Ammonia Scavenger Restores Liver and Muscle Injury in a Mouse Model of Non-alcoholic Steatohepatitis With Sarcopenic Obesity.

Recent studies have revealed that sarcopenia is closely associated with obesity and non-alcoholic steatohepatitis (NASH). However, few attempted to explore the cause-and-effect relationship between sarcopenic obesity and NASH. In this study, we investigated muscular alterations in a rodent NASH model to elucidate their intrinsic relations and explore the potential therapeutic target. Forty-six 8-week-old and twenty 42-week-old male C57BL/6 mice (defined as young and middle-aged mice, respectively) were fed with a high-fat diet (HFD) for 12 or 20 weeks. A subset of young mice was subjected to ammonia lowering treatment by L-ornithine L-aspartate (LOLA). We examined body composition and muscle strength by nuclear magnetic resonance and grip strength meter, respectively. At the end of the 12th week, all HFD-fed mice developed typical steatohepatitis. Meanwhile, sarcopenia occurred in HFD-fed middle-aged mice, whereas young mice only demonstrated decreased grip strength. Until the end of week 20, young mice in the HFD group exhibited significant sarcopenia and obesity phenotypes, including decreased lean body mass and grip strength, and increased body fat mass and percentage body fat. Additionally, plasma ammonia level was markedly increased in HFD-fed mice of both ages at week 20. Plasma ammonia level was negatively associated with muscle strength and myofiber diameter in young mice. LOLA can significantly reduce plasma levels of ammonia, alanine aminotransaminase, aspartate aminotransaminase, and cholesterol in mice fed an HFD. Hepatic infiltration of inflammatory cells and collagen deposition area were significantly decreased in HFD group by LOLA treatment. Meanwhile, LOLA significantly increased lean body mass, grip strength, and average muscle fiber diameter of HFD-fed mice. These findings suggest that the occurrence of NASH precedes sarcopenia in HFD mice, and the steatohepatitis-related hyperammonemia might contribute to the pathogenesis of sarcopenia. LOLA might be an effective drug for both steatohepatitis and sarcopenic obesity.

Escherichia fergusonii Promotes Nonobese Nonalcoholic Fatty Liver Disease by Interfering With Host Hepatic Lipid Metabolism Through Its Own msRNA 23487.

BACKGROUND & AIMS: Gut microbiota and microbial factors regulate the pathogenesis of nonalcoholic fatty liver disease (NAFLD) in patients with obesity and metabolic abnormalities, but little is known about their roles in nonobese NAFLD. Expansion of Escherichia is associated with NAFLD pathogenesis. We aimed to investigate the pathogenic role of Escherichia fergusonii and its products in the development of nonobese NAFLD. METHODS: We characterized the intestinal microbiome signature in a cohort of NAFLD patients and healthy controls by 16S ribosomal RNA sequencing. The role of E fergusonii was estimated in rats after 16 weeks of administration, and features of NAFLD were assessed. E fergusonii-derived microRNA-sized, small RNAs (msRNAs) were analyzed by deep sequencing. RESULTS: We detected an expansion of Escherichia_Shigella in NAFLD patients compared with healthy controls, and its increase was associated with disease severity independent of obesity. E fergusonii, a member of the genus Escherichia, induced the development of nonobese NAFLD characterized by hepatic steatosis and hepatocyte ballooning in rats without obesity. It disturbed host lipid metabolism by inhibiting hepatic lipid ß-oxidation and promoting de novo lipogenesis. We also showed that E fergusonii caused the development of hepatic inflammation and fibrosis in a sizable fraction of animals at an advanced stage of NAFLD. Mechanistically, E fergusonii-derived msRNA 23487 down-regulated host hepatic peroxisome proliferator-activated receptor α expression, which could contribute to lipid accumulation in the liver. CONCLUSIONS: These results suggest that E fergusonii promotes the pathogenesis of steatohepatitis and fibrosis in nonobese rats by secreting msRNA 23487, and it might be a potential biomarker for predicting steatohepatitis in nonobese NAFLD.

Sodium Butyrate Supplementation Inhibits Hepatic Steatosis by Stimulating Liver Kinase B1 and Insulin-Induced Gene.

