Coffee Restores Expression of lncRNAs Involved in Steatosis and Fibrosis in a Mouse Model of NAFLD.

BACKGROUND AND AIM: Coffee intake exerts protective effects against non-alcoholic fatty liver disease (NAFLD), although without fully cleared mechanisms. In this study we aimed to assess whether coffee consumption may influence the expression of long non-coding RNAs (lncRNAs) in the liver. METHODS: C57BL/6J mice were fed a 12-week standard diet (SD), high-fat diet (HFD) or HFD plus decaffeinated coffee solution (HFD + coffee). Expression of specific lncRNAs involved in NAFLD was analyzed by real-time PCR. For the most differentially expressed lncRNAs, the analysis was also extended to their mRNA targets. RESULTS: Decaffeinated coffee intake reduced body weight gain, prevented NAFLD, lowered hyperglycemia and hypercholesterolemia. NAFLD was associated with lower hepatic expression of Gm16551, a lncRNA inhibiting de novo lipogenesis, and higher expression of H19, a lncRNA promoting fibrogenesis. Coffee intake restored Gm16551 to levels observed in lean mice and downregulated gene expression of its targets acetyl coenzyme A carboxylase 1 and stearoyl coenzyme A desaturase 1. Furthermore, coffee consumption markedly decreased hepatic expression of H19 and of its target gene collagen alpha-1(I) chain; consistently, in mice fed HFD + coffee liver expression of αSMA protein returned to levels of mice fed SD. Expression of lncRNA involved in circadian clock such as fatty liver-related lncRNA 1 (FLRL1) and fatty liver-related lncRNA 2 (FLRL2) were upregulated by HFD and were also modulated by coffee intake. CONCLUSION: Hepatoprotective effects of coffee may be depending on the modulation of lncRNAs involved in key pathways of NAFLD onset and progression.

The Combination of Berberine, Tocotrienols and Coffee Extracts Improves Metabolic Profile and Liver Steatosis by the Modulation of Gut Microbiota and Hepatic miR-122 and miR-34a Expression in Mice.

Non-alcoholic-fatty liver disease (NAFLD) is spreading worldwide. Specific drugs for NAFLD are not yet available, even if some plant extracts show beneficial properties. We evaluated the effects of a combination, composed by Berberis Aristata, Elaeis Guineensis and Coffea Canephora, on the development of obesity, hepatic steatosis, insulin-resistance and on the modulation of hepatic microRNAs (miRNA) levels and microbiota composition in a mouse model of liver damage. C57BL/6 mice were fed with standard diet (SD, n = 8), high fat diet (HFD, n = 8) or HFD plus plant extracts (HFD+E, n = 8) for 24 weeks. Liver expression of miR-122 and miR-34a was evaluated by quantitativePCR. Microbiome analysis was performed on cecal content by 16S rRNA sequencing. HFD+E-mice showed lower body weight (p < 0.01), amelioration of insulin-sensitivity (p = 0.021), total cholesterol (p = 0.014), low-density-lipoprotein-cholesterol (p < 0.001), alanine-aminotransferase (p = 0.038) and hepatic steatosis compared to HFD-mice. While a decrease of hepatic miR-122 and increase of miR-34a were observed in HFD-mice compared to SD-mice, both these miRNAs had similar levels to SD-mice in HFD+E-mice. Moreover, a different microbial composition was found between SD- and HFD-mice, with a partial rescue of dysbiosis in HFD+E-mice. This combination of plant extracts had a beneficial effect on HFD-induced NAFLD by the modulation of miR-122, miR-34a and gut microbiome.

Coffee prevents fatty liver disease induced by a high-fat diet by modulating pathways of the gut-liver axis.

