Mechanisms And Significance Of Fat Oxidation In Skeletal Muscle During High-altitude Acclimation

Background:The effect of high-altitude acclimation on metabolic adjustments has caught much attention today, but little is known about the mechanisms responsible for it. To maintain energy balance is the major adaption of metabolic cell during high-altitude acclimation, in which the energy adjustments in skeletal muscle, such as the selection of metabolic fuel, is fundamental for it.As an abundant metabolic source, fat is an important oxidative fuel for resting and contracting muscle. However, the effect of high-altitude acclimation on fat oxidation in skeletal muscle remains uncertain. Classically, it has been suggested that carbohydrate (CHO) oxidation prefer over fat oxidation after high-altitude acclimation for the reason of the"oxygen-wasting effect"of fat, but it is hard to explain some indirect evidences (e.g. a declined degradation rate of muscle glycogen and the increase levels of nonesterified fatty acid and glycerol in plasma) to show a greater dependence on fat oxidation rather than CHO oxidation afte high-altitude acclimation, which is regarded as sparing valuable muscle glycogen.In previous study, we found that the storage of glucose transporters prepared to be translocated or activated, and glucose uptake rate increased in skeletal muscle after high-altitude acclimation,as well as the content of glycogen. All of these suggest that the increase of glucose uptake rate and the storage of glucose transporters induced by high-altitude acclimation are mainly used for the storage of glycogen, and might result in a greater reliance on fat oxidation. However, the effects of high-altitude acclimation on fat and glucose oxidation were undetected. Therefor, the acclimative change of fat oxidation in skeletal muscle after high-altitude acclimation remains unkwown. Study on this can help to realize the characteristics and mechanisms of high-altitude acclimation or adaptation. Objective:To evaluate the characteristics and mechanisms and significance of fat oxidation in skeletal muscle after high-altitude acclimation in rats, the rates of fat oxidation and uptake were observed at a simulated altitude 5,000 m after acclimation for 30 days, and the expression of genes and proteins related to fat oxidation were also measured, as well as exercise endurance. In addition, the results were compared with those at acute high-altitude exposure (24 hours, 5,000 m).Methods:Male Sprague-Dawley rats were divided into four groups randomly: sea level group (H0), hypoxic 1 day (H1), hypoxic 15 days (H15) and hypoxic 30 days (H30). The hypoxia groups were exposed to a simulated altitude 5,000m in a hypobaric hypoxia chamber for 1, 15 or 30 days respectively, 23 hours per day. H0 group was housed in a normobaric pressure (sea level) chamber. At the time of sacrifice for measurements, anesthetized by 10% urethane (1 ml/100 g body weight, ip), the hypoxia groups were killed by bleeding from femoral arteries at 5,000 m in the hypobaric hypoxic chamber while H0 group was in sea level. Arterial blood and keletal muscle from hind leg were collected. [U-14C] palmitinic acid was used to assay the fatty acid uptake and oxidation rates in skeletal muscle, and D-[U-14C] glucose to measure the oxidation rate of glucose. The levels of glucose, glycogen and lactate were assayed by enzyme methods. The contents of phosphate creatine (PCr) and ATP were measured by HLPC. The concentrations of blood and muscle nonesterified fatty acids (NEFA) were detected by colorimetric technique. The mRNA expressions of fatty acid translocase (FAT/CD36), pyruvate dehydrogenase kinase isoenzyme 4 (PDK4), carnitine palmitoyltransferase I (CPT-Ⅰ), acetyl-CoA carboxylase 2 (ACC-2) and peroxisome proliferator activated receptorδ(PPAR-δ) in skeletal muscle were detected by RT-PCR, and the protein expressions of ACC-2 and CPT-Ⅰwere evaluated by western blot, and L-[1-3H] carnitine was used to assay the activity of CPT-Ⅰ. In addition, muscle glycogen was demonstrated by PAS reaction and electron microscope.In another experiment, male SD rats were randomly divided into three groups equally. High-altitude acclimation group (HAA) was exposed to a simulated altitude of 5,000 m in a hypobaric chamber for 30 days, whereas acute high altitude exposure group (AHAE) resided for 24 hours under similar conditions. The sea level group (SL) was housed in a normobaric pressure (sea level) chamber. At the time of sacrifice for measurements, each group was further divided into two subgroups (i.e. the resting group and the exhausting one). The exhausting group was forced to run on the treadmill with 55%~60% of VO2max (treadmill grade 10%, speed 20 m/min) until they refused to run despite mild electrical stimulation at sea level or a simulated 5,000 m altitude. Exercise endurance was evaluated by determining endurance time to exhaustion (ETE). The contents of NEFA, ATP, PCr, lactate and glycogen, the fatty acid uptake and oxidation rates, the oxidation rate of glucose, the activity of CPT-Ⅰ, the mRNA expressions of ACC-2, CPT-Ⅰand PPAR-δ, and the protein expressions of ACC-2 and CPT-Ⅰin skeletal muscle were determined as above mentioned.Results:1. Acclimation was without effect in altering the ATP content in skeletal muscle. The level of PCr and glycogen in H1 were lower than those in H0, H15 and H30 groups, while there was no significant difference among H0, H15 and H30.2. The influence of high-altitude acclimation on fat utilization in skeletal muscle:(1) The blood and skeletal mucle NEFA contents in all hypoxia groups were higher than those in H0 group.(2) The rates of fatty oxidation and uptake in H15 and H30 groups were significantly higher than those in H1 group, while there was no significant difference between H1 and H0.(3) The PPAR-δmRNA level was markedly down-regulated in H1 group relative to that in H0 group, then up-regulated significantly in H15 and H30 groups, while there was no significant difference between H0 and H30 groups.(4) The CD36 mRNA level was markedly up-regulated in H30 group relative to that in H1 group, while there was no significant difference between H1 and H0 groups.(5) The CPT-ⅠmRNA level was markedly up-regulated in H30 and H15 groups relative to that in H1 group. The protein expression of CPT-Ⅰwas down-regulated in H15 group relative to that in H1 group, but up-regulated with hypoxic days prolonged.(6) The activity of CPT-Ⅰwas significantly decreased after high-altitude exposure, but increased with hypoxic days prolonged. The activity of CPT-Ⅰin H30 group was significantly higher than that in H1 group.(7) The ACC-2 mRNA level was markedly down-regulated after high-altitude exposure. The protein expression of ACC-2 was down-regulated in H15 group relative to that in H1 group, but up-regulated with hypoxic days prolonged.3. The effect of high-altitude acclimation on CHO utilization in skeletal muscle:(1) The content of lactate in skeletal muscle was higher in H1 group than those in H0, H15 and H30 groups, while there was no significant difference between H30 and H0 groups.(2) The glucose levels in H0, H15 and H30 groups were significantly higher than that in H1 group, and H30 group was higher than those in H0 and H15 group, while there was no significant difference between H15 and H0 groups.(3) The rate of glucose oxidation in H0 group was significantly higher than those in the hypoxia groups, while there was no significant difference among the hypoxia groups.(4) The levels of glycogen in H0, H15 and H30 groups were higher than that in H1 group, while there was no significant difference between H30 and H0 groups.(5) The PAS reaction in H1 was markedly weakened relative to H0 group, but enhanced in H15 and H30 groups.(6) The result of electron microscope showed that the amount of glycogenosome in skeletal matrix was decreased after 5 days exposure to high-altitude, but increased significently in H15 and H30.(7) The PDK4 mRNA level was markedly up-regulated in H30 group relative to those in H0 and H1 groups, while there was no significant difference between H0 and H1 groups.4. The influence of high-altitude acclimation on exercise intensity:(1) Compared with SL, ETE was significantly reduced in AHAE, and the contents of PCr, ATP and glycogen were also reduced as well as the glucose and fatty acid oxidation rates. However, the content of lactate was increased markedly.(2) Compared with AHAE, ETE was significantly improved in HAA, while there was no marked difference between HAA and SL. Meanwhile, the contents of PCr, ATP, lactate were also increased as well as the rate of oxidation of glucose. However, the content of glycogen was reduced markedly.(3) Compared with AHAE in exhaustive skeletal muscle, after high-altitude acclimation, fatty acid uptake rate and CPT-I activity in HAA were significantly increased, and ETE was positive related to the activity of CPT-I (Y = 28.056 X + 63.399, r = 0.897). (4) Among the exhausting groups, there was no significant difference in the PPARδmRNA and ACC-2 mRNA levels. Compared with AHAE, the CPT-I mRNA level and the protein expressions of CPT-I and ACC-2 were significantly up-regulated in HAA.Conclusion:In summary, the present study provides information that, in rats, 30 days of acclimation to 5,000 m altitude results in an increase of fat oxidation in skeletal muscle, which is correlated with the performance improvement in ETE during short high-intensity exercise after high-altitude acclimation.