www.nature.com/scientificreports
Manoel Benício Teixeira Ribeiro 1 , Vinicius Guzzoni 2 , Jeffrey M. Hord 3 , Gisele Nunes Lopes 4 , Rita de Cássia Marqueti 5 , Rosângela Vieira de Andrade 6 , Heloisa Sobreiro Selistre-de-Araujo & João Luiz Q. Durigan 5
Sarcopenia is a complex multifactorial process, some of which involves fat infiltration. Intramyocellular lipid (IMCL) accumulation is postulated to play a role on sarcopenia during aging, which is believed to be due alterations in glucose homeostasis in the skeletal muscle. Sarcopenia, along with intramuscular lipids, is associated with physical inactivity. Resistance training (RT) has been indicated to minimize the age-induced muscle skeletal adaptations. Thus, we aimed to investigate the effects of RT on mRNA levels of regulatory components related to intramyocellular lipid, glucose metabolism and fiber size in soleus and gastrocnemius muscles of aged rats. Old male rats were submitted to RT (ladder climbing, progressive load, 3 times a week for 12 weeks). Age-induced accumulation of IMCL was attenuated by RT, which was linked to a PPARy-mediated mechanism, concomitant to enhanced regulatory
components of glucose homeostasis (GLUT-4, G6PDH, Hk-2 and Gly-Syn-1). These responses were also linked to decreased catabolic (TNF-α, TWEAK/Fn14 axis; FOXO-1, Atrogin-1 and MuRF1; Myostatin) and increased anabolic intracellular pathways (IGF-1-mTOR-p70S6sk-1 axis; MyoD) in muscles of trained aged rats. Our results point out the importance of RT on modulation of gene expression of intracellular regulators related to age-induced morphological and metabolic adaptations in skeletal muscle.
Sarcopenia is a complex multifactorial process, involving fat infiltration 1 and a reduction in skeletal muscle cross sectional area (CSA) 2 . Intramyocellular lipid (IMCL) accumulation is postulated to play a role in the progression of sarcopenia with aging 3 . Evidence indicates that IMCL accumulation blunts muscle glucose transport activity and glycogen synthesis 4 . Accordingly, age-induced changes in mitochondrial biogenesis may affect fatty acid oxidation and result in accumulation of lipids in skeletal muscle cells leading to an alteration in glucose uptake and glycogen synthesis 5 . However, the mechanisms those mediate IMCL and glucose homeostasis during age-related
muscle loss have yet to be elucidated.
Various transcription factors and intracellular pathways have been implicated in the regulation of fat and glucose metabolism. For example, peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer
binding proteins (C/EBPs), such as C/EBPα, are key early regulators of adipogenesis 6 . PPAR-γ also regulates lipogenesis in skeletal muscle 7 . Additionally, PGC-1α, a PPARγ binding protein plays a role in the transcriptional control of oxidative metabolism 8 and fiber type switching 9 . PGC-1α also increases lipogenesis and lipid
1College of Physical Education, University of Brasília, Distrito Federal, Brazil. 2 Postdoctoral Fellowship, University of Brasília, Distrito Federal, Brazil. 3 Department of Molecular Physiology and Biophysics, Carver College of Medicine, University of Iowa, Iowa City, United States. 4 Department of Physiological Sciences, Center of Biological and Health Science, Federal University of São Carlos, São Carlos, Sao Paulo, Brazil. 5 Graduate program of Rehabilitation Sciences, University of Brasilia, Distrito Federal, Brazil. 6 Graduate program of Genomics and Proteomics, Catholic
University of Brasilia, Distrito Federal, Brazil. Manoel Benício Teixeira Ribeiro and Vinicius Guzzoni contributed equally to this work. Correspondence and requests for materials should be addressed to V.G. (email: vinicius.
