Tho X. Pham

Eliciting the mitochondrial unfolded protein response by nicotinamide adenine dinucleotide repletion reverses fatty liver disease in mice

With no approved pharmacological treatment, nonalcoholic fatty liver disease (NAFLD) is now the most common cause of chronic liver disease in Western countries and its worldwide prevalence continues to increase along with the growing obesity epidemic. Here, we show that a high‐fat high‐sucrose (HFHS) diet, eliciting chronic hepatosteatosis resembling human fatty liver, lowers hepatic nicotinamide adenine dinucleotide (NAD+) levels driving reductions in hepatic mitochondrial content, function, and adenosine triphosphate (ATP) levels, in conjunction with robust increases in hepatic weight, lipid content, and peroxidation in C57BL/6J mice. To assess the effect of NAD+ repletion on the development of steatosis in mice, nicotinamide riboside, a precursor of NAD+ biosynthesis, was added to the HFHS diet, either as a preventive strategy or as a therapeutic intervention. We demonstrate that NR prevents and reverts NAFLD by inducing a sirtuin (SIRT)1‐ and SIRT3‐dependent mitochondrial unfolded protein response, triggering an adaptive mitohormetic pathway to increase hepatic β‐oxidation and mitochondrial complex content and activity. The cell‐autonomous beneficial component of NR treatment was revealed in liver‐specific Sirt1 knockout mice (Sirt1hep−/−), whereas apolipoprotein E‐deficient mice (Apoe−/−) challenged with a high‐fat high‐cholesterol diet affirmed the use of NR in other independent models of NAFLD. Conclusion: Our data warrant the future evaluation of NAD+ boosting strategies to manage the development or progression of NAFLD. (Hepatology 2016;63:1190–1204)

Berry anthocyanins suppress the expression and secretion of proinflammatory mediators in macrophages by inhibiting nuclear translocation of NF-κB independent of NRF2-mediated mechanism

The objectives of this study were to compare the anti-inflammatory effects of anthocyanins from blueberry (BBA), blackberry (BKA), and blackcurrant (BCA) and to determine the relationship between their antioxidant capacity and anti-inflammatory effect in macrophages. Major anthocyanins in BBA, BKA and BCA were malvidin-3-glucoside (16%), cyanidin-3-glucoside (98%) and delphinidin-3-rutinoside (44%), respectively. BKA showed higher total antioxidant capacity than BBA and BCA. RAW 264.7 macrophages were incubated with 0-20 μg/ml of BBA, BKA and BCA, and subsequently activated by lipopolysaccharide (LPS) to measure proinflammatory cytokine production. Interleukin 1β (IL-1β) messenger RNA (mRNA) levels were significantly decreased by all berry anthocyanins at 10 μg/ml or higher. Tumor necrosis factor α (TNFα) mRNA levels and secretion were also significantly decreased in LPS-treated macrophages. The levels of the repression were comparable for all berry anthocyanins. LPS-induced nuclear factor κB (NF-κB) p65 translocation to the nucleus was markedly attenuated by all of the berry anthocyanins. In bone marrow-derived macrophages (BMMs) from nuclear factor E2-related factor 2 wild-type (Nrf2(+/+)) mice, BBA, BKA and BCA significantly decreased cellular reactive oxygen species (ROS) levels with a concomitant decrease in IL-1β mRNA levels upon LPS stimulation. However, in the BMM from Nrf2(-/-) mice, the anthocyanin fractions were able to significantly decrease IL-1β mRNA despite the fact that ROS levels were not significantly affected. In conclusion, BBA, BKA and BCA exert their anti-inflammatory effects in macrophages, at least in part, by inhibiting nuclear translocation of NF-κB independent of the NRF2-mediated pathways.

Astaxanthin-Rich Extract from the Green Alga Haematococcus pluvialis Lowers Plasma Lipid Concentrations and Enhances Antioxidant Defense in Apolipoprotein E Knockout Mice

Dyslipidemia and oxidative stress contribute to atherogenesis. Astaxanthin (ASTX) is a red-colored carotenoid well known for its high antioxidant capacity. However, its effects on lipid metabolism and antioxidant defense mechanisms have received only limited investigation. We fed male apoE knockout (apoE)(-/-) mice, a mouse model for atherosclerosis, a high-fat (15%)/high-cholesterol (0.2%) diet alone (control) or supplemented with ASTX-rich Hematococcus pluvialis extract (0.03% ASTX by weight) for 4 wk. ASTX-fed apoE(-/-) mice had significantly lower plasma total cholesterol and TG concentrations than controls, but body weight and plasma alanine aminotransferase and aspartate aminotransferase did not differ between the groups. qRT-PCR analysis demonstrated significantly greater mRNA levels of LDL receptor (LDLR), 3-hydroxy-3-methylglutaryl CoA reductase, and sterol regulatory element binding protein 2 (SREBP-2) and greater mature SREBP-2 protein in the livers of ASTX-fed mice, indicating that increased LDLR expression may be responsible for the hypocholesterolemic effect of ASTX. Hepatic lipogenic gene expression was not altered, but carnitine palmitoyl transferase 1, acetyl-CoA carboxylase β, and acyl-CoA oxidase mRNA abundance were significantly increased by ASTX supplementation, suggesting the TG-lowering effect of ASTX may be due to increased fatty acid β-oxidation in the liver. Expression of the nuclear factor E2 related factor 2-responsive endogenous antioxidant gene also was induced with concomitantly lower glutathione disulfide levels in the livers of ASTX-fed apoE(-/-) mice compared to controls. In conclusion, these results suggest that supplementation of ASTX-rich H. pluvialis extract improves cholesterol and lipid metabolism as well as antioxidant defense mechanisms, all of which could help mitigate the progression of atherosclerosis.

Dietary Regulation of Histone Acetylases and Deacetylases for the Prevention of Metabolic Diseases

Age-related diseases such as type 2 diabetes, cardiovascular disease, and cancer involve epigenetic modifications, where accumulation of minute changes in the epigenome over time leads to disease manifestation. Epigenetic changes are influenced by life style and diets. This represents an avenue whereby dietary components could accelerate or prevent age-related diseases through their effects on epigenetic modifications. Histone acetylation is an epigenetic modification that is regulated through the opposing action of histone acetylases (HATs) and deacetylases (HDACs). These two families of enzymes play critical roles in metabolic processes and their dysregulation is associated with pathogenesis of several diseases. Dietary components, such as butyrate, sulforaphane, and curcumin, have been shown to affect HAT and HDAC activity, and their health benefits are attributed, at least in part, to epigenetic modifications. Given the decades that it takes to accumulate epigenetic changes, it is unlikely that pharmaceuticals could undo epigenetic changes without side effects. Therefore, long term consumption of dietary components that can alter the epigenome could be an attractive means of disease prevention. The goal of this review is to highlight the roles of diets and food components in epigenetic modifications through the regulation of HATs and HDACs for disease prevention.

Aronia melanocarpa (chokeberry) polyphenol–rich extract improves antioxidant function and reduces total plasma cholesterol in apolipoprotein E knockout mice

We hypothesized that a polyphenol-rich chokeberry extract (CBE) would modulate hepatic lipid metabolism and improve antioxidant function in apolipoprotein E knockout (apoE(-/-)) mice. ApoE(-/-) mice were fed diets containing 15% fat with 0.2% cholesterol alone or supplemented with 0.005% or 0.05% CBE for 4 weeks. CBE polyphenol content was determined by the total phenols, 4-dimethylaminocinnamaldehyde, and ultra high-performance liquid chromatography-mass spectrometry methods. The 0.05% CBE diet provided mice with mean daily doses of 1.2 mg gallic acid equivalents of total phenols, 0.19 mg anthocyanins, 0.17 mg phenolic acids, 0.06 mg proanthocyanidins (as catechin-equivalents), and 0.02 mg flavonols. The 0.05% CBE group had 12% less plasma total cholesterol concentrations than the control. Despite the hypocholesterolemic effect of CBE, hepatic mRNA levels of low-density lipoprotein receptor, hydroxyl-3-methylglutaryl coenzyme A reductase and cholesterol 7α-hydroxylase in CBE-fed mice were not significantly different from controls. Dietary CBE did not alter hepatic lipid content or the hepatic expression of genes involved in lipogenesis and fatty acid β-oxidation such as fatty acid synthase, carnitine palmitoyltransferase 1 and acyl-CoA oxidase. Plasma paraoxonase and catalase activities were significantly increased in mice fed 0.05% CBE. Both CBE diets increased hepatic glutathione peroxidase (GPx) activity but the 0.05% CBE group had 24% less proximal intestine GPx activity relative to controls. Thus, dietary CBE lowered total cholesterol and improved plasma and hepatic antioxidant function at nutritionally-relevant doses in apoE(-/-) mice. Furthermore, the CBE cholesterol-lowering mechanism in apoE(-/-) mice was independent of hepatic expression of genes involved in cholesterol metabolism.

Edible blue-green algae reduce the production of pro-inflammatory cytokines by inhibiting NF-κB pathway in macrophages and splenocytes.

