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

Results

Titration of the in Vitro Conditions for DHA Treatment of Primary Human Adipocytes

DHA concentrations that have been used in other fat cell systems (10–60 μM)[18–20,41] as well as in optimizations by PATHWAY-27 collaborators in SGBS cells, induced LDH activity in conditioned media (after 6 days of stimulation) and increased IL-6 secretion (after 48 h of stimulation), indicating both cytotoxic and pro-inflammatory effects (Additional file 2: Figure S2 https://static-content.springer.com/esm/art%3A10.1186%2Fs12986-016-0064-3/MediaObjects/12986_2016_64_MOESM2_ESM.pptx). These findings are difficult to reconcile with the reported physiological effects of omega-3-fatty acids in vivo.[42] Consequently, the DHA concentration was titrated down and 0.5 μM was selected as the highest non-toxic DHA concentration in our primary culture system.

Decreased Basal Adipocyte Lipolysis After Treatment With Bioactive Compounds

Glycerol release was significantly decreased after all treatments when compared to control, with the exception of AC (p = 0.056) (Fig. 1). Treatment with PI had the strongest attenuating effect on glycerol release compared to control-treated cells which was stronger than that of DHA (p = 0.009). None of the compounds induced cytotoxicity at the selected concentrations, but rather reduced LDH activity in cell cultures after 48 h of treatment (Additional file 3: Figure S3 https://static-content.springer.com/esm/art%3A10.1186%2Fs12986-016-0064-3/MediaObjects/12986_2016_64_MOESM3_ESM.pptx), except for AC in combination with DHA (p = 0.321, due to a large inter-individual variation of the response). LDH activity remained unaltered up after 6 days of treatment with the bioactive compounds.

Figure 1.

Decreased basal adipocyte lipolysis after treatment with bioactive compounds or metabolites. Glycerol release after 48 h of treatment with 0.5 μM docosahexaenoic acid (DHA), anthocyanin (AC), that is 130 nM cyanidin-3-glucoside (C3G) and 13 μM protocatechuic acid (PCA), and 100 μM propionate (PI), relative to control, measured in conditioned medium from human in vitro differentiated adipocytes. Control, n = 9; 0.5 μM DHA, n = 6; AC alone and with DHA, n = 3; 100 μM PI alone and with DHA, n = 4 biological/independent experiments in quadruplicates. Normalized data is adjusted for protein amount and presented as means +/- standard deviation. *p < 0.05, **p < 0.01 and ***p < 0.001 versus control. Statistical significance was determined by one-way ANOVA with Tukey's multiple comparisons post hoc test

Combined Treatment With DHA and AC Increased Insulin-stimulated Adipocyte Lipogenesis

To determine the effects of the bioactive compounds or their metabolites on insulin-stimulated lipogenesis, relative glucose incorporation into lipids was measured in the in vitro differentiated primary adipocytes. Insulin induced lipogenesis in the control and all treatment conditions (Fig. 2). No effect on insulin-stimulated response was found after treatment with any of the compounds alone.

Figure 2.

Combined treatment with DHA and AC increased insulin-stimulated adipocyte lipogenesis. Basal and insulin-stimulated lipogenesis measured in cell lysates after 48 h of treatment with bioactive compounds. Control, n = 8; 0.5 μM DHA, n = 6; AC alone and with DHA, n = 3; 100 μM PI alone, n = 3 and with DHA, n = 4 biological/independent experiments in duplicates. Glucose incorporation into lipids (3-3H counts per minute) is normalized for protein concentration in the cell lysates for each well. Normalized data is presented as means +/- standard deviation. *p < 0.05, **p < 0.01 and ***p < 0.001 counted against basal control with the same bioactive treatment or insulin-stimulated control compared with insulin-stimulated bioactive treated cells. Statistical significance was determined by the non-parametric Kruskal-Wallis test with subsequent Mann-Whitney multiple comparisons post hoc test

However, combined treatment with DHA and AC increased insulin-stimulated glucose uptake compared to the control.

Bioactives Alter Secretion of Pro-inflammatory Adipokines and Adiponectin

To investigate the effect of the bioactive compounds or their metabolites on a panel of the most important pro- (IL-6, MCP-1, TNFα, chemerin) and anti-inflammatory (adiponectin) adipokines, conditioned media from the adipose cells were collected after 48 h of treatment and secretion was measured by ELISA. IL-6, MCP1 and chemerin secretion were suppressed by DHA (Fig. 3a, b and e). The effect of DHA on MCP-1 and chemerin secretion was also statistically significant in combination with PI (Fig. 3b and e). Additionally, DHA combined with PI impeded TNFα secretion, a result that was also obtained with PI alone (Fig. 3d). PI was the only compound that had an effect on adiponectin, contributing to a slightly decreased secretion (Fig. 3c). As the other two compounds, PI suppressed IL-6 secretion (Fig. 3a). AC alone attenuated IL-6 and MCP-1 secretion and in combination with DHA, demonstrated a suppressed secretion of TNFα (Fig. 3a, b and d).

Figure 3.

Bioactives or metabolites alter secretion of pro-inflammatory adipokines and adiponectin. Secretion of adipokines; interleukin-6 (IL-6) (a), monocyte chemoattractant protein-1 (MCP-1) (b), adiponectin (c), tumour necrosis factor α (TNFα) (d) and chemerin (e); after 48 h of treatment with bioactive compounds relative to control in conditioned media. Control, n = 6–9; 0.5 μM DHA, n = 3–6; AC alone and with DHA, n = 3; 100 μM PI alone and with DHA, n = 3–4 in biological/independent experiments in quadruplicates, with the exception of TNFα where n = 2–6 due to levels below minimum detectable dose in four subjects. Normalized data is adjusted for protein amount and presented as means +/- standard deviation. *p < 0.05, **p < 0.01 and ***p < 0.001 versus control. Statistical significance was obtained by one-way ANOVA with Tukey's multiple comparisons post hoc test

No Effects of Bioactives on Adipocyte Differentiation

To measure possible effect of bioactives on adipocyte differentiation and lipid accumulation, cells were treated for 6 days and lipid content was measured by Bodipy staining and normalized per cell based on DNA staining by Hoechst (Fig. 4a). No effects were found of the selected compounds on lipid accumulation after 6 days of treatment (Fig. 4b).

Figure 4.

No effects of bioactives or metabolites on lipid accumulation. Representative pictures of neutral lipid accumulation (green) and cell nuclei (blue) staining after 6 days of treatment with bioactive components (a). Quantified lipid accumulation adjusted for cell amount (control, n = 8; 0.5 μM DHA, n = 8; AC alone and with DHA, n = 4; 100 μM PI alone and with DHA, n = 6 biological/independent experiment in duplicates (b). Normalized data is presented as means +/- standard deviation versus control. The absence of statistical significance was determined by the non-parametric Kruskal-Wallis test

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