Coffee consumption is inversely associated with the risk of non-alcoholic fatty liver disease (NAFLD). A gap in the literature still exists concerning the intestinal mechanisms that are involved in the protective effect of coffee consumption towards NAFLD. In this study, twenty-four C57BL/6J mice were divided into three groups each receiving a standard diet, a high-fat diet (HFD) or an HFD plus decaffeinated coffee (HFD+COFFEE) for 12 weeks. Coffee supplementation reduced HFD-induced liver macrovesicular steatosis (P < 0·01) and serum cholesterol (P < 0·001), alanine aminotransferase and glucose (P < 0·05). Accordingly, liver PPAR- α (P < 0·05) and acyl-CoA oxidase-1 (P < 0·05) as well as duodenal ATP-binding cassette (ABC) subfamily A1 (ABCA1) and subfamily G1 (ABCG1) (P < 0·05) mRNA expressions increased with coffee consumption. Compared with HFD animals, HFD+COFFEE mice had more undigested lipids in the caecal content and higher free fatty acid receptor-1 mRNA expression in the duodenum and colon. Furthermore, they showed an up-regulation of duodenal and colonic zonulin-1 (P < 0·05), duodenal claudin (P < 0·05) and duodenal peptide YY (P < 0·05) mRNA as well as a higher abundance of Alcaligenaceae in the faeces (P < 0·05). HFD+COFFEE mice had an energy intake comparable with HFD-fed mice but starting from the eighth intervention week they gained significantly less weight over time. Data altogether showed that coffee supplementation prevented HFD-induced NAFLD in mice by reducing hepatic fat deposition and metabolic derangement through modification of pathways underpinning liver fat oxidation, intestinal cholesterol efflux, energy metabolism and gut permeability. The hepatic and metabolic benefits induced by coffee were accompanied by changes in the gut microbiota.

Berberis aristata, Elaeis guineensis and Coffea canephora Extracts Modulate the Insulin Receptor Expression and Improve Hepatic Steatosis in NAFLD Patients: A Pilot Clinical Trial.

Non-alcoholic fatty liver disease (NAFLD) is associated with insulin resistance and diabetes. A reduction in insulin receptor (IR) expression has been reported in these patients. The aims of this study were to evaluate the effects of a mixture of plant extracts consisting of Berberis aristata, Elaeis guineensis and decaffeinated green coffee by Coffea canephora on the improvement of glycaemic profile, through the modulation of IR levels, and of hepatic steatosis in NAFLD patients. Forty-nine patients with a grade of steatosis S1-S2 were randomly allocated to the treatment with plant extracts or placebo for six months. Hepatic steatosis was evaluated using transient elastography with CAP (controlled attenuation parameter). Glucose, insulin, and IR levels were measured in serum samples. At the end of the study, patients treated with plant extracts displayed a significant reduction of serum glucose (p < 0.001), insulin levels (p < 0.01), homeostatic model assessment for insulin resistance (HOMA-IR) index (p < 0.001), and CAP value (p < 0.01) compared to placebo. Moreover, the IR expression was increased significantly in the plant extracts group compared to the placebo group (p < 0.05). The combination of plant extracts increases serum IR levels, determining amelioration of glycemic profile and improvement of hepatic steatosis in NAFLD patients.

Dietary supplementation of vitamin D prevents the development of western diet-induced metabolic, hepatic and cardiovascular abnormalities in rats.

BACKGROUND: The western diet high in fat and fructose may cause metabolic disorders and cardiovascular diseases. OBJECTIVE: To evaluate whether long-term daily vitamin D3 supplementation prevents hepatic steatosis and cardiovascular abnormalities and restores insulin sensitivity caused by fat diet in rats without vitamin D deficiency. METHODS: Three groups of rats were fed for 6 months with standard diet (SD), western diet (WD) or WD containing 23 IU/day/rat vitamin D3, respectively. Tail-cuff systolic blood pressure (SBP)measurements in conscious rats and transthoracic echocardiography were performed in basal condition, and after 3 and 6 months of diet. Hepatic steatosis and myocardial fibrosis were assessed in liver and cardiac tissues using standard methods. Serum insulin and 25(OH)D3 concentrations were determined using rat-specific ELISA kits. Insulin resistance was determined according to the homeostasis model assessment of insulin resistance (HOMA-IR) method. RESULTS: Sixty-one per cent of hepatocytes in WD rats had steatotic vacuoles compared with just 27% in rats on a WD plus vitamin D3 (p < 0.05).HOMA-IR was reduced in rats with vitamin D supplementation compared with WD alone (19.4 ± 5.2 vs 41.9 ± 8.9, p < 0.05). Rat blood pressure and left ventricular mass were both reduced by vitamin D3 supplementation. CONCLUSION: In animal models of liver and cardiovascular metabolic damage, the supplementation of vitamin D3 shows liver and cardio-protective effects.

Antifibrotic Effect of Marine Ovothiol in an In Vivo Model of Liver Fibrosis.

