Long Noncoding RNAs in Diet-Induced Metabolic Diseases.

The prevalence of metabolic diseases, including type 2 diabetes and metabolic dysfunction-associated steatotic liver disease (MASLD), is steadily increasing. Although many risk factors, such as obesity, insulin resistance, or hyperlipidemia, as well as several metabolic gene programs that contribute to the development of metabolic diseases are known, the underlying molecular mechanisms of these processes are still not fully understood. In recent years, it has become evident that not only protein-coding genes, but also noncoding genes, including a class of noncoding transcripts referred to as long noncoding RNAs (lncRNAs), play key roles in diet-induced metabolic disorders. Here, we provide an overview of selected lncRNA genes whose direct involvement in the development of diet-induced metabolic dysfunctions has been experimentally demonstrated in suitable in vivo mouse models. We further summarize and discuss the associated molecular modes of action for each lncRNA in the respective metabolic disease context. This overview provides examples of lncRNAs with well-established functions in diet-induced metabolic diseases, highlighting the need for appropriate in vivo models and rigorous molecular analyses to assign clear biological functions to lncRNAs.

MASLD is related to impaired alcohol dehydrogenase (ADH) activity and elevated blood ethanol levels: Role of TNFα and JNK.

Elevated fasting ethanol levels in peripheral blood frequently found in metabolic dysfunction-associated steatohepatitis (MASLD) patients even in the absence of alcohol consumption are discussed to contribute to disease development. To test the hypothesis that besides an enhanced gastrointestinal synthesis a diminished alcohol elimination through alcohol dehydrogenase (ADH) may also be critical herein, we determined fasting ethanol levels and ADH activity in livers and blood of MASLD patients and in wild-type ± anti-TNFα antibody (infliximab) treated and TNFα-/- mice fed a MASLD-inducing diet. Blood ethanol levels were significantly higher in patients and wild-type mice with MASLD while relative ADH activity in blood and liver tissue was significantly lower compared to controls. Both alterations were significantly attenuated in MASLD diet-fed TNFα-/- mice and wild-type mice treated with infliximab. Moreover, alcohol elimination was significantly impaired in mice with MASLD. In in vitro models, TNFα but not IL-1ß or IL-6 significantly decreased ADH activity. Our data suggest that elevated ethanol levels in MASLD patients are related to TNFα-dependent impairments of ADH activity.

Microbiota modulation by dietary oat beta-glucan prevents steatotic liver disease progression.

Background & Aims: Changes in gut microbiota in metabolic dysfunction-associated steatotic liver disease (MASLD) are important drivers of disease progression towards fibrosis. Therefore, reversing microbial alterations could ameliorate MASLD progression. Oat beta-glucan, a non-digestible polysaccharide, has shown promising therapeutic effects on hyperlipidemia associated with MASLD, but its impact on gut microbiota and most importantly MASLD-related fibrosis remains unknown. Methods: We performed detailed metabolic phenotyping, including assessments of body composition, glucose tolerance, and lipid metabolism, as well as comprehensive characterization of the gut-liver axis in a western-style diet (WSD)-induced model of MASLD and assessed the effect of a beta-glucan intervention on early and advanced liver disease. Gut microbiota were modulated using broad-spectrum antibiotic treatment. Results: Oat beta-glucan supplementation did not affect WSD-induced body weight gain or glucose intolerance and the metabolic phenotype remained largely unaffected. Interestingly, oat beta-glucan dampened MASLD-related inflammation, which was associated with significantly reduced monocyte-derived macrophage infiltration and fibroinflammatory gene expression, as well as strongly reduced fibrosis development. Mechanistically, this protective effect was not mediated by changes in bile acid composition or signaling, but was dependent on gut microbiota and was lost upon broad-spectrum antibiotic treatment. Specifically, oat beta-glucan partially reversed unfavorable changes in gut microbiota, resulting in an expansion of protective taxa, including Ruminococcus, and Lactobacillus followed by reduced translocation of Toll-like receptor ligands. Conclusions: Our findings identify oat beta-glucan as a highly efficacious food supplement that dampens inflammation and fibrosis development in diet-induced MASLD. These results, along with its favorable dietary profile, suggest that it may be a cost-effective and well-tolerated approach to preventing MASLD progression and should be assessed in clinical studies. Impact and Implications: Herein, we investigated the effect of oat beta-glucan on the gut-liver axis and fibrosis development in a mouse model of metabolic dysfunction-associated steatotic liver disease (MASLD). Beta-glucan significantly reduced inflammation and fibrosis in the liver, which was associated with favorable shifts in gut microbiota that protected against bacterial translocation and activation of fibroinflammatory pathways. Together, oat beta-glucan may be a cost-effective and well-tolerated approach to prevent MASLD progression and should be assessed in clinical studies.

