Model of nonalcoholic steatohepatitis

¹ From the Section of Liver Disease and Nutrition, Bronx VA Medical Center and Mt Sinai School of Medicine, New York.

² Presented in part at the American Association for the Study of Liver Disease meeting in Boston (1-5 November 2002) and published in abstract form (Hepatology 2002;36:402A).

³ Supported by NIH grant AA11115, the Department of Veterans Affairs, and the Kingsbridge Research Foundation.

⁴ Reprints not available. Address correspondence to CS Lieber, VA Medical Center (151-2), 130 West Kingsbridge Road, Bronx, NY 10468. E-mail: liebercs{at}aol.com.

See corresponding editorial on page 350.

ABSTRACT  
Background: Obesity and diabetes are frequently associated with nonalcoholic steatohepatitis (NASH), but studies have been hampered by the absence of a suitable experimental model.

Objective: Our objective was to create a rat model of NASH.

Design: Sprague-Dawley rats were fed a high-fat, liquid diet (71% of energy from fat, 11% from carbohydrates, 18% from protein) or the standard Lieber-DeCarli diet (35% of energy from fat, 47% from carbohydrates, 18% from protein). The diets were given ad libitum or as two-thirds of the amount consumed ad libitum.

Results: Rats fed the high-fat diet ad libitum for 3 wk developed panlobular steatosis, whereas those fed the standard diet had few fat droplets. Accordingly, total lipid concentrations with the high-fat and standard diets were 129.9 ± 9.1 ( Conclusion: This rat model reproduces the key features of human NASH and provides a realistic experimental model for elucidating its treatment.

Key Words: Model of nonalcoholic steatohepatitis • microsomes • cytochrome P4502E1 • tumor necrosis factor • collagen • 4-hydroxynonenal • insulin resistance • high-fat diet • dietary restriction • oxidative stress • mitochondria

INTRODUCTION  
Obesity and diabetes are common in our aging population and are frequently associated with nonalcoholic fatty liver disease, which includes nonalcoholic fatty liver and nonalcoholic steatohepatitis (NASH). Nonalcoholic fatty liver is usually considered benign, but NASH is increasingly recognized as a precursor to more severe liver disease and sometimes evolves into "cryptogenic" cirrhosis (1). In the general population, the prevalence of nonalcoholic fatty liver disease and NASH averages 20% and 2-3%, respectively (2), which makes these conditions the most common liver diseases in the United States. Fibrosis may or may not be present in persons with these diseases. No therapy for NASH has been proven to be clearly effective (3-6). Indeed, the study of the pathogenic or therapeutic factors involved in NASH has been hampered by the absence of a suitable experimental model: the models available either lack one of the pathogenic factors, such as cytochrome P4502E1 (CYP2E1) induction (7); require rats to be treated for a long time (up to 1 y) (8); use rodents with a genetic defect (9); or involve feeding rats a diet lacking choline and methionine (10), which creates a nutritional deficiency that is not common in patients with NASH but to which rodents are selectively sensitive.

The aim of the present study was to produce a practical and accurate experimental model of NASH by designing a diet that contained all essential nutrients in adequate amounts and that was palatable enough to be consumed by rats in sufficient quantities to lead to increased body weight. We hoped that this model would reproduce the key features of NASH in humans.

MATERIALS AND METHODS  
Animals and diets
Male Sprague-Dawley rats, which were purchased from Charles River Laboratories (Wilmington, MA), were individually housed and fed either a standard liquid diet with 35% of energy derived from fat, 18% from protein, and 47% from carbohydrates (11) or a high-fat liquid diet (12) with 71% of energy derived from fat, 11% from carbohydrates, and 18% from protein. The diets were purchased from Dyets Inc (Bethlehem, PA). The overall compositions of the liquid diets are shown in Table 1. The standard diet has the same fat content as the average "normal" US diet (13, 14), an amount recommended as "healthy" by the Institute of Medicine (15). The rats were fed the diets either ad libitum (22 rats in each diet group) or in an amount restricted to two-thirds of the amount spontaneously consumed (at least 12 rats in each group). They were killed in the fed state either by exsanguination (under light pentobarbital anesthesia) or by decapitation after 3 wk. All animal procedures were carried out in accordance with the Institutional Guidelines.