guzzoni@gmail.com)
SCIEnTIFIC ReporTS | 7: 8593 | DOI:10.1038/s41598-017-09343-6
1www.nature.com/scientificreports/
YS YT OS OT
iBW (g) 295.6 ± 34.6 301.8 ± 30 508.3 ± 76.6 a,b 527.3 ± 75.9 a,b
fBW (g) 508.3 ± 85.6 434 ± 52.7 a 452 ± 86.4 a 480.4 ± 66.5 a,b,c
Gain (%) 57.8 44.4 −6.8 −15
GAS weight (mg) 1.190 ± 0.1 1.121 ± 0.1 1.072 ± 0.1 0.976 ± 0.1
SOL weight (mg) 0.279 ± 0.1 0.248 ± 0.02 0.237 ± 0.04 0.260 ± 0.02
GAS glycogen(mg/100 mg) 15.8 ± 0.4 23.2 ± 0.6 a 15.1 ± 0.3 22.5 ± 0.5 c
SOL glycogen(mg/100 mg) 11.3 ± 0.3/ 23.8 ± 0.3 a 8.8 ± 0.4 23.1 ± 0.4 c
Liver glycogen (mg/100 mg) 29 ± 2.0 58.4 ± 1.8 a 33.6 ± 1.9 60 ± 1.9 c
Table 1. Body weight (BW), percentage of gain of BW, gastrocnemius (GAS) and soleus (SOL) weights,glycogen content of GAS and SOL muscles from experimental groups. iBW = initial body weight; fBW: final body weight; Gain = percentage of increasing or decreasing of BW after 12 weeks; GAS weight = Glycogen content of gastrocnemius; SOL weight = Glycogen content of soleus. Groups: Young Sedentary (YS), Young Trained (YT), Old Sedentary (OS) and Old Trained (OT). Values are expressed as mean ± SEM. Two-way ANOVA, p < 0.05: a vs. YS; b vs. YT; c OS. n = 6/group.
catabolism in skeletal muscle 10 . Another factor is known as lipoprotein lipase (LPL), which is a key enzyme responsible for fatty acid and lipoprotein metabolism in muscle 11 . The regulation of age-related alterations in glucose and fat metabolism have been documented in mice 12 . However, the role of key regulatory components of glucose homeostasis, such as glycogen synthase type 1 (Gly-Syn-1), glucose-6-phosphate dehydrogenase (G6PDH),hexokinase type 2 (Hk-2) and glucose transporter 4 (GLUT-4) with aging is not fully understood. Considering the complexity of the crosstalk between adipogenic transcriptional factors, glycogen metabolism and atrophy/
hypertrophy signaling pathways, a better understanding of molecular pathways that regulate the aging-induced phenotypes is needed.
Skeletal muscle atrophy is complex process that is due in part to proinflammatory cytokines, which includes TNF-α (tumor necrosis factor-α), TWEAK (tumor necrosis factor apoptosis inducing) and its receptor, Fn14
(growth factor-inducible 14 receiver fibroblasts) 13 . Further downstream, activation of forkhead box protein O1 (FOXO-1) promotes the expression of E3 ubiquitin ligases, Atrogin-1 and muscle ring finger protein-1 (MURF-
1), which are key players in the ubiquitin-proteasome system 14 . The loss of muscle mass during aging is also under influence of various growth factors, such as those in the TGF-β (transforming growth factor-β) family, mostly
notably, myostatin 15 . Along with elevated rates of protein degradation, sarcopenia has also been associated with a reduction in muscle protein synthesis 16 . Anabolic signaling is primarily attributed to the activation of anabolic
signaling axis which includes IGF-1 (Insulin-like growth factor 1), mTOR (mammalian target of rapamycin) and activation of p70S6K-1 (p70S6 kinase 1) 17 . Furthermore, altered sensitivity of satellite cells is implicated in sarcopenia. Satellite cells are regulated by myogenic regulatory factors, such as MyoD that promote differentiation of the muscle-specific stem cells 18 .
Resistance training (RT) has been consistently recommended to minimize the age-related muscle adaptations 19 . In this regard, sarcopenia, along with fatty acid infiltration, is associated with physical inactivity 20. Moreover, it has emerged that exercise training confers beneficial effects on glucose homeostasis 21 and modulates IMCL 22 . However, the effects of RT on molecules related to glucose homeostasis and lipogenesis in skeletal muscle with advancing age are unknown. Furthermore, how RT affects intracellular signaling and transcription factors that control glucose homeostasis, lipogenesis and morphology of skeletal muscle in the aging is not understood.