Oral Supplementation of Phosphatidylcholine Attenuates the Onset of a Diet-Induced Metabolic Dysfunction-Associated Steatohepatitis in Female C57BL/6J Mice.

BACKGROUND & AIMS: Changes in phosphatidylcholine levels in the liver have been associated with the development of metabolic dysfunction-associated steatotic liver disease. Here, the effects of supplementing phosphatidylcholine on the development of early signs of metabolic dysfunction-associated steatohepatitis were assessed. METHODS: Male and female C57BL/6J mice were fed a liquid control or a fructose-, fat-, and/or cholesterol-rich diet for 7 or 8 weeks. The diets of female mice were fortified ± phosphatidylcholine (12.5 mg/g diet). In liver tissue and portal blood, indices of liver damage, inflammation, and bacterial endotoxemia were measured. J774A.1 cells and human monocytes preincubated with phosphatidylcholine (0.38 mmol/L) were challenged with lipopolysaccharide (50-100 ng/mL) ± the peroxisome proliferator-activated receptor γ (PPARγ) activator pioglitazone (10 µmol/L) or ± a liver receptor homolog 1 (LRH-1) antagonist 1-(3'-[1-(2-[4-morpholinyl]ethyl)-1H-pyrazol-3-yl]-3-biphenylyl)ethanon (1-10 µmol/L). RESULTS: In fructose-, fat-, and/or cholesterol-rich diet-fed mice the development of fatty liver and the beginning of inflammation were associated with significantly lower hepatic phosphatidylcholine levels when compared with controls. Supplementing phosphatidylcholine significantly attenuated the development of fatty liver and inflammation, being associated with protection against the induction of PPARγ2, and activation of nuclear factor of κ light polypeptide gene enhancer in B-cell inhibitor α whereas Lrh1 expression was unchanged. The protective effects of phosphatidylcholine on the lipopolysaccharide-induced activation of J774A.1 cells and human monocytes were attenuated significantly by the PPARγ activator pioglitazone and the LRH-1 antagonist. CONCLUSIONS: Our data suggest that phosphatidylcholine levels in the liver are lower in early metabolic dysfunction-associated steatohepatitis in mice and that supplementation of phosphatidylcholine can diminish the development of metabolic dysfunction-associated steatotic liver disease through mechanisms involving LRH-1/PPARγ2/ nuclear factor κ-light-chain enhancer of activated B-cell signaling.

Antibiotics attenuate diet-induced nonalcoholic fatty liver disease without altering intestinal barrier dysfunction.

