Hypoxic Training Increases the Concentration of Serum Irisin and Reduces Weight in Diet-induced Obese Rats

Irisin promotes browning of white fat, improves energy metabolism, and-weight loss. In this study, we investigated the effects of different oxygen concentrations during hypoxic training on the serum irisin and the PGC-1α(peroxisome proliferator-activated receptor gamma coactivator 1-alpha)-FNDC5(fibronectin type III domain containing 5)-UCPl(uncoupling protein 1) signaling pathway in the skeletal muscle of obese rats. Male Sprague-Dawley Obese rats (n=80) were randomly divided into 8 groups as follows: the control group (group A, n=10); the endurance exercise group (AE group, n=10), which involved animal treadmill training at slope 0°, 20 m/min, 40 min/d, and 5 d/w; the 16.3% hypoxia exposure group (group B, n=10), 13.3% hypoxia exposure group (group C, n=10), and 11.3% hypoxia exposure group (group D, n=10), which were exposed to a low oxygen environment with oxygen concentrations of 16.3%, 13.3%, and 11.3%, respectively, for 12 h/d; and the 16.3% hypoxic training group (BE group, n=10), 13.3% hypoxic training group (CE group, n=10), and 11.3% hypoxic training group (DE group, n=10) with animal treadmill training during hypoxia exposure. After 8 weeks, the serum irisin concentrations in the AE, BE, CE, and DE groups were significantly higher than that in the A group (p<0.05). Hypoxia exposure and hypoxic training at the three different concentrations significantly increased PGC-1α and FNDC5 gene expression in the skeletal muscle. The PGC-1α and FNDC5 protein contents were significantly higher in the skeletal muscle of the obese rats in the C, AE, and DE groups than those in group A (p<0.05). UCP1 protein expression was significantly higher in groups C, CE, D, and DE than in group A (p<0.05). To conclude, training at oxygen concentrations of 13.3% and 11.3% significantly increased the serum irisin level, and 11.3% hypoxic training enhanced the effects of the PGC-lα-Irisin-UCP1 signaling pathway in skeletal muscle.

11 205 group N (p<0.05), whereas the HDL-C was significantly lower than that in group N (p<0.05). 210 Effects of hypoxic training on weight loss in obese rats 211 Figure 1. Changes in body weight and food intake during the hypoxic training intervention 212 As shown in Figure 1A, the body weights of the rats in group A continued to increase 213 during the intervention. The body weights in groups B, C, and D were still increasing in the 214 first 4 weeks. The increase in the rate of body weight decreased from the 5th week, but without 215 a significant difference compared with group A (p>0.05), indicating that hypoxia exposure 216 inhibited the increased rate of body weight gain in the rats fed the HFD to a certain extent, but 217 the effect was not obvious in the short term. The body weight decreased in the first 4 weeks in 218 the AE group but increased from the 5th week. The body weights continued to reduce from the 219 2nd week in the BE, CE, and DE groups and were significantly different from those in group 220 A (p<0.05). From the 6th week, the body weights of the CE and DE groups were significantly 221 lower than those of the AE group (p<0.05). The results suggest that endurance exercise 12 222 intervention alone can reduce the body weights of rats fed an HFD in a short period of time but 223 that the body weight rebounds in the later stage. Conversely, hypoxic training continuously 224 reduced the body weights of the obese rats, and the training effects were more significant at the 225 13.3% and 11.3% oxygen concentrations. Figure 1B shows that the food intake patterns change 226 after 1 week of intervention. The food intake of group A first increased and then decreased, 227 whereas that of group AE first decreased and then increased. The food intakes of groups B, C, 228 and D were lower than that of group A after 5 weeks of intervention, and group C showed a 229 continuous decline.  Fig 3A). These results indicate that 13.3% hypoxia 256 exposure can significantly reduce the body weights of obese rats fed an HFD, exercise alone is 257 better than hypoxia exposure, the effect of hypoxic training is better than that of hypoxia 258 exposure or endurance training, and a lower oxygen concentration has a more significant effect.
259 Lee's indexes of the rats in groups C and D were significantly higher than those in groups BE 260 and DE (p<0.05), suggesting that hypoxia exposure or endurance exercise did not effectively 261 reduce Lee's index in obese rats, whereas 16.3% and 11.3% hypoxic training was effective in 262 reducing Lee's index in obese rats ( Fig 3B). The percentages of visceral fat in the rats in groups 263 C, AE, BE, CE, and DE were significantly lower than that in group A (p<0.05); the percentage 14 264 was lowest in group DE, and the percentages in groups BE, CE, and DE were significantly 265 lower than that in group AE (p<0.05) and those in the corresponding hypoxia exposure groups 266 (p<0.05). The above results indicate that hypoxia and/or exercise can reduce the percentage of 267 visceral fat to body weight. The effect of 13.3% hypoxia exposure was better than that of 16.3% 268 and 11.3% hypoxia exposure. Hypoxia combined with endurance exercise was more effective 269 than hypoxia exposure or exercise alone, and the lower oxygen concentrations had more 270 significant effects ( Fig 3C).

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The serum irisin content was higher in group CE than in groups A, C, and D (p<0.05).
301 The serum irisin content of group DE was significantly higher than those of groups A, B, C, D, 302 and AE (p<0.05). Hypoxia exposure or endurance training alone did not increase the serum 303 irisin levels, whereas 13.3% and 11.3% oxygen concentration training showed significant 304 effects (Fig 5A). The serum adiponectin concentrations of groups C, D, AE, BE, and DE were 16 305 lower than those of groups A and B (p<0.05). The 13.3% and 11.3% hypoxia exposures reduced 306 the adiponectin concentration, and endurance training and 16.3% and 11.3% hypoxic training 307 also reduced the adiponectin concentration in the rat sera (Fig 5B). The leptin level was higher 308 in group A than in the other groups (p<0.05), was lower in group D than in groups B and C 309 (p<0.05), and was lower in group DE than in groups AE and BE (p<0.05). Hypoxia exposure 310 and/or exercise reduced the serum leptin levels, with 11.3% hypoxia exposure and hypoxic 311 training showing the most pronounced effects (Fig 5C). 325 inversely proportional to the oxygen concentration ( Fig 6A). The HIF-1α mRNA and protein 326 contents were significantly higher in groups D and DE than in group A (p<0.05), with 11.3% 327 hypoxia exposure and/or endurance training showing the most obvious effects (Fig 6A and 7A).

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The three different concentrations of hypoxia exposure and hypoxic training significantly 329 increased PGC-1α and FNDC5 gene expression in the skeletal muscle. The lower oxygen 330 concentrations resulted in higher mRNA transcription levels. Conversely, the effect of 331 endurance exercise alone was not obvious. The DE group showed the highest FNDC5 gene 332 expression level (Fig 6B and 6C). The PGC-1α protein contents in the skeletal muscle were 333 significantly higher in groups C, AE, and DE than in group A (p<0.05). The level in group DE 334 was the highest and was significantly different from that in groups A, B, D, and BE (p<0.05) 335 (Fig. 7B). FNDC5 protein expression was higher in groups C, AE, and DE than in groups A 336 and D (p<0.05), indicating that 13.3% hypoxia exposure, endurance exercise alone, and 11.3% 337 hypoxic training could promote FNDC5 protein expression in the skeletal muscle of obese rats 338 (p<0.05) (Fig 7C)

377
Our study found that hypoxia exposure alone inhibited the excessive growth of body 378 weight to some extent, but the body weight still showed an upward trend compared with the 379 weight before the intervention. Of the three hypoxia exposure concentrations, 13.3% showed 380 the most significant effects. At the end of the 8-week intervention, the body weights of the 13.3% 381 hypoxia exposure group were significantly lower than those of the quiet control group.
382 Therefore, among the three low oxygen concentrations, the moderate 13.3% oxygen 20 383 concentration demonstrated the best control effect on body weight. In addition, the visceral fat 384 percentage of the rats in the 13.3% hypoxia exposure group was lower than those in the other 385 two groups and was significantly lower than that in the quiet control group. Moreover, the 386 percentage of skeletal muscle in this group was higher than those in the other three groups 387 without exercise intervention, although the difference was not significant.