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A high-fat diet leads to the progression of non-alcoholic fatty liver disease in obese rats

Michal Carmiel-Haggai^*,,1, Arthur I. Cederbaum^* and Natalia Nieto^*,1,2

^* Department of Pharmacology and Biological Chemistry;
Division of Liver Diseases, Mount Sinai School of Medicine, New York, New York, USA

² Correspondence: Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1603, New York, NY 10029, USA. E-mail: ny2000{at}hotmail.com

SPECIFIC AIMS

Toxic by-products of lipid peroxidation and oxidative stress associated with increased fat deposition in the liver, cytokine-mediated injury, and hyperglycemia are among proposed mechanisms that trigger fatty liver disease. It is uncertain why only a subgroup of patients with nonalcoholic steatohepatitis (NASH) may progress to liver fibrosis. Fatty livers of obese fa/fa rats are vulnerable to injury when challenged by endotoxin, ischemia-reperfusion, or acute ethanol treatment. Since the prevalence of obesity is expected to rise in the near future due to high saturated fat consumption, the objective of this study was to evaluate whether a high fat diet (HFD) can act as a "second hit" and cause progression to liver injury in obese fa/fa rats compared with lean Fa/? rats.

PRINCIPAL FINDINGS

1. A high saturated fat diet (HFD) contributes to the progression of NASH to fibrosis
Fifteen-wk-old obese fa/fa Zucker rats and their lean littermates, Fa/? rats, were fed a control or a high fat diet (12% vs. 60% total calories, respectively) for 8 wk. Hyperglycemia occurred to the greatest extent in fa/fa rats fed the HFD. Liver injury appeared in fa/fa rats fed the HFD. Hematoxylin and eosin staining showed steatohepatitis, ballooning degeneration, significant macrosteatosis extending beyond the periportal area into the lobule and the central zone, and foci of lobular inflammation only in the fa/fa rats fed the HFD (Fig. 1 A–D). Nonesterified fatty acid levels were elevated 2-fold in fa/fa rats fed the HFD compared with control diet (Fig. 1E) . ALT values were 5-fold higher than in Fa/? rats (Fig. 1F ). Stellate cell-derived TGFß, which actively participates in liver fibrosis and collagen deposition, and plasma TNF increased in fa/fa rats fed HFD compared with fa/fa rats fed control diet or to Fa/? rats fed HFD.

View larger version (129K): [in this window] [in a new window]   Figure 1. A high saturated fat diet contributes to progression of liver disease in obese fa/fa rats. Photomicrographs of liver samples stained with H&E after 8 wk on control diet or HFD. Fa/? rat fed control diet (A). Fa/? rat fed HFD showing minimal central zone micro- and macrovesicular steatosis (B). Micro- and macrovesicular steatosis in the periportal area in a fa/fa rat fed control diet (C). Significant micro- and macrovesicular steatosis in the liver of a fa/fa rat fed the HFD (D). x200. PT, portal tract; CV, central vein. Nonesterified fatty acids (NEFA) are shown in panel (E) and are expressed as mEq/mg of liver protein. Serum ALT (F). Blood was collected from the abdominal aorta, centrifuged at 3000 rpm for 3 min and the serum was immediately frozen until analysis. ALT levels were analyzed using a kit from Sigma (InfinityTM ALT). Data are presented in U/L as mean ± SE(n=4); *P< 0.05 for fa/favs. Fa/?, •P< 0.05, and •••P< 0.001 for HFD vs. control diet (C).

Sirius red/Fast green staining for collagenous proteins showed mild periportal fibrosis only in fa/fa rats fed HFD. Western blot analysis for collagen type I, a marker of liver fibrosis, revealed increased collagen I expression in the liver of fa/fa rats fed HFD; no change was observed with Fa/? rats. -Smooth muscle actin, representative of stellate cell activation, was elevated in fa/fa rats fed HFD. Expression of matrix metalloproteinases (MMPs) that participate in collagen degradation as well as their inhibitors (tissue inhibitors of metalloproteinases, TIMPs) was analyzed. A decrease in pro-MMP13 and active MMP13 was found in fa/fa rats regardless of diet. TIMP1 (which inhibits MMP13, preventing collagen I degradation) was similar in both genotypes fed control diet but elevated in rats fed HFD, especially in fa/fa rats. Along with elevated TGFß production, this may contribute to collagen accumulation in fa/fa rats fed HFD. MMP2 and MMP9 were detected only as latent forms by Western blot analysis; pro-MMP9 was increased 2-fold in fa/fa rats regardless of diet.