To date the role of the alterations of intestinal microbiota in the development of intestinal barrier dysfunction in settings of nonalcoholic fatty liver disease (NAFLD) has not been fully understood. Here, we assessed the effect of antibiotics on development of NAFLD and their impact on intestinal barrier dysfunction. Male C57BL/6J mice were either pair-fed a liquid control diet (C) or fat- and fructose-rich diet (FFr) +/- antibiotics (AB, ampicillin/vancomycin/metronidazole/gentamycin) for 7 weeks. Fasting blood glucose was determined and markers of liver damage, inflammation, intestinal barrier function, and microbiota composition were assessed. The development of hepatic steatosis with early signs of inflammation found in FFr-fed mice was significantly abolished in FFr+AB-fed mice. Also, while prevalence of bacteria in feces was not detectable and TLR4 ligand levels in portal plasma were at the level of controls in FFr+AB-fed mice, impairments of intestinal barrier function like an increased permeation of xylose and iNOS protein levels persisted to a similar extent in both FFr-fed groups irrespective of AB use. Exposure of everted small intestinal tissue sacs of naïve mice to fructose resulted in a significant increase in tissue permeability and loss of tight junction proteins, being not affected by the presence of AB, whereas the concomitant treatment of tissue sacs with the NOS inhibitor aminoguanidine attenuated these alterations. Taken together, our data suggest that intestinal barrier dysfunction in diet-induced NAFLD in mice may not be predominantly dependent on changes in intestinal microbiota but rather that fructose-induced alterations of intestinal NO-homeostasis might be critically involved.

Supplementing L-Citrulline Can Extend Lifespan in C. elegans and Attenuate the Development of Aging-Related Impairments of Glucose Tolerance and Intestinal Barrier in Mice.

L-Citrulline (L-Cit) is discussed to possess a protective effect on intestinal barrier dysfunction but also to diminish aging-associated degenerative processes. Here, the effects of L-Cit on lifespan were assessed in C. elegans, while the effects of L-Cit on aging-associated decline were determined in C57BL/6J mice. For lifespan analysis, C. elegans were treated with ±5 mM L-Cit. Twelve-month-old male C57BL/6J mice (n = 8-10/group) fed a standard chow diet received drinking water ± 2.5 g/kg/d L-Cit or 5 g/kg/d hydrolyzed soy protein (Iso-N-control) for 16 or 32 weeks. Additionally, 4-month-old C57BL/6J mice were treated accordingly for 8 weeks. Markers of senescence, glucose tolerance, intestinal barrier function, and intestinal microbiota composition were analyzed in mice. L-Cit treatment significantly extended the lifespan of C. elegans. The significant increase in markers of senescence and signs of impaired glucose tolerance found in 16- and 20-month-old control mice was attenuated in L-Cit-fed mice, which was associated with protection from intestinal barrier dysfunction and a decrease in NO2- levels in the small intestine, while no marked differences in intestinal microbiota composition were found when comparing age-matched groups. Our results suggest that pharmacological doses of L-Cit may have beneficial effects on lifespan in C. elegans and aging-associated decline in mice.

Cognitive Alterations in Old Mice Are Associated with Intestinal Barrier Dysfunction and Induced Toll-like Receptor 2 and 4 Signaling in Different Brain Regions.

Emerging evidence implicate the 'microbiota-gut-brain axis' in cognitive aging and neuroinflammation; however, underlying mechanisms still remain to be elucidated. Here, we assessed if potential alterations in intestinal barrier function and microbiota composition as well as levels of two key pattern-recognition receptors namely Toll-like receptor (TLR) 2 and TLR4, in blood and different brain regions, and depending signaling cascades are paralleling aging associated alterations of cognition in healthy aging mice. Cognitive function was assessed in the Y-maze and intestinal and brain tissue and blood were collected in young (4 months old) and old (24 months old) male C57BL/6 mice to determine intestinal microbiota composition by Illumina amplicon sequencing, the concentration of TLR2 and TLR4 ligands in plasma and brain tissue as well as to determine markers of intestinal barrier function, senescence and TLR2 and TLR4 signaling. Cognitive function was significantly impaired in old mice. Also, in old mice, intestinal microbiota composition was significantly altered, while the relative abundance of Gram-negative or Gram-positive bacteria in the small and large intestines at different ages was not altered. Moreover, intestinal barrier function was impaired in small intestine of old mice, and the levels of TLR2 and TLR4 ligands were also significantly higher in both portal and peripheral blood. Furthermore, levels of TLR2 and TLR4 ligands, and downstream markers of TLR signaling were higher in the hippocampal and prefrontal cortex of old mice compared to young animals. Taken together, our results suggest that even in 'healthy' aging, cognitive function is impaired in mice going along with an increased intestinal translocation of TLR ligands and alterations of TLR signaling in several brain regions.

