FOXO1 represses PPARα-Mediated induction of FGF21 gene expression.

Fibroblast growth factor 21 (FGF21) has emerged as a metabolic regulator that exerts potent anti-diabetic and lipid-lowering effects in animal models of obesity and type 2 diabetes, showing a protective role in fatty liver disease and hepatocellular carcinoma progression. Hepatic expression of FGF21 is regulated by PPARα and is induced by fasting. Ablation of FoxO1 in liver has been shown to increase FGF21 expression in hyperglycemia. To better understand the role of FOXO1 in the regulation of FGF21 expression we have modified HepG2 human hepatoma cells to overexpress FoxO1 and PPARα. Here we show that FoxO1 represses PPARα-mediated FGF21 induction, and that the repression acts on the FGF21 gene promoter without affecting other PPARα target genes. Additionally, we demonstrate that FoxO1 physically interacts with PPARα and that FoxO1/3/4 depletion in skeletal muscle contributes to increased Fgf21 tissue levels. Taken together, these data indicate that FOXO1 is a PPARα-interacting protein that antagonizes PPARα activity on the FGF21 promoter. Because other PPARα target genes remained unaffected, these results suggest a highly specific mechanism implicated in FGF21 regulation. We conclude that FGF21 can be specifically modulated by FOXO1 in a PPARα-dependent manner.

Nutritional Regulation of Gene Expression: Carbohydrate-, Fat- and Amino Acid-Dependent Modulation of Transcriptional Activity.

The ability to detect changes in nutrient levels and generate an adequate response to these changes is essential for the proper functioning of living organisms. Adaptation to the high degree of variability in nutrient intake requires precise control of metabolic pathways. Mammals have developed different mechanisms to detect the abundance of nutrients such as sugars, lipids and amino acids and provide an integrated response. These mechanisms include the control of gene expression (from transcription to translation). This review reports the main molecular mechanisms that connect nutrients' levels, gene expression and metabolism in health. The manuscript is focused on sugars' signaling through the carbohydrate-responsive element binding protein (ChREBP), the role of peroxisome proliferator-activated receptors (PPARs) in the response to fat and GCN2/activating transcription factor 4 (ATF4) and mTORC1 pathways that sense amino acid concentrations. Frequently, alterations in these pathways underlie the onset of several metabolic pathologies such as obesity, insulin resistance, type 2 diabetes, cardiovascular diseases or cancer. In this context, the complete understanding of these mechanisms may improve our knowledge of metabolic diseases and may offer new therapeutic approaches based on nutritional interventions and individual genetic makeup.

Fibroblast Growth Factor 21 and the Adaptive Response to Nutritional Challenges.

The Fibroblast Growth Factor 21 (FGF21) is considered an attractive therapeutic target for obesity and obesity-related disorders due to its beneficial effects in lipid and carbohydrate metabolism. FGF21 response is essential under stressful conditions and its metabolic effects depend on the inducer factor or stress condition. FGF21 seems to be the key signal which communicates and coordinates the metabolic response to reverse different nutritional stresses and restores the metabolic homeostasis. This review is focused on describing individually the FGF21-dependent metabolic response activated by some of the most common nutritional challenges, the signal pathways triggering this response, and the impact of this response on global homeostasis. We consider that this is essential knowledge to identify the potential role of FGF21 in the onset and progression of some of the most prevalent metabolic pathologies and to understand the potential of FGF21 as a target for these diseases. After this review, we conclude that more research is needed to understand the mechanisms underlying the role of FGF21 in macronutrient preference and food intake behavior, but also in ß-klotho regulation and the activity of the fibroblast activation protein (FAP) to uncover its therapeutic potential as a way to increase the FGF21 signaling.

The Antiobesity Effects of Rosehip (Rosa canina) Flesh by Antagonizing the PPAR Gamma Activity in High-Fat Diet-Fed Mice.

SCOPE: The rosehip (Rosa canina) is a perennial shrub with a reddish pseudofruit that has demonstrated antidiabetic, antiatherosclerotic, and antiobesogenic effects in rodent models but there is low information about the molecular mechanisms underlying these effects on the onset and progression of diet-induced obesity. METHODS AND RESULTS: Four-week-old C57BL/6J male mice are subjected to a high-fat diet (HFD)-supplemented or not with R. canina flesh for 18 weeks. The results indicated that the R. canina flesh exerts a preventive effect on HFD-induced obesity with a significant reduction in body-weight gain and an improvement of hyperglycemia and insulin resistance caused by a HFD. At the tissue level, subcutaneous white adipose tissue exhibits a higher number of smaller adipocytes, with decreased lipogenesis. On its side, the liver shows a significant decrease in lipid droplet content and in the expression of genes related to lipogenesis, fatty acid oxidation, and glucose metabolism. Finally, the data suggest that most of these effects agree with the presence of a putative Perosxisome proliferator-activated receptor gamma (PPARγ) antagonist in the R. canina flesh. CONCLUSIONS: R. canina flesh dietary supplementation slows down the steatotic effect of a HFD at least in part through the regulation of the transcriptional activity of PPARγ.

Fsp27/CIDEC is a CREB target gene induced during early fasting in liver and regulated by FA oxidation rate.

FSP27 [cell death-inducing DFFA-like effector c (CIDEC) in humans] is a protein associated with lipid droplets that downregulates the fatty acid oxidation (FAO) rate when it is overexpressed. However, little is known about its physiological role in liver. Here, we show that fasting regulates liver expression of Fsp27 in a time-dependent manner. Thus, during the initial stages of fasting, a maximal induction of 800-fold was achieved, whereas during the later phase of fasting, Fsp27 expression decreased. The early response to fasting can be explained by a canonical PKA-CREB-CRTC2 signaling pathway because: i) CIDEC expression was induced by forskolin, ii) Fsp27 promoter activity was increased by CREB, and iii) Fsp27 expression was upregulated in the liver of Sirt1 knockout animals. Interestingly, pharmacological (etomoxir) or genetic (Hmgcs2 interference) inhibition of the FAO rate increases the in vivo expression of Fsp27 during fasting. Similarly, CIDEC expression was upregulated in HepG2 cells by either etomoxir or HMGCS2 interference. Our data indicate that there is a kinetic mechanism of autoregulation between short- and long-term fasting, by which free FAs delivered to the liver during early fasting are accumulated/exported by FSP27/CIDEC, whereas over longer periods of fasting, they are degraded in the mitochondria through the carnitine palmitoyl transferase system.

Regulation of human class I alcohol dehydrogenases by bile acids.

Class I alcohol dehydrogenases (ADH1s) are the rate-limiting enzymes for ethanol and vitamin A (retinol) metabolism in the liver. Because previous studies have shown that human ADH1 enzymes may participate in bile acid metabolism, we investigated whether the bile acid-activated nuclear receptor farnesoid X receptor (FXR) regulates ADH1 genes. In human hepatocytes, both the endogenous FXR ligand chenodeoxycholic acid and synthetic FXR-specific agonist GW4064 increased ADH1 mRNA, protein, and activity. Moreover, overexpression of a constitutively active form of FXR induced ADH1A and ADH1B expression, whereas silencing of FXR abolished the effects of FXR agonists on ADH1 expression and activity. Transient transfection studies and electrophoretic mobility shift assays revealed functional FXR response elements in the ADH1A and ADH1B proximal promoters, thus indicating that both genes are direct targets of FXR. These findings provide the first evidence for direct connection of bile acid signaling and alcohol metabolism.

FGF21 mediates the lipid metabolism response to amino acid starvation.

Lipogenic gene expression in liver is repressed in mice upon leucine deprivation. The hormone fibroblast growth factor 21 (FGF21), which is critical to the adaptive metabolic response to starvation, is also induced under amino acid deprivation. Upon leucine deprivation, we found that FGF21 is needed to repress expression of lipogenic genes in liver and white adipose tissue, and stimulate phosphorylation of hormone-sensitive lipase in white adipose tissue. The increased expression of Ucp1 in brown adipose tissue under these circumstances is also impaired in FGF21-deficient mice. Our results demonstrate the important role of FGF21 in the regulation of lipid metabolism during amino acid starvation.

Regulation of N-Myc downstream regulated gene 2 by bile acids.

Here we report that bile acid chenodeoxycholic acid (CDCA) and synthetic farnesoid X receptor (FXR) agonist GW4064 robustly induced tumor suppressor N-Myc downstream regulated gene 2 (NDRG2) expression in human hepatoma cells and primary hepatocytes. Knockdown of FXR abolished the induction by CDCA, whereas overexpression of a constitutively active form of FXR increased NDRG2 expression. A FXR-response element was identified within intronic regions of human and murine genes. Moreover, mice given GW4064 exhibit an increase of Ndrg2 expression in liver and kidney, where both NDRG2 and FXR are enriched. The identification of NDRG2 as a bile acid regulated gene may provide novel knowledge toward the understanding of NDRG2 physiological function and the link between metabolism and cancer.

Activating transcription factor 4-dependent induction of FGF21 during amino acid deprivation.

Nutrient deprivation or starvation frequently correlates with amino acid limitation. Amino acid starvation initiates a signal transduction cascade starting with the activation of the kinase GCN2 (general control non-derepressible 2) phosphorylation of eIF2 (eukaryotic initiation factor 2), global protein synthesis reduction and increased ATF4 (activating transcription factor 4). ATF4 modulates a wide spectrum of genes involved in the adaptation to dietary stress. The hormone FGF21 (fibroblast growth factor 21) is induced during fasting in liver and its expression induces a metabolic state that mimics long-term fasting. Thus FGF21 is critical for the induction of hepatic fat oxidation, ketogenesis and gluconeogenesis, metabolic processes which are essential for the adaptive metabolic response to starvation. In the present study, we have shown that FGF21 is induced by amino acid deprivation in both mouse liver and cultured HepG2 cells. We have identified the human FGF21 gene as a target gene for ATF4 and we have localized two conserved ATF4-binding sequences in the 5' regulatory region of the human FGF21 gene, which are responsible for the ATF4-dependent transcriptional activation of this gene. These results add FGF21 gene induction to the transcriptional programme initiated by increased levels of ATF4 and offer a new mechanism for the induction of the FGF21 gene expression under nutrient deprivation.

Human HMGCS2 regulates mitochondrial fatty acid oxidation and FGF21 expression in HepG2 cell line.

HMGCS2 (hydroxymethylglutaryl CoA synthase 2), the gene that regulates ketone body production, is barely expressed in cultured cell lines. In this study, we restored HMGCS2 expression and activity in HepG2 cells, thus showing that the wild type enzyme can induce fatty acid ß-oxidation (FAO) and ketogenesis, whereas a catalytically inactive mutant C166A did not generate either process. Peroxisome proliferator-activated receptor (PPAR) α expression also induces fatty acid ß-oxidation and endogenous HMGCS2 expression. Interestingly, PPARα-mediated induction was abolished when HMGCS2 expression was down-regulated by RNAi. These results indicate that HMGCS2 expression is both sufficient and necessary to the control of fatty acid oxidation in these cells. Next, we examined the expression pattern of several PPARα target genes in this now "ketogenic" HepG2 cell line. FGF21 (fibroblast growth factor 21) expression was specifically induced by HMGCS2 activity or by the inclusion of the oxidized form of ketone bodies (acetoacetate) in the culture medium. This effect was blunted by SirT1 (sirtuin 1) RNAi, so we propose a SirT1-dependent mechanism for FGF21 induction by acetoacetate. These data suggest a novel feed-forward mechanism by which HMGCS2 could regulate adaptive metabolic responses during fasting. This mechanism could be physiologically relevant, because fasting-mediated induction of liver FGF21 was dependent on SirT1 activity in vivo.

PGC-1ß regulates mouse carnitine-acylcarnitine translocase through estrogen-related receptor α.

Carnitine/acylcarnitine translocase (CACT) is a mitochondrial-membrane carrier proteins that mediates the transport of acylcarnitines into the mitochondrial matrix for their oxidation by the mitochondrial fatty acid-oxidation pathway. CACT deficiency causes a variety of pathological conditions, such as hypoketotic hypoglycemia, cardiac arrest, hepatomegaly, hepatic dysfunction and muscle weakness, and it can be fatal in newborns and infants. Here we report that expression of the Cact gene is induced in mouse skeletal muscle after 24h of fasting. To gain insight into the control of Cact gene expression, we examine the transcriptional regulation of the mouse Cact gene. We show that the 5'-flanking region of this gene is transcriptionally active and contains a consensus sequence for the estrogen-related receptor (ERR), a member of the nuclear receptor family of transcription factors. This sequence binds ERRαin vivo and in vitro and is required for the activation of Cact expression by the peroxisome proliferator-activated receptor gamma coactivator (PGC)-1/ERR axis. We also demonstrate that XTC790, the inverse agonist of ERRα, specifically blocks Cact activation by PGC-1ß in C2C12 cells.

Different fatty acid metabolism effects of (-)-epigallocatechin-3-gallate and C75 in adenocarcinoma lung cancer.

BACKGROUND: Fatty acid synthase (FASN) is overexpressed and hyperactivated in several human carcinomas, including lung cancer. We characterize and compare the anti-cancer effects of the FASN inhibitors C75 and (-)-epigallocatechin-3-gallate (EGCG) in a lung cancer model. METHODS: We evaluated in vitro the effects of C75 and EGCG on fatty acid metabolism (FASN and CPT enzymes), cellular proliferation, apoptosis and cell signaling (EGFR, ERK1/2, AKT and mTOR) in human A549 lung carcinoma cells. In vivo, we evaluated their anti-tumour activity and their effect on body weight in a mice model of human adenocarcinoma xenograft. RESULTS: C75 and EGCG had comparable effects in blocking FASN activity (96,9% and 89,3% of inhibition, respectively). In contrast, EGCG had either no significant effect in CPT activity, the rate-limiting enzyme of fatty acid ß-oxidation, while C75 stimulated CPT up to 130%. Treating lung cancer cells with EGCG or C75 induced apoptosis and affected EGFR-signaling. While EGCG abolished p-EGFR, p-AKT, p-ERK1/2 and p-mTOR, C75 was less active in decreasing the levels of EGFR and p-AKT. In vivo, EGCG and C75 blocked the growth of lung cancer xenografts but C75 treatment, not EGCG, caused a marked animal weight loss. CONCLUSIONS: In lung cancer, inhibition of FASN using EGCG can be achieved without parallel stimulation of fatty acid oxidation and this effect is related mainly to EGFR signaling pathway. EGCG reduce the growth of adenocarcinoma human lung cancer xenografts without inducing body weight loss. Taken together, EGCG may be a candidate for future pre-clinical development.

A Mixture of Pure, Isolated Polyphenols Worsens the Insulin Resistance and Induces Kidney and Liver Fibrosis Markers in Diet-Induced Obese Mice.

Obesity is a worldwide epidemic with severe metabolic consequences. Polyphenols are secondary metabolites in plants and the most abundant dietary antioxidants, which possess a wide range of health effects. The most relevant food sources are fruit and vegetables, red wine, black and green tea, coffee, virgin olive oil, and chocolate, as well as nuts, seeds, herbs, and spices. The aim of this work was to evaluate the ability of a pure, isolated polyphenol supplementation to counteract the pernicious metabolic effects of a high-fat diet (HFD). Our results indicated that the administration of pure, isolated polyphenols under HFD conditions for 26 weeks worsened the glucose metabolism in diet-induced obese mice. The data showed that the main target organ for these undesirable effects were the kidneys, where we observed fibrotic, oxidative, and kidney-disease markers. This work led us to conclude that the administration of pure polyphenols as a food supplement would not be advisable. Instead, the ingestion of complete "whole" foods would be the best way to get the health effects of bioactive compounds such as polyphenols.

Transcriptional regulation of the human acetoacetyl-CoA synthetase gene by PPARgamma.

In the cytosol of lipogenic tissue, ketone bodies are activated by AACS (acetoacetyl-CoA synthetase) and incorporated into cholesterol and fatty acids. AACS gene expression is particularly abundant in white adipose tissue, as it is induced during adipocyte differentiation. In order to elucidate the mechanism controlling the gene expression of human AACS and to clarify its physiological role, we isolated the human promoter, characterized the elements required to initiate transcription and analysed the expression of the gene in response to PPARgamma (peroxisome-proliferator-activated receptor gamma), an inducer of adipogenesis. We show that the human AACS promoter is a PPARgamma target gene and that this nuclear receptor is recruited to the AACS promoter by direct interaction with Sp1 (stimulating protein-1).

Lyophilized Maqui (Aristotelia chilensis) Berry Administration Suppresses High-Fat Diet-Induced Liver Lipogenesis through the Induction of the Nuclear Corepressor SMILE.

The liver is one of the first organs affected by accumulated ectopic lipids. Increased de novo lipogenesis and excessive triglyceride accumulation in the liver are hallmarks of nonalcoholic fatty liver disease (NAFLD) and are strongly associated with obesity, insulin resistance, and type 2 diabetes. Maqui dietary supplemented diet-induced obese mice showed better insulin response and decreased weight gain. We previously described that these positive effects of maqui are partially due to an induction of a brown-like phenotype in subcutaneous white adipose tissue that correlated with a differential expression of Chrebp target genes. In this work, we aimed to deepen the molecular mechanisms underlying the impact of maqui on the onset and development of the obese phenotype and insulin resistance focusing on liver metabolism. Our results showed that maqui supplementation decreased hepatic steatosis caused by a high-fat diet. Changes in the metabolic profile include a downregulation of the lipogenic liver X receptor (LXR) target genes and of fatty acid oxidation gene expression together with an increase in the expression of small heterodimer partner interacting leucine zipper protein (Smile), a corepressor of the nuclear receptor family. Our data suggest that maqui supplementation regulates lipid handling in liver to counteract the metabolic impact of a high-fat diet.

Metabolic Impact of Flavonoids Consumption in Obesity: From Central to Peripheral.

The prevention and treatment of obesity is primary based on the follow-up of a healthy lifestyle, which includes a healthy diet with an important presence of bioactive compounds such as polyphenols. For many years, the health benefits of polyphenols have been attributed to their anti-oxidant capacity as free radical scavengers. More recently it has been described that polyphenols activate other cell-signaling pathways that are not related to ROS production but rather involved in metabolic regulation. In this review, we have summarized the current knowledge in this field by focusing on the metabolic effects of flavonoids. Flavonoids are widely distributed in the plant kingdom where they are used for growing and defensing. They are structurally characterized by two benzene rings and a heterocyclic pyrone ring and based on the oxidation and saturation status of the heterocyclic ring flavonoids are grouped in seven different subclasses. The present work is focused on describing the molecular mechanisms underlying the metabolic impact of flavonoids in obesity and obesity-related diseases. We described the effects of each group of flavonoids in liver, white and brown adipose tissue and central nervous system and the metabolic and signaling pathways involved on them.

Lyophilized Maqui (Aristotelia chilensis) Berry Induces Browning in the Subcutaneous White Adipose Tissue and Ameliorates the Insulin Resistance in High Fat Diet-Induced Obese Mice.

Maqui (Aristotelia Chilensis) berry features a unique profile of anthocyanidins that includes high amounts of delphinidin-3-O-sambubioside-5-O-glucoside and delphinidin-3-O-sambubioside and has shown positive effects on fasting glucose and insulin levels in humans and murine models of type 2 diabetes and obesity. The molecular mechanisms underlying the impact of maqui on the onset and development of the obese phenotype and insulin resistance was investigated in high fat diet-induced obese mice supplemented with a lyophilized maqui berry. Maqui-dietary supplemented animals showed better insulin response and decreased weight gain but also a differential expression of genes involved in de novo lipogenesis, fatty acid oxidation, multilocular lipid droplet formation and thermogenesis in subcutaneous white adipose tissue (scWAT). These changes correlated with an increased expression of the carbohydrate response element binding protein b (Chrebpb), the sterol regulatory binding protein 1c (Srebp1c) and Cellular repressor of adenovirus early region 1A-stimulated genes 1 (Creg1) and an improvement in the fibroblast growth factor 21 (FGF21) signaling. Our evidence suggests that maqui dietary supplementation activates the induction of fuel storage and thermogenesis characteristic of a brown-like phenotype in scWAT and counteracts the unhealthy metabolic impact of an HFD. This induction constitutes a putative strategy to prevent/treat diet-induced obesity and its associated comorbidities.

Green tea catechin inhibits fatty acid synthase without stimulating carnitine palmitoyltransferase-1 or inducing weight loss in experimental animals.

BACKGROUND: The enzyme fatty acid synthase (FASN) is highly expressed in many human carcinomas and its inhibition is cytotoxic to human cancer cells. The use of FASN inhibitors has been limited until now by anorexia and weight loss, which is associated with the stimulation of fatty acid oxidation. MATERIALS AND METHODS: The in vitro effect of (-)-epigallocatechin-3-gallate (EGCG) on fatty acid metabolism enzymes, on apoptosis and on cell signalling was evaluated. In vivo, the effect of EGCG on animal body weight was addressed. RESULTS: EGCG inhibited FASN activity, induced apoptosis and caused a marked decrease of human epidermal growth factor receptor 2 (HER2), phosphatidylinositol 3-kinase (PI3K)/AKT and extracellular (signal)-regulated kinase (ERK) 1/2 proteins, in breast cancer cells. EGCG did not induce a stimulatory effect on CPT-1 activity in vitro (84% of control), or on animal body weight in vivo (99% of control). CONCLUSION: EGCG is a FASN inhibitor with anticancer activity which does not exhibit cross-activation of fatty acid oxidation and does not induce weight loss, suggesting its potential use as an anticancer drug.

Mediterranean Tomato-Based Sofrito Sauce Improves Fibroblast Growth Factor 21 (FGF21) Signaling in White Adipose Tissue of Obese ZUCKER Rats.

SCOPE: Obesity is a fibroblast growth factor 21 (FGF21)-resistant state. Since FGF21 production and signaling are regulated by some bioactive dietary compounds, we analyze the impact of Mediterranean tomato-based sofrito sauce on: (i) the FGF21 expression and signaling in visceral white adipose tissue (vWAT), and (ii) the insulin sensitivity of obese Zucker rats (OZR). METHODS AND RESULTS: OZR are fed with a sofrito-supplemented diet or control diet. Insulin sensitivity and FGF21 signaling are determined. We observed that sofrito is able to improve the responsiveness to both hormones in obese rats. Sofrito-supplemented diet increases FGF21 signaling in vWAT by inducing the expression of the FGF receptors (FGFR1 and FGFR4) that promotes the expression of canonical target genes, like Egr-1, c-Fos and uncoupling protein 1 (Ucp1). CONCLUSIONS: A sofrito-supplemented diet improves insulin and FGF21 sensitivity in OZR, explaining part of sofrito's healthy effects on glucose metabolism. In addition, induction of UCP1 and the unchanged body weight despite the hyperphagic behavior of the sofrito-fed rats suggests that the increase in FGF21 signaling correlates with an increase in energy expenditure (EE). Further studies in humans may help to understand whether sofrito consumption increases the EE in obese individuals.

Ketogenic HMGCS2 Is a c-Myc target gene expressed in differentiated cells of human colonic epithelium and down-regulated in colon cancer.

HMGCS2, the gene that regulates ketone body production, is expressed in liver and several extrahepatic tissues, such as the colon. In CaCo-2 colonic epithelial cells, the expression of this gene increases with cell differentiation. Accordingly, immunohistochemistry with specific antibodies shows that HMGCS2 is expressed mainly in differentiated cells of human colonic epithelium. Here, we used a chromatin immunoprecipitation assay to study the molecular mechanism responsible for this expression pattern. The assay revealed that HMGCS2 is a direct target of c-Myc, which represses HMGCS2 transcriptional activity. c-Myc transrepression is mediated by blockade of the transactivating activity of Miz-1, which occurs mainly through a Sp1-binding site in the proximal promoter of the gene. Accordingly, the expression of human HMGCS2 is down-regulated in 90% of Myc-dependent colon and rectum tumors. HMGCS2 protein expression is down-regulated preferentially in moderately and poorly differentiated carcinomas. In addition, it is also down-regulated in 80% of small intestine Myc-independent tumors. Based on these findings, we propose that ketogenesis is an undesirable metabolic characteristic of the proliferating cell, which is down-regulated through c-Myc-mediated repression of the key metabolic gene HMGCS2.