View this table:
TABLE 1. . Composition of the liquid diets¹

 
Organelle preparation
Livers were homogenized and centrifuged at 8700 x g and 40 °C for 10 min to obtain the mitochondrial pellet. The postmitochondrial supernatant fluid was recentrifuged at 100 000 x g and 4 °C for 1 h to separate the microsomes from the cytosol (16). Aliquots of the homogenates and the microsomes were frozen in liquid nitrogen and stored at -80 °C.

Measurement of CYP2E1, tumor necrosis factor , collagen, and their messenger RNAs
For the measurement of CYP2E1 protein, microsomal proteins (15 µg) were separated by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (17) and electrically transferred to nitrocellulose membranes (18). The Western blots were reacted with rabbit anti-hamster P4502E1 immunoglobulin G, which was produced in our laboratory, and immunostained by using goat anti-rabbit immunoglobulin G conjugated with alkaline phosphatase. The blots were incubated with Immun-Star chemiluminescent substrate and enhancer (Bio-Rad Laboratories, Hercules, CA). The nitrocellulose membranes were then exposed to X-ray film and quantified by using the Evaluating Image Analysis Systems MCID (Imaging Research Inc, St Catherines, Canada). CYP2E1 was expressed in arbitrary units normalized with a standard rat microsomal preparation containing CYP2E1. Total RNA was extracted from the livers by using an RNeasy Mini Kit (Qiagen, Valencia, CA), and CYP2E1 messenger RNA (mRNA) was measured by Northern blot analysis according to Maniatis et al (19). A complementary DNA probe for human CYP2E1 (Oxford Biochemical Research Inc, Oxford, MI) or for ß-actin (American Type Culture Collection, Manassas, VA) was labeled with [³²P]dCTP by using a random priming DNA labeling kit (Amersham, Arlington Heights, IL).

Hepatic tumor necrosis factor (TNF-) was measured by using the Quantikine Rat enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis), and TNF- mRNA was measured by using the Quantikine mRNA Base and Probes and Calibrator kits (R&D Systems) after extraction of total RNA (see above). Collagen type 1 was measured by using an enzyme-linked immunosorbent assay according to Rennard et al (20) and Moshage et al (21), and 1(I) procollagen mRNA was measured by Northern blot hybridization (19) with a complementary DNA probe for rat 1(I) procollagen (American Type Culture Collection) after total RNA extraction (see above).

Amounts of the various mRNAs were quantified by measuring the intensity of the bands on X-ray film as described above for CYP2E1. The data were normalized with ß-actin and expressed in arbitrary units. Protein was measured with the BCA Protein Assay kit (Pierce, Rockford, IL).

Oxidative stress and other chemical measurements
In the liver, oxidative stress was assessed by the concentration of 4-hydroxynonenal, which was measured by using gas chromatography-mass spectrometry according to van Kuijk et al (22) as described before (23). Measurement of total lipids was carried out according to Amenta (24).

Plasma concentrations of alanine aminotransferase and aspartate aminotransferase were measured at 30 °C by using Sigma kits (Sigma Diagnostic, St Louis), and insulin concentrations were measured by using a rat insulin radioimmunoassay kit at 4 °C (Linco Research Inc, St Charles, MO).

Histopathology
Formalin-fixed and paraffin-embedded livers were processed routinely for hematoxylin and eosin, chromotrope aniline blue, and Sirius red staining. Lobular inflammatory activity was determined according to Nouchi et al (25) as 1) focal collections of mononuclear inflammatory cells, 2) diffuse infiltrates of mononuclear inflammatory cells, 3) focal collections of polymorphonuclear cells in addition to mononuclear cell infiltrates, or 4) diffuse infiltrates of polymorphonuclear cells in the parenchymal area. These criteria are modifications of those used by Knodell et al (26) in patients. Tissues were also processed for electron microscopy with standard techniques as previously published (27).

Statistical analysis
Results are expressed as means ± SEMs and were analyzed by using Student's t test (for the data in Table 2) or one-way analysis of variance followed by the Bonferroni multiple comparisons test (for all other analyses). P < 0.05 was considered statistically significant. All statistical analyses were performed with the use of INSTAT version 3 (Graph Pad Software, San Diego).

