Effects of physical activity on the link between PGC-1 a and FNDC 5 in muscle , circulating Ιrisin and UCP 1 of white adipocytes in humans : A systematic review

Exercise may activate a brown adipose-like phenotype in white Background: adipose tissue. The aim of this systematic review was to identify the effects of physical activity on the link between peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1a) and fibronectin type III domain-containing protein 5 (FNDC5) in muscle, circulating Irisin and uncoupling protein one (UCP1) of white adipocytes in humans. Two Methods: databases (PubMed 1966 to 08/2016 and EMBASE 1974 to 08/2016) were searched using an appropriate algorithm. We included articles that examined physical activity and/or exercise in humans that met the following criteria: a) PGC-1a in conjunction with FNDC5 measurements, and b) FNDC5 and/or circulating Irisin and/or UCP1 levels in white adipocytes. We included Results: 51 studies (12 randomised controlled trials) with 2474 participants. Out of the 51 studies, 16 examined PGC-1a and FNDC5 in response to exercise, and only four found increases in both PGC-1a and FNDC5 mRNA and one showed increased FNDC5 mRNA. In total, 22 out of 45 studies that examined circulating Irisin in response to exercise showed increased concentrations when ELISA techniques were used; two studies also revealed increased Irisin levels measured via mass spectrometry. Three studies showed a positive association of circulating Irisin with physical activity levels. One study found no exercise effects on UCP1 mRNA in white adipocytes. The Conclusions: effects of physical activity on the link between PGC-1a, FNDC5 mRNA in muscle and UCP1 in white human adipocytes has attracted little scientific attention. Current methods for Irisin identification lack precision and, therefore, the existing evidence does not allow for conclusions to be made regarding Irisin responses to physical activity. We found a contrast between standardised review methods and accuracy of the measurements used. This should be considered in future systematic reviews. 1,2 1 3 4


Introduction
Brown adipose-like phenotype in white adipose tissue (WAT) may play a role in reducing body weight, and consequently lessen obesity in mammals 1 . Recently, acute and chronic exercise has been found to induce a brown adipose-like phenotype in WAT 2 through a number of sequential steps. Exercise is also known to increase the activation of the peroxisome proliferatoractivated receptor gamma coactivator 1-alpha (PGC-1α) gene in human skeletal muscle 3 . PGC-1α is a co-transcriptional regulator facilitating multiple transcription factors to regulate a complex network of genes 4 and it has been implicated in both the control of tissue mitochondrial content and the program that results in brown adipose tissue (BAT) formation 5 .
While skeletal muscle properly adapts to exercise in the absence of PGC-1α 6 , activation of PGC-1α was proposed to increase the fibronectin type III domain-containing protein 5 (FNDC5) 2 . FNDC5, is a membrane protein expressed in brain and skeletal muscle 7 . It was proposed that FNDC5 was cleaved during exercise, and released into the bloodstream as Irisin -a peptide fragment of FNDC5 measured by western blotting 2 . In vitro, exposure of white adipocytes to Irisin-through an unknown receptor -subsequently led to an increase of the peroxisome proliferator-activated receptor alpha, which in turn increased uncoupling protein one (UCP1) mRNA 2 . The increase in white adipocyte UCP1 mRNA observed with Irisin treatment, presented as fold-change over control, is hard to interpret since white adipocytes in culture do not usually express UCP1 mRNA 8 .
Since, UCP1 is the only contributor to non-shivering thermogenesis that occurs in BAT 9 and it appears that the presence of UCP1 in a white adipocyte is accompanied by "brown-adipocyte like" properties 8,10,11 , it was proposed that increased circulating Irisin in humans after a chronic exercise program may promote increased weight loss and improved metabolic control through induction of UCP1 2 . This hypothesis seemed superficially plausible, as Irisin over-expression stimulated oxygen consumption and has been described to have an inverse association with blood glucose, insulin, total cholesterol and a positive association with adiponectin concentrations 12 . However, other studies have failed to observe such positive associations [13][14][15] , while the effect of exercise on "browning" of the white adipose phenotype remains unclear [16][17][18] .
The exact role of exercise in regulating circulating Irisin concentration remains to be established. Indeed, data indicate that while older adults appear to have a 30% increase in FNDC5 mRNA in muscle compared to younger adults, FNDC5 mRNA was unresponsive to six weeks of endurance training 19 , despite robust increases in mitochondria 20 . In general, results on the effects of exercise on circulating Irisin 17,21-24 have been rather ambiguous; diverse methodology may explain the highly discrepant results 25,26 . Given that Irisin continues to be measured using a variety of methods, an evaluation of the available evidence for its relationship with humans' health is warranted, due to the potential that the browning of white adipocytes may have on human health. In addition, the proposed exercise mechanism that may cause a browning process of WAT in humans must be evaluated. Therefore, the aim of the current review was to systematically identify the effects of physical activity on the link between PGC-1α and FNDC5 in muscle, and circulating Irisin, as well as evidence for regulation of UCP1 in WAT (indicating a browning process) in humans.

