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 N^ω‐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.     

IL-1 Signaling in Obesity-Induced Hepatic Lipogenesis and Steatosis

Non-alcoholic fatty liver disease is prevalent in human obesity and type 2 diabetes, and is characterized by increases in both hepatic triglyceride accumulation (denoted as steatosis) and expression of pro-inflammatory cytokines such as IL-1β. We report here that the development of hepatic steatosis requires IL-1 signaling, which upregulates Fatty acid synthase to promote hepatic lipogenesis. Using clodronate liposomes to selectively deplete liver Kupffer cells in ob/ob mice, we observed remarkable amelioration of obesity-induced hepatic steatosis and reductions in liver weight, triglyceride content and lipogenic enzyme expressions. Similar results were obtained with diet-induced obese mice, although visceral adipose tissue macrophage depletion also occurred in response to clodronate liposomes in this model. There were no differences in the food intake, whole body metabolic parameters, serum β-hydroxybutyrate levels or lipid profiles due to clodronate-treatment, but hepatic cytokine gene expressions including IL-1β were decreased. Conversely, treatment of primary mouse hepatocytes with IL-1β significantly increased triglyceride accumulation and Fatty acid synthase expression. Furthermore, the administration of IL-1 receptor antagonist to obese mice markedly reduced obesity-induced steatosis and hepatic lipogenic gene expression. Collectively, our findings suggest that IL-1β signaling upregulates hepatic lipogenesis in obesity, and is essential for the induction of pathogenic hepatic steatosis in obese mice.     

Combined metabolic activators therapy ameliorates liver fat in nonalcoholic fatty liver disease patients

Nonalcoholic fatty liver disease (NAFLD) refers to excess fat accumulation in the liver. In animal experiments and human kinetic study, we found that administration of combined metabolic activators (CMAs) promotes the oxidation of fat, attenuates the resulting oxidative stress, activates mitochondria, and eventually removes excess fat from the liver. Here, we tested the safety and efficacy of CMA in NAFLD patients in a placebo‐controlled 10‐week study. We found that CMA significantly decreased hepatic steatosis and levels of aspartate aminotransferase, alanine aminotransferase, uric acid, and creatinine, whereas found no differences on these variables in the placebo group after adjustment for weight loss. By integrating clinical data with plasma metabolomics and inflammatory proteomics as well as oral and gut metagenomic data, we revealed the underlying molecular mechanisms associated with the reduced hepatic fat and inflammation in NAFLD patients and identified the key players involved in the host–microbiome interactions. In conclusion, we showed that CMA can be used to develop a pharmacological treatment strategy in NAFLD patients.     

Acetate: Friend or foe against breast tumour growth in the context of obesity?

Acetate is reported as a regulator of fat mass but also as lipogenic source for cancer cells. Breast cancer is surrounded by adipose tissue and has been associated with obesity. However, whether acetate contributes to cancer cell metabolism as lipogenic substrate and/or by changing fat storage and eventually obesity‐induced breast cancer progression remains unknown. Therefore, we studied the contribution of acetate to breast cancer metabolism and progression. In vitro, we found that acetate is not a bioenergetic substrate under normoxia and did not result in a significant change of growth. However, by using lipidomic approaches, we discovered that acetate changes the lipid profiles of the cells under hypoxia. Moreover, while mice fed a high‐fat diet (HFD) developed bigger tumours than their lean counterparts, exogenous acetate supplementation leads to a complete abolishment of fat mass gain without reverting the HFD‐induced obesity‐driven tumour progression. In conclusion, although acetate protects against diet‐induced obesity, our data suggest that it is not affecting HFD‐driven tumour progression.     

