recent studies have reported that oxidative stress may be associated with the multifactorial etiology of insulin resistance , primarily in skeletal muscle tissue , and the subsequent development of t2 dm .
fortunately , several papers have reported that long - term training programs have improved the antioxidant system in young and adult diabetic rats . however , to our knowledge , little attention has been paid to elderly individuals . in addition , it would be of interest to reduce the length of training programs as compared to those previously published .
although extrapolation from animal studies to humans requires caution , shorter training programs may facilitate follow - up , thereby reducing drop - out rates .
the present study was designed to explore whether a short , 6-week , training program can improve the antioxidant system in skeletal muscle of elderly diabetic fatty rats .
the study subjects were 24 male homozygous zucker diabetic fatty ( zdf ) rats ( gmi , fa / fa ) aged 18 weeks with an average weight of 370450 g. animals were housed in single cages in an environmentally controlled laboratory ( temperature 22c ) with a 12:12-h light - dark cycle . a standard rodent chow adjusted to their body weight ( 100 mg / g of weight )
after a 2-week period of environmental adaptation , animals ( n=24 ) were randomly distributed into the exercised group ( n=12 ) that performed a 6-week swimming training protocol , and the sedentary group ( n=12 ) .
the exercised group swam individually in plastic tanks 5010045 cm , once per day ( 1 h ) , 3 days per week ( monday ; wednesday ; friday ) , during the daily dark phase , under red light to enable visual observation . during swimming sessions
however , during the second week of treatment rats had 3% body weight added , increasing 1% each week until the end of the study .
loads were adjusted with the body weight every week . to minimize stress associated with cold or hot water exposure ,
water temperature was monitored and maintained at 32c . in order to separate the effects of exercise and the stress associated with the exercise environment ,
sedentary rats were individually placed in identical swimming tanks , but sat in shallow water at the same temperature , duration and frequency as the exercised rats .
animals were towel - dried and left for 1 h in a heated room to minimize the effects of cold exposure .
animals were sacrificed 24-h after the last exercise session , under anesthesia , using 1 ml of ketamine injection ( 1 g/10 ml ) , by cervical dislocation .
blood samples were collected by cardiac puncture and were centrifuged for 15 min at 3000 rpm .
glycosylated hemoglobin ( hba1c ) was determined using a specific monoclonal antibody with a turbidimetric readout ( dca 200 + analyzer , bayer diagnostics ) .
insulin levels were assessed using the ultrasensitive rat insulin enzyme - linked immunosorbent assay kit ( mercodia , sweden ) .
finally , homa - ir , an index of insulin resistance , was calculated using the following formula : gastrocnemius muscles were excised and immediately frozen in liquid nitrogen and stored at 80c for subsequent use .
generation of ros and other pro - oxidants in muscle mitochondria was evaluated by using the rate of dichlorofluorescein ( dcfh ) oxidation .
briefly , isolated mitochondria were incubated in the respective buffers at 37c for 15 min to allow dcfh - diacetate to cross the mitochondrial membrane .
the solution was then centrifuged at 12,000 g for 8 min , and the supernatant containing excess dcfh - diacetate not crossing the mitochondrial membrane was discarded .
the mitochondrial pellets were resuspended , and 50 l of the suspension ( < 2 mg protein ) were used for assay .
dcf formation was followed at the excitation wavelength of 488 nm and emission wavelength of 525 nm for 30 min by using a hitachi f-2000 fluorescence spectrometer .
the rate of dcfh conversion to dcf was linear for at least 60 min , corrected with the auto - oxidation rate of dcfh without protein .
the units were pmol dcf formed per minute per milligram protein . to determine superoxide - derived changes in protein carbonylation
, we performed an assay for carbonylated proteins provided by chemicon ( oxyblot oxidized protein detection kit ; millipore , amsterdam , netherlands ) .
briefly , carbonyl groups in the protein side chains are derived to 2,4-dinitrophenylhydrazone ( dnp - hydrazone ) by reaction with 2,4-dinitrophenylhydrazine .
the dnp - derived protein samples are subjected to gel electrophoresis and subsequent western blotting .