BACKGROUND AND AIMS: Butyric acid is an intestinal microbiota-produced short-chain fatty acid, which exerts salutary effects on alleviating nonalcoholic fatty liver disease (NAFLD). However, the underlying mechanism of butyrate on regulating hepatic lipid metabolism is largely unexplored. METHODS: A mouse model of NAFLD was induced with high-fat diet feeding, and sodium butyrate (NaB) intervention was initiated at the eighth week and lasted for 8 weeks. Hepatic steatosis was evaluated and metabolic pathways concerning lipid homeostasis were analyzed. RESULTS: Here, we report that administration of NaB by gavage once daily for 8 weeks causes an augmentation of insulin-induced gene (Insig) activity and inhibition of lipogenic gene in mice fed with high-fat diet. Mechanistically, NaB is sufficient to enhance the interaction between Insig and its upstream kinase AMP-activated protein kinase (AMPK). The stimulatory effects of NaB on Insig-1 activity are abolished in AMPKα1/α2 double knockout (AMPK-/-) mouse primary hepatocytes. Moreover, AMPK activation by NaB is mediated by LKB1, as evidenced by the observations showing NaB-mediated induction of phosphorylation of AMPK, and its downstream target acetyl-CoA carboxylase is diminished in LKB1-/- mouse embryonic fibroblasts. CONCLUSIONS: These studies indicate that NaB serves as a negative regulator of hepatic lipogenesis in NAFLD and that NaB attenuates hepatic steatosis and improves lipid profile and liver function largely through the activation of LKB1-AMPK-Insig signaling pathway. Therefore, NaB has therapeutic potential for treating NAFLD and related metabolic diseases.

Neonatal Milk Fat Globule Membrane Supplementation During Breastfeeding Ameliorates the Deleterious Effects of Maternal High-Fat Diet on Metabolism and Modulates Gut Microbiota in Adult Mice Offspring in a Sex-Specific Way.

Exposure to adverse events in early life increases the risk of chronic metabolic disease in adulthood. The objective of this study was to determine the significance of milk fat globule membrane (MFGM)-mediated alterations in the gut microbiome to the metabolic health of offspring in the long-term. Female C57BL/6 mice were fed either a high-fat diet (HFD) or a control diet for 3 weeks before pregnancy and throughout pregnancy and lactation. During lactation, pups from the HFD group were breast-fed with or without 1,000 mg/kg BW/day MFGM supplementation (HFD and HFD-MS group, respectively). After weaning, the offspring in each group were divided into male and female subgroups. The weaned mice were then shifted to a control diet for 8 weeks. At the eleventh week, stool samples were collected for 16S rRNA gene sequencing. Serum biochemical parameters were analyzed, and intraperitoneal glucose and insulin tolerance tests were performed. Neonatal supplementation with MFGM ameliorated metabolic disorder and improved glucose tolerance in offspring exposed to maternal HFD in a sex-specific manner. Furthermore, maternal HFD induced gut microbiota perturbation in offspring in adulthood. Neonatal MFGM supplementation significantly enriched g-Parabacteroides, g-Bifidobacterium, g-Faecalibaculum, and g-Lactobacillus in male offspring exposed to maternal HFD, while significantly enriched g-Parabacteroides and g-Alistipes in female offspring exposed to maternal HFD. These bacteria may be associated with the favorable changes in metabolism that occur in adulthood. Sex differences in the changes of metagenomic pathways related to oxidative phosphorylation, citrate cycle, electron transfer carries, and ubiquinone biosynthesis were also observed in the offspring. Maternal HFD has an adverse effect on the metabolism of offspring in later life. Neonatal MFGM supplementation could modulate the structure of gut microbiota communities and may have long-term protective effects on lipid and glucose metabolism, but these effects are sex dimorphic.

Milk Fat Globule Membrane Supplementation During Suckling Ameliorates Maternal High Fat Diet-Induced Hepatic Steatosis in Adult Male Offspring of Mice.