Polydatin inhibits ZEB1‐invoked epithelial‐mesenchymal transition in fructose‐induced liver fibrosis

High fructose intake is a risk factor for liver fibrosis. Polydatin is a main constituent of the rhizome of Polygonum cuspidatum, which has been used in traditional Chinese medicine to treat liver fibrosis. However, the underlying mechanisms of fructose‐driven liver fibrosis as well as the actions of polydatin are not fully understood. In this study, fructose was found to promote zinc finger E‐box binding homeobox 1 (ZEB1) nuclear translocation, decrease microRNA‐203 (miR‐203) expression, increase survivin, activate transforming growth factor β1 (TGF‐β1)/Smad signalling, down‐regulate E‐cadherin, and up‐regulate fibroblast specific protein 1 (FSP1), vimentin, N‐cadherin and collagen I (COL1A1) in rat livers and BRL‐3A cells, in parallel with fructose‐induced liver fibrosis. Furthermore, ZEB1 nuclear translocation‐mediated miR‐203 low‐expression was found to target survivin to activate TGF‐β1/Smad signalling, causing the EMT in fructose‐exposed BRL‐3A cells. Polydatin antagonized ZEB1 nuclear translocation to up‐regulate miR‐203, subsequently blocked survivin‐activated TGF‐β1/Smad signalling, which were consistent with its protection against fructose‐induced EMT and liver fibrosis. These results suggest that ZEB1 nuclear translocation may play an essential role in fructose‐induced EMT in liver fibrosis by targeting survivin to activate TGF‐β1/Smad signalling. The suppression of ZEB1 nuclear translocation by polydatin may be a novel strategy for attenuating the EMT in liver fibrosis associated with high fructose diet.     

Paracrine activation of hepatic CB1 receptors by stellate cell-derived endocannabinoids mediates alcoholic fatty liver

Alcohol-induced fatty liver, a major cause of morbidity, has been attributed to enhanced hepatic lipogenesis and decreased fat clearance of unknown mechanism. Here we report that the steatosis induced in mice by a low-fat, liquid ethanol diet is attenuated by concurrent blockade of cannabinoid CB1 receptors. Global or hepatocyte-specific CB1 knockout mice are resistant to ethanol-induced steatosis and increases in lipogenic gene expression and have increased carnitine palmitoyltransferase 1 activity, which, unlike in controls, is not reduced by ethanol treatment. Ethanol feeding increases the hepatic expression of CB1 receptors and upregulates the endocannabinoid 2-arachidonoylglycerol (2-AG) and its biosynthetic enzyme diacylglycerol lipase beta selectively in hepatic stellate cells. In control but not CB1 receptor-deficient hepatocytes, coculture with stellate cells from ethanol-fed mice results in upregulation of CB1 receptors and lipogenic gene expression. We conclude that paracrine activation of hepatic CB1 receptors by stellate cell-derived 2-AG mediates ethanol-induced steatosis through increasing lipogenesis and decreasing fatty acid oxidation.     

Protective effect of bile acids on the onset of fructose-induced hepatic steatosis in mice

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 fructose-induced 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 fructose-induced 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)α, 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 levels, and markers of Kupffer cell activation to the level of water controls. Taken together, these data suggest that bile acids prevent fructose-induced hepatic steatosis in mice through mechanisms involving protection against the fructose-induced translocation of intestinal bacterial endotoxin.     

CD36-deficient mice are resistant to alcohol- and high-carbohydrate-induced hepatic steatosis

CD36 is a scavenger receptor with multiple ligands and cellular functions, including facilitating cellular uptake of free fatty acids (FFAs). Chronic alcohol consumption increases hepatic CD36 expression, leading to the hypothesis that this promotes uptake of circulating FFAs, which then serve as a substrate for triglyceride (TG) synthesis and the development of alcoholic steatosis. We investigated this hypothesis in alcohol-fed wild-type and Cd36-deficient (Cd36^−/−) mice using low-fat/high-carbohydrate Lieber-DeCarli liquid diets, positing that Cd36^−/− mice would be resistant to alcoholic steatosis. Our data show that the livers of Cd36^−/− mice are resistant to the lipogenic effect of consuming high-carbohydrate liquid diets. These mice also do not further develop alcoholic steatosis when chronically fed alcohol. Surprisingly, we did not detect an effect of alcohol or CD36 deficiency on hepatic FFA uptake; however, the lower baseline levels of hepatic TG in Cd36^−/− mice fed a liquid diet were associated with decreased expression of genes in the de novo lipogenesis pathway and a lower rate of hepatic de novo lipogenesis. In conclusion, Cd36^−/− mice are resistant to hepatic steatosis when fed a high-carbohydrate liquid diet, and they are also resistant to alcoholic steatosis. These studies highlight an important role for CD36 in hepatic lipid homeostasis that is not associated with hepatic fatty acid uptake.     

