obesity as a common problem in menopausal women is a major risk factor for the development of cardiovascular diseases .
this is due to the importance of estrogen in the determination of body fat distribution .
previous studies showed that deficiency of this hormone leads to an accumulation of visceral fat , and consequently , a decrease in insulin sensitivity .
obesity is often associated with cardiovascular disease risk factors such as hypertension , dyslipoproteinemia , diabetes , and elevated inflammation markers [ 79 ] .
accumulating evidence suggests that altered vitamin d homeostasis may also contribute to an increased cardiovascular disease risk in obese subjects .
there are positive correlations between low 25-hydroxyvitamin d [ 25(oh)d ; < 43 nmol / l ] and obesity   as well as between 25(oh)d < 3337.5 nmol / l and cardiovascular mortality .
vitamin d has a protective effect on bones , anticancer   and anti inflammatory   effects and its receptors are present in pancreatic b cells and insulin sensitive tissues including skeletal muscle tissues .
vitamin d repletion improves insulin and glucose homeostasis in animal models of vitamin d deficiency .
it has also been reported that vitamin d deficiency in the blood is greatly associated with various metabolic factors related to diabetes such as hyperglycemia and insulin resistance , but data supporting this relationship are sparse .
there are some studies reporting physical activity as an important environmental factor associated with body weight regulation and increasing physical activity has become an important aspect of non - pharmacological strategies to control obesity , weight gain and blood glucose in addition to preventing progression from impaired glucose tolerance . due to this connection ,
the american diabetes association ( ada ) recommends aerobic exercise of medium intensity such as 150 minutes of walking per week , or 75 minutes of high intensity aerobic exercise per week for patients with type 2 diabetes .
previous studies showed that only 28% of postmenopausal women perform regular physical activity . despite the beneficial effects of physical activity , there are many complications to achieving the predicted improvement in health .
first of all , menopausal women are predisposed to osteoporosis and tend to avoid involvement in different kinds of physical activity .
second , it was recently reported that exercise is not that effective in some obese people .
the mechanisms by which the central nervous system or hormones interact with metabolic pathways and restrict weight loss are not yet understood . here
, we assumed that vitamin d could be one of the determining factors in achieving the health benefits of exercise .
therefore , the authors of this paper intended to investigate the effects of implementing vitamin d intake for 8 weeks in addition to aerobic exercise training on the plasma lipid profiles and insulin resistance in ovariectomized rats .
forthy female wistar rats weighing 240255 g were housed four per cage and fed standard - pellet rat chow and tap water ad libitum .
the 12-h:12-h light cycle ( in which all fluorescent lighting was shielded , preventing the endogenous production of vitamin d3 ) began at 07.00 and the room temperature was maintained at 2124c .
the experiments described in this study were conducted according to the policy of the iranian convention for the protection of vertebrate animals used for experimental and other scientific purposes , and the protocol was approved by the ethics committee of guilan university of medical sciences , rasht , iran ( irb number ; 88038 ) . body weight , food intake and visceral fat were measured using a scale ( sartorius , germany ) accurate to 0.1 g. all rats were weighed once a week , between 09.00 and 11.30 .
also , food intake was monitored weekly . once a week , the food was weighed before it was given to the animals and again at the same time the next day , after it was removed from the cages . to measure the food intake , an equal amount of food ( 20 g / day / rat )
was given to each rat in each cage in each identical group , and food consumption was measured by subtracting the weight of the remaining uneaten food .
the differences between the weights were divided by the number of rats in the cage .
after 8 weeks of exercise , rats were anesthetised for the measurement of body length ( nose - to - anus ) .
two month dietary manipulation of vitamin d was carried out using cholecalciferol ( vit d3 ) added to an isocaloric diet in the following amounts ( based on pilot studies intended to mimic the range of human levels ) : aerobic training with vitamin d supplementation = 10000 iu / kg ( 9000 iu by injection and 1000 iu by food intake ) per week , vitamin d supplementation = 10000 iu / kg
( 9000 iu by injection and 1000 iu by food intake ) per week , control vit d = 1000 iu / kg food a week , aerobic training vit d = 1000 iu / kg food per week .
