a growing body of evidence supports the predominant role of chronic , low - grade inflammation in mediating insulin resistance and all stages of atherosclerosis in type 2 diabetes mellitus ( t2 dm ) . among pro - inflammatory mediators associated with cardiovascular diseases ( cvd ) , adipose - tissue derived cytokines ,
known as adipokines , have emerged as potential key factors of atherosclerosis - related complications .
apelin , an adipocyte - secreted factor upregulated by insulin , has been recently identified as a contributor to glucose homeostasis . moreover , previous trials have suggested apelin as a potent regulator of cardiovascular function .
we and other investigators have recently documented the inverse relationship of circulating apelin levels with coronary artery disease . until present ,
available data concerning pharmaceutical and non - pharmaceutical regulation of apelin have been extremely limited .
the pathophysiological role of ghrelin derives from the association of low plasma ghrelin levels with elevated fasting insulin levels and insulin resistance . in parallel ,
numerous investigations suggest the anti - inflammatory properties of ghrelin in the presence of advanced lesions of atherosclerosis . taken together , ghrelin manipulation , at least theoretically , could be useful for the treatment of t2 dm and its complications .
however , it is still unknown whether exercise training per se , without weight loss , can produce beneficial effects on ghrelin levels .
physical exercise has been recommended world - wide as one of the mainstays of treatment of t2 dm , along with diet and medications .
reduced cardiovascular morbidity and mortality has been associated with increased regular physical activity in the diabetic population .
in addition to the favorable modulation of  classical  cardiovascular risk factors ( eg , dyslipidemia and hypertension ) , numerous studies have indicated the anti - inflammatory effect of long - term exercise training .
however , the influence of chronic exercise on human circulating adipokines is not well - documented .
taken together , the modulation of apelin and ghrelin seems quite promising , because they interact with numerous pathophysiological mechanisms in t2 dm and cardiovascular diseases . the present study aimed to investigate the effect of long - term aerobic exercise on circulating levels of novel adipokines , including apelin and ghrelin , in type 2 diabetic patients without any history of cvd .
we enrolled 54 overweight ( bmi > 25 kg / m ) caucasian men and women with t2 dm , aged 5070 years .
all participants had poor glycemic control ( hba1c > 6.5% ) with oral anti - diabetic medications ( sulfonylureas , metformin , for at least 4 months ) .
suitable patients , identified from review of case notes and/or computerized clinic registers , were contacted by the investigators in person or by telephone .
patients were excluded if they had any of the following conditions : a history of ketoacidosis within 6 months before study enrollment , excessive hyperglycemia ( hba1c > 10% ) , diabetic micro - vascular complications , diabetic neuropathy , impaired hepatic function ( plasma aminotransferase and/or -glutamyltransferase level higher than the upper limit of normal for age and sex ) , impaired renal function ( serum creatinine level higher than the upper limit of normal for age and sex ) , severe anaemia , uncontrolled hypertension ( blood pressure > 170/100 mmhg ) , orthopaedic problems limiting exercise , malignancy , or autoimmune diseases .
patients with cardiovascular disease ( new york heart association class i  iv congestive heart failure or a history of myocardial infarction ) , overt cardiac - related symptoms or history of cerebrovascular events also were excluded .
active patients reporting sport activities > 1 per week and insulin - treated patients were considered ineligible .
patients were randomly assigned to either a supervised , systemic , exercise training group ( eg , n=27 ) or a control group ( cg , n=27 ) . randomization process , based on computer software , created sex - matched groups .
the supervised exercise training program was similar to that performed in another exercise - study in our department .
each session included 10 min of warm - up , 30 min of aerobic activity ( eg , walking / or running on treadmill , cycling , calisthenics of the upper and lower limbs ) and 5 min of cool - down .
the intensity and the duration of each session gradually increased across the first 4 weeks .
after that , intensity achieved 6075% of maximal heart rate and the duration of each session was constant at 60 min .
the control group was orally instructed , at baseline , to follow american diabetes association guidelines for cardiovascular diseases prevention .
in particular , the control patients were encouraged to perform a total of 150 minutes of self - controlled , moderate - intensity exercise , per week .
