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.
Over the last decades, obesity has become a global epidemic and an important public health problem in many countries [
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 [
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 [
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. Blood samples were taken by cardiac puncture and stored at −20°C. 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. Fresh tissue samples were collected to evaluate antioxidant enzymes activity.
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. A rat belonging to the CG20 died and was excluded from all analyses.
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 (Moça, Nestlé, Brazil), 7% of sucrose (refined sugar, União, Brazil), and 27% of water [
The diet was prepared daily, weighed, fractionated in portions, and stored in the feeder for 8–10 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 (cm²), was calculated [
All animals were acclimatized to exercise on the motor-driven treadmill (Gaustec, Brazil) by running at a speed of 10 m·min−1 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 m·min−1, 5% inclination, during 60 minutes/day. The achievement of this exercise intensity ensures that a significant endurance training effect is produced [
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. [
Catalase (CAT) activity was measured in the supernatant of liver homogenate as described by Nelson and Kiesow [
Histological sections were prepared from the material embedded in paraffin and stained with hematoxylin-eosin. The histological analysis was performed simultaneously by two examiners. 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 = 33–66%; 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 98°C, 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
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
Comparison of anthropometric parameters between experimental and control groups.
Time (weeks) | Groups |
| |
---|---|---|---|
Experimental | Control | ||
ΔBMI (kg/cm2)† | |||
5 | 0.25 (±0.09) | 0.36 (±0.07) | 0.032 |
10 | 0.26 (±0.14) | 0.27 (±0.08) | 0.082 |
20 | 0.48 (±0.09) | 0.40 (±0.10) | 0.193 |
30 | 0.50 (±0.15) | 0.34 (±0.06) | 0.003 |
|
|||
ΔThoracic circumference (cm)† | |||
5 | 6.67 (±0.45) | 6.67 (±0.80) | 1.000 |
10 | 10.48 (±1.86) | 8.32 (±2.04) | 0.083 |
20 | 12.00 (±1.27) | 9.02 (±1.03) | 0.002 |
30 | 13.82 (±2.84) | 9.85 (±1.30) | 0.031 |
Comparison of biochemical parameters between experimental and control groups.
Time (weeks) | Groups |
| |
---|---|---|---|
Experimental | Control | ||
Glucose (mg/dL)† | |||
5 | 272.3 (±92.5) | 176.2 (±27.1) | 0.035 |
10 | 434.5 (±214.5) | 307.2 (±121.9) | 0.235 |
20 | 324.2 (±53.4) | 279.4 (±133.8) | 0.468 |
30 | 463.1 (±101.7) | 299.8 (±78.6) | <0.001 |
|
|||
Total cholesterol (mg/dL)† | |||
5 | 83.5 (±18.0) | 72.8 (±20.6) | 0.363 |
10 | 64.5 (±13.1) | 73.2 (±7.9) | 0.194 |
20 | 108.0 (±33.3) | 85.8 (±21.0) | 0.231 |
30 | 104.3 (±33.6) | 80.6 (±15.0) | 0.099 |
|
|||
HDL-cholesterol (mg/dL)‡ | |||
5 | 49.1 (22.9–65.3) | 39.2 (22.9–64.1) | 0.779 |
10 | 34.8 (23.0–52.8) | 42.3 (37.7–49.7) | 0.689 |
20 | 49.6 (44.0–69.5) | 57.9 (56.8–63.7) | 0.315 |
30 | 65.8 (51.8–75.3) | 64.0 (51.1–79.4) | 1.000 |
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|||
VLDL-cholesterol (mg/dL)‡ | |||
5 | 20.5 (14.3–25.9) | 13.7 (10.9–18.9) | 0.149 |
10 | 17.4 (14.1–35.7) | 17.9 (13.5–19.2) | 0.522 |
20 | 17.1 (11.8–24.3) | 11.0 (9.0–14.1) | 0.083 |
30 | 26.3 (22.1–49.8) | 11.0 (9.1–17.0) | <0.001 |
|
|||
LDL-cholesterol (mg/dL)† | |||
5 | 24.8 (±21.6) | 24.8 (±12.2) | 1.000 |
10 | 23.7 (±26.5) | 12.7 (±6.0) | 0.343 |
20 | 34.6 (±22.6) | 19.5 (±16.6) | 0.246 |
30 | 38.4 (±18.1) | 15.0 (±10.7) | 0.341 |
|
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Triglycerides (mg/dL)‡ | |||
5 | 102.5 (71.5–129.5) | 68.5 (54.5–94.3) | 0.150 |
10 | 87.0 (70.5–178.3) | 89.5 (67.3–95.8) | 0.522 |
20 | 85.5 (58.8–121.5) | 55.0 (45.0–70.5) | 0.083 |
30 | 131.5 (110.5–248.8) | 55.0 (45.3–85.0) | <0.001 |
Comparison of hormonal levels and enzyme activity between experimental and control groups.
