Hyperbaric Oxygen Ameliorates Insulin Sensitivity by Increasing GLUT4 Expression in Skeletal Muscle and Stimulating UCP1 in Brown Adipose Tissue in T2DM Mice

Figures

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  Figure 1. Schedule diagram. (A) Type 2 diabetic model building. (B) HBO treatment protocol.

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  Figure 2. The differences between HFD and T2DM mice in terms of blood glucose levels, body weight, OGTT, ITT, insulin concentration, and HOMA-IR. (A) Blood glucose in HFD and T2DM mice under 12-h fasting conditions after successful induction of diabetes. (B) Changes in body weight over 46 days. (C) Oral glucose tolerance tests (OGTT) in mice fed with high-fat diet. (D) Insulin tolerance tests (ITT) in HFD and T2DM mice. (E) Insulin concentration and (F) HOMA-IR values between HFD and T2DM mice. Data are presented as means ± SEM (HFD: n = 13; T2DM: n = 15). *P < 0.05, **P < 0.01 (compared with HFD group), #P < 0.05, ##p < 0.01 (compared with 0 min blood glucose in T2DM group), and ∧P < 0.05, ∧∧P < 0.01 (compared with 0 min blood glucose in HFD group).

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  Figure 3. Changes in blood glucose, body weight gained after 7-day HBO or NBO treatment. (A) The blood glucose changes pre- and post HBO treatment. (B) HBO-induced body weight gained after 7-day HBO treatment. Data are presented as means ± SEM (HFD: n = 6; HFD+HBO: n = 7; T2DM: n = 7; T2DM+HBO: n = 8). **P < 0.01 (compared with T2DM group), ##P < 0.05 (compared with HFD group).

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  Figure 4. HBO influenced feeding responses and central NPY-positive neurons expression in the Arc. (A) After 12-h food deprivation, the HBO increased nocturnal cumulative food intake in the last 2 h of monitoring in T2DM. (B) NPY immunoreactive cell detection in Arc (200×, picture in upper-lift corner is 400×). Numbers of NPY-positive neurons were counted. Data are presented as means ± SEM(HFD: n = 6; HFD+HBO: n = 7; T2DM: n = 7; T2DM+HBO: n = 8). Normobaric oxygen (NBO). Scale bar = 100 μm. *P < 0.05 (compared with T2DM group), ##P < 0.01 (compared with HFD+HBO group).

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  Figure 5. Effects of HBO on insulin sensitivity and HOMA-IR coefficient. (A) Insulin tolerance tests (ITT) in HFD and T2DM mice after HBO treatment. (B) The insulin-resistance coefficient. HOMA IR = fasting plasma insulin (mU/ml) × fasting plasma glucose (mmol/L)/22.5. Data are presented as means ± SEM (HFD: n = 6; HFD+HBO: n = 7; T2DM: n = 7; T2DM+HBO: n = 8). *P < 0.05, **P < 0.01 (compared with T2DM group), ∧P < 0.05 (compared with HFD+HBO group), ##P < 0.01 (compared with HFD group).

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  Figure 6. Effect of HBO on β cell mass. Pancreas were cut into 5-μm sections stained with an anti-insulin antibody by immunohistochemical staining (400×) and area ratio of β cell mass were counted. Data are presented as means ± SEM (HFD: n = 6; HFD+HBO: n = 7; T2DM: n = 7; T2DM+HBO: n = 8). *P < 0.05 (compared with T2DM group), #P < 0.05 (compared with HFD group).

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  Figure 7. Effect of HBO on levels of GLUT4 in skeletal muscle, Akt, and AMPK phosphorylation. (A) GLUT4 expression levels detected by immunofluorescence in skeletal muscle (400×) and measured intensity per arbitrary units. Arrows point to GLUT4 immunoreactive cells. Blue is DAPI (HFD: n = 3; HFD+HBO: n = 3; T2DM: n = 4; T2DM+HBO: n = 4). (B) GLUT4 levels in skeletal muscle measuring by western blot. (C) Western blot of p-Akt/Akt level in skeletal muscle (n = 3 per group). (D) Western blot of p-AMPK/AMPK level in skeletal muscle (n = 3 per group). Scale bar = 200 μm. Data are presented as means ± SEM. *P < 0.05, **P < 0.01 (compared with T2DM group), #P < 0.05 (compared with HFD+HBO group), ∧P < 0.05 (compared with HFD group).

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  Figure 8. (A) Morphology of BAT and cell number counting. BAT morphology and UCP1 protein analysis after HBO. Partial BATs were cut into 5-μm sections stained using hematoxylin-eosin staining (400×) and numbers of cell from the same unit areas were counted (HFD: n = 3; HFD+HBO: n = 4; T2DM: n = 3; T2DM+HBO: n = 4). (B) UCP1 protein expression levels in BAT were measured (HFD: n = 3; HFD+HBO: n = 3; T2DM: n = 4; T2DM+HBO: n = 4). Data are presented as means ± SEM. Normal baric oxygen (NBO). Scale bar = 200 μm. *P < 0.05, **P < 0.01 (compared with T2DM group), #P < 0.05 (compared with HFD group), ∧P < 0.05 (compared with HFD+HBO group).