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Fatty acid composition of foetal bovine serum (FBS) and the phospholipids of FHM cells maintained in L-15 medium containing 10% (v/v) FBS
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![Table 3 Incorporation of [1-14 C]-labelled fatty acids into total lipid...](profile/Melissa-Gregory-2/publication/51084946/figure/tbl2/AS:601749161271314@1520479588640/ncorporation-of-1-14-C-labelled-fatty-acids-into-total-lipid-and-lipid-classes-in-FHM_Q320.jpg)
Fish oils are rich in omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA), predominantly 20:5n-3 and 22:6n-3, whereas vegetable oils contain abundant C(18)-PUFA, predominantly 18:3n-3 or 18:2n-6. We hypothesized that replacement of fish oils with vegetable oils would increase the oxidative stability of fish lipids. Here we have used the lo...
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Context 1
... fatty acid composition of the FHM cell phospholipids closely resembled that of the FBS in the growth medium, with the exceptions that 16:0 and 18:0 were less abundant and 18:1n-9 was more abundant (Table 1). Increasing the concentration of FBS in the growth medium from 2 to 20% (v/v) significantly increased the concentrations of 22:5n-3, 22:6n-3 and 20:4n-6 in the cell phospholipids (Fig. 2). ...
Context 2
... showed that 18:3n-3 and 20:5n-3 were more readily oxidized than 16:0, 18:2n-6 or 22:6n-3 which in turn were more readily oxidized than 18:1n-9. This result was consistent with the high retention levels for 18:1n-9 in the cell phospholipids (Table 1). ...
Context 3
... incorporation into all lipid classes was similar for 18:1n-9, 18:2n-6 and 20:5n-3 but significantly greater for 18:3n-3 and significantly less for 16:0 and 22:6n-3 (Table 3). The lower incorporation rate for 16:0 was con- sistent with its lower steady state level in the FHM cell phospholipids as compared with FBS (Table 1). This contrasts with the higher steady state level for 18:1n-9 which was apparently due to a lower rate of b-oxidation. ...
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Backgrounds:
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Citations
... respectively, of total plasma lipids by weight (Edelstein, 1986;Glaser et al., 2010;Bradbury et al., 2011). Similar values of 0.1-0.4% and 0.8-1.4% for C12:0 and C14:0, respectively, were found in bovine serum (Evans et al., 1961;Gregory et al., 2011). Plasma concentrations of C12:0 and C14:0 were less frequently reported. ...
Trypanosoma brucei spp. causes African Sleeping Sickness in humans and nagana, a wasting disease, in cattle. As T. brucei goes through its life cycle in its mammalian and insect vector hosts, it is exposed to distinct environments that differ in their nutrient resources. One such nutrient resource is fatty acids, which T. brucei uses to build complex lipids or as a potential carbon source for oxidative metabolism. Of note, fatty acids are the membrane anchoring moiety of the glycosylphosphatidylinositol (GPI)-anchors of the major surface proteins, Variant Surface Glycoprotein (VSG) and the Procyclins, which are implicated in parasite survival in the host. While T. brucei can synthesize fatty acids de novo, it also readily acquires fatty acids from its surroundings. The relative contribution of parasite-derived vs. host-derived fatty acids to T. brucei growth and survival is not known, nor have the molecular mechanisms of fatty acid uptake been defined. To facilitate experimental inquiry into these important aspects of T. brucei biology, we addressed two questions in this review: (1) What is known about the availability of fatty acids in different host tissues where T. brucei can live? (2) What is known about the molecular mechanisms mediating fatty acid uptake in T. brucei? Finally, based on existing biochemical and genomic data, we suggest a model for T. brucei fatty acid uptake that proposes two major routes of fatty acid uptake: diffusion across membranes followed by intracellular trapping, and endocytosis of host lipoproteins.
... Instead, we allowed for an unconstrained influx of stearate, palmitate, oleate, linolenate, linoleate, and arachidonate. These fatty acids have previously been shown to make up the majority of lipids present in fetal calf serum [62,63] which was used as a growth medium supplement. ...
Background:
Mass spectrometry-based metabolomics approaches provide an immense opportunity to enhance our understanding of the mechanisms that underpin the cellular reprogramming of cancers. Accurate comparative metabolic profiling of heterogeneous conditions, however, is still a challenge.
