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World J Gastroenterol. 2010 May 14; 16(18): 2223–2226.
Published online 2010 May 14. doi:  10.3748/wjg.v16.i18.2223
PMCID: PMC2868214

Understanding mechanisms of the pathogenesis of nonalcoholic fatty liver disease


A central issue in the understanding of the pathogenesis of nonalcoholic fatty liver disease is the problem of the underlying mechanisms which are not fully understood. In the setting of excessive central adiposity, insulin resistance is the major underlying cause of fat accumulation in hepatocytes. Because of the difficulties with human trials, several animal models have been developed for this purpose mainly characterized as follows: genetically disturbed or murine fatty liver, methionine-choline deficient diet fed or murine steatohepatitis, and high-fat or sucrose diet fed models. Although these animal models have provided useful information, none of them accurately reflect genetic, metabolic and biochemical characteristics of the human disease.

Keywords: Nonalcoholic fatty liver disease, Pathogenesis, Rat, Rodents


Nonalcoholic fatty liver disease (NAFLD) is one of the most prevalent forms of chronic liver disease[1-4]. The reported prevalence of NAFLD in developed countries is 30% and 13% in adults and children, respectively. It is likely that in type 2 diabetes mellitus, NAFLD is one of the consequences of obesity. The prevalence of NAFLD in the obese population is nearly 95%. Factors contributing to NAFLD include sedentary life style, and increased consumption of foods with high-fat and high fructose corn syrup content. A cafeteria style diet which includes both high fat and fructose corn syrup is the leading cause of obesity in the population. Furthermore, in the setting of excessive central adiposity, insulin resistance is the major underlying cause of fat accumulation in the liver[5-11]. NAFLD is characterized by hepatic fat accumulation in hepatocytes (> 5% of liver weight).


Nonalcoholic steatohepatitis (NASH), as a subgroup of NAFLD, is characterized by chronic, progressive liver pathology which has the ability to lead to advanced fibrosis, cirrhosis, hepatocellular carcinoma, and liver-related death[1,2,4,5]. The prevalence of NASH is 3% among adults. The mechanisms underlying NASH pathogenesis are not fully understood. One of the hypotheses has been termed the “two-hit” theory[5,6]. According to this paradigm, NAFLD is a result of inappropriate fat storage or ectopic fat accumulation and the primary abnormality is most likely insulin resistance which leads to the accumulation of triglycerides (TGs) within the hepatocytes. After the first hit of steatosis, the second hit of oxidative stress leads to hepatocyte injury and inflammation.

Animal models

There are several types of animal model used for NAFLD studies, and these are mainly characterized as follows: genetically disturbed or murine fatty liver, methionine-choline deficient (MCD) diet fed mice or murine steatohepatitis model and feeding high-fat and/or sucrose diets with or without high caloric intake model[12-24]. Manipulation of the mouse genome and production of new animal models, such as leptin-deficient ob/ob mice, leptin-resistant db/db rats or knockout mice regarding a special character, have given us great opportunities for research[12-16]. However, it is not possible to extrapolate all the information gained from these animal models into knowledge on the human species as there are some fundamental differences regarding their genetics, mediators, and the mechanisms of the events. For example, although obese patients have higher circulating leptin levels, ob/ob mice exhibit complete leptin deficiency. Additionally, leptin has some immunologic functions, besides its metabolic regulatory capacity. Secondly, although insulin resistance is a universal feature of patients with NASH, the MCD model is not insulin-resistant and not obese[15]. MCD mice have increased insulin hypersensitivity, and their serum has both insulin and glucose levels lower than mice fed a standard diet.

High caloric intake models

With regard to the problems mentioned above, it is reasonable to try to find a suitable model for investigating the mechanism behind human NASH. Actually, there have been several attempts to mimic human species by feeding mice with high fat and/or sucrose diets with or without high caloric intake in order to produce obesity, insulin resistance, and NASH[16-23]. The type and amount of fat, and total daily caloric intake on these diets are very important and there has been no standardized diet reported by study groups. In this regard, Lieber et al[25] fed Sprague-Dawley rats with a high-fat, liquid diet (71% of energy from fat which included corn, olive, and safflower oil) for 3 wk and developed a steatohepatitis model in murines which resembled human NASH. These mice displayed obesity and insulin resistance together with increased hepatic tumor necrosis factor (TNF)-α, TNF-α messenger RNA, cytochrome 2E1, cytochrome 2E1 mRNA, increased oxidative stress and lipid peroxidation, fatty liver histopathology, mononuclear cell infiltration, abnormal mitochondria and increased collagen in the liver. Bruce et al[26] used a high fat diet (45% kcal from fat, 20% kcal protein, 35% kcal carbohydrate) and Tetri et al[27] fed mice with 45% calories in the chow from fat and 30% of the fat in the form of partially hydrogenated vegetable oil (28% saturated, 57% monounsaturated fatty acids, 13% polyunsaturated fatty acids). Both research groups developed mice displaying obesity, insulin resistance and NASH.

