Hsp90 modulates PPARγ activity in a mouse model of nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is a highly prevalent complication of obesity, yet cellular mechanisms that lead to its development are not well defined. Previously, we have documented hepatic steatosis in mice carrying a mutation in the Sec61a1 gene. Here we examined the mechanism behind NAFLD in Sec61a1 mutant mice. Livers of mutant mice exhibited upregulation of Pparg and its target genes Cd36, Cidec, and Lpl, correlating with increased uptake of fatty acid. Interestingly, these mice also displayed activation of the heat shock response (HSR), with elevated levels of heat shock protein (Hsp) 70, Hsp90, and heat shock factor 1. In cell lines, inhibition of Hsp90 function reduced Pparγ signaling and protein levels. Conversely, overexpression of Hsp90 increased Pparγ signaling and protein levels by reducing degradation. This may occur via a physical interaction as Hsp90 and Pparγ coimmunoprecipitated in vivo. Furthermore, inhibition of Hsp90 in Sec61a1 mutant hepatocytes also reduced Pparγ protein levels and signaling. Finally, overexpression of Hsp90 in liver cell lines increased neutral lipid accumulation, and this accumulation was blocked by Hsp90 inhibition. Our results show that the HSR and Hsp90 play an important role in the development of NAFLD, opening new avenues for the prevention and treatment of this highly prevalent disease.     

The PPARα agonist fenofibrate suppresses B-cell lymphoma in mice by modulating lipid metabolism

Obesity is associated with an increased risk for malignant lymphoma development. We used Bcr/Abl transformed B cells to determine the impact of aggressive lymphoma formation on systemic lipid mobilization and turnover. In wild-type mice, tumor size significantly correlated with depletion of white adipose tissues (WAT), resulting in increased serum free fatty acid (FFA) concentrations which promote B-cell proliferation in vitro. Moreover, B-cell tumor development induced hepatic lipid accumulation due to enhanced hepatic fatty acid (FA) uptake and impaired FA oxidation. Serum triglyceride, FFA, phospholipid and cholesterol levels were significantly elevated. Consistently, serum VLDL/LDL-cholesterol and apolipoprotein B levels were drastically increased. These findings suggest that B-cell tumors trigger systemic lipid mobilization from WAT to the liver and increase VLDL/LDL release from the liver to promote tumor growth. Further support for this concept stems from experiments where we used the peroxisome proliferator-activated receptor α (PPARα) agonist and lipid-lowering drug fenofibrate that significantly suppressed tumor growth independent of angiogenesis and inflammation. In addition to WAT depletion, fenofibrate further stimulated FFA uptake by the liver and restored hepatic FA oxidation capacity, thereby accelerating the clearance of lipids released from WAT. Furthermore, fenofibrate blocked hepatic lipid release induced by the tumors. In contrast, lipid utilization in the tumor tissue itself was not increased by fenofibrate which correlates with extremely low expression levels of PPARα in B-cells. Our data show that fenofibrate associated effects on hepatic lipid metabolism and deprivation of serum lipids are capable to suppress B-cell lymphoma growth which may direct novel treatment strategies. This article is part of a Special Issue entitled Lipid Metabolism in Cancer.     

The marine-derived furanone reduces intracellular lipid accumulation in vitro by targeting LXRα and PPARα

Recent studies have demonstrated that commercially available lipid-lowering drugs cause various side effects; therefore, searching for anti-hyperlipidaemic compounds with lower toxicity is a research hotspot. This study was designed to investigate whether the marine-derived compound, 5-hydroxy-3-methoxy-5-methyl-4-butylfuran-2(5H)-one, has an anti-hyperlipidaemic activity, and the potential underlying mechanism in vitro. Results showed that the furanone had weaker cytotoxicity compared to positive control drugs. In RAW 264.7 cells, the furanone significantly lowered ox-LDL-induced lipid accumulation (~50%), and its triglyceride (TG)-lowering effect was greater than that of liver X receptor (LXR) agonist T0901317. In addition, it significantly elevated the protein levels of peroxisome proliferator-activated receptors (PPARα) and ATP-binding cassette (ABC) transporters, which could be partially inhibited by LXR antagonists, GSK2033 and SR9243. In HepG2 cells, it significantly decreased oleic acid-induced lipid accumulation, enhanced the protein levels of low-density lipoprotein receptor (LDLR), ABCG5, ABCG8 and PPARα, and reduced the expression of sterol regulatory element-binding protein 2 (~32%). PPARα antagonists, GW6471 and MK886, could significantly inhibit the furanone-induced lipid-lowering effect. Furthermore, the furanone showed a significantly lower activity on the activation of the expression of lipogenic genes compared to T0901317. Taken together, the furanone exhibited a weak cytotoxicity but had powerful TC- and TG-lowering effects most likely through targeting LXRα and PPARα, respectively. These findings indicate that the furanone has a potential application for the treatment of dyslipidaemia.     

