Citizen science in environmental health research: A comparison with conventional approaches and creation of a guidance tool issued from the LILAS initiative.

CONTEXT: Public interest for citizen science (CS) in environmental health is growing. The goals of environmental health research projects are diverse, as are the methods used to reach these goals. Opportunities for greater implication of the civil society and related challenges differ at each step of such projects. These methodological aspects need to be widely shared and understood by all stakeholders. The LILAS initiative (acronym for "application of citizen science approaches such as LIving LAbS to research on environmental exposures and chronic risks") aimed to 1) favor a mutual understanding of the main issues and research methods in environmental health, of their stakes for different actors, but also of the requirements, strengths and limitations of these methods and to 2) identify expected benefits and points of attention related to stronger degrees of participation as part of environmental health research projects. METHODS: The LILAS initiative gathered institutional researchers, academics and civil society representatives interested in environmental exposures. Five meetings allowed to collectively identify different types of environmental health research studies and reflect about the benefits, limitations, and methodological issues related to the introduction of growing citizen participation as part of such studies. An analytic table matrix summarizing these aspects was co-created and filled by participants, as a tool devoted to help stakeholders with the definition of future CS research projects in environmental health. RESULTS: For different fields of research (e.g.: studies for assessment of environmental exposures, interventions on these exposures, quantitative risk assessment, epidemiological studies), the matrix lists expected benefits for various stakeholders, the fundamental principles of research methods and related practical constraints, but also advantages and limitations related to the use of CS or conventional research approaches. CONCLUSION: The LILAS initiative allowed to develop a tool which provides consolidated grounds for the co-creation of research projects on environmental exposures involving CS.

SDHi fungicides: An example of mitotoxic pesticides targeting the succinate dehydrogenase complex.

Succinate dehydrogenase inhibitors (SDHi) are fungicides used to control the proliferation of pathogenic fungi in crops. Their mode of action is based on blocking the activity of succinate dehydrogenase (SDH), a universal enzyme expressed by all species harboring mitochondria. The SDH is involved in two interconnected metabolic processes for energy production: the transfer of electrons in the mitochondrial respiratory chain and the oxidation of succinate to fumarate in the Krebs cycle. In humans, inherited SDH deficiencies may cause major pathologies including encephalopathies and cancers. The cellular and molecular mechanisms related to such genetic inactivation have been well described in neuroendocrine tumors, in which it induces an oxidative stress, a pseudohypoxic phenotype, a metabolic, epigenetic and transcriptomic remodeling, and alterations in the migration and invasion capacities of cancer cells, in connection with the accumulation of succinate, an oncometabolite, substrate of the SDH. We will discuss recent studies reporting toxic effects of SDHi in non-target organisms and their implications for risk assessment of pesticides. Recent data show that the SDH structure is highly conserved during evolution and that SDHi can inhibit SDH activity in mitochondria of non-target species, including humans. These observations suggest that SDHi are not specific inhibitors of fungal SDH. We hypothesize that SDHi could have toxic effects in other species, including humans. Moreover, the analysis of regulatory assessment reports shows that most SDHi induce tumors in animals without evidence of genotoxicity. Thus, these substances could have a non-genotoxic mechanism of carcinogenicity that still needs to be fully characterized and that could be related to SDH inhibition. The use of pesticides targeting mitochondrial enzymes encoded by tumor suppressor genes raises questions on the risk assessment framework of mitotoxic pesticides. The issue of SDHi fungicides is therefore a textbook case that highlights the urgent need for changes in regulatory assessment.

New approach methodologies to facilitate and improve the hazard assessment of non-genotoxic carcinogens-a PARC project.

Carcinogenic chemicals, or their metabolites, can be classified as genotoxic or non-genotoxic carcinogens (NGTxCs). Genotoxic compounds induce DNA damage, which can be detected by an established in vitro and in vivo battery of genotoxicity assays. For NGTxCs, DNA is not the primary target, and the possible modes of action (MoA) of NGTxCs are much more diverse than those of genotoxic compounds, and there is no specific in vitro assay for detecting NGTxCs. Therefore, the evaluation of the carcinogenic potential is still dependent on long-term studies in rodents. This 2-year bioassay, mainly applied for testing agrochemicals and pharmaceuticals, is time-consuming, costly and requires very high numbers of animals. More importantly, its relevance for human risk assessment is questionable due to the limited predictivity for human cancer risk, especially with regard to NGTxCs. Thus, there is an urgent need for a transition to new approach methodologies (NAMs), integrating human-relevant in vitro assays and in silico tools that better exploit the current knowledge of the multiple processes involved in carcinogenesis into a modern safety assessment toolbox. Here, we describe an integrative project that aims to use a variety of novel approaches to detect the carcinogenic potential of NGTxCs based on different mechanisms and pathways involved in carcinogenesis. The aim of this project is to contribute suitable assays for the safety assessment toolbox for an efficient and improved, internationally recognized hazard assessment of NGTxCs, and ultimately to contribute to reliable mechanism-based next-generation risk assessment for chemical carcinogens.

Cigarette smoke and tumor microenvironment copromote aggressiveness of human breast cancer cells.

Breast cancer is a major public health issue and the role of pollutants in promoting breast cancer progression has recently been suggested. We aimed to assess if a mixture of pollutants, cigarette smoke, could favor the aggressivity of breast cancer cells. We also evaluated the impact of the tumor microenvironment, largely represented by adipocytes, in mediating this modification of cell phenotype. Breast cancer cells lines, MCF-7 were cultured using a transwell coculture model with preadipocytes hMADS cells or were cultured alone. Cells were treated with cigarette smoke extract (CSE) and the four conditions: control, treated by CSE, coculture, and coexposure (coculture and CSE) were compared. We analyzed morphological changes, cell migration, resistance to anoikis, stemness, epithelial-to-mesenchymal transition (EMT), and the presence of hormonal receptors in each condition. A complete transcriptomic analysis was carried out to highlight certain pathways. We also assessed whether the aryl hydrocarbon receptor (AhR), a receptor involved in the metabolism of xenobiotics, could mediate these modifications. Several hallmarks of metastasis were specific to the coexposure condition (cell migration, resistance to anoikis, stemness characterized by CD24/CD44 ratios and ALDH1A1 and ALDH1A3 rates) whereas others (morphological changes, EMT, loss of hormonal receptors) could be seen in the coculture condition and were aggravated by CSE (coexposure). Moreover, MCF-7 cells presented a decrease in hormonal receptors, suggesting an endocrine treatment resistance. These results were confirmed by the transcriptomic analysis. We suggest that the AhR could mediate the loss of hormonal receptor and the increase in cell migration.

Identification of non-validated endocrine disrupting chemical characterization methods by screening of the literature using artificial intelligence and by database exploration.

BACKGROUND: Exposure to endocrine disrupting chemicals (EDCs) represents a critical public health threat. Several adverse health outcomes (e.g., cancers, metabolic and neurocognitive/neurodevelopmental disorders, infertility, immune diseases and allergies) are associated with exposure to EDCs. However, the regulatory tests that are currently employed in the EU to identify EDCs do not assess all of the endocrine pathways. OBJECTIVE: Our objective was to explore the literature, guidelines and databases to identify relevant and reliable test methods which could be used for prioritization and regulatory pre-validation of EDCs in missing and urgent key areas. METHODS: Abstracts of articles referenced in PubMed were automatically screened using an updated version of the AOP-helpFinder text mining approach. Other available sources were manually explored. Exclusion criteria (computational methods, specific tests for estrogen receptors, tests under validation or already validated, methods accepted by regulatory bodies) were applied according to the priorities of the French Public-privatE Platform for the Pre-validation of Endocrine disRuptors (PEPPER) characterisation methods. RESULTS: 226 unique non-validated methods were identified. These experimental methods (in vitro and in vivo) were developed for 30 species using diverse techniques (e.g., reporter gene assays and radioimmunoassays). We retrieved bioassays mainly for the reproductive system, growth/developmental systems, lipogenesis/adipogenicity, thyroid, steroidogenesis, liver metabolism-mediated toxicity, and more specifically for the androgen-, thyroid hormone-, glucocorticoid- and aryl hydrocarbon receptors. CONCLUSION: We identified methods to characterize EDCs which could be relevant for regulatory pre-validation and, ultimately for the efficient prevention of EDC-related severe health outcomes. This integrative approach highlights a successful and complementary strategy which combines computational and manual curation approaches.