rats in the at group were trained to run on a motor - driven rodent treadmill ( iranian model , seven lanes , designed by sports sciences research center , tehran , iran ) from the 3rd week of experiment ( 16 weeks old ) . during the first week of training , rats began to practice walking at the speed of 6 m / min , 10 min / day , with no inclination of the treadmill .
exercise training consisted of running on the treadmill for 3 days / week for 8 weeks .
rats progressed from running for 15 min / day at 15 m / min , 0% slope , up to 40 min / day at 25 m / min , 10% slope for the last 4 weeks .
two weeks after acclimatization , 16-week - old rats were separated into ovx ( n = 32 ) and sham - operated control ( sham ; n = 8) groups .
ovx rats were subdivided into four groups : aerobic training ( ovx + at ; n = 8) , vitamin d supplementation ( ovx + vit d ; n = 8) , aerobic training with vitamin d supplementation ( ovx + at + vit d ; n = 8) and control ( ovx + c ; n = 8) group .
rats were ovariectomized and sham - operated under general anesthesia with an intraperitoneal injection of ketamine ( 50 mg / ml ; rotex medica , germany ) and xylazine ( 20 mg / ml ; alfasan , the netherlands ) at a ratio of 4:1 , according to the technique described by shinoda and colleagues .
all trained rats were restrained from training and food was removed from the animals cages at least 12 h before sacrifice . after complete anesthesia ,
the abdominal cavity was rapidly opened and blood samples were drawn from the inferior vena cava .
the serum was separated by centrifugation ( 4000 rpm for 15 min ) and stored at 80c for later biochemical and hormonal measurements .
the concentrations of triglyceride , total cholesterol , hdl - cholesterol   and ldl - cholesterol   in the plasma were measured using enzymatic analysis kits ( asan pharmaceuticals , hwasung , korea ) .
the serum glucose concentration was determined with an enzymatic ( god - pap , glucose oxidaseamino antipyrine ) colorimetric assay ( pars azmoun , tehran , iran ) and the serum insulin concentration was measured with a rat insulin elisa kit ( drg , usa ) .
homa - ir ( fasting insulin ( m/ml)fasting glucose ( mmol / l)/22.5 ) was used to estimate the insulin resistance .
all data are presented as mean  standard error . before statistical analysis , the normal distribution and homogeneity of the variances
comparisons within groups ( after 8 weeks ) were performed by repeated measure anova followed by tukey s post - hoc test .
the sas software version 9.1 was used for data analysis , with the level of significance set at 5% ( p < 0.05 ) .
forthy female wistar rats weighing 240255 g were housed four per cage and fed standard - pellet rat chow and tap water ad libitum .
the 12-h:12-h light cycle ( in which all fluorescent lighting was shielded , preventing the endogenous production of vitamin d3 ) began at 07.00 and the room temperature was maintained at 2124c .
the experiments described in this study were conducted according to the policy of the iranian convention for the protection of vertebrate animals used for experimental and other scientific purposes , and the protocol was approved by the ethics committee of guilan university of medical sciences , rasht , iran ( irb number ; 88038 ) .
body weight , food intake and visceral fat were measured using a scale ( sartorius , germany ) accurate to 0.1 g. all rats were weighed once a week , between 09.00 and 11.30 .
also , food intake was monitored weekly . once a week , the food was weighed before it was given to the animals and again at the same time the next day , after it was removed from the cages . to measure the food intake ,
an equal amount of food ( 20 g / day / rat ) was given to each rat in each cage in each identical group , and food consumption was measured by subtracting the weight of the remaining uneaten food .
the differences between the weights were divided by the number of rats in the cage .
after 8 weeks of exercise , rats were anesthetised for the measurement of body length ( nose - to - anus ) .
two month dietary manipulation of vitamin d was carried out using cholecalciferol ( vit d3 ) added to an isocaloric diet in the following amounts ( based on pilot studies intended to mimic the range of human levels ) : aerobic training with vitamin d supplementation = 10000 iu / kg ( 9000 iu by injection and 1000 iu by food intake ) per week , vitamin d supplementation = 10000 iu / kg ( 9000 iu by injection and 1000 iu by food intake ) per week , control vit d = 1000 iu / kg food a week , aerobic training vit d = 1000 iu / kg food per week .