View this table:
TABLE 2. Energy consumption, body weight, and liver weight of rats fed the standard or the high-fat diet at libitum¹

 

RESULTS  
Effects of standard and high-fat diets given ad libitum
There were no significant differences between the 2 diet groups in total calories consumed and starting and final body weight as analyzed by Student's t test (Table 2). The weight gain achieved with these 2 liquid diets fed ad libitum was greater than that observed with a nonpurified stock diet (28). The rats that were fed the standard diet for 3 wk developed modest steatosis (Figure 1 A), whereas steatosis was much more striking in the rats that were fed the high-fat diet (Figure 1B). This higher steatosis in the high-fat diet group was confirmed by total lipid concentrations, which were 66.7 ± 4.6 and 129.9 ± 9.1 mg/g liver in the standard and high-fat diet groups, respectively (P < 0.001). Inflammation was also more prominent with the high-fat diet (Figure 1D) than with the standard diet (Figure 1C). This was corroborated by morphologic analysis of the abundance of inflammatory cells (2.6 ± 0.3 and 1.4 ± 0.2 arbitrary units in the high-fat and standard diet groups, respectively; P < 0.005). The greater inflammation in the high-fat diet group was accompanied by significantly higher concentrations of hepatic TNF- (Figure 2) and its mRNA (477.9 ± 13.3 and 841.0 ± 31.2 amol/mg liver in the standard and high-fat diet groups, respectively; P < 0.001).

View larger version (158K):
FIGURE 1.. Hepatic pathology in rats fed liquid diets ad libitum. The livers of rats fed the standard diet showed an accumulation of glycogen and minimal fat (A) and few inflammatory cells (C). The livers of the rats fed the high-fat diet showed pronounced hepatic steatosis (B) and abundant mononuclear inflammatory cells (D). The liver samples were stained with hematoxylin and eosin, and the magnification is x95.

 

View larger version (33K):
FIGURE 2.. Mean (± SEM) hepatic concentrations of tumor necrosis factor (TNF-). TNF- concentrations were significantly higher in the rats fed the high-fat diet than in those fed the standard diet (P < 0.001) even when the diets were given in restricted amounts (P < 0.001). However, restriction diminished the effect of the high-fat diet, and TNF- concentrations were significantly lower in the rats fed the high-fat diet in restricted amounts than in the rats fed the same diet ad libitum (P < 0.001).

 
In the rats fed the high-fat diet, electron microscopy showed abnormal mitochondria with degenerative changes, including rarefied matrix and loss of cristae (Figure 3 A). The higher number of abnormal mitochondria in the high-fat diet group than in the standard diet group was confirmed by morphometry, which showed frequencies of abnormal mitochondria of 60.0 ± 13.8% and 15.0 ± 7.5% in the high-fat and standard diet groups, respectively (P < 0.05). Herniated mitochondria were also observed in the rats fed the high-fat diet (data not shown).

View larger version (101K):
FIGURE 3.. Electron microscopy of hepatic mitochondria. Mitochondria in the rats fed the high-fat diet ad libitum (A) showed degenerative changes with rarefied matrix and loss of cristae (arrows). These changes were prominent in most of the mitochondria in the rats fed the high-fat diet but were rare in the mitochondria in the rats fed the standard diet ad libitum (B). The magnification is x25 000.

 
Compared with the standard diet, the high-fat diet also resulted in significantly higher hepatic concentrations of CYP2E1, as measured on Western blots (1.31 ± 0.08 compared with 0.80 ± 0.07 arbitrary units; P < 0.001). These higher concentrations were associated with significantly higher concentrations of CYP2E1 mRNA (Figure 4). As expected, the CYP2E1 induction was associated with oxidative stress, as indicated by the significantly higher hepatic concentrations of 4-hydroxynonenal in the high-fat diet group than in the standard diet group (Figure 5).

View larger version (36K):
FIGURE 4.. Mean (± SEM) hepatic concentrations of CYP2E1 messenger RNA (mRNA). The significantly higher hepatic concentrations of CYP2E1 protein in the rats fed the high-fat diet than in those fed the standard diet (see Results) were associated with significantly higher mRNA concentrations (P < 0.001). Dietary restriction of the high-fat diet attenuated this difference, and CYP2E1 mRNA concentrations were significantly lower in the rats fed the high-fat diet in restricted amounts than in those given the same diet ad libitum (P < 0.001). With the standard diet, there was no significant difference between the rats fed ad libitum and those fed a restricted diet.

 

View larger version (31K):
FIGURE 5.. Mean (± SEM) hepatic concentrations of 4-hydroxynonenal (4-HNE). This variable of oxidative stress was significantly higher with the high-fat diet than with the standard diet whether the diets were fed ad libitum (P < 0.001) or in restricted amounts (P < 0.001).

