|Year : 2017 | Volume
| Issue : 3 | Page : 141-145
Role of high-fructose diet in experimental induction of nonalcoholic steatohepatitis in rats
Assmaa M.F. Abdel-Aziz El Sheikh, Sobhy A El-Hamid Hassan, Manal M El-Batch, Soha S Zakareia
Department of Medical Biochemistry, Tanta Faculty of Medicine, Tanta, Egypt
|Date of Submission||11-Oct-2016|
|Date of Acceptance||11-Apr-2017|
|Date of Web Publication||29-Nov-2017|
Assmaa M.F. Abdel-Aziz El Sheikh
Department of Medical Biochemistry, Tanta Faculty of Medicine, Tanta, 31121
Background Nonalcoholic steatohepatitis (NASH) is the most common chronic liver disease in the Western world and has rapidly become an important cause of liver failure and hepatocellular carcinoma. Obesity is considered the primary risk factor for NASH.
Aim The aim of this study was to clarify the role of high-fructose diet in the pathogenesis of steatohepatitis.
Materials and methods This study was conducted on 40 male albino rats, equally divided into two groups. Group 1 (control) received standard caloric diet and plain drinking water. Group 2 (induced NASH group) received standard caloric diet and had free access to 70% (weight/volume) fructose-enriched drinking water for 5 weeks. All rats were weighed and killed, and then serum was collected for estimating triglycerides, total cholesterol, serum alanine aminotransferase, and serum aspartate aminotransferase levels, as well as liver tissue sampling was used for histopathologic examination and for estimation of tissue triglycerides level.
Results Compared with the control group, rats with induced NASH group showed an increased weight gain and significant hypertriglyceridemia and hypercholesterolemia, as well as significant increases in serum alanine aminotransferase and aspartate aminotransferase levels, which were associated with increased hepatic triglycerides level.
Conclusion NASH can be experimentally induced in rats using a high-fructose diet. Steatohepatitis is associated with obesity as indicated by significant increase in body weight, hypertriglyceridemia, hypercholesterolemia, and hepatic hypertriglyceridemia.
Keywords: fructose, nonalcoholic steatohepatitis, steatohepatitis, steatosis
|How to cite this article:|
El Sheikh AM, El-Hamid Hassan SA, El-Batch MM, Zakareia SS. Role of high-fructose diet in experimental induction of nonalcoholic steatohepatitis in rats. Tanta Med J 2017;45:141-5
|How to cite this URL:|
El Sheikh AM, El-Hamid Hassan SA, El-Batch MM, Zakareia SS. Role of high-fructose diet in experimental induction of nonalcoholic steatohepatitis in rats. Tanta Med J [serial online] 2017 [cited 2018 Mar 24];45:141-5. Available from: http://www.tdj.eg.net/text.asp?2017/45/3/141/219440
| Introduction|| |
Nonalcoholic steatohepatitis (NASH) is the most common chronic liver disease in the Western world and has rapidly become an important cause of liver failure and hepatocellular carcinoma . NASH represents an advanced stage and the most severe form of nonalcoholic fatty liver disease (NAFLD). In NASH, fatty liver, hepatic inflammation, and hepatocyte injury with or without fibrogenesis, are associated, and the condition may eventually lead to cirrhosis .
It is now well established that the prevalence of NAFLD in developed countries is associated with the prevalence of metabolic syndrome. In Europe, NAFLD is the underlying cause of liver damage in 40–60% patients with mild fibrosis, and in the USA, where one-third of the population is overweight, it is estimated that nine million patients have different degrees of NASH. Although there is paucity of NASH screening in Middle East countries, it has been recorded that ∼30% of Israeli population experience NASH ,.