Mitochondrial Dysfunction as a Hallmark of Environmental Injury.

Environmental factors including diet, sedentary lifestyle and exposure to pollutants largely influence human health throughout life. Cellular and molecular events triggered by an exposure to environmental pollutants are extremely variable and depend on the age, the chronicity and the doses of exposure. Only a fraction of all relevant mechanisms involved in the onset and progression of pathologies in response to toxicants has probably been identified. Mitochondria are central hubs of metabolic and cell signaling responsible for a large variety of biochemical processes, including oxidative stress, metabolite production, energy transduction, hormone synthesis, and apoptosis. Growing evidence highlights mitochondrial dysfunction as a major hallmark of environmental insults. Here, we present mitochondria as crucial organelles for healthy metabolic homeostasis and whose dysfunction induces critical adverse effects. Then, we review the multiple mechanisms of action of pollutants causing mitochondrial toxicity in link with chronic diseases. We propose the Aryl hydrocarbon Receptor (AhR) as a model of "exposome receptor", whose activation by environmental pollutants leads to various toxic events through mitochondrial dysfunction. Finally, we provide some remarks related to mitotoxicity and risk assessment.

Evolutionarily conserved susceptibility of the mitochondrial respiratory chain to SDHI pesticides and its consequence on the impact of SDHIs on human cultured cells.

Succinate dehydrogenase (SDH) inhibitors (SDHIs) are used worldwide to limit the proliferation of molds on plants and plant products. However, as SDH, also known as respiratory chain (RC) complex II, is a universal component of mitochondria from living organisms, highly conserved through evolution, the specificity of these inhibitors toward fungi warrants investigation. We first establish that the human, honeybee, earthworm and fungal SDHs are all sensitive to the eight SDHIs tested, albeit with varying IC50 values, generally in the micromolar range. In addition to SDH, we observed that five of the SDHIs, mostly from the latest generation, inhibit the activity of RC complex III. Finally, we show that the provision of glucose ad libitum in the cell culture medium, while simultaneously providing sufficient ATP and reducing power for antioxidant enzymes through glycolysis, allows the growth of RC-deficient cells, fully masking the deleterious effect of SDHIs. As a result, when glutamine is the major carbon source, the presence of SDHIs leads to time-dependent cell death. This process is significantly accelerated in fibroblasts derived from patients with neurological or neurodegenerative diseases due to RC impairment (encephalopathy originating from a partial SDH defect) and/or hypersensitivity to oxidative insults (Friedreich ataxia, familial Alzheimer's disease).

A dual mixture of persistent organic pollutants modifies carbohydrate metabolism in the human hepatic cell line HepaRG.

Individuals as well as entire ecosystems are exposed to mixtures of Persistent Organic Pollutants (POPs). Previously, we showed, by a non-targeted approach, that the expression of several genes involved in carbohydrate metabolism was almost completely inhibited in the human hepatic cell line HepaRG following exposure to a mixture of the organochlorine insecticide alpha-endosulfan and 2,3,7,8 tetrachlorodibenzo-p-dioxin. In this European HEALS project, which studies the effects of the exposome on human health, we used a Physiologically Based BioKinetic model to compare the concentrations previously used in vitro with in vivo exposures for humans. We investigated the effects of these POPs on the levels of proteins, on glycogen content, glucose production and the oxidation of glucose into CO2 and correlated them to the expression of genes involved in carbohydrate metabolism as measured by RT-qPCR. Exposure to individual POPs and the mixture decreased the expression of the proteins investigated as well as glucose output (up to 82%), glucose oxidation (up to 29%) and glycogen content (up to 48%). siRNAs that specifically inhibit the expression of several xenobiotic receptors were used to assess receptor involvement in the effects of the POPs. In the HepaRG model, we demonstrate that the effects are mediated by the aryl hydrocarbon receptor and the estrogen receptor alpha, but not the pregnane X receptor or the constitutive androstane receptor. These results provide evidence that exposure to combinations of POPs, acting through different signaling pathways, may affect, more profoundly than single pollutants alone, metabolic pathways such as carbohydrate/energy metabolism and play a potential role in pollutant associated metabolic disorders.