 
Compared with the rats fed the standard diet, those fed the high-fat diet had significantly higher concentrations of collagen type 1 (917.8 ± 53.2 compared with 620.7 ± 57.3 pg/mg protein; P < 0.01) and 1(I) procollagen mRNA (Figure 6). The rats fed the high-fat diet also had significantly higher plasma insulin concentrations (Figure 7), which reflected insulin resistance. There were no significant differences between the standard and high-fat diet groups in plasma concentrations of alanine aminotransferase or aspartate aminotransferase (data not shown).

View larger version (38K):
FIGURE 6.. Mean (± SEM) hepatic concentrations of 1(I) procollagen messenger RNA (mRNA). 1(I) Procollagen mRNA concentrations in the rats fed the high-fat diet ad libitum were double those in the rats fed the standard diet ad libitum (P < 0.001). Dietary restriction eliminated this difference between the diets (NS). 1(I) Procollagen mRNA concentrations were significantly lower in the rats fed the high-fat diet in a restricted amount than in those fed the same diet ad libitum (P < 0.001).

 

View larger version (31K):
FIGURE 7.. Mean (± SEM) plasma insulin concentrations. Plasma insulin concentrations were significantly higher in the rats fed the high-fat diet ad libitum than in those fed the standard diet ad libitum (P < 0.01). Plasma insulin concentrations were significantly lower in the rats fed the high-fat diet in a restricted amount than in those fed the same diet ad libitum (P < 0.01).

 
Effects of dietary restriction
As expected, compared with ad libitum feeding, dietary restriction led to lower gains in body weight: 4.5 ± 0.1 compared with 8.4 ± 1.2 g/d (P < 0.001) with the standard diet and 4.7 ± 0.1 compared with 8.2 ± 1.1 g/d (P < 0.001) with the high-fat diet. Dietary restriction also attenuated the hepatopathology (Figure 8): only modest fat accumulation and inflammation occurred with the restricted high-fat diet (Figure 8B), whereas florid lesions developed when the same diet was fed ad libitum (panels B and D in Figure 1 and insets in Figure 8). With the restricted standard diet, liver histology was normal (Figure 8A).

View larger version (139K):
FIGURE 8.. Dietary restriction and liver histology. With the restricted standard diet, livers were normal (A), as also illustrated in the corresponding inset at higher magnification. With the restricted high-fat diet, there was only modest fat accumulation and inflammation (B) compared with that observed with the high-fat diet fed ad libitum (see Figure 1, B and D). The liver samples were stained with hematoxylin and eosin. The magnification in the main panels is x54, and the magnification in the insets is x96.

 
Consistent with the lower inflammation, TNF- concentrations in the high-fat diet group were not as high when the diets were restricted as when they were given ad libitum (Figure 2). Dietary restriction produced a similar trend in TNF- mRNA concentrations, which were lower when the diets were restricted than when they were given ad libitum [333.8 ± 31.0 compared with 477.9 ± 13.3 amol/mg liver (NS) with the standard diet and 718.8 ± 59.5 compared with 841.0 ± 31.2 amol/mg liver (NS) with the high-fat diet]. No significant differences in aspartate aminotransferase and alanine aminotransferase concentrations were observed between the ad libitum and the restricted diets (data not shown).

There were fewer abnormal mitochondria with the restricted high-fat diet (frequency of 18.0 ± 11.4%) than with the ad libitum high-fat diet (frequency of 60.0 ± 13.8%; P < 0.05). No abnormal mitochondria were seen with the restricted standard diet.

Similarly, CYP2E1 induction with the high-fat diet was lower when the diet was restricted than when it was given ad libitum (see above; 0.98 ± 0.08 compared with 1.31 ± 0.08 arbitary units; P < 0.05), and the same effect was seen for CYP2E1 mRNA concentrations (Figure 4). As expected, the lower induction of CYP2E1 with dietary restriction was associated with lower 4-hydroxynonenal concentrations for both diets (Figure 5), but a significant difference remained between the high-fat and standard diets even when both were restricted: lower 4-hydroxynonenal concentrations were observed with the standard diet.