TNFα is a key trigger of inflammation in diet-induced non-obese MASLD in mice.

Tumor necrosis factor alpha (TNFα) is thought to be a critical factor in the development of metabolic dysfunction-associated steatotic liver disease (MASLD). Here, we determined the effects of a treatment with the anti-TNFα antibody infliximab and a genetic deletion of TNFα, respectively, in the development of non-obese diet-induced early metabolic dysfunction-associated steatohepatitis (MASH) in mice. The treatment with infliximab improved markers of liver damage in mice with pre-existing early MASH. In TNFα-/- mice, the development of early signs of MASH and insulin resistance was significantly attenuated compared to wild-type animals. While mRNA expression of proinflammatory cytokines like interleukin 1ß (Il1b) and interleukin 6 (Il6) were significantly lower in livers of MASH-diet-fed TNFα-/- mice compared to wild-type mice with early MASH, markers of intestinal barrier function were similarly impaired in both MASH-diet-fed groups compared to controls. Our data suggest that TNFα is a key regulator of hepatic inflammation and insulin resistance associated with the development of early non-obese MASH.

Impairments of intestinal arginine and NO metabolisms trigger aging-associated intestinal barrier dysfunction and 'inflammaging'.

Aging is considered a state of low grade inflammation, occurring in the absence of any overt infection often referred to as 'inflammaging'. Maintaining intestinal homeostasis may be a target to extend a healthier status in older adults. Here, we report that even in healthy older men low grade bacterial endotoxemia is prevalent. In addition, employing multiple mouse models, we also show that while intestinal microbiota composition changes significantly during aging, fecal microbiota transplantation to old mice does not protect against aging-associated intestinal barrier dysfunction in small intestine. Rather, intestinal NO homeostasis and arginine metabolism mediated through arginase and NO synthesis is altered in small intestine of aging mice. Treatment with the arginase inhibitor norNOHA prevented aging-associated intestinal barrier dysfunction, low grade endotoxemia and delayed the onset of senescence in peripheral tissue e.g., liver. Intestinal arginine and NO metabolisms could be a target in the prevention of aging-associated intestinal barrier dysfunction and subsequently decline and 'inflammaging'.

GW9662, a peroxisome proliferator-activated receptor gamma antagonist, attenuates the development of non-alcoholic fatty liver disease.

BACKGROUND AND AIMS: Insulin resistance is among the key risk factors for the development of non-alcoholic fatty liver disease (NAFLD). Recently, it has been reported that GW9662, shown to be a potent peroxisome proliferator-activated receptor gamma (PPARγ) antagonist, may improve insulin sensitivity in settings of type 2 diabetes. Here, we determined the effects of GW9662 on the development of NAFLD and molecular mechanisms involved. METHODS: Female C57BL/6J mice were pair-fed either a liquid control diet (C) or a fat-, fructose- and cholesterol-rich diet (FFC) for 8 weeks while either being treated with GW9662 (1 mg/kg body weight; C+GW9662 and FFC+GW9662) or vehicle (C and FFC) i.p. three times weekly. Indices of liver damage and inflammation, parameters of glucose metabolism and portal endotoxin levels were determined. Lipopolysaccharide (LPS)-challenged J774A.1 cells were treated with 10 µM GW9662. RESULTS: Despite similar caloric intake the development of NAFLD and insulin resistance were significantly attenuated in FFC+GW9662-treated mice when compared to FFC-fed animals. Bacterial endotoxin levels in portal plasma were almost similarly increased in both FFC-fed groups while expressions of toll-like receptor 4 (Tlr4), myeloid differentiation primary response 88 (Myd88) and interleukin 1 beta (Il1b) as well as nitrite (NO2-) concentration in liver were significantly higher in FFC-fed mice than in FFC+GW9662-treated animals. In J774A.1 cells, treatment with GW9662 significantly attenuated LPS-induced expression of Il1b, interleukin 6 (Il6) and inducible nitric oxide synthase (iNos) as well as NO2- formation. CONCLUSION: In summary, our data suggest that the PPARγ antagonist GW9662 attenuates the development of a diet-induced NAFLD and that this is associated with a protection against the activation of the TLR4 signaling cascade.

The Intestinal Barrier Dysfunction as Driving Factor of Inflammaging.

The intestinal barrier, composed of the luminal microbiota, the mucus layer, and the physical barrier consisting of epithelial cells and immune cells, the latter residing underneath and within the epithelial cells, plays a special role in health and disease. While there is growing knowledge on the changes to the different layers associated with disease development, the barrier function also plays an important role during aging. Besides changes in the composition and function of cellular junctions, the entire gastrointestinal physiology contributes to essential age-related changes. This is also reflected by substantial differences in the microbial composition throughout the life span. Even though it remains difficult to define physiological age-related changes and to distinguish them from early signs of pathologies, studies in centenarians provide insights into the intestinal barrier features associated with longevity. The knowledge reviewed in this narrative review article might contribute to the definition of strategies to prevent the development of diseases in the elderly. Thus, targeted interventions to improve overall barrier function will be important disease prevention strategies for healthy aging in the future.

Alterations of nitric oxide homeostasis as trigger of intestinal barrier dysfunction in non-alcoholic fatty liver disease.

Changes in intestinal nitric oxide metabolism are discussed to contribute for the development of intestinal barrier dysfunction in non-alcoholic fatty liver disease (NAFLD). To induce steatosis, female C57BL/6J mice were pair-fed with a liquid control diet (C) or a fat-, fructose- and cholesterol-rich diet (FFC) for 8 weeks. Mice received the diets ± 2.49 g L-arginine/kg bw/day for additional 5 weeks. Furthermore, mice fed C or FFC ± L-arginine/kg bw/day for 8 weeks were concomitantly treated with the arginase inhibitor Nω -hydroxy-nor-L-arginine (nor-NOHA, 0.01 g/kg bw). Liver damage, intestinal barrier function, nitric oxide levels and arginase activity in small intestine were assessed. Also, arginase activity was measured in serum from 13 patients with steatosis (NAFL) and 14 controls. The development of steatosis with beginning inflammation was associated with impaired intestinal barrier function, increased nitric oxide levels and a loss of arginase activity in small intestine in mice. L-arginine supplementation abolished the latter along with an improvement of intestinal barrier dysfunction; nor-NOHA attenuated these effects. In patients with NAFL, arginase activity in serum was significantly lower than in healthy controls. Our data suggest that increased formation of nitric oxide and a loss of intestinal arginase activity is critical in NAFLD-associated intestinal barrier dysfunction.

Effects of different standard and special diets on cognition and brain mitochondrial function in mice.

Objectives: Human nutrition plays an important role in prevention or at least slowing down the progression of age- and diet-related diseases. Thereby, mitochondrial dysfunction represents one common underlying mechanism, which is being investigated in mouse models. However, the influence of the selected diets in preclinical studies on cognition and mitochondrial function has not yet been reported cohesively.Methods: Therefore, we present the results of three different studies that addressed this question. First, we investigated the influence of two standard control chow diets and a special diet low in antioxidants over 6 months in aged NMRI mice. Additionally, a 70% high-fat (HF) chow diet as well as a western-style diet (WSD) rich in lard and fructose were examined in C57/BL6 mice. Cognitive performance, mitochondrial function and bioenergetics in the brain were investigated. Moreover, cerebral expression of genes involved in biogenesis and antioxidant defence (citrate synthase, complex I, complex IV, SOD2, Cat1, GPx-1) were quantified.Results: The results show that a modified, low antioxidant diet increased ATP levels in the brain of aged mice, while cognitive functions remained largely unaffected. A HF diet also showed significant effects on ATP levels and gene expression levels of relevant antioxidant markers, while the WSD had marginal effects on mitochondrial function and bioenergetics in the brain.Discussion: Our results indicate that standard- and special diets have an impact on cognition and mitochondrial function in the brain. Thus, appropriate caution is warranted when selecting a suitable diet for preclinical studies in mice.

Toll-like receptor 1 as a possible target in non-alcoholic fatty liver disease.

Toll-like receptors (TLRs) in the liver compartment have repeatedly been attributed to the development of non-alcoholic fatty liver disease (NAFLD). Knowledge on TLR expression in blood cells and their relation to intestinal microbiota and NAFLD development is limited. Here, we determined TLR expression patterns in peripheral blood mononuclear cells (PBMCs) of NAFLD patients and controls, their relation to intestinal microbiota and the impact of TLRs found altered in NAFLD development. Markers of intestinal permeability in blood and TLR mRNA expression in PBMCs were determined in 37 NAFLD patients and 15 age-matched healthy controls. Fecal microbiota composition was evaluated in 21 NAFLD patients and 9 controls using 16S rRNA gene amplicon sequencing. Furthermore, TLR1-/- and C57BL/6 mice (n = 5-6/group) were pair-fed a liquid control or a fat-, fructose- and cholesterol-rich diet. Intestinal microbiota composition and markers of intestinal permeability like zonulin and bacterial endotoxin differed significantly between groups with the latter markers being significantly higher in NAFLD patients. Expression of TLR1-8 and 10 mRNA was detectable in PBMCs; however, only TLR1 expression, being higher in NAFLD patients, were significantly positively correlated with the prevalence of Holdemanella genus while negative correlations were found with Gemmiger and Ruminococcus genera. TLR1-/- mice were significantly protected from the development of diet-induced NAFLD when compared to wild-type mice. While intestinal microbiota composition and permeability differed significantly between NAFLD patients and healthy subjects, in PBMCs, only TLR1 expression differed between groups. Still, targeting these alterations might be a beneficial approach in the treatment of NAFLD in some patients.

Markers of Intestinal Permeability Are Rapidly Improved by Alcohol Withdrawal in Patients with Alcohol-Related Liver Disease.

Changes in intestinal microbiome and barrier function are critical in the development of alcohol-related liver disease (ALD). Here, we determined the effects of a one-week alcohol withdrawal on parameters of intestinal barrier function in heavy drinkers with ALD in comparison to healthy non-drinkers (controls). In serum samples of 17 controls (m = 10/f = 7) and 37 age-matched ALD patients (m = 26/f = 11) undergoing a one-week alcohol withdrawal, markers of liver health and intestinal barrier function were assessed. Liver damage, e.g., fibrosis and hepatic steatosis, were assessed using FibroScan. Before alcohol withdrawal, markers of liver damage, lipopolysaccharide binding protein (LBP) and overall TLR4/TLR2 ligands in serum were significantly higher in ALD patients than in controls, whereas intestinal fatty acid binding protein (I-FABP) and zonulin protein concentrations in serum were lower. All parameters, with the exception of LBP, were significantly improved after alcohol withdrawal; however, not to the level of controls. Our data suggest that one-week of abstinence improves markers of intestinal barrier function and liver health in ALD patients.

Fortifying Butterfat with Soybean Oil Attenuates the Onset of Diet-Induced Non-Alcoholic Steatohepatitis and Glucose Intolerance.