2. Oxidative stress in obese fa/fa rats fed HFD
Formation of malondialdehyde, a lipid peroxidation end product, was similar in both genotypes when fed a control diet, but there was an increase of 60% in fa/fa rats fed HFD vs. fa/fa rats fed control diet. These results suggest that the steatotic liver in fa/fa rats may be vulnerable to further peroxidation and injury when challenged by additional insults such as a HFD. Carbonyl formation, an early marker for protein oxidation, increased in fa/fa rats fed HFD compared with corresponding controls. fa/fa rats fed control diet had about half the levels of GSH as Fa/? rats; GSH levels were further decreased by HFD in association with an increase in GSSG. Catalase, glutathione peroxidase, glutathione reductase, and total superoxide dismutase (SOD) activity was lower in fa/fa rats fed control diet than in Fa/? rats, and all four enzymes were further decreased in fa/fa rats fed HFD. Thus, antioxidant defense was lowest in fa/fa rats fed HFD.

3. NADPH oxidase is a key source of ROS production in obese fa/fa rats fed a high saturated fat diet
A potential major source of O₂^.– in the liver is NADPH oxidase whose activity can be modulated by proinflammatory cytokines (e.g., TNF). Fa/? and fa/fa rats showed no difference in NADPH oxidase activity when fed control diet; moreover, HFD did not alter NADPH oxidase activity in Fa/? rats. However, fa/fa rats fed HFD showed an 2.5-fold increase in NADPH oxidase activity (Fig. 2 ). This increase was blocked by diphenyleneiodonium, a NADPH oxidase inhibitor, but not by rotenone, an inhibitor of the mitochondrial respiratory chain. That O₂^.– was a product generated by NADPH oxidase was validated by inhibition of product formation by SOD. Expression and activity of cytochrome P450 2E1 and xanthine oxidase activity were down-regulated in fa/fa compared with Fa/? rats.

View larger version (19K): [in this window] [in a new window]   Figure 2. NADPH oxidase activity. NADPH oxidase activity was evaluated in the absence or presence of 200 U/mL of SOD, 100 µM diphenyleneiodonium (DPI), or 10 µM rotenone. Data are expressed as arbitrary units of chemiluminescence per milligram of protein. Results are average values ±SE (n=4). ***P< 0.001 for fa/favs. Fa/? rats; •••P< 0.001 for HFD vs. control (C).

CONCLUSIONS AND SIGNIFICANCE

In the present study we evaluated whether a high saturated fat diet could induce pathogenesis in the obese fa/fa rat compared with lean controls. The rationale of exposing Zucker fa/fa rats with preexisting liver steatosis to HFD was to accelerate liver damage and promote a controlled "second hit" in order to study factors and mechanisms in the process. A lipid overload example of an already pathological lipid metabolism mimics the worldwide situation in which HFD is being consumed by an increasingly overweight, hyperlipidemic, and insulin-resistant population. fa/fa rats fed HFD for 8 wk showed a 2-fold increase in serum glucose levels compared with fa/fa rats fed control diet or Fa/? rats fed HFD. Significant steatohepatitis, periportal collagen accumulation, activation of stellate cells, increased ALT levels, and elevated stellate cell-derived TGFß and serum TNF levels were observed in fa/fa rats fed HFD. Thus, liver injury was induced in fa/fa rats fed HFD. To our knowledge, the current report is the first study connecting hepatitis and fibrosis induced by HFD in an animal model of metabolic syndrome.

Oxidative stress occurred in fa/fa rats fed HFD as evident by elevated levels of malondialdehyde and protein carbonyls, suggesting increased lipid peroxidation and protein oxidation. Hepatic GSH levels declined in all rats fed HFD but were especially low in fa/fa rats; GSSG levels were elevated in the latter. Levels of critical antioxidant enzymes were lowest in fa/fa rats fed HFD, which is likely to exacerbate existing oxidative stress. To prevent oxidative stress, there is an ongoing balance between intrahepatic antioxidants and ROS. When there is an imbalance, ROS may accumulate and trigger steatohepatitis by lipid peroxidation, protein oxidation, and cytokine induction (TNF, TGFß, and IL-8), accompanied by increased collagen synthesis and cell death.