1(I) Procollagen mRNA concentrations with the high-fat diet were also lower when the diet was restricted than when it was given ad libitum (Figure 6). There was a similar trend for collagen type 1 concentrations, which were lower when the diets were restricted than when they were given ad libitum [538.9 ± 58.8 compared with 620.7 ± 57.3 pg/mg protein (NS) with the standard diet and 718.8 ± 59.5 compared with 917.8 ± 53.2 pg/mg protein (NS) with the high-fat diet]. In addition, the plasma insulin concentrations of the rats fed the restricted high-fat diet were significantly lower than those of the rats fed the high-fat diet ad libitum but were comparable with those of the rats fed the standard diet ad libitum or in a restricted amount (Figure 7).

DISCUSSION  
Progress in the understanding and treatment of NASH has been hampered by the lack of a practical experimental model that reproduces the key features of the disease. This gap has been filled in the present study. By feeding rats a high-fat liquid diet ad libitum, we reproduced the typical hepatic lesions of NASH, namely, steatosis (Figure 1B), inflammation (Figure 1D), and early fibrosis, as indicated by a significant increase in collagen type 1 (see Results) and in 1(I) procollagen mRNA (Figure 6). The model also reproduces the most probable pathogenic factors. Indeed, although the pathogenesis of NASH has not yet been fully elucidated, a popular mechanism is the "two-hit" theory (12), in which the first hit is the accumulation of fatty acids in the liver due to several causes (such as obesity). One major promoting factor of this first hit is insulin resistance, which is present in most patients with NASH (29). The second hit is the peroxidation of these fatty acids because of the oxidative stress produced by different factors, such as CYP2E1 induction (30). CYP2E1 is the key enzyme of the microsomal ethanol oxidizing system, and the activity of CYP2E1 increases as part of the induction of the microsomal ethanol oxidizing system with chronic alcohol consumption (31). CYP2E1 has been shown to play a key role in the pathogenesis of alcoholic liver injury, including alcoholic steatohepatitis, because of the oxidative stress it generates (32, 33). Its pathogenic role in NASH has been recognized by a number of experts (1, 34-36). Indeed, CYP2E1 concentrations increase not only in experimental animals (8) but also in men (37, 38). CYP2E1 concentrations are invariably elevated in the liver of patients with NASH (37) because fatty acids (which increase in obesity) and ketones (which increase in diabetes) are also substrates for CYP2E1 (32); their excess upregulates CYP2E1. The resulting oxidative stress and liver injury are exacerbated by a diet low in carbohydrates and rich in fat, including unsaturated lipids, which promote CYP2E1 induction (39, 40). The diet of obese subjects is typically high in saturated fat, but it is of interest that hepatic fatty acid analyses in NASH patients indicate a significant accumulation not only of saturated fatty acids but also of unsaturated fatty acids in subjects with morbid obesity (41). In the present study, the diet rich in unsaturated fat and low in carbohydrates may have contributed to the rapid development of the pathologic changes of NASH within 3 wk. It is likely that more time may be needed for NASH to develop with diets containing less unsaturated fatty acids and more carbohydrates.

The model also reproduces mitochondrial lesions, and mitochondrial dysfunction contributes to oxidative stress (42). Note that many features are common to nonalcoholic fatty liver and NASH, but NASH alone is associated with mitochondrial structural defects (42). Oxidative stress causes various types of functional and structural damage and commonly increases TNF- production in NASH (43). Obese patients with NASH also have enhanced expression of TNF- mRNA, whereas obese patients without NASH do not (44). TNF- increases in our model as well (Figure 2), and this proinflammatory cytokine probably contributes to the histologic inflammation (Figure 1D) and the steatosis, as shown in alcoholic steatohepatitis (45). TNF- concentrations increased less after the restricted than after the ad libitum high-fat diet, probably because this increase in TNF- is part of the inflammatory response, which decreases strikingly after dietary fat restriction, as illustrated in Figure 8.

In our model there was no significant increase in alanine aminotransferase concentrations, a finding that is also not uncommon in the human disease, for which a significant increase in alanine aminotransferase concentrations has been reported in less than one-third of patients (46). As with human NASH, histologic inflammation was generally present in our rat experimental model. Moreover, it was recently reported that a low normal alanine aminotransferase value does not guarantee freedom from underlying steatohepatitis, even when there is advanced fibrosis (47). In any event, taken together, the present data show that the 2 hits postulated for the pathogenesis of NASH (ie, significant hepatic steatosis and oxidative stress), as well as insulin resistance, were reproduced in the experimental model of NASH described here.