Search strategy
Using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines 27-29 , two databases (PubMed and EMBASE) were searched up until 19 th August 2016. Two investigators (PCD and IML) independently conducted two identical searches in both databases using appropriate search algorithms (PubMed: Supplementary File 1; EMBASE: Supplementary File 2). The lists of the included articles were reviewed to identify publications that were relevant to the topic under review.

Selection criteria
We included studies that met at least one of the following eligibility criteria: a) measurements of PGC-1a (mRNA and/or protein concentrations) in conjunction with measurements of FNDC5; b) measurements of FNDC5, and/or Irisin concentrations and/or UCP1 in WAT, along with the following criteria: c) measurements of physical activity levels and/or exercise interventions, and d) human participant study. No other eligibility criteria were set (e.g., language, date of publication). From the included studies, we retrieved outcomes regarding the effects of physical activity on PGC-1a in conjunction with FNDC5 in muscle, FNDC5 in muscle, Irisin in the bloodstream and UCP1 in WAT. We report the studies' design, the participants' characteristics, the Irisin identification and other outcome methods and study outcomes. We have also recorded the secondary associations in the included studies, i.e. associations between FNDC5 and/or circulating Irisin and several health-related phenotypes [e.g. energy expenditure, blood pressure, waist to hip ratio, body mass index (BMI)].
Risk of bias assessment and quality of reporting data Two independent reviewers (PCD and GSM) evaluated the risk of bias of the studies included in the current review via the "Cochrane Collaboration's tool for assessing risk of bias" 30 . Conflicts in the risk of bias assessment were resolved by IL and ADF. We also evaluated independently (PCD and GSM) the quality of reporting in the included randomised controlled trials (RCTs), controlled trials (CTs) and single group design studies (SGS) using the Consolidated Standards of Reporting Trials (CONSORT)

Amendments from Version 1
We have implemented additional information regarding Irisin identification in content with the already existing argument in the text while we removed the detailed discussion of the most likely invalid ELISA. We have also included all the suggested information from the reviewers, regarding the differences that the existing studies displayed in the identification of the molecular weight of circulating Irisin. We removed repetition in the discussion section and redundancy in the results section. We have corrected minor errors in the Table 1 and the presented references in the text. As per the reviewers' suggestions, we have included in the discussion two very important recent papers , Montes-Nieto et al., 2016.

See referee reports
REVISED checklist 31 , which is a 25-item checklist and we provided a score for each study included. For CTs and SGS, we used a modified CONSORT checklist comprised of 18 items, given that these studies are not RCTs and therefore, seven out of the 25 items of the CONSORT checklist are not applicable for CTs and SGS (i.e. randomization, blinding). We also evaluated independently (PCD and GSM) the quality of the reporting data of the included cross sectional studies (CSS) using the 22-item checklist of the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) and we also provided a score for each study included 32 . Disagreements on studies' CONSORT and STROBE scores were arbitrated by IL and ADF. JT and PS then reviewed the molecular and genomic content of the review independent of the search process.

Results
The reporting of the available information in this systematic review is shown in a PRISMA checklist in Supplementary  Table 1.

Searching procedure results
The initial searching date was the 14 th September 2015 while weekly alerts were received from both databases up until the 19 th August 2016. Overall, the searching procedure revealed 51 studies that involved 2474 participants and met the inclusion criteria, and were therefore included in this systematic review. The reference lists of these studies did not result in the identification of additional relevant articles. The searching outcome is presented in a PRISMA flow diagram in Supplementary Figure 1.

Characteristics of the included studies
The characteristics and the results of the included studies can be found in Table 1. From the 51 eligible studies, 12 (23.5%) were RCTs, of which four were cross-over RCTs, eight (15.7%) were CTs, 23 (45%) were SGS, and eight (15.7%) were CSS. One of the included RCTs 33 reported the effect of resistance exercise training versus the effects of resistance exercise training combined with Ursolic supplementation, because for the latter group the effects of resistance exercise cannot be isolated, we will report only the results from the resistance exercise training group. Furthermore, one of the CTs 34 will be included in the results of both CTs and CSS because this study consisted of a controlled trial nested within a CSS. Eight of the included studies examined overweight/obese adults and children 17,35-41 , while 11 studies included a clinical population, including patients with chronic obstructive pulmonary disease (COPD) 23,34,42 , heart failure 43 , metabolic syndrome 44 , haemodialysis 45 , osteoporotic 46 , anorexia nervosa 36,47 , pre-diabetes 16 and diabetes type II 48 .

