Nonalcoholic fatty liver disease (NAFLD) is considered a hepatic manifestation of metabolic syndrome. In this study, we investigated histological and biochemical changes in NAFLD and the gene expression involving
Nonalcoholic fatty liver disease (NAFLD) is presently well recognized as a hepatic manifestation of metabolic syndrome [
Many NAFLD studies have used animal models such as the rat model of methionine and choline deficiency- (MCD-) induced NASH [
The OLETF rat is a well-established model of metabolic syndrome, characterized by abdominal obesity, insulin resistance, hypertension, and hyperlipidemia [
In previous studies, OLETF rats at 32 weeks of age were used to investigate the effect of rosiglitazone on hepatic steatosis [
The aim of this study was to establish the time course of the development of NAFLD in the OLETF rat model. We therefore investigated the histological and biochemical changes associated with NAFLD in OLETF rats up to 50 weeks of age, as well as expression of genes involved in
This study was performed in compliance with the ARRIVE guidelines for research [
The experiment was initiated when the rats were 10 weeks of age. OLETF rats were killed for histological analysis at 4-week intervals up to 50 weeks of age, while LETO rats were killed only at 50 weeks of age. All rats were fasted for 12 h prior to being killed, and blood was collected from tail veins. Body weights were measured at 4-week intervals.
Serum was obtained from blood by centrifugation and stored at −70°C. Serum glucose, total cholesterol (TC), triglyceride (TG), insulin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and free fatty acid (FFA) levels were measured using an autoanalyzer (Olympus GmbH, Germany) [
Livers were fixed in 10% buffered formalin and embedded in paraffin. The severity of histological changes was assessed by hematoxylin and eosin (H&E) and Masson’s trichrome (MT) staining. Oil Red O staining was performed as previously described [
RNA was isolated from 40 mg samples of liver tissue using Qiazol reagent (QIAgen, USA). RNA concentration was measured with a NanoDrop ND-2000 UV/Vis spectrophotometer (Thermo Fisher Scientific Inc., USA), and RNA purity was determined by measuring the ratio of absorbance at 260 and 280 nm, which ranged from 1.8 to 2.0.
Total RNA was extracted from 20 mg samples of liver tissue using Qiazol reagent (QIAgen, Valencia, CA) following the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 3
Sequences of primers.
Primer | Sequences (5′ to 3′) | Size |
---|---|---|
SREBP-1c | GCT ACC GTT CCT CTA TCA ATG ACA A | 81 |
CAG ATT TAT TCA GCT TTG CCT CAG T | ||
SCD-1 | TTC TTG AGA TAC ACT CTG GTG CTC A | 97 |
GAG ATT GAA TGT TCT TGT CGT AGG G | ||
chREBP | CAG TAT GTG GCT TCG TAA CTC CTC T | 89 |
CCA GTA ATT ACC CTC CAA GAC AAC A | ||
FAS | TCC ACA GCT CTT ACA GTG AGA ATC A | 99 |
CTT CTC CAG GGT GGG GAC CAG | ||
ACC | AGA GTG AGT GCT CTC AAT TCT GTC C | 97 |
GTC CTT CTT CTT TCC CGA TAA TGT C | ||
GAPDH | CCT TCT CTT GTG ACA AAG TGG ACA T | 96 |
CGT GGG TAG AGT CAT ACT GGA ACA T |
SREBP: sterol regulatory element-binding protein; SCD: stearoyl-CoA; chREBP: carbohydrate response element-binding protein; FAS: fatty acid synthase; ACC: acetyl-CoA carboxylase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.
All data are presented as means ± SD, except for the Oil Red O staining data obtained with the image analysis system, which are presented as mean ± SE. Comparisons between groups were made using one-way analysis of variance followed by a post hoc Tukey’s test using Statistical Program for the Social Sciences (SPSS software version 17.0 (SPSS, Inc., USA)). Side-to-side comparisons within the same group were made using Student’s
Body weight was significantly higher in the OLETF rats than in the LETO rats until 30 weeks of age and fell below that of the LETO rats by 42 weeks (Figure
Biochemical analyses of serum obtained from the LETO and OLETF rats at 50 weeks of age.
LETO | OLETF | |
---|---|---|
Insulin (uIU/mL) | 6.16 ± 1.61 | 6.32 ± 0.39 |
Alanine aminotransferase (U/L) | 44.20 ± 4.66 | 43.60 ± 9.02 |
Aspartate aminotransferase (U/L) | 74.20 ± 14.34 | 122.20 ± 8.23** |
Free fatty acid (µEq/L) | 712.00 ± 25.11 | 840.80 ± 83.05* |
OLETF: Otsuka Long-Evans Tokushima fatty rats; LETO: Long-Evans Tokushima rats. All data are expressed as means ± SE.
HOMA-IR level of the LETO and OLETF rats at 30 and 50 weeks of age.