patients were asked to perform their preferred type of exercise ( eg , walking , swimming ) 35 times per week , with a minimum duration of 30 minutes per time .
the latter modality of exercise intervention was selected as control group , because it is very common in clinical practice of a large general hospital .
finally , control patients were asked to record the weekly amount of physical activity at the end of the study . before starting the study
, all patients underwent an initial clinical evaluation including medical history , physical examination , measurements of body mass index ( bmi ) , waist - hip ratio ( whr ) and blood pressure ( bp ) , as previously described .
the duration of the study was 12 weeks and clinical assessment was repeated at the end .
cardiorespiratory fitness was estimated at the beginning and at the end of the study by ergospirometry , using an electronically - controlled ergocycle .
exercise testing protocol was supervised by an experienced physician as previously described . during ergospirometry ,
gas exchanges were continuously measured by an analyzer ( cosmed k4 , rome , italy ) , using facemask and breath - by - breath technique .
patients were asked to maintain their eating patterns , anti - diabetic and anti - hypertensive medications throughout the study .
dietary habits of all patients were recorded at baseline by an expert dietician using a structured questionnaire .
additional weight control diet instructions were not provided throughout the study and patients were asked not to alter their dietary habits . during the study ,
the study design was approved by the ethics committee of hippokratio general hospital of thessaloniki , greece and the study was conducted in accordance with the helsinki declaration .
blood samples were collected in the morning after a 12 h overnight fast . fasting plasma glucose ( fpg ) and lipid parameters
were quantified using standard enzymatic methods ( roche / hitachi 912 automatic analyzer ; roche diagnostics , basel , switzerland ) . for low - density lipoprotein cholesterol ( ldl - c ) calculation
measurements of hba1c were taken by high - performance liquid chromatography ( menarini diagnostics , florence , italy ) .
after centrifugation of blood samples ( 5000 g for 7 min ) , serum was collected and kept frozen at 80c until analysis in the same assay . using commercially available enzyme immunoassay ( eia ) kits ( phoenix pharmaceuticals , belmont , ca
, usa ) we quantified serum levels of apelin ( human apelin-12 ) and ghrelin .
the intra - assay coefficients of variance ( cvs ) were 5% and < 14% for apelin and ghrelin , respectively .
the inter - assay cvs for the latter adipocytokines were 14% and 7.5% , respectively .
we measured plasma insulin using elisa method ( drg diagnostics , marburg , germany ) .
the homeostasis model assessment ( homa - ir ) index was determined as a surrogate index of insulin resistance and was calculated with the following formula : homa - ir = fasting insulin ( mu / l )  fasting glucose ( mg / dl)/405 . nephelometric procedure ( dade behrin , bnii , marburg , germany ) was used for serum high - sensitivity crp ( hscrp ) assay .
paired - samples t test and student s t test were used for comparisons of parametric variables within and between groups , respectively .
the correlations of apelin and ghrelin with the other continuous variables at baseline , as well as with their changes post - treatment , were calculated by pearson correlation coefficients .
standard multiple regression analysis , adjusted for sex and age , was performed to determine the independent correlations of either of the 2 novel adipokines with the other parameters .
statistical analyses were performed using the computer software package spss ( version 16.0 ; spss inc , chicago , il , usa ) .
we enrolled 54 overweight ( bmi > 25 kg / m ) caucasian men and women with t2 dm , aged 5070 years .
all participants had poor glycemic control ( hba1c > 6.5% ) with oral anti - diabetic medications ( sulfonylureas , metformin , for at least 4 months ) .
suitable patients , identified from review of case notes and/or computerized clinic registers , were contacted by the investigators in person or by telephone .
patients were excluded if they had any of the following conditions : a history of ketoacidosis within 6 months before study enrollment , excessive hyperglycemia ( hba1c > 10% ) , diabetic micro - vascular complications , diabetic neuropathy , impaired hepatic function ( plasma aminotransferase and/or -glutamyltransferase level higher than the upper limit of normal for age and sex ) , impaired renal function ( serum creatinine level higher than the upper limit of normal for age and sex ) , severe anaemia , uncontrolled hypertension ( blood pressure > 170/100 mmhg ) , orthopaedic problems limiting exercise , malignancy , or autoimmune diseases .
