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.

By now, non-alcoholic fatty liver disease (NAFLD) is considered to be among the most common liver diseases world-wide. NAFLD encompasses a broad spectrum of pathological conditions ranging from simple steatosis to steatohepatitis, fibrosis and finally even cirrhosis; however, only a minority of patients progress to end-stages of the disease, and the course of the disease progression to the later stages seems to be slow, developing progressively over several years. Key risk factors including overweight, insulin resistance, a sedentary life-style and an altered dietary pattern, as well as genetic factors and disturbances of the intestinal barrier function have been identified in recent years. Despite intense research efforts that lead to the identification of these risk factors, knowledge about disease initiation and molecular mechanisms involved in progression is still limited. This review summarizes diet-induced and genetic animal models, as well as cell culture models commonly used in recent years to add to the understanding of the mechanisms involved in NAFLD, also referring to their advantages and disadvantages.

To test the hypothesis that metformin protects against fructose-induced steatosis, and if so, to elucidate underlying mechanisms, C57BL/6J mice were either fed 30% fructose solution or plain water for 8 weeks. Some of the animals were concomitantly treated with metformin (300 mg/kg body weight/day) in the drinking solution. While chronic consumption of 30% fructose solution caused a significant increase in hepatic triglyceride accumulation and plasma alanineaminotransferase levels, this effect of fructose was markedly attenuated in fructose-fed mice concomitantly treatment with metformin. The protective effects of the metformin treatment on the onset of fructose-induced non-alcoholic fatty liver disease (NAFLD) were associated with a protection against the loss of the tight junction proteins occludin and zonula occludens 1 in the duodenum of fructose-fed mice and the increased translocation of bacterial endotoxin found in mice only fed with fructose. In line with these findings, in metformin-treated fructose-fed animals, hepatic expression of genes of the toll-like receptor-4-dependent signalling cascade as well as the plasminogen-activator inhibitor/cMet-regulated lipid export were almost at the level of controls. Taken together, these data suggest that metformin not only protects the liver from the onset of fructose-induced NAFLD through mechanisms involving its direct effects on hepatic insulin signalling but rather through altering intestinal permeability and subsequently the endotoxin-dependent activation of hepatic Kupffer cells.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide with universally accepted treatments still lacking. Oral supplementation of sodium butyrate (SoB) has been suggested to attenuate liver damage of various aetiologies. Our study aimed to further delineate mechanisms involved in the SoB-dependent hepatic protection using a mouse model of fructose-induced NAFLD and in in vitro models. C57BL/6J mice were either pair-fed a fructose-enriched liquid diet ±0·6 g/kg body weight per d SoB or standard chow for 6 weeks. Markers of liver damage, intestinal barrier function, glucose metabolism, toll-like receptor-4 (TLR-4) and melatonin signalling were determined in mice. Differentiated human carcinoma colon-2 (Caco-2) and J774A.1 cells were used to determine molecular mechanisms involved in the effects of SoB. Despite having no effects on markers of intestinal barrier function and glucose metabolism or body weight gain, SoB supplementation significantly attenuated fructose-induced hepatic TAG accumulation and inflammation. The protective effects of SoB were associated with significantly lower expression of markers of the TLR-4-dependent signalling cascade, concentrations of inducible nitric oxide synthase (iNOS) protein and 4-hydroxynonenal protein adducts in liver. Treatment with SoB increased melatonin levels and expression of enzymes involved in melatonin synthesis in duodenal tissue and Caco-2 cells. Moreover, treatment with melatonin significantly attenuated lipopolysaccharide-induced expression of iNOS and nitrate levels in J774A.1 cells. Taken together, our results indicated that the protective effects of SoB on the development of fructose-induced NAFLD in mice are associated with an increased duodenal melatonin synthesis and attenuation of iNOS induction in liver.

Elevated dietary fructose intake, altered intestinal motility, and barrier function may be involved in the development of nonalcoholic fatty liver disease (NAFLD). Because intestinal motility and permeability are also regulated through the bioavailability of serotonin (5-HT), we assessed markers of hepatic injury in serotonin reuptake transporter knockout (SERT(-/-)) and wild-type mice chronically exposed to different monosaccharide solutions (30% glucose or fructose solution) or water for 8 wk. The significant increase in hepatic triglyceride, TNF-alpha, and 4-hydroxynonenal adduct as well as portal endotoxin levels found in fructose-fed mice was associated with a significant decrease of SERT and the tight-junction occludin in the duodenum. Similar effects were not found in mice fed glucose. In contrast, in SERT(-/-) mice fed glucose, portal endotoxin levels, concentration of occludin, and indices of hepatic damage were similar to those found in wild-type and SERT(-/-) mice fed fructose. In fructose-fed mice treated with a 5-HT3 receptor antagonist, hepatic steatosis was significantly attenuated. Our data suggest that a loss of intestinal SERT is a critical factor in fructose-induced impairment of intestinal barrier function and subsequently the development of steatosis.

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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To test the hypothesis that the inducible nitric oxide synthase (iNOS) is involved in mediating the toll-like receptor 4-dependent effects on the liver in the onset of fructose-induced steatosis, wild-type and iNOS knockout (iNOS(-/-)) mice were either fed tap water or 30% fructose solution for 8 weeks. Chronic consumption of 30% fructose solution led to a significant increase in hepatic steatosis and inflammation as well as plasma alanine-aminotransferase levels in wild-type mice. This effect of fructose feeding was markedly attenuated in iNOS(-/-) mice. Hepatic lipidperoxidation, concentration of phospho-IκB, nuclear factor κB activity, and tumor necrosis factor-α mRNA level were significantly increased in fructose-fed wild-type mice, whereas in livers of fructose-fed iNOS(-/-) mice, lipidperoxidation, phospho-IκB, nuclear factor κB activity, and tumor necrosis factor-α expression were almost at the level of controls. However, portal endotoxin levels and hepatic myeloid differentiation factor 88 expression were significantly higher in both fructose-fed groups compared to controls. Taken together, these data suggest that (i) the formation of reactive oxygen species in liver is a key factor in the onset of fatty liver and (ii) iNOS is involved in mediating the endotoxin/toll-like receptor 4-dependent effects in the development of fructose-induced fatty liver.