Chronic Intermittent Hypoxia-Induced Dysmetabolism Is Associated with Hepatic Oxidative Stress, Mitochondrial Dysfunction and Inflammation

Abstract

The association between obstructive sleep apnea (OSA) and metabolic disorders is well-established; however, the underlying mechanisms that elucidate this relationship remain incompletely understood. Since the liver is a major organ in the maintenance of metabolic homeostasis, we hypothesize that liver dysfunction plays a crucial role in the pathogenesis of metabolic dysfunction associated with obstructive sleep apnea (OSA). Herein, we explored the underlying mechanisms of this association within the liver. Experiments were performed in male Wistar rats fed with a control or high fat (HF) diet (60% lipid-rich) for 12 weeks. Half of the groups were exposed to chronic intermittent hypoxia (CIH) (30 hypoxic (5% O₂) cycles, 8 h/day) that mimics OSA, in the last 15 days. Insulin sensitivity and glucose tolerance were assessed. Liver samples were collected for evaluation of lipid deposition, insulin signaling, glucose homeostasis, hypoxia, oxidative stress, antioxidant defenses, mitochondrial biogenesis and inflammation. Both the CIH and HF diet induced dysmetabolism, a state not aggravated in animals submitted to HF plus CIH. CIH aggravates hepatic lipid deposition in obese animals. Hypoxia-inducible factors levels were altered by these stimuli. CIH decreased the levels of oxidative phosphorylation complexes in both groups and the levels of SOD-1. The HF diet reduced mitochondrial density and hepatic antioxidant capacity. The CIH and HF diet produced alterations in cysteine-related thiols and pro-inflammatory markers. The results obtained suggest that hepatic mitochondrial dysfunction and oxidative stress, leading to inflammation, may be significant factors contributing to the development of dysmetabolism associated with OSA.

Keywords: chronic intermittent hypoxia; inflammation; insulin resistance; metabolic disorders; mitochondrial dysfunction; obstructive sleep apnea; oxidative stress.

Figure 1

Schematic representation of the study experimental design. Male Wistar rats (12 weeks) were divided into a control group (CTL), fed with a standard diet, and an obese group (HF), fed with a 60% lipid-enriched diet for 12 weeks. From weeks 10 to 12, half of the animals from both groups were submitted to a CIH protocol (CIH and HFIH) consisting of 30 intermittent hypoxia cycles/h, 8 h/day. During the CIH protocol, the remaining age-matched control or high-fat animals were in the same room and exposed to a normal air atmosphere, to experience similar conditions.

Figure 2

Effects of chronic intermittent hypoxia (CIH) and high-fat diet (HF) on whole-body metabolic parameters, insulin sensitivity (A), and glucose tolerance (B,C); and on liver lipid deposition (D). Panel (A) shows the effect of CIH and HF diet on insulin sensitivity evaluated using HOMA-IR index. Panel (B) shows, on the left, the glucose excursion curves of the intra-peritoneal glucose tolerance test (ipGTT) and on the right, the correspondent area under the curve (AUC). Panel (D) shows from the left to the right representative images of H&E staining from CTL, CIH, HF, and HFIH animals. Visual analysis of H&E staining shows an increase in lipid deposition in the HF group, which is aggravated by exposure to chronic intermittent hypoxia (Scale bar 50 μm). Animal groups: CTL-control; CIH-chronic intermittent hypoxia; HF-high-fat diet; HFIH-high-fat plus CIH; Data are presented as means ± SEM. Two-way ANOVA with Bonferroni and Sidak’s multiple comparison tests: * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 compared with control animals; $ p < 0.05, $$ p < 0.01, $$$$ p < 0.0001 compared with control animals submitted to the CIH protocol.

Figure 3

Effects of chronic intermittent hypoxia (CIH) and high-fat (HF) diet on HIF-1α (A–C) and HIF-2α (D–F) levels in the liver. Panel (A,D) depicts the levels of HIF-1α (120 kDa) and HIF-2α (118 kDa), respectively, in the liver. Protein levels were normalized to the loading control β-actin (42 kDa). On the top of each graph are presented the representative Western blots for each protein studied. Panel (B,F) show the fluorescence quantification for HIF-1α and HIF-2α (orange fluorescence), respectively, normalized for the number of nuclei present in the liver labelled with DAPI staining (blue fluoresecence). Panel (C,F) are representative immunohistochemistry images from liver labelled with HIF-1α and HIF-2α, respectively, and the merged images with DAPI for each hypoxia marker (scale bar: 50 μm). Animal groups: CTL-control; CIH-chronic intermittent hypoxia; HF-high-fat diet; HFIH-high-fat plus CIH; data are presented as means ± SEM. Two-way ANOVA with Bonferroni and Sidak’s multiple comparison tests, respectively: * p < 0.05, ** p < 0.01, *** p < 0.001 compared with CTL animals; $ p < 0.05, $$$ p < 0.001, $$$$ p < 0.0001 compared with CTL animals submitted to the CIH protocol.

