over the last decades , obesity has become a global epidemic and an important public health problem in many countries .
this condition is largely due to excessive consumption of saturated fats and simple sugars [ 2 , 3 ] , which , associated with sedentarism , represent the modern lifestyle .
obesity is recognized as a risk factor for many disorders including type-2 diabetes and nonalcoholic fatty liver disease ( nafld ) .
nafld encompasses a spectrum of increasingly severe clinicopathological conditions ranging from fatty liver to steatohepatitis ( nash ) with or without hepatic fibrosis / cirrhosis .
recent evidence suggests that nafld is also associated with cardiovascular and chronic kidney disease   and increased risk of hepatocellular carcinoma [ 58 ] .
it has been considered that insulin resistance and hyperinsulinemia play a key role in the pathogenesis of nalfd ( first causative step ) .
excessive deposition of fat in adipocytes and muscles determines insulin resistance with subsequent accumulation of fat in the liver , which , in turn , increases the rate of mitochondrial beta - oxidation of fatty acids and ketogenesis that can promote lipid peroxidation and accumulation of reactive oxygen species ( ros ) in the hepatocytes [ 10 , 11 ] .
these compounds generate a variety of cellular stimulations with subsequent inflammatory response , which has been recognized as the causal factor of nash / fibrosis ( second causative step ) [ 12 , 13 ] . in spite of
growing knowledge , several aspects of nafld pathogenesis are still unknown . considering the difficulty in developing human studies to evaluate the influence of nutrition in the development of nafld and associated metabolic abnormalities , experimental models constitute a reliable alternative way .
different animal models of nafld / nash have been developed , but few of them replicate the entire human phenotype [ 12 , 14 ] .
these models may be classified into three basic categories : those caused by either spontaneous or induced genetic mutation ; those produced by either dietary or pharmacological manipulation ; and those involving genetic mutation and dietary or chemical challenges .
the dietary manipulations used in these last two types of models usually do not resemble human dietary pattern . in the present study , we developed a model of obesity and obesity - related nafld in nongenetically
modified wistar rats using a simple carbohydrate - rich diet , which resembles the current dietary pattern of humans , and followed the sequence of the pathophysiologic events and their clinical and metabolic consequences . in this context
, it should be noted that , in the vast majority of studies on nafld in which animal models were employed , the description of the sequence of the pathophysiologic events and their consequences have not been addressed , as their key goal is usually the evaluation of a specific aspect such as a therapeutic intervention .
furthermore , we evaluated the impact of physical training on the metabolic abnormalities associated with this disorder .
sixty male wistar rats , approximately 28 days old ( after weaning ) , were housed individually and had free access to water and rat diet .
the animals were randomly separated into the following groups : experimental group ( eg ) , fed with highly palatable diet ( see below ) during 5 ( eg5 , 6 rats ) , 10 ( eg10 , 6 rats ) , 20 ( eg20 , 6 rats ) , and 30 ( eg30 , 12 rats ) weeks , and control group ( cg ) , fed with standard rat chow during 5 ( cg5 , 6 rats ) , 10 ( cg10 , 6 rats ) , 20 ( cg20 , 6 rats ) , and 30 ( cg30 , 12 rats ) weeks . from week 25
to week 30 , 12 animals belonging to the eg30 ( 6 rats ) and cg30 ( 6 rats ) were submitted to physical training ( see below ) . at the end of each experimental period , after fasting for 10 hours , the animals were sacrificed .
the livers were immediately removed and fragments of about 1  mm thickness were fixed in 4% formaldehyde , dehydrated , immersed in xylene , and then embedded in paraffin for histology .
all experiments were approved by the ethics committee of the universidade federal de minas gerais for the care and use of laboratory animals ( cetea 53/2007 ) and were carried out in accordance with the regulations described in the committee 's guiding principles manual .