Risk of bias and quality of reporting data
The estimated risk of bias assessment results can be found in Table 2, and a summary is displayed in Supplementary Figure 2. Five RCTs 44,49-52 , and all the included CTs and CSS, as well as 22 of the 23 SGS, displayed a high risk of bias due to inadequate generation of a randomised sequence, while four RCTs 23,33,42,53 showed low risk of bias, and three RCTs 41,54,55 , as well as one SGS 56 , showed unclear risk of bias because there was no description of the method  used for allocation (even though the participants were said to be "randomly" assigned). Six RCTs 23,42,49,50,52,53 displayed low risk of bias for "allocation concealment", while two 44,54 showed unclear risk of bias because of the lack of description of the randomization allocation. Also, four RCTs 33,41,51,55 , and all the included CTs and SGS, as well as CSS, showed high risk of bias due to the lack of concealment of allocations before assignment. In "blinding of participants and personnel", all RCTs, CTs, SGS and CSS displayed high risk of bias because the exercise interventions could not be blinded to the participants.
In "blinding of outcome assessment", three RCTs displayed low risk of bias 23 All the included studies showed low risk of bias of "selective reporting" because they reported all the outcomes measured, and all the included studies displayed low risk of bias in "other bias".
The results of our evaluation in the quality of the reporting data showed a mean score of 13.6 out of 25 (54.4%) for the included RCTs, 10.56 out of 18 (58.68%) for the included CTs and 10.52 out of 18 (58.44%) for the included SGS (Table 3). The CSS displayed a mean score of 13.37 out of 22 (60.8%) ( Table 4). The score represents the number of items (with percentage of items) on the checklist that were reported satisfactorily in each study. Therefore, a high score represents a high adherence to reporting guidelines, while a low score represents low adherence to reporting guidelines.

Reporting of the outcomes
The link between PGG-1a and FNDC5 in muscle in response to physical activity/exercise Acute effects of exercise Five studies 16,17,50,61,62 investigating the link between PGC-1α with FNDC5 in muscle in response to acute exercise showed an increase of the PGC-1α mRNA in muscle; however, only two studies 16,62 also found an increase in muscle FNDC5 mRNA, while one study 43 detected a positive association of PGC-1α with FNDC5 in muscle. More specifically, a study found that an aerobic (2.1±0.8-fold over baseline, p=0.05) and a resistance (3.5±0.9-fold over baseline, p=0.01) training session increased PGC-1α splice variant1 but it did not change FNDC5 mRNA in the muscle of healthy adults 50 . Similarly, a resistance training session increased PGC-1α splice variant1 four hours post exercise (200%, over baseline and over control, p<0.05), but it did not change FNDC5 mRNA in the muscle of healthy adults 61 . A 45-minute endurance exercise session increased Exon 11 of PGC-1α mRNA in muscle (7.4-fold over baseline, p<0.05), but it did not change FNDC5 mRNA in muscle in both healthy and pre-diabetic adults, while a positive association between PGC-1α Table 3. Quality of the reporting of the results using the CONSORT checklist. Score represents the number of items (with percentage of items) on the checklist that were reported satisfactorily in each study. Therefore, a high score represents a high adherence to reporting guidelines, while a low score represents low adherence to reporting guidelines.  and FNDC5 mRNA was found at baseline (r=0.82, p<0.01) when data of the two groups were combined 16 . Furthermore, PGC-1α mRNA in muscle increased (>6-fold over baseline, p<0.05) in response to acute exercise; however, FNDC5 mRNA in muscle was not altered in sedentary overweight and obese adults 17 . Also, a resistance exercise session increased Exon 11 of PGC-1α mRNA in muscle of both young (4-fold over baseline, p<0.05) and older (2-fold over baseline, p<0.05) healthy adults, while it increased FNDC5 mRNA in muscle only in young (1.4-fold over baseline, 95% Confidence Interval=0.3-2.2, p<0.05) healthy adults 62 . Finally, PGC-1α mRNA in muscle was positively associated with FNDC5 mRNA in muscle (r=0.56, p<0.05) in a sub-set of 24 patients with heart failure 43 ; stratification was ad hoc.