rats in the at group were trained to run on a motor - driven rodent treadmill ( iranian model , seven lanes , designed by sports sciences research center , tehran , iran ) from the 3rd week of experiment ( 16 weeks old ) . during the first week of training , rats began to practice walking at the speed of 6 m / min , 10 min / day , with no inclination of the treadmill .
exercise training consisted of running on the treadmill for 3 days / week for 8 weeks .
rats progressed from running for 15 min / day at 15 m / min , 0% slope , up to 40 min / day at 25 m / min , 10% slope for the last 4 weeks .
two weeks after acclimatization , 16-week - old rats were separated into ovx ( n = 32 ) and sham - operated control ( sham ; n = 8) groups .
ovx rats were subdivided into four groups : aerobic training ( ovx + at ; n = 8) , vitamin d supplementation ( ovx + vit d ; n = 8) , aerobic training with vitamin d supplementation ( ovx + at + vit d ; n = 8) and control ( ovx + c ; n = 8) group .
rats were ovariectomized and sham - operated under general anesthesia with an intraperitoneal injection of ketamine ( 50 mg / ml ; rotex medica , germany ) and xylazine ( 20 mg / ml ; alfasan , the netherlands ) at a ratio of 4:1 , according to the technique described by shinoda and colleagues .
all trained rats were restrained from training and food was removed from the animals cages at least 12 h before sacrifice .
after complete anesthesia , the abdominal cavity was rapidly opened and blood samples were drawn from the inferior vena cava .
the serum was separated by centrifugation ( 4000 rpm for 15 min ) and stored at 80c for later biochemical and hormonal measurements .
the concentrations of triglyceride , total cholesterol , hdl - cholesterol   and ldl - cholesterol   in the plasma were measured using enzymatic analysis kits ( asan pharmaceuticals , hwasung , korea ) .
the serum glucose concentration was determined with an enzymatic ( god - pap , glucose oxidaseamino antipyrine ) colorimetric assay ( pars azmoun , tehran , iran ) and the serum insulin concentration was measured with a rat insulin elisa kit ( drg , usa ) .
homa - ir ( fasting insulin ( m/ml)fasting glucose ( mmol / l)/22.5 ) was used to estimate the insulin resistance .
all data are presented as mean  standard error . before statistical analysis , the normal distribution and homogeneity of the variances
comparisons within groups ( after 8 weeks ) were performed by repeated measure anova followed by tukey s post - hoc test .
the sas software version 9.1 was used for data analysis , with the level of significance set at 5% ( p < 0.05 ) .
the results for body weight and food intake changes between and within groups are shown in figs . 1 and 2 respectively .
initial body weights and food intakes in the ovx groups were not significantly different in this study . compared with the sham rats ,
the ovx rats were found to have higher body weight intakes ( p  0.05 ) .
three weeks after the beginning of the study , there was significant difference in body weight between the groups ( fig .
1 ) . the repeated measures anova test showed a significant reduction in body weight in ovx + at + vit d , ovx + at and ovx + vit d groups during the experiment , while the body weights of the ovx - c and sham groups significantly increased after 8 weeks compared with the initial body weights . also , food intake significantly increased in the ovx + c and sham groups over time , but significantly decreased in the ovx + at + vit d group ( fig .
interestingly , the mean body weight and food intake from the third week in the control group was significantly higher than in the other groups ( fig . 1 and 2 ) . the tc , tg , hdl - c and ldl - c levels are shown in table 1 .
the tc levels were significantly higher in the c group than in the other groups ( 11.74% , p = 0.002 ) , while the tc levels were significantly lower in the ovx + at + vit d group than in the other groups ( 11.99% , p = 0.001 ) .
the ovx + at + vit d group displayed significantly lower tg levels than the other groups ( 11.30% , p = 0.001 ) . similarly
, the ovx + at , ovx + vit d and sham groups had significantly lower tg levels than the c group ( 15.01% , p = 0.001 ; 1.51% , p = 0.003 ; 4.34% , p = 0.002 ) .
the hdl - c levels were significantly higher in the ovx + at + vit d and ovx + at groups than in the other groups ( 22.40% , p = 0.001 and 12.06% , p = 0.043 ) ( table 1 ) .
furthermore , the hdl - c levels were significantly lower in the c group compared with the other groups ( 17.48% , p = 0.002 ) .
