Bempedoic Acid Restores Liver H2S Production in a Female Sprague-Dawley Rat Dietary Model of Non-Alcoholic Fatty Liver.

We previously demonstrated that treatment with BemA (bempedoic acid), an inhibitor of ATP citrate lyase, significantly reduces fatty liver in a model of liver steatosis (HFHFr-female Sprague-Dawley rat fed a high-fat high-fructose diet). Since the hepatic production of the gasotransmitter H2S is impaired in liver disorders, we were interested in determining if the production of H2S was altered in our HFHFr model and whether the administration of BemA reversed these changes. We used stored liver samples from a previous study to determine the total and enzymatic H2S production, as well as the expression of CBS (cystathionine ß-synthase), CSE (cystathionine γ-lyase), and 3MST (3-mercaptopiruvate sulfurtransferase), and the expression/activity of FXR (farnesoid X receptor), a transcription factor involved in regulating CSE expression. Our data show that the HFHFr diet reduces the total and enzymatic production of liver H2S, mainly by decreasing the expression of CBS and CSE. Furthermore, BemA treatment restored H2S production, increasing the expression of CBS and CSE, providing evidence for the involvement of FXR transcriptional activity and the mTORC1 (mammalian target of rapamycin1)/S6K1 (ribosomal protein S6 kinase beta-1)/PGC1α (peroxisome proliferator receptor gamma coactivator1α) pathway.

Bempedoic acid as a PPARα activator: new perspectives for hepatic steatosis treatment in a female rat experimental model. / El ácido bempedoico como activador PPARα: Nuevas perspectivas para el tratamiento de la esteatosis hepática en un modelo experimental de rata hembra.

INTRODUCTION: In its initial stages, nonalcoholic fatty liver disease presents hypertriglyceridemia and accumulation of lipids in the liver (hepatic steatosis). Bempedoic acid is an ATP:citrate lyase inhibitor that promotes a dual inhibition of the synthesis of cholesterol and fatty acids. However, its effect in the prevention / treatment of hepatic steatosis and hypertriglyceridemia has not been investigated. The aim of our work has been to elucidate whether bempedoic acid, through a mechanism other than ATP:citrate lyase inhibition, reverses these metabolic alterations. EXPERIMENTAL DESIGN: The study was carried out in female Sprague-Dawley rats fed, for three months, with a high fat diet supplemented with fructose (10% w/v) in drinking water. During the last month, bempedoic acid (30mg/kg/day) was administered to a group of animals. Zoometric and plasmatic parameters were analyzed, gene and protein expression analysis were performed in liver samples and PPAR-PPRE binding activity was determined. RESULTS: Our interventional model developed hepatic steatosis and hypertriglyceridemia. Despite an increase in total caloric intake, there was no increase in body weight of the animals. The administration of bempedoic acid significantly reduced hepatic steatosis and promoted a marked hepatocyte hypertrophy. There was a 66% increase in the liver weight of the animals treated with the drug that was not accompanied by modifications in the markers of inflammation, oxidative stress, or endoplasmic reticulum stress. Bempedoic acid activated the peroxisome proliferator activated nuclear receptor (PPARα) and its target genes. CONCLUSIONS: Bempedoic acid could be an effective therapy for the treatment of fatty liver and associated cardiovascular risk. Bempedoic acid has other mechanisms of action besides the inhibition of ATP: citrate lyase, such as the activation of PPARα, which could explain the reduction in hepatic steatosis and the increase in liver weight observed in animals treated with the drug.

Chronic liquid fructose supplementation does not cause liver tumorigenesis but elicits clear sex differences in the metabolic response in Sprague-Dawley rats.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) has increased over the last decades and may evolve into hepatocellular carcinoma (HCC). As HCC is challenging to treat, knowledge on the modifiable risk factors for NAFLD/HCC (e.g. hyper caloric diets rich in fructose) is essential. OBJECTIVE AND DESIGN: We used a model of diethyl nitrosamine-induced hepatocarcinogenesis to investigate the liver cancer-promoting effects of a diet supplemented with 10% liquid fructose, administered to male and female rats for 11 months. A subset of the fructose-supplemented rats received resveratrol (RVT) in the last 4 months of treatment. RESULTS AND DISCUSSION: Rat livers showed no de visu or histological evidence of liver tumorigenesis. However, we observed metabolic abnormalities that could be related to cancer development mainly in the female fructose-supplemented rats, such as increases in weight, adiposity and hepatic triglyceride levels, as well as hyperglycaemia, hyperuricemia, hyperleptinemia and a reduced insulin sensitivity index, which were partially reversed by RVT. Therefore, we performed a targeted analysis of 84 cancer-related genes in the female liver samples, which revealed expression changes associated with cancer-related pathways. Analysis of individual genes indicated that some changes increased the risk of hepatocarcinogenesis (Sfrp2, Ccl5, Socs3, and Gstp1), while others exerted a protective/preventive effect (Bcl2 and Cdh1). CONCLUSION: Our data clearly demonstrate that chronic fructose supplementation, as the sole dietary intervention, does not cause HCC development in rats.

