cardiovascular diseases are the leading cause of morbidity and mortality in diabetic patients ,    and it is likely that vascular abnormalities may be responsible for the higher incidence of cardiovascular diseases in diabetes . although it is suggested that endothelial dysfunction is an important contributor to the vascular complications of diabetes [ 2 , 3 ] , the exact mechanisms of impaired endothelial function are unclear .  
lifestyle modification , especially exercise , is routinely recommended for the management of human type 2 diabetes [ 4 , 5 ] .
exercise is thought to improve vascular function by reducing plasma lipids and blood glucose level , oxidative stress , and increasing insulin sensitivity .
chronic low - grade inflammation , as reflected by elevated plasma levels of crp , is an independent predictor of cardiovascular disease [ 9 , 10 ] and diabetes .
crp has a number of roles in several cardiovascular diseases , and levels of crp are positively correlated with obesity and insulin resistance .
many studies suggest that a chronic inflammatory process promotes the progression of endothelial dysfunction . in this study , we hypothesized that moderate - intensity exercise improves endothelial function by decreasing low - grade inflammation in db / db mice , a frequently used animal model of type 2 diabetes .
six - week - old control wild type and diabetic db / db mice ( bks.cg-m + /+ lepr db / j ) were purchased from jackson laboratory ( bar harbor , me , usa ) .
all experiments were performed according to the guidelines of the university of british columbia animal care committee .
after one week of acclimatization , animals were randomly divided into four groups ( n = 10 each ) : two groups each of control ( control sedentary and control exercised ) and diabetic mice ( diabetic sedentary , diabetic exercised ) .
the animals were housed ten per cage under conditions of a 12-hour light / dark cycle , 22c temperature , and with free access to food and water .
mice were exercised using a running wheel ( lafayette instruments , lafayette , in , usa ) as previously described in [ 2 , 15 ] .
mice assigned to the exercise groups were placed in individual running wheels for one hour of daily exercise at a speed of 5.2  m / min ( which represents a daily forced exercise of 312  m ) for 6 weeks . during the training period ( two weeks ) , mice were exercised daily at a set time each day for 5 days a week . the sedentary db / db or control groups were placed in nonrotating wheels for one hour per day .  
animals were anaesthetized with pentobarbital ( 50  mg / kg , i.p . ) combined with heparin ( 50  u / kg ) .
blood samples were taken at baseline ( 6 weeks old ) , after two weeks of exercise following a two - week training period ( the 10th week ) and at the end of study ( the 14th week ) .
plasma crp and tnf- levels were measured using elisa kits ( alpco diagnostic , usa ) .
thoracic aortas were removed and placed in ice - cold physiological salt solution ( pss ) and cleaned of connective tissue .
segments of aorta were threaded with stainless steel wire ( 0.04  mm diameter ) and attached to the tissue holders of a four - channel wire myograph ( jp trading , aarhus , denmark ) .
tissues were allowed to equilibrate for 60 minutes at 37c during which time the pss was replaced at 20-minute intervals . during the equilibration period ,
the resting tension was gradually increased to 5.5  mn and kept at this level for 20 to 30 minutes .
each tissue was maximally activated with a solution of kcl ( 80  mmol / l ) that was prepared by equimolar substitution of nacl . following washout with fresh pss and return of tension to basal preload , phenylephrine ( 1 
thereafter , cumulative additions of acetylcholine ( ach ) ( 1  nmol / l to 10 
vasodilatory responses were recorded on a computer using myodaq acquisition software ( version 2.01 ; danish myotechnology , aarhus , denmark ) and expressed as percent dilation of phenylephrine - induced constriction . to document the efficacy of an endurance - trained state , citrate synthase activity levels were measured in skeletal muscle .
thigh adductor muscles were gently removed after sacrificing the animal , and citrate synthase activity was measured as previously described in .  
the composition of the pss ( mm ) was nacl ( 119 ) , kcl ( 4.7 ) , kh2po4 ( 1.18 ) , mgso4 ( 1.17 ) , nahco3 ( 24.9 ) , edta ( 0.023 ) , cacl2 ( 1.6 ) , and dextrose ( 11.1 ) .
isotonic substitutions ( replacement of na with equimolar concentrations of k ) were used when using pss solutions with increased k concentrations .  
data analysis was done using ncss-2000 software and graphpad prism ( version 3.02 - 2000 ) .
after six weeks of exercise , db / db exercised had lower body weights compared to the sedentary group ( table 1 ) .
analysis of baseline blood parameters ( 6 weeks old ) , after two weeks ( 10 weeks old ) and six weeks ( 14 weeks old ) of exercise are shown in table 1 .
diabetic mice had higher fasting blood glucose levels at all time points , and while two weeks exercise did not alter blood glucose levels in db / db mice , six weeks of exercise reduced blood glucose levels in diabetic mice ( p < .05 ) .   
baseline plasma crp levels were higher in db / db mice compared to control ( 3.81  0.23 versus 1.83  0.30 ) ( p < .05 ) .
plasma crp levels in db / db mice were not affected by two weeks of exercise but were significantly reduced after 6 weeks exercise ( at the 14th week ) ( 3.59  0.41 versus 5.12  0.25 ) ( p < .05 )
. plasma levels of crp were significantly correlated with body weight ( r = 0.5855 , p < .0001 ) and blood glucose ( r = 0.4821 , p = 0.0003 ) when analyzed by the pearson test .