BACKGROUND: Exposure to a maternal high-fat diet (HFD) predisposes offspring to nonalcoholic fatty liver disease. OBJECTIVES: The aim of this study was to explore whether milk fat globule membrane (MFGM) supplementation during suckling exerts a long-term protective effect on hepatic lipid metabolism in adult offspring exposed to maternal HFD. METHODS: We fed 5-week-old female C57BL/6J mice either a HFD (60% kcal fat) or control diet (CD; 16.7% kcal fat) for 3 weeks before mating, as well as throughout gestation and lactation. After delivery, male offspring from HFD dams were supplemented with 1 g/(kg body weight·day) MFGM (HFD + MFGM group) or the same volume of vehicle (HFD group) during suckling. Male offspring from CD dams were also supplemented with vehicle during suckling (CD group). All offspring were weaned onto CD for 8 weeks. Histopathology, metabolic parameters, lipogenic level, oxidative stress, and mitochondria function in the liver were analyzed. A 1-way ANOVA and a Kruskal-Wallis test were used for multi-group comparisons. RESULTS: As compared to the CD group, the HFD group had more lipid droplets in livers, and exhibited ∼100% higher serum triglycerides, ∼38% higher hepatic triglycerides, ∼75% higher serum aspartate aminotransferase, and ∼130% higher fasting blood glucose (P < 0.05). The changes of these metabolic parameters were normalized in the HFD + MFGM group. Phosphorylated mammalian targets of rapamycin and AKT were downregulated, but phosphorylated adenosine monophosphate-activated protein kinase was upregulated in the HFD + MFGM group as compared to the HFD group (P < 0.05). As compared to the CD group, the HFD group showed an ∼80% higher malondialdehyde level, and ∼20% lower superoxide dismutase activity (P < 0.05), which were normalized in the HFD + MFGM group. Additionally, mitochondria function was also impaired in the HFD group and normalized in the HFD + MFGM group. CONCLUSIONS: MFGM supplementation during suckling ameliorates maternal HFD-induced hepatic steatosis in mice via suppressing de novo lipogenesis, reinforcing antioxidant defenses and improving mitochondrial function.

Folic acid attenuates high-fat diet-induced steatohepatitis via deacetylase SIRT1-dependent restoration of PPARα.

BACKGROUND: Folic acid has been shown to improve non-alcoholic steatohepatitis (NASH), but its roles in hepatic lipid metabolism, hepatic one-carbon metabolism, and gut microbiota are still unknown. AIM: To demonstrate the role of folic acid in lipid metabolism and gut microbiota in NASH. METHODS: Twenty-four Sprague-Dawley rats were assigned into three groups: Chow diet, high-fat diet (HFD), and HFD with folic acid administration. At the end of 16 wk, the liver histology, the expression of hepatic genes related to lipid metabolism, one-carbon metabolism, and gut microbiota structure analysis of fecal samples based on 16S rRNA sequencing were measured to evaluate the effect of folic acid. Palmitic acid-exposed Huh7 cell line was used to evaluate the role of folic acid in hepatic lipid metabolism. RESULTS: Folic acid treatment attenuated steatosis, lobular inflammation, and hepatocellular ballooning in rats with HFD-induced steatohepatitis. Genes related to lipid de novo lipogenesis, ß-oxidation, and lipid uptake were improved in HFD-fed folic acid-treated rats. Furthermore, peroxisome proliferator-activated receptor alpha (PPARα) and silence information regulation factor 1 (SIRT1) were restored by folic acid in HFD-fed rats and palmitic acid-exposed Huh7 cell line. The restoration of PPARα by folic acid was blocked after transfection with SIRT1 siRNA in the Huh7 cell line. Additionally, folic acid administration ameliorated depleted hepatic one-carbon metabolism and restored the diversity of the gut microbiota in rats with HFD-induced steatohepatitis. CONCLUSION: Folic acid improves hepatic lipid metabolism by upregulating PPARα levels via a SIRT1-dependent mechanism and restores hepatic one-carbon metabolism and diversity of gut microbiota, thereby attenuating HFD-induced NASH in rats.

Lipotoxic Hepatocyte-Derived Exosomal MicroRNA 192-5p Activates Macrophages Through Rictor/Akt/Forkhead Box Transcription Factor O1 Signaling in Nonalcoholic Fatty Liver Disease.