Simple sugar intake and cancer incidence, cancer mortality and all-cause mortality: A cohort study from the PREDIMED trial.

OBJECTIVE: To examine associations between intake of simple sugars and cancer incidence, cancer mortality, and total mortality in a prospective cohort study based on the PREDIMED trial conducted from 2003 to 2010. METHODS: Participants were older individuals at high cardiovascular risk. Exposures were total sugar, glucose and fructose from solid or liquid sources, and fructose from fruit and 100% fruit juice. Cancer incidence was the primary outcome; cancer mortality and all-cause mortality were secondary outcomes. Multivariable-adjusted, time-dependent Cox proportional hazard models were used. RESULTS: Of 7447 individuals enrolled, 7056 (94.7%) were included (57.6% women, aged 67.0 ± 6.2 years). 534 incident cancers with 152 cancer deaths and 409 all-cause deaths were recorded after a median follow-up of 6 years. Intake of simple sugars in solid form was unrelated to outcomes. Higher cancer incidence was found per 5 g/day increase in intake of liquid sugars, with multivariable-adjusted HR of 1.08 (95% CI, 1.03-1.13) for total liquid sugar, 1.19 (95% CI, 1.07-1.31) for liquid glucose, 1.14 (95% CI, 1.05-1.23) for liquid fructose, and 1.39 (95% CI, 1.10-1.74) for fructose from fruit juice. Cancer and all-cause mortality increased to a similar extent with intake of all sugars in liquid form. In categorical models, cancer risk was dose-related for all liquid sugars. CONCLUSIONS: Simple sugar intake in drinks and fruit juice was associated with an increased risk of overall cancer incidence and mortality and all-cause mortality. This suggests that sugary beverages are a modifiable risk factor for cancer and all-cause mortality.

The Addition of Liquid Fructose to a Western-Type Diet in LDL-R-/- Mice Induces Liver Inflammation and Fibrogenesis Markers without Disrupting Insulin Receptor Signalling after an Insulin Challenge.

A high consumption of fat and simple sugars, especially fructose, has been related to the development of insulin resistance, but the mechanisms involved in the effects of these nutrients are not fully understood. This study investigates the effects of a Western-type diet and liquid fructose supplementation, alone and combined, on insulin signalling and inflammation in low-density lipoprotein (LDL) receptor-deficient mice (LDL-R-/-). LDL-R-/- mice were fed chow or Western diet ±15% fructose solution for 12 weeks. Plasma glucose and insulin, and the expression of genes related to inflammation in the liver and visceral white adipose tissue (vWAT), were analysed. V-akt murine thymoma viral oncogene homolog-2 (Akt) activation was measured in the liver of the mice after a single injection of saline or insulin. None of the dietary interventions caused inflammation in vWAT, whereas the Western diet induced hepatic inflammation, which was further enhanced by liquid fructose, leading also to a significant increase in fibrogenesis markers. However, there was no change in plasma glucose or insulin, or insulin-induced Akt phosphorylation. In conclusion, hepatic inflammation and fibrogenesis markers induced by a Western diet supplemented with liquid fructose in LDL-R-/- mice are not associated with a significant impairment of hepatic insulin signalling.

Fructose, but not glucose, impairs insulin signaling in the three major insulin-sensitive tissues.

