Hyperbaric oxygen treatment improves pancreatic β‑cell function and hepatic gluconeogenesis in STZ‑induced type‑2 diabetes mellitus model mice

Abstract

Type‑2 diabetes mellitus (T2DM) causes several complications that affect the quality of life and life span of patients. Hyperbaric oxygen therapy (HBOT) has been used to successfully treat several diseases, including carbon monoxide poisoning, ischemia, infections and diabetic foot ulcer, and increases insulin sensitivity in T2DM. The present study aimed to determine the effect of HBOT on β‑cell function and hepatic gluconeogenesis in streptozotocin (STZ)‑induced type‑2 diabetic mice. To establish a T2DM model, 7‑week‑old male C57BL/6J mice were fed a high‑fat diet (HFD) and injected once daily with low‑dose STZ for 3 days after 1‑week HFD feeding. At the 14th week, HFD+HBOT and T2DM+HBOT groups received 1‑h HBOT (2 ATA; 100% pure O₂) daily from 5:00 to 6:00 p.m. for 7 days. The HFD and T2DM groups were maintained under normobaric oxygen conditions and used as controls. During HBOT, the 12‑h nocturnal food intake and body weight were measured daily. Moreover, blood glucose was measured by using a tail vein prick and a glucometer. After the final HBO treatment, all mice were sacrificed to conduct molecular biology experiments. Fasting insulin levels of blood samples of sacrificed mice were measured by an ultrasensitive ELISA kit. Pancreas and liver tissues were stained with hematoxylin and eosin, while immunohistochemistry was performed to determine the effects of HBOT on insulin resistance. TUNEL was used to determine the effects of HBOT on β‑cell apoptosis, and immunoblotting was conducted to determine the β‑cell apoptosis pathway. HBOT notably reduced fasting blood glucose and improved insulin sensitivity in T2DM mice. After HBOT, β‑cell area and β‑cell mass in T2DM mice were significantly increased. HBOT significantly decreased the β‑cell apoptotic rate in T2DM mice via the pancreatic Bcl‑2/caspase‑3/poly(ADP‑ribose) polymerase (PARP) apoptosis pathway. Moreover, HBOT improved the morphology of the liver tissue and increased hepatic glycogen storage in T2DM mice. These findings suggested that HBOT ameliorated the insulin sensitivity of T2DM mice by decreasing the β‑cell apoptotic rate via the pancreatic Bcl‑2/caspase‑3/PARP apoptosis pathway.

Keywords: Bcl-2/caspase-3/poly(ADP-ribose) polymerase pathway; hyperbaric oxygen; type-2 diabetes mellitus; β‑cell apoptosis.

Figure 1.

Blood glucose levels after streptozotocin injection in T2DM mice. (A) Non-fasting blood glucose and (B) fasting blood glucose levels in the T2DM and HFD groups. Data are presented as the mean ± SEM. **P<0.01. T2DM, type-2 diabetes mellitus; HFD, high-fat diet.

Figure 2.

Effects of HBOT on nocturnal feeding, body weight and body weight gain. Effects of 7-day HBOT on (A) nocturnal food intake and (B) body weight. (C) HBO-induced body weight gain after 7-day HBO intervention. Data are presented as the mean ± SEM. HBO, hyperbaric oxygen; HFD, high-fat diet; T2DM, type-2 diabetes mellitus; HBOT, hyperbaric oxygen therapy.

Figure 3.

Effects of HBOT on blood glucose and insulin sensitivity. (A) Fasting blood glucose pre- and post-7-day HBOT. (B) Intraperitoneal glucose tolerance test in T2DM mice after 7-day HBOT. (C) AUC in the T2DM and T2DM+HBOT groups after 7-day HBOT. (D) HOMA-IR and (E) HOMA-β values in the HFD and T2DM groups. Data are presented as the mean ± SEM. *P<0.05 and **P<0.01; ^#P<0.05, ^##P<0.01 and ^###P<0.001; ^&P<0.05 and ^&&&P<0.001. HBO, hyperbaric oxygen; T2DM, type-2 diabetes mellitus; AUC, area under the blood glucose curve; HOMA-IR, homeostatic model assessment of insulin resistance; HOMA-β, homeostatic model assessment of β-cell function; HFD, high-fat diet; HBOT, hyperbaric oxygen therapy.

Figure 4.

Effects of HBOT on fat and pancreas weight. Effects of HBOT on the weight of (A) inguinal fat, (B) epididymal fat and (C) the sum weight of the inguinal fat and epididymal fat. (D) Pancreas weight in T2DM mice and HFD mice. (E) Ratio of pancreas weight to the 8th body weight. Data are presented as the mean ± SEM. *P<0.05 and **P<0.01. HBO, hyperbaric oxygen; T2DM, type-2 diabetes mellitus; HFD, high-fat diet; HBOT, hyperbaric oxygen therapy; iWAT, inguinal white adipose tissue; eWAT, epididymal white adipose tissue.

Figure 5.

Effects of HBOT on pancreatic β-cell morphology and structure. (A) H&E staining of the pancreas (scale bar, 68 µm). Effects of HBOT on the number of islets. (B) Representative immunohistochemistry images of insulin staining in pancreatic tissues (Scale bar, 142 µm). Effects of HBOT on β-cell area. (C) Effects of HBOT on β-cell mass. Data are presented as the mean ± SEM. *P<0.05 and **P<0.01; ^#P<0.05 and ^###P<0.001; ^&P<0.05 and ^&&&P<0.001. HBO, hyperbaric oxygen; HFD, high-fat diet; HBOT, hyperbaric oxygen therapy; T2DM, type-2 diabetes mellitus.

Figure 6.

Effects of HBOT on β-cell apoptosis. (A) TUNEL staining of β-cell apoptosis. Red fluorescence indicates pancreatic β-cells and green fluorescence indicates apoptotic cells (scale bar, 252 µm), white arrows indicate TUNEL⁺ β-cells (%). (B) Effects of HBOT on the expression levels of apoptosis-related proteins, including Bax/Bcl-2, Casp-3 and PARP, in pancreatic tissue. Data are presented as the mean ± SEM. *P<0.05, **P<0.01 and ***P<0.001. HBO, hyperbaric oxygen; PARP, poly(ADP-ribose) polymerase; HFD, high-fat diet; HBOT, hyperbaric oxygen therapy; T2DM, type-2 diabetes mellitus; Casp, caspase.

Figure 7.

Effects of HBOT on hepatic gluconeogenesis. (A) Representative H&E staining of the liver (Scale bar, 272 µm). (B) Representative H&E staining of glycogen in the liver. Green arrows point to glycogen (Scale bar, 272 µm). HBO, hyperbaric oxygen; HFD, high-fat diet; HBOT, hyperbaric oxygen therapy; T2DM, type-2 diabetes mellitus.