There is substantial diversity within and between mouse strains resembling phenotypic variations in human populations. C57 BL6J mice have usually been chosen by NASH researchers because of their predisposition to develop insulin resistance by means of diet and the availability of genetically manipulated mice[14,27-29]. These features of the strain are explained by a strong influence of the genetic background on the susceptibility to diet-induced obesity and insulin resistance[14].


For NASH pathogenesis, it is a prerequisite firstly to develop fatty liver which is then unusually vulnerable to various second hits or injury[5,6]. Although insulin resistance is a universal finding for both simple steatosis and NASH, only a small group of insulin-resistant patients develop NASH, even in patients with metabolic syndrome (MS). MS is the most severe form of insulin resistance and might instigate more advanced NASH. It is believed that oxidative stress and lipid peroxidation might play a central role in the transition of simple steatosis to NASH[30].

Increased production of ROS and lipid peroxidation of hepatocyte membranes and organelles promote necroinflammation, satellite cell activation and fibrosis in the liver. In this context, elevated free fatty acids (FFAs) in both circulation and the liver, mitochondrial abnormalities (dysfunction and structural abnormalities such as mega mitochondria with true crystalline inclusions), gut-derived endotoxins, ethanol secondary to gut and liver interaction, and disturbed production of adipokines should be major concerns in the development of steatohepatitis in an animal model.

Insulin resistance and peripheral lipolysis cause an increased FFA pool in the circulation[7,21,30]. This pool is one of the major sources of hepatic TGs. FFAs are also the major source of hepatic mitochondrial, peroxisomal and microsomal ROS production. It has been reported that increased hepatic and serum FFA concentrations promote hepatic and systemic insulin resistance by the activation of PKCtheta; and serine phosphorylation of insulin receptor substrates. Feldstein et al[30] reported that FFAs promote lysosomal permeabilization, release cathepsin B which is a lysosomal protease within hepatocytes, and cause hepatocyte apoptosis and injury. In addition to these aspects, increased serum concentrations of FFAs due to obesity were found to be correlated with the severity of fibrosis in patients with NASH[31]. FFAs play a major role in the transition from simple steatosis to NASH.

High-fat diet-induced obesity and insulin resistance have been reported in Wistar rats[32,33], Sprague-Dawley rats[34,35], F344 rats[36], and in Long-Evans rats[37]. Additionally, prolonged feeding periods with high fat (59% fat), such as a 3 mo duration, promoted a greater degree of insulin resistance in Wistar rats[33]. Borst and Conover induced obesity and developed an insulin-resistant animal model by feeding them for 39 d with a high-fat (50%) inclusive diet[38]. In this model, daily caloric intake was not increased, and it was even slightly less than for the normal rat chow (12.4% fat). These mice developed increased visceral and subcutaneous fat mass, insulin resistance, elevated fasting serum insulin, decreased insulin-stimulated glucose transport in skeletal muscle, increased TNF-α expression on visceral adipose tissue, and an undetectable serum TNF-α concentration. Most importantly, serum FFA concentration was not increased significantly in both mice fed high-fat diet and controls, while liver TNF-α expression was not affected. This observation is interesting, as obesity is strongly associated with increased concentration of FFAs in the circulation.

It was previously reported that a five-fold increase of plasma FFAs caused up to 100-fold increase in plasma insulin concentrations[26,30]. Thus, FFAs are more important than TNF-α for inducing insulin resistance. TNF-α expressed in visceral adipose tissue macrophages, and maybe TNF-α in muscle, appears the major cause of systemic insulin resistance in these animal models, since FFA levels were not increased. Oxidative stress is one of the most popular proposed mechanisms of hepatocellular injury in NASH in both animal experiments and human trials. It has been reported that obesity correlates with systemic oxidative stress. Obese adults with MS have a higher plasma concentration of oxidative stress biomarkers than obese adults without MS[2,6,7]. Increased ROS production has been selectively shown in adipose tissue of obese mice. There is also some indirect evidence as to the benefit of antioxidants such as vitamin E, S-adenosylmethionine and betaine, phlebotomy to remove iron, and N-acetylcysteine for treating NAFLD. However, a causal relationship or a pathogenic link between NASH and oxidative stress has so far not been established.