patients with cardiovascular disease ( new york heart association class i  iv congestive heart failure or a history of myocardial infarction ) , overt cardiac - related symptoms or history of cerebrovascular events also were excluded .
active patients reporting sport activities > 1 per week and insulin - treated patients were considered ineligible .
patients were randomly assigned to either a supervised , systemic , exercise training group ( eg , n=27 ) or a control group ( cg , n=27 ) . randomization process , based on computer software , created sex - matched groups .
the supervised exercise training program was similar to that performed in another exercise - study in our department .
each session included 10 min of warm - up , 30 min of aerobic activity ( eg , walking / or running on treadmill , cycling , calisthenics of the upper and lower limbs ) and 5 min of cool - down .
the intensity and the duration of each session gradually increased across the first 4 weeks .
after that , intensity achieved 6075% of maximal heart rate and the duration of each session was constant at 60 min .
the control group was orally instructed , at baseline , to follow american diabetes association guidelines for cardiovascular diseases prevention . in particular , the control patients were encouraged to perform a total of 150 minutes of self - controlled , moderate - intensity exercise , per week .
patients were asked to perform their preferred type of exercise ( eg , walking , swimming ) 35 times per week , with a minimum duration of 30 minutes per time .
the latter modality of exercise intervention was selected as control group , because it is very common in clinical practice of a large general hospital .
finally , control patients were asked to record the weekly amount of physical activity at the end of the study .
before starting the study , all patients underwent an initial clinical evaluation including medical history , physical examination , measurements of body mass index ( bmi ) , waist - hip ratio ( whr ) and blood pressure ( bp ) , as previously described .
the duration of the study was 12 weeks and clinical assessment was repeated at the end .
cardiorespiratory fitness was estimated at the beginning and at the end of the study by ergospirometry , using an electronically - controlled ergocycle .
exercise testing protocol was supervised by an experienced physician as previously described . during ergospirometry ,
gas exchanges were continuously measured by an analyzer ( cosmed k4 , rome , italy ) , using facemask and breath - by - breath technique .
patients were asked to maintain their eating patterns , anti - diabetic and anti - hypertensive medications throughout the study .
dietary habits of all patients were recorded at baseline by an expert dietician using a structured questionnaire .
additional weight control diet instructions were not provided throughout the study and patients were asked not to alter their dietary habits . during the study ,
the study design was approved by the ethics committee of hippokratio general hospital of thessaloniki , greece and the study was conducted in accordance with the helsinki declaration .
fasting plasma glucose ( fpg ) and lipid parameters were quantified using standard enzymatic methods ( roche / hitachi 912 automatic analyzer ; roche diagnostics , basel , switzerland ) . for low - density lipoprotein cholesterol ( ldl - c ) calculation
measurements of hba1c were taken by high - performance liquid chromatography ( menarini diagnostics , florence , italy ) .
after centrifugation of blood samples ( 5000 g for 7 min ) , serum was collected and kept frozen at 80c until analysis in the same assay . using commercially available enzyme immunoassay ( eia ) kits ( phoenix pharmaceuticals , belmont , ca
, usa ) we quantified serum levels of apelin ( human apelin-12 ) and ghrelin .
the intra - assay coefficients of variance ( cvs ) were 5% and < 14% for apelin and ghrelin , respectively .
the inter - assay cvs for the latter adipocytokines were 14% and 7.5% , respectively .
we measured plasma insulin using elisa method ( drg diagnostics , marburg , germany ) .
the homeostasis model assessment ( homa - ir ) index was determined as a surrogate index of insulin resistance and was calculated with the following formula : homa - ir = fasting insulin ( mu / l )  fasting glucose ( mg / dl)/405 .
nephelometric procedure ( dade behrin , bnii , marburg , germany ) was used for serum high - sensitivity crp ( hscrp ) assay .
paired - samples t test and student s t test were used for comparisons of parametric variables within and between groups , respectively .
