br Methods br Results br Discussion Exercise results in a
Discussion Exercise results in a remarkable redistribution of blood flow, which increases in active skeletal muscles but decreases in the splanchnic circulation. The regional blood flow in the kidney, spleen, stomach and intestine was measured by using the microsphere technique in rats. Regional vascular resistance of the intestine was 29.5 mm Hg/mL/min/g before exercise and increased to 84.5 mm Hg/mL/min/g after exercise, and the results showed that the intestinal blood flow was decreased by exercise.18, 19 The effect of exercise on gastric mucosal perfusion adequacy has been investigated using air tonometry in athletes, with the results suggesting that GI system ischemia was present in all athletes during maximum intensity exercise and in 50% of the athletes during submaximal exercise. Athletes with GI symptoms, such as stomach pain, diarrhea and constipation, had an increased susceptibility to developing ischemia during exercise. However, the relationship between exercise-induced lower blood perfusion and hypoxia has rarely been studied in mouse models. Sufficient blood perfusion is important to maintain GI system function, deliver oxygen and nutrients and remove the products of metabolism. The small intestine has a unique oxygenation characteristic, that is, regular fluctuations in blood perfusion. Additionally, in the small intestine, steep O2 gradient is present from the villi to the crypt and from the Benzamil lumen to the epithelial mucosa, leading to graded hypoxia. Intestinal epithelial cells are positioned between the anaerobic lumen and the highly metabolic lamina propria and therefore are located in a physiologically hypoxic environment with a steep O2 gradient. In the lumen, PO2 is less than 1 mmHg.20 In the present study, we investigated tissue hypoxia distribution in the internal organs of exercised mice and sedentary mice. The local PO2 was detected using pimonidazole HCl. In the control group, the pimonidazole HCl staining results were consistent with those of other studies23, 24 and showed an oxygen gradient in the small intestine (Fig. 1) and tissue hypoxia in the liver and the kidney (Fig. 2). A single bout of moderate exercise exacerbated the hypoxia in small intestine the kidney, liver, and colon (Figs. 1 and 2). In the small intestine, both the location and extent of hypoxic tissue were altered (Fig. 1). However, positive staining was not found before or after exercise in the heart, skeletal muscle and spleen tissues (Fig. 3). HIF-1α is pivotal for survival, metabolism, and oxygen homeostasis. PHDs hydroxylate a prolyl residue in the amino- and the carboxy-terminal ODD domains. Factor-inhibiting HIF (FIH) hydroxylates an asparagine in the carboxy-terminal activation domain. The regulation of both PHDs and FIH results in the destruction of the HIFα subunit and inactivation of transcriptional activity. During hypoxia, these processes are inhibited, and a transcriptionally active complex is formed. Under normoxic conditions, PHDs, which are HIF hydroxylases, are activated. The HIF-1α protein level in the brains of mice exposed to 6% O2 for 75 min was half of its maximum level 15 min after the mice were returned to normoxic conditions and decreased to normoxic levels 60 min later, indicating a rapid degradation rate of HIF-1α in vivo. Another study showed that the HIF-1α protein is unstable because it has a short half-life of 5 min, which increases the difficulty of detecting HIF-1α under normoxic conditions. Therefore, we used ODD-Luc mice that provided a bioluminescent reporter consisting of firefly luciferase fused to a region of HIF-1α. Many applications of HIF-1α have been described, including gene regulation, tumor growth and inflammation.29, 30 In this study, a HIF-1α reporter mouse was used to further study the influence of exercise on HIF-1α distribution. We found that moderate exercise increased HIF-1α in the abdominal area in vivo (Fig. 4), and we further confirmed that the expression of HIF-1α was increased in the small intestine (Figs. 5A and 5B). We evaluated the expression of HIF-1α in the small intestine by using 3 exercise models. However, the increase in HIF-1α level was not affected by the exercise intensity (Fig. 4). Additionally, we measured the photon output at different times after exercise and found a gradual reduction. The level of photons emitted at the 0th h, 2nd h, 4th h and 6th h was greater in the ME group than that in the control group (Fig. 6). Previous studies have shown that the duration of hypoxia influences the reoxygenation response. The delay in HIF-1α protein degradation after exercise may reflect the necessity of retaining this protein until its target genes are upregulated, whereas the HIF-1α and HIF-1β mRNA levels were unaffected by changes in oxygen tension. Considering the role of PHDs in HIF-1α degradation under normoxic conditions, one possible reason for the time-dependent change in HIF-1α level after an increases in PO2 could be the recovery rate of PHD activity.