Effects of Selected Bioactive Food Compounds on Human White Adipocyte Function

Christel Björk; Uta Wilhelm; Susanne Mandrup; Bjørk Ditlev Larsen; Alessandra Bordoni; Per Hedén; Mikael Rydén; Peter Arner; Jurga Laurencikiene

Disclosures

Nutr Metab. 2016;13(4) 

In This Article

Methods

Cell Cultures of Primary Human Adipocytes

Primary human pre-adipocytes for in vitro differentiation from 22 female subjects and 2 male subjects between 26–65 (mean 42) years of age with a BMI ranging from 21–31 kg/m2 (mean 25) were isolated from the stromal vascular fraction of subcutaneous WAT, which was obtained as a waste product from cosmetic liposuction. Isolation, culture and differentiation into adipocytes were performed as previously described.[36] The subjects were healthy according to self-report and not selected for age, sex or BMI. Briefly, intact pieces of adipose tissue were cut into 3–4 mm pieces and digested with collagenase for 90 min at 37 °C, whereas material obtained from liposuctions was digested with collagenase for 45 min at 37 °C. Thereafter the cell suspension was filtered, centrifuged at 200 × g for 10 min. The remaining pre-adipocytes and mesenchymal stem cells in the stromal vascular fraction were then suspended in erythrocyte lysis buffer for 10 min and centrifuged. The supernatant was discarded, the cells suspended in proliferation medium containing Dulbecco's Modified Eagle Medium (DMEM)/F12 (Biochrome AG, Berlin, Germany/Gibco, Life Technologies) supplemented with 10 % fetal bovine serum (Gibco, Life Technologies) and 2 % Pen-Strep (Gibco, Life Technologies) and subsequently filtered through a 70 μm cell strainer (BD BioSciences, Durham, NC) .

To study the effects of bioactives on fat cell metabolism and adipokine secretion, the cells were plated in proliferation medium at a density of 115 000 to 120 000 cells/well in 24-well plates. After 24 h, the medium was changed to DMEM/F12 supplemented with 15 mM HEPES (Gibco, Life Technologies), 100 μg/ml penicillin-streptomycin (Gibco, Life Technologies), 66 nM human insulin, 1 nM triiodo-L-thyronine, 10 μg/ml human transferrin, 33 μM biotin, 17 μM panthothenate, 100 nM cortisol (all from Sigma St. Louis, MO), 1.25 μg/ml amphotericin B (Gibco, Life Technologies) and 10 μM rosiglitazone (BRL49653) (GlaxoSmithKline, Durham, UK) to induce differentiation. Rosiglitazone was included during the first 3–4 days and then removed from the differentiation medium. Medium was changed every 2–3 days and the cells were kept until full differentiation (12–14 days). Forty-eight hours before full differentiation, the compounds were added in adipogenic medium.

To investigate the effects of bioactives on lipid accumulation, 20 000 cells/well were seeded in 96-well plates. Medium was supplemented with the compounds after 6 days in differentiation medium and treatment was performed for 6 days until day 12 of differentiation. Adipose tissue stromal vascular fraction, which we use to in vitro differentiate adipocytes, contains various cell types including immune cells. At day 6 of differentiation, immune cells are almost entirely washed away and absent and this is the earliest time point in differentiation, where effects unspecific for adipocytes could be excluded. Each experiment was repeated in cells isolated separately from at least three individuals in duplicates (lipid accumulation and lipogenesis) or quadruplicates (lipolysis and adipokine secretion). Cytotoxicity of compounds was measured for all treatment conditions. The grade of differentiation was determined under the microscope and cells with differentiation grade below 80 % were discarded. The study was approved by the Ethics Committee at Karolinska Institutet (Sweden). All subjects were informed about this study and provided their written informed consent.

