A link between dietary fructose intake, gut-derived endotoxemia, and nonalcoholic fatty liver disease (NAFLD) has been suggested by the results of human and animal studies. To further investigate the role of gut-derived endotoxin in the onset of fructose-induced NAFLD, Toll-like receptor (TLR-) 4-mutant (C3H/HeJ) mice and wildtype (C3H/HouJ) mice were either fed plain water or water enriched with 30% fructose for 8 weeks. Hepatic steatosis, plasma alanine aminotransferase (ALT), and markers of insulin resistance as well as portal endotoxin levels were determined. Hepatic levels of myeloid differentiation factor 88 (MyD88), interferon regulatory factor (IRF) 3 and 7, and tumor necrosis factor alpha (TNF␣) as well as markers of lipid peroxidation were assessed. Chronic intake of 30% fructose solution caused a significant increase in hepatic steatosis and plasma ALT levels in wildtype animals in comparison to water controls. In fructose-fed TLR-4 mutant mice, hepatic triglyceride accumulation was significantly reduced by Ϸ40% in comparison to fructose-fed wildtype mice and plasma ALT levels were at the level of water-fed controls. No difference in portal endotoxin concentration between fructose-fed wildtype and TLR-4-mutant animals was detected. In contrast, hepatic lipid peroxidation, MyD88, and TNF␣ levels were significantly decreased in fructose-fed TLR-4-mutant mice in comparison to fructose-fed wildtype mice, whereas IRF3 and IRF7 expression remained unchanged. Markers of insulin resistance (e.g., plasma TNF␣, retinol binding protein 4, and hepatic phospho-AKT) were only altered in fructose-fed wildtype animals. Conclusion: Taken together, these data further support the hypothesis that in mice the onset of fructose-induced NAFLD is associated with intestinal bacterial overgrowth and increased intestinal permeability, subsequently leading to an endotoxin-dependent activation of hepatic Kupffer cells.

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Studies in animals and human subjects indicate that gut-derived bacterial endotoxins may play a critical role in the development of nonalcoholic fatty liver disease (NAFLD). In the present study, we investigated if the liver is also sensitised by other microbial components during the onset of fructose-induced steatosis in a mouse model. C57BL/6 mice were either fed with 30 % fructose solution or tap water (control) with or without antibiotics for 8 weeks. Expression of toll-like receptors (TLR)1-9, TNF-a, inducible NO synthase (iNOS), myeloid differentiation factor 88 (MyD88) and number of F4/80 positive cells in the liver were assessed. Occludin protein, DNA of microbiota in the small and large intestine and retinol binding protein 4 (RBP4) in plasma were analysed using Western blot, DNA fingerprinting and ELISA, respectively. F4/80 positive cells were determined by immunohistochemistry. The accumulation of TAG found in the livers of fructose-fed mice was associated with a significant induction of TLR 1 -4 and 6-8. Plasma RBP4 concentration and hepatic mRNA expression levels of TNF-a, iNOS, MyD88 and number of F4/80 positive cells of fructose-fed animals were significantly higher than those of controls; however, these effects of fructose were attenuated in antibiotic-treated mice. Whereas protein concentration of occludin was lower in the duodenum of fructose-treated mice, no systematic alterations of microbiota were found in this part of the intestine. Taken together, these data support the hypothesis that (1) an increased intestinal translocation of microbial components and (2) an increased number of F4/80 positive cells and induction of several TLR and dependent pathways (e.g. MyD88 and iNOS) may be involved in the onset of fructose-induced NAFLD.