BACKGROUND AND AIMS: Hepatic macrophages can be activated by many factors such as gut-derived bacterial components and factors released from damaged hepatocytes. Macrophage polarization toward a proinflammatory phenotype (M1) represents an important event in the disease progression of nonalcoholic fatty liver disease (NAFLD). However, the underlying molecular mechanisms remain incompletely understood. Exosomes have been identified as important mediators for cell-cell communication by transferring various biological components such as microRNAs (miRs), proteins, and lipids. The role of exosomes in crosstalk between hepatocytes and macrophages in disease progression of NAFLD is yet to be explored. APPROACH AND RESULTS: In the present study, we reported that lipotoxic injury-induced release of hepatocyte exosomes enriched with miR-192-5p played a critical role in the activation of M1 macrophages and hepatic inflammation. Serum miR-192-5p levels in patients with NAFLD positively correlated with hepatic inflammatory activity score and disease progression. Similarly, the serum miR-192-5p level and the number of M1 macrophages, as well as the expression levels of the hepatic proinflammatory mediators, were correlated with disease progression in high-fat high-cholesterol diet-fed rat models. Lipotoxic hepatocytes released more miR-192-5p-enriched exosomes than controls, which induced M1 macrophage (cluster of differentiation 11b-positive [CD11b+ ]/CD86+ ) activation and increase of inducible nitric oxide synthase, interleukin 6, and tumor necrosis factor alpha expression. Furthermore, hepatocyte-derived exosomal miR-192-5p inhibited the protein expression of the rapamycin-insensitive companion of mammalian target of rapamycin (Rictor), which further inhibited the phosphorylation levels of Akt and forkhead box transcription factor O1 (FoxO1) and resulted in activation of FoxO1 and subsequent induction of the inflammatory response. CONCLUSIONS: Hepatocyte-derived exosomal miR-192-5p plays a critical role in the activation of proinflammatory macrophages and disease progression of NAFLD through modulating Rictor/Akt/FoxO1 signaling. Serum exosomal miR-192-5p represents a potential noninvasive biomarker and therapeutic target for nonalcoholic steatohepatitis.

Therapeutic effect and autophagy regulation of myriocin in nonalcoholic steatohepatitis.

BACKGROUND: Ceramide plays pathogenic roles in nonalcoholic fatty liver disease (NAFLD) via multiple mechanisms, and as such inhibition of ceramide de novo synthesis in the liver may be of therapeutically beneficial in patients with NAFLD. In this study, we aimed to explore whether inhibition of ceramide signaling by myriocin is beneficial in animal model of NAFLD via regulating autophagy. METHODS: Sprague Dawley rats were randomly divided into three groups: standard chow (n = 10), high-fat diet (HFD) (n = 10) or HFD combined with oral administration of myriocin (0.3 mg/kg on alternate days for 8 weeks) (n = 10). Liver histology and autophagy function were measured. HepG2 cells were incubated with fatty acid with or without myriocin treatment. Lipid accumulation and autophagy markers in the HepG2 cells were analyzed. Serum ceramide changes were studied in 104 subjects consisting healthy adults, liver biopsy-proven patients with NAFLD and liver biopsy-proven patients with chronic hepatitis B (CHB). RESULTS: Myriocin reversed the elevated body weight and serum transaminases and alleviated dyslipidemia in HFD fed rats. Myriocin treatment significantly attenuated liver pathology including steatosis, lobular inflammation and ballooning. By qPCR analysis, it was revealed that myriocin corrected the expression pattern of fatty acid metabolism associated genes including Fabp1, Pparα, Cpt-1α and Acox-2. Further, myriocin also restored the impaired hepatic autophagy function in rats with HFD-induced NASH, and this has been verified in HepG2 cells. Among the sphingolipid species that we screened in lipidomic profiles, significantly increased ceramide was observed in NASH patients as compared to the controls and non-NASH patients, regardless of whether or not they have active CHB. CONCLUSIONS: Ceramide may play an important regulatory role in the autophagy function in the pathogenesis of NASH. Hence, blockade of ceramide signaling by myriocin may be of therapeutically beneficial in NASH. TRIAL REGISTRATION: Registration ID: ChiCTR-DDT-13003983 . Data of registration: 13 May, 2013, retrospectively registered.

Indole-3-propionic acid inhibits gut dysbiosis and endotoxin leakage to attenuate steatohepatitis in rats.