Multiple possible sources of oxidant stress in the fatty liver that may constitute the ‘second hit’ for cellular injury in NASH have been identified. These include mitochondrial and peroxisomal ß-oxidation, mitochondrial electron leak, NADPH oxidases, xanthine oxidase, cytochrome P450 isoforms, and recruited inflammatory cells. We evaluated the role of some of these pathways in contributing to oxidative stress in obesity in the presence of a high saturated fat diet. Cytochrome P450 could contribute to cellular injury and pathogenesis of NASH, particularly when antioxidant reserves are depleted, as occurs in fa/fa rats. CYP2E1 is a major microsomal source of H₂O₂ and NADPH-dependent lipid peroxidation and is capable of reducing O₂ to O₂^.– and H₂O₂ due to its uncoupled turnover. However, fa/fa rats showed lower CYP2E1 content and there was no induction of CYP2E1 by HFD. In terms of CYP2E1 regulation, insulin has a repressive effect, and lipid peroxidation derived by-products, which are up-regulated in fa/fa rats fed HFD, can cause nonselective destruction of cytochrome P450 enzymes.

Activity and expression of NADPH oxidase are regulated by proinflammatory cytokines such as TNF. Our results suggest a possible role for the NADPH oxidase system in oxidant stress observed in fa/fa rats fed HFD, since NADPH oxidase activity increased 2.5-fold; this effect was prevented by addition of diphenyleneiodonium, a NADPH oxidase inhibitor. In liver, NADPH oxidase activity is high in Kupffer cells. fa/fa rats show evidence of Kupffer cell alterations since they exhibit reduced hepatic clearance of intraperitoneally administered fluorescent-labeled microspheres. If macrophages do not trigger fibrosis onset, they most likely worsen the fibrosis process once initiated.

Elevated expression of -sma and collagen I suggests that hepatic stellate cells were activated in fa/fa rats fed HFD. Stellate cell-derived TGFß was elevated in fa/fa rats fed HFD. Tissue fibrosis results from a relative imbalance between synthesis and degradation of extracellular matrix. MMPs are the major enzymes that degrade extracellular matrix and display substrate specificity; MMP-13 degrades interstitial collagens whereas MMP-2 and MMP-9 degrade collagen type IV and fibronectin. TIMPs, important regulatory molecules in tissue remodeling and repair, inhibit MMP activity.

A putative glucose response element has been identified in the promoter of TGFß₁. Increased immunostaining for stellate cell-derived TGFß₁ was detected in liver sections from fa/fa rats fed HFD. The latter may play a role in activating stellate cells to produce increased -sma and collagen I. It is also possible that TGFß₁ in the liver is activated by a glucose-sensing mechanism, such as the hexosamine biosynthetic pathway.

In summary, an HFD caused liver injury in fa/fa rats leading to periportal fibrosis, but no injury was found in lean control rats. Hyperglycemia and steatohepatitis occurred in fa/fa rats on HFD, as did elevated cytokine production and oxidative stress. The latter may be due to a combination of increased ROS production via the elevated activity of NADPH oxidase coupled with low antioxidant defense, which was further diminished by HFD.

View larger version (16K): [in this window] [in a new window]   Figure 3. Schematic diagram of proposed mechanism. Obese fa/fa Zucker rats fed a high saturated fat diet show hyperglycemia and elevated oxidative injury due to low antioxidant defense and increased NADPH oxidase activity, leading to liver injury, as indicated by increased transaminases and liver pathology. Elevated cytokine production such as TGFß can directly induce stellate cell activation and collagen I production or elevate collagen I due to transcriptional regulation. TGFß up-regulates TIMP-1, an inhibitor of MMP-13, the metalloproteinase that degrades collagen I, contributing to net accumulation of matrix. ALT, alanine aminotransferase; TGFß, transforming growth factor ß; MMP-13, matrix metalloproteinase-13.

FOOTNOTES

¹ These authors contributed equally to the experimental part of this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2291fje;

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