Based on our assumptions, we hypothesized that RT could protect against the age-induced IMCL accumulation, glucose homeostasis and muscle atrophy via downregulation of adipogenic factors (CEPB-α, LPL, PPARγ and PGC-1α,) and atrophy-associated molecules (TNF-α, TWEAK/Fn14 axis; FOXO-1, Atrogin1, MURF-1 and myostatin) concomitant to increases in hypertrophy-associated factors (IGF-1/mTOR/p70S6k-1/MyoD) and glucose homeostasis (Gly-Syn-1, G6PDH, GLUT-4, Hk-2). Thus, we sought to investigate the effects of RT on mRNA levels of atrophy/hypertrophy signaling, intracellular fatty infiltration and glucose metabolism in the soleus and gastrocnemius muscles of aged rats.
Results
Body weight in old and trained rats. The iBW and fBW are shown in Table 1. The iBW of old rats was higher than young groups at the beginning of experiment - first day of RT session. After 12 weeks of RT, fBW of
YS and YT rats were 57.8% and 44.4% higher in comparison to their matched iBW. On the other hand, fBW of OS and OT groups decreased (6.8% and 15%, respectively) when compared to their matched iBW.
CSA and IMCL and glycogen content of gastrocnemius (GAS) and soleus (SOL) muscles in old and trained rats. RT alleviated the age-induced decrease of CSA in SOL and GAS muscles (OT vs. OS) (Figs 1A and 2A), as well as the age-related increase of IMCL content in SOL muscle (Fig. 1B). Unlike SOL, CSA
of GAS was larger in YT than in YS rats (Fig. 2A) and IMCL content was greater in OS than in YS group (OS vs.YS), with no effect of RT (Fig. 2B). On the other hand, RT enhanced tissue glycogen content in SOL and GAS, in
both young and old groups (OT vs. OS and YT vs. YS) (Table 1).
Figure 1. Light microscope images of hematoxilin-eosin stained sections of SOL muscle at 10X magnification.* and α means atrophied and hypertrophied fibers respectively (A). Quantification of cross sectional area (CSA) normalized by fBW (B). Light microscope images of intramyocellular lipids (IMCL) of SOL muscle at 20X magnification (C). Quantification of IMCL content (D). Groups: young sedentary (YS), young trained (YT), old sedentary (OS) and old trained (OT) rats. Values are expressed as means ± SEM. Two-way ANOVA, p < 0.05: a vs. YS; b vs. YT; c vs. OS. n = 6/group.
mRNA levels of adipogenic factors in response to aging and exercise in SOL muscle. CEBP-α, LPL, PPAR-γ and PGC-1α mRNA levels were elevated with aging (Fig. 3A–D). In response to exercise training, RT elevated mRNA levels of CEBP-α and PGC-1α in SOL muscle (Fig. 3A and D), whereas PPAR-γ and LPL
were reduced in OT in relation to OS animals (Fig. 3B and C). In young rats, RT led to decreases in CEBP-α and PPAR-y mRNA levels, even though PGC-1α was increased in YT rats in relation to their age-matched
counterparts.
mRNA levels of glucose metabolism regulators of SOL muscle in response to aging and exercise. Aging did not affect GLUT-4, G6PDH, Hk-2 and Gly-synt-1 mRNA levels in SOL muscle (Fig. 3E–H).However, all those transcripts were elevated in trained rats when compared with their matched sedentary group (OT vs. OS and OT vs. YS).
mRNA levels of atrophy and hypertrophy-related factors of SOL muscle in response to aging and resistance training. RT was effective at mitigating the age-associated increase of TNF-α, TWEAK, Atrogin-1 and MURF-1 mRNA levels in SOL muscle (OT vs. OS) (Fig. 3I,J,M and N). Likewise, RT decreased
FOXO-1 and myostatin in OT rats when compared with OS and YS animals (Fig. 3L and O). Moreover, TWEAK, Fn-14, FOXO-1, MURF-1, and myostatin mRNA levels were reduced after RT in young rats (YT vs. YS) (Fig. 3J,K,L,N and O). Interestingly, Fn-14 transcript was lower in OS and OT rats in relation to YS (Fig. 3K). On the other hand, IGF-1, mTOR and MyoD mRNA levels were not affected by aging (Fig. 3P,Q and S), even though SCIEnTIFIC ReporTS | 7: 8593 | DOI:10.1038/s41598-017-09343-6
(Continua...)
Artigo Completo em PDF deixem email nos comentarios que envio.