First author
Chronic effects of exercise Of the eleven eligible studies 2,16-19,40,62-67 that examined the link between PGC-1α with FNDC5 in muscle in response to chronic exercise, only two 16,66 showed that chronic exercise increased PGC-1α and FNDC5 mRNA in muscle, while four studies 18,19,62,63 showed no effect of chronic exercise on PGC-1α and FNDC5 mRNA in muscle. In the five studies that only measured FNDC5 in muscle, one study 2 found increased and four 17,40,64,65 showed no effect of chronic exercise on FNDC5 mRNA in muscle.
A 12-week of endurance and resistance combined exercise training increased Exon 11 of PGC-1α mRNA in muscle (1.2-fold in healthy and 1.6-fold in pre-diabetic adults over baseline, p<0.05) and FNDC5 mRNA in muscle (1.4-fold in healthy and 2-fold in pre-diabetic adults over baseline, p<0.05) 16 . Furthermore, an 8-week sprints exercise program increased PGC-1a and FNDC5 mRNA in muscle (p<0.05) in healthy adults 66 . Finally, Bostrom et al. (2012) showed that in eight older participants selected from a larger group of 27 participants, chronic exercise increased FNDC5 mRNA in muscle (p<0.05) 2 .
A 21-week endurance and resistance combined exercise program in healthy adults did not alter PGC-1α and FNDC5 mRNA in muscle 62 . One of the included studies 19 found no effect of chronic exercise on PGC1a or FNDC5 mRNA in younger adults (despite detecting significant changes in ~1,000 other mRNAs and finding mitochondrial enzyme activity was increased in ~25%) 68 . Similarly, an 8-week resistance exercise program did not alter PGC-1α or FNDC5 mRNA in muscle of young healthy adults 63 . In addition, 12 weeks of resistance training did not alter PGC-1α splice vari-ant1 mRNA, and it did not change the FNDC5 mRNA in muscle in untrained young females 18 . Also, a 12-week aerobic and resistance exercise combined program 17 and an 8-week aerobic exercise program 40 did not alter FNDC5 mRNA in muscle of sedentary obese adults, while chronic exercise had no effect on FNDC5 mRNA in muscle of healthy adults 64 . Finally, a 3-week sprint interval training program did not alter FNDC5 mRNA in muscle of healthy adults 65 . A resistance training session did not change FNDC5 mRNA in the muscle of healthy adults and circulating Irisin increased (p<0.001) over the following 24-hour 50 , indicating no short-term association between FNDC5 and Irisin. Furthermore, an aerobic exercise session increased circulating Irisin (p=0.04) and Irisin concentrations were measured at ~355-459 ng/ml 50 , greater than recent mass spectrometry measurements 73 . Similarly, a running exercise session in healthy individuals 49 and an aerobic exercise session, as well as a resistance exercise session, in healthy individuals and in metabolic syndrome patients 44 increased circulating Irisin (p<0.05). In the latter studies, Irisin concentrations measured at ~99-175 ng/ml 49 and ~80-94.6 ng/ml 44 , respectively, which is greater than recent mass spectrometry measurements 73 . Also, an acute resistance exercise session increased circulating Irisin (p<0.05) as oppose to aerobic and combined (aerobic and resistance) sessions that did not alter circulating Irisin in healthy males (Irisin concentrations ~18-151 ng/ml) 52 . Furthermore, a 90-minute aerobic exercise session increased circulating Irisin during (54 th minute) the exercise session (20.4% compared to baseline, F (3,36) =5.28, p=0.004), but circulating Irisin decreased after the exercise session (p=0.021) in healthy male adults 69 . In the latter study, the aerobic exercise session also increased circulating Irisin during (54 th minute) the exercise session (F (3,24) =5.03, p=0.01) in healthy female adults 69  A 6-month resistance training program increased circulating Irisin in healthy controls (p<0.01), but not in the exercisers 54 , while an 8-day vibration exercise increased circulating Irisin in COPD patients (p=0.01) 23 . Notably, the Irisin concentrations in the latter study 23 were ~785-1196 ng/ml, a lot greater than recent mass spectrometry based detection of Irisin concentrations 73 . Furthermore, a 12-week resistance exercise increased circulating Irisin in elderly healthy females (Irisin concentrations ~61-83 ng/ml, p<0.05,) 51 . In addition, a 12-week of endurance and resistance combined exercise training in both healthy and pre-diabetic adults increased FNDC5 mRNA in muscle, while it decreased circulating Irisin (p<0.05) when the data of both healthy and prediabetic groups were combined 16 . In the latter study, Irisin concentrations were detected at 160 ng/ml at baseline and 143 ng/ml after the exercise program, a lot greater than recent mass spectrometry based detection of Irisin concentrations 73 . In addition, an 8-week endurance training program increased circulating Irisin only in middle-aged and not in young healthy adults (Irisin concentrations ~140-168 ng/ml, p<0.05) 74 , while an 8-week chronic exercise program in COPD patients increased circulating Irisin (p<0.05) 34 . Finally, a 12-month physical activity intervention increased circulating Irisin by ~12% (p=0.001) in obese children 39 . Notably, in the latter study, Irisin concentrations were 111 ng/ml, a lot greater than recent mass spectrometry based detection of Irisin concentrations 73 .