the correlations of apelin and ghrelin with the other continuous variables at baseline , as well as with their changes post - treatment , were calculated by pearson correlation coefficients .
standard multiple regression analysis , adjusted for sex and age , was performed to determine the independent correlations of either of the 2 novel adipokines with the other parameters .
statistical analyses were performed using the computer software package spss ( version 16.0 ; spss inc , chicago , il , usa ) .
anthropometric and metabolic data ( means standard deviations ) are presented in tables 1 and 2 and figure 1 . among initially recruited patients , only 1 stopped in the eg due to time constraint . throughout the study
, most pharmaceutical regimens remained constant ; in 3 patients in the eg group the dosage of the anti - diabetic medications was reduced .
exercise - treated patients showed high compliance , since they attended at least 85% of the scheduled sessions . on the other hand
, only 22.22% of the control patients achieved the target of increased physical activity at the end of the study , suggesting a lack of motivation .
no significant difference between groups was detected at baseline for any clinical characteristics , including sex , age , diabetes duration , smoking habits and current anti - diabetic , anti - hypertensive and lipid - lowering drug therapy .
fitness parameters , traditional cv risk factors and inflammatory biomarkers were also similar among groups . although all patients were asked to maintain their dietary habits , the exercise - treated patients reported an almost 20% increase in water and energy intake compared to their untreated counterparts .
thus , the 12-week exercise intervention did not significantly affect body weight and anthropometric characteristics ( p>0.05 ) , but the intervention considerably reduced most lipid parameters , fpg and hba1c compared to either baseline values or control group ( p<0.05 ) .
significant reductions ( p<0.01 ) were noted in fasting insulin and homa - ir levels after the exercise training program . as expected , exercise training yielded a significant increase in the cardiorespiratory capacity , expressed by vo2peak ( p<0.001 ) ( table 2 ) . as noted in figure 1 , after the exercise intervention there was a significant increment in apelin serum concentrations in the eg ( from 0.2090.102 ng / ml to 0.2910.135 ng / ml ; p<0.001 ) , while there was a non - significant decrease in the cg ( from 0.2000.088 ng / ml to 0.1860.109 ng / ml ; p=0.781 ) ( p<0.001 ) . at the end of the study
, the alterations in ghrelin concentrations did not reach statistically significant levels in either group ( p>0.05 ) .
however , ghrelin was significantly reduced after exercise training in the women s sub - group ( from 2.140.71 ng / ml to 3.871.07 ng / ml , p=0.042 ) compared to control group women ( from 2.201.36 ng / ml to 2.101.40 ng / ml , p=0.779 ) ( p=0.038 ) .
pearson correlations were calculated for the novel adipokines at baseline as well as their changes post - exercise . at baseline ,
apelin correlated with age ( r=0.311 , p=0.024 ) , homa - ir ( r=0.245 , p=0.019 ) , ldl - c ( r=0.521 , p=0.022 ) and fpg ( r=0.431 , p=0.041 ) .
baseline values of ghrelin significantly correlated with bmi ( r=0.469 , p=0.038 ) and homa - ir ( r=0.399 , p=0.010 ) .
the exercise - induced upregulation in apelin was inversely associated with changes in ldl ( r=0.363 , p=0.002 ) and homa - ir ( r=0.411 , p=0.003 ) .
the latter parameters retained their significant correlations with apelin in standard multiple regression analysis ( r=0.391 , p=0.011 ) .
no significant correlations were found between the change of ghrelin and the rest of the variables within the exercise group .
although all patients were asked to maintain their dietary habits , the exercise - treated patients reported an almost 20% increase in water and energy intake compared to their untreated counterparts .
thus , the 12-week exercise intervention did not significantly affect body weight and anthropometric characteristics ( p>0.05 ) , but the intervention considerably reduced most lipid parameters , fpg and hba1c compared to either baseline values or control group ( p<0.05 ) .
significant reductions ( p<0.01 ) were noted in fasting insulin and homa - ir levels after the exercise training program .
as expected , exercise training yielded a significant increase in the cardiorespiratory capacity , expressed by vo2peak ( p<0.001 ) ( table 2 ) .