BACKGROUND Chronic inflammation contributes to the development of pathological disorders including insulin resistance and atherosclerosis. Identification of anti-inflammatory natural products can prevent the inflammatory diseases. METHODS Anti-inflammatory effects of blue-green algae (BGA), i.e., Nostoc commune var. sphaeroides Kützing (NO) and Spirulina platensis (SP), were compared in RAW 264.7 and mouse bone marrow-derived macrophages (BMM) as well as splenocytes from apolipoprotein E knockout (apoE(-/-)) mice fed BGA. RESULTS When macrophages pretreated with 100μg/ml NO lipid extract (NOE) or SP lipid extract (SPE) were activated by lipopolysaccharide (LPS), expression and secretion of pro-inflammatory cytokines, such as tumor necrosis factor α (TNFα), interleukin 1β (IL-1β), and IL-6, were significantly repressed. NOE and SPE also significantly repressed the expression of TNFα and IL-1β in BMM. LPS-induced secretion of IL-6 was lower in splenocytes from apoE(-/-) fed an atherogenic diet containing 5% NO or SP for 12weeks. In RAW 264.7 macrophages, NOE and SPE markedly decreased nuclear translocation of NF-κB. The degree of repression of pro-inflammatory gene expression by algal extracts was much stronger than that of SN50, an inhibitor of NF-κB nuclear translocation. Trichostatin A, a pan histone deacetylase inhibitor, increased basal expression of IL-1β and attenuated the repression of the gene expression by SPE. SPE significantly down-regulated mRNA abundance of 11 HDAC isoforms, consequently increasing acetylated histone 3 levels. CONCLUSION NOE and SPE repress pro-inflammatory cytokine expression and secretion in macrophages and splenocytes via inhibition of NF-κB pathway. Histone acetylation state is likely involved in the inhibition. GENERAL SIGNIFICANCE This study underscores natural products can exert anti-inflammatory effects by epigenetic modifications such as histone acetylation.

Lipopolysaccharide represses the expression of ATP-binding cassette transporter G1 and scavenger receptor class B, type I in murine macrophages

Objective and designTo investigate the regulation of cholesterol transporters, including ATP-binding cassette transporter A1 (ABCA1), ABCG1 and scavenger receptor class B, type I (SR-BI), by inflammatory stimuli in macrophages.Materials and treatmentsRAW 264.7 macrophages and mouse peritoneal macrophages were treated with inflammatory stimuli with or without rosiglitazone, a peroxisome proliferator activated receptor γ (PPARγ) agonist, or T0901317, a liver X receptor (LXR) agonist.MethodsReal-time PCR and Western blotting for cholesterol transporters as well as cellular cholesterol efflux to high-density lipoprotein 2 (HDL2) were determined.ResultsIn RAW 264.7 macrophages, lipopolysaccharide (LPS) significantly reduced ABCG1 and PPARγ as well as cholesterol efflux to HDL2. Rosiglitazone and T0901317 induced ABCA1 and ABCG1 several-fold, but LPS reduced only ABCG1. ABCG1 and SR-BI proteins, but not ABCA1, were decreased by LPS. In mouse peritoneal macrophages, LPS, tumor necrosis factor α and interleukin-1β decreased ABCG1, SR-BI, LXRα and PPARγ mRNA. The agonists increased ABC transporter expression but LPS reduced mRNA of T0901317-induced ABCA1 as well as basal and agonists-induced ABCG1. SR-BI protein was increased by rosiglitazone but LPS decreased the levels.ConclusionThe data suggest that inflammatory insults repress ABCG1 and SR-BI expression partly dependent on PPARγ with a minimal effect on ABCA1 expression.

Astaxanthin lowers plasma TAG concentrations and increases hepatic antioxidant gene expression in diet-induced obesity mice