the ldl - c levels were significantly lower in the ovx + at + vit d group than in the other groups ( 19.74% , p = 0.001 ) and were significantly much higher in the c group compared with the other groups ( 25.74% , p = 0.001 ) .
the visceral fat level was significantly lower in the ovx + at + vit d , ovx + vit d , ovx + at and sham groups compared with the control group ( 13.10% , p = 0.001 ; 2.83% , p = 0.003 ; 10.48% , p = 0.004 and 10.69% , p = 0.003 , respectively ) .
glucose , serum insulin and homa - ir levels decreased significantly by 5.82% , 15.56% and 20.94% , respectively , in ovx + at + vit d compared with the other groups ( p = 0.001 ; table 1 ) .
the purpose of this study was to examine the interaction between aerobic exercise training and vitamin d supplementation and its effect on plasma lipid profiles and insulin resistance in ovariectomized rats . in the study ,
ovariectomy resulted in a significant increase in body weight and also an increase in visceral fat , confirming previous findings , this indicates that the lack of estrogen following ovariectomy influences body weight , partially by increasing visceral fat .
estrogen plays a crucial role in distributing visceral fat , probably by changing lipoprotein lipase activity .
our study revealed that exercise training decreases body weight , which has also been reported in many studies . on the other hand
, there is evidence showing lack of weight reduction after exercise , especially in menopausal women .
it has been reported that the concentration of serum vitamin d has an effect on muscular activity , the number and size of muscular fibers and also the fat free mass .
the present study showed that treatment with ovx - at & vit d for 8 weeks leads to more weight loss ( 21% ) compared with exercise alone ( 7% ) .
additionally , mean food intake from week three in the control group was significantly higher than other groups .
reported that in a study comparing exercise training and vitamin d intake ( 400 iu / day ) over 9 months in elderly women , the fat free mass and the muscle strength in the exercise training group significantly increased , but there were no significant changes in fat free mass and muscle strength in the vitamin d intake group .
it has been reported that unlike vitamin d intake , regular aerobic training effects positive changes in body weight .
exercise training targeting patients with diabetes improve their health and has positive effects on body weight .
on the other hand , there have also been research results indicating that significant changes in body weight were not apparent despite participation in exercise training . according to body weight and food intake results obtained in this study , the most significant reduction was in the group with aerobic training combined with vitamin d. the mechanism by which an increase in the concentration of vitamin d in the body affects body composition is difficult to explain . however , some studies reported no effect from vitamin d on body weight change and energy expenditure and little improvement in cardiovascular risks with cholecalciferol .
interestingly , vitamin d is fat - soluble and is readily stored in adipose tissue ; it is sequestered after absorption and stored in tissues such as fat and muscle .
this fate of vitamin d has been demonstrated by injecting radio - labeled vitamin d3 into individuals and monitoring the highest levels of biological activity and radioactivity in the fat tissue .
1,25-dihydroxyvitamin d , inversely , modulates adipokine expression , inhibits anti - inflammatory cytokine expression , reduces monocyte recruitment by human pre - adipocytes , and restores glucose uptake in adipocytes .
1,25-dihydroxyvitamin d has a role in human adipose tissue because it is the active form of the vitamin d3 metabolite .
vitamin d receptors ( vdr ) present in adipocytes allow the suppression of parathyroid hormone ( pth ) levels .
the pth excess observed in menopausal women with both primary and secondary hyperparathyroidism may promote weight gain by impeding catecholamine - induced lipolysis .
a previous study showed that vit d has a positive effect on muscle mass , especially on fat free mass , and also on muscle activity .
we assumed that an increase in muscle mass could elevate metabolism and decrease fat mass .
regarding the one - way anova test , we showed that the tg , tc , and ldl - c levels were significantly decreased with increased aerobic training and vitamin d intake .
also , the hdl - c levels were significantly higher in the ovx + at + vit d and ovx + at groups compared with the other groups .
several studies have revealed the importance of nutritional education aimed at preventing cardiovascular disease ( cvd ) in postmenopausal women .
the present study confirmed this with the observation of a significant decline in body weight , food intake , tg , tc , and ldl - c levels as well as increases in the hdl - c level by aerobic training and vit d. interestingly , weight loss parallels the reduction of cardiovascular risk factors ( tg , tc , ldl - c ) and hdl - c levels were improved in the exercise with vitamin d supplemented group compared with the exercise only group .