Human studies support the relationship between high intake of fructose-sweetened beverages and type 2 diabetes, but there is a debate on whether this effect is fructose-specific or it is merely associated to an excessive caloric intake. Here we investigate the effects of 2 months' supplementation to female rats of equicaloric 10% w/v fructose or glucose solutions on insulin sensitivity in target tissues. Fructose supplementation caused hepatic deposition of triglycerides and changed the fatty acid profile of this fraction, with an increase in monounsaturated and a decrease in polyunsaturated species, but did not cause inflammation and oxidative stress. Fructose but not glucose-supplemented rats displayed an abnormal glucose tolerance test, and did not show increased phosphorylation of V-akt murine thymoma viral oncogene homolog-2 (Akt) in white adipose tissue and liver after insulin administration. In skeletal muscle, phosphorylation of Akt and of Akt substrate of 160 kDA (AS160) was not impaired but the expression of the glucose transporter type 4 (GLUT4) in the plasma membrane was reduced only in fructose-fed rats. In conclusion, fructose but not glucose supplementation causes fatty liver without inflammation and oxidative stress and impairs insulin signaling in the three major insulin-responsive tissues independently from the increase in energy intake.

Hypercholesterolemia and neurodegeneration. Comparison of hippocampal phenotypes in LDLr knockout and APPswe/PS1dE9 mice.

Previous studies suggest that Alzheimer's disease (AD) neurobiology could not be explained solely by an increase in ß-amyloid levels. Recently, it has been proposed that alterations in brain cholesterol metabolism may contribute to the pathogenesis of AD. In the present work, we focus on early changes in the hippocampal phenotypes of two mouse models in which cognitive impairments were previously described: a) the hypercholesterolemic LDL receptor knockout (LDLr -/-) and b) the APPswe/PS1dE9 (APP/PS1) transgenic model of familial AD. Our initial analysis, subsequent validation and additional experiments at the mRNA and protein levels demonstrate some parallels between the hippocampal phenotypes of these 2 mouse models, however our data suggest that the molecular mechanisms leading to cognitive decline are distinct in LDLr -/- and APP/PS1 animals. Genes related to cytokine signaling were significantly down-regulated in LDLr -/- mice when compared to both the wild-type and APP/PS1 mice, and these include prostaglandin-endoperoxide synthases 1 and 2 (ptgs1 and 2) and nerve grow factor (ngf). We have also detected reduced expression of genes related to lipid metabolism in LDLr -/- mice: peroxisome proliferator activated receptor gamma (pparg), pro-opiomelanocortin-alpha (pomc) and of protein kinase, AMP-activated, alpha 1 catalytic subunit of AMPK (prkaa1). Our array data also indicate that transcriptional activity of early genes involved in memory process, such as FBJ osteosarcoma oncogene (Fos) and the activity regulated cytoskeletal-associated protein (Arc) gene, are increased in the hippocampus of LDLr -/- mice. Several proteins like insulin degrading enzyme (IDE), PGC-1α, OXPHOS 1, NMDAR1 and cyclic AMP response element binding protein (CREB) are up-regulated in the LDLr -/- mice, while in the APP/PS1 mouse model only OXPHOS complexes 2, 3 and 5 are slightly downregulated. Further studies are necessary to understand the molecular pathways involved in memory loss in hypercholesterolemic LDLr -/- mice.

Simple sugar intake and hepatocellular carcinoma: epidemiological and mechanistic insight.

Sugar intake has dramatically increased during the last few decades. Specifically, there has been a clear trend towards higher consumption of fructose and high fructose corn syrup, which are the most common added sugars in processed food, soft drinks and other sweetened beverages. Although still controversial, this rising trend in simple sugar consumption has been positively associated with weight gain and obesity, insulin resistance and type 2 diabetes mellitus and non-alcoholic fatty liver disease. Interestingly, all of these metabolic alterations have also been related to the development of hepatocellular carcinoma. The purpose of this review is to discuss the evidence coming from epidemiological studies and data from animal models relating the consumption of simple sugars, and specifically fructose, with an increased risk of hepatocellular carcinoma and to gain insight into the putative molecular mechanisms involved.

Liquid fructose down-regulates liver insulin receptor substrate 2 and gluconeogenic enzymes by modifying nutrient sensing factors in rats.

High consumption of fructose-sweetened beverages has been linked to a high prevalence of chronic metabolic diseases. We have previously shown that a short course of fructose supplementation as a liquid solution induces glucose intolerance in female rats. In the present work, we characterized the fructose-driven changes in the liver and the molecular pathways involved. To this end, female rats were supplemented or not with liquid fructose (10%, w/v) for 7 or 14 days. Glucose and pyruvate tolerance tests were performed, and the expression of genes related to insulin signaling, gluconeogenesis and nutrient sensing pathways was evaluated. Fructose-supplemented rats showed increased plasma glucose excursions in glucose and pyruvate tolerance tests and reduced hepatic expression of several genes related to insulin signaling, including insulin receptor substrate 2 (IRS-2). However, the expression of key gluconeogenic enzymes, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, was reduced. These effects were caused by an inactivation of hepatic forkhead box O1 (FoxO1) due to an increase in its acetylation state driven by a reduced expression and activity of sirtuin 1 (SIRT1). Further contributing to FoxO1 inactivation, fructose consumption elevated liver expression of the spliced form of X-box-binding-protein-1 as a consequence of an increase in the activity of the mammalian target of rapamycin 1 and protein 38-mitogen activated protein kinase (p38-MAPK). Liquid fructose affects both insulin signaling (IRS-2 and FoxO1) and nutrient sensing pathways (p38-MAPK, mTOR and SIRT1), thus disrupting hepatic insulin signaling without increasing the expression of key gluconeogenic enzymes.

Liquid fructose downregulates Sirt1 expression and activity and impairs the oxidation of fatty acids in rat and human liver cells.

Fructose ingestion is associated with the production of hepatic steatosis and hypertriglyceridemia. For fructose to attain these effects in rats, simultaneous induction of fatty acid synthesis and inhibition of fatty acid oxidation is required. We aimed to determine the mechanism involved in the inhibition of fatty acid oxidation by fructose and whether this effect occurs also in human liver cells. Female rats were supplemented or not with liquid fructose (10% w/v) for 7 or 14 days; rat (FaO) and human (HepG2) hepatoma cells, and human hepatocytes were incubated with fructose 25mM for 24h. The expression and activity of the enzymes and transcription factors relating to fatty acid ß-oxidation were evaluated. Fructose inhibited the activity of fatty acid ß-oxidation only in livers of 14-day fructose-supplemented rats, as well as the expression and activity of peroxisome proliferator activated receptor α (PPARα). Similar results were observed in FaO and HepG2 cells and human hepatocytes. PPARα downregulation was not due to an osmotic effect or to an increase in protein-phosphatase 2A activity caused by fructose. Rather, it was related to increased content in liver of inactive and acetylated peroxisome proliferator activated receptor gamma coactivator 1α, due to a reduction in sirtuin 1 expression and activity. In conclusion, fructose inhibits liver fatty acid oxidation by reducing PPARα expression and activity, both in rat and human liver cells, by a mechanism involving sirtuin 1 down-regulation.

Regulation of gene expression in atherosclerosis: insights from microarray studies in monocytes/macrophages.

Atherosclerosis is a pathological phenomenon in which the walls of large arteries thicken and lose elasticity as a result of the growth of atheromatous lesions. It is a complex, multifactorial disease that involves several cell types and various pathobiological processes. Its genetic basis has not yet been deciphered, but it is related to complex multigene patterns influenced by environmental interactions. In this review, we focus specifically on the application of microarrays to atherosclerosis research using monocytes and monocyte-derived macrophages, as these are key cells in all phases of atherosclerosis, from the formation of foam cells to the destabilization and rupture of the atherosclerotic plaque. These studies have provided relevant information on genes involved in atherosclerosis development, contributing to our understanding of the molecular mechanisms that underlie this complex disease.

Type II interleukin-1 receptor expression is reduced in monocytes/macrophages and atherosclerotic lesions.

Type II interleukin-1 receptor (IL-1R2) is a non-signaling decoy receptor that negatively regulates the activity of interleukin-1 (IL-1), a pro-inflammatory cytokine involved in atherogenesis. In this article we assessed the relevance of IL-1R2 in atherosclerosis by studying its expression in monocytes from hyperlipidemic patients, in THP-1 macrophages exposed to lipoproteins and in human atherosclerotic lesions. Our results showed that the mRNA and protein expression of IL-1R2 was reduced in monocytes from patients with familial combined hyperlipidemia (-30%, p<0.05). THP-1 macrophages incubated with increasing concentrations of acetylated low density (ac-LDL) and very low density (VLDL) lipoproteins also exhibit a decrease in IL-1R2 mRNA and protein levels. Pre-incubation with agents that block intracellular accumulation of lipids prevents the decrease in IL-1R2 mRNA caused by lipoproteins. Lipoproteins also prevented the increase in IL-1R1 and IL-1R2 caused by a 4-h stimulation with LPS and reduced protein expression of total and phosphorylated IL-1 receptor-associated kinase-1. Finally, IL-1R2 expression in human atherosclerotic vessels was markedly lower than in non-atherosclerotic arteries (-80%, p<0.0005). Overall, our results suggest that under atherogenic conditions, there is a decrease in IL-1R2 expression in monocytes/macrophages and in the vascular wall that may facilitate IL-1 signaling.

