Diabetes Metab J.  2019 Oct;43(5):659-674. 10.4093/dmj.2018.0196.

Low-Frequency Intermittent Hypoxia Suppresses Subcutaneous Adipogenesis and Induces Macrophage Polarization in Lean Mice

• Wang Y
• Lee MY
• Mak JC
• Ip MS

• KMID: 2460959
• DOI: http://doi.org/10.4093/dmj.2018.0196

Abstract

BACKGROUND
The relationship between obstructive sleep apnoea (OSA) and metabolic disorders is complex and highly associated. The impairment of adipogenic capacity in pre-adipocytes may promote adipocyte hypertrophy and increase the risk of further metabolic dysfunction. We hypothesize that intermittent hypoxia (IH), as a pathophysiologic feature of OSA, may regulate adipogenesis by promoting macrophage polarization.
METHODS
Male C57BL/6N mice were exposed to either IH (240 seconds of 10% Oâ‚‚ followed by 120 seconds of 21% Oâ‚‚, i.e., 10 cycles/hour) or intermittent normoxia (IN) for 6 weeks. Stromal-vascular fractions derived from subcutaneous (SUB-SVF) and visceral (VIS-SVF) adipose tissues were cultured and differentiated. Conditioned media from cultured RAW 264.7 macrophages after air (Raw) or IH exposure (Raw-IH) were incubated with SUB-SVF during adipogenic differentiation.
RESULTS
Adipogenic differentiation of SUB-SVF but not VIS-SVF from IH-exposed mice was significantly downregulated in comparison with that derived from IN-exposed mice. IH-exposed mice compared to IN-exposed mice showed induction of hypertrophic adipocytes and increased preferential infiltration of M1 macrophages in subcutaneous adipose tissue (SAT) compared to visceral adipose tissue. Complementary in vitro analysis demonstrated that Raw-IH media significantly enhanced inhibition of adipogenesis of SUB-SVF compared to Raw media, in agreement with corresponding gene expression levels of differentiation-associated markers and adipogenic transcription factors.
CONCLUSION
Low frequency IH exposure impaired adipogenesis of SAT in lean mice, and macrophage polarization may be a potential mechanism for the impaired adipogenesis.

Keyword

Adipogenesis; Inflammation; Hypoxia; Macrophages

MeSH Terms

Adipocytes
Adipogenesis*
Animals
Anoxia*
Culture Media, Conditioned
Gene Expression
Humans
Hypertrophy
In Vitro Techniques
Inflammation
Intra-Abdominal Fat
Macrophages*
Male
Mice*
Subcutaneous Fat
Transcription Factors
Culture Media, Conditioned
Transcription Factors

Figure

• 

  Fig. 1 Adipogenic 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. 2 Intermittent 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. 3 Intermittent 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. 4 Intermittent 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. 5 Conditioned 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. 6 A 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.

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