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Plasminogen activator inhibitor-1 (PAI-1) is an acute-phase protein known to be involved in alcoholic liver disease and hepatic fibrosis. In the present study, the hypothesis that PAI-1 is causally involved in the onset of fructose-induced hepatic steatosis was tested in a mouse model. Wild-type C57BL/6J and PAI-1⁻/⁻ mice were fed with 30% fructose solution or water for 8 weeks. Markers of hepatic steatosis, expression of PAI-1, apolipoprotein B (ApoB), cluster of differentiation 1d (CD1d), markers of natural killer T (NKT) cells, protein levels of phospho-c-Met and tumor necrosis factor-α (TNF-α) were determined. Activity of the microsomal triglyceride transfer protein (MTTP) was measured in liver tissue. In comparison with water controls, chronic intake of 30% fructose solution caused a significant increase in hepatic triglycerides, PAI-1 expression and plasma alanine aminotransferase levels in wild-type mice. This effect of fructose feeding was markedly attenuated in PAI-1⁻/⁻ mice. Despite no differences in portal endotoxin levels and hepatic TNF-α protein levels between fructose-fed groups, the protective effect of the loss of PAI-1 against the onset of fructose-induced steatosis was associated with a significant increase in phospho-c-Met, phospho Akt, expression of ApoB and activity of MTTP in livers of PAI-1⁻/⁻ mice in comparison with fructose-fed wild types. Moreover, in PAI-1⁻/⁻ mice, expressions of CD1d and markers of CD1d-reactive NKT cells were markedly higher than in wild-type mice; however, expression of markers of activation of CD1d-reactive NKT cells (eg, interleukin-15 and interferon-γ) were only found to be increased in livers of fructose-fed PAI-1⁻/⁻ mice. Taken together, these data suggest that PAI-1 has a causal role in mediating the early phase of fructose-induced liver damage in mice through signaling cascades downstream of Kupffer cells and TNF-α.

This article is available online at http://www.jlr.org phase of NAFLD ( 2 ). Furthermore, results of recent studies suggest that steatosis may play a critical role not only in the onset of NAFLD but also in its progression to later stages of the disease (e.g., fi brosis and cirrhosis ( 3 )). Therefore, therapies protecting against the onset of NAFLD may also be benefi cial for the later stages of the disease.Results of several epidemiologic and clinical studies indicate that besides a general over-nutrition dietary intake of carbohydrates and fructose consumption in particular may play a critical role in the development of NAFLD in humans ( 4 ). The hypothesis that a diet rich in mono-and disaccharides, such as fructose and sucrose, might play a critical role in the pathogenesis of NAFLD is also supported by a number of studies performed in animals. In these studies, it was shown that an increased consumption of fructose (e.g., up to 60% of daily calories derived from fructose) resulted in an increased lipid accumulation in the liver, which was accompanied by insulin resistance, elevated plasma triglyceride levels, and oxidative stress ( 5-9 ). Recently, our group was able to show that hepatic steatosis resulting from chronic intake of fructose is associated with a loss of the tight junction protein occludin in the duodenum, an increased translocation of bacterial endotoxins from the intestine, and an induction of tumor necrosis factor (TNF) ␣ in the liver of mice ( 7, 10, 11 ). However, while plasma levels of TNF ␣ and retinol binding protein 4 were both increased under this feeding regimen, suggesting that fructose-fed mice were insulin-resistant, glucose levels in blood of food-deprived, fructose-fed mice did not differ from those of water-fed controls ( 7, 10 ). In these studies, the concomitant treatment with antibiotics Abstract Fructose intake is being discussed as a key dietary factor in the development of nonalcoholic fatty liver disease (NAFLD). Bile acids have been shown to modulate energy metabolism. We tested the effects of bile acids on fructoseinduced hepatic steatosis. In C57BL/6J mice treated with a combination of chenodeoxycholic acid and cholic acid (100 mg/kg body weight each) while drinking water or a 30% fructose solution for eight weeks and appropriate controls, markers of hepatic steatosis, portal endotoxin levels, and markers of hepatic lipogenesis were determined. In mice concomitantly treated with bile acids, the onset of fructoseinduced hepatic steatosis was markedly attenuated compared to mice only fed fructose. The protective effects of the bile acid treatment were associated with a downregulation of tumor necrosis factor (TNF) ␣ , sterol regulatory element-binding protein (SREBP)1, FAS mRNA expression, and lipid peroxidation in the liver, whereas hepatic farnesoid X receptor (FXR) or short heterodimer partner (SHP) protein concentration did not differ between groups fed fructose. Rather, bile acid treatment normalized occludin protein concentration in the duodenum, portal endotoxin...

Taken together, these data suggest that female mice are also more susceptible to acute alcohol-induced liver steatosis and that this involves an increased activation of TLR-4-dependent signaling pathways in the liver.