as noted in figure 1 , after the exercise intervention there was a significant increment in apelin serum concentrations in the eg ( from 0.2090.102 ng / ml to 0.2910.135 ng / ml ; p<0.001 ) , while there was a non - significant decrease in the cg ( from 0.2000.088 ng / ml to 0.1860.109 ng / ml ; p=0.781 ) ( p<0.001 ) . at the end of the study , the alterations in ghrelin concentrations did not reach statistically significant levels in either group ( p>0.05 ) .
however , ghrelin was significantly reduced after exercise training in the women s sub - group ( from 2.140.71 ng / ml to 3.871.07 ng / ml , p=0.042 ) compared to control group women ( from 2.201.36 ng / ml to 2.101.40 ng / ml , p=0.779 ) ( p=0.038 ) .
pearson correlations were calculated for the novel adipokines at baseline as well as their changes post - exercise . at baseline , apelin correlated with age ( r=0.311 , p=0.024 ) , homa - ir ( r=0.245 , p=0.019 ) , ldl - c ( r=0.521 , p=0.022 ) and fpg ( r=0.431 , p=0.041 ) .
baseline values of ghrelin significantly correlated with bmi ( r=0.469 , p=0.038 ) and homa - ir ( r=0.399 , p=0.010 ) .
the exercise - induced upregulation in apelin was inversely associated with changes in ldl ( r=0.363 , p=0.002 ) and homa - ir ( r=0.411 , p=0.003 ) .
the latter parameters retained their significant correlations with apelin in standard multiple regression analysis ( r=0.391 , p=0.011 ) .
no significant correlations were found between the change of ghrelin and the rest of the variables within the exercise group .
in the present study 12-week intensive aerobic exercise training significantly improved metabolic profile and cardiorespiratory capacity in overweight patients with t2 dm . furthermore , we conclusively demonstrated the positive effects of exercise training on serum concentrations of the novel adipokines , apelin and ghrelin .
the latter results seemed to be mediated by the insulin - sensitizing effects of exercise , outlining a novel atheroprotective mechanism . in our study body - weight and waist
this was not surprising , since exercise - treated patients increased food intake compensatory to increased energy expenditure .
consistent with previous studies , blood pressure , insulin sensitivity , glycemic and lipid control were substantially improved in exercise - treated patients , despite the absence of significant variations in anthropometric parameters .
it is well documented that strenuous exercise can ameliorate plasma levels of various adipokines , including il-6 , resistin and tnfa in type 2 diabetic individuals . to our knowledge
this is the first study demonstrating a significant upregulation of serum apelin levels after structured exercise treatment in the diabetic population .
apelin and its receptor ( apj ) are localized in cardiomyocytes and vascular cells and seem to play a key role in the regulation of vascular tone and cardiovascular function .
therefore , the observed apelin increase after exercise supports the hypothesis that an exercise training - induced increase in apelin might contribute to the beneficial effects of exercise training on cardiovascular disease risk .
( 2009 )   reported an almost 4-fold increase in apelin levels after a 12-week therapeutic lifestyle program combining diet and self - controlled exercise .
the study enrolled non - diabetic , statin - free , non - obese ( bmi < 30 kg / m ) individuals with dyslipidemia .
consistent with this , we observed ldl reduction was an independent predictor of the exercise - induced alterations in apelin levels .
in addition to this , we found that insulin resistance attenuation was independently associated with apelin increase in our exercise - treated group .
a growing body of evidence , derived from experimental and clinical trials , supports the interaction between apelin and insulin sensitivity .
notably , a significant increment in serum apelin levels could be obtained in diabetic patients after treatment with insulin sensitizers , metformin and rosiglitazone .
however , the activation of amp - activated protein kinase provides the underlying link between exercise - mediated insulin sensitization and apelin changes .
future studies will be needed to verify the latter hypothesis in different stages of insulin resistance .
the novel adipokine ghrelin induces weight gain not only by stimulating appetite , but also by decreasing fat utilization .
conversely , significant up - regulation of serum ghrelin levels has been recorded after weight loss interventions .