densitometry of the 2,4-dinitrophenylhydrazine - derived bands in the gel was performed and normalized to a standard control sample loaded on all gels and expressed as arbitrary units ( au ) , as previously stated . total antioxidant status ( tas )
gsh and glutathione disulfide ( gssg ) were analyzed by using hplc method , as previously described .
government principles for the utilization and care of vertebrate animals used in testing , research , and training , and was approved by our institutional ethics committee .
the statistical analysis of data was performed using analysis of variance ( one - way anova ) .
the study subjects were 24 male homozygous zucker diabetic fatty ( zdf ) rats ( gmi , fa / fa ) aged 18 weeks with an average weight of 370450 g. animals were housed in single cages in an environmentally controlled laboratory ( temperature 22c ) with a 12:12-h light - dark cycle . a standard rodent chow adjusted to their body weight ( 100 mg / g of weight )
after a 2-week period of environmental adaptation , animals ( n=24 ) were randomly distributed into the exercised group ( n=12 ) that performed a 6-week swimming training protocol , and the sedentary group ( n=12 ) .
the exercised group swam individually in plastic tanks 5010045 cm , once per day ( 1 h ) , 3 days per week ( monday ; wednesday ; friday ) , during the daily dark phase , under red light to enable visual observation . during swimming sessions
however , during the second week of treatment rats had 3% body weight added , increasing 1% each week until the end of the study .
loads were adjusted with the body weight every week . to minimize stress associated with cold or hot water exposure ,
water temperature was monitored and maintained at 32c . in order to separate the effects of exercise and the stress associated with the exercise environment ,
sedentary rats were individually placed in identical swimming tanks , but sat in shallow water at the same temperature , duration and frequency as the exercised rats .
animals were towel - dried and left for 1 h in a heated room to minimize the effects of cold exposure .
animals were sacrificed 24-h after the last exercise session , under anesthesia , using 1 ml of ketamine injection ( 1 g/10 ml ) , by cervical dislocation .
blood samples were collected by cardiac puncture and were centrifuged for 15 min at 3000 rpm .
glycosylated hemoglobin ( hba1c ) was determined using a specific monoclonal antibody with a turbidimetric readout ( dca 200 + analyzer , bayer diagnostics ) .
insulin levels were assessed using the ultrasensitive rat insulin enzyme - linked immunosorbent assay kit ( mercodia , sweden ) . finally , homa - ir , an index of insulin resistance , was calculated using the following formula :
gastrocnemius muscles were excised and immediately frozen in liquid nitrogen and stored at 80c for subsequent use .
generation of ros and other pro - oxidants in muscle mitochondria was evaluated by using the rate of dichlorofluorescein ( dcfh ) oxidation .
briefly , isolated mitochondria were incubated in the respective buffers at 37c for 15 min to allow dcfh - diacetate to cross the mitochondrial membrane .
the solution was then centrifuged at 12,000 g for 8 min , and the supernatant containing excess dcfh - diacetate not crossing the mitochondrial membrane was discarded .
the mitochondrial pellets were resuspended , and 50 l of the suspension ( < 2 mg protein ) were used for assay .
dcf formation was followed at the excitation wavelength of 488 nm and emission wavelength of 525 nm for 30 min by using a hitachi f-2000 fluorescence spectrometer .
the rate of dcfh conversion to dcf was linear for at least 60 min , corrected with the auto - oxidation rate of dcfh without protein .
the units were pmol dcf formed per minute per milligram protein . to determine superoxide - derived changes in protein carbonylation
, we performed an assay for carbonylated proteins provided by chemicon ( oxyblot oxidized protein detection kit ; millipore , amsterdam , netherlands ) .
briefly , carbonyl groups in the protein side chains are derived to 2,4-dinitrophenylhydrazone ( dnp - hydrazone ) by reaction with 2,4-dinitrophenylhydrazine .
the dnp - derived protein samples are subjected to gel electrophoresis and subsequent western blotting .
densitometry of the 2,4-dinitrophenylhydrazine - derived bands in the gel was performed and normalized to a standard control sample loaded on all gels and expressed as arbitrary units ( au ) , as previously stated . total antioxidant status ( tas )
gsh and glutathione disulfide ( gssg ) were analyzed by using hplc method , as previously described .