Treatment With Bioactive Compounds

The cells were treated with DHA (D8768, sodium salt, Sigma, St. Louis, MO), C3G (1201–1, chloride salt, Polyphenols Laboratories AS, Sandes, Norway) together with its metabolite PCA (03930590, Sigma) or the BG metabolite PI (P1880, sodium salt, Sigma). To study possible synergistic, additive or antagonistic effects with DHA, the bioactive compound was also studied in combination with AC or PI. Compound concentrations were based on relevant literature[27,37] and unpublished treatment optimizations in the human Simpson-Golabi-Behmel syndrome (SGBS) pre-adipocyte cell line or in a hepatocyte cell line by collaborators in the PATHWAY-27 consortium. DHA was coupled to bovine serum albumin (BSA; A6003, Fraction V, Sigma) in a 4:1 ratio. Firstly, BSA was dissolved in 150 mM NaCl in a 37 °C water bath and filtered via a sterile 0.2 μm polypropylene membrane (VWR International, USA). Shortly after, DHA was dissolved in water and heated to 70 °C in a water bath. The coupling was performed through mixing both heated solutions and stirring for 1 h at 37 °C (pH 7.4). The final DHA concentration in the cell culture medium was 0.5–60 μM. C3G was dissolved in phosphate-buffered saline (PBS, Gibco, Life Technologies) whereas its main metabolite PCA was diluted in 50 % v/v solution of ethanol in water. According to agreements within the PATHWAY-27 consortium, C3G and PCA were added together to represent AC in a final concentration of 130 nM and 13 μM, respectively. PI (P1880, sodium salt, Sigma) was dissolved in 50 % v/v solution of ethanol in water and added to the cells at a final concentration of 100 μM. Corresponding concentrations of BSA and EtOH were added the control samples. As higher concentrations of BSA has been reported to have stimulatory effect on inflammatory cytokine secretion,[38] we have performed control experiments comparing BSA and EtOH (at concentrations used in all our treatments) to untreated cells. Such treatment did not affect release of IL-6 and glycerol (Additional file 1: Figure S1 https://static-content.springer.com/esm/art%3A10.1186%2Fs12986-016-0064-3/MediaObjects/12986_2016_64_MOESM1_ESM.pptx).

Cytotoxicity Measured as Lactate Dehydrogenase (LDH) Activity in Conditioned Medium

Medium from bioactive treated cells was collected when full differentiation was reached. LDH activity, as a measure of damaged cells, was quantified according to the manufacturer's instructions (Cat. No. 11 644 793 001, Roche Diagnostics GmbH, Mannheim, Germany).

Lipolysis Assay

Conditioned medium was collected after 48 h of treatment with the bioactive compounds. Basal lipolytic activity was measured as glycerol release with a bioluminescence method using Free Glycerol determination kit from Sigma (FG100) in combination with the fluorescence probe Amplex ultra red reagent (Cat. No. A36006, Molecular Probes, Life Technologies).[39,40] Glycerol release was normalized to protein concentration in each cell culture well.

Lipogenesis

Determination of basal and insulin-stimulated glucose incorporation into lipids was made when cells reached full differentiation that is after 48 h of bioactive treatment. Cells were initially washed twice with insulin- and glucose free DMEM (BioChromeAG) and incubated in DMEM containing 1 μM glucose. After 3 h, 3-3H glucose (37 MBq/ml; Perkin Elmer-Cetus, Norwalk, CT) diluted 1:1000 in DMEM containing 1 μM glucose with was added. For stimulated lipogenesis, insulin was added to a final concentration of 100 nM. After 2 h of incubation, the cells were washed three times with 4 °C PBS and thereafter lysed in aqueous 0.1 % SDS (sodium dodecyl sulfate, Sigma) solution. An aliquot was saved for protein determination and the rest of the lysate was transferred to scintillation tubes. Toluene scintillation liquid (toluene with 5 g/l 2,5-diphenyloxazol and 0.3 g/l 1,4-bis (4-methyl-5-phemyl-2-oxazolyl)-benzene; Sigma-Aldrich) was added and the samples were incubated overnight to extract the lipids. Glucose incorporation into lipids as a proxy of lipogenesis was quantified as 3-3H counts per minute (CPM) using a LS 6500 Multi-Purpose scintillation counter (Beckman, USA).

Adipokine Quantification by Enzyme-linked Immunosorbent Assay (ELISA)

To quantify the secretion of pro- (IL-6, MCP-1, chemerin, and TNFα) and anti-inflammatory (adiponectin) adipokines, cells were treated with bioactive compounds for 48 h and conditioned medium was collected for adipokine quantification by ELISA according to the manufacturer's instructions. The samples were diluted 5–60 fold for IL-6 detection (Quantikine ELISA Human IL-6 Immunoassay; R&D Systems, Minneapolis, MN, Intra-assay precision: CV <5 %, Inter-assay precision: CV <4 %), 3–10 fold for MCP-1 (Quantikine ELISA Human CCL3/MCP-1 Immunoassay; R&D Systems, Intra- and Inter-assay precision: CV <6 %) and 2–10 fold for chemerin (Quantikine ELISA Human Chemerin Immunoassay; R&D Systems, Intra-assay precision: CV ≤3 %, Inter-assay precision: CV <6 %) to match the standard curve. Adiponectin (Mercodia Adiponectin ELISA; Mercodia AB, Uppsala, Sweden, Intra-assay precision: CV <5 %, Inter-assay precision: CV <8 %) and TNFα (Quantiglo ELISA Human TNFα Immunoassay; R&D Systems, Intra-assay precision: CV <6 %, Inter-assay precision: CV <9 %) secretion were measured in undiluted samples. Levels of TNFα were detectable in adipocyte cultures treated with AC from only two subjects.

Protein Quantification

Protein amount was quantified in SDS lysates from the lipogenesis experiments using Pierce™ BCA Protein Assay Kit (Cat. No. 23225 Pierce, Rockford, IL) according to the manufacturer's instructions. The protein concentration for each well was used to normalize glucose incorporation into lipids as well as glycerol release, LDH activity and adipokine secretion for cell amount.

Neutral Lipid and DNA Staining

The cells were differentiated in 96-well plates and treated with the bioactive compounds for 6 days before full differentiation. Medium supplemented with the compounds was added to the cell cultures every second day. Before staining, the cells were washed with PBS and fixed with 4 % paraformaldehyde solution (PFA) containing 0.123 M NaH2PO4xH2O, 0.1 M NaOH and 0.03 M glucose for 10 min at room temperature. Fixed cells were washed with PBS and stained with Hoechst 33342 (2 μg/ml; staining cell nuclei, Cat. No. H3570, Molecular probes) and Bodipy 493/503 (0.2 μg/ml; staining neutral lipids, Cat. No. D-3922, Molecular probes) diluted in PBS for 20 min at room temperature. After washing with PBS, accumulation of intracellular lipid droplets and cell number (stained nuclei) were quantified with Acumen eX3 imager (TTP Labtech, Hertfordshire, UK). Bodipy lipid droplet fluorescence was normalized to the amount of nuclei in each well (Hoechst).

Statistical Analysis

The impact of DHA, AC and PI on cytotoxicity, lipid accumulation, adipokine secretion and metabolic outcomes (lipolysis/lipogenesis) was compared to control cells without bioactives. If statistical significance was reached, the effect was also compared with the other bioactives having significant outcomes. Additionally, to investigate possible combinatorial (synergistic, additive or antagonistic) effects of DHA exposure, the impact of DHA alone was compared to that obtained in combination with AC or PI. If a difference was found, AC and PI exposure alone was compared to the effect of the respective bioactive in combination with DHA. The data was tested for normal distribution with the Shapiro-Wilk test. If the criteria for normal distribution were fulfilled, one-way analysis of variance (ANOVA) with subsequent Tukey's HSD post-hoc test was utilized. When the criteria for normal distribution were not achieved, the non-parametric Kruskal-Wallis and Mann-Whitney pair-wise comparison tests were used. The level of statistical significance was set as 0.05 with *p < 0.05, **p < 0.01 and ***p < 0.001. Bars are shown as mean ± standard deviation.

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