The presence and magnitude of the oxidative stress was also corroborated by increased lipid peroxidation, as shown by the appearance of corresponding breakdown products such as 4-hydroxynonenal. Hydroxynonenal is known to stimulate collagen production (48), as also reflected in the present study (Figure 5).

The availability of this new experimental model should facilitate further elucidation of the pathogenesis of NASH and the establishment of effective treatment of NASH. One such application is illustrated by the results obtained with dietary restriction, which has been shown previously to promote health, such as by delaying aging (49). The present study shows that most key features of the NASH produced by the high-fat diet were significantly attenuated by dietary restriction but that the pathology was not fully abolished (Figure 8B). By contrast, feeding the same restricted amount of the standard diet fully normalized the morphology of the liver (Figure 8A) and much of its biochemistry. Thus, future studies using the liquid diet model of NASH described here will not only be of interest for further pathogenic studies of this disorder, but they may also shed some light on desirable dietary therapies. Indeed, dietary approaches to treat and prevent NASH and obesity are still the subject of debate. The prevailing view that a low-fat, high-carbohydrate diet is the most appropriate diet is now being challenged by the rising popularity of a high-fat, high-protein, and low-carbohydrate (Atkins-type) diet, and it is presently not clear which diet is preferable. Recent reports showed that an Atkins-type diet had some success in relatively short-term weight-loss studies (50). However, concern remains about the long-term effects, not only in terms of sustained body weight, but also regarding negative cardiovascular side effects of a high-fat diet. Such side effects were not observed in another relatively short-term study (51), but the question whether a long-term high-fat diet leads to adverse cardiovascular effects remains. In addition, the present study also showed that possible negative effects of a high-fat diet on the liver should not be ignored. Obviously, long-term clinical trials are now needed, and it might be desirable that they be preceded by experimental studies. To that effect, variants of our present model may be useful. Indeed, our totally synthetic liquid diet provides the versatility needed to establish an optimal mix of fat, protein, and carbohydrates for achieving the desired goal while avoiding unwarranted hepatic and other side effects. In any event, the present study not only shows that a high-fat diet with 71% of energy derived from fat is more deleterious to the liver than is a normal diet (with 35% of energy from fat), but it also illustrates that even when restricted, the high-fat diet produces some undesirable liver changes that are not present with equivalent amounts of the standard diet.

The key features of human NASH were successfully reproduced in rats by feeding them a high-fat, low-carbohydrate Lieber-DeCarli liquid diet ad libitum for 3 wk. This high-fat diet was more deleterious to the liver than was a calorically equivalent diet with normal fat and carbohydrate contents. Severe hepatic changes were prevented with both of the diets when their intake was restricted, but with the high-fat diet, some pathology nevertheless developed, albeit less than that observed when the diet was fed ad libitum. By contrast, the livers were normal when the standard diet was restricted. The techniques described here provide a useful tool for further studies to develop the best diet for preventing NASH.

ACKNOWLEDGMENTS  
The skillful technical assistance of L Shoichet is gratefully acknowledged.

CSL contributed to the design of the experiment, analysis of the data, and the writing of the manuscript. MAL contributed to the collection and analysis of the data and to the writing of the manuscript. KMM, QC, and AP contributed to the collection and analysis of the data, and YX, CR, and LMD contributed to the collection of the data. There were no financial or other conflicts of interest to disclose for any of the authors.

