesis on hormonal regulation of PUFA metabolism, which should be taken into account for specific nutritional recommendations.

Differential effects of steroids on the synthesis of polyunsaturated fatty acids by human neuroblastoma cells.

Polyunsaturated fatty acids (PUFA) are crucial for proper functioning of cell membranes, particularly in brain. Biologically important PUFA include docosahexaenoic acid (n-3 series) and arachidonic acid (n-6 series) which can be formed from their respective dietary essential precursors, alpha-linolenic acid (ALA) and linoleic acid (LA). Steroid hormones are thought to modulate PUFA synthesis in humans but whether they regulate PUFA status in brain and/or in neural membranes is unknown. In human neuroblastoma SH-SY5Y cells, we compared the effect of estradiol, testosterone, and progesterone on PUFA synthesis. Cells were incubated with ALA and/or LA 7 microM in combination with estradiol, testosterone, or progesterone at 10 nM without serum. The fatty acid composition was determined by gas chromatography and the mRNA expression of genes involved in PUFA metabolism by real-time RT-PCR. Estradiol affected both the n-3 and the n-6 PUFA conversion, the n-3 PUFA pathway being more sensitive to the estradiol treatment. In ALA-supplemented cells, estradiol increased while testosterone decreased the long-chain n-3 PUFA content (+17% and -15%, respectively) and the mRNA expression of the Delta5-desaturase (+11% and -9%), these two events being strongly correlated. Progesterone did not affect the PUFA composition. The positive effect of estradiol was blocked by the estrogen receptor antagonist ICI-182,780. We conclude that steroids have differential effects on PUFA synthesis and that their mode of action could involve the modulation of the Delta5-desaturase mRNA expression in neuroblastoma cells. These results help our understanding of the regulation of brain PUFA metabolism by steroid hormones.

Estradiol favors the formation of eicosapentaenoic acid (20:5n-3) and n-3 docosapentaenoic acid (22:5n-3) from alpha-linolenic acid (18:3n-3) in SH-SY5Y neuroblastoma cells.

Whether neurosteroids regulate the synthesis of long chain polyunsaturated fatty acids in brain cells is unknown. We examined the influence of 17-beta-estradiol (E2) on the capacity of SH-SY5Y cells supplemented with alpha-linolenic acid (ALA), to produce eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). Cells were incubated for 24 or 72 h with ALA added alone or in combination with E2 (ALA + E2). Fatty acids were analyzed by gas chromatography of ethanolamine glycerophospholipids (EtnGpl) and phosphatidylcholine (PtdCho). Incubation for 24 h with ALA alone increased EPA and DPA in EtnGpl, by 330 and 430% compared to controls (P < 0.001) and DHA by only 10% (P < 0.05). Although DHA increased by 30% (P < 0.001) in ALA + E2-treated cells, the difference between the ALA and ALA + E2 treatments were not significant after 24 h (Anova-1, Fisher's test). After 72 h, EPA, DPA and DHA further increased in EtnGpl and PtdCho of cells supplemented with ALA or ALA + E2. Incubation for 72 h with ALA + E2 specifically increased EPA (+34% in EtnGpl, P < 0.001) and DPA (+15%, P < 0.001) compared to ALA alone. Thus, SH-SY5Y cells produced membrane EPA, DPA and DHA from supplemental ALA. The formation of DHA was limited, even in the presence of E2. E2 significantly favored EPA and DPA production in cells grown for 72 h. Enhanced synthesis of ALA-elongation products in neuroblastoma cells treated with E2 supports the hypothesis that neurosteroids could modulate the metabolism of PUFA.

Changes of the transcriptional and fatty acid profiles in response to n-3 fatty acids in SH-SY5Y neuroblastoma cells.

Synthesis of docosahexaenoic acid (DHA) from its metabolic precursors contributes to membrane incorporation of this FA within the central nervous system. Although cultured neural cells are able to produce DHA, the membrane DHA contents resulting from metabolic conversion do not match the high values of those resulting from supplementation with preformed DHA. We have examined whether the DHA precursors down-regulate the incorporation of newly formed DHA within human neuroblastoma cells. SH-SY5Y cells were incubated with gradual doses of alpha-linolenic acid (alpha-LNA), EPA, or docosapentaenoic acid (DPA), and the incorporation of DHA into ethanolamine glycerophospholipids was analyzed as a reflection of synthesizing activity. The incorporation of EPA, DPA, and preformed DHA followed a dose-response saturating curve, whereas that of DHA synthesized either from alpha-LNA, EPA, or DPA peaked at concentrations of precursors below 15-30 microM and sharply decreased with higher doses. The mRNA encoding for six FA metabolism genes were quantified using real-time PCR. Two enzymes of the peroxisomal beta-oxidation, L-bifunctional protein and peroxisomal acyl-CoA oxidase, were expressed at lower levels than fatty acyl-CoA ligase 3 (FACL3) and delta6-desaturase (delta6-D). The delta6-D mRNA slightly increased between 16 and 48 h of culture, and this effect was abolished in the presence of 70 microM EPA. In contrast, the EPA treatment resulted in a time-dependent increase of FACL3 mRNA. The terminal step of DHA synthesis seems to form a "metabolic bottleneck," resulting in accretion of EPA and DPA when the precursor concentration exceeds a specific threshold value. We conclude that the critical precursor- concentration window of responsiveness may originate from the low basal expression level of peroxisomal enzymes.