Methods:
Measuring both intracellular and extracellular metabolite concentrations, we constrain four instances of a thermodynamic genome-scale metabolic model of the HCT116 colorectal carcinoma cell line to compare the metabolic flux profiles of cells that are either sensitive or resistant to ruthenium- or platinum-based treatments with BOLD-100/KP1339 and oxaliplatin, respectively.
Results:
Normalizing according to growth rate and normalizing resistant cells according to their respective sensitive controls, we are able to dissect metabolic responses specific to the drug and to the resistance states. We find the normalization steps to be crucial in the interpretation of the metabolomics data and show that the metabolic reprogramming in resistant cells is limited to a select number of pathways.
Conclusions:
Here, we elucidate the key importance of normalization steps in the interpretation of metabolomics data, allowing us to uncover drug-specific metabolic reprogramming during acquired metal-drug resistance.
... As demonstrated in this study, by testing the impact of a range of fatty acid supplements in cell culture, the CL compositions as well as the RCxI activities respond to changes in the lipid content and composition in the medium. While the content of linoleic acid in human plasma is approximately 27% of free fatty acids, with total polyunsaturated fatty acid levels of 35-40% (45, 46), the typical lipid content of FBS mainly includes saturated and monounsaturated fatty acids with a linoleic acid fraction of as little as 5-8% (47,48). This results in the general linoleic acid-deprived "CL phenotype" found across many different cultured cell lines (8). ...
The molecular assembly of cells depends not only on the balance between anabolism and catabolism, but to a large degree on the building blocks available in the environment. For cultured mammalian cells, this is largely determined by the composition of the applied growth medium. Here we study the impact of lipids in the medium on mitochondrial membrane architecture and function by combining LC-MS/MS lipidomics and functional tests with lipid supplementation experiments in an otherwise serum- and lipid-free cell culture model. We demonstrate that the composition of mitochondrial cardiolipins strongly depends on the lipid environment in cultured cells and favours the incorporation of essential linoleic acid over other fatty acids. Simultaneously, the mitochondrial respiratory Complex I activity was altered, whereas the matrix-localized enzyme citrate synthase was unaffected. This raises the question on a link between membrane composition and respiratory control. In summary, we found a strong dependency of central mitochondrial features on the type of lipids contained in the growth medium. This underlines the importance of considering these factors when using and establishing cell culture models in biomedical research.
... The copyright holder for this preprint this version posted June 10, 2021. of stearate, palmitate, oleate, linolenate, linoleate, and arachidonate. These fatty acids have previously been shown to make up the majority of lipids present in fetal calf serum [61,62] which was used as a growth medium supplement. ...
Background
Mass spectrometry-based metabolomics approaches provide an immense opportunity to enhance our understanding of the mechanisms that underpin the cellular reprogramming of cancers. Accurate comparative metabolic profiling of heterogeneous conditions, however, is still a challenge.
Methods
Measuring both intracellular and extracellular metabolite concentrations, we constrain four instances of a thermodynamic genome-scale metabolic model of the HCT116 colorectal carcinoma cell line to compare the metabolic flux profiles of cells that are either sensitive or resistant to ruthenium- or platinum-based treatments with BOLD-100/KP1339 and oxaliplatin, respectively.
Results
Normalizing according to growth rate and normalizing resistant cells according to their respective sensitive controls, we are able to dissect metabolic responses specific to the drug and to the resistance states. We find the normalization steps to be crucial in the interpretation of the metabolomics data and show that the metabolic reprogramming in resistant cells is limited to a select number of pathways.
Conclusions
Here we elucidate the key importance of normalization steps in the interpretation of metabolomics data, allowing us to uncover drug-specific metabolic reprogramming during acquired metal-drug resistance.
... As demonstrated in this study, by testing the impact of a range of fatty acid supplements in cell culture, the CL compositions as well as the respiratory complex I activities respond to changes in the lipid content and composition in the medium. While the content of linoleic acid in human plasma is approximately 27%, with total polyunsaturated fatty acid levels of 35-40% [33,34], the typical lipid content of FBS mainly includes saturated and monounsaturated fatty acids with a linoleic acid fraction of as little as 5-8% [35,36]. This results in the general linoleic acid-deprived "CL phenotype" found across many different cultured cell lines [7]. ...
The molecular assembly of cells depends not only on their balance between anabolism and catabolism, but to a large degree also on the building blocks available in the environment. For cultivated mammalian cells, this is largely determined by the composition of the growth medium used. Here we study the impact of medium lipids on mitochondrial membrane architecture and function by combining LC-MS/MS lipidomics and functional tests with lipid supplementation experiments in an otherwise serum- and lipid-free cell culture model. We demonstrate that the composition of mitochondrial cardiolipins (CL) strongly depends on the lipid environment in cultured cells and prefers the incorporation of essential linoleic acid over other fatty acids. Simultaneously, the mitochondrial respiratory complex I activity was altered, whereas the matrix-localized enzyme citrate synthase was unaffected. This suggests a link between membrane composition and respiratory capacity. In summary, we find a strong dependency of central mitochondrial features on the type of lipids contained in the growth medium. Thus, this underlines the importance of considering these factors when using and establishing cell culture models in biomedical research.
Abstract Figure
... Recently, replacement of fish meal in aqua feed with alternative protein sources has been a major goal for aquaculture nutritionist (Fuchs et al. 2015;Liu et al. 2014). Numerous studies have been carried out, from feeding trials (Lee et al. 2015;Wang et al. 2016a) to primary cell culture studies (Gregory et al. 2011;Minghetti et al. 2011), to explore the molecular mechanism involved in the limited usage of alternative protein sources. However, the results were highly influenced by feeding environments (Boeuf and Payan 2001), animal health (Oliva-Teles 2012), diet palatability (Yamamoto et al. 2015), and digestibility (Eusebio et al. 2004). ...
A continuous fibroblast-like cell line, TMF (turbot muscle fibroblasts), was established from juvenile turbot Scophthalmus maximus muscle with the method of trypsin digestion. It has been subcultured more than 60 passages for over 150 days. The TMF cells were cultured in L-15 medium supplemented with HEPES, fetal bovine serum (FBS), GlutaMAX, and basic fibroblast growth factor (bFGF). The optimal temperature for TMF culture was 24 °C. TMF cells were predominantly composed of fibroblastic-like cells, and the transcription factor 4 (TCF-4) was highly expressed in TMF cells. Chromosome analysis revealed that it had a diploid chromosome number of 2n = 44. The transfection efficiency achieved 54.95 ± 6.59%, and the cell mortality rate was about 8.70% when transfected with the nucleofection method. Meanwhile, the TMF cells showed a sensitive response to amino acid levels and activation target of rapamycin (TOR) signaling pathway. These results indicate that TMF was a potential tool to explore the signal transduction of teleost in vitro.
... EPA and DHA have much lower melting points, higher fluidity and more steps for β-oxidation than MUFA and SFA (Mori et al., 2000). However, they could exhibit higher levels of malondialdehyde (MDA) generated from lipid peroxidation compared with SFA and MUFA (Gregory et al., 2011). The DHA/EPA ratios could modify the gene expression of lipid metabolism to regulate the larval growth, body fatty acid structures and health. ...
... But LC-n-3 HUFA could exhibit higher levels of MDA generated from lipid peroxidation compared with SFA and MUFA; especially, 18:1n-9 can be efficiently β-oxidized to produce ATP, whereas PUFA is primarily esterified into phospholipids (Gregory et al., 2011;Wang et al., 2017). MDA is directly related to the level of lipid peroxidation. ...