The effects of physical activity/exercise on Irisin
A 3-week sprint interval training program did not alter FNDC5 mRNA in muscle and showed a gender difference in circulating Irisin, which was decreased in healthy males and increased in healthy females (p<0.05) 65 73 . This study used mass spectrometry and detected circulating Irisin at 3.6 ng/ml in controls and 4.3 ng/ml in exercisers, which was significantly different between the two groups (p=0.04). No details regarding training or control of hydration in the training group were reported 73 .
The effects of physical activity/exercise on UCP1 in WAT We located only one study that examined the effects of exercise on UCP1 mRNA in subcutaneous WAT in humans. This study found that a 12-week intervention of endurance and resistance combined exercise in both healthy and pre-diabetic adults had no significant effect on UCP1 mRNA in subcutaneous WAT, even though UCP1 mRNA was increased (1.82-fold over baseline, p<0.05) when data from both groups were combined 16 . Also, UCP1 mRNA did not associate with FNDC5 mRNA in muscle (r=0.28, p=0.18) and circulating Irisin (r=-0.11, p=0.60) 16 .

Results for associations of Irisin with secondary outcome measures
The secondary results of the included studies can be found in  70 , all of which indicate unfavourable effects of Irisin on human health. Nevertheless, some secondary evidence suggests that circulating Irisin was positively associated with fat free mass 36,70 , muscle mass 41 and energy expenditure 36 , and Irisin that was incubated within white adipocytes in vitro increased glucose and fatty acids uptake 66 . Furthermore, circulating Irisin after a maximal workload was significantly greater in individuals with higher VO 2 max than individuals with lower VO 2 max 21 . However, circulating Irisin was not associated with VO 2 peak before and post exercise in healthy females 57 and sedentary overweight and obese individuals, while it was inversely correlated with VO 2 peak (p<0.05) in healthy males 60 .

Discussion
The aim of the current review was to systematically identify the effects of physical activity on the link between PGC-1a and FNDC5 in muscle and circulating Irisin, as well as evidence for regulation of UCP1 in WAT (indicating a browning process) in humans.
Overall completeness and applicability of evidence We were unable to find strong evidence that links PGC-1α and FNDC5 mRNA in muscle in response to exercise training or increased physical activity levels. Notably, we located only one study that examined the effects of exercise on UCP1 in WAT, and this found no effect 16 . Despite PGC-1α being firmly placed as a central regulator of adaptation to exercise in mice and humans, numerous aspects of the literature are contradictory or incomplete. For example, previous evidence indicates that PGC-1α mRNA accumulates with endurance training, while studies of PGC-1α protein reflect various antibodies that measure distinct molecular entities ranging from 70 to >110 kDa [76][77][78] . Furthermore, mice lacking PGC-1α adapt normally to endurance exercise training, and in humans the PGC-1α regulated gene network does not correlate with aerobic adaptation 68 . Thus any argument that places Irisin as part of the core PGC-1α regulated exercise adaptation program needs to reflect, on both technical and theoretical grounds, that there is great uncertainty of the nature and importance of PGC-1α in exercise and health 79 .
When PGC-1α protein content is measured (albeit with uncertainty over protein identities) exercise training increases PGC-1α protein in skeletal muscle or causes nuclear translocation of protein [80][81][82][83] . However, the studies included in the current review only relied on measuring PGC-1α mRNA to determine the effects of exercise on PGC-1α, and the time-course of mRNA and protein responses to exercise are distinct. Thus, the link between PGC-1α and FNDC5 in skeletal muscle may reflect measurement of mRNA dynamics and this may explain inconsistent findings for PGC-1α. Also, the proposed mechanism by Bostrom et al. (2012) indicates that induction of PGC-1α mRNA and then protein would activate the transcription of FNDC5, and hence, if this theory was correct, it would be expected that a strong correlation between PGC-1α mRNA and FNDC5 mRNA would exist. However, previous evidence showed that FNDC5 mRNA in muscle is not regularly increased by exercise or differently regulated between those with and without insulin resistance 19 , and was only modestly increased in a subset of older people following chronic exercise training 19 . If we focus on more reliable mRNA measures of PGC-1α and FDNC5, then the variable findings may be explained by the different characteristics of the populations examined and the different exercise protocols used.
An interesting aspect brought forward in the included studies showed that the start codon of the FNDC5 gene displays a variation in humans due to the non-ATG start codon 64 . In humans, ATG is usually the first codon to lead to efficient protein production, and therefore, the latter may suggest that Irisin, if produced, would be done so in an inefficient manner 64 . However, this notion has been questioned by a subsequent study, which supports that human Irisin is mainly translated from its non-ATG start codon, while the molecular weight of the protein is similar to that of important proteins in human body, such as insulin, leptin and resistin 73 , indicating a biological role of Irisin.
The various commercially available antibodies used in the ELISA kits of the studies included in the current systematic review, yield a protein concentration that appears to be ~5-278 times greater than a more recent mass spectrometry data (data that may require independent validation), and still far above what others have found 84  According to the results of the current systematic review, two studies have measured circulating Irisin via mass spectrometry in response to exercise in humans. In the study by Jedrychowski et al. (2015), blood samples for Irisin identification were collected only after the exercise program from a small number of participants who were sedentary (n=4) or aerobic exercisers (n=6) 73  . While the studies that utilised mass spectrometry do not agree 24,73 , reflecting issues of sensitivity and methodology, the latest identification and analysis of Irisin 24,73 indicates that Irisin may circulate in blood and probably has a similar or identical structure to the mouse structure; however, whether it has genuine biological activity remains to be elucidated.

