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

Abstract

Hyperbaric oxygen (HBO) therapy is a treatment modality useful for diseases. Hypoxia could stimulate the induction of insulin resistance. Therefore, we sought to determine whether hyperbaric oxygen would ameliorate insulin sensitivity by promoting glucose transporter type 4 (GLUT4) expression in muscle and by stimulating UCP1 in brown adipose tissue (BAT) in a streptozocin (STZ)-induced type 2 diabetes mellitus (T2DM) mouse model. Male C57BL/6J mice were treated three times with low-dose of streptozocin (60 mg/kg, i.p.) and were fed with high-fat diets (HFD) to establish the T2DM model. HBO was administered daily as 100% oxygen at 2.0 atmosphere absolute (ATA) for 1 h for a week. We found that HBO significantly reduced blood glucose levels and attenuated insulin resistance in T2DM mice. HBO modulated food intake by influencing the activity of neuropeptide Y (NPY)-positive neurons in the arcuate nucleus (Arc). HBO treatment increased GLUT4 amount and level of phosphorylated Akt (p-Akt) in muscles of T2DM mice whereas this treatment stimulated the phosphorylation of AMPK in muscles of both T2DM and HFD mice. The morphological staining of BAT and the increased expression of uncoupling of protein 1 (UCP1) demonstrated the promotion of metabolism after HBO treatment. These findings suggest that HBO ameliorates insulin sensitivity of T2DM mice by stimulating the Akt signaling pathway and by promoting GLUT4 expression in muscle, and by increasing UCP1 expression in BAT.

Keywords: UCP1; glucose transporter type 4; hyperbaric oxygen; insulin sensitivity; type 2 diabetes mellitus.

Figure 1

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

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).

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).

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).

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).

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).

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).

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).