Hepatocyte mitochondria are the main site of β-oxidation of FFAs and adenosine triphosphate (ATP) production is one of the crucial issues in the understanding of NASH pathogenesis. It was previously reported that mitochondrial structural abnormalities, depletion of mitochondrial DNA and ATP, and mitochondrial dysfunction are characteristics of NASH patients as well as of diet-induced and animal models[39-41]. Increased oxidative stress and lipid peroxidation products are integral components of the pathway progressing to NASH from fatty liver. Specifically, rats fed a high-fat diet have been shown to have reduced electron transport chain capacity and increased oxidative stress in liver mitochondria[39,41].

Excessive fatty acids might be used as an alternative pathway to produce mitochondrial injury rather than the mitochondrial β-oxidation pathway. These possible routes of injury include the peroxisomal and microsomal oxidation systems[42]. Alternative fatty acid oxidation systems produce more hydrogen peroxide and thus may contribute to oxidant stress. Increased cytochrome P450 2E1 expression and induction have been well-established previously in both murine SH and human NASH studies.

In a very recent study, researchers developed a novel model which suggested maternal fat intake contributes toward the NAFLD progression in adult offspring; mediated through impaired hepatic mitochondrial metabolism and up-regulated hepatic lipogenesis[26]. Large-scale gene expression study, particularly a whole genome array analysis, is a very powerful technique which was used to measure the differences between the samples in this model[26]. This technique was able to measure the mRNA expressed in the tissue analyzed, assessing parameters such as relative expression of genes involved in inflammation, oxidative stress, lipogenesis, and beta-oxidation.


One of the areas for ongoing research is the understanding of how much calorie intake and composition of the diet affect the development of NASH. In conclusion, although these animal NAFLD models are already in use and further improve our knowledge, the underlying mechanisms in NAFLD pathogenesis need further research.


Peer reviewers: Dr. MH Ahmed, MD, PhD, Chemical Pathology Department, Southampton University Hospital NHS trust, Mail point 6, Level D, South Academic Block, Southampton SO16 6YD, United Kingdom; Maarten Tushuizen, MD, Department of Gastroenterology & Hepatology, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands; Dr. Mihaela Petrova, MD, PhD, Clinic of Gastroenterology, Medical Institute, Ministry of Interior, Sofia 1606, Bulgaria

