Riboflavin Reduces Pro-Inflammatory Activation of Adipocyte-Macrophage Co-culture. Potential Application of Vitamin B2 Enrichment for Attenuation of Insulin Resistance and Metabolic Syndrome Development

Figures

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  Figure 1. Effects of various riboflavin concentrations (10.4; 300; 500; 1000 nM) on LPS-induced: tumor necrosis factor-alpha (TNFα; a), Interleukin 6 (IL-6; b), monocyte chemotactic protein 1 (MCP-1; c) and interleukin 1 beta (IL-1β; d) cytokine mRNA expression and release of cytokines by adipocyte-macrophage co-cultures. Fully differentiated adipocytes 3T3 L1 and macrophages RAW 264.7 were cultured in 12-well dishes with inserts (0.4 μm pore size) for 6 h (mRNA expression; rtPCR) or 24 h (cytokine release; ELISA tests) after LPS stimulation (100 ng/mL). CTR – control group without LPS stimulation. The results are expressed as means + SE of five independent experiments (* p < 0.05 significance compared with 10.4 group for mRNAs expression; # p < 0.05 significance compared with 10.4 group for cytokines release to supernatant). Statistical significances were determined with the Duncan t-test.

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  Figure 2. Effects of various riboflavin concentrations (10.4; 300; 500; 1000 nM) on LPS-induced adipokines: leptin (a) and adiponectin (b) mRNA expression by adipocytes 3T3 L1 and adipokine release by adipocyte-macrophage co-cultures. Fully differentiated adipocytes 3T3 L1 and macrophages RAW 264.7 were cultured in 12-well dishes with inserts (0.4 μm pore size) for 6 h (mRNA expression) or 24 h (cytokine release) after LPS stimulation (100 ng/mL). CTR – control group without LPS stimulation. The results are expressed as means + SE of five independent experiments (* p < 0.05 significance compared with 10.4 group for adipokine mRNAs expression; # p < 0.05 significance compared with 10.4 group for adipokine release to supernatant). Statistical differences were determined with the Duncan t-test.

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  Figure 3. Effects of various riboflavin concentrations (10.4; 300; 500; 1000 nM) on LPS-induced HMGB1 mRNA expression and release by adipocyte-macrophage co-cultures. Fully differentiated adipocytes 3T3 L1 and macrophages RAW 264.7 were cultured in 12-well dishes with inserts (0.4 μm pore size) for 6 h (mRNA expression) or 24 h (HMGB1 release) after LPS stimulation (100 ng/mL). CTR – control group without LPS stimulation. The results are expressed as means + SE of five independent experiments (* p < 0.05 significance compared with 10.4 group for HMGB1 mRNA expression; # p < 0.05 significance compared with 10.4 group for HMGB1 release to supernatant). Statistical differences were determined with the Duncan t-test.

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  Figure 4. Effects of various riboflavin concentrations (10.4; 300; 500; 1000 nM) on LPS-induced metalloproteinase-9 (MMP-9; a,c) and its inhibitor TIMP-1 (b) mRNA expression and release by adipocyte-macrophage co-cultures. Fully differentiated adipocytes 3T3 L1 and macrophages RAW 264.7 were cultured in 12-well dishes with inserts (0.4 μm pore size) for 6 h (mRNA expression) or 24 h (cytokine release) after LPS stimulation (100 ng/mL). CTR − control group without LPS stimulation. (d) Macrophages RAW 264.7 migration to supernatants from adipocytes cultured in various riboflavin concentrations for 24 h and migration to various riboflavin concentrations (RF solution). Normal medium was used as a negative control (CTR−), fMLP was used as a positive control (CTR+). The results are expressed as means + SE of four independent experiments (* p < 0.05 significance compared with 10.4 group for mRNAs expression; # p < 0.05 significance compared with 10.4 group for MMP-9 activity, TIMP-1 release and migration to supernatant). Statistical significances were determined with the Duncan t-test.

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  Figure 5. Effects of various riboflavin concentrations (10.4; 300; 500; 1000 nM) on LPS-induced nitric oxide synthase (iNOS) mRNA and protein expression (a) and nitric oxide release (NO; b) by RAW 264.7 macrophages cultured in adipocyte-macrophage co-cultures. Fully differentiated adipocytes 3T3 L1 and macrophages RAW 264.7 were cultured in 12-well dishes with inserts (0.4 μm pore size) for 6 h (mRNA, protein expression) or 24 h (NO release) after LPS stimulation (100 ng/mL). CTR – control group without LPS stimulation. The results are expressed as means + SE of five independent experiments (* p < 0.05 significance compared with 10.4 group for iNOS mRNA expression; # p < 0.05 significance compared with 10.4 group for iNOS protein expression and NO release to supernatant). Statistical differences were determined with the Duncan t-test.

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  Figure 6. Effects of various riboflavin concentrations (10.4; 300; 500; 1000 nM) on LPS-induced phosphorylation of nuclear factor κB (p-NFκB) in co-cultured RAW 264.7 macrophages and 3T3 L1 adipocytes. Fully differentiated adipocytes and macrophages were cultured in 12-well dishes with inserts (0.4 μm pore size) upon LPS stimulation (100 ng/mL). CTR – control group without LPS stimulation. The results are expressed as means + SE of five independent experiments (* p < 0.05 significance compared with 10.4 group). Statistical differences were determined with the Duncan t-test.