few studies have investigated the relationship between serum levels of vitamin d and lipid profiles .
major et al . reported that in obese middle - aged women taking 200 iu of vitamin d per day for 15 weeks , tc , ldl - c , and hdl - c concentrations were significantly reduced .
rejnmark and colleagues performed a study among 82 healthy postmenopausal women who had been treated with either 40 mg / day simvastatin or a placebo for 1 year , in which vitamin d , tg , and ldl levels were measured at baseline and after 26 weeks of treatment . in this study , simvastatin showed no effect on vitamin d status , but reduced the serum levels of tg and ldl .
these results suggest that the serum concentration of tg is inversely associated with the serum level of 25(oh ) d .
in contrast , kota and colleagues showed no relationship between serum levels of 25(oh ) d and tg or hdl cholesterol in healthy subjects .
it was suggested that vitamin d has both direct and indirect effects on modifying the lipid profile and that the effect of vitamin d on decreasing serum levels of tg , tc and ldl may occur through regulatory action that increases the activity of lipoprotein lipase in adiposity . in our study , exercise led to improvements in lipids profile as well . in agreement with our results ,
greene et al . also reported that obese middle - aged men and women participating in endurance exercise at 70% of vo2max , 3 times per week for 12 weeks had significant improvements in lipid profiles .
sung & bae reported a similar finding in diabetic elderly men participating in walking exercise at 6575% of hrmax for 50 minutes a day , 3 times a week for 24 weeks .
taken together , aerobic training is considered effective for inducing positive changes in blood lipid .
several mechanisms are suggested to explain the effect of vitamin d on lipids , including its reducing role in fatty acid absorption via the formation of insoluble calcium - fatty complexes in the gut . by decreased absorption of fat ,
particularly saturated fatty acids , it is expected that serum levels of total and ldl cholesterol will be reduced .
in addition , vitamin d can increase the conversion of cholesterol to bile acids due to its ability to bind with bile acids .
however , the effect of enteric vitamin d and calcium on lipid absorption is very limited and it does not have a significant effect on lipid profiles . on the other hand , in vitro studies have demonstrated that pth reduces lipolysis ( through increasing the level of cytosolic calcium )   and increases the expression of fatty acid synthesis .
based on existing studies , it is known that the accumulation of visceral fat in postmenopausal women is the main cause of insulin resistance and metabolic syndrome .
it has been reported that the accumulation of visceral fat increases the secretion of adipocytokine and has a negative effect on hypertension , diabetes and cardiovascular diseases .
previous studies reported that vitamin d deficiency often appeared in cases of obesity and the concentration of 25(oh)d shows a negative correlation with visceral fat . in our study ,
visceral fat showed more reduction in ovx + at + d compared with other groups .
interestingly , the reduction of visceral fat in the group receiving co - treatment of exercise and vit d was much higher than with exercise alone .
in this regard , bacchi et al . reported that for middle - aged men and women with diabetes conducting aerobic exercise using a tread mill or cycle at 6065% of hrr , 60 minutes per day , 3 times per week for 16 weeks , the abdominal visceral fat area and subcutaneous fat area were significantly reduced . while inconsistent , wamberg et al . reported that women taking vitamin d ( 7,000 iu per day over 26 weeks ) showed no significant changes in subcutaneous fat mass and visceral fat mass
generally , the research results show that aerobic exercise training and adequate intake of vitamin d positively affect the body weight , visceral fat and plasma lipid profiles during menopause and also reduce metabolic syndrome risk factors .
in addition , the results of our study showed a significant improvement in insulin resistance in the ovx + d + at group ( fig 2 , b and , d ) .
reported that people who have vit d insufficiency ( < 30 ngml ) and people with deficiency ( < 20 ngml ) < incomplete sentence > . also , vonhurst et al . reported that as a result of taking 4,000 iu of vitamin d every day for 26 weeks , the insulin and blood glucose levels of patients with diabetes were significantly reduced .
although our previous studies   and others showed adequate effectiveness by regular physical activity in improving insulin resistance , ovx + at + d showed a better response .
there are some mechanisms for the effects of vitamin d : the presence of vitamin d receptors on pancreatic  cells , vitamin d - activating 1hydroxylase is expressed in pancreatic  cells , the vitamin d response element in the insulin gene due to the presence of vitamin d receptors in skeletal muscle   and the fact that 1,25(oh)d increases the transcription of insulin receptor genes   and also suppresses the renin gene , reducing hyperglycemic - induced increases in renin levels in pancreatic  cells .
blockade of renin - angiotensin activity has been proposed as a novel target for the treatment of diabetes .
the protective effects of vitamin d on obesity may be due to well known effects of vitamin d such as its anti - inflammatory properties , its effects on calcium and phosphorus metabolism and its regulation of the insulin receptor gene .
it seems that vitamin d increases the calcium content of cells , in turn leading to the increased transport of glucose into the muscle .
vitamin d also regulates nuclear ppar ( peroxisome proliferative activated receptor ) , which has an important role in insulin sensitivity .
it would be useful though to undertake further studies to discover more about the mechanism and the effect of vitamin d on both alpha and islet beta - cell functions and also on the mechanisms that determine insulin resistance .
aerobic training and vitamin d supplementation contribute to increases in the metabolic rate by improving cardiovascular capacity and suppressing pro - inflammatory cytokine production , in addition to increasing the release of nitric oxide and the consumption of free fatty acids , thus enhancing insulin sensitivity .
in general , inadequate vitamin d may promote greater adiposity through other metabolic effects , such as the regulation of pth and the modulation of adipogenesis .
moderate to severe vitamin d deficiency leads to increased pth , which may promote an increase in free intracellular calcium in adipocytes and thereby enhance lipogenesis .
the question that needs to be answered is whether or not vitamin d deficiency prevents the beneficial effects of exercise especially after menopause .
recently , numerous interventional and epidemiological studies have shown that calcium and vitamin d consumption effectively reduce abdominal fat and metabolic syndrome risk .
it has been reported that vitamin d plays an essential role in the homeostasis control of calcium .
vitamin d was closely connected to the metabolic mechanism of calcium , and increased blood calcium concentration consequently affects blood lipid concentration and insulin resistance .
one limitation of the present study was the lack of a group of ovariectomized animals treated with hormone replacement therapy to confirm the positive effects of at and vit d on plasma lipid profiles and insulin resistance .
further studies should be conducted to more accurately elucidate and characterize the metabolic energy imposed by this aerobic training protocol and vitamin d supplementation in rats and humans .
in conclusion , aerobic training and vitamin d supplementation successfully attenuate some metabolic syndrome profiles and might prevent cardiovascular risk factors caused by ovariectomy . from the clinical point of view
, this study suggests that vit d supplementation is a very useful strategy to prevent obesity and menopause - associated metabolic disturbances .
 purposethe purpose of this study was to determine the interaction effects of aerobic exercise training and vitamin d supplementation on indices of obesity and plasma lipid profiles in ovariectomized ( ovx ) rats.methodsforty female wistar rats were divided into 5 groups : aerobic training ( 3 days / week for 8 weeks ; at ; n = 8) , aerobic training and vitamin d supplementation ( ovx + at + vit d ; n = 8) , vitamin d supplementation ( ovx + vit d ; n = 8) , ovariectomized control 
 ( ovx + c , n = 8) and sham ( n = 8) . 
 after blood sampling , visceral fat was taken from the abdominal cavity and weighed immediately . 
 data was statistically analyzed by one - way anova and repeated measure anova tests with a 0.05 significance level.resultsbody weight , visceral fat , bmi and food intake decreased significantly in ovx + at + vit d ( p < 0.001 ) ; whereas these variables increased significantly in ovx + c ( p < 0.001 ) and sham ( p < 0.023 ) groups . at the end of two - months of follow - up , we observed significant differences in tc , tg , hdl - c , ldl - c , glucose , insulin , and homa - ir in all groups.conclusionit seems that aerobic training with vitamin d , due to the involvement of muscle mass and exposure to dynamic pressure on the bones and muscles , increased energy expenditure , stimulated insulin exudation and glucose homeostasis , decreased insulin resistance and improved the lipid profile in ovariectomized rats . 
INTRODUCTION
METHODS
Animal care
Body weight and food intake
Vitamin D supplementation
Protocol for exercise training
Groups and surgery
Blood analysis
Assessment of concentrations of plasma lipids
Statistical analysis
RESULTS
DISCUSSION
CONCLUSIONS