Liver Clock Protein BMAL1 Promotes de Novo Lipogenesis through Insulin-mTORC2-AKT Signaling

The clock protein BMAL1 (brain and muscle Arnt-like protein 1) participates in circadian regulation of lipid metabolism, but its contribution to insulin AKT-regulated hepatic lipid synthesis is unclear. Here we used both Bmal1^−/− and acute liver-specific Bmal1-depleted mice to study the role of BMAL1 in refeeding-induced de novo lipogenesis in the liver. Both global deficiency and acute hepatic depletion of Bmal1 reduced lipogenic gene expression in the liver upon refeeding. Conversely, Bmal1 overexpression in mouse liver by adenovirus was sufficient to elevate the levels of mRNA of lipogenic enzymes. Bmal1^−/− primary mouse hepatocytes displayed decreased levels of de novo lipogenesis and lipogenic enzymes, supporting the notion that BMAL1 regulates lipid synthesis in hepatocytes in a cell-autonomous manner. Both refed mouse liver and insulin-treated primary mouse hepatocytes showed impaired AKT activation in the case of either Bmal1 deficiency or Bmal1 depletion by adenoviral shRNA. Restoring AKT activity by a constitutively active mutant of AKT nearly normalized de novo lipogenesis in Bmal1^−/− hepatocytes. Finally, Bmal1 deficiency or knockdown decreased the protein abundance of RICTOR, the key component of the mTORC2 complex, without affecting the gene expression of key factors of insulin signaling. Thus, our study uncovered a novel metabolic function of hepatic BMAL1 that promotes de novo lipogenesis via the insulin-mTORC2-AKT signaling during refeeding.     

Isocaloric manipulation of macronutrients within a high-carbohydrate/moderate-fat diet induces unique effects on hepatic lipogenesis,steatosis and liver injury

Diets containing excess carbohydrate and fat promote hepatic steatosis and steatohepatitis in mice. Little is known, however, about the impact of specific carbohydrate/fat combinations on liver outcome. This study was designed to determine whether high-energy diets with identical caloric density but different carbohydrate and fat composition have unique effects on the liver. Four experimental diets were formulated with 60% kcal carbohydrate and 20% kcal fat, each in nearly pure form from a single source: starch-oleate, starch-palmitate, sucrose-oleate and sucrose-palmitate. The diets were fed to mice for 3 or 12 weeks for analysis of lipid metabolism and liver injury. All mice developed hepatic steatosis over 12 weeks, but mice fed the sucrose-palmitate diet accumulated more hepatic lipid than those in the other three experimental groups. The exaggerated lipid accumulation in sucrose-palmitate-fed mice was attributable to a disproportionate rise in hepatic de novo lipogenesis. These mice accrued more hepatic palmitate and exhibited more evidence of liver injury than any of the other experimental groups. Interestingly, lipogenic gene expression in mice fed the custom diets did not correlate with actual de novo lipogenesis. In addition, de novo lipogenesis rose in all mice between 3 and 12 weeks, without feedback inhibition from hepatic steatosis. The pairing of simple sugar (sucrose) and saturated fat (palmitate) in a high-carbohydrate/moderate-fat diet induces more de novo lipogenesis and liver injury than other carbohydrate/fat combinations. Diet-induced liver injury correlates positively with hepatic de novo lipogenesis and is not predictable by isolated analysis of lipogenic gene expression.     

Combination of honokiol and magnolol inhibits hepatic steatosis through AMPK-SREBP-1?c pathway

Honokiol and magnolol, as pharmacological biphenolic compounds of Magnolia officinalis, have been reported to have antioxidant and anti-inflammatory properties. Sterol regulatory element binding protein-1 c (SREBP-1 c) plays an important role in the development and processing of steatosis in the liver. In the present study, we investigated the effects of a combination of honokiol and magnolol on SREBP-1 c-dependent lipogenesis in hepatocytes as well as in mice with fatty liver due to consumption of high-fat diet (HFD). Liver X receptor α (LXRα) agonists induced activation of SREBP-1 c and expression of lipogenic genes, which were blocked by co-treatment of honokiol and magnolol (HM). Moreover, a combination of HM potently increased mRNA of fatty acid oxidation genes. HM induced AMP-activated protein kinase (AMPK), an inhibitory kinase of the LXRα-SREBP-1 c pathway. The role of AMPK activation induced by HM was confirmed using an inhibitor of AMPK, Compound C, which reversed the ability of HM to both inhibit SREBP-1 c induction as well as induce genes for fatty acid oxidation. In mice, HM administration for four weeks ameliorated HFD-induced hepatic steatosis and liver dysfunction, as indicated by plasma parameters and Oil Red O staining. Taken together, our results demonstrated that a combination of HM has beneficial effects on inhibition of fatty liver and SREBP-1 c-mediated hepatic lipogenesis, and these events may be mediated by AMPK activation.     

Involvement of AMP-activated protein kinase in beneficial effects of betaine on high-sucrose diet-induced hepatic steatosis

Although simple steatosis was originally thought to be a pathologically inert histological change, fat accumulation in the liver may play a critical role not only in disease initiation, but also in the progression to nonalcoholic steatohepatitis and cirrhosis. Therefore, prevention of fat accumulation in the liver may be an effective therapy for multiple stages of nonalcoholic fatty liver disease (NAFLD). Promising beneficial effects of betaine supplementation on human NAFLD have been reported in some pilot clinical studies; however, data related to betaine therapy in NAFLD are limited. In this study, we examined the effects of betaine on fat accumulation in the liver induced by high-sucrose diet and evaluated mechanisms by which betaine could attenuate or prevent hepatic steatosis in this model. Male C57BL/6 mice weighing 20 +/- 0.5 g (means +/- SE) were divided into four groups (8 mice per group) and started on one of four treatments: standard diet (SD), SD+betaine, high-sucrose diet (HS), and HS + betaine. Betaine was supplemented in the drinking water at a concentration of 1% (wt/vol) (anhydrous). Long-term feeding of high-sucrose diet to mice caused significant hepatic steatosis accompanied by markedly increased lipogenic activity. Betaine significantly attenuated hepatic steatosis in this animal model, and this change was associated with increased activation of hepatic AMP-activated protein kinase (AMPK) and attenuated lipogenic capability (enzyme activities and gene expression) in the liver. Our findings are the first to suggest that betaine might serve as a therapeutic tool to attenuate hepatic steatosis by targeting the hepatic AMPK system.     

Omega-3 phospholipids from fish suppress hepatic steatosis by integrated inhibition of biosynthetic pathways in dietary obese mice

Non-alcoholic fatty liver disease (NAFLD) accompanies obesity and insulin resistance. Recent meta-analysis suggested omega-3 polyunsaturated fatty acids DHA and EPA to decrease liver fat in NAFLD patients. Anti-inflammatory, hypolipidemic, and insulin-sensitizing effects of DHA/EPA depend on their lipid form, with marine phospholipids showing better efficacy than fish oils. We characterized the mechanisms underlying beneficial effects of DHA/EPA phospholipids, alone or combined with an antidiabetic drug, on hepatosteatosis. C57BL/6N mice were fed for 7 weeks an obesogenic high-fat diet (cHF) or cHF-based interventions: (i) cHF supplemented with phosphatidylcholine-rich concentrate from herring (replacing 10% of dietary lipids; PC), (ii) cHF containing rosiglitazone (10 mg/kg diet; R), or (iii) PC + R. Metabolic analyses, hepatic gene expression and lipidome profiling were performed. Results showed that PC and PC + R prevented cHF-induced weight gain and glucose intolerance, while all interventions reduced abdominal fat and plasma triacylglycerols. PC and PC + R also lowered hepatic and plasma cholesterol and reduced hepatosteatosis. Microarray analysis revealed integrated down-regulation of hepatic lipogenic and cholesterol biosynthesis pathways by PC, while R-induced lipogenesis was fully counteracted in PC + R. Gene expression changes in PC and PC + R were associated with preferential enrichment of hepatic phosphatidylcholine and phosphatidylethanolamine fractions by DHA/EPA. The complex down-regulation of hepatic lipogenic and cholesterol biosynthesis genes and the antisteatotic effects were unique to DHA/EPA-containing phospholipids, since they were absent in mice fed soy-derived phosphatidylcholine. Thus, inhibition of lipid and cholesterol biosynthesis associated with potent antisteatotic effects in the liver in response to DHA/EPA-containing phospholipids support their use in NAFLD prevention and treatment.     