the standard rat chow ( nuvilab - cr1 nuvital - colombo , brazil ) had the following nutrient composition : protein , 22% ; fat , 4% ; carbohydrate , 42% ; minerals , 10% ; phosphorus , 0.8% ; vitamins , 1% ; fiber , 8% ; water , 12.5% .
the chemical analysis revealed that 100  g of this diet contained 309  kcal , 24.8  g of protein , 3.4  g of fat , 44.8  g of carbohydrates , 8.2  g of fixed mineral residue , and 18.8  g of dietary fiber .
the diet known as effective in inducing obesity in rats and described as highly palatable was composed of what follows : 33% of standard rat chow compacted to powder , 33% of condensed milk ( moa , nestl , brazil ) , 7% of sucrose ( refined sugar , unio , brazil ) , and 27% of water .
the condensed milk was nutritionally composed of carbohydrate , 56.7% ; fat , 8.3% ; protein , 6.7% ; water , 28.3% . according to the chemical analysis ,
100  g of dried highly palatable diet contained 339  kcal , 16.1  g of protein , 3.4  g of fat , 61  g of carbohydrates ( 18% of simple carbohydrates ) , 5.1  g of fixed mineral residue , and 14.4  g of dietary fiber .
the diet was prepared daily , weighed , fractionated in portions , and stored in the feeder for 810 hours .
the remaining food in the feeder was weighed to calculate the final amount of ingested food .
the water content of the drinking bottles was renewed daily . on a weekly basis ,
the body weight , thoracic circumference ( tc ) ( measured between the foreleg and hind leg ) , and nasoanal length were measured .
body mass index ( bmi ) , that is , the ratio between body weight ( g ) and the square of body length ( cm ) , was calculated .
all animals were acclimatized to exercise on the motor - driven treadmill ( gaustec , brazil ) by running at a speed of 10  mmin at 5% inclination for 5 minutes / day , during 5 consecutive days .
after exercise familiarization , trained rats were submitted to the physical training protocol , which consisted of running sessions with gradual increase in intensity across 5 weeks , 5 days / week .
the speed and duration of the exercise bouts were increased until the rats were able to run at 25  mmin , 5% inclination , during 60 minutes / day .
the achievement of this exercise intensity ensures that a significant endurance training effect is produced . in order to ensure that all animals were subjected to the same handling stress , untrained group
was submitted to running exercise on the same days of physical training , at the same speed , but for 2 minutes only .
measurement of glucose , total cholesterol , very low - density lipoprotein- ( vldl- ) cholesterol , low - density lipoprotein ( ldl- ) cholesterol , high - density lipoprotein- ( hdl- ) cholesterol , and triglycerides was performed as recommended by the manufacturer ( bioclin , quimbasa , basic chemistry ltda , brazil ) using an autoanalyzer ( statplus 2300 , yellow spring inst , usa ) .
serum concentrations of leptin and insulin were determined by radioimmunoassay ( rat leptin ria kit , rat insulin ria kit , linco research , usa ) using a gamma - ray counter ( mor - abbot , usa ) .
the minimum detection value was 0.5  ng / ml . the determination of superoxide dismutase ( sod ) activity was adapted from dieterich et al . .
briefly , fresh liver samples were homogenized in 50  mm sodium phosphate buffer ( 1  ml , ph 7.8 , 37c ) and 1  mm of diethylenetriamine pentaacetic acid ( dtpa ) , immediately after their removal .
the reaction was initiated by addition of pyrogallol acid ( 0.2  mm / l , 37c for 3 minutes ) and the absorbance measured at 420  nm .
sod activity was calculated as u / mg protein , where 1  u of the enzyme was defined as the amount required to inhibit the oxidation of pyrogallol by 50% .
catalase ( cat ) activity was measured in the supernatant of liver homogenate as described by nelson and kiesow .
briefly , 0.04  ml of h2o2 , 0.06  ml of liver homogenate , and 1.9  ml of potassium phosphate buffer ( 50  mm , ph 7.0 ) were mixed to give a final concentration of 6  mm of h2o2 .
