Reagents and materials
Murine fibroblast (3T3-L1, cat no. CL-173) and macrophage (RAW 264.7, cat no. TIB-71) immortalized cell lines were purchased from ATCC (Manassass, VA). Dulbecco’s modified Eagle’s medium (DMEM), phosphate buffered saline (PBS), fetal bovine serum (FBS), penicillin–streptomycin, and polystyrene 6-well plates were purchased from Fisher Scientific (Pittsburg, PA). The IL-6 (DY406-05-Duo Set for preliminary experiments and SM6000B-quantikine for all other experiments) and leptin (SMOB00B) enzyme-linked immunosorbent assays (ELISA) were purchased from R&D systems (Minneapolis, MN), while the Griess assay for nitric oxide (NO) quantification was obtained from Promega (Madison, WI). Trypan blue, insulin, dexamethasone (DEX), d-biotin, 3-isobutyl-1-methylxanthine (IBMX), trypsin–EDTA 0.25%, oil red O dye, and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (St Louis, MO). Transwell permeable supports (0.4 μm pore size, 12 mm diameter, polyester membrane) and 12-well plates (polystyrene) were obtained from Corning Costar (Corning, NY).
3T3-L1 cell maintenance, culture, and differentiation into mature adipocytes
The 3T3-L1 cell line must be differentiated from a fibroblast phenotype into its final, mature adipocyte phenotype containing lipid droplets. To do this, the cells were incubated in 12-well plates, in a 5% CO2 humidified atmosphere, and were kept in the undifferentiated fibroblast phenotype at less than 50% confluency during sub-culturing. Detachment of cell monolayer for sub-culturing was performed via trypsinization. Growth medium for 3T3-L1 cells consisted of DMEM, 10% (v/v) heat-inactivated FBS, 1% antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin), and 0.008 μg/ml D-biotin. Differentiation into the adipocyte phenotype was performed as described by Zebisch et al. (2012). Briefly, three days after cells reached ~ 90% confluency and started to clump together and lose fibroblast morphology, they were washed with 1X PBS and treated with a differentiation cocktail consisting of growth medium supplemented with 0.5 mM IBMX, 1 μM DEX, and 20 μg/ml insulin. Forty-eight hours after addition of the differentiation cocktail, the cells were washed with 1X PBS and treated with post-differentiation medium consisting of growth medium supplemented with 20 μg/ml insulin. Treatment with post-differentiation medium was performed every forty-eight hours for a total of four times. At the end of the differentiation period, lipid droplets inside the adipocytes could be visualized using an inverted microscope. In preliminary experiments, lipid droplet deposition was quantified through spectrophotometric analysis of Oil Red O staining as described by Manickam et al. (2010) to confirm the differentiation of the fibroblasts into the mature adipocyte phenotype. Briefly, both differentiated and undifferentiated cells were washed with cold PBS (pH 7.4) and subsequently fixed with a 4% paraformaldehyde solution. The cells were then stained for 30 min with Oil Red O dye (0.5% w/v) prepared in isopropanol and diluted to a working solution at a 3:2 ratio of dye/water. Cells were then washed thoroughly with water, and then, the dye in the lipid droplets was dissolved with isopropanol. The isopropanol-dye solution was analyzed spectrophotometrically at A520 nm, and values obtained for undifferentiated cells were compared to those of differentiated cells (Additional file 1: Fig. S1).
RAW 264.7 cell maintenance and culture
RAW 264.7 cells were grown in polystyrene 6-well plates with DMEM supplemented with 10% (v/v) heat-inactivated FBS and 1% antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin). Cells were incubated in a 95% O2 and 5% CO2 humidified atmosphere. During initial expansion, the medium was changed every two days after washing cells with 1× PBS. Cells were not grown beyond 80% confluency during expansion. Detachment of cell monolayer for sub-culturing was performed with the cell scraping method. When cells reached 80% confluency, they were transferred to transwell inserts to commence the co-incubation experiments.
RAW 264.7 cell activation
A concentration of 0.01 μg/ml of LPS was used, and three methods of LPS challenge for co-incubation experiments were tested: macrophages in 6-well plate were washed, resuspended (via scraping) in fresh medium, transferred to transwell inserts and challenged with LPS that was added into the transwell compartment (3T3 + RAW + LPS); macrophages were challenged with LPS for 24 h while in well of 6-well plate, were then washed, resuspended (via scraping) in fresh medium, and transferred to transwell insert (3T3 + StimRAW); macrophages were challenged with LPS for 24 h while in well of 6-well plate and then resuspended (via scraping) and transferred to transwell insert along with the conditioned media (3T3 + StimRAW + CondMed). Non-LPS-challenged macrophages were co-incubated with adipocytes as a control (3T3 + RAW). The LPS concentration (0.01 mg/ml) and incubation time (6 h) were selected based on preliminary experiments testing the effects of low LPS doses that resulted in high production of IL-6 and NO by the macrophages (Additional file 1: Fig. S2). The highest IL-6 production was observed when incubating the cells with 0.01 mg/ml of LPS for 6 h (Additional file 1: Fig. S2a). While 0.1 mg/ml of LPS resulted in significantly higher NO production by the macrophages after 6 and 24 h of incubation compared to 0.01 mg/ml (Additional file 1: Fig. S2b), both LPS doses caused the macrophages to produce IL-6 concentrations beyond the limit of detection of the assay (IL-6 DuoSet ELISA). Based on these results, we selected the lowest dose that resulted in a strong response in order to utilize a concentration that falls within the bounds of what is considered physiologically and clinically relevant (Guo et al. 2013). The macrophages did not produce quantifiable levels of leptin under any of the LPS concentrations or incubation times (data not shown).
Quantification of nitric oxide (NO) production
Macrophage activation was quantified via measurement of NO levels. Nitric oxide production by the macrophages was determined through quantifying nitrite levels in cell-conditioned media using the Griess assay. Briefly, 50 μL of cell-conditioned media was added in triplicate to a 96-well plate and mixed with 50 μL of sulfanilamide solution and allowed to incubate for 10 min. Following the incubation, 50 μL of N-1-naphthylethylenediamine (NED) was added to each well, followed by a 10 min incubation. After the second incubation, absorbance was measured at 530 nm. Nitrite concentrations were determined by extrapolating absorbance measurements from a 0–100 μM standard curve. An Epoch plate reader (BioTek Instruments, Winooski, VT) was used for absorbance measurements (Chen et al. 2017).
Co-incubation of RAW 264.7 and 3T3-L1 cells using transwell inserts
Several co-incubation methodologies were tested to determine if they resulted in different activation level of the macrophages, as measured by NO and IL-6 levels in cell-conditioned medium. Additionally, leptin levels in cell-conditioned medium were measured to test if activation state of the macrophages affected production of this adipokine by the mature adipocytes. Three methods of macrophage stimulation/co-incubation were tested. Mature adipocytes (differentiated according to the steps described in “3T3-L1 cell maintenance, culture, and differentiation into mature adipocytes” section) in 12-well plates were co-incubated with macrophages stimulated with LPS as described in the “RAW 264.7 cell activation” section: 1. 3T3 + RAW + LPS, 2. 3T3 + StimRAW, and 3. 3T3 + StimRAW + CondMed. Additionally, adipocytes were co-incubated with unstimulated RAW264.7 cells resuspended in fresh medium (3T3 + RAW) as a control. On average, 8.0 × 105 macrophages were plated onto each transwell insert in a total of 500 μl. Twenty-four hours after co-incubation commenced, media was collected separately from the transwell inserts and wells, transferred to 1.5 ml microcentrifuge tubes, and stored at − 20 °C until used for IL-6, leptin, and NO quantification. Wells were run in duplicate, and experiments were performed four times.
Quantification of adipokine production
Analyses of cell-conditioned media for determination of adipokine levels were done using IL-6 and leptin sandwich ELISAs according to manufacturer’s instructions. Samples were tested in triplicate, and a standard curve was produced and used to extrapolate the cytokine concentrations in the samples. Experimental samples were analyzed using the quantikine IL-6 ELISA, and samples measuring > 500 pg/ml (highest standard) were diluted, re-quantified, and results were adjusted taking the dilution factor into account. Samples from LPS dose/time response preliminary experiments were analyzed using the DuoSet IL-6 ELISA, and samples measuring above the 1000 pg/ml highest standard of the DuoSet IL-6 ELISA were analyzed as “1000 pg/ml.” An Epoch plate reader was used for absorbance measurements.
Statistical analyses
Statistical analyses were done using JMP Pro 13 (Cary, NC). Non-normal data were normalized using a log transformation. Matched paired t tests were used to determine differences in NO, IL-6, and leptin levels between cell-conditioned medium collected from transwell inserts containing macrophages and wells containing adipocytes. In order to determine differences in NO, IL-6, and leptin produced by control and LPS-challenged cells, Student’s t tests were used. Linear regression analyses were used to test relationships between IL-6, NO, and leptin. Interleukin-6, leptin, and NO concentrations in cell-conditioned media were analyzed using the general mixed linear model. All statistical analyses were conducted using JMP Pro 15 (SAS, Cary, North Carolina). The sources of variation included experiment, treatment, experiment x treatment interaction, and well nested within experiment x treatment interaction. The experiment, treatment × experiment interaction, and well nested within experiment x treatment interaction were considered as random variables. When treatment effects were detected, means were separated using Tukey’s HSD. The level of significance was defined at p < 0.05. Experimental results are expressed as mean ± SE.