The exact pathogenesis of NASH is still not fully understood; however, the development of NASH has been linked to a complex interaction between the environment, represented by a sedentary lifestyle and excessive intake of calories, and individual predisposition, such as type 2 diabetes mellitus, metabolic syndrome, rapid weight loss, and hypertriglyceridemia . Obesity is considered the primary risk factor for NASH; the prevalence of simple steatosis in obese patients reaches 60%, and among those, 20–25% will develop NASH. Simple (or ‘bland’) steatosis is considered a relatively benign condition owing to its extremely low likelihood to progress to cirrhosis. NASH, however, is associated with the potential to progress to the entire clinical picture of advanced liver disease. It is believed that fat accumulation in the liver generates inflammatory signals and reactive oxygen species that can amplify liver injury and stimulates fibrosis. This is similar to the picture observed in different etiologies of chronic liver injury, such as chronic hepatitis C virus or hepatitis B virus infection, where the spectrum of alterations may range from a near-normal liver to severe hepatitis and cirrhosis ,.
Patients with simple steatosis progress to NASH according to the ‘two-hit’ theory. Liver steatosis (the first hit) sensitizes hepatocytes to the second hits leading to hepatocyte damage, inflammation, and fibrosis. These second insults may result from increased oxidative stress and lipid peroxidation, mitochondrial dysfunction, cytokine/adipokine imbalance, lipotoxicity of free fatty acids, hepatic accumulation of cholesterol and lipopolysaccharide derived from the gut, and the activation of innate immunity ,.
NASH progression results from parallel events originating from the liver as well as from the adipose tissue, the gut, and the gastrointestinal tract. Thus, dysfunction of the adipose tissue through enhanced flow of free fatty acids and release of adipocytokines generate proinflammatory signals that underlie NASH progression. Additional ‘extrahepatic hits’ include dietary factors including high-fructose diet and gastrointestinal hormones. Within the liver, hepatocyte autophagy and apoptosis, endoplasmic reticulum stress, and oxidative stress are key contributors to hepatocellular injury. The steatosis-induced oxidative stress promotes cell death through the activation of stress-related signaling pathways such as c-Jun N-terminal kinase or p38 mitogen-activated protein kinase. Reactive oxygen species can stimulate Kupffer cells to release proinflammatory and profibrogenic cytokines, which induce activation of hepatic stellate cells ,. In addition, lipotoxic mediators and danger signals activate Kupffer cells which initiate and perpetuate the inflammatory response by releasing inflammatory mediators that contribute to inflammatory cell recruitment and development of fibrosis ,,.
| Materials and methods|| |
This study was conducted on 40 male albino rats of ∼100–140 g body weight. During this study, animals were housed in wire mesh cages, kept at a room temperature of 22–24°C, and were exposed to 12/12 h light–dark cycle.
After 1-week adaptation period, rats were randomly equally divided into two groups: group 1 was designated as control group and received standard caloric diet (consisting of 59.7% carbohydrates, 10.6% fat, and 27.3% protein) and had free access to plain water. Group 2 (NASH-induced group) received standard caloric diet and had free access to water containing 70% (w/v) fructose for 5 weeks to induce NASH . d-Fructose was supplied by Sigma-Aldrich Co. LLC (St. Louis, MO, USA). The fructose-enriched drinking water was changed twice a week.
All experiments were carried out according to the guidelines of the ethical committee of Tanta University, Faculty of Medicine.
- At the end of the experimental period (after 5 weeks) and following 16 h of fasting, blood was collected into dry sterile centrifuge tubes by heart puncture; the rats were then killed. The blood was allowed to clot at room temperature and then was centrifuged at 1000g for 20 min at 4°C; serum was separated for determination of the following:
- Serum alanine aminotransferase and aspartate aminotransferase by spectrophotometric method, as monitors for liver functions .
- Serum triglycerides (TGs) and total cholesterol .
- Liver was removed and washed with ice-cold saline to remove extraneous materials and was divided into two specimens. The first part was used for the assessment of hepatic TGs by spectrophotometric method according to the procedure of Allain et al. . Bradford method was used for quantitative estimation of proteins in liver tissue homogenates . The second part was used for the histopathological examination of liver tissue specimens to confirm the presence of NASH.
| Results|| |
This current study has been conducted to evaluate the role of high-fructose diet in the induction of NASH in rats.