Nonalcoholic fatty liver disease impairs the cytochrome P-450-dependent metabolism of α-tocopherol (vitamin E)

This study aims to investigate in in vivo and in vitro models of nonalcoholic fatty liver disease (NAFLD) the enzymatic metabolism of α-tocopherol (vitamin E) and its relationship to vitamin E-responsive genes with key role in the lipid metabolism and detoxification of the liver. The experimental models included mice fed a high-fat diet combined or not with fructose (HFD+F) and HepG2 human hepatocarcinoma cells treated with the lipogenic agents palmitate, oleate or fructose. CYP4F2 protein, a cytochrome P-450 isoform with proposed α-tocopherol ω-hydroxylase activity, decreased in HFD and even more in HFD+F mice liver; this finding was associated with increased hepatic levels of α-tocopherol and decreased formation of the corresponding long-chain metabolites α-13-hydroxy and α-13-carboxy chromanols. A decreased expression was also observed for PPAR-γ and SREBP-1 proteins, two vitamin E-responsive genes with key role in lipid metabolism and CYP4F2 gene regulation. A transient activation of CYP4F2 gene followed by a repression response was observed in HepG2 cells during the exposure to increasing levels of the lipogenic and cytotoxic agent palmitic acid; such gene repression effect was further exacerbated by the co-treatment with oleic acid and α-tocopherol and was also observed for PPAR-γ and the SREBP isoforms 1 and 2. Such gene response was associated with increased uptake and ω-hydroxylation of α-tocopherol, which suggests a minor role of CYP4F2 in the enzymatic metabolism of vitamin E in HepG2 cells. In conclusion, the liver metabolism and gene response of α-tocopherol are impaired in experimental NAFLD.     

Ganglioside metabolism in a transgenic mouse model of Alzheimer's disease: expression of Chol-1α antigens in the brain

The accumulation of Aβ (amyloid β-protein) is one of the major pathological hallmarks in AD (Alzheimer''s disease). Gangliosides, sialic acid-containing glycosphingolipids enriched in the nervous system and frequently used as biomarkers associated with the biochemical pathology of neurological disorders, have been suggested to be involved in the initial aggregation of Aβ. In the present study, we have examined ganglioside metabolism in the brain of a double-Tg (transgenic) mouse model of AD that co-expresses mouse/human chimaeric APP (amyloid precursor protein) with the Swedish mutation and human presenilin-1 with a deletion of exon 9. Although accumulation of Aβ was confirmed in the double-Tg mouse brains and sera, no statistically significant change was detected in the concentration and composition of major ganglio-N-tetraosyl-series gangliosides in the double-Tg brain. Most interestingly, Chol-1α antigens (cholinergic neuron-specific gangliosides), such as GT1aα and GQ1bα, which are minor species in the brain, were found to be increased in the double-Tg mouse brain. We interpret that the occurrence of these gangliosides may represent evidence for generation of cholinergic neurons in the AD brain, as a result of compensatory neurogenesis activated by the presence of Aβ.     

Scutellarin ameliorates nonalcoholic fatty liver disease through the PPARγ/PGC-1α-Nrf2 pathway

Nonalcoholic fatty liver disease (NAFLD) is characterised by excessive accumulation of hepatic lipids and oxidative injury of hepatocytes. Scutellarin is a flavonoid glycoside having antioxidative stress activity. Our current study aims to investigate the molecular mechanism of scutellarin ameliorating NAFLD. Scutellarin treatment was applied to male C57BL/6 mice maintained on a high-fat diet (HFD) and HepG2 cells challenged with oleic acid. The antioxidation biochemical indicators and lipid levels in the liver and cells were detected by kits. Liver pathology was observed by light microscope, Oil Red O staining, and transmission electron microscope (TEM). In addition, quantitative real-time polymerase chain reactions (qRT-PCR) and western blot assays were employed to detect the mRNA and protein levels of various antioxidative-related genes in the presence or absence of peroxisome proliferator-activated receptor gamma (PPARγ); inhibitor GW9662. Our results showed that scutellarin could significantly reduce blood lipid levels and enhance antioxidative capacities in both the models. In addition, scutellarin treatment conspicuously activated PPARγ, peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), nuclear factor erythroid-2-related factor (Nrf2), haem oxygenase-1 (HO-1), glutathione S-transferase (GST), and NAD(P)H quinone dehydrogenase one (NQO1), while it significantly inhibited nuclear factor kappa B (NF-κB), Kelch-like ECH-associated protein 1 (Keap1) at both the mRNA and protein levels. However, after interfered by GW9662, scutellarin effect was significantly decreased. The experimental data demonstrated that scutellarin showed strong hypolipidaemic, antioxidative, and liver protective activity which could be attributed to its regulating activity in the PPARγ/PGC-1α-Nrf2 signaling pathway.     

β-Casomorphin increases fat deposition in broiler chickens by modulating expression of lipid metabolism genes

β-Casomorphin is an opioid-like bioactive peptide derived from β-casein of milk that plays a crucial role in modulating animal’s feed intake, growth, nutrient utilization and immunity. However, the effect of β-casomorphin on lipid metabolism in chickens and its mechanism remain unclear. The aim of this study was to investigate the effects of β-casomorphin on fat deposition in broiler chickens and explore its mechanism of action. A total of 120 21-day-old Arbor Acres male broilers (747.94±8.85 g) was chosen and randomly divided into four groups with six replicates of five birds per replicate. Three groups of broilers were injected with 0.1, 0.5 or 1.0 mg/kg BW of β-casomorphin in 1 ml saline for 7 days, whereas the control group received 1 ml saline only. The results showed that subcutaneous administration of β-casomorphin to broiler chickens increased average daily gain, average daily feed intake and fat deposition, and decreased feed : gain ratio (P<0.05). The activity of malate dehydrogenase in the pectoral muscle, liver and abdominal adipose tissue was also increased along with the concentrations of insulin, very-low-density lipoprotein and triglyceride in the plasma (P<0.05). The activity of hormone-sensitive lipase in the liver and abdominal adipose tissue and the concentration of glucagon in the plasma were decreased by injection with β-casomorphin (P<0.05). Affymetrix gene chip analysis revealed that administering 1.0 mg/kg BW β-casomorphin caused differential expression of 168 genes in the liver with a minimum of fourfold difference. Of those, 37 genes are directly involved in lipid metabolism with 18 up-regulated genes such as very low density lipoprotein receptor gene and fatty acid synthase gene, and 19 down-regulated genes such as lipoprotein lipase gene and low density lipoprotein receptor gene. In conclusion, β-casomorphin increased growth performance and fat deposition of broilers. Regulation of fat deposition by β-casomorphin appears to take place through changes in hormone secretion and enzyme activities by controlling the gene expression of lipid metabolism and feed intake, increasing fat synthesis and deposition.     

PPARα deficiency augments a ketogenic diet-induced circadian PAI-1 expression possibly through PPARγ activation in the liver