The addition of plant oils such as soybean oil (S) to a diet rich in saturated fatty acids is discussed as a possible route to prevent or diminish the development of metabolic disease. Here, we assessed whether a butterfat-rich diet fortified with S affects the development of early non-alcoholic steatohepatitis (NASH) and glucose intolerance. Female C57BL/6J mice were fed a standard-control diet (C); a fat-, fructose-, and cholesterol-rich diet (FFC, 25E% butterfat, 50% (wt./wt.) fructose, 0.16% (wt./wt.) cholesterol); or FFC supplemented with S (FFC + S, 21E% butterfat + 4E% S) for 13 weeks. Indicators of liver damage, inflammation, intestinal barrier function, and glucose metabolism were measured. Lipopolysaccharide (LPS)-challenged J774A.1 cells were incubated with linolenic and linoleic acids (ratio 1:7.1, equivalent to S). The development of early NASH and glucose intolerance was significantly attenuated in FFC + S-fed mice compared to FFC-fed mice associated with lower hepatic toll-like receptor-4 mRNA expression, while markers of intestinal barrier function were significantly higher than in C-fed mice. Linolenic and linoleic acid significantly attenuated LPS-induced formation of reactive nitrogen species and interleukin-1 beta mRNA expression in J774A.1 cells. Our results indicate that fortifying butterfat with S may attenuate the development of NASH and glucose intolerance in mice.

Microbiota profiling in aging-associated inflammation and liver degeneration.

BACKGROUND: The number of people above the age of 60 years is raising world-wide being associated with an increase in the prevalence of aging-associated impairments and even diseases. Recent studies suggest that aging is associated with alterations in bacterial endotoxin levels and that these changes may add to low-grade inflammation, the so-called 'inflammaging', and aging-associated liver degeneration. However, mechanisms involved, and especially, the interaction of intestinal microbiota and barrier in the development of aging-associated inflammation and liver degeneration have not been fully understood. OBJECTIVE: The aim of the present study was to determine if intestinal microbiota composition changes with age and if these alterations are associated with changes of markers of intestinal barrier function and the development of inflammation and liver degeneration. METHODS: Blood, liver, small and large intestinal tissue of male 2-, 15-, 24- and 30-months old C57BL/6 mice fed standard chow were obtained. Intestinal microbiota composition, expression levels of antimicrobial peptides in small intestine and markers of intestinal barrier function were measured. Furthermore, indices of liver damage, inflammation and expression levels of lipopolysaccharide binding protein (Lbp) as well as of toll-like receptors (Tlr) 1-9 in liver tissue were assessed. RESULTS: Pairwise comparisons of the microbial community in the small intestine showed differences between 2- and 24-, 15- and 24-, as well as 15- and 30-months old animals while Shannon's diversity, species richness and evenness indexes did not differ in both small and large intestine, respectively, between age groups. Concentrations of nitric oxide were significantly lower in small intestine of 15-, 24- and 30-months old mice compared to 2-months old mice while mRNA expression of the antimicrobial peptides defensin alpha 1 and lysozyme 1 was unchanged. In contrast, in liver tissue, older age of animals was associated with increasing inflammation and the development of fibrosis in 24- and 30-months old mice. Numbers of inflammatory foci and neutrophils in livers of 24- and 30-months old mice were significantly higher compared to 2-months old mice. These alterations were also associated with higher endotoxin levels in plasma as well as an increased mRNA expression of Lbp and Tlr1, Tlr2, Tlr4, Tlr6 and Tlr9 in livers in older mice. CONCLUSION: Despite no consistent and robust changes of microbiota composition in small and/or large intestine of mice of different age were observed, our data suggest that alterations of markers of intestinal barrier function in small intestine are associated with an induction of several Tlrs and beginning hepatic inflammation in older mice and increase with age.

Citrulline supplementation attenuates the development of non-alcoholic steatohepatitis in female mice through mechanisms involving intestinal arginase.