This study has been conducted on two groups of white albino rats: group 1 consisted of 20 rats that received standard caloric diet and plain drinking water. Group 2 consisted of 20 rats that received standard caloric diet and had free access to 70% (weight/volume) fructose-enriched drinking water for 5 weeks.
Histopathological examination revealed the presence of steatohepatitis in fructose-fed group, indicating a role of increased fructose intake in initiating obesity that is associated with steatohepatitis ([Figure 2]), compared with normal hepatic tissue with no inflammatory changes or steatosis in the control group ([Figure 1]).
|Figure 2 Micrograph from group 2 (nonalcoholic steatohepatitis-induced group) showing small fat vacuoles filling hepatocytes cytoplasm (marked microvesicular steatosis), with nuclei pushed to the periphery, with thickened portal tracts, and mild to moderate inflammatory lymphocytic cellular infiltration (hematoxylin and eosin, ×400).|
Click here to view
|Figure 1 Micrograph from group 1 (control group) showing normal hepatocytes with a well-preserved cytoplasm and well-defined nuclei, with no steatosis or lymphocytic infiltration (hematoxylin and eosin, ×400).|
Click here to view
The presence of obesity has been confirmed by significant increase in body weight in fructose-fed rats when compared with control rats. At the beginning of the experiment, the mean body weight of all rats was 112.7 g. After 5 weeks of the experiment, the mean body weight of the control group was 154.25 g showing a percent increase of almost 36.9%, whereas rats of fructose-induced NASH showed a mean body weight of 270.75±20.6 g, with a percent increase of almost 140.2%, which was statistically significantly higher than that of the control group (t=20.42, P<0.001) ([Table 1]).
|Table 1 Comparison regarding the final body weights (g) and percent increase in mean body weight between the studied groups|
Click here to view
Moreover, the results of our study revealed that rats with induced NASH showed hypertriglyceridemia and hypercholesterolemia, which were consistent with the presence of hepatic hypertriglyceridemia. There was statistically significant increase in serum TG levels (t=5.61, P<0.001), serum cholesterol levels (t=2.64, P<0.005), and liver tissue TG levels (t=7.87, P<0.001) in NASH-induced group when compared with control group ([Table 2]).
|Table 2 Comparison between both the studied groups regarding all the studied parameters|
Click here to view
A statistically significant increase in both serum aspartate aminotransferase and alanine aminotransferase levels was noted in NASH-induced group when compared with the control group (t=10.16 and 12.61, respectively, P<0.001).
Thus, it can be concluded that NASH can be induced in rats with excess fructose intake in diet. Fructose in the diet causes obesity and fatty liver disease that commonly progress to NASH. This is typically associated with hypertriglyceridemia, hypercholesterolemia, and hepatic hypertriglyceridemia.
| Discussion|| |
NAFLD is the most common hepatic alteration in both affluent and developing countries . The most severe form of this disease is NASH, which affects 20–30% of adults and is characterized by inflammatory infiltration and hepatocellular damage with or without fibrosis .
In this current study, NASH has been induced in 20 male albino rats by keeping those rats on 70% fructose intake for 5 weeks. Fructose-induced obesity has been recorded in all rats, and the percent increase in the mean body weight was reported to be double that of the control rats. Histopathological examination of liver specimens of those rats showed severe changes, with the predominant lesions being marked microvesicular steatosis (small fat vacuoles filling hepatocytes cytoplasm, with nuclei pushed to the periphery), with thickened portal tracts, and moderate to marked inflammatory lymphocytic cellular infiltration. Meanwhile, hypertriglyceridemia and hypercholesterolemia were also reported in the sera of NASH-induced group.