Quality of evidence and limitations
Based on the studies selected for the purposes of the current review, we cannot reach precise conclusions regarding the effects of acute and chronic exercise on PGC-1α in conjunction with FNDC5 mRNA in muscle; this is mainly due to the inconsistency of the findings and the different population characteristics examined. Most of the RCTs 33,44,49-52 display high risk of bias, due to inadequate generation of a randomised sequence and a lack of concealment of allocations before assignment, while all the RCTs exhibit high risk of bias since the exercise interventions could not be blinded to the participants. In addition, four RCTs 44,49,50,52 display unclear risk of bias because of the lack of information regarding the blinding procedures. Therefore, the risk of bias assessment of the included RCTs indicates that they may provide imprecise results (Table 2). In addition, the CTs and SGS display a high risk of bias due to the absence of generation of a randomised sequence, inadequate concealment of allocations before assignment and knowledge of the allocated interventions by the outcome assessors. They also display unclear risk of bias due to knowledge of the allocated interventions by the investigators during the study (Table 2). Finally, the included CSS display high risk of bias due to inadequate generation of a randomised sequence, lack of concealment of allocations before assignment and knowledge of the allocated interventions by the assessors, while they display unclear risk of bias for "incomplete outcome data" because of the lack of information of the participants who were excluded from the analysis. This evidence indicates that the CTs, SGS and CSS may also provide imprecise results. Furthermore, quality of reporting, as expressed through the adherence guidelines (i.e. CON-SORT and STROBE), showed low scores of the required results that should have been reported (54.4% for RCTs, 58.68% for CTs, 58.44% for SGS and 60.8% for CSS) by the included studies in the current review. This shows inadequate reporting of the results of the included studies that may not aid the critical appraisal and interpretation of their outcomes.