Non-alcoholic fatty liver disease (NAFLD) is by now the most prevalent liver disease worldwide. The non-proteogenic amino acid l-citrulline (L-Cit) has been shown to protect mice from the development of NAFLD. Here, we aimed to further assess if L-Cit also attenuates the progression of a pre-existing diet-induced NAFLD and to determine molecular mechanisms involved. Female C57BL/6J mice were either fed a liquid fat-, fructose- and cholesterol-rich diet (FFC) or control diet (C) for 8 weeks to induce early stages of NASH followed by 5 more weeks with either FFC-feeding +/- 2.5 g L-Cit/kg bw or C-feeding. In addition, female C57BL/6J mice were either pair-fed a FFC +/- 2.5 g L-Cit/kg bw +/- 0.01 g/kg bw i.p. N(ω)-hydroxy-nor-l-arginine (NOHA) or C diet for 8 weeks. The protective effects of supplementing L-Cit on the progression of a pre-existing NAFLD were associated with an attenuation of 1) the increased translocation of bacterial endotoxin and 2) the loss of tight junction proteins as well as 3) arginase activity in small intestinal tissue, while no marked changes in intestinal microbiota composition were prevalent in small intestine. Treatment of mice with the arginase inhibitor NOHA abolished the protective effects of L-Cit on diet-induced NAFLD. Our results suggest that the protective effects of L-Cit on the development and progression of NAFLD are related to alterations of intestinal arginase activity and intestinal permeability.

Exchanging dietary fat source with extra virgin olive oil does not prevent progression of diet-induced non-alcoholic fatty liver disease and insulin resistance.

Dietary fat is discussed to be critical in the development of non-alcoholic fatty liver disease. Here, we assess the effect of exchanging dietary fat source from butterfat to extra virgin olive oil on the progression of an already existing diet-induced non-alcoholic fatty liver disease in mice. Female C57BL/6J mice were fed a liquid butterfat-, fructose- and cholesterol-rich diet (BFC, 25E% from butterfat) or control diet (C, 12%E from soybean oil) for 13 weeks. In week 9, fat sources of some BFC- and C-fed mice were switched either to 25E% or 12E% olive oil (OFC and CO). Glucose and insulin tolerance tests were performed, and markers of liver damage and glucose metabolism were assessed. After 6 weeks of feeding, BFC-fed mice had developed marked signs of insulin resistance, which progressed to week 12 being not affected by the exchange of fat sources. Liver damage was similar between BFC- and OFC-fed mice. Markers of lipid metabolism and lipid peroxidation in liver and of insulin signaling in liver and muscle were also similarly altered in BFC- and OFC-fed mice. Taken together, our data suggest that exchanging butterfat with extra virgin olive oil has no effect on the progression of non-alcoholic fatty liver disease and glucose tolerance in mice.

Fortifying diet with rapeseed oil instead of butterfat attenuates the progression of diet-induced non-alcoholic fatty liver disease (NAFLD) and impairment of glucose tolerance.

BACKGROUND: Absolute dietary fat intake but even more so fatty acid pattern is discussed to be critical in the development of non-alcoholic fatty liver disease (NAFLD). Here, we determined if switching a butterfat enriched diet to a rapeseed oil (RO) enriched diet affects progression of an existing NAFLD and glucose intolerance in mice. METHODS: For eight weeks, female C57Bl/6J mice were either fed a liquid control (C) or a butterfat-, fructose- and cholesterol-rich diet (BFC, 25E% butterfat) to induce early signs of steatohepatitis and glucose intolerance in mice. For additional five weeks mice received either BFC or C or a fat-, fructose- and cholesterol-rich and control diet, in which butterfat was replaced with RO (ROFC and CRO). Markers of glucose metabolism, liver damage and intestinal barrier were assessed. RESULTS: Exchanging butterfat with RO attenuated the progression of BFC diet-induced NAFLD and glucose intolerance. Beneficial effects of RO were associated with lower portal endotoxin levels and an attenuation of the induction of the toll-like receptor-4-dependent signaling cascades in liver. Peroxisome proliferator-activated receptor γ activity was induced in small intestine of ROFC-fed mice. CONCLUSION: Taken together, exchanging butterfat with RO attenuated the progression of diet-induced steatohepatitis and glucose intolerance in mice.