An increasing body of evidence indicates that fructose in the diet causes obesity and fatty liver disease that commonly progress to NASH. Previous studies in rodents have demonstrated various histological alterations of liver tissue after fructose consumption including focal inflammation and microvascular and macrovascular steatosis in the periportal region ,. Tetri et al.  have showed that increased fructose consumption in American lifestyle-induced obesity mice models was associated with adverse alteration of plasma lipid profiles. In those mice models, fructose was administered in the form of high-fructose corn syrup in amounts relevant to that consumed by some Americans who consume ‘fast food’ or ‘cafeteria food’.
In humans, consumption of fructose-rich diet in the form of soft drinks and other carbohydrate-sweetened beverages has been linked to increased prevalence of obesity, type 2 diabetes, and fatty liver ,.
In this study, an elevated level of hepatic TGs has been detected in liver tissue of NASH-induced rats when compared with the control group. This finding is consistent with those reported by Koliwad et al.  and Yamguchi et al.  who were able to detect the accumulation of TGs in obese mice developing hepatic steatosis.
Increased accumulation of fat has been attributed to increased hepatic de-novo lipogenesis, inhibition of fatty acid β-oxidation, impaired TG clearance, and reduced VLD export .
The major pathway of fructose metabolism involves the formation of fructose-1-P, under the effect of fructokinase, which is subsequently splitted to glyceraldehyde and dihydroxyacetone phosphate which both are converted to glyceraldehyde-3-P and ultimately converted to pyruvate and then acetyl-CoA, thus bypassing the regulatory mechanisms imposed on phosphofructokinase-1. Excess acetyl-CoA will be forwarded for de-novo synthesis of fatty acids and cholesterol .
De-novo lipogenesis has been explained by Koo et al.  on the basis that fructose administration induces the carbohydrate response element binding protein and acts synergistically with sterol response element binding protein, where both proteins increase the expression of lipogenic genes, including those encoding acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturatase enzymes. Meanwhile, Choli and Diehl  reported that hepatic acyl-CoA diacylglycerol acyltransferase enzyme that catalyzes the final step in TG synthesis is often activated in states of energy excess such as fructose-induced obesity.After fatty acids are converted into TGs by esterification, TGs can be exported from the liver by very low density lipoprotein particles, which are formed by the incorporation of TGs into apolipoprotein B (apoB). The degradation of apoB is dramatically reduced when the supply of fatty acids (and TG biosynthesis) is increased; thus, the apoB level is increased in fructose metabolism .
| Conclusion|| |
NASH can be experimentally induced in rats using a high-fructose diet. Steatohepatitis is associated with obesity as indicated by significant increase in body weight, hypertriglyceridemia, hypercholesterolemia, and hepatic hypertriglyceridemia.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Williams CD, Stengel J, Asike MI, Torres DM, Shaw J, Contreras M et al.
Prevalence of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis among a largely middle-aged population utilizing ultrasound and liver biopsy: a prospective study. Gastroenterology 2011; 140:124–131.
Garcia-Monzon C, Vargas-Castrillon J, Porrero JL, Alonso MT, Bonachia O, Castillo MJ et al.
Prevalence and risk factors for biopsy-proven non-alcoholic fatty liver disease and non-alcoholic steatohepatitis in a prospective cohort of adult patients with gallstones. Liver Int 2015; 35:1983–1991.
Rau M, Weiss J, Geier A. Non-alcoholic fatty liver disease (NAFLD). Dtsch Med Wochenschr 2015; 140:1051–1055.
Ono M, Okamoto N, Saibara T. The latest idea in NAFLD/NASH pathogenesis. Clin J Gastroenterol 2010; 3:263–270.
Marra F, Lotersztajn S. Pathophysiology of NASH: perspectives for a targeted treatment. Curr Pharm Des 2013; 19:5250–5269.
Schattenberg JM, Schuppan D. Nonalcoholic steatohepatitis: the therapeutic challenge of a global epidemic. Curr Opin Lipidol 2011; 22:479–488.