the level of plasma tnf- in sedentary db / db mice at 14 weeks old ( 18.62  2.11  pg / ml ) tends to be higher than in control mice ( 14.88  0.35  pg / ml ) ; however , it does not reach statistical significance ( p > .05 ) . acetylcholine ( ach )
responses to ach vasodilation were impaired in aortic rings from db / db mice compared with control counterparts ( figure 1 ) .
moderate - intensity exercise in db / db mice for either two or six weeks restored endothelium - dependent vasodilation ( figure 1 ) .
the maximal vasodilatation ( % loss of induced tone ) and sensitivity ( ec50 ) is shown in figure 2 .   as shown in table 2 ,   tissue levels of citrate synthase activity
were significantly increased in the thigh adductor muscles of db / db and control exercised mice compared to the sedentary groups at both time points ( after two and six weeks of   exercise ) ( p < .01 , n = 10 ) .  
this study examined the effects of moderate levels of exercise on vascular endothelial function and plasma crp levels in control and type 2 diabetic ( db / db ) mice .
we report that endothelial function ( endothelium - dependent relaxation ) was significantly impaired in db / db mice , as also reported in other studies [ 1719 ] .
there is much evidence to support the notion that endothelial dysfunction precedes the development of type 2 diabetes [ 20 , 21 ] . two - week and six - week of moderate - intensity exercise both
our results show that diabetic mice initially have higher crp levels compared to control animals .
for example , plasma levels of plasma crp and icam levels are higher in diabetic subjects [ 2325 ] , and it is likely that increases in crp levels also occur in patients with impaired glucose tolerance .
thus , hyperglycemia may be one reason for endothelial dysfunction and low - grade inflammation in db / db mice .
hyperglycemia is thought to activate the immune and macrophage - monocyte systems and so stimulate the production of cytokines and acute phase proteins , which are also proposed to reduce endothelial dependent vasodilation [ 22 , 28 ] . moreover , highly - glycated haemoglobin impairs no - mediated vascular responses by a mechanism involving superoxide anions but not cyclooxygenase derivatives [ 7 , 29 ] .
in addition , db / db mice are obese , and there is also a close association between adiposity and crp [ 13 , 30 ] .
adipose tissue secretes inflammatory mediators ( especially il-6 ) which stimulates crp synthesis in the liver .
crp is related to insulin resistance and is a marker of endothelial dysfunction .   in our experiments ,
exercise improved endothelium - dependent relaxation in db / db mice after two - week exercise independently of reductions in weight , blood glucose , or plasma crp levels ; our data shows a lack of a correlation between improved vasodilatation to ach and decreased plasma crp levels after two weeks of exercise as shown by the nonsignificant ( p = .1941 ) pearson correlation coefficient for the relationship between maximal ach dilation and plasma crp levels .
however , six weeks of exercise improved ach - mediated vasodilatation while also reducing plasma crp levels in db / db mice ; this was associated with a significant correlation between plasma crp levels and body weight , a finding that is consistent with other reports in experimental   and human diabetes .  
our results indicate that crp levels are increased in control mice that underwent a period of forced - exercise .
this finding is in keeping with recent studies in healthy humans indicating that there were significant increases in plasma crp and tnf alpha following a two - week bout of exercise .
in addition ,   exercise has also been shown to stimulate a marked but transient increases in inflammatory markers such as il-6 and cortisol ( which subsequently stimulate hepatocytes to generate the synthesis of acute phase proteins such as crp ) , responses that may reflect   muscle injury [ 36 , 37 ] .  
since crp can a cause dose - dependent decrease in no production in endothelial cells , it is possible that this effect is time - dependent and occurs independently of inflammation as reported by crp levels .
other studies have reported that that crp directly inhibits the endothelium - dependent no mediated dilation of porcine retinal arterioles , and down - regulates enos protein to decrease no release .
the plasma levels of tnf- in sedentary db / db mice tends to be higher than in control mice ;   however , it does not reach statistical significance .  
previous reports have failed to demonstrate a parallelism between changes in plasma levels of crp , il-6 , and tnf- under pathological conditions [ 4143 ] .
overweight adolescent boys had higher tnf- , but not crp or il-6 levels compared to   normal weight controls .
however , crp is the marker of chronic inflammation most frequently studied   and has been shown to predict cardiovascular diseases more than other cytokines . in conclusion ,
we report a reciprocal association between endothelial dysfunction and crp levels in diabetic db / db mice .
short - term exercise improves endothelial function without changing plasma crp levels ( two weeks of exercise ) .
longer periods of exercise ( six weeks ) reduce plasma crp levels and maintain improved endothelial function in diabetic mice .
 the aim of this study was to evaluate the effects of moderate - intensity exercise on plasma levels of c - reactive protein ( crp ) and tumor necrosis factor - alpha ( tnf- ) as markers of low - grade inflammation and endothelial function in diabetic ( db / db ) mice . 
 control and 
                  db / db mice were divided into sedentary and exercised groups . 
 aortic endothelial function was evaluated after two- and six - week exercises using a wire myograph . 
 plasma crp levels were measured at baseline , and after two and six weeks of exercise . 
 baseline plasma crp levels were significantly higher in db / db mice compared to control ( p < .05 ) . 
 after two weeks of exercise , aortic endothelial function was significantly improved without affecting body weight or plasma crp levels . 
 six weeks of exercise not only improved endothelial function , but also significantly reduced body weight and plasma crp levels in db / db mice . thus short - term exercise has beneficial effect on endothelial function without reducing low - grade inflammation while more prolonged exercise periods are required to reduce inflammatory markers . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion