Marie-Hélène Perruchot

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 downregulate the incorporation of newly formed DHA within human neuroblastoma cells. SH-SY5Y cells were incubated with gradual doses of α-linolenic acid (α-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 α-LNA, EPA, or DPA peaked at concentrations of precursors below 15–30 μM 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 β-oxidation, L-bifunctional protein and peroxisomal acyl-CoA oxidase, were expressed at lower levels than fatty acyl-CoA ligase 3 (FACL3) and Δ6-desaturase (Δ6-D). The Δ6-D mRNA slightly increased between 16 and 48 h of culture, and this effect was abolished in the presence of 70 μM 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.

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.

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.

Docosahexaenoic acid membrane content and mRNA expression of acyl-CoA oxidase and of peroxisome proliferator-activated receptor-δ are modulated in Y79 retinoblastoma cells differently by low and high doses of α-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‐δ (PPAR‐δ), 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 α‐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‐δ relative expression levels increased 2.3 and 3.4 times, respectively, upon dosing of cells with 7 μM 18:3n‐3, whereas AOX cDNA abundance decreased by 50% upon dosing with 70 μM 18:3n‐3. Concurrently, the DHA content increased by 23% in the membrane ethanolamine‐phosphoglycerides from cells dosed with 7 μM 18:3n‐3, whereas it decreased by 38% upon dosing with 70 μM 18:3n‐3. The DHAs upstream precursors (20:5n‐3 and 22:5n‐3) both accumulated in cells dosed with 7 or 70 μM 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 μM 18:3n‐3‐induced increase of mRNAs encoding for AOX and for PPAR‐δ suggests that 18:3n‐3 (or its metabolites) at low concentration could trigger its proper conversion to DHA, possibly through activation of PPAR‐δ‐mediated transcription of AOX. Decreased membrane DHA content and mRNA expression level of AOX in 70‐μM 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.

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.

Differential gene expressions in subcutaneous adipose tissue pointed to a delayed adipocytic differentiation in small pig fetuses compared to their heavier siblings

Intra-uterine growth retardation in piglets is associated to neonatal losses and a greater susceptibility to fat deposition in the long term. Dietary l-arginine supplementation to gilts during early gestation has been proposed as a way to enhance fetal survival. This study aims to investigate the effects of variation in fetal growth within litters and dietary l-arginine treatment during early gestation in pregnant sows on expression levels of several genes involved in early adipose tissue development and lipid deposition in the fetuses. At day 75 of pregnancy, sows fed a standard gestation diet throughout pregnancy and sows fed 26g L-arginine daily from days 14 to 28 of gestation in supplement to the standard diet were sacrificed. Six pairs of littermates in each dietary group with the smallest or the heaviest fetal weights within each litter were collected (total: 24 fetuses). Expression levels of DLK1/PREF1 and FZD7 were significantly greater in subcutaneous backfat of the smallest fetuses. Conversely, transcriptional adipogenic regulators PPARG, SREBP1, and CEBPA, and genes involved in terminal adipocytic differentiation LPL, ME1, and FABP4 were less expressed in those piglets. Fetal weight has no effect on expression levels of genes involved in cell cycle progression and DNA content in subcutaneous adipose tissue. Maternal dietary L-arginine treatment did not affect subcutaneous adipose tissue features in 75-day old fetuses. The gene expression changes observed in the smallest fetuses are likely associated to a lower body fat content at birth, and could predispose to catch-up fat growth during the postnatal period.

Invited review: Pre- and postnatal adipose tissue development in farm animals: from stem cells to adipocyte physiology.

Both white and brown adipose tissues are recognized to be differently involved in energy metabolism and are also able to secrete a variety of factors called adipokines that are involved in a wide range of physiological and metabolic functions. Brown adipose tissue is predominant around birth, except in pigs. Irrespective of species, white adipose tissue has a large capacity to expand postnatally and is able to adapt to a variety of factors. The aim of this review is to update the cellular and molecular mechanisms associated with pre- and postnatal adipose tissue development with a special focus on pigs and ruminants. In contrast to other tissues, the embryonic origin of adipose cells remains the subject of debate. Adipose cells arise from the recruitment of specific multipotent stem cells/progenitors named adipose tissue-derived stromal cells. Recent studies have highlighted the existence of a variety of those cells being able to differentiate into white, brown or brown-like/beige adipocytes. After commitment to the adipocyte lineage, progenitors undergo large changes in the expression of many genes involved in cell cycle arrest, lipid accumulation and secretory functions. Early nutrition can affect these processes during fetal and perinatal periods and can also influence or pre-determinate later growth of adipose tissue. How these changes may be related to adipose tissue functional maturity around birth and can influence newborn survival is discussed. Altogether, a better knowledge of fetal and postnatal adipose tissue development is important for various aspects of animal production, including neonatal survival, postnatal growth efficiency and health.

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.

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.