Cusi K. Role of obesity and lipotoxicity in the development of nonalcoholic steatohepatitis: pathophysiology and clinical implications. Gastroenterology 2012; 142:711–725.e6.
Kucera O, Cervinkova Z. Experimental models of non-alcoholic fatty liver disease in rats. World J Gastroenterol 2014; 20:8364–8376.
Sanches SCL, Ramalho LNZ, Augusto MJ, da Silva DM, Ramalho FS. Nonalcoholic steatohepatitis: a search for factual animal models. BioMed Res Int 2015; 2015:574832.
Parola M, Marra F. Adipokines and redox signaling: impact on fatty liver disease. Antioxid Redox Signal 2011; 15:461–483.
Fuchs CD, Claudel T, Kumari P, Haemmerle G, Pollheimer MJ, Stojakovic T et al.
Absence of adipose triglyceride lipase protects from hepatic endoplasmic reticulum stress in mice. Hepatology 2012; 56:270–280.
Takahashi Y, Soejima Y, Fukusato T. Animal models of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. World J Gastroenterol 2012; 18:2300–2308.
Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am J Clin Pathol 1957; 28:56–63.
Allain CC, Poon LS, Chan CS, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clin Chem 1974; 20:470–475.
Kruger NJ. The Bradford method for protein quantitation. Methods Mol Biol 1994; 32:9–15.
Sanchez-Lozada LG, Mu W, Roncal C, Sautin YY, Abdelmalek M, Reungjui S et al.
Comparison of free fructose and glucose to sucrose in the ability to cause fatty liver. Eur J Nutr 2010; 49:1–9.
Koteish A, Diehl AM. Animal models of steatosis. Semin Liver Dis 2001; 21:89–104.
Tetri LH, Basaranoglu M, Brunt EM, Yerian LM, Neuschwander-Tetri BA. Severe NAFLD with hepatic necroinflammatory changes in mice fed trans fats and a high-fructose corn syrup equivalent. Am J Physiol Gastrointest Liver Physiol 2008; 295:G987–995.
Malik VS, Popkin BM, Bray GA, Després JP, Willett WC, Hu FB. Sugar-sweetened beverages and risk of metabolic syndrome and type 2 diabetes: a meta-analysis. Diabetes Care 2010; 33:2477–2483.
Dhingra R, Sullivan L, Jacques PF, Wang TJ, Fox CS, Meigs JB et al.
Soft drink consumption and risk of developing cardiometabolic risk factors and the metabolic syndrome in middle-aged adults in the community. Circulation 2007; 116:480–488.
Koliwad SK, Streeper RS, Monetti M, Cornelissen I, Chan L, Terayama K et al.
DGAT1-dependent triacylglycerol storage by macrophages protects mice from diet-induced insulin resistance and inflammation. J Clin Invest 2010; 120:756–767.
Yamaguchi K, Yang L, McCall S, Huang J, Yu XX, Pandey SK et al.
Inhibiting triglyceride synthesis improves hepatic steatosis but exacerbates liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis. Hepatology 2007; 45:1366–1374.
Nomura K, Yamanouchi T. The role of fructose-enriched diets in mechanisms of nonalcoholic fatty liver disease. J Nutr Biochem 2012; 23:203–208.
Devlin TM. Textbook of biochemistry: with clinical correlations. 18th ed. Hoboken, NJ: John Wiley & Sons; 2011.
Uyeda K, Repa JJ. Carbohydrate response element binding protein, ChREBP, a transcription factor coupling hepatic glucose utilization and lipid synthesis. Cell Metab 2006; 4:107–110.
Choi SS, Diehl AM. Hepatic triglyceride synthesis and nonalcoholic fatty liver disease. Curr Opin Lipidol 2008; 19:295–300.
Kawano Y, Cohen D. Mechanisms of hepatic triglyceride accumulation in non-alcoholic fatty liver disease. J Gastroenterol 2013; 48:434–441.
[Figure 1], [Figure 2]
[Table 1], [Table 2]