Tissue factor pathway inhibitor 2 is induced by thrombin in human macrophages.

Tissue factor pathway inhibitor 2 (TFPI2) is a serine protease inhibitor critical for the regulation of extracellular matrix remodeling and atherosclerotic plaque stability. Previously, we demonstrated that TFPI2 expression is increased in monocytes from patients with familial combined hyperlipidemia (FCH). To gain insight into the molecular mechanisms responsible for this upregulation, we examined TFPI2 expression in THP-1 macrophages exposed to lipoproteins and thrombin. Our results showed that TFPI2 expression was not affected by treatment with very low density lipoproteins (VLDL), but was induced by thrombin (10 U/ml) in THP-1 (1.9-fold increase, p<0.001) and human monocyte-derived macrophages (2.3-fold increase, p<0.005). The specificity of the inductive effect was demonstrated by preincubation with the thrombin inhibitors hirudin and PPACK, which ablated thrombin effects. TFPI2 induction was prevented by pre-incubation with MEK1/2 and JNK inhibitors, but not by the EGF receptor antagonist AG1478. In the presence of parthenolide, an inhibitor of NFκB, but not of SR-11302, a selective AP-1 inhibitor, thrombin-mediated TFPI2 induction was blunted. Our results also show that thrombin treatment increased ERK1/2, JNK and IκBα phosphorylation. Finally, we ruled out the possibility that TFPI2 induction by thrombin was mediated by COX-2, as preincubation with a selective COX-2 inhibitor did not prevent the inductive effect. In conclusion, thrombin induces TFPI2 expression by a mechanism involving ERK1/2 and JNK phosphorylation, leading finally to NFkB activation. In the context of atherosclerosis, thrombin-induced macrophage TFPI2 expression could represent a means of avoiding excessive activation of matrix metalloproteases at sites of inflammation.

Liver AMP/ATP ratio and fructokinase expression are related to gender differences in AMPK activity and glucose intolerance in rats ingesting liquid fructose.

Women, but not men, show an association between fructose consumption and an increased risk of Type 2 diabetes mellitus. As rats are considered a model for human fructose metabolism, we sought to determine whether such a gender-related difference is present in Sprague-Dawley rats and to analyze the molecular mechanism behind. Male and female Sprague-Dawley rats had free access to water or to a 10% w/v fructose solution for 14 days. Plasma analytes, liver triglycerides and enzyme activities and the expression of enzymes and transcription factors related to fatty acid metabolism, insulin signaling and glucose tolerance were determined. Fructose-fed rats had hypertriglyceridemia, steatosis and reduced fatty acid oxidation activity, although the metabolic pattern of fructose-fed female rats was different to that observed for male rats. Fructose-fed female, but not male rats, showed no change in plasma leptin; they had hyperinsulinemia, an altered glucose tolerance test and less liver insulin receptor substrate-2. Further, only fructose-fed female rats had increased adenosine 5'-monophosphate (AMP)-activated protein kinase activity, resulting in a decreased expression of hepatic nuclear factor 4 and sterol response element binding protein 1. These differences were related to the fact that liver expression of the enzyme fructokinase, controlling fructose metabolism, was markedly induced by fructose ingestion in female, but not in male rats, resulting in a significant increase in the AMP/adenosine 5'-triphosphate (ATP) ratio and, thus, AMP-activated protein kinase activation, in female rats only. The difference in fructokinase induction could explain the higher metabolic burden produced by fructose ingestion in the livers of female Sprague-Dawley rats.

Reduction of liver fructokinase expression and improved hepatic inflammation and metabolism in liquid fructose-fed rats after atorvastatin treatment.