Agreements and disagreements with other studies or reviews
To the best of our knowledge, this is the first systematic review that examines the effects of physical activity on the link between PGC-1α and FNDC5 in muscle, circulating Irisin and on UCP1 of WAT in humans. We compared our results with a recent metaanalysis that aimed to identify the effects of exercise on circulating Irisin 86 . This meta-analysis concluded that chronic exercise may decrease circulating Irisin in the RCTs while the non-RCTs cannot form any conclusion. However, the latter meta-analysis did not take into consideration the issues raised regarding the validity of the methods used for Irisin identification 26 . In contrast, while we considered the methods used for Irisin identification in the studies included in the current review, our review had a different aim, to systematically identify the effects of physical activity on the link between PGC-1α and FNDC5 in muscle, circulating Irisin and find evidence for regulation of UCP1 in WAT in humans. Regarding circulating Irisin, we also report that we cannot form any firm conclusion of the effects of exercise on circulating Irisin. Our review highlights previous evidence showing that circulating Irisin may only be detected in humans via mass spectrometry 25,26,73 , while we suggest that the previous available data coming from methods that have not been previously validated for circulating Irisin identification should not be used. This is because recent evidence questioned the antibodies used in the commercial ELISA kits given the polyclonal nature of these antibodies that may attract crossreacting proteins 26 . However, publications that use commercial ELISA that have not been previously validated to detect human Irisin continue at an alarming rate. Therefore, our review indicates to consider using only valid methods for human circulating Irisin identification in the future. Furthermore, our results are in accordance with a previous review that showed equivocal results among studies examining circulating Irisin due to the methodological variations for Irisin detection 87 . In this critical review, the authors examined the commercial antibodies and ELISA used to measure circulating Irisin and concluded that the currently available antibodies should be tested for cross-reacting antigens detection 87 . Additionally, another recent review showed that the previous measurements for circulating Irisin identification differs greatly, given that they displayed a molecular weight of the protein between 0.01 ng/ml and 2000 ng/ml 88 . The latter critical review concluded that it is necessary to establish accurate methods for irisin measurements. Our systematic review analysis, agrees with the latter conclusion given that the Irisin measurements in the included studies via commercial ELISA kits, displayed a molecular weight of the protein ranging between 22 ng/ml to 1196 ng/ml.
Initially, Irisin was proposed to have a therapeutic effect given the potential to cause a browning formation of WAT that may have anti-obesity and antidiabetic effects 2 . This was mainly suggested when Irisin administered in obese mice improved glucose homeostasis and caused weight loss 2 . Also, the browning formation that Irisin may cause could lead to reduced weight gain, up-regulated insulin sensitivity, reduced risk of diabetes type II and other metabolic disorders as animal studies indicate [89][90][91][92][93] , as well as increase daily resting energy expenditure in humans 94,95 . The available evidence from the included studies in the current review revealed that the available commercial ELISA kits for Irisin identification either were found to be invalid 26,87 or they should be tested for validity 87 . Thus, we cannot confirm a favourable effect of Irisin on human metabolism. Finally, none of the included studies in the current review examined associations of circulating Irisin with indices indicate a therapeutic role of the protein using western blotting and/or mass spectrometry methods.

Potential biases in the review process
The current review has a number of strengths. For instance, we used the PubMed and the EMBASE databases using appropriate algorithms with standardized indexing terms. Standardized indexing terms can retrieve records that may use different words to describe the same concept and information beyond that may be contained in the words of the title and abstract 96 . Furthermore, the current review used a systematic manner to identify articles according to previous methodology 27-29 , and we used wellestablished tools 30-32 to evaluate the included studies. To reduce bias, two investigators worked independently on the screening of the included studies for eligibility, risk of bias assessment, and in the provision of CONSORT and STROBE scores. Also, we have not excluded studies based on language. However, a limitation of the current review includes the use of only published literature; we did not include grey literature searching. In this light, there is a potential of publication bias in the current review. Nevertheless, the inclusion of grey literature may itself introduce bias and one reason to include grey literature would be the absence of peerreview sources 96 .

Conclusions
We found little evidence to determine the link between PGC-1a mRNA and FNDC5 mRNA in human muscle, and there was limited evidence on the effects of physical activity on UCP1 in subcutaneous WAT. We also found a heterogeneity in the populations examined, high risk of bias by the selected studies and a relatively small number of RCTs (n=12) with inconsistent findings regarding the link between physical activity, PGC-1a, FNDC5, and UCP1.
Mass spectrometry detection of Irisin of exercise effects were compromised by the methodological limitations of the existed studies (i.e. post exercise comparisons, lack of control, small samples). The current systematic review highlights previous evidence that indicates via mass spectrometry that Irisin is present in human blood at concentrations that are ~5-278 folds lower than those detected by commercial ELISA kits. Therefore, we are unable to conclude on the circulating Irisin response to physical activity due to methodological limitations. In this regard, our systematic review used well-established methodology (i.e. PRISMA and Cochrane Library guidelines). However, we have also considered the validity and accuracy of the measurements of Irisin protein concentrations in the included studies. This additional analysis completely redirected our conclusion compared to the conclusion that a wellestablished systematic review methodology would provide. Therefore, we suggest that future systematic reviews should also take into consideration the validity and accuracy of the measurements of the included studies, to avoid misleading conclusions. We also suggest that future studies should only consider currently valid methods for human circulating Irisin (i.e. mass spectrometry), until new methods are introduced. The latter also implies that future studies should re-examine the biological role for human Irisin and the effects of physical activity/exercise on the link between PGC-1a and FNDC5 in muscle, circulating Irisin and UCP1 in WAT.
Author contributions PCD and IML formed the paper, developed the algorithms and conducted the searching procedure. PCD and GSM performed the risk of bias and the quality of the reporting of the results assessments. Disagreements in the assessment of both risk of bias and the quality of the reporting of the results was arbitrated by IML and ADF. JT and PS contributed in the data extraction from the selected studies, reviewed and modified the molecular and genomic content of the paper. YK contributed in the data extraction from the selected studies, reviewed and modified the content of the manuscript. All authors approved the submitted version.

