Fig. 1Adipogenic differentiation of subcutaneous and visceral stromal-vascular fractions (SUB-SVF and VIS-SVF). (A) Oil red O staining was carried out to identify the degree of differentiation of SUB-SVF and VIS-SVF. (B–E) The mRNA expression of differentiation markers (B, fatty acid-binding proteins [Fabp4]; C, glucose transporter type 4 [Glut4]) and transcription factors (D, peroxisome proliferator-activator-γ [Pparg]; E, CCAAT/enhancer binding protein-α [Cebpa]) were detected in SUB-SVF and VIS-SVF after adipogenic differentiation. Intermittent hypoxia (IH; receiving exposure to 4 minutes of 10% O2 followed by 2 minutes of 21% O2) inhibited adipogenic differentiation of SUB-SVF but not VIS-SVF. The results are expressed as the fold change of the mean±standard error of the mean with respect to the intermittent normoxia (IN; receiving exposure to room air) group (n=5 to 6). S, SUB-SVF; V, VIS-SVF. aP<0.05.
Fig. 2Intermittent hypoxia (IH)-induced hypertrophied adipocytes in (A) subcutaneous adipose tissue (SAT) but not (B) visceral adipose tissue (VAT). Quantitative analysis of adipocyte size in SAT and VAT after haematoxylin and eosin (H&E) staining (n=4 to 6). IN, intermittent normoxia. aP<0.001.
Fig. 3Intermittent hypoxia (IH)-induced M1 macrophages in subcutaneous adipose tissue (SAT) but not visceral adipose tissue (VAT). (A) Immunofluorescence double labelling for F4/80 (macrophages) and inducible nitric oxide synthase (iNOS; M1 macrophages) in SAT and VAT. The merge view indicates activated M1 macrophages. The mRNA expression levels of (B) nitric oxide synthase 2 (Nos2)/F4/80 and (C) Nos2/arginase 1 (Arg1) in SUB-SVF and VIS-SVF. (D) The mRNA expression of a M1 macrophage marker (tumor necrosis factor-α [Tnfa]) in SUB-SVF and VIS-SVF or (E) a M2 macrophage marker (Chi313) in SUB-SVF and VIS-SVF. The results are expressed as the fold change of the mean±standard error of the mean with respect to the intermittent normoxia (IN) group (n=4 to 5). S, SUB-SVF (subcutaneous stromal-vascular fraction); V, VIS-SVF (visceral stromal-vascular fraction). aP<0.05.
Fig. 4Intermittent hypoxia (IH) promoted M1-secreted mediators in SUB-SVF. (A, B) Monocyte chemoattractant protein-1 (MCP-1) and (C, D) interleukin-6 (IL-6) were measured in conditioned media of SUB-SVF and VIS-SVF. The results are expressed as the mean±standard error of the mean (n=4 to 5). S, SUB-SVF (subcutaneous stromal-vascular fraction); V, VIS-SVF (visceral stromal-vascular fraction). aP<0.05.
Fig. 5Conditioned medium from intermittent hypoxia (IH)-exposed RAW 264.7 (Raw-IH) inhibited adipogenic differentiation of subcutaneous stromal-vascular fraction (SUB-SVF). (A) Oil red O staining was applied to identify the degree of differentiation of SUB-SVF in the absence and presence of conditioned media from air-exposed RAW 264.7 (Raw) and Raw-IH. (B–E) The mRNA expression of differentiation markers (B, fatty acid-binding proteins [Fabp4]; C, glucose transporter type 4 [Glut4])—and transcription factors (D, peroxisome proliferator-activator-γ [Pparg]; E, CCAAT/enhancer binding protein-α [Cebpa]) were detected in SUB-SVF during adipogenic differentiation. Cycle 1 (Cy1) indicates the SUB-SVF completing the first cycle of differentiation (i.e., 1 cycle), and Cycle 2 (Cy2) indicates completing both the first and the second cycles (i.e., a total of 2 cycles) of differentiation. The results are expressed as the fold change of the mean±standard error of the mean with respect to Cy1-Con group (n=4). Con, control; NS, not significant. aP<0.05, bP<0.01, cP<0.001.
Fig. 6A schematic diagram showing the involvement of macrophages in intermittent hypoxia (IH)-regulated adipogenic differentiation in subcutaneous stromal-vascular fraction. IH induced M1 macrophage polarization in subcutaneous adipose tissue and promoted the release of pro-inflammatory mediators and chemoattractants, leading to the inhibition of adipogenic differentiation and hypertrophy of adipocytes. TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; MCP-1, monocyte chemoattractant protein-1.