the decomposition of h2o2 by cat was evaluated by the change in absorbance at 240  nm .
cat activity was expressed as mmol of h2o2 decomposed per minute per milligram of protein .
this procedure was adopted to avoid the possibility of interference in the activity of glutathione peroxidase , once the necessary cofactors were not present in the reaction medium .
histological sections were prepared from the material embedded in paraffin and stained with hematoxylin - eosin .
the criteria established by brunt et al . were used to describe the histological lesions . according to these criteria , macrovesicular steatosis
is quantified based on the percentage of involved hepatocytes ( 0 = absent ; 1 < 33% ; 2 = 3366% ; 3 > 66% ) , and its zonal distribution and the presence of microvesicular steatosis are noted ; hepatocellular ballooning is evaluated for zonal location , and the estimate of its severity ( mild , marked ) is based on the numbers of hepatocytes showing this abnormality .
hepatic expression of malondialdehyde ( mda ) , leptin , and the leptin receptor ob - r was evaluated by immunohistochemistry in the animals sacrificed at weeks 20 and 30 . from paraffin
embedded tissues , sections on salinized slides ( 4  mm ) were collected , deparaffinized , and hydrated . for immunohistochemistry ,
antigen reaction with ethylenediaminetetraacetic acid ( edta ) at ph 8.0 , no steamer for 30 minutes at 98c , was conducted , followed by tris hcl ph 7.6 washing .
the whole procedure was performed using polymer detection system kit ( novolink polymer detection system , novocastra , usa ) .
the primary antibodies used were anti - mda monoclonal antibody ( 1f83 ) ( cosmo bio co. , ltd . ,
japan ) diluted in 0.5  ml ; anti - ob ( a-20 ) sc-84 ; and anti - ob - r ( h-300 ) sc-8325 ( santa cruz biotechnology inc . , usa ) at a dilution of 1  :  250 and 1  :  100 , respectively .
data are presented as frequencies and percentages , mean  standard deviation ( sd ) , and median and interquartile range ( iqr ) . for each quantitative response 's variables
, we developed linear regression models in which all variables with p value 0.25 at univariate analysis would be included initially .
however , due to the high level of correlation between the explanatory covariates , we opted to adjust the final model with the following covariates : group , physical training , variation in bmi ( bmi ) , and variation in the amount of ingested calories ( kcal ) .
the adequacy of the models was assessed by analysis of the residues . for the categorical variables ,
logistic regression models were developed , with inclusion of the variables that showed on the univariate analysis a p value 0.25 , and also clinical significance .
the results of the anthropometric parameters , lipid and glucose profile , hormones levels , and antioxidant enzymes activity , as well the results of their comparative analyses between eg and cg along the time of follow - up , are described in tables 1 , 2 , and 3 .
insulin ( figure 1(a ) ) and leptin ( figure 1(b ) ) serum levels varied inversely over time in the eg .
liver histology was normal ( figure 2(a ) ) in the cg in all times of the experiment .
steatosis and hepatocellular ballooning ( figures 2(b ) and 2(c ) ) were observed only in the eg , from week 10 .
steatosis was macro- and microvacuolar , located predominantly in zone 3 of the liver acinus .
the intensity of the macrovacuolar steatosis varied from mild ( involvement of less than 33% of the hepatocytes ) to severe ( involvement of more than 66% of the hepatocytes ) regardless of the time of the experiment .
ballooning was localized in zones 2 and 3 of the acinus , ranging from mild to marked and mismatched with the time of experiment .
the reaction for identifying mda ( figure 2(d ) ) was positive and intense , of cytoplasmic localization in zone 3 of the hepatic acinus , around the central vein , in eg20 and eg30 .
leptin ( figure 2(e ) ) was identified in the cytoplasm especially in zone 3 of the acinus , in eg20 and eg30 . in cg rats ,
ob - r was expressed as a weak cytoplasmic reaction predominantly in zone 3 of the acinus , in the rats of both groups , at weeks 20 and 30 .
the comparison of the different variables between physical trained and untrained groups showed higher serum levels of hdl - cholesterol in the first group : medians 75  mg / dl and 52.2  mg / dl , respectively ( p = 0.007 ) .
no other clinical or metabolic variable was significantly different between the groups after the physical training . 