Consumption of beverages that contain fructose favors the increasing prevalence of metabolic syndrome alterations in humans, including non-alcoholic fatty liver disease (NAFLD). Although the only effective treatment for NAFLD is caloric restriction and weight loss, existing data show that atorvastatin, a hydroxymethyl-glutaryl-CoA reductase inhibitor, can be used safely in patients with NAFLD and improves hepatic histology. To gain further insight into the molecular mechanisms of atorvastatin's therapeutic effect on NAFLD, we used an experimental model that mimics human consumption of fructose-sweetened beverages. Control, fructose (10% w/v solution) and fructose+atorvastatin (30 mg/kg/day) Sprague-Dawley rats were sacrificed after 14 days. Plasma and liver tissue samples were obtained to determine plasma analytes, liver histology, and the expression of liver proteins that are related to fatty acid synthesis and catabolism, and inflammatory processes. Fructose supplementation induced hypertriglyceridemia and hyperleptinemia, hepatic steatosis and necroinflammation, increased the expression of genes related to fatty acid synthesis and decreased fatty acid ß-oxidation activity. Atorvastatin treatment completely abolished histological signs of necroinflammation, reducing the hepatic expression of metallothionein-1 and nuclear factor kappa B binding. Furthermore, atorvastatin reduced plasma (x 0.74) and liver triglyceride (x 0.62) concentrations, decreased the liver expression of carbohydrate response element binding protein transcription factor (x 0.45) and its target genes, and increased the hepatic activity of the fatty acid ß-oxidation system (x 1.15). These effects may be related to the fact that atorvastatin decreased the expression of fructokinase (x 0.6) in livers of fructose-supplemented rats, reducing the metabolic burden on the liver that is imposed by continuous fructose ingestion.

Suppressor of cytokine signaling-3 (SOCS-3) and a deficit of serine/threonine (Ser/Thr) phosphoproteins involved in leptin transduction mediate the effect of fructose on rat liver lipid metabolism.

UNLABELLED: There is controversy regarding whether fructose in liquid beverages constitutes another dietary ingredient of high caloric density or introduces qualitative changes in energy metabolism that further facilitate the appearance of metabolic diseases. Central to this issue is the elucidation of the molecular mechanism responsible for the metabolic alterations induced by fructose ingestion. Fructose administration (10% wt/vol) in the drinking water of Sprague-Dawley male rats for 14 days induced hyperleptinemia and hepatic leptin resistance. This was caused by impairment of the leptin-signal transduction mediated by both janus-activated kinase-2 and the mitogen-activated protein kinase pathway. The subsequent increase in activity in the liver of the unphosphorylated and active form of the forkhead box O1 nuclear factor, which transrepresses peroxisome proliferator-activated receptor alpha activity, and a lack of activation of the adenosine monophosphate-activated protein kinase, led to hypertriglyceridemia and hepatic steatosis. These alterations are attributable to two key events: (1) an increase in the amount of suppressor of cytokine signaling-3 protein, which blocks the phosphorylation and activation of janus-activated kinase-2 and Tyr(985) on the long form of the leptin receptor; and (2) a common deficit of phosphorylation in serine/threonine residues of key proteins in leptin-signal transduction pathways. The latter is probably produced by the early activation of protein phosphatase 2A, and further sustained by the accumulation in liver tissue of ceramide, an activator of protein phosphatase 2A, due to incomplete oxidation of fatty acids. CONCLUSION: Our data indicate that fructose ingestion as a liquid solution induces qualitative changes in liver metabolism that lead to metabolic diseases.

Monocyte gene-expression profile in men with familial combined hyperlipidemia and its modification by atorvastatin treatment.

AIM: The genetic origin of familial combined hyperlipidemia (FCH) is not well understood. We used microarray profiling of peripheral blood monocytes to search novel genes and pathways involved in FCH. METHODS: Fasting plasma for determination of lipid profiles, inflammatory molecules and adipokines was obtained and peripheral blood monocytes were isolated from male FCH patients basally and after 4 weeks of atorvastatin treatment. Sex-, age- and adiposity-matched controls were also studied. Gene-expression profiles were analyzed using Affymetrix Human Genome U133A 2.0 GeneChip arrays. RESULTS: Analysis of gene expression by cDNA microarrays showed that 82 genes were differentially expressed in FCH monocytes compared with controls. Atorvastatin treatment modified the expression of 86 genes. Pathway analysis revealed the over-representation of the complement and coagulation cascades, the hematopoietic cell lineage and the arachidonic acid metabolism pathways. Changes in the expression of some genes, confirmed by real-time RT-PCR, (CD36, leucine-rich repeats and immunoglobulin-like domains-1, tissue factor pathway inhibitor 2, myeloid cell nuclear differentiation antigen, tumor necrosis factor receptor superfamily, member 25, CD96 and lipoprotein lipase), may be related to a proinflammatory environment in FCH monocytes, which is partially reversed by atorvastatin. Higher plasma levels of triglycerides and free fatty acids and lower levels of adiponectin in FCH patients could also trigger changes in gene expression that atorvastatin cannot modify. CONCLUSION: Our results show clear differences in gene expression in FCH monocytes compared with those of matched healthy controls, some of which are influenced by atorvastatin treatment.