An increased level of plasminogen activator inhibitor-1 (PAI-1) is considered a risk factor for cardiovascular diseases, and PAI-1 gene expression is under the control of molecular circadian clocks in mammals. We recently showed that PAI-1 expression is augmented in a phase-advanced circadian manner in mice fed with a ketogenic diet (KD). To determine whether peroxisome proliferator-activated receptor α (PPARα) is involved in hypofibrinolytic status induced by a KD, we examined the expression profiles of PAI-1 and circadian clock genes in PPARα-null KD mice. Chronic administration of bezafibrate induced the PAI-1 gene expression in a PPARα-dependent manner. Feeding with a KD augmented the circadian expression of PAI-1 mRNA in the hearts and livers of wild-type (WT) mice as previously described. The KD-induced mRNA expression of typical PPARα target genes such as Cyp4A10 and FGF21 was damped in PPARα-null mice. However, plasma PAI-1 concentrations were significantly more elevated in PPARα-null KD mice in accordance with hepatic mRNA levels. These observations suggest that PPARα activation is dispensable for KD-induced PAI-1 expression. We also found that hyperlipidemia, fatty liver, and the hepatic expressions of PPARγ and its coactivator PCG-1α were more effectively induced in PPARα-null, than in WT mice on a KD. Furthermore, KD-induced hepatic PAI-1 expression was significantly suppressed by supplementation with bisphenol A diglycidyl ether, a PPARγ antagonist, in both WT and PPARα-null mice. PPARγ activation seems to be involved in KD-induced hypofibrinolysis by augmenting PAI-1 gene expression in the fatty liver.     

Role of tumor necrosis factor α (TNFα) in the onset of fructose-induced nonalcoholic fatty liver disease in mice

Tumor necrosis factor α (TNFα) is known to be involved in dysregulation of hepatic lipid metabolism and insulin signaling. However, whether TNFα also plays a casual role in the onset of fructose-induced nonalcoholic fatty liver disease (NAFLD) has not yet been determined. Therefore, wild-type and TNFα receptor 1 (TNFR1)−/− mice were fed with either 30% fructose solution or plain tap water. Hepatic triglycerides, markers of inflammation and ATP concentration as well as plasma ALT levels were determined. Hepatic PAI-1, SREBP-1, FAS mRNA expression was assessed by real-time RT-PCR. Furthermore, lipid peroxidation and indices of insulin resistance were determined in liver tissue and plasma. In comparison to water controls, chronic intake of 30% fructose solution caused a significant ∼5-fold increase in triglyceride accumulation and neutrophil infiltration in livers of wild-type mice and a ∼8-fold increase in plasma ALT levels. In TNFR1−/− mice, hepatic steatosis was attenuated and neutrophil infiltration in the liver as well as plasma ALT levels was similar to water controls. The protective effect of the TNFR1 deletion against the onset of fructose-induced steatosis was associated with increased phospho AMPK and Akt levels, decreased SREBP-1 and FAS expression in the liver and decreased RBP4 plasma levels, whereas hepatic lipid peroxidation, iNOS protein and ATP levels were similar between wild-type and TNFR1−/− mice fed fructose. Taken together, these data suggest that TNFα plays a casual role in the onset of fructose-induced liver damage as well as insulin resistance in mice through signaling cascades downstream of TNFR1.     

A case–control study on the effects of the apolipoprotein E genotypes in nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) has reached epidemic proportions being the most common cause of chronic liver disease in Western countries. The Apolipoprotein E (ApoE) gene has three major isoforms encoded by the ε2, ε3, and ε4 alleles, with the ε4 allele associated with hypercholesterolemia and the ε2 allele with the opposite effect. The role of apoE genotypes on NAFLD has been previously investigated with conflicting results. Our hospital-based case-control study conducted in Italy aims to explore the effect of the apoE genotypes on NAFLD risk and their effect on the clinical features of NAFLD patients. 310 NAFLD cases and 422 controls were genotyped for apoE. Adjusted odds ratios (ORs) and 95% confidence intervals (CI) from logistic regression were used to explore the relationship between NAFLD and apoE genotypes, as well as their interaction with selected demographic and lifestyle factors. ApoE ε4 allele carriers showed a statistically significant two-fold reduction of NAFLD risk (OR = 0.51, 95% CI: 0.28-0.93) compared with ε3 homozygotes. A statistically significant lower HDL cholesterol level was observed for ApoE ε4 carriers if compared with ε3/ε3 genotype or ApoE ε2 carriers with a nearly linear decreasing trend from ApoE ε2 to ApoE ε4 carriers. Our study reports for the first time a protective effect of the ε4 allele towards NAFLD that might be attributable to its role in the regulation of hepatic triglycerides rich very low-density lipoproteins secretion.     

An increase in liver PPARγ2 is an initial event to induce fatty liver in response to a diet high in butter: PPARγ2 knockdown improves fatty liver induced by high-saturated fat

The effects of a diet rich in saturated fat on fatty liver formation and the related mechanisms that induce fatty liver were examined. C57BL/6J mice were fed butter or safflower oil as a high-fat (HF) diet (40% fat calories) for 2, 4, 10, or 17 weeks. Although both HF diets induced similar levels of obesity, HF butter-fed mice showed a two to threefold increase in liver triacylglycerol (TG) concentration compared to HF safflower oil-fed mice at 4 or 10 weeks without hyperinsulinemia. At 4 weeks, increases in peroxisome proliferator-activated receptor γ2 (PPARγ2), CD36, and adipose differentiation-related protein (ADRP) mRNAs were observed in HF butter-fed mice; at 10 weeks, an increase in sterol regulatory element-binding protein-1c (SREBP-1c) was observed; at 17 weeks, these increases were attenuated. At 4 weeks, a single injection of adenoviral vector-based short hairpin interfering RNA against PPARγ2 in HF butter-fed mice reduced PPARγ protein and mRNA of its target genes (CD36 and ADRP) by 43%, 43%, and 39%, respectively, with a reduction in liver TG concentration by 38% in 5 days. PPARγ2 knockdown also reduced mRNAs in lipogenic genes (fatty-acid-synthase, stearoyl-CoA desaturase 1, acetyl-CoA carboxylase 1) without alteration of SREBP-1c mRNA. PPARγ2 knockdown reduced mRNAs in genes related to inflammation (CD68, interleukin-1β, tumor necrosis factor-α, and monocyte chemoattractant protein-1). In conclusion, saturated fatty acid-rich oil induced fatty liver in mice, and this was triggered initially by an increase in PPARγ2 protein in the liver, which led to increased expression of lipogenic genes. Inactivation of PPARγ2 may improve fatty liver induced by HF saturated fat.     

Tumor necrosis factor-α accelerates apoptosis of steatotic hepatocytes from a murine model of non-alcoholic fatty liver disease

Non-alcoholic steatohepatitis (NASH) develops in a subset of patients with non-alcoholic fatty liver disease (NAFLD), but the exact mechanisms involved in the progression of NAFLD to NASH remain poorly understood. We investigated the role of tumor necrosis factor-α (TNF-α) in the apoptosis of hepatocytes that is related to the severity of NASH. We separated primary hepatocytes from the NAFLD liver caused by a high-fat diet. The production of intracellular reactive oxygen species was increased in steatotic hepatocytes, which were also sensitive to TNF-α. This factor induced significant apoptosis through the signal-regulating kinase 1 (ASK1) and c-Jun N-terminal kinase (JNK) pathway. We describe here a novel culture model of steatotic hepatocytes separated from the NAFLD liver, and demonstrate that TNF-α induces their apoptosis in vitro.     

Increased peroxisomal fatty acid β-oxidation and enhanced expression of peroxisome proliferator-activated receptor-α in diabetic rat liver

To determine whether the increased fatty acid -oxidation in the peroxisomes of diabetic rat liver is mediated by a common peroxisome proliferation mechanism, we measured the activation of long-chain (LC) and very long chain (VLC) fatty acids catalyzed by palmitoyl CoA ligase (PAL) and lignoceryl CoA ligase and oxidation of LC (palmitic acid) and VLC (lignoceric acid) fatty acids by isotopic methods. Immunoblot analysis of acyl-CoA oxidase (ACO), and Northern blot analysis of peroxisome proliferator-activated receptor (PPAR-), ACO, and PAL were also performed. The PAL activity increased in peroxisomes and mitochondria from the liver of diabetic rats by 2.6-fold and 2.1-fold, respectively. The lignoceroyl-CoA ligase activity increased by 2.6-fold in diabetic peroxisomes. Palmitic acid oxidation increased in the diabetic peroxisomes and mitochondria by 2.5-fold and 2.7-fold, respectively, while lignoceric acid oxidation increased by 2.0-fold in the peroxisomes. Immunoreactive ACO protein increased by 2-fold in the diabetic group. The mRNA levels for PPAR-, ACO and PAL increased 2.9-, 2.8- and 1.6-fold, respectively, in the diabetic group. These results suggest that the increased supply of fatty acids to liver in diabetic state stimulates the expression of PPAR- and its target genes responsible for the metabolism of fatty acids.