table 4 shows the results of the final linear and logistic regressions models . in summary ,
blood glucose levels were 49% higher in eg rats than in cg rats , and the rats studied for 10 and 30 weeks had an increase of 49% and 65% , respectively , in serum glucose compared to those studied for 5 weeks .
total cholesterol was 19.2  mg / dl higher in the eg in comparison with the cg .
rats undergoing physical training showed an average of 27.1  mg / dl increase in hdl - cholesterol than those that did not exercise ; and each increase of 1 unit in kcal intake caused an average reduction of 0.03  mg / dl in hdl - cholesterol levels .
regarding ldl - cholesterol , there was an average increase of 60.2  mg / dl for each increase of 1 unit in bmi .
the first , composed by the time ( categorical ) and groups of rats , showed that the eg20 and eg30 had , respectively , lower insulin values of 83% and 89% compared to eg5 .
furthermore , the animals of eg had an average insulin levels increased by 123% compared to the cg . the second model , including time ( quantitative form ) , groups of rats , and kcal intake , showed that , for each increase of 1 unit in time , the average value of insulin decreased by 7% and , for each increase of 1 unit in kcal intake , the average value of insulin increased by 0.2% .
the eg rats had an average insulin level increased by 100% compared to those of the cg .
in eg20 and eg30 , the leptin values were 33% and 40% higher , respectively , compared to the rats followed for 5 weeks .
the eg had a mean value of leptin increased by 267% compared to the cg , and for every increase of 1 unit in bmi the average value of leptin increased by 124.6% .
the amount of sod was 24% lower in the animals followed for 30 weeks in relation to those studied for 5 weeks . in the eg , the mean values of sod were 11% lower compared to the cg ; and , for each increase of 1 unit in bmi , the mean sod values decreased by 54% .
the rats studied for 20 weeks presented an average of 2.4 less cat units than those studied for 5 weeks , and in the eg an average of 1.8 less units of cat relative to the cg was observed .
concerning the histological findings , it was found that , for each increase of 1 unit in the tc , the chance of expressing ballooning and steatosis increased by 50% .
this study demonstrates that a diet with high amount of simple carbohydrates , which resembles the current human dietary pattern , was able to induce obesity - related nafld , here characterized histologically by hepatic steatosis and hepatocyte ballooning , clinically by increased tc and bmi associated with hyperleptinemia , and metabolically by hyperglycemia , hyperinsulinemia ( with subsequent insulin return to baseline levels ) , hypertriglyceridemia , increased serum levels of vldl - cholesterol , depletion of antioxidants liver enzymes , and increased levels of mda , an oxidative stress marker .
furthermore , rats that underwent physical training showed a significant increase in hdl - cholesterol in comparison to those that did not exercise .
high - fat and methionine choline - deficient diets are widely used to produce hepatic steatosis and nash in experimental animals [ 12 , 14 , 2126 ] . however
, these diets do not reflect the usual dietary pattern of humans regarding their composition .
diets high in both saturated fat and simple carbohydrate have also been commonly used in genetically modified or wild - type animals in experimental models of nafld [ 2735 ] .
animal models in which nafld was induced by simple carbohydrate - rich diets ( usually fructose ) are less numerous , and in most of them only hepatic steatosis was observed [ 28 , 3644 ] .
although the animal models that combine naturally occurring or induced genetic mutations associated with dietary or chemical challenges resemble the histopathology and pathophysiology of human nafld more closely , the dietary challenge is usually performed by high - fat or methionine choline - deficient diets [ 12 , 14 , 4547 ] .
