| Fatigue refers to physical exhaustion as well as mental and cognitive reactions associated with physical exhaustion, such as memory loss and decreased attention. Fatigue is a wide spread problem, and occurs in 22% of the working population. A report by NASA estimated that 21% of aviation incidents are fatigue-related. Numerous researches have also clearly established fatigue as a major contributor to medical errors.Fatigue is a non-specific symptom associated with many medical conditions such as multiple sclerosis, cancer, stroke, systemic lupus erythematosus, ischemic heart disease, and major depression. Severe, unexplained fatigue often precedes the development of fatigue-related illnesses, such as chronic fatigue syndrome (CFS). In this study, we examined protein expression profile in the liver after a fatigue paradigm, i.e., water immersion in rodents, as previously described. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI- TOF MS) revealed that fatigue substantially up-regulated orosomucoid 1 (ORM 1). ORM 1 binds to a variety of basic and neutral lipophilic substances of both endogenous and exogenous origins. It is synthesized mainly by the liver.Under physiological conditions, ORM 1 was barely expressed. Fatigue increased ORM 1 expression in the liver by almost 10 folds. Serum ORM 1 was also dramatically increased upon fatigue. In addition to the liver, ORM 1 was produced by skeletal muscle in response to―fatigue‖. To the best of our knowledge, ORM 1 expression in skeletal muscle has not been reported previously, probably due to the use of tissue under resting conditions. To further assess the relationship between ORM 1 expression and the severity of fatigue, serum ORM 1 was examined with ELISA during the 5-day fatigue paradigm as well as the 3-day period of recovery afterwards. The results demonstrated that increased ORM 1 expression occurs but only after 3 days of water immersion. Returning of ORM 1 towards the baseline was also a gradual process, taking approximately 3 days.To determine the cause-effect relationship between increased ORM 1 expression and fatigue, we examined potential effects of purified ORM 1 on swimming time in C57 mice. The experiments clearly demonstrated that purified ORM 1 could increase the swimming time. Inhibiting the synthesis of ORM 1 with a siRNA, in contrast, decreased the swimming time.Macrolide antibiotics such as erythromycin could increase ORM 1 expression at both the transcriptional and translational levels. In our experiments, treatment of C57 mice with erythromycin (5 - 60 mg/kg for 3 consecutive days) dose-dependently increased the serum ORM 1 level. Consistent with the experiments using purified ORM1, erythromycin treatment also increased the swimming time in a dose-dependent manner. RNA interference attenuated the effects of erythromycin on swimming time.Experiments with clarithromycin and roxithromycin yielded similar results: increased ORM 1 expression and swimming time. Spiroketal, an erythromycin analogue without anti-bacterial activity, also significantly increased ORM 1 expression and extended swimming time in mice, suggesting that the effects of macrolide antibiotics are not related to their antimicrobial activity. High glycogen levels improve endurance, whereas reduced levels of muscle glycogen results in impaired performance. Glucose transporter 4 (GLUT4) is responsible for the bulk of glucose transportation into skeletal muscle. We therefore speculated that ORM 1 might exert its anti-fatigue effects by inducing the translocation of GLUT4 from the intracellular pool to plasma membrane. Western blotting revealed no significant difference in total GLUT4 among control, erythromycin-treated, fatigue and erythromycin-treated + siRNA groups. Fatigue significantly increased GLUT4 protein in the membrane fraction. The same pattern also occurred with erythromycin treatment. Treatment with a siRNA against ORM 1 decreased GLUT4 in the membrane fraction. Fatigue increased the expression of glycogen synthase (GS, active form) but decreased phosphorylated-GS (p-GS, inactive form) in the skeletal muscle. Erythromycin treatment produced a significant increase in GS protein. The si-RNA against ORM 1, in contrast, decreased GS protein and increased p-GS. Glycogen level increased significantly both in the fatigue and erythromycin groups. RNA interference attenuated the effects of erythromycin on the expression of ORM 1 and glycogen storage in skeletal muscle. These results provided support to our hypothesis that ORM 1 could produce anti-fatigue effects by stimulating GLUT4 translocation and subsequently increasing glycogen storage.AMP-activated protein kinase (AMPK) is a regulator of energy balance. Once activated by low energy status, AMPK could stimulate ATP-producing catabolic pathways. In skeletal muscle, AMPK complexes containingα2 catalytic subunit are predominant. Fatigue and erythromycin significantly increased the level of p-AMPKα, the active form of AMPK, but not total AMPK in mice. In AMPK-α-/- mice, the effects of erythromycin on swimming time was completely abolished although the induced expression of ORM 1 by erythromycin could still be observed. In order to figure out whether ORM-1 mediated anti-fatigue effects were associated with p-AMPK, we compared the differences of protein level induced by erythromycin in two different kinds of mice: wild-type and AMPK-α-/-. The level of membrane-GLUT4 and GS in skeletal muscle was significantly increased by erythromycin in wild-type but not in AMPK-α-/- mice despite of increasing level of ORM 1 in both groups. The effect of erythromycin on the level of glycogen was also abolished in AMPK-α-/- mice. These results suggested that the anti-fatigue effect of ORM 1 is mainly mediated by the following sequence of events in skeletal muscle: phosphorylation of AMPK-α, GLUT4 translocation to cell membrane and finally increased glycogen storage.Chronic fatigue syndrome (CFS) was first defined in 1988 by the US Centers for Disease Control and Prevention,and is characterized by clinically unexplained fatigue, lasting for at least six months and is not substantially alleviated by rest. The current study examined serum level of ORM 1 in 46 patients diagnosed with CFS vs. a group of 38 healthy volunteers. The results demonstrated a robust increase in serum ORM 1 in the CFS group when compared with control group, about 2.78 mg/ml vs. 0.44 mg/ml.Female are reported more susceptibale to fatigue when compared with men. No specific reason has been found to illustrate it. Previous researches have found estrogen could inhibit the expression of ORM 1. Therefore, we detected the level of ORM 1 both in female and male fatigue rats and found the expression of ORM 1 in livers of fatigue female rats was significantly less than that of males. Besides, the expression of ORM 1 could increase sharply in ovarectomized female rats when compared with their sham control ones. Consistant with these results, the swimming time was also significantly increased in ovarectomized female rats.Here are our conclusions:1. ORM 1 is an endogenous anti-fatigue protein;2. ORM 1 could be used as a potential marker in patients with chronic fatigue syndrome;3. Sex differences in fatigue is probably due to gender disparity in orosomucoid 1 production...
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