Vanadate inhibits transcription of the rat insulin receptor gene via a proximal sequence of the 5'flanking region.

Vanadate, a protein tyrosine phosphatase inhibitor which elicits insulin-like effects, has previously been shown to inhibit expression of the insulin receptor gene at the transcriptional level in rat hepatoma cells. In an attempt to identify the DNA sequence and transcription factors potentially involved in this effect, a fragment of the proximal 5'flanking region of the IR gene (-1143/-252 upstream the ATG codon) has been cloned and functionally characterized. RNase protection allowed the identification of several transcription start sites in the conserved region of the gene, among which two major sites at -455 and -396. Upon fusion to the luciferase gene and transient transfection into hepatoma cells, the -1143/-252 fragment showed promoter activity. This was unaffected by deletion of the -1143/-761 sequence, but markedly decreased (90%) by additional deletion of the -760/-465 sequence. Treatment of hepatoma cells with vanadate led to a dose-dependent decrease in promoter activity of the 1143/-252, -760/-252 and -464/-252 constructs (change relative to untreated cells, 40, 55 and 23% at 125 µM, and 70, 85 and 62% at 250 µM, respectively). These data suggest that although the entire DNA sequence upstream the transcription start sites is probably involved in vanadate-induced inhibition, the short sequence downstream of position -464 and is sufficient for inhibition. Potential targets of vanadate are the transcription factors FoxO1 and HMGA1, two downstream targets of the insulin signaling pathway which have been shown to mediate the inhibitory effect of insulin on IR gene expression.

A comprehensive characterization of the impact of mycophenolic acid on the metabolism of Jurkat T cells.

Metabolic reprogramming is critical for T cell fate and polarization and is regulated by metabolic checkpoints, including Myc, HIF-1α, AMPK and mTORC1. Our objective was to determine the impact of mycophenolic acid (MPA) in comparison with rapamycin (Rapa), an inhibitor of mTORC1, on the metabolism of Jurkat T cells. We identified a drug-specific transcriptome signature consisting of the key enzymes and transporters involved in glycolysis, glutaminolysis or nucleotide synthesis. MPA produced an early and transient drop in the intracellular ATP content related to the inhibition of de novo synthesis of purines, leading to the activation of the energy sensor AMPK. MPA decreases glycolytic flux, consistent with a reduction in glucose uptake, but also in the oxidation of glutamine. Additionally, both drugs reduce aerobic glycolysis. The expression of HIF-1α and Myc, promoting the activation of glycolysis and glutaminolysis, was inhibited by MPA and Rapa. In conclusion, we report that MPA profoundly impacts the cellular metabolism of Jurkat T cells by generating an energetic distress, decreasing the glycolytic and glutaminolytic fluxes and by targeting HIF-1α and Myc. These findings open interesting perspectives for novel combinatorial therapeutic strategies targeting metabolic checkpoints to block the proliferation of T cells.

Resveratrol reverses the Warburg effect by targeting the pyruvate dehydrogenase complex in colon cancer cells.

Resveratrol (RES), a polyphenol found in natural foods, displays anti-oxidant, anti-inflammatory and anti-proliferative properties potentially beneficial in cancers, in particular in the prevention of tumor growth. However, the rapid metabolism of resveratrol strongly limits its bioavailability. The molecular mechanisms sustaining the potential biological activity of low doses of resveratrol has not been extensively studied and, thus, needs better characterization. Here, we show that resveratrol (10 µM, 48 hr) induces both a cell growth arrest and a metabolic reprogramming in colon cancer cells. Resveratrol modifies the lipidomic profile, increases oxidative capacities and decreases glycolysis, in association with a decreased pentose phosphate activity and an increased ATP production. Resveratrol targets the pyruvate dehydrogenase (PDH) complex, a key mitochondrial gatekeeper of energy metabolism, leading to an enhanced PDH activity. Calcium chelation, as well as the blockade of the mitochondrial calcium uniport, prevents the resveratrol-induced augmentation in oxidative capacities and the increased PDH activity suggesting that calcium might play a role in the metabolic shift. We further demonstrate that the inhibition of the CamKKB or the downstream AMPK pathway partly abolished the resveratrol-induced increase of glucose oxidation. This suggests that resveratrol might improve the oxidative capacities of cancer cells through the CamKKB/AMPK pathway.

6-mercaptopurine promotes energetic failure in proliferating T cells.

The anticancer drug 6-mercaptopurine (6-MP) inhibits de novo purine synthesis and acts as an antiproliferative agent by interfering with protein, DNA and RNA synthesis and promoting apoptosis. Metabolic reprogramming is crucial for tumor progression to foster cancer cells growth and proliferation, and is regulated by mechanistic target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) as well as the oncogenes Myc and hypoxia inducible factor 1α (HIF-1α). We hypothesized that 6-MP impacts metabolic remodeling through its action on nucleotide synthesis. The aim of our study is to provide a comprehensive characterization of the metabolic changes induced by 6-MP in leukemic T cells. Our results indicate that exposition to 6-MP rapidly reduces intracellular ATP concentration, leading to the activation of AMPK. In turn, mTOR, an AMPK target, was inhibited, and the expression of HIF-1α and Myc was reduced upon 6-MP incubation. As a consequence of these inhibitions, glucose and glutamine fluxes were strongly decreased. Notably, no difference was observed on glucose uptake upon exposition to 6-MP. In conclusion, our findings provide new insights into how 6-MP profoundly impacts cellular energetic metabolism by reducing ATP production and decreasing glycolytic and glutaminolytic fluxes, and how 6-MP modifies human leukemic T cells metabolism with potential antiproliferative effects.

Role for the ATPase inhibitory factor 1 in the environmental carcinogen-induced Warburg phenotype.

Most tumors undergo metabolic reprogramming towards glycolysis, the so-called Warburg effect, to support growth and survival. Overexpression of IF1, the physiological inhibitor of the F0F1ATPase, has been related to this phenomenon and appears to be a relevant marker in cancer. Environmental contributions to cancer development are now widely accepted but little is known about the underlying intracellular mechanisms. Among the environmental pollutants humans are commonly exposed to, benzo[a]pyrene (B[a]P), the prototype molecule of polycyclic aromatic hydrocarbons (PAHs), is a well-known human carcinogen. Besides apoptotic signals, B[a]P can also induce survival signals in liver cells, both likely involved in cancer promotion. Our previous works showed that B[a]P elicited a Warburg-like effect, thus favoring cell survival. The present study aimed at further elucidating the molecular mechanisms involved in the B[a]P-induced metabolic reprogramming, by testing the possible involvement of IF1. We presently demonstrate, both in vitro and in vivo, that PAHs, especially B[a]P, strongly increase IF1 expression. Such an increase, which might rely on ß2-adrenergic receptor activation, notably participates to the B[a]P-induced glycolytic shift and cell survival in liver cells. By identifying IF1 as a target of PAHs, this study provides new insights about how environmental factors may contribute to related carcinogenesis.

Paradoxical Inhibition of Glycolysis by Pioglitazone Opposes the Mitochondriopathy Caused by AIF Deficiency.

Mice with the hypomorphic AIF-Harlequin mutation exhibit a highly heterogeneous mitochondriopathy that mostly affects respiratory chain complex I, causing a cerebral pathology that resembles that found in patients with AIF loss-of-function mutations. Here we describe that the antidiabetic drug pioglitazone (PIO) can improve the phenotype of a mouse Harlequin (Hq) subgroup, presumably due to an inhibition of glycolysis that causes an increase in blood glucose levels. This glycolysis-inhibitory PIO effect was observed in cultured astrocytes from Hq mice, as well as in human skin fibroblasts from patients with AIF mutation. Glycolysis inhibition by PIO resulted from direct competitive inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Moreover, GAPDH protein levels were reduced in the cerebellum and in the muscle from Hq mice that exhibited an improved phenotype upon PIO treatment. Altogether, our results suggest that excessive glycolysis participates to the pathogenesis of mitochondriopathies and that pharmacological inhibition of glycolysis may have beneficial effects in this condition.

Early induction of pyruvate dehydrogenase kinase 4 by retinoic acids in adipocytes.

SCOPE: Vitamin A and its metabolites, such as retinoic acids (RA), are related to metabolic diseases, in particular insulin resistance and obesity. Here, we studied the roles of 9-cis RA and all-trans RA on the regulation of pyruvate dehydrogenase kinase 4 (PDK4), an enzyme involved in fatty acid reesterification, which is a crucial metabolic pathway in adipose tissue (AT) lipid homeostasis. METHODS AND RESULTS: 9-cis RA and all-trans RA treatment of human and murine AT explants, as well as adipocytes (3T3-F442A cell line) induces PDK4 expression both at the mRNA and the protein level, via a transcriptional mechanism. Using site-directed mutagenesis and chomatin immuno-precipitation, we showed that this activation involves two new RA responsive elements in the Pdk4 promoter, RAREa (DR1: -125/-112) and RAREb (DR1: -86/-73), specific to AT. Furthermore, even though endogeneous Pdk4 gene was upregulated by RA in Fao cells, a rat hepatoma cell line, the induction did not occur through the newly found RAREs. CONCLUSION: In this study, we showed that adipocyte PDK4 gene is a new target of the vitamin A derived RA and might participate to the reduced fatty acid efflux from the adipocyte, a step that plays an important role in the developement of metabolic diseases.

The environmental carcinogen benzo[a]pyrene induces a Warburg-like metabolic reprogramming dependent on NHE1 and associated with cell survival.

Cancer cells display alterations in many cellular processes. One core hallmark of cancer is the Warburg effect which is a glycolytic reprogramming that allows cells to survive and proliferate. Although the contributions of environmental contaminants to cancer development are widely accepted, the underlying mechanisms have to be clarified. Benzo[a]pyrene (B[a]P), the prototype of polycyclic aromatic hydrocarbons, exhibits genotoxic and carcinogenic effects, and it is a human carcinogen according to the International Agency for Research on Cancer. In addition to triggering apoptotic signals, B[a]P may induce survival signals, both of which are likely to be involved in cancer promotion. We previously suggested that B[a]P-induced mitochondrial dysfunctions, especially membrane hyperpolarization, might trigger cell survival signaling in rat hepatic epithelial F258 cells. Here, we further characterized these dysfunctions by focusing on energy metabolism. We found that B[a]P promoted a metabolic reprogramming. Cell respiration decreased and lactate production increased. These changes were associated with alterations in the tricarboxylic acid cycle which likely involve a dysfunction of the mitochondrial complex II. The glycolytic shift relied on activation of the Na(+)/H(+) exchanger 1 (NHE1) and appeared to be a key feature in B[a]P-induced cell survival related to changes in cell phenotype (epithelial-to-mesenchymal transition and cell migration).

The pyruvate dehydrogenase complex in cancer: An old metabolic gatekeeper regulated by new pathways and pharmacological agents.

Cancer cells exhibit an altered metabolism which is characterized by a preference for aerobic glycolysis more than mitochondrial oxidation of pyruvate. This provides anabolic support and selective growth advantage for cancer cells. Recently, a new concept has arisen suggesting that these metabolic changes may be due, in part, to an attenuated mitochondrial function which results from the inhibition of the pyruvate dehydrogenase complex (PDC). This mitochondrial complex links glycolysis to the Krebs cycle and the current understanding of its regulation involves the cyclic phosphorylation and dephosphorylation by specific pyruvate dehydrogenase kinases (PDKs) and pyruvate dehydrogenase phosphatases (PDPs).

Butyrate elicits a metabolic switch in human colon cancer cells by targeting the pyruvate dehydrogenase complex.

Butyrate, a short-chain fatty acid produced by the colonic bacterial fermentation is able to induce cell growth inhibition and differentiation in colon cancer cells at least partially through its capacity to inhibit histone deacetylases. Since butyrate is expected to impact cellular metabolic pathways in colon cancer cells, we hypothesize that it could exert its antiproliferative properties by altering cellular metabolism. We show that although Caco2 colon cancer cells oxidized both butyrate and glucose into CO(2) , they displayed a higher oxidation rate with butyrate as substrate than with glucose. Furthermore, butyrate pretreatment led to an increase cell capacity to oxidize butyrate and a decreased capacity to oxidize glucose, suggesting that colon cancer cells, which are initially highly glycolytic, can switch to a butyrate utilizing phenotype, and preferentially oxidize butyrate instead of glucose as energy source to produce acetyl coA. Butyrate pretreated cells displayed a modulation of glutamine metabolism characterized by an increased incorporation of carbons derived from glutamine into lipids and a reduced lactate production. The butyrate-stimulated glutamine utilization is linked to pyruvate dehydrogenase complex since dichloroacetate reverses this effect. Furthermore, butyrate positively regulates gene expression of pyruvate dehydrogenase kinases and this effect involves a hyperacetylation of histones at PDK4 gene promoter level. Our data suggest that butyrate exerts two distinct effects to ensure the regulation of glutamine metabolism: it provides acetyl coA needed for fatty acid synthesis, and it also plays a role in the control of the expression of genes involved in glucose utilization leading to the inactivation of PDC.

Down-regulation of the phosphoenolpyruvate carboxykinase gene in human colon tumors and induction by omega-3 fatty acids.

The polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) reduces proliferation of several cell types, including colon tumor cells, and regulates gene expression in a cell- and gene-selective manner. In hepatocytes, the fatty acid synthase (FAS) gene is down-regulated by DHA whereas the carnitine palmitoyltransferase-1 (CPT-1) gene is up-regulated. In adipocytes but not in hepatocytes, the expression of the cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) gene is stimulated by unsaturated FA, including DHA. We monitored the expression of the FAS, CPT-1 and PEPCK-C genes in rat and human colon and in colonic tumors from humans. The ratio of PEPCK-C to FAS transcripts was in favor of PEPCK-C in human and rat colon, whereas the opposite occurred in Caco2 tumoral cells. FAS gene expression declined from proliferative to differentiated Caco2 cells, while in contrast the expression of PEPCK-C and CPT-1 genes increased. DHA strongly induced expression of the PEPCK-C and CPT-1 genes, in correlation with decreased cell growth, while, as expected, it reduced FAS mRNA. We assessed the relative expression of PEPCK-C, CPT-1 and FAS genes in fragments of colonic tumors and adjacent non-tumoral tissue from a series of 10 patients. PEPCK-C and CPT-1 mRNAs are more abundant in non-tumoral tissues than in the tumoral counterpart, whereas the opposite occurred for the FAS gene. Therefore, the PEPCK-C gene can be defined as a new negative marker for colonic tumors and a target for the anti-tumorigenic action of omega-3 PUFAs.

Pyruvate dehydrogenase kinase 4: regulation by thiazolidinediones and implication in glyceroneogenesis in adipose tissue.

OBJECTIVE: Pyruvate dehydrogenase complex (PDC) serves as the metabolic switch between glucose and fatty acid utilization. PDC activity is inhibited by PDC kinase (PDK). PDC shares the same substrate, i.e., pyruvate, as glyceroneogenesis, a pathway controlling fatty acid release from white adipose tissue (WAT). Thiazolidinediones activate glyceroneogenesis. We studied the regulation by rosiglitazone of PDK2 and PDK4 isoforms and tested the hypothesis that glyceroneogenesis could be controlled by PDK. RESEARCH DESIGN AND METHODS: Rosiglitazone was administered to Zucker fa/fa rats, and then PDK4 and PDK2 mRNAs were examined in subcutaneous, periepididymal, and retroperitoneal WAT, liver, and muscle by real-time RT-PCR. Cultured WAT explants from humans and rats and 3T3-F442A adipocytes were rosiglitazone-treated before analyses of PDK2 and PDK4 mRNA and protein. Small interfering RNA (siRNA) was transfected by electroporation. Glyceroneogenesis was determined using [1-(14)C]pyruvate incorporation into lipids. RESULTS: Rosiglitazone increased PDK4 mRNA in all WAT depots but not in liver and muscle. PDK2 transcript was not affected. This isoform selectivity was also found in ex vivo-treated explants. In 3T3-F442A adipocytes, Pdk4 expression was strongly and selectively induced by rosiglitazone in a direct and transcriptional manner, with a concentration required for half-maximal effect at 1 nmol/l. The use of dichloroacetic acid or leelamine, two PDK inhibitors, or a specific PDK4 siRNA demonstrated that PDK4 participated in glyceroneogenesis, therefore altering nonesterified fatty acid release in both basal and rosiglitazone-activated conditions. CONCLUSIONS: These data show that PDK4 upregulation in adipocytes participates in the hypolipidemic effect of thiazolidinediones through modulation of glyceroneogenesis.