No abstract

Overnutrition, insulin resistance and an impaired intestinal barrier function are discussed as critical factors in the development of nonalcoholic fatty liver disease. Not only butyrate-producing probiotics as well as supplementation of sodium butyrate (SoB) have been suggested to bear protective effects on liver damage of various aetiologies. However, whether an oral consumption of SoB has a protective effect on Western-style diet (WSD)-induced non-alcoholic steatohepatitis (NASH) and if so molecular mechanism involved has not yet been determined. Eight-week-old C57BL/6J mice were pair-fed either a liquid control or WSD ± 0·6 g/kg body weight SoB. After 6 weeks, markers of liver damage, inflammation, toll-like receptor (TLR)-4 signalling, lipid peroxidation and glucose as well as lipid metabolism were determined in the liver tissue. Tight junction protein levels were determined in the duodenal tissue. SoB supplementation had no effects on the body weight gain or liver weight of WSD-fed mice, whereas liver steatosis and hepatic inflammation were significantly decreased (e.g. less inflammatory foci and neutrophils) when compared with mice fed only a WSD. Tight junction protein levels in duodenum, hepatic mRNA expression of TLR-4 and sterol regulatory element-binding protein 1c were altered similarly in both WSD groups when compared with controls, whereas protein levels of myeloid differentiation primary response gene 88, inducible nitric oxide synthase, 4-hydroxynonenal protein adducts and F4/80 macrophages were only significantly induced in livers of mice fed only the WSD. In summary, these data suggest that an oral supplementation of SoB protects mice from inflammation in the liver and thus from the development of WSD-induced NASH.

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.

NCT01306396.

Universally accepted therapeutic strategies for the treatment of nonalcoholic steatohepatitis (NASH) are still lacking. Studies suggest a preventive effect of oral Gln supplementation on the development of NASH; however, whether Gln also has therapeutic potential for pre-existing NASH has not yet been clarified. The aim of the present study was to determine whether Gln prevents the progression of diet-induced NASH in mice. For 8 wk, female C57BL/6J mice (6-8 wk old) were pair-fed a liquid Western-style diet [WSD, 25% of energy from fat, 50% wt:wt fructose, 0.16% wt:wt cholesterol] or control diet (C diet) to induce liver damage. From week 8 to 13, they were pair-fed the C diet or WSD alone or supplemented with l-Gln to provide 2.1 g/kg body weight (C diet + Gln or WSD + Gln). Energy intake was adjusted to the group with the lowest energy intake. Indexes of liver damage and inflammation, intestinal barrier function, and toll-like receptor 4 () signaling in the liver were determined. The liver histology scores significantly increased from 8 to 13 wk (+31%) in WSD-fed mice and were significantly higher than in controls ( ≤ 0.05 for both time comparisons), whereas scores did not differ between C diet-fed and WSD + Gln-fed mice after 13 wk of feeding. The occludin protein concentrations in the small intestinal tissue were similarly reduced in both WSD-fed groups when compared with controls [WSD compared with C diet (-53%) and C diet + Gln (-42%), ≤ 0.05; WSD + Gln compared with C diet + Gln (-34%), ≤ 0.05] after 13 wk, whereas the expression of myeloid differentiation primary response gene 88 mRNA and concentration of inducible nitric oxide synthase and 4-hydroxynonenal protein adducts were significantly higher only in livers of WSD-fed mice ( ≤ 0.05 for the WSD group compared with all other groups; WSD + Gln group compared with the C diet groups: NS). Taken together, our data suggest that oral Gln supplementation protects mice from the progression of pre-existing, WSD-induced NASH.

Sodium butyrate (SoB) supplementation has been suggested to attenuate the development of non-alcoholic fatty liver disease (NAFLD). Here, we determined the therapeutic potential of SoB on NAFLD progression and molecular mechanism involved. Eight-week old C57BL/6J mice were pair-fed a fat-, fructose- and cholesterol-rich diet (FFC) or control diet (C). After 8 weeks, some mice received 0.6g SoB/kg bw in their respective diets (C+SoB; FFC+SoB) or were maintained on C or FFC for the next 5 weeks of feeding. Liver damage, markers of glucose metabolism, inflammation, intestinal barrier function and melatonin metabolism were determined. FFC-fed mice progressed from simple steatosis to early non-alcoholic steatohepatitis, along with significantly higher TNFα and IL-6 protein levels in the liver and impaired glucose tolerance. In FFC+SoB-fed mice, disease was limited to steatosis associated with protection against the induction of Tlr4 mRNA and iNOS protein levels in livers. SoB supplementation had no effect on FFC-induced loss of tight junction proteins in the small intestine but was associated with protection against alterations in melatonin synthesis and receptor expression in the small intestine and livers of FFC-fed animals. Our results suggest that the oral supplementation of SoB may attenuate the progression of simple steatosis to steatohepatitis.

Intestinal microbiota and barrier functions seem to play an important role in the development of non-alcoholic fatty liver disease (NAFLD). However, whether these changes are an early event in the development of NAFLD or are primarily associated with later stages of the disease, has not yet been clarified. Using a pair-feeding model, we determined the effects of a short-term intake of a fat-, fructose- and cholesterol-rich diet (FFC) on the development of early hepatic steatosis and markers of intestinal barrier function in mice treated with and without non-resorbable antibiotics (AB). For four days, C57BL/6J mice were either pair-fed a control diet or a FFC diet ± AB (92 mg/kg body weight (BW) polymyxin B and 216 mg/kg BW neomycin). Hepatic steatosis and markers of inflammation, lipidperoxidation and intestinal barrier function were assessed. Lipid accumulation and early signs of inflammation found in the livers of FFC-fed mice were markedly attenuated in FFC + AB-fed animals. In FFC-fed mice the development of NAFLD was associated with a significant loss of tight junction proteins and an induction of matrix metalloproteinase-13 in the upper parts of the small intestine as well as significantly higher portal endotoxin levels and an induction of dependent signaling cascades in the liver. As expected, portal endotoxin levels and the expression of dependent signaling cascades in liver tissue were almost at the level of controls in FFC + AB-fed mice. However, FFC + AB-fed mice were also protected from the loss of zonula occludens-1 and partially of occludin protein in small intestine. Our data suggest that the development of early diet-induced hepatic steatosis in mice at least in part results from alterations of intestinal barrier function.

Our data suggest that the protective effects of oral Gln supplementation on the development of WSD-induced NASH in mice are associated with protection against the induction of iNOS and lipid peroxidation in the liver.