30 weeks of age | 50 weeks of ages | |
---|---|---|
LETO | 14.74 ± 6.73 | 10.70 ± 2.46 |
OLETF | 27.87 ± 5.60 | 15.20 ± 1.12* |
HOMA-IR: homeostasis model assessment of insulin resistance; HOMA-IR
Animal characteristics and biochemical analyses. Body weights ((a),
The progression of NAFLD was confirmed in the liver tissue of OLETF rats by H&E (4-week intervals), Oil Red O, and MT staining (at 20-week intervals). Microvesicular steatosis was observed in OLETF rats at 18 weeks, and micro- and macrovesicular steatosis and hepatocyte ballooning became evident from 22–38 weeks of age and declined after 42 weeks (Figure
Histological parameter.
Hepatic Oil Red O (% staining) | ||
---|---|---|
Age (week) | LETO | OLETF |
10 | 0.17 ± 0.07 | 0.99 ± 0.36* |
30 | 0.44 ± 0.09 | 17.61 ± 0.81** |
50 | 0.24 ± 0.13 | 0.36 ± 0.13 |
OLETF: Otsuka Long-Evans Tokushima fatty rats; LETO: Long-Evans Tokushima rats. All data are expressed as means ± SE.
Liver histology by hematoxylin and eosin (H&E) staining. Histological changes in liver tissue observed by H&E staining in OLETF rats at 4-week intervals up to 50 weeks of age (magnification, ×200).
Liver histology. Representative images of Oil Red O (a) and Masson’s trichrome (MT) staining (b) of the livers at 10, 30, and 50 weeks of age (magnification, ×400).
Levels of SREBP-1c, SCD-1, chREBP, FAS, and ACC mRNA in the liver of rats were measured by real-time PCR (OLETF, at 20-week intervals; LETO, at 50 weeks of age) (Figure
Expression of genes related to lipogenesis. Levels of SREBP-1c, SCD-1, chREBP, FAS, and ACC mRNA in the liver at 10, 30, and 50 weeks of age. ((a)–(e)) Sterol regulatory element-binding protein (SREBP) 1c, stearoyl-CoA- (SCD-) 1, carbohydrate response element-binding protein (chREBP), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC) mRNA levels, respectively. Mean values were obtained from livers of 3 separate animals. Polymerase chain reactions performed in duplicate. The transcript levels normalized relative to GAPDH expression. Data are expressed as means ± SD. *
In this study, we examined the development with age of NAFLD in OLETF rats. We demonstrated that hepatic steatosis was evident in OLETF rats in the absence of dietary manipulation between 22 and 38 weeks of age and declined after 42 weeks. Fibrosis and collagen deposition in perivenular regions did not appear within 50 weeks. Further, the expression of genes related to
OLETF rats spontaneously develop insulin resistance, type 2 diabetes [
We found that OLETF rats had higher serum levels of glucose, triglyceride, and total cholesterol than LETO rats (Figures
As expected, hepatic micro- and macrovesicular steatosis and hepatocyte ballooning were not observed in the OLETF rats before 14 weeks of age; they appeared at 18 weeks and were very evident between 22 and 38 weeks. Surprisingly, however, they disappeared by 42 weeks (Figure
Fibrosis and collagen deposition in the perivenular regions of the livers of OLETF rats did not increase over time (Figure
We showed that the expression of genes related to
This study has several limitations. First, the mechanism responsible for the reduction of hepatic steatosis after 42 weeks of age in the OLETF rats is unclear. Second, our study involved only a small number of animals. Finally, time course data of food intake in the OLETF rats is limited. However, we suppose that the body weight loss in the OLETF rats is not due to reduction of food intake, because previous studies have shown that the food intake of OLETF rat does not decline until at least 38 weeks of age [
In summary, we have demonstrated that hepatic steatosis in OLETF rats in the absence of dietary manipulation increases from 22–38 weeks of age and declines after 42 weeks. We recommend the use of OLETF rats at 22–38 weeks of age as animal models of hepatic steatosis, but these rats are not useful as models of NASH in the absence of dietary manipulation. Our findings provide insight into the use of OLETF rats as models of NAFLD and define the optimal time for observing hepatic steatosis. We believe these data will contribute to the study of NAFLD using OLETF rats.
Nonalcoholic fatty liver disease
Otsuka Long-Evans Tokushima fatty
Long-Evans Tokushima Otsuka
Nonalcoholic steatohepatitis
Methionine and choline deficiency
Carbon tetrachloride
Cholecystokinin
Sterol regulatory element-binding protein
Stearoyl-CoA
Carbohydrate response element-binding protein
Fatty acid synthase
Acetyl-CoA carboxylase
Total cholesterol
Triglyceride
Alanine aminotransferase
Aspartate aminotransferase
Free fatty acids
Hematoxylin and eosin
Masson’s trichrome.
The authors declare that they have no conflict of interests.