government principles for the utilization and care of vertebrate animals used in testing , research , and training , and was approved by our institutional ethics committee .
the statistical analysis of data was performed using analysis of variance ( one - way anova ) .
serum metabolic profile in both exercised and unexercised zdf rats is listed in table 1 .
regarding oxidant production in muscle mitochondria as revealed by dcfh oxidation rate , we found no significant differences between the exercised and sedentary groups ( 96.47.7 vs. 93.07.2 pmol dcf / min / mg ; p=0.64 ) .
total antioxidant status ( tas ) in gastrocnemius tissue was significantly increased in exercised rats as compared with sedentary counterparts ( 0.190.002 vs. 0.130.002 g / mg protein ; p<0.001 ) .
the expression of antioxidant enzyme mnsod was also increased in zdf rats that performed our 6-week training program as compared with sedentary controls ( 847190 vs. 6258102 u/g protein ; p=0.003 ) .
in contrast , no significant differences were found in zn , cusod expression between exercised rats and sedentary controls ( 101.67.3 vs. 97.68.1 u/g protein ; p=0.87 ) .
regarding gssg / gsh ratio in gastrocnemius muscle , we found significant differences between exercised and sedentary rats ( 0.340.006 vs. 0.400.004 ; p=0.032 ) , suggesting that sedentary animals are more vulnerable to oxidative stress . in a more detailed way , we found reduced glutathione ( gsh ) content was significantly higher in exercised rats as compared with sedentary diabetic fatty rats ( 0.950.012 vs. 0.800.006 nmol / mg protein ; p=0.020 ) .
conversely , no significant changes were observed when assessing oxidized glutathione ( gssg ) levels in exercised and sedentary fatty diabetic rats ( 0.330.008 vs. 0.320.006 nmol / mg protein ; p=0.52 ) .
finally , the improvement of antioxidant system was enough to reduce carbonylated proteins in exercised diabetic fatty rats as compared with sedentary animals ( 1.370.33 vs. 1.620.58 ua ; p=0.011 ) .
firstly , it should be pointed out that the zucker diabetic fatty ( zdf ) rat is a good animal model , not only for studying t2 dm physiopathology , but also for assessing the effects of therapeutic options such as intervention programs based on exercise .
in fact , the development of diabetes is quite similar to what is seen in obese humans , since it results from hyperphagic behavior due to a leptin receptor mutation ( fa gene ) . and secondly , we chose swimming because it is widely used to identify biochemical and molecular responses to exercise .
swimming is a uniform type of activity that is less traumatic to animals than other forms of exercise .
further , we did not find significant changes in body weight , in agreement with previous studies .
interestingly , a study that employed forced running as the exercise regime showed that body weight increased in exercised animals despite a lack of differences in muscle mass or fat mass .
although we did not measure glycosuria in our study , it has been reported that 2530% of the caloric intake is excreted in untreated zdf rats .
with respect to biochemical indices , diabetic fatty rats that performed our training protocol had significantly improved glycemic and lipid profiles .
it should be emphasized that our protocol induced similar effects and lasted just 6 weeks .
we also found hyperinsulinemia was significantly reduced in exercised rats when compared to sedentary counterparts .
however , our results suggest that exercised rats were still insulin resistant on a systemic level , as indicated by their elevated plasma insulin concentrations
. this hyperinsulinemia could be a reflection of insulin resistance in the liver that was not ameliorated by swimming training protocol .
several studies have reported that reactive oxygen species ( ros ) are produced in various tissues under diabetic conditions by several mechanisms , such as non - enzymatic glycosylation reactions , electron transport chain in the mitochondria , and membrane - bound nicotinamide adenine dinucleotide phosphate ( nadph ) oxidase .
fortunately , we found no significant differences in oxidant production in muscle mitochondria between exercised and sedentary groups . on the contrary ,
several studies have demonstrated unequivocally that a single session of strenuous exercise significantly enhanced free radical generation in working muscle and oxidative damage in aging rats .