although each of these models is valuable , they fail to address key aspects of the process in humans . for example , few humans have diets that are deficient in methionine and choline .
moreover , rodents exposed to methionine- and choline - deficient diets are not obese ; rather , they lose weight and become more insulin - sensitive . on the other hand , the diet used in our investigation was balanced in terms of its content in proteins , lipids , carbohydrates , vitamins , and minerals , in addition to being highly palatable , normocaloric , and fiber containing .
furthermore , it was administered in solid consistency , as pellets , during a relatively long period of time .
what has usually been described in the other animal investigations is a rapid induction of obesity due to the administration , in a short period of time , of a high - caloric high - fructose and/or high - fat diet , as liquid in troughs or via a nasogastric tube . in synthesis , we sought to feed the animals with a diet as similar as possible to a normal diet regarding its content as well as its form of administration . in our study , free access to the sucrose - rich diet and high food consumption caused obesity / abdominal obesity in the eg rats from week 10 .
obesity was associated with increased serum levels of glucose , triglycerides , vldl - cholesterol , and insulin , which are manifestations of insulin resistance [ 9 , 49 ] .
the hyperinsulinemia led to increased hepatic synthesis of fatty acids , triglyceride accumulation in the hepatocytes , with subsequent steatosis .
the de novo hepatic lipogenesis , which is aggravated by diets with higher carbohydrate content than fat , plays an important role in glucose homeostasis and development of hypertriglyceridemia and hyperinsulinemia [ 50 , 51 ] .
for example , when the amount of ingested carbohydrate exceeds the total calorie needs , the rate of de novo hepatic lipogenesis increases by 10 times . likewise , this rate increases 27 times with the ingestion of a diet with high carbohydrate content compared to low - carbohydrate diets and fasting .
a positive correlation between increase in serum levels of leptin and bmi was another finding of this study that corroborates human observations .
the hyperleptinemia may be not only a consequence of hyperphagia and obesity , but also a result of the fructose component of the diet , which is in agreement with the study by vil et al . , which demonstrated induction of hyperleptinemia by fructose . in humans ,
increased levels of leptin are observed in obese individuals and in patients with nafld / nash .
it is suggested that this increase may reflect a state of leptin resistance at central level as well in the muscles and liver [ 56 , 57 ] . in an attempt to understand the action of leptin in the liver and its possible role in the pathogenesis of nafld / nash
, we evaluated the expression of leptin and ob - r in the hepatic parenchyma and found intense leptin reaction in eg30 , whereas ob - r was observed in both groups , without difference between them .
a possible role of leptin as an inducer of hepatic mitochondrial beta - oxidation has been postulated .
huang et al . demonstrated that leptin in vivo enhances the activity of the fatty acid oxidative pathway in the liver , thus contributing to the reduction of triglycerides and vldl - cholesterol in rats without leptin resistance . on the other hand , some authors observed increased mitochondrial beta - oxidation in the liver of leptin deficient mice ( ob / ob ) with severe steatosis .
cao et al . showed that leptin , in the long term , can cause hepatic fibrosis due to the increase of the local levels of oxidative stress .
therefore , it is possible to hypothesize that leptin may play a protective role in the early stages of nafld ; and , at later stages , it may contribute to the development of fibrosis .
further studies are necessary to clarify the biological function of leptin in the normal liver and its possible role in diet - induced nafld .
early stages of nafld  were present in all liver samples of the eg from week 10 . at the final stage of the investigation , although more exuberant steatosis was expected , the pattern was similar to that observed at week 10 .
the duration of the study may not have been long enough to allow the development of more severe steatosis and the histological changes that characterize nash . as the hepatic lesions that occur in nash
are associated with the expression of proinflammatory cytokines in the liver , it is possible that their investigation could have demonstrated nash at an early stage .
it is also possible to speculate that the high levels of leptin could be exerting a protective effect .