furthermore , low plasma ghrelin levels have been associated with obesity , insulin resistance , inflammation and atherosclerosis . despite the emerging atheroprotective role of ghrelin
a limited number of studies have demonstrated increased total ghrelin levels after long - term exercise associated with weight loss . in our study exercise training had negligible effects on body weight and concomitant serum levels of ghrelin in the whole study population . on the other hand , sub - group analysis revealed a considerable increase in ghrelin concentrations in exercise - treated women , despite the absence of weight loss .
our finding is consistent with results of a recent study showing increased ghrelin levels in non - diabetic , physically active women , but not in men , and that effect was independent of energy equilibrium .
looking for the underlying mechanisms , we initially hypothesized sex - related differences in appetite and energy homeostasis . after assessing the energy balance throughout the study
, we observed an equivalent increase in energy expenditure and compensatory increased energy intake in both sexes .
another plausible explanation for the aforementioned ghrelin sex - based fluctuation may center on differences in sex hormones .
our findings indicate another sex - dependent cardioprotective mechanism and suggest that therapeutic interventions targeting ghrelin should optimally achieve weight loss .
further studies are needed to determine the magnitude of exercise - induced energy deficit resulting in positive effects on adipokines .
thus , the recorded diet changes in the exercise group might have moderated our findings , and the final results could have been even better .
an additional consideration is that exercise - treated subjects had apparently more contact with study staff during supervised exercise training sessions than did the control group , raising concerns about the effect on other behaviors ( eg , daily physical activities ) during the study . since unsupervised exercise was not monitored , no data are available from which to evaluate the potential role of such changes . finally , we measured total ghrelin levels .
the acylated form of ghrelin is thought to be more potent than non - acylated ghrelin in stimulating appetite .
our study might have had different results if we had assessed the latter form of ghrelin .
in conclusion , in patients with t2 dm , exercise training led to significant increase of apelin and ghrelin ( only in women ) serum concentrations , despite the absence of body weight change . increased insulin sensitivity and lowered ldl - c after training were most strongly related to changes in apelin , suggesting the multi - factorial ,  pleiotropic  effects of exercise .
 summarybackgroundaccumulating data support the atheroprotective role of the novel adipokines , apelin and ghrelin . 
 the aim of the present randomized study was to investigate the effects of aerobic exercise training on these adipokines in patients with type 2 diabetes mellitus ( t2dm).material / methodsfifty - four overweight ( bmi > 25 kg / m2 ) patients with t2 dm , but without vascular complications , were randomized to either the aerobic exercise training group ( eg , n=27 ) , 4 times / week , 4560 min / session ; or to the control group ( cg , n=27 ) , orally instructed to increase physical activity . clinical glycemic and lipid parameters , exercise capacity ( vo2peak ) , insulin , homa - ir , and serum levels of apelin and ghrelin were assessed at baseline and after 12 weeks.resultsaerobic exercise significantly improved lipid and glycemic profile and insulin sensitivity compared to cg ( p<0.05 ) . 
 furthermore , between - groups comparison showed a considerable exercise - induced upregulation in apelin ( p=0.007 ) and vo2peak ( p<0.001 ) levels . 
 negligible changes in body - weight , waist - hip ratio and ghrelin concentrations were detected within and between groups after the completion of the study ( p>0.05 ) . 
 however , subgroup analysis revealed a considerable increment in ghrelin levels only in the exercise - treated women compared to their control counterparts ( p=0.038 ) . 
 ldl and homa - ir reduction were found to be independent predictors of apelin increment in multiple regression analysis ( r2=0.391 , p=0.011).conclusionsin patients with t2 dm , systemic , long - term , aerobic exercise exerts positive effects on apelin and ghrelin ( only in women ) , even in the absence of significant weight loss , suggesting its pleiotropic effects . 
Background
Material and Methods
Patients
Study design  Exercise intervention
Clinical evaluation
Blood assays
Statistical analysis
Results
Anthropometric characteristics, lipid and glycemic profile, insulin resistance indexes
Adipokines
Discussion
Conclusions