Phosphorylation of eIF2α signaling pathway attenuates obesity-induced non-alcoholic fatty liver disease in an ER stress and autophagy-dependent manner

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disorder and frequently exacerbates in postmenopausal women. In NAFLD, the endoplasmic reticulum (ER) plays an important role in lipid metabolism, in which salubrinal is a selective inhibitor of eIF2α de-phosphorylation in response to ER stress. To determine the potential mechanism of obesity-induced NAFLD, we employed salubrinal and evaluated the effect of ER stress and autophagy on lipid metabolism. Ninety-five female C57BL/6 mice were randomly divided into five groups: standard chow diet, high-fat (HF) diet, HF with salubrinal, HF with ovariectomy, and HF with ovariectomy and salubrinal. All mice except for SC were given HF diet. After the 8-week obesity induction, salubrinal was subcutaneously injected for the next 8 weeks. The expression of ER stress and autophagy markers was evaluated in vivo and in vitro. Compared to the normal mice, the serum lipid level and adipose tissue were increased in obese mice, while salubrinal attenuated obesity by blocking lipid disorder. Also, the histological severity of hepatic steatosis and fibrosis in the liver and lipidosis was suppressed in response to salubrinal. Furthermore, salubrinal inhibited ER stress by increasing the expression of p-eIF2α and ATF4 with a decrease in the level of CHOP. It promoted autophagy by increasing LC3II/I and inhibiting p62. Correlation analysis indicated that lipogenesis in the development of NAFLD was associated with ER stress. Collectively, we demonstrated that eIF2α played a key role in obesity-induced NAFLD, and salubrinal alleviated hepatic steatosis and lipid metabolism by altering ER stress and autophagy through eIF2α signaling.Subject terms: Obesity, Metabolic syndrome, Outcomes research     

GCN2 deficiency protects against high fat diet induced hepatic steatosis and insulin resistance in mice

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid deposition and oxidative stress. It has been demonstrated that general control nonderepressible 2 (GCN2) is required to maintain hepatic fatty acid homeostasis under conditions of amino acid deprivation. However, the impact of GCN2 on the development of NAFLD has not been investigated. In this study, we used Gcn2^?/? mice to investigate the effect of GCN2 on high fat diet (HFD)-induced hepatic steatosis. After HFD feeding for 12?weeks, Gcn2^?/? mice were less obese than wild-type (WT) mice, and Gcn2^?/? significantly attenuated HFD-induced liver dysfunction, hepatic steatosis and insulin resistance. In the livers of the HFD-fed mice, GCN2 deficiency resulted in higher levels of lipolysis genes, lower expression of genes related to FA synthesis, transport and lipogenesis, and less induction of oxidative stress. Furthermore, we found that knockdown of GCN2 attenuated, whereas overexpression of GCN2 exacerbated, palmitic acid-induced steatosis, oxidative & ER stress, and changes of peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FAS) and metallothionein (MT) expression in HepG2 cells. Collectively, our data provide evidences that GCN2 deficiency protects against HFD-induced hepatic steatosis by inhibiting lipogenesis and reducing oxidative stress. Our findings suggest that strategies to inhibit GCN2 activity in the liver may provide a novel approach to attenuate NAFLD development.