Endogenous Metabolite; Na+/K+ ATPase

The most prevalent unsaturated fertilizer (FA) in human fat cells and other tissues is oleic acid. Oleic acid increases β-oxidation-mediated AMPK activation-mediated cell proliferation and migration of highly metastatic cells. Oleic acid inhibits the growth and death of low-proliferation tumors, including the breast cancer MCF-7 cell line, as well as SGC7901 [1].

Dietary oleic acid intake improved running endurance with the changes of muscle fiber type shares in mice. This study elucidated a novel functionality of oleic acid in skeletal muscle fiber types. Further studies are required to elucidate the underlying mechanisms. Our findings have the potential to contribute to the field of health and sports science through nutritional approaches, such as the development of supplements aimed at improving muscle function.[3]

Oil Red O Staining and Triglyceride Assay[2]
The lipid droplets were stained by Oil red O. A stock solution was prepared in 2-propanol (0.3%), and a working solution was freshly prepared by diluting the stock solution with water (3∶2). After fixation, the cells were washed twice in PBS and stained with Oil red O and hematoxylin for 15 min and 1 min, respectively. The cells were washed with PBS, and images were obtained under a light contrast microscope. The intracellular triglycerides were assayed using a triglyceride assay kit (GPO-POD) according to the manufacturer’s recommended protocol.

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Determination of Oxygen Consumption[2]
Five million cells were resuspended in 1 ml of fresh warm medium pre-equilibrated with 21% oxygen and placed in a sealed respiration chamber equipped with a thermostat control, micro stirring device and Clark-type oxygen electrode disc. The oxygen content in the cell suspension medium was constantly monitored for 10 min, and the oxygen consumption rate was recorded.

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ATP Assays[2]
The intracellular level of ATP was measured with an ATPlite assay kit. The cells were incubated in a serum-free medium with or without 400 µM of OA for 48 h, washed with PBS three times, lysed in ATP extraction buffer and centrifuged in 4°C. ATP was measured by luminometric methods using commercially available luciferin/luciferase reagents on a luminometer (TD-20/20) according to the manufacturer’s instructions. The data were normalised to total protein.

Cell Viability Assay[2]
The cells in 48-well plates at a density of 10,000 cells per well were treated with different stressors. The cell viability was measured using the 3-[4,5-dimethylthiazol-2-yl]-2,5-dephenyl tetrazolium bromide (MTT) assay, according to the manufacturer’s protocol.

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BrdU Incorporation Assay[2]
The cell proliferation was determined by measuring the BrdU incorporation using a BrdU incorporation assay, according to the manufacturer’s instructions. Briefly, 5,000 cells/well seeded in a 96-well plate were pulse-labelled for 2 h with 10-uM BrdU. The cells were incubated for 30 min with a diluted, peroxidase-conjugated anti-BrdU antibody. The absorbance values were measured at 450 nm using an ELISA reader.

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Migration Assay[2]
The cell migration assays were performed using Transwell chambers (8-µm pore size polycarbonate membrane). A total of 50 K cells were plated into the insert in 200 µl serum-free medium containing BSA or 400 µM BSA-bound oleic acid and allowed to migrate from the upper compartment to the lower compartment toward a 15% FBS gradient for the indicated time period. After the migration, the non-migratory cells on the upper membrane surface were removed by scrubbing, and the membrane was fixed in buffered 4% paraformaldehyde and stained with 0.1% crystal violet at room temperature. The migrated cells were then enumerated. The migration values were expressed as the average number of migrated cells per microscopic field over six fields per assay from three independent membrane experiments.

All animal experiments were conducted in strict accordance with the Guidelines for Proper Conduct of Animal Experiments published by the Science Council of Japan and with the approval of the Animal Care and Use Committee of Kitasato University (approval no. 20-007). And all methods were performed in accordance with guidelines and regulations at Kitasato University, as well as complying with ARRIVE guidelines for the reporting of animal experiments. Eight-week-old male C57BL/6JJcl mice were purchased from CLEA Japan, Inc. The experimental design is illustrated in Fig. 5. The mice were housed in plastic cages in an animal room at 22 ± 2 °C and 50 ± 10% humidity under an artificial lighting system of 12-h light/12-h dark cycle. They were acclimated to the environment for one week. Following the acclimatization period, the mice were fed a CE-2 diet supplemented with 10% (w/w) palmitic acid (control diet) or oleic acid for 4 weeks. Palmitic acid was chosen as control fatty acid because it is a representative saturated fatty acid commonly found in soybean oil and olive oil, which has been used in previous studies. Although we considered using linoleic acid as a control, we decided against it because unsaturated fatty acids have ligand activity for PPARs. In our previous study, we reported the results of an 8-week feeding test, and in a preliminary study we confirmed similar changes in skeletal muscle characteristics even with a 4-week period. Therefore, we chose a shorter period of 4 weeks for this study. Nutritional and fatty acid compositions of experimental diets are shown in Table 2 and 3, respectively. Running endurance tests and serum biochemical analyses were performed on the last day of week 3 and the first day of week 4, respectively. After a 4-week feeding period, the mice were euthanized by cervical dislocation under inhalation anesthesia with isoflurane. The adipose tissues, liver, and skeletal muscles (soleus, EDL, and gastrocnemius muscles) were harvested and weighed immediately. The growth performance and tissue weight are shown in Table 1. All tissues were stored at –80 °C until further analysis [3].

[1]. Jack-Hays MG, et al. Activation of Na+/K(+)-ATPase by fatty acids, acylglycerols, and related amphiphiles: structure-activity relationship. Biochim Biophys Acta. 1996 Feb 21;1279(1):43-8.
[2]. Li S, et al. High metastaticgastric and breast cancer cells consume oleic acid in an AMPK dependent manner. PLoS One. 2014 May 13;9(5):e97330.
[3]. Sci Rep. 2024 Jan 8;14(1):755. doi: 10.1038/s41598-023-50464-y.

C18H34O2

282.468

282.2559

C, 76.54; H, 12.13; O, 11.33

112-80-1

Sodium oleate;143-19-1;Oleic acid-13C;82005-44-5;Oleic acid-d2;5711-29-5;Glycerol Monoleate;25496-72-4;Oleic acid-13C18;287100-82-7;Oleic acid-d17;223487-44-3;Oleic acid-13C-1;2483735-58-4;Oleic acid-d9;2687960-84-3

Colorless to light yellow liquid

Endogenous Metabolite

7.7

37.3

O([H])C(C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])/C(/[H])=C(/[H])\C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H])=O

ZQPPMHVWECSIRJ-KTKRTIGZSA-N

InChI=1S/C18H34O2/c1-2-3-4-5-6-7-8-9-10-11-12-13-14-15-16-17-18(19)20/h9-10H,2-8,11-17H2,1H3,(H,19,20)/b10-9-

9-Octadecenoic acid (9Z)-

D 100 Oleic acid9-cis-Octadecenoic acid9Z-Octadecenoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Ethanol : ~100 mg/mL (~354.03 mM)
0.1 M NaOH : ~100 mg/mL (~354.03 mM)
DMSO : ≥ 62.5 mg/mL (~221.27 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.85 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.85 mM) (saturation unknown) in 10% EtOH + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.85 mM) (saturation unknown) in 10% EtOH + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 2.08 mg/mL (7.36 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 5: ≥ 2.08 mg/mL (7.36 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 6: ≥ 2.08 mg/mL (7.36 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)