S- Editor Wang YR L- Editor Logan S E- Editor Zheng XM


1. Caldwell SH, Oelsner DH, Iezzoni JC, Hespenheide EE, Battle EH, Driscoll CJ. Cryptogenic cirrhosis: clinical characterization and risk factors for underlying disease. Hepatology. 1999;29:664–669. [PubMed]
2. Poonawala A, Nair SP, Thuluvath PJ. Prevalence of obesity and diabetes in patients with cryptogenic cirrhosis: a case-control study. Hepatology. 2000;32:689–692. [PubMed]
3. Charlton M, Kasparova P, Weston S, Lindor K, Maor-Kendler Y, Wiesner RH, Rosen CB, Batts KP. Frequency of nonalcoholic steatohepatitis as a cause of advanced liver disease. Liver Transpl. 2001;7:608–614. [PubMed]
4. Ratziu V, Bonyhay L, Di Martino V, Charlotte F, Cavallaro L, Sayegh-Tainturier MH, Giral P, Grimaldi A, Opolon P, Poynard T. Survival, liver failure, and hepatocellular carcinoma in obesity-related cryptogenic cirrhosis. Hepatology. 2002;35:1485–1493. [PubMed]
5. Marchesini G, Brizi M, Morselli-Labate AM, Bianchi G, Bugianesi E, McCullough AJ, Forlani G, Melchionda N. Association of nonalcoholic fatty liver disease with insulin resistance. Am J Med. 1999;107:450–455. [PubMed]
6. Marchesini G, Brizi M, Bianchi G, Tomassetti S, Bugianesi E, Lenzi M, McCullough AJ, Natale S, Forlani G, Melchionda N. Nonalcoholic fatty liver disease: a feature of the metabolic syndrome. Diabetes. 2001;50:1844–1850. [PubMed]
7. Seppälä-Lindroos A, Vehkavaara S, Häkkinen AM, Goto T, Westerbacka J, Sovijärvi A, Halavaara J, Yki-Järvinen H. Fat accumulation in the liver is associated with defects in insulin suppression of glucose production and serum free fatty acids independent of obesity in normal men. J Clin Endocrinol Metab. 2002;87:3023–3028. [PubMed]
8. Pagano G, Pacini G, Musso G, Gambino R, Mecca F, Depetris N, Cassader M, David E, Cavallo-Perin P, Rizzetto M. Nonalcoholic steatohepatitis, insulin resistance, and metabolic syndrome: further evidence for an etiologic association. Hepatology. 2002;35:367–372. [PubMed]
9. Marchesini G, Bugianesi E, Forlani G, Cerrelli F, Lenzi M, Manini R, Natale S, Vanni E, Villanova N, Melchionda N, et al. Nonalcoholic fatty liver, steatohepatitis, and the metabolic syndrome. Hepatology. 2003;37:917–923. [PubMed]
10. Bugianesi E, Zannoni C, Vanni E, Marzocchi R, Marchesini G. Non-alcoholic fatty liver and insulin resistance: a cause-effect relationship? Dig Liver Dis. 2004;36:165–173. [PubMed]
11. Reaven G. The metabolic syndrome or the insulin resistance syndrome? Different names, different concepts, and different goals. Endocrinol Metab Clin North Am. 2004;33:283–303. [PubMed]
12. Koteish A, Diehl AM. Animal models of steatosis. Semin Liver Dis. 2001;21:89–104. [PubMed]
13. Anstee QM, Goldin RD. Mouse models in non-alcoholic fatty liver disease and steatohepatitis research. Int J Exp Pathol. 2006;87:1–16. [PubMed]
14. Haluzik M, Colombo C, Gavrilova O, Chua S, Wolf N, Chen M, Stannard B, Dietz KR, Le Roith D, Reitman ML. Genetic background (C57BL/6J versus FVB/N) strongly influences the severity of diabetes and insulin resistance in ob/ob mice. Endocrinology. 2004;145:3258–3264. [PubMed]
15. Rinella ME, Green RM. The methionine-choline deficient dietary model of steatohepatitis does not exhibit insulin resistance. J Hepatol. 2004;40:47–51. [PubMed]
16. Buettner R, Ottinger I, Schölmerich J, Bollheimer LC. Preserved direct hepatic insulin action in rats with diet-induced hepatic steatosis. Am J Physiol Endocrinol Metab. 2004;286:E828–E833. [PubMed]
17. Buettner R, Parhofer KG, Woenckhaus M, Wrede CE, Kunz-Schughart LA, Schölmerich J, Bollheimer LC. Defining high-fat-diet rat models: metabolic and molecular effects of different fat types. J Mol Endocrinol. 2006;36:485–501. [PubMed]
18. Gauthier MS, Couturier K, Latour JG, Lavoie JM. Concurrent exercise prevents high-fat-diet-induced macrovesicular hepatic steatosis. J Appl Physiol. 2003;94:2127–2134. [PubMed]
19. Jürgens H, Haass W, Castañeda TR, Schürmann A, Koebnick C, Dombrowski F, Otto B, Nawrocki AR, Scherer PE, Spranger J, et al. Consuming fructose-sweetened beverages increases body adiposity in mice. Obes Res. 2005;13:1146–1156. [PubMed]
20. Marchesini G, Ridolfi V, Nepoti V. Hepatotoxicity of fast food? Gut. 2008;57:568–570. [PubMed]