Time (weeks) | Groups |
| |
---|---|---|---|
Experimental | Control | ||
Insulin ( |
|||
5 | 7.6 (3.8–11.1) | 1.6 (1.5–2.1) | 0.005 |
10 | 4.7 (3.4–8.2) | 1.7 (1.6–3.1) | 0.013 |
20 | 0.9 (0.6–1.1) | 0.3 (0.3–0.4) | 0.008 |
30 | 0.4 (0.3–0.8) | 0.2 (0.2–0.4) | 0.043 |
|
|||
Leptin ( |
|||
5 | 14.3 (11.7–18.9) | 6.6 (5.8–7.6) | 0.001 |
10 | 13.7 (9.6–20.9) | 4.4 (3.6–4.8) | 0.005 |
20 | 18.2 (10.3–25.9) | 5.1 (3.3–8.1) | 0.021 |
30 | 24.7 (13.1–37.0) | 2.6 (1.8–5.7) | <0.001 |
|
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Superoxide dismutase (U/mg protein)† | |||
5 | 1.5 (±0.1) | 1.6 (±0.03) | 0.090 |
10 | 1.6 (±0.02) | 1.6 (±0.1) | 1.000 |
20 | 1.6 (±0.1) | 1.5 (±0.1) | 0.288 |
30 | 0.9 (±0.3) | 1.4 (±0.1) | <0.001 |
|
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Catalase | |||
(mmol of H2O2 decomposed/minute/milligram of protein)† | |||
5 | 15.8 (±2.4) | 15.9 (±2.7) | 0.948 |
10 | 14.3 (±3.1) | 15.9 (±2.7) | 0.411 |
20 | 11.8 (±1.4) | 15.1 (±2.9) | 0.037 |
30 | 14.6 (±1.9) | 16.7 (±3.5) | 0.111 |
Levels of insulin and leptin over time. Mean and standard deviation of (a) insulin and (b) leptin serum concentrations, over time, in the experimental (EG) and control (CG) groups.
Liver histology was normal (Figure
Liver histology and immunohistochemistry. (a) Rats fed with standard diet (control group) at 30 weeks; normal histology, hematoxylin and eosin stain ×10. (b, c, d, and e) Rats fed a sucrose-rich diet (experimental group) at 30 weeks; (b) macro and micro vacuolar steatosis, and hepatocellular ballooning, hematoxylin and eosin stain, ×40; (c) macro and micro vacuolar steatosis, and hepatocellular ballooning, hematoxylin and eosin stain, ×10. (d) Intense reaction to malondialdehyde ×40; (e) reaction to leptin ×20.
The reaction for identifying MDA (Figure
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 (
Table
Linear and logistic regression models for the response variables.