Incorporation of docosahexaenoic acid into nerve membrane phospholipids: bridging the gap between animals and cultured cells.

BACKGROUND: Functional maturation of nervous tissues depends on membrane accretion of docosahexaenoic acid (DHA). Animal studies have shown that incorporation of dietary DHA into membrane phospholipids is dose dependent. The molecular effects of DHA are commonly studied in cultured cells, but questions remain about the physiologic connection between animal and cell models. OBJECTIVE: We developed a linear model for comparing the responses of rat nervous tissues to dietary DHA with the responses of human cell lines to DHA in medium. DESIGN: Rats were rendered chronically deficient in n-3 fatty acids by being reared on a peanut oil diet. DHA status was replenished in the F2 generation by using increasing supplements of a microalgal oil. Human retinoblastoma and neuroblastoma cells were dosed with unesterified DHA. DHA accumulation into phospholipids was defined by the plateau of the dose-response curve (DHA(max)) and by the supplement required to produce one-half the DHA(max) (DHA(50)). RESULTS: The DHA(max) values for 4 brain regions and 2 neuroblastoma lines were similar, and the value for the retinoblastoma line was similar to the retinal value. Expressing the DHA input as micro mol/10 g diet and as micro mol/L medium resulted in similar values for the ratio of DHA(max) to DHA(50) in the 4 brain regions and the 3 cell lines. The DHA(max)-DHA(50) ratios in the ethanolamine phosphoglyceride and phosphatidylcholine fractions in retinal phospholipids were 6 and 10 times, respectively, those in the brain and cultured cells. CONCLUSIONS: The dose-dependent responses of cells and the brain to DHA supplements can be compared by using DHA(max)-DHA(50) ratios. We propose a counting frame that allows the comparison of the dose responses of the brain and cells to exogenous DHA.

Docosahexaenoic acid membrane content and mRNA expression of acyl-CoA oxidase and of peroxisome proliferator-activated receptor-delta are modulated in Y79 retinoblastoma cells differently by low and high doses of alpha-linolenic acid.

The mRNA expression levels of acyl-CoA oxidase (AOX), a key enzyme in very-long-chain fatty acid peroxisomal oxidation, and of peroxisome proliferator-activated receptor-delta (PPAR-delta), a nuclear receptor possibly involved in the gene regulation of brain lipid metabolism, were determined in human Y79 retinoblastoma cells by using real-time quantitative polymerase chain reaction. Cells were dosed with alpha-linolenic acid (18:3n-3), the essential metabolic precursor of the n-3 polyunsaturated fatty acid series that normally gives rise through terminal peroxisomal oxidation to the synthesis of membrane docosahexaenoic acid (22:6n-3, or DHA). The AOX and PPAR-delta relative expression levels increased 2.3 and 3.4 times, respectively, upon dosing of cells with 7 microM 18:3n-3, whereas AOX cDNA abundance decreased by 50% upon dosing with 70 microM 18:3n-3. Concurrently, the DHA content increased by 23% in the membrane ethanolamine-phosphoglycerides from cells dosed with 7 microM 18:3n-3, whereas it decreased by 38% upon dosing with 70 microM 18:3n-3. The DHA's upstream precursors (20:5n-3 and 22:5n-3) both accumulated in cells dosed with 7 or 70 microM 18:3n-3. The 18:3n-3-induced changes in membrane phospholipid fatty acid composition support the hypothesis that the terminal peroxisomal step of n-3 conversion is rate limiting in the Y79 line. The concurrent 7 microM 18:3n-3-induced increase of mRNAs encoding for AOX and for PPAR-delta suggests that 18:3n-3 (or its metabolites) at low concentration could trigger its proper conversion to DHA, possibly through activation of PPAR-delta-mediated transcription of AOX. Decreased membrane DHA content and mRNA expression level of AOX in 70-microM 18:3n-3-dosed cells corroborated the relationship between AOX expression and DHA synthesis and suggested that simultaneous down-regulating events occurred at high concentrations of 18:3n-3.