REFERENCES  

1. James OFW, Day CP. Non-alcoholic steatohepatitis (NASH): a disease of emerging identity and importance. J Hepatol 1998;29:495-501.
2. Yu AS, Keeffe EB. Nonalcoholic fatty liver disease. Rev Gastroenterol Disord 2002;2:11-9.
3. Lee RG, Keeffe EB. Non-alcoholic fatty liver: causes and complications. In: Bircher J, Benhamou JP, McIntyre M, et al, eds. Oxford textbook of clinical hepatology. 2nd ed. Oxford, United Kingdom: Oxford University Press, 1999:1251-7.
4. Kumar KS, Malet PF. Nonalcoholic steatohepatitis. Mayo Clin Proc 2000;75:733-9.
5. Matteoni CA, Younossi ZM, Gramich T, Boparai N, Liu YC, McCullough AJ. Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity. Gastroenterology 1999;116:1413-9.
6. Fong DG, Nehra V, Lindor KD, Buchman AL. Metabolic and nutritional considerations in non-alcoholic fatty liver. Hepatology 2000;32:3-10.
7. Enriquez A, Leclercq I, Farrell GC, Robertson G. Altered expression of hepatic CYP2E1 and CYP4A in obese, diabetic ob/ob mice, and fa/fa Zucker rats. Biochem Biophys Res Commun 1999;255:300-6.
8. Raucy JL, Lasker JM, Kramer JC, Salazar DE, Lieber CS, Corcoran GB. Induction of P45OIIE1 in the obese rat. Mol Pharmacol 1991;39:275-80.
9. Irizar A, Barnett CR, Flatt PR, Ioannides C. Defective expression of cytochrome P450 proteins in the liver of the genetically obese Zucker rats. Eur J Pharmacol 1995;293:385-93.
10. Weltman MD, Farrell GC, Liddle C. Increased hepatocyte CYP2E1 expression in a rat nutritional model of hepatic steatosis with inflammation. Gastroenterology 1996;111:1645-53.
11. Lieber CS, DeCarli LM. Animal models of chronic ethanol toxicity. In: Packer L, ed. Methods in enzymology: oxygen radical in biological systems. Vol 233. Orlando, FL: Academic Press, Inc, 1994:585-94.
12. Lieber CS, Leo MA, Mak KM, et al. A model of non-alcoholic fatty liver disease (NAFLD), including steatohepatitis (NASH), in rats fed a high fat Lieber-DeCarli diet. Hepatology 2002;36:402A.
13. Taubes G. The soft science of dietary fat. Science 2001;291:2536-45.
14. Dietary intake of macronutrients, micronutrients, and other dietary constituents: United States, 1988-94. Vital Health Stat 11 2002;245.
15. Couzin J. IOM panel weighs in on diet and health. Science 2002;297:1788-9.
16. Lieber CS, DeCarli LM. Effect of drug administration on the activity of the hepatic microsomal ethanol oxidizing system. Life Sci 1970;9:267-76.
17. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-5.
18. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 1979;76:4350-4.
19. Maniatis T, Fritch EF, Sambrook J. Gel electrophoresis. In: Maniatis T, Fritch EF, Sambrook J, eds. Molecular cloning: a laboratory manual. 1st ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1982:161-75.
20. Rennard SI, Berg R, Martin GR, Foidart JM, Robey PG. Enzyme-linked immunoassay (ELISA) for connective tissue components. Anal Biochem 1980;104:205-14.
21. Moshage H, Casini A, Lieber CS. Acetaldehyde selectively stimulates collagen production in cultured rat liver fat-storing cells but not in hepatocytes. Hepatology 1990;12:511-8.
22. van Kuijk FJGM, Siakotos AN, Fong LG, Stephens RJ, Thomas DW. Quantitative measurement of 4-hydroxyalkenals in oxidized low-density lipoprotein by gas chromatography-mass spectrometry. Anal Biochem 1995;224:420-4.
23. Lieber CS, Leo MA, Aleynik S, Aleynik M, DeCarli LM. Polyenylphosphatidylcholine decreases alcohol-induced oxidative stress in the baboon. Alcohol Clin Exp Res 1997;21:375-9.
24. Amenta JS. A rapid chemical method for quantification of lipids separated by thin-layer chromatography. J Lipid Res 1964;5:270-2.
25. Nouchi T, Worner TM, Sato S, Lieber CS. Serum procollagen type III N-terminal peptides and laminin P1 peptide in alcoholic liver disease. Alcohol Clin Exp Res 1987;11:287-91.
26. Knodell RG, Ishak KG, Black WC, et al. Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Hepatology 1981;1:431-5.
27. Leo MA, Kim CI, Lowe N, Lieber CS. Interaction of ethanol with ß-carotene: delayed blood clearance and enhanced hepatotoxicity. Hepatology 1992;15:883-91.