Competing interests
No competing interests were disclosed. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Grant information
In this manuscript, the authors analyzed the effects of physical activity on the connection between muscle peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and fibronectin type III domain-containing protein 5 (FNDC5), circulating Irisin and uncoupling protein one (UCP1) of white adipocytes in humans through a systematic review. The authors review the evidence connecting PGC-1α, FNDC5, Irisin and UCP1 with physical exercise, which might play an important role in diabetes and obesity. Moreover, they have addressed a controversial topic since, though Irisin was initially described as a hormone potentially regulated by exercise and also with a potential role in obesity and diabetes, conflicting results have been reported. There is an ongoing intense debate on different results from different Irisin assays. In effect, in 2014, we were the first to underline those discrepancies and inconsistencies. For that reason, I feel this review is timed appropriately and is interesting for the scientific community. The review is effectively organized and the sequence of points is logical, and follows PRISMA guideline.
In my opinion, both results and discussion are properly presented, although the discussion section should be slightly shortened. Also, references 8 and 87 are not needed. I recommend changing PGC-1a by PGC-1α throughout the manuscript. Furthermore, the following manuscript that was recently published deserves to be included and discussed:  .

Introduction
Reference 8 is not suited to support this statement because it comments the results of Boström et al. and is no independent confirmation Table 1, page 5 Aydin 2013: They used EK-067-52 for measurement of serum irisin. The given product H-067-17 is an antibody for immuno-histochemistry page 6 Moienneia 2016: The correct name of the company is CUSABIO and it is based in China -the name and affiliation is wrong in the original publication. Taken together, this systematic review is a valuable contribution to guide through the confusing literature concerning the relationship between exercise and the proposed PGC1-alpha -FNDC5 -irisin -axis. The evaluation of the included studies with well-acknowledged quality measures adds additional value and makes the article unique in the reviewed field. No competing interests were disclosed.

Competing Interests:
We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.
Author Response 22 May 2017 , FAME LABORATORY, UNIVERSITY OF THESSALY, Greece

PETROS DINAS
We have implemented the suggested information regarding Irisin identification in content with the already existing argument in the text. Also, we removed the detailed discussion of the most likely invalid ELISA (Page 37). We would like to mention that we completely agree with the Reviewer invalid ELISA (Page 37). We would like to mention that we completely agree with the Reviewer that a meaningful discussion regarding the effects of physical activity on Irisin is hardly possible, given the problematic methods used for Irisin identification. In this regard, we conclude that we cannot form any firm conclusion. Furthermore, we highlight the fact (Conclusions section) that even though we used a well-established methodology for systematic reviews, we had to additionally consider the validity and accuracy of the methods used in the included studies to avoid misleading conclusions. Finally, we have mentioned all this information that was suggested by the reviewer including the reference by Montes-Nieto et al. (2016) (Page 35).
We have implemented the information (Page 36) that the studies using mass spectrometry delivered contradictory results.
The results section is formed based on the mechanism that this systematic review examined (i.e. PGC-1a and FNDC5 in muscle, Irisin and UCP1 in white adipocytes) to directly reflect to the aim of the study. In addition, we report the results considering the different kind of exercises (i.e. acute and chronic exercise), while we separately present the relationships of the examined factors with physical activity levels. We believe that this is particularly important to increase clarity in the presented outcomes in line with the aim of our systematic review. However, considering your comment, we removed the repetition from the results section (i.e. values for Irisin determined with mass spectrometry) (Pages 31-32). We also removed repetition from the discussion section as per your suggestion (Pages 34).
We removed the reference 8 from the relevant statement. We have corrected the Table 1 (Pages 9, 10, 11, 15 and 18) Thank you for bringing to our attention that reference 87 was mixed up with reference 25. We have now corrected this in the text.
We have removed the reference 26 that was mixed up with reference 25.
. No competing interests were disclosed. Competing Interests: