Fig. 4

AS differentiated with InRo medium have increased expression of adipogenic markers and leptin secretion compared to adipocyte monolayers and increased glycerol secretion in response to isoprenaline stimulation. A Quantification of mRNA levels relative to adult mouse WAT (dotted line). Data are presented as mean ± standard deviation. N = 4 per group. Two-way ANOVA with Tukey's post-test. B Leptin concentration in conditioned medium (48 h) normalized to total DNA. Data are presented as mean ± standard deviation. N = 4 per group. C Differentiated adipocyte monolayers and AS at day 15 of differentiation were incubated with DMEM-F12 medium plus 10% SFB in the absence of insulin and rosiglitazone for 48 h. Next, cells were incubated for additional 48 h with DMEM-F12 medium plus 10% SFB in the presence or absence of 2,000 nM insulin. The conditioned medium (48 h) was harvested for leptin quantification and normalized by total DNA. Data are presented as mean ± standard deviation. N = 4 per group. A significant effect for culture format was determined (F (1, 12) = 21.93; p = 0.0005). D 2D adipocytes and AS were differentiated with InRo protocol and incubated by 3 h in color-free DMEM-F12 medium with or without 1 μM isoprenaline. Conditioned medium was collected for glycerol determination by colorimetric methods and AS. Cells were collected for total DNA extraction and quantification. Data are represented as mean ± standard deviation. N = 3 for monolayer cultures and 5 for AS groups. There was a significant increment in glycerol secretion in response to isoprenaline stimulation (F (1, 11) = 8.357; P = 0.0147). Two-way ANOVA with Tukey's post-test. * p < 0.05; ** p < 0.01; ns: not significant; N.D.: non-detected