regarding the antioxidant system , we found total antioxidant status ( tas ) was significantly higher in exercised rats when compared with controls .
furthermore , a 15-week swimming protocol significantly improved antioxidant enzyme activities in the soleus muscle of exercised rats .
rats used in our study were older and consequently would be expected to have higher oxidative damage , as well as different antioxidant capacities , when compared with younger animals . in any case , the improvements in insulin sensitivity induced by our 6-week training program appear to be related , at least in part , to changes in these pro - oxidants and antioxidants balance .
as hypothesized , the expression of antioxidant enzyme mnsod was also increased in zdf rats that performed our 6-week training program when compared with sedentary controls .
previous data have demonstrated that the major antioxidant enzyme , mnsod , is most consistently upregulated by exercise training . in this respect ,
the ability of exercise training to upregulate protective mnsod protein may explain , at least in part , the improvement of total antioxidant status ( tas ) that we also found in diabetic exercised rats .
this may be explained , at least in part , since transcriptional response to oxidative stress is impaired in skeletal muscle from aged animals , as previously reported .
we have also found significant differences when assessing gssg / gsh ratio , suggesting that sedentary animals are more vulnerable to oxidative stress . because mitochondria lack the enzymes in the -glutamyl cycle , and gsh is transported into the mitochondria via a membrane - borne energy - dependent system , our results suggest that mitochondrial gsh transport in skeletal muscle may be enhanced in exercised animals , as was reported previously .
it should be emphasized that this improvement of the antioxidant system was able to reduce the presence of carbonylated proteins in exercised diabetic fatty rats , when compared with sedentary controls .
this finding is of particular interest , since protein oxidation has been involved in the pathogenesis of insulin resistance and complications associated with t2 dm .
finally , although there are differences between animal and human conditions , our results suggest that regular exercise is an attractive and healthy non - pharmacological therapeutic measure in t2 dm .
a short swimming program improved metabolic profile and the antioxidant system in zucker diabetic fatty ( zdf ) rats .
in addition , our training protocol significantly increased total antioxidant status , glutathione system and mnsod expression .
fortunately , the improvement in the antioxidant system was enough to reduce oxidative damage , expressed as protein oxidation .
a major finding of this study was that our training protocol lasted just 6 weeks , in contrast to longer protocols based on exercise that were previously published in the literature .
 summarybackgroundit is widely accepted that oxidative stress is associated with the physiopathology of type 2 diabetes mellitus . 
 in fact , it has been pointed out as a therapeutic target in t2 dm . 
 fortunately , several papers have reported that long - term training programs improved the antioxidant system in young and adult diabetic rats . 
 accordingly , this study was designed to assess the influence of a shorter training program in elderly diabetic fatty rats.material/methodsstudy subjects were 24 male homozygous zucker diabetic fatty rats ( gmi , fa / fa ) aged 18 weeks with an average weight of 370450 g. after a 2-week period of environmental adaptation , animals were randomly distributed into the exercised group ( n=12 ) that performed a 6-week swimming training protocol and the sedentary group ( n=12 ) . 
 animals were sacrificed under anesthesia 24 h after the last exercise session . 
 serum metabolic profile was determined . 
 total antioxidant status ( tas ) , mnsod expression , glutathione status and ros generation were assayed in gastrocnemius muscle.resultswhen compared with controls , exercised rats significantly improved their metabolic profile . 
 total antioxidant status ( 0.190.002 vs. 0.130.002 g / mg protein ; p<0.001 ) and mnsod expression ( 847190 vs. 6258102 u/g protein ; 
 p=0.003 ) were also increased in exercised rats.conclusionsa 6-week swimming training program improved the antioxidant system in elderly fatty diabetic rats . 
 fortunately , this improvement was enough to reduce oxidative damage , expressed as protein oxidation . 
 a major finding of this study was that our training protocol lasted just 6 weeks , in contrast to longer protocols previously published . 
Background
Material and Methods
Animals
Swimming training program
Metabolic profile
ROS generation, muscle protein oxidation, total antioxidant status, MnSOD expression and GSSG/GSH ratio assessment
Ethics and statistic assessment
Results
Discussion
Conclusions