Manoel Benício Teixeira Ribeiro 1 , Vinicius Guzzoni 2 , Jeffrey M. Hord 3 , Gisele Nunes Lopes 4 , Rita de Cássia Marqueti 5 , Rosângela Vieira de Andrade 6 , Heloisa Sobreiro Selistre-de-Araujo & João Luiz Q. Durigan 5
Sarcopenia is a complex multifactorial process, some of which involves fat infiltration. Intramyocellular lipid (IMCL) accumulation is postulated to play a role on sarcopenia during aging, which is believed to be due alterations in glucose homeostasis in the skeletal muscle. Sarcopenia, along with intramuscular lipids, is associated with physical inactivity. Resistance training (RT) has been indicated to minimize the age-induced muscle skeletal adaptations. Thus, we aimed to investigate the effects of RT on mRNA levels of regulatory components related to intramyocellular lipid, glucose metabolism and fiber size in soleus and gastrocnemius muscles of aged rats. Old male rats were submitted to RT (ladder climbing, progressive load, 3 times a week for 12 weeks). Age-induced accumulation of IMCL was attenuated by RT, which was linked to a PPARy-mediated mechanism, concomitant to enhanced regulatory
components of glucose homeostasis (GLUT-4, G6PDH, Hk-2 and Gly-Syn-1). These responses were also linked to decreased catabolic (TNF-α, TWEAK/Fn14 axis; FOXO-1, Atrogin-1 and MuRF1; Myostatin) and increased anabolic intracellular pathways (IGF-1-mTOR-p70S6sk-1 axis; MyoD) in muscles of trained aged rats. Our results point out the importance of RT on modulation of gene expression of intracellular regulators related to age-induced morphological and metabolic adaptations in skeletal muscle.
Sarcopenia is a complex multifactorial process, involving fat infiltration 1 and a reduction in skeletal muscle cross sectional area (CSA) 2 . Intramyocellular lipid (IMCL) accumulation is postulated to play a role in the progression of sarcopenia with aging 3 . Evidence indicates that IMCL accumulation blunts muscle glucose transport activity and glycogen synthesis 4 . Accordingly, age-induced changes in mitochondrial biogenesis may affect fatty acid oxidation and result in accumulation of lipids in skeletal muscle cells leading to an alteration in glucose uptake and glycogen synthesis 5 . However, the mechanisms those mediate IMCL and glucose homeostasis during age-related
muscle loss have yet to be elucidated.
Various transcription factors and intracellular pathways have been implicated in the regulation of fat and glucose metabolism. For example, peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer
binding proteins (C/EBPs), such as C/EBPα, are key early regulators of adipogenesis 6 . PPAR-γ also regulates lipogenesis in skeletal muscle 7 . Additionally, PGC-1α, a PPARγ binding protein plays a role in the transcriptional control of oxidative metabolism 8 and fiber type switching 9 . PGC-1α also increases lipogenesis and lipid
1College of Physical Education, University of Brasília, Distrito Federal, Brazil. 2 Postdoctoral Fellowship, University of Brasília, Distrito Federal, Brazil. 3 Department of Molecular Physiology and Biophysics, Carver College of Medicine, University of Iowa, Iowa City, United States. 4 Department of Physiological Sciences, Center of Biological and Health Science, Federal University of São Carlos, São Carlos, Sao Paulo, Brazil. 5 Graduate program of Rehabilitation Sciences, University of Brasilia, Distrito Federal, Brazil. 6 Graduate program of Genomics and Proteomics, Catholic
University of Brasilia, Distrito Federal, Brazil. Manoel Benício Teixeira Ribeiro and Vinicius Guzzoni contributed equally to this work. Correspondence and requests for materials should be addressed to V.G. (email: vinicius.