Liver fibrosis is a complex process caused by chronic hepatic injury, which leads to an excessive increase in extracellular matrix protein accumulation and fibrogenesis. Several natural products, including sulfur-containing compounds, have been investigated for their antifibrotic effects; however, the molecular mechanisms underpinning their action are partially still obscure. In this study, we have investigated for the first time the effect of ovothiol A, π-methyl-5-thiohistidine, isolated from sea urchin eggs on an in vivo murine model of liver fibrosis. Mice were intraperitoneally injected with carbon tetrachloride (CCl4) to induce liver fibrosis and treated with ovothiol A at the dose of 50 mg/kg 3 times a week for 2 months. Treatment with ovothiol A caused a significant reduction of collagen fibers as observed by histopathological changes and serum parameters compared to mice treated with control solution. This antifibrotic effect was associated to the decrease of fibrogenic markers involved in liver fibrosis progression, such as the transforming growth factor (TGF-ß), the α-smooth muscle actin (α-SMA), and the tissue metalloproteinases inhibitor (TIMP-1). Finally, we provided evidence that the attenuation of liver fibrosis by ovothiol A treatment can be regulated by the expression and activity of the membrane-bound γ-glutamyl-transpeptidase (GGT), which is a key player in maintaining intracellular redox homoeostasis. Overall, these findings indicate that ovothiol A has significant antifibrotic properties and can be considered as a new marine drug or dietary supplement in potential therapeutic strategies for the treatment of liver fibrosis.

Silibinin Restores NAD⁺ Levels and Induces the SIRT1/AMPK Pathway in Non-Alcoholic Fatty Liver.

Nicotinamide adenine dinucleotide (NAD⁺) homeostasis is emerging as a key player in the pathogenesis of non-alcoholic fatty liver disease (NAFLD) and is tightly linked to the SIRT1/5'-AMP-activated protein kinase (AMPK) pathway. Silibinin, the main component of silymarin, has been proposed as a nutraceutical for the treatment of NAFLD. In this study, we aimed to identify whether silibinin may influence the NAD⁺/SIRT1 axis. To this end, C57BL/6 mice were fed a high fat diet (HFD) for 16 weeks, and were treated with silibinin or vehicle during the last 8 weeks. HepG2 cells were treated with 0.25 mM palmitate for 24 h with silibinin 25 µM or vehicle. HFD and palmitate administration led to oxidative stress, poly-(ADP-ribose)-polymerase (PARP) activation, NAD⁺ consumption, and lower SIRT1 activity. In mice fed the HFD, and in HepG2 treated with palmitate, we consistently observed lower levels of phospho-AMPKThr172 and phospho-acetyl-CoA carboxylaseSer79 and higher levels of nuclear sterol regulatory element-binding protein 1 activity, indicating de novo lipogenesis. Treatment of mice and HepG2 with silibinin abolished oxidative stress, and inhibited PARP activation thus restoring the NAD⁺ pool. In agreement with preserved NAD⁺ levels, SIRT1 activity and AMPK phosphorylation returned to control levels in mice and HepG2. Our results further indicate silibinin as a promising molecule for the treatment of NAFLD.

Effect of Red Wine Polyphenols on the Expression of Transthyretin in Murine Choroid Plexus.

Plasmatic transthyretin may be regarded as a suitable candidate biomarker for the onset, severity, and progression of Alzheimer disease. The aim of the present experimental work was to evaluate the effect of red wine polyphenols (RWPs) on the expression of transthyretin in murine choroid plexus. In contrast to what generally reported in literature for polyphenols, our experimental results indicated a correlation between RWPs assumption and a decrease of transthyretin expression, with a non-dose dependent trend. The present study would point out the attention on the possible pro-oxidant effects of red wine polyphenols at certain doses, although further in vitro, in vivo, and clinical experiments must be performed in order to clarify the mechanisms of action at the base of observed results.

Coffee and liver health.

Coffee is one of the most widely used beverages in the world. It includes a wide array of components that can have potential implications for health. Several epidemiological studies associate coffee consumption with a reduced incidence of various chronic diseases such as diabetes, cardiovascular diseases, and neurodegenerative diseases. Over the past 20 years, an increasing number of epidemiological and experimental studies have demonstrated the positive effects of coffee on chronic liver diseases. Coffee consumption has been inversely associated with the activity of liver enzymes in subjects at risk, including heavy drinkers. Coffee favours an improvement in hepatic steatosis and fibrosis, and a reduction in cirrhosis and the risk of hepatocellular carcinoma. The mechanisms of action through which it exerts its beneficial effects are not fully understood. Experimental studies show that coffee consumption reduces fat accumulation and collagen deposition in the liver and promotes antioxidant capacity through an increase in glutathione as well as modulation of the gene and protein expression of several inflammatory mediators. Animal and in vitro studies indicate that cafestol and kahweol, 2 diterpens, can operate by modulating multiple enzymes involved in the detoxification process of carcinogens causing hepatocellular carcinoma. It is unclear whether the benefits are significant enough to "treat" patients with chronic liver disease. While we await clarification, moderate daily unsweetened coffee use is a reasonable adjuvant to therapy for these patients.

Application of PTR-TOF-MS to investigate metabolites in exhaled breath of patients affected by coeliac disease under gluten free diet.

Coeliac disease (CD) is a common chronic inflammatory disorder of the small bowel induced in genetically susceptible people by the exposure to gliadin gluten. Even though several tests are available to assist the diagnosis, CD remains a biopsy-defined disorder, thus any non-invasive or less invasive diagnostic tool may be beneficial. The analysis of volatile metabolites in exhaled breath, given its non-invasive nature, is particularly promising as a screening tool of disease in symptomatic or non-symptomatic patients. In this preliminary study the proton transfer reaction time of flight mass spectrometry coupled to a buffered end-tidal on-line sampler to investigate metabolites in the exhaled breath of patients affected by coeliac disease under a gluten free diet was applied. Both H3O(+) or NO(+) were used as precursor ions. In our investigation no differences were found in the exhaled breath of CD patients compared to healthy controls. In this study, 33 subjects were enrolled: 16 patients with CD, all adhering a gluten free diet, and 17 healthy controls. CD patients did not show any symptom of the disease at the time of breath analysis; thus the absence of discrimination from healthy controls was not surprising.

Eicosapentaenoic acid free fatty acid prevents and suppresses colonic neoplasia in colitis-associated colorectal cancer acting on Notch signaling and gut microbiota.

Inflammatory bowel diseases are associated with increased risk of developing colitis-associated colorectal cancer (CAC). Epidemiological data show that the consumption of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) decreases the risk of sporadic colorectal cancer (CRC). Importantly, recent data have shown that eicosapentaenoic acid-free fatty acid (EPA-FFA) reduces polyp formation and growth in models of familial adenomatous polyposis. However, the effects of dietary EPA-FFA are unknown in CAC. We tested the effectiveness of substituting EPA-FFA, for other dietary fats, in preventing inflammation and cancer in the AOM-DSS model of CAC. The AOM-DSS protocols were designed to evaluate the effect of EPA-FFA on both initiation and promotion of carcinogenesis. We found that EPA-FFA diet strongly decreased tumor multiplicity, incidence and maximum tumor size in the promotion and initiation arms. Moreover EPA-FFA, in particular in the initiation arm, led to reduced cell proliferation and nuclear ß-catenin expression, whilst it increased apoptosis. In both arms, EPA-FFA treatment led to increased membrane switch from ω-6 to ω-3 PUFAs and a concomitant reduction in PGE2 production. We observed no significant changes in intestinal inflammation between EPA-FFA treated arms and AOM-DSS controls. Importantly, we found that EPA-FFA treatment restored the loss of Notch signaling found in the AOM-DSS control and resulted in the enrichment of Lactobacillus species in the gut microbiota. Taken together, our data suggest that EPA-FFA is an excellent candidate for CRC chemoprevention in CAC.

Rapid "breath-print" of liver cirrhosis by proton transfer reaction time-of-flight mass spectrometry. A pilot study.

UNLABELLED: The aim of the present work was to test the potential of Proton Transfer Reaction Time-of-Flight Mass Spectrometry (PTR-ToF-MS) in the diagnosis of liver cirrhosis and the assessment of disease severity by direct analysis of exhaled breath. Twenty-six volunteers have been enrolled in this study: 12 patients (M/F 8/4, mean age 70.5 years, min-max 42-80 years) with liver cirrhosis of different etiologies and at different severity of disease and 14 healthy subjects (M/F 5/9, mean age 52.3 years, min-max 35-77 years). Real time breath analysis was performed on fasting subjects using a buffered end-tidal on-line sampler directly coupled to a PTR-ToF-MS. Twelve volatile organic compounds (VOCs) resulted significantly differently in cirrhotic patients (CP) compared to healthy controls (CTRL): four ketones (2-butanone, 2- or 3- pentanone, C8-ketone, C9-ketone), two terpenes (monoterpene, monoterpene related), four sulphur or nitrogen compounds (sulfoxide-compound, S-compound, NS-compound, N-compound) and two alcohols (heptadienol, methanol). Seven VOCs (2-butanone, C8-ketone, a monoterpene, 2,4-heptadienol and three compounds containing N, S or NS) resulted significantly differently in compensate cirrhotic patients (Child-Pugh A; CP-A) and decompensated cirrhotic subjects (Child-Pugh B+C; CP-B+C). ROC (Receiver Operating Characteristic) analysis was performed considering three contrast groups: CP vs CTRL, CP-A vs CTRL and CP-A vs CP-B+C. In these comparisons monoterpene and N-compound showed the best diagnostic performance. CONCLUSIONS: Breath analysis by PTR-ToF-MS was able to distinguish cirrhotic patients from healthy subjects and to discriminate those with well compensated liver disease from those at more advanced severity stage. A breath-print of liver cirrhosis was assessed for the first time.

Garlic extract attenuating rat liver fibrosis by inhibiting TGF-ß1.

BACKGROUND & AIMS: We previously demonstrated the efficacy of garlic extract (GE) in the prevention of rat liver fibrosis by inhibiting tissue transglutaminase (tTG) activity. In the present study we aimed to evaluate the potential of GE in the regression of liver fibrosis and the underlining mechanism. METHODS: Male Wistar rats were i.p. injected, twice a week, for 7 weeks, with CCl(4) to develop liver fibrosis. Successively, a group was immediately sacrificed, while the remaining two groups received the GE or the vehicle, respectively, over the following 2 wks. A group of normal rats was also included in the study. Liver function, histology, and collagen deposition in parallel with gene and protein expression of α-SMA, tTG, TGF-ß1, SEMA-7A, and metalloproteinase inhibitor 1 (TIMP1) as well as measure of active by total TGF-ß1 were assessed. RESULTS: CCl(4) administration increased alanine-aminotransferase (ALT) activity, hepatic collagen deposition and gene and protein expression of all monitored markers. GE, but not the sole vehicle, restored liver histology and function by decreasing fibrogenesis markers (α-SMA, tTG, TGF-ß1, SEMA-7A and TIMP1). Active by total TGF-ß1 was significantly reduced (p < 0.05) in GE treated rats compared to the CCl(4) at 7 weeks, and vehicle rats. CONCLUSIONS: These findings concurrently suggested that GE elicited therapeutic effect against liver fibrosis. Regression of liver fibrosis occurred by reducing myofibroblasts (through modulation of HSCs activation mechanisms), remodelling extracellular matrix (through increase of its degradation) and regenerating liver tissue and functions: three processes regulated by fine mechanisms where active TGF-ß1 and tTG play a central role.

Analysis of breath by proton transfer reaction time of flight mass spectrometry in rats with steatohepatitis induced by high-fat diet.

Breath testing has been largely used as a diagnostic tool, but the difficulties in data interpretation and sample collection have limited its application. We developed a fast (< 20 s), on-line, non-invasive method for the collection and analysis of exhaled breath in awake rats based on proton transfer reaction time of flight mass spectrometry (PTR-ToF-MS) and applied it to investigate possible relationships between pathologies induced by dietary regime and breath composition. As a case study, we investigated rats with dietary induced non-alcoholic steatohepatitis (NASH) and modifications induced by coffee addition to the diet. We considered two different diets (standard and high fat) complemented with two different drinking possibilities (water or decaffeinated coffee) for a total of four groups with four rats each. Several spectrometric peaks were reliable markers for both dietary fat content and coffee supplementation. The high resolution and accuracy of PTR-ToF-MS allowed the identification of related compounds such as methanol, dimethyl sulphide, dimethyl sulphone and ammonia. In conclusion, the rapid and minimally invasive breath analysis of awake rats permitted the identification of markers related to diet and specific pathologic conditions and provided a useful tool for broader metabolic investigations.