Microbial metabolites have emerged as critical components that mediate the metabolic effects of the gut microbiota. Here, we show that indole-3-propionic acid (IPA), a tryptophan metabolite produced by gut bacteria, is a potent anti-non-alcoholic steatohepatitis (NASH) microbial metabolite. Here, we demonstrate that administration of IPA modulates the microbiota composition in the gut and inhibits microbial dysbiosis in rats fed a high-fat diet. IPA induces the expression of tight junction proteins, such as ZO-1 and Occludin, and maintains intestinal epithelium homeostasis, leading to a reduction in plasma endotoxin levels. Interestingly, IPA inhibits NF-κB signaling and reduces the levels of proinflammatory cytokines, such as TNFα, IL-1ß, and IL-6, in response to endotoxin in macrophages to repress hepatic inflammation and liver injury. Moreover, IPA is sufficient to inhibit the expression of fibrogenic and collagen genes and attenuate diet-induced NASH phenotypes. The beneficial effects of IPA on the liver are likely mediated through inhibiting the production of endotoxin in the gut. These findings suggest a protective role of IPA in the control of metabolism and uncover the gut microbiome and liver cross-talk in regulating the intestinal microenvironment and liver pathology via a novel dietary nutrient metabolite. IPA may provide a new therapeutic strategy for treating NASH.

Trimethylamine N-oxide attenuates high-fat high-cholesterol diet-induced steatohepatitis by reducing hepatic cholesterol overload in rats.

BACKGROUND: Trimethylamine N-oxide (TMAO) has been shown to be involved in cardiovascular disease (CVD). However, its role in nonalcoholic steatohepatitis (NASH) is unknown. AIM: To determine the effect of TMAO on the progression of NASH. METHODS: A rat model was induced by 16-wk high-fat high-cholesterol (HFHC) diet feeding and TMAO was administrated by daily oral gavage for 8 wk. RESULTS: Oral TMAO intervention attenuated HFHC diet-induced steatohepatitis in rats. Histological evaluation showed that TMAO treatment significantly alleviated lobular inflammation and hepatocyte ballooning in the livers of rats fed a HFHC diet. Serum levels of alanine aminotransferase and aspartate aminotransferase were also decreased by TMAO treatment. Moreover, hepatic endoplasmic reticulum (ER) stress and cell death were mitigated in HFHC diet-fed TMAO-treated rats. Hepatic and serum levels of cholesterol were both decreased by TMAO treatment in rats fed a HFHC diet. Furthermore, the expression levels of intestinal cholesterol transporters were detected. Interestingly, cholesterol influx-related Niemann-Pick C1-like 1 was downregulated and cholesterol efflux-related ABCG5/8 were upregulated by TMAO treatment in the small intestine. Gut microbiota analysis showed that TMAO could alter the gut microbial profile and restore the diversity of gut flora. CONCLUSION: These data suggest that TMAO may modulate the gut microbiota, inhibit intestinal cholesterol absorption, and ameliorate hepatic ER stress and cell death under cholesterol overload, thereby attenuating HFHC diet-induced steatohepatitis in rats. Further studies are needed to evaluate the influence on CVD and define the safe does of TMAO treatment.

Sodium butyrate reduces high-fat diet-induced non-alcoholic steatohepatitis through upregulation of hepatic GLP-1R expression.

Glucagon-like peptide-1 (GLP-1) has a broad spectrum of biological activity by regulating metabolic processes via both the direct activation of the class B family of G protein-coupled receptors and indirect nonreceptor-mediated pathways. GLP-1 receptor (GLP-1R) agonists have significant therapeutic effects on non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) in animal models. However, clinical studies indicated that GLP-1 treatment had little effect on hepatic steatosis in some NAFLD patients, suggesting that GLP-1 resistance may occur in these patients. It is well-known that the gut metabolite sodium butyrate (NaB) could promote GLP-1 secretion from intestinal L cells. However, it is unclear whether NaB improves hepatic GLP-1 responsiveness in NAFLD. In the current study, we showed that the serum GLP-1 levels of NAFLD patients were similar to those of normal controls, but hepatic GLP-1R expression was significantly downregulated in NAFLD patients. Similarly, in the NAFLD mouse model, mice fed with a high-fat diet showed reduced hepatic GLP-1R expression, which was reversed by NaB treatment and accompanied by markedly alleviated liver steatosis. In addition, NaB treatment also upregulated the hepatic p-AMPK/p-ACC and insulin receptor/insulin receptor substrate-1 expression levels. Furthermore, NaB-enhanced GLP-1R expression in HepG2 cells by inhibiting histone deacetylase-2 independent of GPR43/GPR109a. These results indicate that NaB is able to prevent the progression of NAFL to NASH via promoting hepatic GLP-1R expression. NaB is a GLP-1 sensitizer and represents a potential therapeutic adjuvant to prevent NAFL progression to NASH.