guzzoni@gmail.com)
SCIEnTIFIC ReporTS | 7: 8593 | DOI:10.1038/s41598-017-09343-6
1www.nature.com/scientificreports/
YS YT OS OT
iBW (g) 295.6 ± 34.6 301.8 ± 30 508.3 ± 76.6 a,b 527.3 ± 75.9 a,b
fBW (g) 508.3 ± 85.6 434 ± 52.7 a 452 ± 86.4 a 480.4 ± 66.5 a,b,c
Gain (%) 57.8 44.4 −6.8 −15
GAS weight (mg) 1.190 ± 0.1 1.121 ± 0.1 1.072 ± 0.1 0.976 ± 0.1
SOL weight (mg) 0.279 ± 0.1 0.248 ± 0.02 0.237 ± 0.04 0.260 ± 0.02
GAS glycogen(mg/100 mg) 15.8 ± 0.4 23.2 ± 0.6 a 15.1 ± 0.3 22.5 ± 0.5 c
SOL glycogen(mg/100 mg) 11.3 ± 0.3/ 23.8 ± 0.3 a 8.8 ± 0.4 23.1 ± 0.4 c
Liver glycogen (mg/100 mg) 29 ± 2.0 58.4 ± 1.8 a 33.6 ± 1.9 60 ± 1.9 c
Table 1. Body weight (BW), percentage of gain of BW, gastrocnemius (GAS) and soleus (SOL) weights,glycogen content of GAS and SOL muscles from experimental groups. iBW = initial body weight; fBW: final body weight; Gain = percentage of increasing or decreasing of BW after 12 weeks; GAS weight = Glycogen content of gastrocnemius; SOL weight = Glycogen content of soleus. Groups: Young Sedentary (YS), Young Trained (YT), Old Sedentary (OS) and Old Trained (OT). Values are expressed as mean ± SEM. Two-way ANOVA, p < 0.05: a vs. YS; b vs. YT; c OS. n = 6/group.
catabolism in skeletal muscle 10 . Another factor is known as lipoprotein lipase (LPL), which is a key enzyme responsible for fatty acid and lipoprotein metabolism in muscle 11 . The regulation of age-related alterations in glucose and fat metabolism have been documented in mice 12 . However, the role of key regulatory components of glucose homeostasis, such as glycogen synthase type 1 (Gly-Syn-1), glucose-6-phosphate dehydrogenase (G6PDH),hexokinase type 2 (Hk-2) and glucose transporter 4 (GLUT-4) with aging is not fully understood. Considering the complexity of the crosstalk between adipogenic transcriptional factors, glycogen metabolism and atrophy/
hypertrophy signaling pathways, a better understanding of molecular pathways that regulate the aging-induced phenotypes is needed.
Skeletal muscle atrophy is complex process that is due in part to proinflammatory cytokines, which includes TNF-α (tumor necrosis factor-α), TWEAK (tumor necrosis factor apoptosis inducing) and its receptor, Fn14
(growth factor-inducible 14 receiver fibroblasts) 13 . Further downstream, activation of forkhead box protein O1 (FOXO-1) promotes the expression of E3 ubiquitin ligases, Atrogin-1 and muscle ring finger protein-1 (MURF-
1), which are key players in the ubiquitin-proteasome system 14 . The loss of muscle mass during aging is also under influence of various growth factors, such as those in the TGF-β (transforming growth factor-β) family, mostly
notably, myostatin 15 . Along with elevated rates of protein degradation, sarcopenia has also been associated with a reduction in muscle protein synthesis 16 . Anabolic signaling is primarily attributed to the activation of anabolic
signaling axis which includes IGF-1 (Insulin-like growth factor 1), mTOR (mammalian target of rapamycin) and activation of p70S6K-1 (p70S6 kinase 1) 17 . Furthermore, altered sensitivity of satellite cells is implicated in sarcopenia. Satellite cells are regulated by myogenic regulatory factors, such as MyoD that promote differentiation of the muscle-specific stem cells 18 .
Resistance training (RT) has been consistently recommended to minimize the age-related muscle adaptations 19 . In this regard, sarcopenia, along with fatty acid infiltration, is associated with physical inactivity 20. Moreover, it has emerged that exercise training confers beneficial effects on glucose homeostasis 21 and modulates IMCL 22 . However, the effects of RT on molecules related to glucose homeostasis and lipogenesis in skeletal muscle with advancing age are unknown. Furthermore, how RT affects intracellular signaling and transcription factors that control glucose homeostasis, lipogenesis and morphology of skeletal muscle in the aging is not understood.
