2Department of education and health, Federal University of Sergipe, Lagarto/SE, Brazil
3Department of physiological sciences, Federal University of São Carlos, São Paulo/SP, Brazil
4Postgraduate education in Human Motricity Sciences, Department of physical education, São Paulo State University, Rio Claro/SP, Brazil
5Department of Clinical Analysis, School of Pharmaceutical Sciences, São Paulo State University, Araraquara/SP, Brazil
Propose: This study investigated the effects of irisin signaling pathway induced by Resistance Training (RT) in ovariectomized (Ovx) rats.
Methods: Thirty-two Holztman rats were randomly distributed to four experimental groups: Sham-Sedentary (Sed); Ovx-Sed; Sham-RT and Ovx-RT. The RT protocol demanded from the animals a vertical climb. Each session consisted of 4 to 12 climbs with 2 min. of rest during 12 weeks. To quantify mRNA expression the ΔΔCt method was applied, protein expression was verified by Western Blotting and the analysis of irisin was determined by ELISA. When group averages were different (p ≤ 0.05), a Tukey post-hoc test was applied.
Results: The Ovx-RT group had higher expression of PGC1α FNDC5, irisin levels, and UCP1 compared to Ovx-Sed.
Conclusion: RT was led to higher expression of the irisin signaling pathway in the Ovx group showing that the RT seems to be an excellent strategy to counteract the ovariectomy-induced metabolic disorders.
Keywords: PGC1-Α, FNDC5; Adipose Tissue; UCP1; Resistance Training
Physical exercise can be used as an intervention to improve the health of postmenopausal women given that that Resistance Training (RT) has been associated to decreases in fat mass and increases in lean body mass to prevent sarcopenia, cardiovascular diseases, type 2 diabetes and obesity [9-14]. These benefits occur in part because skeletal muscle is an endocrine organ and exercise stimulates skeletal muscle to produce and release myokines, which have endocrine functions [15]. Irisin is a remarkable myokine and its concentration seems to increase in response to both endurance training and RT [16-20]. During physical exercise, the release of this moykine is induced by the activation of PPARγ co-activator 1 alpha (PGC1-α), which stimulates the fibronectin type III domain containing 5 (FNDC5) to cleave and release irisin into the blood [16].
Irisin is bound to unidentified receptors in the surface of white fat adipocytes and positively regulates the release of UCP1 (Uncoupling Protein 1), which provokes uncoupling in mitochondrial respiration and loss of energy in the form of heat and browning of the White Adipose Tissue (WAT) [16,21,22]. In addition, irisin could stimulate energy expenditure through modulation of hypothalamic neuropeptides and neurotransmitters involved in feeding control [23]. Thus, the thermogenic changes in white adipose tissue may play an important protective role against metabolic disorders, such as cardiovascular diseases, type-2 diabetes and obesity [24,25]. In view of these benefits, the activation of the irisin pathway could play an important protective role against the deleterious effects caused by reduced levels of estrogen. However, to date there are no studies that demonstrate the effect of RT on the irisin signaling pathway in Ovariectomized (Ovx) rats. Our hypotheses in the present study were: 1) the irisin signaling pathway would be lower in Ovx rats, and 2) the Sham and Ovx rats would have greater irisin signaling pathway after RT.
Gene |
Forward Primer |
Reverse Primer |
Access number |
PGC-1α |
GGCCCGGTACAGTGAGTGTT |
ATTGCTCCGGCCCTTTCTT |
NM_031347.1 |
FNDC5 |
CTCTTCATGTGGGCAGGTGTC |
GCTGGTCTCTGATGCACTCTTG |
NM_001270981.1 |
UCP1 |
GGCATCCAGAGGCAAATCAGC |
CCAATGAATACCGCCACGCC |
NM_012682.2 |
GAPDH |
GATGCTGGTGCTGAGTATGTCG |
GTGGTGCAGGATGCATTGCTGA |
NM_017008.3 |
Experimental Groups |
Sham-Sed |
Ovx-Sed |
Sham-RT |
Ovx-RT |
Body Weight (g) |
312.71 ± 16.66 |
373.98 ± 20.70* |
311.29 ± 8.75 |
355.93 ± 15.44* &# |
Left Gastrocnemius (g) |
1.56 ± 0.14 |
1.66 ± 0.12 |
1.65 ± 0.10 |
1.85 ± 0.10* &# |
Mesenteric Fat (g) |
2.99 ± 0.064 |
3.45 ± 0.33* |
1.92 ± 0.33* |
3.08 ± 0.55& |
17β-Estradiol (pg/ml) |
34.05 ± 0.28 |
14.91 ± 0.56* |
34.13 ± 0.28 |
14.63 ± 0.51* & |
Uterus Mass (mg) |
0.65 ± 0.14 |
0.09 ± 0.02* |
0.62 ± 0.06 |
0.09 ± 0.02* & |
The groups that performed RT presented higher (p ≤ 0.05) PGC1-α mRNA expression when compared to their sedentary control groups. The Ovx-RT group presented higher (p ≤ 0.05) PGC1-α mRNA expression compared to Sham-Sed groups (Figure 1). FNDC5 mRNA expression (Figure 2a) was higher (p ≤ 0.05) on trained animals compared to their sedentary control groups. The date of FNDC5 protein expression (Figure 2b) was also higher (p ≤ 0.05) on trained animals compared to their sedentary control groups.
*(p < 0.05) compared to Sham-Sed;
& (p < 0.05) compared to Sham-RT;
# (p < 0.05) compared to Ovx-Sed.
*(p < 0.05) compared to Sham-Sed;
& (p < 0.05) compared to Sham-RT;
# (p < 0.05) compared to Ovx-Sed.
*(p < 0.05) compared to Sham-Sed;
# (p < 0.05) compared to Ovx-Sed.
When Ovx-RT was compared to Sham-Sed group the irisin concentration was significantly higher (p ≤ 0.05). UCP1 protein expression in mesenteric fat (Figure 4b) presented higher values (p ≤ 0.05) in Sham-RT group compared to Sham-Sed. The Ovx-RT group presented higher (p ≤ 0.05) mRNA and protein expression of UCP1 compared to the Ovx-Sed group. Finally, Ovx- RT presented higher (p ≤ 0.05) protein expression of UCP1 compared to Sham-Sed (Figure 4).
The results on gene expression of PGC1-α in the Ovx-Sed group represent that low estrogen levels could have a negative impact on skeletal muscle energy metabolism as mitochondria are estrogen-sensitive organelles [36]. This result corroborates with the findings of Barbosa, Shiguemoto, et al. which seems to be the first study to present some changes in mitochondrial function in an experimental model of menopause [37]. Our results strengthen the evidence that reduction of estrogen levels is a problem of female physiology that requires further investigation. Thus, our results suggest a link between ovariectomy and reduction of PGC1-α expression in the muscle.
*(p < 0.05) compared to Sham-Sed;
& (p < 0.05) compared to Sham-RT;
# (p < 0.05) compared to Ovx-Sed.
The results of UCP1 gene and protein expression in mesenteric fat of the Ovx-Sed group (Figure 4a,b) were lower when compared to the Sham group. This result shows part of the deleterious effects caused by reduced estrogen, once UCP1 expression is related to changes in the thermogenesis of WAT [24]. Nowadays, there are no studies in the literature that show these results, however, according to Pedersen, Bruun, et al. ovariectomy induces lower expression of the UCP1 gene in BAT [47]. Furthermore, our study confirms these findings on the effects of ovariectomy leading to lower UCP1 expression in the WAT of Ovx-Sed rats. Our results show that in the Ovx-Sed group, even in normal irisin concentrations it does not produce the same stimulatory effect of UCP1 expression. As demonstrated in a previous study, the lower expression of UCP1 in brown adipose tissue seems to be associated with reduced levels of estrogen in rats [47]. Our study is the first to demonstrate that despite the fact that irisin levels are not impaired by the reduction of estradiol, UCP1 expression is reduced in Ovx-Sed rats. Thus, we can speculate that the Ovx- Sed group did not have a thermogenic effect of WAT, once body and mesenteric fat weights were significantly greater compared to the Sham-Sed group (Table 2).
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