Non-alcoholic fatty liver disease (NAFLD) is significantly associated with hyperlipidaemia and oxidative stress. We have previously reported that astaxanthin (ASTX), a xanthophyll carotenoid, lowers plasma total cholesterol and TAG concentrations in apoE knockout mice. To investigate whether ASTX supplementation can prevent the development of NAFLD in obesity, male C57BL/6J mice (n 8 per group) were fed a high-fat diet (35%, w/w) supplemented with 0, 0.003, 0.01 or 0.03% of ASTX (w/w) for 12 weeks. The 0.03% ASTX-supplemented group, but not the other groups, exhibited a significant decrease in plasma TAG concentrations, suggesting that ASTX at a 0.03% supplementation dosage exerts a hypotriacylglycerolaemic effect. Although there was an increase in the mRNA expression of fatty acid synthase and diglyceride acyltransferase 2, the mRNA levels of acyl-CoA oxidase 1, a critical enzyme in peroxisomal fatty acid β-oxidation, exhibited an increase in the 0.03% ASTX-supplemented group. There was a decrease in plasma alanine transaminase (ALT) and aspartate transaminase (AST) concentrations in the 0.03% ASTX-supplemented group. There was a significant increase in the hepatic mRNA expression of nuclear factor erythroid 2-related factor 2 and its downstream genes, which are critical for endogenous antioxidant mechanism, in the 0.03% ASTX-supplemented group. Furthermore, there was a significant decrease in the mRNA abundance of IL-6 in the primary splenocytes isolated from the 0.03% ASTX-supplemented group upon lipopolysaccharide (LPS) stimulation when compared with that in the splenocytes isolated from the control group. In conclusion, ASTX supplementation lowered the plasma concentrations of TAG, ALT and AST, increased the hepatic expression of endogenous antioxidant genes, and rendered splenocytes less sensitive to LPS stimulation. Therefore, ASTX may prevent obesity-associated metabolic disturbances and inflammation.

Polyphenol-rich blackcurrant extract exerts hypocholesterolaemic and hypoglycaemic effects in mice fed a diet containing high fat and cholesterol

Obesity is associated with an increased risk of metabolic abnormalities, such as hyperlipidaemia and hyperglycaemia. We investigated whether polyphenol-rich blackcurrant extract (BCE) can prevent high fat/high cholesterol (HF/HC) diet-induced metabolic disturbances in mice. Male C57BL/6J mice were fed a modified AIN-93M diet containing HF/HC (16% fat, 0·25% cholesterol, w/w) or the same diet supplemented with 0·1% BCE (w/w) for 12 weeks. There were no differences in total body weight and liver weight between groups. Plasma total cholesterol (TC) and glucose levels were significantly lower in BCE group than in controls, while plasma TAG levels were not significantly different. There was a decreasing trend in hepatic TAG levels, and histological evaluation of steatosis grade was markedly lower in the livers of mice fed BCE. Although the mRNA levels of major regulators of hepatic cholesterol metabolism, i.e. 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) and LDL receptor (LDLR), were not significantly altered by BCE supplementation, protein expression of mature sterol-regulatory element-binding protein and LDLR was significantly increased with no change in HMGR protein. The expression of proprotein convertase subtilisin/kexin type 9 that facilitates LDLR protein degradation, as well as one of its transcriptional regulators, i.e. hepatocyte nuclear factor 4α, was significantly decreased in the livers of mice fed BCE. Taken together, BCE supplementation decreased plasma TC and glucose, and inhibited liver steatosis, suggesting that this berry may be consumed to prevent metabolic dysfunctions induced by diets high in fat and cholesterol.

Astaxanthin inhibits inflammation and fibrosis in the liver and adipose tissue of mouse models of diet-induced obesity and nonalcoholic steatohepatitis

The objective of this study was to determine if astaxanthin (ASTX), a xanthophyll carotenoid, can prevent obesity-associated metabolic abnormalities, inflammation and fibrosis in diet-induced obesity (DIO) and nonalcoholic steatohepatitis (NASH) mouse models. Male C57BL/6J mice were fed a low-fat (6% fat, w/w), a high-fat/high-sucrose control (HF/HS; 35% fat, 35% sucrose, w/w), or a HF/HS containing ASTX (AHF/HS; 0.03% ASTX, w/w) for 30 weeks. To induce NASH, another set of mice was fed a HF/HS diet containing 2% cholesterol (HF/HS/HC) a HF/HS/HC with 0.015% ASTX (AHF/HS/HC) for 18 weeks. Compared to LF, HF/HS significantly increased plasma total cholesterol, triglyceride and glucose, which were lowered by ASTX. ASTX decreased hepatic mRNA levels of markers of macrophages and fibrosis in both models. The effect of ASTX was more prominent in NASH than DIO mice. In epididymal fat, ASTX also decreased macrophage infiltration and M1 macrophage marker expression, and inhibited hypoxia-inducible factor 1-α and its downstream fibrogenic genes in both mouse models. ASTX significantly decreased tumor necrosis factor α mRNA in the splenocytes from DIO mice upon lipopolysaccharides stimulation compared with those from control mice fed an HF/HS diet. Additionally, ASTX significantly elevated the levels of genes that regulate fatty acid β-oxidation and mitochondrial biogenesis in the skeletal muscle compared with control obese mice, whereas no differences were noted in adipose lipogenic genes. Our results indicate that ASTX inhibits inflammation and fibrosis in the liver and adipose tissue and enhances the skeletal muscles capacity for mitochondrial fatty acid oxidation in obese mice.