21. Milagro FI, Campión J, Martínez JA. Weight gain induced by high-fat feeding involves increased liver oxidative stress. Obesity (Silver Spring) 2006;14:1118–1123. [PubMed]
22. Mozaffarian D, Katan MB, Ascherio A, Stampfer MJ, Willett WC. Trans fatty acids and cardiovascular disease. N Engl J Med. 2006;354:1601–1613. [PubMed]
23. Almind K, Kahn CR. Genetic determinants of energy expenditure and insulin resistance in diet-induced obesity in mice. Diabetes. 2004;53:3274–3285. [PubMed]
24. Ip E, Farrell GC, Robertson G, Hall P, Kirsch R, Leclercq I. Central role of PPARalpha-dependent hepatic lipid turnover in dietary steatohepatitis in mice. Hepatology. 2003;38:123–132. [PubMed]
25. Lieber CS, Leo MA, Mak KM, Xu Y, Cao Q, Ren C, Ponomarenko A, DeCarli LM. Model of nonalcoholic steatohepatitis. Am J Clin Nutr. 2004;79:502–509. [PubMed]
26. Bruce KD, Cagampang FR, Argenton M, Zhang J, Ethirajan PL, Burdge GC, Bateman AC, Clough GF, Poston L, Hanson MA, et al. Maternal high-fat feeding primes steatohepatitis in adult mice offspring, involving mitochondrial dysfunction and altered lipogenesis gene expression. Hepatology. 2009;50:1796–1808. [PubMed]
27. 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–G995. [PubMed]
28. Hesse D, Dunn M, Heldmaier G, Klingenspor M, Rozman J. Behavioural mechanisms affecting energy regulation in mice prone or resistant to diet- induced obesity. Physiol Behav. 2010;99:370–380. [PubMed]
29. Brownlow BS, Petro A, Feinglos MN, Surwit RS. The role of motor activity in diet-induced obesity in C57BL/6J mice. Physiol Behav. 1996;60:37–41. [PubMed]
30. Feldstein AE, Werneburg NW, Canbay A, Guicciardi ME, Bronk SF, Rydzewski R, Burgart LJ, Gores GJ. Free fatty acids promote hepatic lipotoxicity by stimulating TNF-alpha expression via a lysosomal pathway. Hepatology. 2004;40:185–194. [PubMed]
31. Nehra V, Angulo P, Buchman AL, Lindor KD. Nutritional and metabolic considerations in the etiology of nonalcoholic steatohepatitis. Dig Dis Sci. 2001;46:2347–2352. [PubMed]
32. Chalkley SM, Hettiarachchi M, Chisholm DJ, Kraegen EW. Long-term high-fat feeding leads to severe insulin resistance but not diabetes in Wistar rats. Am J Physiol Endocrinol Metab. 2002;282:E1231–E1238. [PubMed]
33. Singh MK, Krisan AD, Crain AM, Collins DE, Yaspelkis BB 3rd. High-fat diet and leptin treatment alter skeletal muscle insulin-stimulated phosphatidylinositol 3-kinase activity and glucose transport. Metabolism. 2003;52:1196–1205. [PubMed]
34. Brown JL, Spicer MT, Spicer LJ. Effect of high-fat diet on body composition and hormone responses to glucose tolerance tests. Endocrine. 2002;19:327–332. [PubMed]
35. Axen KV, Dikeakos A, Sclafani A. High dietary fat promotes syndrome X in nonobese rats. J Nutr. 2003;133:2244–2249. [PubMed]
36. Yasuda K, Nishikawa W, Iwanaka N, Nakamura E, Seino Y, Tsuda K, Ishihara A. Abnormality in fibre type distribution of soleus and plantaris muscles in non-obese diabetic Goto-Kakizaki rats. Clin Exp Pharmacol Physiol. 2002;29:1001–1008. [PubMed]
37. Woods SC, Seeley RJ, Rushing PA, D'Alessio D, Tso P. A controlled high-fat diet induces an obese syndrome in rats. J Nutr. 2003;133:1081–1087. [PubMed]
38. Borst SE, Conover CF. High-fat diet induces increased tissue expression of TNF-alpha. Life Sci. 2005;77:2156–2165. [PubMed]
39. Begriche K, Igoudjil A, Pessayre D, Fromenty B. Mitochondrial dysfunction in NASH: causes, consequences and possible means to prevent it. Mitochondrion. 2006;6:1–28. [PubMed]
40. Pérez-Carreras M, Del Hoyo P, Martín MA, Rubio JC, Martín A, Castellano G, Colina F, Arenas J, Solis-Herruzo JA. Defective hepatic mitochondrial respiratory chain in patients with nonalcoholic steatohepatitis. Hepatology. 2003;38:999–1007. [PubMed]
41. Serviddio G, Bellanti F, Tamborra R, Rollo T, Romano AD, Giudetti AM, Capitanio N, Petrella A, Vendemiale G, Altomare E. Alterations of hepatic ATP homeostasis and respiratory chain during development of non-alcoholic steatohepatitis in a rodent model. Eur J Clin Invest. 2008;38:245–252. [PubMed]
42. Bradbury MW, Berk PD. Lipid metabolism in hepatic steatosis. Clin Liver Dis. 2004;8:639–671, xi. [PubMed]

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