28. Charles River Laboratories. Life. Science, Products. Services. Wilmington, MA: Charles River Laboratories, 2002:6-10.
29. Chitturi S, Abeygunasekera S, Farrell GC, et al. NASH and insulin resistance: insulin hypersecretion and specific association with the insulin resistance syndrome. Hepatology 2002;35:373-9.
30. Angulo P, Lindor KD. Insulin resistance and mitochondrial abnormalities in NASH: a cool look into a burning issue. Gastroenterology 2001;120:1281-4.
31. Lieber CS, DeCarli LM. Ethanol oxidation by hepatic microsomes: adaptive increase after ethanol feeding. Science 1968;162:917-8.
32. Lieber CS. Cytochrome P-4502E1: its physiological and pathological role. Physiol Rev 1997;77:517-44.
33. Lieber CS, ed. Medical and nutritional complications of alcoholism: mechanisms and management. New York: Plenum Press, 1992:579.
34. Chalasani N, Gorski JC, Asghar MS, et al. Hepatic cytochrome P450 2E1 activity in nondiabetic patients with nonalcoholic steatohepatitis. Hepatology 2003;37:544-50.
35. Harrison SA, Kadakia S, Lang KA, Schenker S. Nonalcoholic steatohepatitis: what we know in the new millennium. Am J Gastroenterol 2002;97:2714-24.
36. Leclercq IA, Farrell GC, Field J, Bell DR, Gonzalez FJ, Robertson GR. CYP2E1 and CYP4A as microsomal catalysts of lipid peroxides in murine nonalcoholic steatohepatitis. J Clin Invest 2000;105:1067-75.
37. Weltman MD, Farrell GC, Hall P, lngelman-Sundberg M, Liddle C. Hepatic cytochrome P4502E1 is increased in patients with nonalcoholic steatohepatitis. Hepatology 1998;27:128-33.
38. Emery MG, Fisher JM, Chien JY, et al. CYP2E1 activity before and after weight loss in morbidly obese subjects with nonalcoholic fatty liver disease. Hepatology 2003;38:428-35.
39. Teschke R, Moreno F, Petrides AS. Hepatic microsomal ethanol oxidizing system (MEOS): respective roles of ethanol and carbohydrates for the enhanced activity after chronic alcohol consumption. Biochem Pharmacol 1981;30:1745-51.
40. Yoo JSH, Ning SM, Pantuck CB, Pantuck EJ, Yang CS. Regulation of hepatic miscrosomal cytochrome P450IIE1 level by dietary lipids and carbohydrates in rats. J Nutr 1991;121:959-65.
41. Mavrelis PG, Ammon HV, Gleysteen JJ, Komorowski RA, Charaf UK. Hepatic free fatty acids in alcoholic liver disease and morbid obesity. Hepatology 1983;3:226-31.
42. Sanyal AJ, Campbell-Sargent C, Mirshahi F, et al. Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities. Gastroenterology 2001;120:1183-92.
43. Pessayre D, Mansouri A, Fromenty B. Nonalcoholic steatosis and steatohepatitis v. mitochondrial dysfunction in steatohepatitis. Am J Physiol 2002;282:G193-9.
44. Crespo J, Cayon A, Fernandez-Gil P, et al. Gene expression of tumor necrosis factor and TNF-receptors, p55 and p75, in nonalcoholic steatohepatitis patients. Hepatology 2001;34:1158-63.
45. Yin M, Wheeler MD, Kono H, et al. Essential role of tumor necrosis factor in alcohol-induced liver injury in mice. Gastroenterology 1999;117:942-52.
46. Diehl AM, Goodman Z, Ishak KG. Alcohollike liver disease in nonalcoholics. A clinical and histologic comparison with alcohol-induced liver injury. Gastroenterology 1988;95:1056-62.
47. Mofrad P, Conton MJ, Haque M, et al. Clinical and histologic spectrum of nonalcoholic fatty liver disease associated with normal ALT values. Hepatology 2003;37:1286-92.
48. Houglum K, Filip M, Witztun JL, Chojkier M. Malondialdehyde and 4-hydroxynonenal protein adducts in plasma and liver of rats with iron overload. J Clin Invest 1990;86:1991-8.
49. Stern JS, Gaades MD, Wheeldon CM, Borchers AT. Calorie restriction in obesity: prevention of kidney disease in rodents. J Nutr 2001;131:913S-7S.
50. Samaha FF, Iqbal N, Seshadri P, et al. A low-carbohydrate as compared with a low-fat diet in severe obesity. N Engl J Med 2003;348:2074-81.
51. Foster GD, Wyatt HR, Hill JO, et al. A randomized trial of a low-carbohydrate diet for obesity. N Engl J Med 2003;348:2082-90.

Related articles in AJCN:

Another animal model for nonalcoholic steatohepatitis: how close to the human condition?

Amin A Nanji
AJCN 2004 79: 350-351. [Full Text]