Based on our assumptions, we hypothesized that RT could protect against the age-induced IMCL accumulation, glucose homeostasis and muscle atrophy via downregulation of adipogenic factors (CEPB-α, LPL, PPARγ and PGC-1α,) and atrophy-associated molecules (TNF-α, TWEAK/Fn14 axis; FOXO-1, Atrogin1, MURF-1 and myostatin) concomitant to increases in hypertrophy-associated factors (IGF-1/mTOR/p70S6k-1/MyoD) and glucose homeostasis (Gly-Syn-1, G6PDH, GLUT-4, Hk-2). Thus, we sought to investigate the effects of RT on mRNA levels of atrophy/hypertrophy signaling, intracellular fatty infiltration and glucose metabolism in the soleus and gastrocnemius muscles of aged rats.
Results
Body weight in old and trained rats. The iBW and fBW are shown in Table 1. The iBW of old rats was higher than young groups at the beginning of experiment - first day of RT session. After 12 weeks of RT, fBW of
YS and YT rats were 57.8% and 44.4% higher in comparison to their matched iBW. On the other hand, fBW of OS and OT groups decreased (6.8% and 15%, respectively) when compared to their matched iBW.
CSA and IMCL and glycogen content of gastrocnemius (GAS) and soleus (SOL) muscles in old and trained rats. RT alleviated the age-induced decrease of CSA in SOL and GAS muscles (OT vs. OS) (Figs 1A and 2A), as well as the age-related increase of IMCL content in SOL muscle (Fig. 1B). Unlike SOL, CSA
of GAS was larger in YT than in YS rats (Fig. 2A) and IMCL content was greater in OS than in YS group (OS vs.YS), with no effect of RT (Fig. 2B). On the other hand, RT enhanced tissue glycogen content in SOL and GAS, in
both young and old groups (OT vs. OS and YT vs. YS) (Table 1).
Figure 1. Light microscope images of hematoxilin-eosin stained sections of SOL muscle at 10X magnification.* and α means atrophied and hypertrophied fibers respectively (A). Quantification of cross sectional area (CSA) normalized by fBW (B). Light microscope images of intramyocellular lipids (IMCL) of SOL muscle at 20X magnification (C). Quantification of IMCL content (D). Groups: young sedentary (YS), young trained (YT), old sedentary (OS) and old trained (OT) rats. Values are expressed as means ± SEM. Two-way ANOVA, p < 0.05: a vs. YS; b vs. YT; c vs. OS. n = 6/group.
mRNA levels of adipogenic factors in response to aging and exercise in SOL muscle. CEBP-α, LPL, PPAR-γ and PGC-1α mRNA levels were elevated with aging (Fig. 3A–D). In response to exercise training, RT elevated mRNA levels of CEBP-α and PGC-1α in SOL muscle (Fig. 3A and D), whereas PPAR-γ and LPL
were reduced in OT in relation to OS animals (Fig. 3B and C). In young rats, RT led to decreases in CEBP-α and PPAR-y mRNA levels, even though PGC-1α was increased in YT rats in relation to their age-matched
counterparts.
mRNA levels of glucose metabolism regulators of SOL muscle in response to aging and exercise. Aging did not affect GLUT-4, G6PDH, Hk-2 and Gly-synt-1 mRNA levels in SOL muscle (Fig. 3E–H).However, all those transcripts were elevated in trained rats when compared with their matched sedentary group (OT vs. OS and OT vs. YS).
mRNA levels of atrophy and hypertrophy-related factors of SOL muscle in response to aging and resistance training. RT was effective at mitigating the age-associated increase of TNF-α, TWEAK, Atrogin-1 and MURF-1 mRNA levels in SOL muscle (OT vs. OS) (Fig. 3I,J,M and N). Likewise, RT decreased
FOXO-1 and myostatin in OT rats when compared with OS and YS animals (Fig. 3L and O). Moreover, TWEAK, Fn-14, FOXO-1, MURF-1, and myostatin mRNA levels were reduced after RT in young rats (YT vs. YS) (Fig. 3J,K,L,N and O). Interestingly, Fn-14 transcript was lower in OS and OT rats in relation to YS (Fig. 3K). On the other hand, IGF-1, mTOR and MyoD mRNA levels were not affected by aging (Fig. 3P,Q and S), even though SCIEnTIFIC ReporTS | 7: 8593 | DOI:10.1038/s41598-017-09343-6
(Continua...)
Artigo Completo em PDF deixem email nos comentarios que envio.
Nenhum comentário:
Postar um comentário