Peroxisome proliferator-activated receptor alpha down-regulation is associated with enhanced ceramide levels in age-associated cardiac hypertrophy.

We used an experimental murine model of accelerated aging, the senescence-accelerated mouse (SAM), to examine the effect of age-associated cardiac hypertrophy on peroxisome proliferator-activated receptor alpha (PPARalpha) expression and activity in the heart. Senescence-accelerated prone mice (SAM-P8) showed cardiac hypertrophy compared with senescence-accelerated resistant mice (SAM-R1). Furthermore, a decrease in PPARalpha messenger RNA (mRNA; 28% reduction, p<.001) and protein (47%, p<.05) levels and in PPAR DNA-binding activity was observed in SAM-P8 hearts. Increased protein-protein interaction between PPARalpha and the p65 subunit of nuclear factor-kappaB (NF-kappaB) was found, suggesting that this mechanism may prevent PPARalpha from binding to its response elements. The mRNA levels of PPARalpha target genes involved in fatty acid use were strongly suppressed in SAM-P8, which was consistent with the accumulation of ceramide in SAM-P8 hearts (2.5-fold induction, p<.05). These findings suggest that NF-kappaB activation in SAM-P8 heart prevents PPARalpha from binding to its response elements leading to changes in gene expression that may lead to ceramide accumulation in the aged heart.

PGC-1beta down-regulation is associated with reduced ERRalpha activity and MCAD expression in skeletal muscle of senescence-accelerated mice.

Mitochondrial dysfunction is involved in the development of aging. Here, we examined the effect of aging on the skeletal muscle expression of two isoforms of the transcriptional peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator-1 (PGC-1) in an experimental murine model of accelerated aging, the senescence-accelerated mouse (SAM). The senescence-accelerated prone mice (SAM-P8) showed no changes in PGC-1alpha, but a decrease in PGC-1beta expression (52% reduction, p <.001) was observed compared to the senescence-accelerated resistant mice (SAM-R1). In agreement with the proposed role of PGC-1beta as an estrogen-related receptor (ERR) protein ligand, the expression of the ERRalpha target gene medium-chain acyl-coenzyme A dehydrogenase was strongly suppressed (85%, p <.001) in SAM-P8. The decrease in the expression of medium-chain acyl-coenzyme A dehydrogenase was consistent with the reduction in ERRalpha DNA-binding activity of SAM-P8. These findings indicate that the age-mediated decrease in PGC-1beta expression in SAM-P8 skeletal muscle affects the expression of genes involved in mitochondrial fatty acid oxidation.

Ageing introduces a complex pattern of changes in several rat brain transcription factors depending on gender and anatomical localization.

As ageing changes the activity of several transcription factors in the rat cortex, we were interested in determining whether similar changes also appear in the hippocampus of old rats. We determined by electrophoretic gel shift assays the binding activity of nuclear factor kappa B (NFkappaB), activator protein-1 (AP-1), peroxisome proliferator-activated receptor (PPAR), and liver X receptor (LXR) in cortex and hippocampus samples from young (3-month-old), and old (18-month-old) male and female Sprague-Dawley rats. NFkappaB activity increased in old male and female rats, though only in cortex samples, while AP-1 activity decreased only in the cortex and hippocampus of old female animals. LXR activity decreased in all conditions, except in old male cortexes; whereas PPAR activity only decreased in the hippocampus of old female rats. Decreases in AP-1 and PPAR activities restricted to old female rats did not result from an age-related decline in plasma 17beta-estradiol concentration, as their activities did not change in samples obtained from ovariectomized young female rats. Our results indicate that ageing induces a complex pattern of changes in the brain-binding activity of NFkappaB, AP-1, PPAR and LXR, depending on the anatomical origin of the samples (cortex or hippocampus), and the sex of the animals studied.