Chronic Intermittent Hypoxia Triggers a Senescence-like Phenotype in Human White Preadipocytes

Figures

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  Figure 1. Intermittent hypoxia (IH) induces senescence in cultured preadipocytes. Exposure to 7 days of IH was associated with a higher prevalence of senescence associated β-galactosidase (SA-β-gal) positive preadipocytes (n = 6 independent experiments) (a). Compared to tissue grown in continuous normoxia (NO), chronic IH exposure was also associated with stronger green coloration of SA-β-gal staining in subcutaneous adipose tissue explants (ex vivo) (b). Representative images of SA-β-gal staining in cells exposed to 7 days of IH (c). Green marks indicate SA-β-gal positive (senescent) cells; Etoposide (ETO) treatment served as a positive control. Data are presented as mean ± SEM. P-values determined by one-tailed paired t-test compared to the NO control.

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  Figure 2. P16 and γH2AX expression is increased with chronic exposure to intermittent hypoxia (IH). Representative Western bolts and graphs showing upregulation of p16 (a) and γH2AX (b) protein expression with IH treatment. Representative confocal images showing increased nuclear localization of p16 (green) and γH2AX (red) in cells exposed to IH (c). Nuclei are counterstained blue (DAPI). White arrows indicate positive nuclei. Quantitation of cells positive for nuclear p16 (d), γH2AX (e) and p16&γH2AX (f) in preadipocytes grown in continuous normoxia (NO) versus cells grown with intermittent exposure to hypoxia (n = 4 independent experiments). Cells treated with etopside (ETO) were used as positive control. Data are presented as mean ± SEM. P-values determined by one-tailed paired t-test compared to the NO control.

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  Figure 3. Intermittent hypoxia (IH) alters preadipocyte phenotype and replicative capacity. Chronic IH was associated with a characteristic set of phenotypic changes such as flattened and enlarged cells (a) and a reduced multiplication ratio (MR) (b). Etoposide (ETO) was used as a positive control. Data are presented as mean ± SEM. P-values determined by one-tailed paired t-test compared to the normoxia (NO) control (n = 6 independent experiments).

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  Figure 4. Exposure of preadipocytes to chronic intermittent hypoxia (IH) attenuates lipid accumulation during differentiation. Representative images of Oil-Red-O stained preadipocytes differentiated for 6 days (a). Quantitation of lipid content accumulated in differentiated cells (b) (n = 4 independent experiments). Compared to preadipocytes grown in continuous normoxia (NO), preadipocytes exposed to IH show less lipid accumulation at day 6 of differentiation. Representative Western Blots and graphs showing protein expression of peroxisome proliferator-activated receptor γ (PPARγ) (c) (n = 7 independent experiments), cAMP response element-binding (CREB) (d) (n = 8 independent experiments), and hypoxia-inducible factor-1α (HIF-1α) (e) (n = 7 independent experiments) in 6 day differentiated preadipocytes. IH elevated expression of PPARγ and CREB even in absence of exposure to differentiating media. HIF-1α expression was elevated in preadipocytes exposed to IH prior to treatment with differentiating media. 1: NO; 2: NO + differentiation in NO; 3: IH; 4: IH + differentiation in NO; 5: IH + differentiation in IH; 6: etopside + differentiation in NO. Data are presented as mean ± SEM. P-values…

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  Figure 5. Mitochondrial reactive species generated during chronic intermittent hypoxia (IH) exposure in preadipocytes is attenuated in presence of aspirin, atorvastatin and drugs targeting renin-angiotensin system. Intervention with aspirin, atorvastatin, angiotensin II type-1 receptor antagonist (losartan), and angiotensin converting enzyme inhibitor (captopril) reduced IH-induced mitochondrial reactive oxygen species generation while angiotensinII (AngII) increased mitochondrial reactive oxygen species generation as indicated by MitoSox staining (a) Representative images of MitoSox staining with red color indicating increased ROS (b). Cells treated with menadione (150 µM) served as a positive control and cells treated with N-acetyl-L-cysteine (50 µM) served as a negative control. Data are presented as mean ± SEM, *P < 0.05 determined by one-tailed paired t-test (n = 3–5 independent experiments) as compared to IH. NO: normoxia.

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  Figure 6. Increases in nuclear H2AX foci during intermittent hypoxia (IH) is reduced by concomitant exposure to aspirin, atorvastatin and drugs targeting renin-angiotensin system. Treatment with aspirin, atorvastatin, angiotensin II type-1 receptor antagonist (losartan), and angiotensin converting enzyme inhibitor (captopril) was associated with reduced IH-induced DNA damage as indicated by H2AX phosphorylation (a). Angiotensin II (AngII) treatment resulted in increased DNA damage in both normoxia (NO) as well as IH. Representative confocal images of immunofluorescence staining with red color indicating phosphorylated H2AX foci (b). Data are presented as mean ± SEM, *P < 0.05 determined by one-tailed paired t-test (n = 3–5 independent experiments) as compared to IH.

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  Figure 7. Intermittent hypoxia (IH)-induced senescence is attenuated by treatment with aspirin, atorvastatin, and renin-angiotensin system targeted drugs. IH mediated increased prevalence of SA-β-gal positive cells was attenuated by concomitant treatment with aspirin, atorvastatin, angiotensin II type-1 receptor antagonist (losartan), and angiotensin converting enzyme inhibitor (captopril) (a). Representative images of SA-β-gal staining with green marks indicating senescent cells (b). Data are presented as mean ± SEM, *P < 0.05 determined by one-tailed paired t-test (n = 3–5 independent experiments) as compared to IH. NO: normoxia; AngII: angiotensinII.

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  Figure 8. Adipose tissue of OSA patients show increased expression of nuclear p16 and γH2AX. OSA was associated with a higher prevalence of cells with senescence-like phenotype in subcutaneous adipose tissue as determined by nuclear localization of both γH2AX and p16 (a). The prevalence of dual positive nuclei was not attenuated by CPAP usage but was altered by concomitant use of medications. Representative images of immunofluorescence staining with white arrows indicating γH2AX positive nuclei (b). OSA – obstructive sleep apnea, CPAP – continuous positive airway pressure, med – statins, aspirin and/or renin-angiotensin system inhibitors, (+/−) – present or absent. Data are presented as mean ± SEM. P-values determined by linear regression model adjusted for age and BMI.