Sodium butyrate attenuates high-fat diet-induced steatohepatitis in mice by improving gut microbiota and gastrointestinal barrier.

AIM: To investigate whether gut microbiota metabolite sodium butyrate (NaB) is an effective substance for attenuating non-alcoholic fatty liver disease (NAFLD) and the internal mechanisms. METHODS: Male C57BL/6J mice were divided into three groups, normal control were fed standard chow and model group were fed a high-fat diet (HFD) for 16 wk, the intervention group were fed HFD for 16 wk and treated with NaB for 8 wk. Gut microbiota from each group were detected at baseline and at 16 wk, liver histology were evaluated and gastrointestinal barrier indicator such as zonula occluden-1 (ZO-1) were detected by immunohistochemistry and realtime-PCR, further serum or liver endotoxin were determined by ELISA and inflammation- or metabolism-associated genes were quantified by real-time PCR. RESULTS: NaB corrected the HFD-induced gut microbiota imbalance in mice, while it considerably elevated the abundances of the beneficial bacteria Christensenellaceae, Blautia and Lactobacillus. These bacteria can produce butyric acid in what seems like a virtuous circle. And butyrate restored HFD induced intestinal mucosa damage, increased the expression of ZO-1 in small intestine, further decreased the levels of gut endotoxin in serum and liver compared with HF group. Endotoxin-associated genes such as TLR4 and Myd88, pro-inflammation genes such as MCP-1, TNF-α, IL-1, IL-2, IL-6 and IFN-γ in liver or epididymal fat were obviously downregulated after NaB intervention. Liver inflammation and fat accumulation were ameliorated, the levels of TG and cholesterol in liver were decreased after NaB intervention, NAS score was significantly decreased, metabolic indices such as FBG and HOMA-IR and liver function indicators ALT and AST were improved compared with HF group. CONCLUSION: NaB may restore the dysbiosis of gut microbiota to attenuate steatohepatitis, which is suggested to be a potential gut microbiota modulator and therapeutic substance for NAFLD.

miR-192-5p regulates lipid synthesis in non-alcoholic fatty liver disease through SCD-1.

AIM: To evaluate the levels of miR-192-5p in non-alcoholic fatty liver disease (NAFLD) models and demonstrate the role of miR-192-5p in lipid accumulation. METHODS: Thirty Sprague Dawley rats were randomly divided into three groups, which were given a standard diet, a high-fat diet (HFD), and an HFD with injection of liraglutide. At the end of 16 weeks, hepatic miR-192-5p and stearoyl-CoA desaturase 1 (SCD-1) levels were measured. MiR-192-5p mimic and inhibitor and SCD-1 siRNA were transfected into Huh7 cells exposed to palmitic acid (PA). Lipid accumulation was evaluated by oil red O staining and triglyceride assays. Direct interaction was validated by dual-luciferase reporter gene assays. RESULTS: The HFD rats showed a 0.46-fold decrease and a 3.5-fold increase in hepatic miR-192-5p and SCD-1 protein levels compared with controls, respectively, which could be reversed after disease remission by liraglutide injection (P < 0.01). The Huh7 cells exposed to PA also showed down-regulation and up-regulation of miR-192-5p and SCD-1 protein levels, respectively (P < 0.01). Transfection with miR-192-5p mimic and inhibitor in Huh7 cells induced dramatic repression and promotion of SCD-1 protein levels, respectively (P < 0.01). Luciferase activity was suppressed and enhanced by miR-192-5p mimic and inhibitor, respectively, in wild-type SCD-1 (P < 0.01) but not in mutant SCD-1. MiR-192-5p overexpression reduced lipid accumulation significantly in PA-treated Huh7 cells, and SCD-1 siRNA transfection abrogated the lipid deposition aggravated by miR-192-5p inhibitor (P < 0.01). CONCLUSION: This study demonstrates that miR-192-5p has a negative regulatory role in lipid synthesis, which is mediated through its direct regulation of SCD-1.