Figure 4

Effects of chronic intermittent hypoxia (CIH) and high-fat (HF) diet on the levels of proteins involved in insulin signaling and glucose transport in the liver. Panels (A–D) depicts the levels of insulin Receptor (IR, 90 kDa), protein kinase B (Akt, 60 kDa), insulin degrading enzyme (IDE, 118 kDa, and glucose transporter 2 (Glut2, 60 kDa), respectively. Protein levels were normalized to the loading control β-actin (42 kDa). On the top of each graph are presented the representative Western blots for each protein studied. Animal groups: CTL—control; CIH—chronic intermittent hypoxia; HF—high-fat diet; HFIH—high-fat plus CIH. Data are presented as means ± SEM. Two-way ANOVA with Bonferroni and Sidak’s multiple comparison tests, respectively: * p < 0.05 compared with control animals.

Figure 5

Effects of chronic intermittent hypoxia (CIH) and high-fat (HF) diet on the mitochondrial density, evaluated by Mitotracker assay; oxidative phosphorylation complexes levels, evaluated by Western blot analysis of OXPHOS complexes levels; and activities, namely citrate synthase, complex I, and complex II activity assessment. Panels (A,B) present the representative immunohistochemistry staining of Mitotracker (red fluorescence) and merge with DAPI (blue fluoresence) and the fluorescent intensity quantification, respectively (scale bar 50 μm). In panel (C), the graph depicts the levels of complex I (20 kDa), II (30 kDa), III (48 kDa), IV (40 kDa), and V (55 kDa). Proteins levels were normalized to the loading control, calnexin (90 KDa). On the right side of the graph, we show representative Western blots for each protein evaluated. On panel (D–F), the activity of citrate synthase, mitochondrial complex I, and mitochondrial complex II, respectively, is represented. The enzymatic activity of each complex was normalized by citrate synthase activity. Animal groups: CTL—control; CIH—chronic intermittent hypoxia; HF—high-fat diet; HFIH—high-fat plus CIH. Data are presented as means ± SEM. Two-way ANOVA with Bonferroni and Sidak’s multiple comparison tests, * p < 0.05, ** p < 0.01 compared with CTL animals, # p < 0.05, ## p < 0.01, ### p < 0.001 compared with HF animals, and $ p < 0.05 compared with CTL animals submitted to the CIH protocol.

Figure 6

Effects of chronic intermittent hypoxia (CIH) and high-fat (HF) diet on ROS levels, lipid peroxidation (LPO), and on the antioxidant capacity of the liver. Panel (A) depicts the intracellular levels of ROS in the liver measured by CM-H2DCFDA labelling. Panel (B) presents the FRAP assay showing the overall antioxidant capacity of the tissue. Panel (C) depicts the levels of LPO shown by the index of lipid peroxidation measured per g of liver tissue. Panels (D,E) depicts the levels of catalase (60 KDa) and superoxide Dismutase 1 (SOD-1, 23 KDa), respectively. Protein levels were normalized to the loading control β-actin (42 kDa) and calnexin (90 KDa), respectively. Top of the graphs show representative Western blots for each protein studied. Panels (F–H) represent the levels of cysteine-related thiols in the liver, namely Cys on panel (F), CysGly in panel (G), and GSH in panel (H). In each of these panels, it is shown on the left the total fractions of each thiol, in the middle the free total thiol fraction, and on the right the protein-bounded thiol fraction. Animal groups: CTL—control; CIH—chronic intermittent hypoxia; HF—high-fat diet; HFIH—high-fat plus CIH. Data are presented as means ± SEM. Two-way ANOVA with Bonferroni and Sidak’s multiple comparison tests: * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with CTL animals; # p < 0.05 and #### p < 0.0001 compared with HF animals; $ p < 0.05 and $$ p < 0.01 compared with CIH animals.

Figure 7

Effects of chronic intermittent hypoxia (CIH) and high-fat (HF) diet on the levels of inflammatory markers in the liver. Panel (A–F) depicts, respectively, the levels of Arginase I (35 KDa), NF-κB (65 KDa), IL-6 Receptor (IL-6R, 80 KDa), IL-1 Receptor (IL-1R, 80 KDa), tumor necrosis factor alpha (TNF-α, 17 KDa), and TNF-α Receptor (TNF-αR, 55 KDa). Protein levels were normalized to the loading control β-actin (42 kDa). Top of the graphs show representative Western blots for each protein studied. Panel (G,H) depicts, respectively, the mean fluorescence of F4/80 per area (% of control), immunofluorescence images for positive staining for F4/80 (green fluorescence, top panel), and merge with the DAPI (blue fluorescence, bottom panels) (scale bar 25 μm). Animal groups: CTL—control; CIH—chronic intermittent hypoxia; HF—high-fat diet; HFIH—high-fat plus CIH. Data are presented as means ± SEM. Two-way ANOVA with Bonferroni and Sidak’s multiple comparison tests: * p < 0.05 and ** p < 0.01 compared with CTL animals; $ p < 0.05 compared with CIH animals; # p < 0.05 compared with HF animals.