A 30‐day growth study was conducted to evaluate the impacts of the dietary DHA/EPA ratios on growth performance, antioxidant and lipid metabolism of Siberian sturgeon larvae. Four isonitrogenous and isoenergetic microcapsuled diets were formulated with various DHA/EPA ratios as 0.73 (R0.73), 1.25 (R1.25), 1.75 (R1.75) and 2.33 (R2.33). The results showed that the final body length, final body weight, specific growth rate and survival in the group R2.33 were highest (p < 0.05), while there were no significant differences on viscerosomatic index and condition factor among all groups. Visceral catalase activity of groups R2.33 and R1.75 was significantly higher than that of groups R0.73 and R1.25, but superoxide dismutase, total antioxidant capacity, malondialdehyde, total cholesterol, triglycerides and high‐density cholesterol were not different among groups. The gene expression of PPARα and lipoprotein lipase had no difference among groups, but the expression of hepatic lipase gene in groups R1.75 and R2.33 was significantly lower than that in group R0.73 as a negative feedback of high dietary DHA intake. In conclusion, growth performance was improved but kept stable antioxidant response in visceral mass with increased levels of DHA/EPA ratios from 0.73 to 2.33 for Siberian sturgeon larvae. Improving DHA/EPA ratios is a benefit for larvae to selectively deposit more DHA, then EPA and ARA than MUFA in the body to meet the requirement for early development.
... Attached differentiated THP-1 cells (macrophages) were washed in complete RPMI and incubated with complete RPMI without added PMA for > 2 days to allow resting. Two hours before challenge with S. pneumoniae, a subset of macrophages was treated with 5 µM AA (Sigma-Aldrich), which represents a concentration appropriate for treatment of cell cultures (Caruso et al., 1994;Ghioni et al., 1997;Hughes-Fulford et al., 2005;Gregory et al., 2011). The AA was pre-incubated with fatty acid free BSA (Sigma) at a 4:1 ratio (AA:BSA) to allow for efficient delivery of AA to THP-1 cells, following standard fatty acid supplementation procedures (Ghioni et al., 1997). ...
Free fatty acids hold dual roles during infection, serving to modulate the host immune response while also functioning directly as antimicrobials. Of particular importance are the long chain polyunsaturated fatty acids, which are not commonly found in bacterial organisms, that have been proposed to have antibacterial roles. Arachidonic acid (AA) is a highly abundant long chain polyunsaturated fatty acid and we examined its effect upon Streptococcus pneumoniae. Here, we observed that in a murine model of S. pneumoniae infection the concentration of AA significantly increases in the blood. The impact of AA stress upon the pathogen was then assessed by a combination of biochemical, biophysical and microbiological assays. In vitro bacterial growth and intra-macrophage survival assays revealed that AA has detrimental effects on pneumococcal fitness. Subsequent analyses demonstrated that AA exerts antimicrobial activity via insertion into the pneumococcal membrane, although this did not increase the susceptibility of the bacterium to antibiotic, oxidative or metal ion stress. Transcriptomic profiling showed that AA treatment also resulted in a dramatic down-regulation of the genes involved in fatty acid biosynthesis, in addition to impacts on other metabolic processes, such as carbon-source utilization. Hence, these data reveal that AA has two distinct mechanisms of perturbing the pneumococcal membrane composition. Collectively, this work provides a molecular basis for the antimicrobial contribution of AA to combat pneumococcal infections.
... Within one week, cells had absorbed individual PUFAs at high levels and incorporated them with some biotransformation products in their phospholipids. These observations are in line with our previous results with the same cell line (Ferain et al., 2016) and emphasize, once more, the importance of the fatty acid composition of growth media in defining the fatty acid profile of fish cells in culture (Buzzi et al., 1996;Gregory et al., 2011;Tocher, 1990). Noteworthy, the range of n3/n6 ratio resulting from the different PUFA enrichment strategies (at fixed PUFA concentrations and incubation periods), encompassed a 200-fold range. ...
... In pigs, ractopamine supplementation promotes reduction of body fat, particular subcutaneous and intermuscular (CArr et al., 2005). Decreased fat deposition can reduce lipid oxidation rates, improving pork quality (GreGory et al., 2011). In the present experiment, however, the treatments did not alter lipid oxidation, corroborating with the results obtained by In the present study, interaction also revealed that use of the 10-20 ppm ractopamine plan with non-adjusted diets increased (P<0.05) ...
This study evaluated the effects that gradual ractopamine supplementation in diets with nutritional adjustments on pig meat. Were used 80 finishing crossbred barrows in a randomized block design with a 2×5 factorial arrangement (two diets: with and without nutritional adjustment; five levels of ractopamine supplementation: 5-5, 10-10, 20-20, 5-10 and 10-20 ppm) in the 14 initial and 14 final study days, four replicates with two animals by experimental unit. Higher shear force values (P