mda , a marker of lipid peroxidation , presented exuberant expression in the eg , whereas this reaction was negative in the cg .
oxidative stress induced by lipid peroxidation is a result of oxidant / antioxidant system imbalance .
cellular stimulation by ros and the subsequent inflammatory response have been described as the  second hit  that culminate with the development of nash [ 62 , 63 ] . in this context
, we found in eg30 a reduction in the levels of the antioxidants enzymes sod and cat .
this observation suggests that during the initial phases of the experiment there was a balance between antioxidants / prooxidants constituents ; however , over time , an imbalance in favor of prooxidants was developed .
the use of diets with high amounts of simple carbohydrates induces hypertriglyceridemia resulting in reduction of the antioxidants reserves [ 64 , 65 ] .
although we observed hepatocellular ballooning denoting cell injury , one limitation of our study is the fact of not detecting nash histologically .
this was also a finding in several of the previous models in which nafld was induced by a simple carbohydrate - rich diet [ 28 , 3644 ] .
as stated above , it is possible that the time of the experiment was not long enough to enable the development of the histological characteristics of nash , which may require higher levels of ros and/or longer exposure to the offending agent , in addition to liver susceptibility probably related to genetically determined factors , such as preexisting defects in mitochondrial oxidative phosphorylation [ 66 , 67 ] . in the presence of intense and sustained production ,
ros can cause damage to cell membranes , proteins , and dna , leading to the release of proinflammatory cytokines , activation of hepatic stellate cells , fibrogenesis , and direct liver damage .
the physical training used in this study was effective in increasing hdl - cholesterol , corroborating the findings from a study in zucker rats .
on the other hand , other authors found no significant effect on hdl - cholesterol in rats or mice submitted to physical training [ 71 , 72 ] .
no other metabolic parameter suffered alteration in response to physical exercise , which could have been due , at least partially , to the time not long enough of the physical training .
in this context , 12 weeks of regular exercise reduced liver triglyceride content and serum levels of ldl - cholesterol in the kk / ta mice fed a high - sucrose diet . in humans , evidence suggests that regular exercise reduces the risk factors for nash [ 1 , 8 ] .
our study demonstrated that a diet enriched with sucrose induced obesity , insulin resistance , diabetes , oxidative stress , and subsequent hepatic steatosis and hepatocellular ballooning .
the lack of histologically evident inflammation and fibrosis in the liver parenchyma may have been due to the insufficient time of the experiment .
 the pathogenesis of nonalcoholic fatty liver disease ( nafld ) is not fully understood , and experimental models are an alternative to study this issue . 
 we investigated the effects of a simple carbohydrate - rich diet on the development of obesity - related nafld and the impact of physical training on the metabolic abnormalities associated with this disorder . 
 sixty wistar rats were randomly separated into experimental and control groups , which were fed with sucrose - enriched ( 18% simple carbohydrates ) and standard diet , respectively . at the end of each experimental period ( 5 , 10 , 20 , and 30 weeks ) , 6 animals from each group were sacrificed for blood tests and liver histology and immunohistochemistry . from weeks 25 to 30 
 , 6 animals from each group underwent physical training . 
 the experimental group animals developed obesity and nafld , characterized histopathologically by steatosis and hepatocellular ballooning , clinically by increased thoracic circumference and body mass index associated with hyperleptinemia , and metabolically by hyperglycemia , hyperinsulinemia , hypertriglyceridemia , increased levels of very low - density lipoprotein- ( vldl- ) cholesterol , depletion of the antioxidants liver enzymes superoxide dismutase and catalase , and increased hepatic levels of malondialdehyde , an oxidative stress marker . 
 rats that underwent physical training showed increased high - density lipoprotein- ( hdl- ) cholesterol levels . in conclusion , 
 a sucrose - rich diet induced obesity , insulin resistance , oxidative stress , and nafld in rats . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion
5. Conclusion