Variable/model | Coefficient |
Coefficient |
|
---|---|---|---|
Glucose | |||
Constant | 5.2 | <0.001 | |
Time (weeks) | |||
5 | |||
10 | 0.4 | 1.49 (1.1; 2.0) | 0.005 |
20 | 0.3 | 1.35 (1.0; 1.8) | 0.090 |
30 | 0.5 | 1.65 (1.3; 2.1) | <0.001 |
Group | |||
EG | 0.4 | 1.49 (1.3; 1.8) | <0.001 |
CG | |||
|
|||
Total cholesterol | |||
Constant | 177.7 (139.3; 216.1) | <0.001 | |
Time (weeks) | |||
5 | |||
10 | −25.2 (−46.8; 3.6) | 0.026 | |
20 | −50.9 (−107.7; 5.9) | 0.085 | |
30 | −40.2 (−84.8; 4.4) | 0.083 | |
Group | |||
EG | 19.2 (4.4; 31.2) | 0.003 | |
CG | |||
ΔKcal | −0.1 (−0.2; −0.03) | 0.014 | |
|
|||
HDL-cholesterol | |||
Constant | 53.1 (46.7; 59.5) | <0.001 | |
Exercise | |||
Yes | 26.1 (12.9; 39.4) | <0.001 | |
No | |||
ΔKcal | −0.03 (−0.05; −0.01) | 0.011 | |
|
|||
LDL-cholesterol | |||
Constant | 2.8 (−16.3; 21.9) | 0.774 | |
ΔBMI | 60.2 (11.9; 108.6) | 0.018 | |
|
|||
Insulin (first model) | |||
Constant | 0.8 | <0.001 | |
Time (weeks) | |||
5 | |||
10 | −0.005 | 1.00 (0.67; 1.47) | 0.980 |
20 | −1.8 | 0.17 (0.11; 0.24) | <0.001 |
30 | −2.2 | 0.11 (0.07; 0.16) | <0.001 |
Group | |||
EG | 0.8 | 2.23 (0.18; 2.70) | <0.001 |
CG | |||
|
|||
Insulin (second model) | |||
Constant | 0.7 | 0.011 | |
Time (quantitative) | −0.07 | 0.93 (0.91; 0.95) | <0.001 |
Group | |||
EG | 0.7 | 2.01 (1.65; 2.45) | <0.001 |
CG | |||
ΔKcal | 0.002 | 1.002 (1.001; 1.003) | 0.004 |
|
|||
Leptin | |||
Constant | 0.8 | <0.001 | |
Time (weeks) | |||
5 | |||
10 | −0.1 | 0.90 (0.61; 1.33) | 0.450 |
20 | −0.4 | 0.67 (0.45; 0.99) | 0.047 |
30 | −0.5 | 0.60 (0.41; 0.90) | 0.004 |
Group | |||
EG | 1.3 | 3.67 (3.01; 4.46) | <0.001 |
CG | |||
ΔBMI | 2.6 | 13.46 (5.1; 35.87) | <0.001 |
|
|||
Superoxide dismutase | |||
Constant | 0.7 | <0.001 | |
Time (weeks) | |||
5 | |||
10 | −0.03 | 0.97 (0.85; 1.11) | 0.606 |
20 | 0.10 | 1.11 (0.96; 1.27) | 0.156 |
30 | −0.27 | 0.76 (0.68; 0.86) | <0.001 |
Group | |||
EG | −0.12 | 0.89 (0.82; 0.96) | 0.006 |
CG | |||
ΔIMC | −0.77 | 0.46 (0.32; 0.67) | <0.001 |
|
|||
Catalase | |||
Constant | 16.6 (15.0; 18.3) | <0.001 | |
Time (weeks) | |||
5 | |||
10 | −0.7 (−2.9; 1.6) | 0.564 | |
20 | −2.4 (−4.6; 0.2) | 0.041 | |
30 | −0.1 (−2.1; 1.8) | 0.902 | |
Group | |||
EG | −1.8 (−3.2; 0.4) | 0.016 | |
CG | |||
|
|||
Model/variable | Odds ratio (95% CI) |
| |
|
|||
Steatosis | |||
ΔTC | 1.50 (1.10; 1.90) | 0.002 | |
|
|||
Hepatocellular ballooning | |||
ΔTC | 1.50 (1.10; 1.90) | 0.002 |
EG, experimental group; CG, control group; Kcal, amount of calorie intake; TC, thoracic circumference.
Two models were adjusted for the dependent variable insulin. 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 Δkcal 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 Δkcal 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 ΔBMI 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 ΔBMI, 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 ΔTC, 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 [
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 [
A positive correlation between increase in serum levels of leptin and BMI was another finding of this study that corroborates human observations [
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
Hepatic steatosis and hepatocellular ballooning—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. In addition, genetic factors could be acting. 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 [
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 [
Exercise is considered an effective resource for controlling metabolic changes associated with obesity [
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 authors declare that there is no conflict of interests regarding the publication of this paper.
The study was supported by the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) grant (CDS463/2006).